Wound healing compositions and uses thereof

Abstract

The present disclosure provides a pharmaceutical composition comprising an Agrin fragment or a derivative thereof, and uses of the pharmaceutical composition. The present disclosure also provides a method of producing the pharmaceutical composition.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to Singapore Provisional Application No. 10202007138Y, filed on 24 July 2020, the contents of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0003] This invention generally relates to the fields of molecular biology and biochemistry. In particular, this invention relates to compositions for treating wounds. Background Technology

[0004] It is estimated that more than 400 million people worldwide have diabetes (http: / / www.idf.org). In Singapore alone, it is estimated that 11% of people aged 20-80 have diabetes, second only to the United States, and this number is expected to rise significantly.

[0005] A major complication of diabetes is non-healing wounds. What begins as a small scratch can develop into a large, unhealing wound, in some cases requiring amputation. In Singapore, non-healing wounds are a leading cause of non-traumatic lower limb amputations, with more than four amputations occurring daily. Non-healing wounds or ulcers can persist for 12 months or longer and have a very high recurrence rate of 65%. It is estimated that 33% of the annual diabetes budget is spent on diabetic foot ulcers. In recent years, the inability to effectively treat non-healing wounds has led to a dramatic increase in wound care costs in Singapore, currently estimated to exceed S$700 million annually.

[0006] Despite the prevalence of non-healing wounds and the significant medical burden they impose, there remains no effective treatment. Therefore, the need for compositions and methods for treating non-healing wounds remains unmet. Summary of the Invention

[0007] In one aspect, a pharmaceutical composition comprising an Agrin fragment or a derivative thereof is provided, wherein the Agrin fragment or derivative thereof comprises the LG3 domain of Agrin and an eight-amino acid insert ELANEIPV (SEQ ID NO:1) at the z-site of the LG3 domain.

[0008] In another aspect, a vector is provided comprising a nucleic acid molecule encoding an Agrin fragment or a derivative thereof, wherein the Agrin fragment or derivative thereof comprises the LG3 domain of Agrin and an eight-amino acid insert ELANEIPV (SEQ ID NO:1) at the z site of the LG3 domain.

[0009] In another aspect, host cells containing vectors as disclosed herein are provided.

[0010] In another aspect, hydrogels or scaffolds comprising pharmaceutical compositions as disclosed herein are provided.

[0011] In another aspect, drugs, hydrogels, or stents, as disclosed herein, are provided for treatment.

[0012] On the other hand, a method for treating a wound is provided, the method comprising administering to a subject in need a pharmaceutically effective amount of a pharmaceutical composition, hydrogel, or scaffold disclosed herein.

[0013] On the other hand, the use of pharmaceutically effective amounts of pharmaceutical compositions, hydrogels or scaffolds, such as those disclosed herein, in the preparation of medicaments for the treatment of wounds is provided. Attached Figure Description

[0014] The invention will be better understood when considered in conjunction with non-limiting examples and the accompanying drawings, and with reference to the detailed description, in which:

[0015] Figure 1The results of assays investigating the effects of skin injury on the expression of multiple proteins, including Agrin, are presented. (a) Gene expression analysis of wound features based on ECM from single-cell transcriptomics of injured cells in a wound-healing mouse model (Student's t-test, *p<0.05, **p<0.005, respectively). (b) Confluent cells were scraped from the wound and allowed to migrate. After specified time points, mRNA isolated from migrating cells was analyzed by RT-PCR for specified genes. Data are presented as mean + / - sd. Multiple t-tests were performed on all genes to compare different stages after injury; p-values ​​are presented in tabular form (grey values ​​indicate decrease). (c) Western blot analysis of migrating skin cells (Figure b) for Agrin expression. GAPDH was used as a loading control. The optical density quantification of Agrin levels in specified cells is illustrated. Data are presented as mean + / - sd (n = 3 experiments, multiple t-test, p-values ​​as shown in the figure). (d) Western blot analysis of the specified protein in uninjured keratinocytes and fibroblasts. GAPDH was used as a loading control. The optical density quantification of Agrin levels in the specified cells is illustrated (n = 3 experiments, **p = 0.001, *p = 0.02, Student's t' test). (e) Confocal microscopy analysis of Agrin immunofluorescence in mouse skin perforation wound biopsy sections at specified days, counterstained with DAPI. Scale bar: 50 μm. The mean fluorescence intensity (MFU) of Agrin staining in mouse skin sections is quantified graphically. Data are presented as mean + / - sd (n = 3 mice, three sections analyzed per analysis, **p = 0.003, Student's t' test). White dashed lines represent the epidermal and dermal boundaries, respectively. (f) Immunohistochemical sections of mouse skin perforation wound biopsies at specified days post-injury show relative Agrin expression. Scale bar: 100 μm, EP - epidermis, D - dermis, d - WAT - white adipose tissue in the dermis. Dashed lines indicate the boundary between the epidermis and dermis. Mean Agrin intensity in mouse skin sections was graphically quantified using ImageJ. Data are presented as mean + / - sd (n = 3 mice, three sections analyzed at each time point, ANOVA and Student's t test, p-values ​​are shown in the figure). Results showed that Agrin expression was triggered during mechanical injury to the skin.

[0016] Figure 2This study investigated the effects of AGRN and GPC1 knockdown on keratinocyte migration after trauma. (a) RT-PCR analysis showed AGRN and GPC1 knockdown in HaCaT cells. (b) Wound scraping assays were performed on control, Agrin-depleted, and GPC1-knockdown cells. Representative bright-field images showing relative migration were presented at specified time points. Mean unmigrated area + / - sd was quantified using ImageJ (n=3, Student's t-test, **p=0.005, ****p=0.000009, ****p=0.00002, respectively). Scale bar: 50 μm. (c) Comparison of migration rates of Agrin- and GPC1-knockdown HaCaT cells over a 24-hour period following wound scraping (n=3, Student's t-test, ****p<0.00001). The results indicate that Agrin and Glypican-1 play a role in keratinocyte migration after trauma.

[0017] Figure 3This study demonstrates the effect of Agrin knockdown on wound healing in vivo. (a) The protocol shows Agrin siRNA treatment before injury and the application of Aquaphore ointment containing siRNA to injured mouse skin. (b) Western blots of wound lysates from perforated biopsies of mice 72 hours after intradermal siRNA injection on day 0 of wound injury, with and without fixation (left) and with fixation (right). Agrin was detected in mouse skin lysates. Actin or GAPDH was used as loading controls. (c) Confocal microscopy images of Agrin immunostaining in injured mouse skin treated with control and specified Agrin siRNA in an unfixed wound on day 5 post-injury. Scale bar: 50 μm. White dashed lines indicate migrating epidermis. (d) Mouse skin sections stained with hematoxylin and eosin after treatment with control and Agrin siRNA #1 on day 8 post-injury (n = 4 mice / group). Double arrows indicate uncovered wound areas. An asterisk (*) indicates a migrating epithelial tongue covering the wound. Scale bar: 50 μm. (e) Western blot confirming Akrin knockdown in HEK cells induced by a stealth siRNA targeting human Akrin. GAPDH was used as a loading control. (f) Confocal microscopy images of Akrin immunostaining in human skin explants treated with control and Akrin siRNA #1. A batch of Akrin siRNA-treated explants was subsequently rescued with sAgrin starting on day 2. Images are shown on day 8 post-traumatic injury. Cell nuclei were stained with DAPI. Arrows indicate uncovered wound areas. Scale bar: 50 μm. (g) Representative hematoxylin and eosin stained sections of human skin explants treated with 75 nM control or Akrin siRNA #1 on day 8 post-traumatic injury. The third figure consists of skin explant sections treated with Agrin siRNA#1 ointment on day 0, but subsequently treated with additional ointments containing sAgrin (20 μg) on ​​days 2, 4, and 6, respectively. Arrows indicate migrating epithelial tongues covering the wound area in control skin sections. Double arrows indicate uncovered wound areas. Scale bar: 500 μm. The results show that silencing Agrin delayed wound healing in vivo.

[0018] Figure 4Results of an investigation into the effect of Agrin depletion on skin wound healing are presented. (a) Mouse skin was treated with randomized or AgrinsiRNA #1-3. Three days later, the siRNA-treated areas were perforated, followed by treatment with an Aquaphore-based topical ointment containing 75 nM of the specified siRNA every 2–3 days. Representative photographs of the perforated wounds of mouse skin treated with the aforementioned siRNA-containing topical ointment at specified days post-traumatic incision are shown. Mean wound diameter is plotted against specified days post-traumatic incision. Data are presented as mean + / - sd (n = 4 mice / group, two-way ANOVA, *p = 0.02, 0.03, and 0.01, respectively). Scale bar: 1 mm. (b) Confocal micrographs of K17 and DAPI staining in migrating epithelial tongues in mouse skin sections treated with the specified siRNA are shown on day 5 post-traumatic incision. Scale bar: 50 μm. Relative K17 staining intensity is expressed as mean + / - sd (n = 3 slices / group, Student's t' test, p = 0.01, p = 0.002, respectively). (c) Photographs of the fixation wound dressing protocol used in the ICR mouse strain are shown. Wound diameters of fixed (closed) and unfixed (open) wounds were quantified on day 7 (n = 3 mice per group, *p = 0.05, Student's t' test). (d) Mouse skin was treated with randomized or Agrin siRNA #1. Three days later, the siRNA-treated areas were perforated and subsequently treated every 2–3 days with an Aquaphore-based topical ointment containing 75 nM of the specified siRNA. Wounds secured with 10 mm fixation tethers were covered with Tegaderm. Representative photographs are shown at specified days of dressing and siRNA ointment application. Mean wound diameter is plotted against specified days post-traumatic wounding. Data are presented as mean ± sd (n = 3 mice / group, ANOVA p = 0.003). (e) Confocal micrographs of K17 and DAPI staining in migrating epithelial tongue in mouse skin sections treated with the specified siRNA are shown on day 7 post-traumatic injury. Scale bar: 50 μm. White arrows indicate weakened epithelial tongue. Relative K17 occupancy is presented as mean ± sd (n = 3 sections / group, Student's t test, p = 0.001). (f) Sirius red staining shows the relative collagen distribution in the wound bed of control and Agrin siRNA-treated mouse skin on day 7. Representative bright-field and polarized light images are shown. Boxed areas indicate magnified views. Scale bar: 100 μm. Relative collagen fibers were quantified using ImageJ (analysis of n = 4 sections from 3 mice per group, Student's t test, *p = 0.02).(g) Human skin explants were perforated and treated every three days with an ointment containing either a 75 nM randomized control or Agrin siRNA#1. For group 3, 20 μg sAgrin was incorporated into the ointment after day 2 and applied every two days. Representative photographs of the skin wounds on day 0 and day 10 are shown. Relative wound diameters were calculated on day 10 post-injury and expressed as mean + / - sd (n = 3 / group, Student's t-test, ***p = 0.0001, *p = 0.03, respectively). Scale bar: 0.5 mm. (h) Representative confocal images showing K17 staining in migrating keratinocytes in the wound bed of the covered control, Agrin-depleted, and those rescued by sAgrin skin explants. Arrows mark the leading edge of the migrating keratinocytes. White lines indicate the wound boundary. Scale bar: 50 μm. The results show that Agrin depletion impairs skin wound healing.

[0019] Figure 5This study demonstrates the effect of Agrin knockdown on the 2D migration of keratinocytes and dermal fibroblasts. (a) RT-PCR analysis of Agrin gene expression in BJ and HaCaT cells treated with control or Agrin siRNA. Data are expressed as mean + / - sd. These knockdown cells were then used in a transwell migration assay. A batch of Agrin-depleted cells was analyzed for migration in response to sAgrin (10 μg / ml) as a chemical inducer in the bottom chamber (Figure 3). Migration was quantified using ImageJ and expressed as mean + / - sd (n = 3, Student's t' test, p = 0.0004, 0.0003, and 0.0005, respectively). Scale bar: 20 μm. (b) Scraping wound assays of control, Agrin-depleted, and Agrin-knockdown BJ or primary mouse dermal fibroblast (DF) cells pretreated with 10 μg / ml sAgrin for 18 h. Representative bright-field images showing relative migration were presented at specified time points. Mean unmigrated area + / - sd was quantified using ImageJ (n=3, Student's t-test, *p<0.05, **p<0.004, ***p<0.0005, ****p=0.000002, ****p<0.000001, respectively). Scale bar: 20 μm. Western blots validating Agrin knockdown in DF are also shown, with GAPDH as a loading control. (c) Scratching wound assays in specified keratinocytes that were Agrin depleted and Agrin knockdown pretreated with 10 μg / ml sAgrin for 18 h. Representative bright-field images are shown. Scale bar: 20 μm. RT-PCR analysis confirming Agrin expression in HEK cells also presented as mean + / - sd (middle panel), while Western blot confirming Agrin knockdown was observed in primary mouse keratinocytes (far right panel). Results were quantified as shown in Figure (b) (n=3, Student's t-test, ****p<0.000001, **p<0.005, *p<0.05, all other p-values ​​are shown in the figure). (d) Comparison of migration rates of specified cells treated in Figures (b) and (c) at 15 hours post-traumatic injury (n=4 independent migration events, Student's t-test, p-values ​​shown in the figure). Results showed that Agrin knockdown inhibited 2D migration of keratinocytes and dermal fibroblasts.

[0020] Figure 6Agrin affects keratinocyte proliferation after wound injury. (a) Control, Agrin-depleted specified keratinocytes, and those rescued with 10 μg / ml sAgrin for 18 hours were labeled with 20 μM BrdU for 12 hours before wound-scraping assays. Cells were fixed 4 hours after scraping and stained with anti-BrdU and K17 antibodies. Cell nuclei were counterstained with DAPI. Representative confocal images at the wound margin (leader) and at least 200 μm from the wound margin (follower) are shown. The relative percentage of proliferating cells for each condition is shown graphically (Student's t-test for n = 5–6 images from three replicates of each condition, **p = 0.006, *p - 0.003, p = 0.005, *** = 0.0001, respectively). Scale bar: 10 μm. (bc) Schematic diagram showing the proliferative status at the migrating wound margin. Immunohistochemical sections stained with Ki67 were obtained from wound margins and wound beds in control and Agrin-depleted areas on day 7 post-traumatic injury. Solid arrows indicate the base of the epidermis (Ep), while arrows indicate the wound margin. An asterisk (*) indicates loss of Ki67 proliferation at the wound margin. Scale bar: 100 μm. The percentage of proliferating cells was quantified using Image J (analysis of n=4 sections from two separate mice, Student's t-test, **p=0.001). The results show that Agrin affects keratinocyte proliferation following wound injury.

[0021] Figure 7The results of assays investigating whether Agrin generates a mechanically competent environment conducive to collective keratinocyte migration and wound closure are shown. (a) Confluent treated HaCaT cells were cultured for 18 h in a single soft substrate or in a soft substrate supplemented with sAgrin (10 μg / ml). After removal of the barrier stencil, the collective migration of cells was imaged at 0 h and 24 h under a 4X bright-field microscope. Untreated HaCaT cells cultured on a hard substrate served as a control. Representative wound areas co-covered by HaCaT cells at different ECM stiffness are shown as mean + / - sd (n = 3, Student's t' test, **p < 0.001). Scale bar: 50 μm. (b) Untreated or sAgrin (10 μg / ml) HaCaT cells were cultured in a soft substrate and allowed to migrate as in (a). Cells were fixed and stained with K17 4 h after barrier removal. Representative confocal images show K17 and DAPI staining in the migration front. Scale bar: 10 μm. Quantitative relative K17 intensity was used to represent the leader cell fraction in untreated and sAgrin-treated HaCaT cells 4 h post-migration (n = 10 images / group, Student's t-test, *p = 0.01). White arrows indicate leader cells. Dashed lines indicate wound edges. (c) Control and Agrin-depleted HaCaT cells were grown in separate hard substrates for 18 h, and their migration potential was analyzed at 0 and 24 h after barrier removal. A batch of Agrin-depleted cells was cultured in a hard substrate containing 10 μg / ml sAgrin for 18 h, followed by migration analysis as described above. Representative bright-field images and quantitative migration areas covered by cells for each condition are shown as mean + / - sd. Scale bar: 50 μm. Western blots show Agrin knockdown in HaCaT cells. GAPDH was used as a loading control. (d) Cells treated in the same manner as in (c) were fixed 4 hours after barrier removal and stained for K17 and DAPI. Representative confocal images are shown. Scale bar: 10 μm. Dashed lines indicate wound edges. The relative percentage of K17-expressing leader cells is quantified and expressed as mean + / - sd (n = 10 images per condition, Student's t-test, **p = 0.002 and 0.001, respectively). (e) A 3D stiffness-dependent migration assay of mouse primary keratinocytes relative to dermal fibroblasts on days 0 and 5 post-traumatic stress in the presence or absence of 10 μg / ml sAgrin in the soft matrix (0.8 kPa). Representative bright-field images of migrating cells are shown. Scale bar: 100 μm.Relative migration area was quantified by ImageJ (n = 3 images / group, Student's t-test, *p = 0.02). The black dashed line indicates the original wound area. (f) Representative bright-field images of migration of primary mouse keratinocytes and dermal fibroblasts individually or in the presence of sAgrin embedded in a hard matrix (30 kPa) at specified days, control, and Agrin-depleted cells. Scale bar: 100 μm. Relative migration area was quantified by ImageJ (n = 3 images per group, Student's t-test, *p = 0.05, **p = 0.005, **p = 0.002, and ***p = 0.0005, respectively). Western blot images reveal the degree of Agrin knockdown in specified cells. GAPDH was used as a loading control. (g) A schematic diagram of skin extracted from 3-day-old mouse pups, which was subsequently cultured in separate soft or hard ECMs or ECMs containing sAgrin. (h) Bright-field micrographs showing keratinocyte outgrowth from mouse skin explants grown on soft ECMs alone or supplemented with 20 μg / ml sAgrin on day 5. Outgrowth area was measured by ImageJ and is graphically represented as mean + / - sd (n = 3 explants / group, Student's t' test, *p = 0.03). Scale bar: 50 μm. (i) Mouse skin explants cultured on hard ECMs were treated with mouse controls or Agrin siRNA for 3 days. A batch of Agrin-depleted mouse explants was then cultured for another 2 days on hard ECMs conditioned with 20 μg / ml sAgrin. Representative RT-PCR analysis of Agrin expression on day 5 is shown as mean + / - sd (n = 3 mouse explants per group). Bright-field images showing keratinocyte outgrowth are also shown. Scale bar: 50 μm. The growth area was quantified using ImageJ and expressed as mean + / - sd (n = 3 explants per group, Student's t-test, *p = 0.019 and 0.04, respectively). (j) AFM measurements of cell stiffness in migrating controls, Agrin-depleted cells, and those rescued with 10 μg / ml sAgrin at 4 hours post-traumatic stress. Relative stiffness is shown in Pascals (n = 10–15 cells per group, triple replicates, Student's t-test, *p = 0.01 and *p = 0.008, respectively). (k) Thermographs showing traction forces in migrating controls, Agrin-depleted cells, and rescued HaCaT cells on the microfabricated substrate. White arrows point to the leading edge. Scale bar: 100 μm. Calculations of leading edge traction forces under all conditions (n ​​= 6–10 images from three experiments, Student's t-test, *p = 0.02 and 0.04, respectively).(l) PIV analysis shows the velocity vectors in the migration control, Agrin-depleted, and salvaged HaCaT on the PDMS substrate. Green arrows indicate the velocity field. White asterisks indicate eddy regions. Scale bar: 100 μm. Quantified velocities are presented graphically (n=3, Student's t-test, ***p=0.0001). The results indicate that Agrin sensitizes collective migration under ECM stiffness by enhancing traction stress and hydrodynamics.

[0022] Figure 8 The effect of Agrin on the stiffness of migrating keratinocytes is shown. (a) Scratching assays were performed on primary mouse keratinocytes pretreated with 10 μg / ml sAgrin for 18 h, including control, Agrin-depleted, and Agrin-knockdown cells. Cells at the leading edge (white line) were analyzed by AFM 4 h after scraping. A representative Western blot validating Agrin knockdown is shown, with GAPDH as a loading control. Scale bar: 10 μm. (b) AFM stiffness maps under specified conditions are shown. Force scale represents 0–7 kPa. The results show that Agrin induced stiffness in migrating keratinocytes.

[0023] Figure 9The results of assays investigating whether Agrin modulates cellular mechanics during wound injury by coordinating cytoskeleton structure are shown. (a) Confocal images of the wound margins of confluent controls, Agrin-depleted cells, and cells treated with 10 μg / ml sAgrin for 18 hours at specified time points after injury. Cells were immunostained for phosphorylated myosin light chain (pMLC) and F-actin and counterstained with DAPI. Scale bar: 10 μm. (b) Confluent cultures of control, Agrin siRNA-treated, and Agrin-depleted HEK cells treated with 10 μg / ml sAgrin for 18 hours were scraped. Cell lysates from the migration region were collected at specified time points, and pMLC activation was analyzed by Western blotting. Agrin expression validated the knockdown efficacy, while actin was used as a loading control. (cd) Confluent HEK cells untreated or nutrient-treated with 10 μg / ml sAgrin for 18 hours. Prior to wound scraping, a batch of sAgrin-nurtured HEK cells were further subjected to 10 μm blebbistatin for 2 hours and allowed to migrate for specified time periods, either alone or in the presence of sAgrin and blebbistatin. Following scraping, cells were fixed at specified time points and stained for pMLC, F-actin, and DAPI. Representative confocal images are shown. Scale bar: 10 μm (c). For Figure d, migrating cells were fixed 18 hours post-traumatically and visualized under a bright-field microscope. Relative wound area for cell migration under each condition is expressed as mean + / - sd (n = 3, Student's t-test, **p = 0.001, ***p = 0.0001, respectively). (e) HEK cells treated with control and Agrin siRNA #1 were coated with 4.5 μm magnetic beads conjugated with 20 μg BSA or sAgrin at 4 °C for 30 min. The confluent cells were then scraped and allowed to migrate for 30 minutes with or without a permanent magnet applied. At 30 minutes post-traumatic initiation, the cells were fixed and immunostained against pMLCs and actin, and the nuclei were visualized using DAPI. Representative confocal images of sAgrin-coated magnetic beads are shown. Scale bar: 10 μm. White circles indicate beads. White arrows indicate enriched pMLC-actin structures. (f) Quantification of pMLC intensity near sAgrin beads in Agrin-depleted HEK cells as mean intensity from three independent experiments (n = 8–15 cells analyzed, Student's t-test, *p = 0.04, ***p = 0.0001, respectively). Scale bar: 10 μm. White circles indicate beads, while blue dashed circles indicate regions of interest (ROI) quantified around each bead. 200 μm 2 Area. (g) HEK cells were individually treated with a small (800 μ) 2 ) or large (1600μ)2 (h) Confocal images of pMLC intensity maps of HEK cells plated in micropatterns coated with fibronectin or 20 μg / ml sAgrin for 4–6 h. Representative confocal images of cross-shaped cells immunostained for pMLC and F-actin and counterstained with DAPI are shown. The relative number of F-actin stress fibers is quantified as the mean + / - sd from three independent experiments (n = 10 cells per group, Student's t test, **p = 0.005, *p = 0.01, respectively). Scale bar: 10 μm. (h) Confocal images of pMLC intensity maps of HEK cells plated in micropatterns coated with macrofibronectin or fibronectin-sAgrin for 4 h. Scale bar: 10 μm. Quantification of the stress curvature of the transverse arc of the cross-shaped cells is expressed as the mean + / - sd from three experiments (n = 10–15 cells / group, Student's t test, **p = 0.002). (i) Control or Agrin-depleted HEK cells were plated for 4 h on fibronectin patterns or on larger micropatterns coated with fibronectin and sAgrin. The resulting cross-shaped cells were fixed and immunostained with pMLC and F-actin, with nuclei labeled with DAPI. Representative confocal images are shown. Scale bar: 10 μm. The stress curvature at the corresponding transverse arc of the cross-shaped cells, indicated by pMLC intensity, was quantified as the mean + / - sd from three experiments (n = 10 cells per group, Student's t-test, **p = 0.001, **p = 0.005, respectively). The results indicate that Agrin modulates cell mechanics during wound injury.

[0024] Figure 10 The effect of Agrin-induced force recognition on actomyosin binding is shown. (a) Magnetic force transfer to injured keratinocytes. Briefly, keratinocytes (HaCaT) were coated with magnetic beads pre-conjugated with control or sAgrin at 4°C for 30 min. Cells were then scraped and allowed to migrate at 37°C under standard culture conditions or under a 200 pN force applied for 30 min with a permanent magnet placed 6 mm above the cells. (b) SDS-PAGE gels show the individual conjugation of 20 μg of ligands sAgrin, bovine serum albumin (BSA), and fibronectin (FN) to the magnetic beads. Following covalent conjugation, the corresponding free proteins in the bead-free supernatant (detected in the supernatant prior to incubation with the magnetic beads) were reduced. (c) HaCaT cells coated with BSA, sAgrin, or FN beads for 30 min were then migrated for another 30 min under standard conditions or under a permanent magnet force. Cells were then fixed and stained for active integrin β1 (HUTS-4 clone) and Agrin. Nuclei were counterstained with DAPI. White circles indicate the location of the magnetic beads. Scale bar: 10 μm. Quantification was performed in three experiments within 200 μm of each bead. 2Relative intensity at area, expressed as mean + / - sd (n = 10-15 cells / group). (de) HEK cells treated with control and Agrin siRNA #1 were subjected to force transduction as in (b) 72 hours after siRNA treatment with sAgrin and FN (d) or multiligand glycan 4 beads (e). Proteins adhered to the beads were magnetically separated and resolved in Western blot analysis for probing pMLC and total MLC. Agrin was probing each cell lysate, with GAPDH used as a loading control. (fh) Representative confocal images of Agrin-depleted HEK cells probing with sAgrin (f), FN (g), or BSA (h) coated magnetic beads under standard conditions (no force) or with applied permanent magnet force. Cells were fixed and immunostained for pMLC and F-actin, respectively. Cell nuclei were labeled with DAPI. White circles represent beads. Scale bar: 10 μm. (i) Agrin-depleted HaCaT cells were exposed to increased concentrations of sAgrin-coated magnetic beads for 30 minutes. Following trauma, cells were allowed to migrate for 30 minutes under no force or with applied permanent magnet force, followed by fixation and staining for pMLCs. Cell nuclei were stained with DAPI. Block arrows indicate enhanced pMLC recruitment near the beads. Cells were stained with DAPI around 200 μm of the beads according to three experiments (n = 10–15 cells). 2 The pMLC intensity of the region was quantified and expressed as mean + / - sd. Scale bar: 10 μm. (j) Agrin-depleted HaCaT cells were treated with sAgrin beads and allowed to migrate at specified time points under sustained force application. The resulting cells were treated and quantified as in (i). The results showed that the force recognition of injured keratinocytes by Agrin introduced the involvement of actomyosin.

[0025] Figure 11 The results of transcriptomic analysis of Agrin-depleted keratinocytes are shown. (a) RNA-Seq, revealing loss of Agrin gene expression (n = 3 replicates). MDS plot of large populations from RNA-Seq of siControl and siAgrin HaCaT (n = 3 samples). (b) Gene Ontology (GO) analysis in Agrin-knockdown HaCaT cells shows the most significantly downregulated gene clusters. (c) GSEA analysis at Agrin knockdown reveals a set of genes belonging to ECM structural components that are highly downregulated. (d) Upregulated and downregulated gene clusters in the NABA matrix (ECM-related) after Agrin depletion in HaCaT cells. (e) RT-PCR analysis validated a selected set of significantly upregulated and downregulated genes.

[0026] Figure 12The results of assays identifying downstream effectors of Agrin-mediated mechanotropic stress in keratinocytes following wound injury are shown. (a) Volcano plot showing differentially expressed genes in control and Agrin-depleted HaCaT cells cultured in hard plastic plates. Inset shows MMP levels. (n = 3 replicates). (b) GSEA analysis shows downregulated clusters corresponding to genes mediating positive regulation of wound. (c) HaCaT cells plated on a soft substrate were treated with increasing concentrations of sAgrin for 18 hours (left panel) or with 10 μg / ml sAgrin for a specified time (right panel). Western blot analysis was performed to detect MMP12 and Agrin levels. GAPDH was used as a loading control. (d) Control or Agrin siRNA-treated HaCaT cells were plated on a hard substrate. Multiple batches of Agrin-depleted cells were further plated on hard substrates containing increasing concentrations of sAgrin and cultured for 18 hours. Subsequently, specified proteins in cell lysates were analyzed by Western blot. Actin was used as a loading control. Densitometry results for MMP12 in three independent assays were quantified as mean + / - sd (Student's t-test, *p<0.03, **p<0.005, respectively). (e) Western blots are presented to detect MMP12 levels in control and MMP12 siRNA-treated HaCaT or mouse keratinocytes (KRT) used in the scraping assay. The scraping assay used control, Agrin-depleted, and Agrin-depleted HaCaT / mouse KRT pretreated with 10 μg / ml Agrin for 18 h. Representative bright-field images at specified time points post-traumatic injury are shown. Results were quantified as the area of ​​unmigrated wounds left in each group, expressed as mean + / - sd (n=3, Student's t-test, **p<0.005, ***p=0.0001, respectively). Scale bar: 50 μm. (f) Western blots showing MMP12 knockdown in the corresponding mouse KRT and DF are displayed. GAPDH was used as a loading control. Representative bright-field images of primary mouse keratinocytes and dermal fibroblasts, depleted of MMP12, migrating individually or in the presence of sAgrin at 10 μg / ml embedded in a soft matrix (0.8 kPa), at specified days. Scale bar: 100 μm. Relative migration area was quantified by ImageJ (n = 4 wounds per group, multiple 't' tests, ***p = 0.0001 and ****p = 0.00006, respectively). (g) Mouse skin explants placed on a separate soft collagen substrate or a soft collagen substrate with 20 μg / ml sAgrin were treated with solvent (DMSO) or MMP408 (5 nM) for 5 days. Representative bright-field images showing keratinocyte growth from the explants on day 5 are shown. Scale bar: 50 μm.The growth area was quantified using ImageJ and expressed as mean + / - sd (n = 4 explants per group, Student's t test, **p = 0.002). (h) HaCaT cells were treated with control and MMP12 siRNA. Three days later, a batch of MMP12-depleted cells was treated with 10 μg / ml sAgrin for 18 hours. Subsequently, confluent cells were scraped and fixed at designated time points for staining against pMLC, actin, and DAPI. Confocal images of migrating cells are shown. Scale bar: 10 μm. White arrows point to actomyosin cables. (i) Migration of HEK cells treated in the same manner as in (h) at designated time points. Designated proteins from cell lysates of migrating cells were tested by Western blot analysis. GAPDH was used as a loading control. (j) Thermal maps showing traction stress in control and MMP12-depleted HaCaT cells with or without 10 μg / ml sAgrin for 18–24 hours. Scale bar: 100 μm. Leading edge stress in Pascals (Pa) is presented graphically (n = 6–10 images from three experiments, Student's t-test, *p = 0.007, ns p = 0.06, after outlier removal *-siControl vs siMMP12*p = 0.01, siControl vs siMMP12+sAgrin**p = 0.001, Student's t-test, *p = 0.007 respectively). (k) Before wound-scraping, MMP12-depleted HaCaT cells were labeled with sAgrin or FN-conjugated magnetic beads at 4 °C for 30 min. After trauma, migrating cells were fixed individually or under permanent magnets at 37 °C for 30 min and stained for pMLC, actin, and DAPI, respectively. Representative confocal images are shown. Scale bar: 10 μm. White circles represent beads. 200 μ around each bead from three independent experiments (n = 10 cells / group, Student's t test, ns). 2 Quantitative mean pMLC intensity + / - sd in the region. Results show that MMP12 is a downstream effector of Agrin mechanosensing.

[0027] Figure 13The effects of MMP1 and MMP10 depletion on wound healing and cellular mechanical tension were shown. (a) RT-PCR analysis confirmed the inhibition of MMP1 and MMP10 in HaCaT cells. Wound scraping assays were performed on control, MMP1 and MMP10 depleted HaCaT cells, and those pretreated with 10 μg / ml sAgrin for 18 hours. Representative bright-field images showing relative migration were presented at specified time points. Mean unmigrated area + / - sd was quantified using ImageJ (n = 3, Student's t' test, p-values ​​are shown in the figure). Scale bar: 50 μm. (b) The migration rate of specified knockdown cells treated alone or with 10 μg / ml sAgrin was compared at 15 hours post-traumatic injury (n = 4, Student's t' test, ****p = 0.00001, ****p = 0.0001, **p = 0.001, respectively). (c) RT-PCR analysis showed the mRNA expression of MMP1 and MMP10 in HaCaT cells at specified time points post-traumatic injury. (d) Control, MMP1, and MMP10-depleted HaCaT cells were scraped and allowed to migrate for 4 hours. Cells were then fixed and immunostained against pMLC and F-actin. Representative confocal images are shown. Nuclei were counterstained with DAPI. White arrows indicate actomyosin cables at the leading edge. Scale bar: 10 μm. (e) Control, MMP1, and MMP10-depleted HaCaT cells were plated on a large cross pattern for 6 hours. Cells were then fixed and stained against pMLC and F-actin, with nuclei labeled with DAPI. White arrows indicate transverse arcs enriched in pMLC. The associated stress curvature of pMLC-enriched cells was quantified as the mean + / - sd from three independent experiments (n = 6 cells / group, Student's t-test, p = 0.5 and p = 0.16, respectively). The results showed that MMP1 and MMP10 depletion impaired wound healing without affecting cellular mechanical tension.

[0028] Figure 14The effects of Agrin on MMP12 expression, cell mechanics, and collective fluid migration are shown. (a) MMP12 and Agrin expression in cell lysates of control and Agrin siRNA-treated cells were analyzed by Western blotting. Actin and GAPDH were used as loading controls, respectively. (b) Catalytic activity of MMP12 in supernatants of control and Agrin-depleted HaCaT cells was detected by gelatin and casein protease proteolysis. The catalytic activity of MMP12 in supernatants of control, Agrin siRNA-treated cells alone, or cells treated with an increased dose of sAgrin for 24 hours was analyzed by gelatin (left) or casein (right) gel digestion. The appearance of the MMP12 band (~54 kDa) is a result of gelatin digestion of MMP12. Representative images from one of the three independent experiments are shown. The relative MMP12 band intensity was quantified using ImageJ (n=3, Student's t' test, *p=0.02 and 0.03, respectively, multiple t-test, p values ​​are shown in the figure). (c) RT-PCR analysis of MMP12 mRNA in migrating HaCaT cells at 0 h and 24 h post-wound injury. Data are presented as mean + / - sd. Wound-specified cells were allowed to migrate. At specified time points, cell lysates from migrating cells were collected and analyzed for MMP12 by Western blotting. GAPDH was used as a loading control. (d) Confluent HaCaT cells were either untreated or pretreated with sAgrin (10 μg / ml) for 18 h, then scraped and allowed to migrate for 6 h. At specified time points, cells were fixed and immunostained for MMP12 and Agrin, respectively. Representative confocal images are shown. White arrows indicate leader cells at the migration margin. Scale bar: 10 μm. (e) Representative confocal micrographs of mouse skin sections from controls and Agrin-depleted mice, showing MMP12 expression at the wound margin. White dashed lines indicate migrating epithelial tongues. Cell nuclei were counterstained with DAPI. Scale bar: 50 μm. (f) Western blot analysis showing MMP12 levels in control and Agrin-depleted mouse skin tissue on day 4 under fixation conditions. GAPDH was used as a loading control. (g) Control and Agrin-depleted HaCaT cells cultured on a hard substrate. A batch of Agrin-depleted cells was cultured on a hard substrate containing 20 μg / ml sAgrin. Western blot confirmed the loss of MMP12 expression in HaCaT cells. GAPDH was used as a loading control. After confluence, cells were scraped and allowed to migrate for 4 hours. Cells were then fixed and immunostained against K17. Dashed lines indicate the migration front. K17 intensity in leader cells was quantified as mean + / - sd from two independent experiments (n = 20–30 cells, Student's t' test, *p = 0.009 and 0.04, respectively).(hi) HaCaT cells were either untreated or pretreated with 10 μg / ml sAgrin for 18 h. Subsequently, cells were pretreated with a solvent (DMSO) or a 5 nM MMP408 inhibitor for 2 h, then wounded and allowed to migrate again for 4 h in the presence of the solvent or inhibitor. Confocal images showing K17 cells and nuclei stained with DAPI(g) are shown. White dashed lines indicate the migration front. Scale bar: 10 μm. Bright-field micrographs at specified times show migrating cells (h). Uncovered wound area was quantified using ImageJ according to three independent experiments, expressed as mean + / - sd (Student's t' test, *p<0.05, **p<0.005). Scale bar: 50 μm. (j) Confocal images show K17 immunostained growth of mouse skin explants cultured for 5 days on individual soft ECMs or those supplemented with sAgrin in the presence of DMSO or 5 nM MMP408 (n = 2 independent experiments, 3 skin explants per group). White dashed lines indicate the growth area. Scale bar: 50 μm. (k) Control and MMP12-depleted HaCaT cells were plated on large FN-coated micropatterns for 4–6 hours. Additionally, a batch of MMP12-depleted cells was plated on FN patterns containing 20 μg / ml sAgrin for 4–6 hours. Cells were then fixed and stained for pMLC and F-actin, with nuclei labeled with DAPI. White arrows indicate transverse arcs of pMLC enrichment. The relevant stress curvature of pMLC enrichment was quantified as the mean + / - sd from three independent experiments (n = 15–20 cells / group, Student's t test, **p = 0.001–0.004). Scale bar: 10 μm. (l) AFM calculation of cell stiffness in primary mouse keratinocytes in control, MMP12-depleted, and sAgrin-treated MMP12-knockdown cells (n = 10–15 cells from three replicate analyses, Student's t test, *p = 0.01). (m) PIV analysis of fluid velocity during collective migration in control, MMP12-knockdown, and sAgrin-treated cells. Results from one of the three independent experiments are shown. Green arrows indicate velocity vectors. Scale bar: 100 μm. Migration velocity was quantified as the mean + / - sd from four independent experiments (Student's t test, ***p < 0.0001). Agrin-regulated MMP12 enhanced cell mechanics and collective fluid migration.

[0029] Figure 15Demonstrates MMP12 knockdown in mouse skin. (a) Mice were treated with either control or MMP12-specific siRNA. Three days later, skin tissue was collected with or without 200 μg sAgrin, and MMP12 levels were Western blotted. GAPDH was used as a loading control. (b) Confocal imaging of skin tissue from mice treated in the same manner as in (a) on day 10 post-wound injury. White dashed lines indicate the epidermal and dermal boundaries. Scale bar: 10 μm.

[0030] Figure 16Results of an investigation into the role of MMP12 depletion in Agrin-induced wound healing are shown. (a) The intended wound site on mouse skin after injection of control siRNA or mouse-specific MMP12 siRNA. Three days later, the site was wounded, and an Aquaphore-based ointment containing control or MMP12 siRNA was applied to the immobilized wound every other day, with or without 200 μgs of Agrin. Photographs of mouse skin wounds at specified days are presented. Scale bar: 1 mm. Wound diameter was quantified at specified days (n = 3–4 mice per group, ANOVA, *p = 0.007, *p = 0.01, ***p = 0.0001, respectively). (b) Representative hematoxylin and eosin stained sections of mouse skin treated with the siRNA combination shown in Figure (a) on day 10 post-traumatic injury. Areas coded by black arrows indicate unhealed areas. Scale bar: 100 μm. (c) Representative confocal images showing pMLC staining in control and MMP12-depleted mouse skin with or without sAgrin treatment. Scale bar: 10 μm. pMLC intensity was quantified using Zeiss Fluoview (n = 3 independent mouse sections per group, Student's t test, *p = 0.01, **p = 0.005, and 0.002, respectively). (d) Sirius red staining images of designated mouse skin sections at day 10 post-traumatic injury. Representative bright-field and polarized views are presented. Scale bar: 100 μm. WB represents the wound bed. Distribution of various collagen fiber types was performed using ImageJ and presented graphically (n = 3 sections / group, Student's t test, p-values ​​are presented in the table). (e) Angiogenesis was detected by CD-31 immunohistochemical analysis in the wound beds of control and MMP12-deficient mouse skin with or without sAgrin treatment at day 10 post-injury. The number and diameter of blood vessels are presented graphically as mean + / - sd (n=3 mice per group, 30-60 vessels per image from each of the three mice in each group were analyzed, ANOVA, *p=0.02, multiple t-tests, *p=0.02, p=0.01, p=0.008, **p=0.0009, respectively). The results indicate that MMP12 is required for sAgrin-induced wound healing in vivo.

[0031] Figure 17The results of assays investigating the potential use of sAgrin as a bio-additive material for skin wound healing are shown. (a) Protein sequence of the C-terminal Agrin fragment used as a bio-additive. Gel filter plot of purified sAgrin. Recombinant sAgrin was tested in a Coomassie-stained gel and by Western blotting using an Agrin antibody. (b) BrDu proliferation assay of mouse keratinocytes treated with escalating concentrations of sAgrin for specified days (n=3 experiments, Student's t-test, **p=0.001 and *p=0.01, respectively). (c) Scratching wound assay of HaCaT / mouse keratinocytes treated with escalating doses of sAgrin. Representative bright-field images are shown at specified time points post-traumatic injury. Scale bar: 20 μm. The area of ​​uncovered wounds obtained was quantified using ImageJ and is expressed as mean + / - sd (n=3, Student's t-test, *p<0.01). (d) Untreated or sAgrin (10 μg / ml) HaCaT cells were scraped and allowed to migrate for 4 hours. Cells were then fixed and immunostained for K17, with the nuclei stained with DAPI. Representative confocal images are presented. White arrows indicate the direction of migration. White dashed lines represent the migration front. Scale bar: 10 μm. (e) Confocal images showing K17 staining from mouse skin explants cultured for 5 days on a separate soft substrate or on a soft substrate supplemented with sAgrin (20 μg / ml). Ex indicates the explant, and the dashed lines around Ex indicate the explant boundary. The areas between the dashed lines represent the growth areas. Scale bar: 50 μm. The experiment was performed three times, with three explants per group. (f) Agrin-depleted HaCaT cells were treated with sAgrin or FN-coated beads for 30 minutes and then traumatized. Cells were placed individually or under magnetic force and allowed to migrate for 30 minutes, then fixed and immunostained for MMP12 and pMLC. Cell nuclei were counterstained with DAPI. Representative confocal images are shown (n = 2 experimental replicates). Scale bar: 10 μm. (g) Every 2–3 days, a petrolatum-based ointment containing 200 μg BSA (control) or an increased amount of sAgrin was applied topically to perforated wounds of mouse skin. Representative photographs of the treated mouse wound areas are shown at specified days. Scale bar: 1 mm. The relative diameter of the wound was measured at specified days and expressed as the mean + / - sd according to an experimental graph (n = 3 mice / group, one-way ANOVA, *p<0.04, **p<0.004, respectively). (h) Ki67-stained sections of mouse skin treated with BSA or sAgrin on day 7 post-injury. The extent of proliferating cells was quantified using ImageJ (n = 3 sections analyzed from two independent mouse wounds, Student's t' test, *p = 0.01).(i) Sirius red staining images of mouse skin sections (without fixation model) treated with BSA and sAgrin on day 7 post-traumatic injury. Representative bright-field and polarized views are presented. Scale bar: 100 μm. White arrows indicate unhealed areas. Quantification of various collagen fiber types was performed using ImageJ (n = 3 sections analyzed per group, Student's t-test, *p = 0.01 and 0.05, respectively). The results indicate that sAgrin accelerates wound healing in vitro and in vivo, and has clinical relevance as a wound healing biomaterial.

[0032] Figure 18The results of an assay investigating the effect of Agrin-based topical biomaterials on skin wound healing are shown. (ab) Pluronic-F-127-based ointments incorporating BSA or Agrin (200 μg) were applied to perforated wounds in mice every two days. Wounds receiving the specified ointment were left uncovered in the figure (a - not fixed). For Figure b, 200 μg of rat tail collagen was used as an additional control along with sAgrin and BSA. The wounds were secured with a 10 mm clear nylon fixation and covered with Tegaderm (fixation). Representative photographs show the perforated wound areas in mice receiving the above treatment. Scale bar: 1 mm. Mean wound area + / - sd plotted over time since wound initiation (n = 4 mice per group, ANOVA, p < 0.0001, p = 0.001, p = 0.0001, respectively). (c) Bright-field images of hematoxylin and eosin stained wound margins of control and Agrin-treated wounds on day 2 post-injury. An asterisk (*) indicates migrating keratinocytes forming an epithelial tongue at the wound margin. Double-headed black arrows (without fixation) show the length of migrating keratinocytes on day 2 post-injury. Black arrows (with fixation) indicate unhealed areas on day 10 post-injury. Scale bar: 100 μm. (d-) Immunofluorescence confocal images showing the occupancy of K17 expression in mouse skin wounds treated with BSA, collagen, or sAgrin under either the no-fixation condition (day 2) or the fixation condition (day 7). Relative K17 intensities are shown graphically (n = 3–6 slices analyzed from 3 mice per group, Student's t-test, *p = 0.01, **p = 0.005, ***p = 0.0001, respectively). (ef) Representative confocal immunofluorescence images of MMP12 (e) and pMLC (f) within the wound margin and wound bed of mouse skin treated with BSA, collagen, or sAgrin ointment on day 2 post-injury (in the non-fixation model) or day 7 (fixation model). Relative staining intensities are expressed as the mean + / - sd from three to four sections from three mice (Student's t-test, *p = 0.02, p = 0.04, p = 0.03, p = 0.01, and ***p = 0.001, respectively). Scale bar: 100 μm. White dashed lines represent the thickness of migrating epidermis, while white solid lines separate keratinocytes from the underlying dermal region. (g) Sirius red staining images of mouse skin sections (fixation model) treated with BSA, collagen, and sAgrin on day 10 post-injury. Representative bright-field and polarized views are presented. Scale bar: 100 μm. WB represents the wound bed. White arrows indicate unhealed areas. Quantification of various types of collagen fibers was performed using ImageJ (n = 3 slices analyzed from 3 mice in each group, Student's t test, p-values ​​shown in the figure).(h) Immunohistochemical analysis of CD31 in mouse skin wound beds treated with BSA, collagen, and Agrin at specified days post-injury, under both fixation-free and fixation-free conditions. Arrows indicate blood vessels. The number and diameter of blood vessels are presented graphically as mean + / - sd. (n = 5–10 slides analyzed from three mice per group, Student's t-tests, *p = 0.01, *p = 0.04, ANOVA *p = 0.02, and ***p = 0.0001, respectively). The results indicate that Agrin-based topical biomaterials accelerate skin wound healing.

[0033] Figure 19 A profile of pro-inflammatory proteins induced by sAgrin during the early stages of wound healing is shown. (a) Heatmap showing mRNA expression of cytokines and chemokines typically induced at 4 and 48 hours post-traumatic stress in mouse skin treated with BSA, collagen, or sAgrin. Photographs of representative wounds for each condition are shown above. The corresponding p-values ​​for groups of selected significant genes are presented in tabular form (n = 3 mice / group, multiple t-tests). (b) Western blot analysis of mouse skin treated as in (a), showing protein expression of selected proteins. GAPDH was used as a loading control (n = 2 independent mouse wounds analyzed per group).

[0034] Figure 20 The results of the study on the effect of Agrin on angiogenesis are shown. (a) Western blot analysis of BJ-GFP cells treated with Agrin siRNA and HDMECs labeled with Cell Tracker Blue for 18 hours. GAPDH was used as a loading control. Cells treated as shown in Figure (a) were co-cultured for 24 hours to form spheroids. Bright-field images of budding are shown for each specified condition. The corresponding fibroblasts and HDMEC spheroids are depicted by green and blue fluorescence, respectively. Scale bar: 100 μm. Budding was quantified using ImageJ (n = 3 spheroids per condition, Student's t' test, *p = 0.05 and p = 0.02, respectively). The results show that Agrin mediates angiogenesis by promoting the interaction between dermal fibroblasts and endothelial cells.

[0035] Figure 21A working model explaining how the Agrin microenvironment, triggered by wound injury, facilitates a productive healing process is presented. In short, Agrin sensitizes injured cells to ECM stiffness, force recognition, and geometric constraints, resulting in improved traction stress, elasticity, and cytoskeletal tension to promote enhanced fluid-like collective migration at the wound site. Furthermore, Agrin deploys MMP12 as its downstream effector to enhance mechanosensory perception and actomyosin integrity of keratinocytes during migration. The cumulative effect of the Agrin-driven mechanosensitive wound healing microenvironment leads to greater collective migration and wound closure by promoting re-epithelialization, optimal ECM deposition, and angiogenesis.

[0036] Figure 22 An experimental setup for imaging traction forces in co-migrating cells after trauma is shown. (a) An image of the 3D-printed mold and the cast hydrogel block is shown. (b) An image showing the intended use of the polyacrylamide block for creating an artificial wound on a glass-bottomed culture dish. Detailed Implementation

[0037] Wound healing proceeds in an organized manner, following four processes: hemostasis, inflammation, proliferation, and maturation. Hemostasis is the process of closing the wound through blood clotting. Inflammation is the second stage of wound healing; it controls bleeding and prevents infection. Proliferation is the stage where the wound is reconstructed with new tissue composed of collagen and extracellular matrix (ECM). The maturation stage occurs when collagen remodels from type III to type I and the wound is completely closed. The proliferation stage includes angiogenesis, fibrosis and granulation tissue formation, collagen deposition, re-epithelialization, and contraction, and is a critical stage of wound healing. Re-epithelialization is a key step in the proliferation stage. Keratinocytes are the main cells responsible for re-epithelialization. Activated keratinocytes at the wound edge migrate across the wound bed until they meet the epithelial tongue at the center of the wound. From there, keratinocytes differentiate to form a new skin barrier. When the epidermis is injured, keratinocytes at the wound edge undergo a transition from a passive epithelial state to a mesenchymal-like state, in which they lose cell-cell contact and become motile. Migrating cells reorganize their actin cytoskeleton and secrete proteases to remodel the ECM and enable it to migrate across the wound. Directly behind the migrating cells, keratinocytes proliferate rapidly to provide sufficient cells to cover the wound. Non-healing wounds are characterized by defective keratinocyte migration, resulting in the inability of re-epithelialization to occur.

[0038] As demonstrated in this disclosure, Agrin expression is significantly triggered in the epidermis and dermis of the skin following mechanical injury, and supplementation with sAgrin (a C-terminal recombinant protein fragment of Agrin having binding sites for its receptors lipoprotein-associated receptor-4 (LRP4) and integrin) significantly rescues wound healing and keratinocyte migration. Therefore, in a first aspect, the present invention relates to pharmaceutical compositions comprising an Agrin fragment or a derivative thereof, wherein the Agrin fragment or derivative thereof comprises the LG3 domain of Agrin and an eight-amino acid insert ELANEIPV (SEQ ID NO: 1) at the z-site of the LG3 domain. The Agrin fragment or derivative thereof, as defined above, contains binding sites for lipoprotein-associated receptor-4 (LRP4) and integrin.

[0039] In some instances, Agrin fragments or their derivatives also contain the LG2 domain of Agrin.

[0040] Agrin is a large heparin proteoglycan with a molecular weight of 400-600 kDa. The protein core of Agrin consists of approximately 2000 amino acids and has a mass of approximately 225 kDa. Agrin is a multidomain protein composed of 9 K (kunitz-type) domains, 2 LE (lamin-EGF-like) domains, 1 SEA (sperm protein, enterokinase, and agrin) domain, 4 EG (epidermal growth factor-like) domains, and 3 LG (lamin globular) domains. Agrin exists in several splice variants and can be expressed as a secreted protein containing an N-terminal NtA (N-terminal Agrin) domain, which is the most abundant form of Agrin. The C-terminal 75 kDa portion of Agrin begins with the first EG domain. Several binding sites for Agrin's interacting partner proteins (including α-dystrophin, heparin, some integrins, and LRP4) are localized to the C-terminal region. In the C-terminal region of human agrin, there are two alternative splicing sites, y and z. The y site is located within the LG2 domain, and the z site is located within the LG3 domain. At the y site, there can be inserts of 0, 4, 17, or 21 (4+17) amino acids; while at the z site, there can be inserts of 0, 8, 11, or 19 (8+11) amino acids.

[0041] Lipoprotein receptor-related protein 4 (LRP-4), also known as low-density lipoprotein receptor-related protein 4, is a protein encoded by the LRP4 gene in humans. LRP-4 is a member of the lipoprotein receptor-related protein family and can be a regulator of Wnt signaling.

[0042] Integrins are transmembrane receptors that promote cell-cell and cell-ECM adhesion. Upon ligand binding, integrins activate signal transduction pathways that mediate cellular signals such as cell cycle regulation, intracellular cytoskeleton organization, and the migration of new receptors to the cell membrane. The presence of integrins allows for rapid and flexible responses to events at the cell surface.

[0043] As used herein, the terms “LG2” and “LG3” refer to the second and third laminin globular domains of Agrin. LG2 and LG3 should encompass all possible different splicing variants of these domains. In one example, the LG2 domain of Agrin without any insertion at the y-site has the following sequence:

[0044] PFLADFNGFSHLELRGLHTFARDLGEKMALEVVFLARGPSGLLLYNGQKTDGKGDFVSLALRDRRLEFRYDLGKGAAVIRSREPVTLGAWTRVSLERNGRKGALRVGDGPRVLGESPVPHTVLNLKEPLYVGGAPDFSKLARAAAVSSGFDGAIQLVSLGGRQLLTPEHVLRQVDVTSFAGHPC (SEQ ID NO: 2). In one example, the LG2 domain of the Agrin insert with a 4-amino acid at the y site has the following sequence:

[0045] PFLADFNGFSHLELRGLHTFARDLGEKMALEVVFLARGPSGLLLYNGQKTDGKGDFVSLALRDRRLEFRYDLGKGAAVIRSREPVTLGAWTRVSLERNGRKGALRVGDGPRVLGESPVPKSRKHTVLNLKEPLYVGGAPDFSKLARAAAVSSGFDGAIQLVSLGGRQLLTPEHVLRQVDVTSFAGHPC (SEQ ID NO: 3), wherein the sequence of the insert is KSRK (SEQ ID NO: 4). In the above LG2 domain, the y site begins at P(proline)119. In one example, the Agrin fragment or derivative described herein contains the LG2 domain of Agrin without any insert at the y site. In one example, the LG3 domain of Agrin without any insert at the z site has the sequence:

[0046] EYLNAVTESEKALQSNHFELSLRTEATQGLVLWSGKATERADYVALAIVDGHLQLSYNLGSQPVVLRSTVPVNTNRWLRVVAHREQREGSLQVGNEAPVTGSSPLGATQLDTDGALWLGGLPELPVGPALPKAYGTGFVGCLRDVVVGRHPLHLLEDAVTKPELRPC (SEQ ID NO: 5). In another example, the LG3 domain of Agrin, an insert with 8 amino acids at the z-site, has the following sequence:

[0047] EYLNAVTESELANEIPVEKALQSNHFELSLRTEATQGLVLWSGKATERADYVALAIVDGHLQLSYNLGSQPVVLRSTVPVNTNRWLRVVAHREQREGSLQVGNEAPVTGSSPLGATQLDTDGALWLGGLPELPVGPALPKAYGTGFVGCLRDVVVGRHPLHLLEDAVTKPELRPC (SEQ ID NO: 6), wherein the sequence of the insert is ELANEIPV (SEQ ID NO: 1). In the above LG3 domain, the z-site is between S (serine) 9 and E (glutamate) 10. In some instances, Agrin fragments or derivatives thereof containing the LG3 domain of Agrin and an eight-amino acid insert at the z-site of the LG3 domain have the following sequence: DTLAFDGRTFVEYLNAVTESELANEIPVEKALQSNHFELSLRTEATQGLVLWSGKATERADYVALAIVDGHLQLSYNLGSQPVVLRSTVPVNTNRWLRVVAHREQREGSLQVGNEAPVTGSSPLGATQLDTDGALWLGGLPELPVGPALPKAYGTGFVGCLRDVVGRHPLHLLEDAVTKPELRPCPTP (SEQ ID NO: 7).

[0048] In some instances, Agrin fragments or derivatives thereof containing the LG2 and LG3 domains of Agrin and an eight-amino acid insert at the z-site of the LG3 domain have the following sequence:

[0049]

[0050] Since other sequence variations that do not affect biological activity are possible, the present invention should not be limited to different splicing variants of the LG2 and LG3 domains of the illustrated sequences. In some instances, the Agrin fragment or a derivative thereof comprises a sequence having at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with the exemplary LG2 and / or LG3 domain sequences provided herein.

[0051] As used herein, the term "derivative" refers to a polypeptide derived from a base sequence through modification, including the deletion or addition of amino acids to the polypeptide or variant, and modification of the side chain, wherein the derivative retains the activity of the base protein. The resulting derivative will retain at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% homology to the base sequence of the original polypeptide. The derivative will also exhibit effects similar in nature to the unmodified polypeptide.

[0052] In some instances, the Agrin fragment or its derivatives additionally include at least one additional domain naturally present in Agrin, including but not limited to LG1 and EGF-like domains EG1-4. Exemplary sequences of the LG1 and EG1-4 domains are as follows: LG1 domain: APVPAFEGRSFLAFPTLRAYHTLRLALEFRALEPQGLLLYNGNARGKDFLALALLDGRVQLRFDTGSGPAVLTSAVPVEPGQWHRLELSRHWRRGTLSVDGETPVLGESPSGTDGLNLDTDLFVGGVPEDQAAVALERTFVGAGLRGCIRLLDVNNQRLELGIGPGAATRGSGVGEC (SEQ ID NO: 9). EG1 domain: PPKPCDSQPCFHGGTCQDWALGGGFTCSCPAGRGGAVCE (SEQ ID NO: 10). EG2 domain: GDHPCLPNPCHGGAPCQNLEAGRFHCQCPPGRVGPTCA (SEQ ID NO: 11). EG3 domain: EKSPCQPNPCHGAAPCRVLPEGGAQCECPLGREGTFCQ (SEQ ID NO: 12). EG4 domain: AGHPCTRASGHPCLNGASCVPREAAYVCLCPGGFSGPHCE (SEQ ID NO: 13).

[0053]

[0054] In some instances, vectors containing nucleic acid molecules encoding the Agrin fragment or derivatives thereof disclosed herein are provided. As used herein, the term "vector" includes vectors that can be used to express DNA sequences contained therein, wherein such DNA sequences are operatively linked to other sequences (e.g., promoter / operon sequences) capable of achieving their expression. Typically, expression vectors useful in recombinant DNA technologies are often in the form of "plasmids," which refer to circular double-stranded DNA loops in vector form that do not bind to chromosomes. Various expression vectors known in the art can be used to obtain nucleic acid molecules encoding the Agrin fragment or derivatives thereof disclosed herein, including but not limited to bacterial expression vectors such as pET28, pUC19, pBR327, pBR322, pET3a, pEXP4-DEST, pSP72, pET SUMO, pBAD TOPO, pGEX-4T2, pQE-30, and pACYC177, and mammalian expression vectors such as pACT, pBIND, pG5luc, pTNT, pTarget, pRegne, pCat3-Basic, pSI, pcDBA, and pCMV. In one example, the expression vector is the pET28 vector. In a specific example, the expression vector is a pET28 vector containing a His tag.

[0055] This disclosure also provides a host cell comprising a vector containing a nucleic acid molecule encoding the Agrin fragment or a derivative thereof disclosed herein. Several prokaryotic and eukaryotic expression systems are suitable for producing the Agrin fragment or a derivative thereof as disclosed herein. Prokaryotic expression systems include, but are not limited to, expression in *Escherichia coli* (E. coli). Eukaryotic expression systems include expression in mouse myeloma cells, baculovirus-mediated expression in insect cells, expression in human embryonic kidney (HEK) cells, transient expression in Chinese hamster ovary (CHO) cells, and stable expression in *Pichia pastoris*. These systems have the advantages that they can be easily adapted to serum-free conditions to reduce the amount of contaminating proteins in the supernatant and can be adapted for large-scale production. Furthermore, a variety of cell lines can be used, including HEK293T and HEK293 cells, COS cells, CHO cells, HeLa cells, H9 cells, Jurkat cells, NIH3T3 cells, C127 cells, CV1 cells, CAP cells, or SF cells. Therefore, in one instance, this disclosure provides a host cell comprising a vector containing a nucleic acid molecule encoding the Agrin fragment or a derivative thereof disclosed herein. In a specific instance, the host cell is an *E. coli* cell. This disclosure also provides a method for producing the Agrin fragment or a derivative thereof as disclosed herein, the method comprising culturing a host cell as disclosed herein to express the Agrin fragment or a derivative thereof, and harvesting the resulting Agrin fragment or a derivative thereof. In some instances, the method further includes purifying the obtained Agrin fragment or a derivative thereof.

[0056] To purify the obtained Agrin fragment or its derivatives, standard protein purification techniques can be applied. His-tagged proteins can be purified using IMAC, and affinity purification using ion exchange chromatography or heparin columns can also be used. Purification can also be performed using antibodies generated targeting the C-terminal portion of Agrin. The eluted protein can then be further purified using, for example, hydroxyapatite columns or by gel filtration.

[0057] The Agrin fragments or derivatives thereof disclosed in this application may be in secretory or transmembrane form. In some instances, the Agrin fragments or derivatives thereof are in secretory form. In some instances, the Agrin fragments or derivatives thereof are soluble.

[0058] The Agrin fragments or derivatives thereof disclosed in this application may be from any source. In some instances, the Agrin fragments or derivatives thereof are derived from humans, non-human primates, mice, rats, hamsters, rabbits, goats, or other mammal species. In some specific instances, the Agrin fragments or derivatives thereof are derived from humans or mice.

[0059] In some instances, the pharmaceutical compositions disclosed herein comprise about 0.01% to about 25%, or about 0.01% to about 10%, or about 0.03% to about 1%, or about 0.03% to about 5%, or about 1% to about 10% w / v, or about 6%, 8%, 10%, 15% or 20% w / v of an Agrin fragment or a derivative thereof.

[0060] In some instances, the pharmaceutical compositions disclosed herein also contain other active agents that synergistically act on wounds to promote wound healing or wound closure or treat non-healing wounds.

[0061] In some instances, the pharmaceutical composition also contains a pharmaceutically acceptable carrier and / or other pharmaceutically acceptable inert agents.

[0062] As used in this article, "pharmaceutical acceptable" means an ingredient, reagent, or composition that is suitable for drug administration without excessive toxicity, incompatibility, instability, irritation, allergic reactions, etc.

[0063] As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, medium, or other inert agent that is applied together with an Agrin fragment or its derivative.

[0064] Examples of pharmaceutically acceptable carriers include, but are not limited to, sugars, starches, celluloses, excipients, oils, ethylene glycols, polyols, esters, agars, and buffers. These are non-limiting examples of carriers. Pharmaceutically acceptable carriers can be readily formulated by those skilled in the art.

[0065] Examples of excipients include, but are not limited to, solvents, emollients and / or emulsifiers, oily bases, preservatives, antioxidants, tension modifiers, penetration enhancers and solubilizers, chelating agents, buffers, surfactants, one or more polymers, and combinations thereof. Suitable solvents for aqueous or hydrophilic topical formulations include water; ethanol; isopropanol; mixtures of water and ethanol and / or isopropanol; glycerin; ethylene glycol, propylene glycol, or butylene glycol; DMSO; and mixtures thereof. Suitable solvents for hydrophobic topical formulations include mineral oils, vegetable oils, and silicone oils. If desired, the pharmaceutical composition as described herein can be dissolved or dispersed in a hydrophobic oil phase, which can then be emulsified in an aqueous phase containing water alone or in combination with lower alcohols, glycerin, and / or ethylene glycol. Suitable emollients include hydrocarbon oils and waxes, such as mineral oil, petrolatum, paraffin, pure ceresin, microcrystalline wax, polyethylene, squalene, perhydrosqualene, silicone oil, triglycerides, acetylated glycerides, such as acetylated monoglycerides; ethoxylated glycerides, such as ethoxylated monostearate; and alkyl esters of fatty acids or dicarboxylic acids. Suitable silicone oils for use as emollients include dimethylpolysiloxane, methyl(phenyl)polysiloxane, and water-soluble and alcohol-soluble siloxane diol copolymers. Suitable triglycerides for use as emollients include vegetable and animal fats and oils, including castor oil, safflower oil, cottonseed oil, corn oil, olive oil, cod liver oil, almond oil, avocado oil, palm oil, sesame oil, and soybean oil. Suitable esters of carboxylic acids or dicarboxylic acids for use as emollients include methyl esters, isopropyl esters, and butyl esters of fatty acids. Specific examples of alkyl esters include hexyl laurate, isohexyl laurate, isohexyl palmitate, isopropyl palmitate, decyl oleate, isodecyl oleate, hexadecyl stearate, decyl stearate, isopropyl isostearate, dilauryl lactate, myristyl lactate, and cetyl lactate; and alkenyl esters of fatty acids, such as oleyl myristate, oleyl stearate, and oleyl oleate. Specific examples of alkyl esters of diacids include diisopropyl adipate, diisohexyl adipate, bis(hexyldecyl) adipate, and diisopropyl sebacate. Other suitable classes of emollients or emulsifiers for use in topical formulations include fatty acids, fatty alcohols, fatty alcohol ethers, ethoxylated fatty alcohols, fatty acid esters of ethoxylated fatty alcohols, and waxes. Specific examples of fatty acids used as emollients include nonanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, isostearic acid, hydroxystearic acid, oleic acid, linoleic acid, ricinoleic acid, arachidic acid, behenic acid, and erucic acid. Specific examples of fatty alcohols used as emollients include lauryl alcohol, myristic alcohol, cetyl alcohol, hexadecyl alcohol, stearyl alcohol, isostearyl alcohol, hydroxystearyl alcohol, oleyl alcohol, ricinoleyl alcohol, behenyl alcohol, and garnet alcohol, as well as 2-octyldodecyl alcohol.Specific examples of waxes suitable for use as emollients include lanolin and its derivatives, including lanolin oil, lanolin wax, lanolin alcohol, lanolin fatty acids, isopropyl lanolinate, ethoxylated lanolin, ethoxylated lanolin alcohol, ethoxylated cholesterol, propoxylated lanolin alcohol, acetylated lanolin, acetylated lanolin alcohol, lanolin alcohol linoleate, lanolin alcohols recinoleate, lanolin alcohol acetate, lanolin alcohol acetate, ethoxylated alcohol acetate, hydrogenated lanolin, hydrogenated lanolin, ethoxylated hydrogenated lanolin, ethoxylated sorbitol lanolin, and liquid and semi-solid lanolin. Also suitable for use as waxes include hydrocarbon waxes, ester waxes, and amide waxes. Available waxes include wax esters such as beeswax, cetearyl, myristate, and stearate; beeswax derivatives such as polyoxyethylene sorbitan beeswax; and plant waxes, including carnauba wax and candelilla wax. Polyols and polyether derivatives can be used as solvents and / or surfactants in topical formulations. Suitable polyols and polyethers include propylene glycol, dipropylene glycol, polypropylene glycol 2000 and 4000, poly(ethylene oxide co-oxypropylene) glycol, glycerol, sorbitol, ethoxylated sorbitol, hydroxypropyl sorbitol, polyethylene glycol 200-6000, methoxy polyethylene glycol 350, 550, 750, 2000 and 5000, poly[ethylene oxide] homopolymers (100,000-5,000,000), polyalkylene glycols and derivatives, hexanediol, 2-methyl-2,4-pentanediol, 1,3-butanediol, 1,2,6-hexanetriol, 2-ethyl-1,3-hexanediol, ortho-diols having 15 to 18 carbon atoms, and polyoxypropylene derivatives of trimethylolpropane. Polyol esters can be used as emulsifiers or emollients. Suitable polyol esters include ethylene glycol mono- and di-fatty acid esters, diethylene glycol mono- and di-fatty acid esters, polyethylene glycol (200-6000) mono- and di-fatty acid esters, propylene glycol mono- and di-fatty acid esters, polypropylene glycol 2000 monooleate, polypropylene glycol 2000 monostearate, ethoxylated propylene glycol monostearate, glyceryl mono- and di-fatty acid esters, polyglyceryl polyfatty acid esters, ethoxylated glyceryl monostearate, 1,3-butanediol monostearate, 1,3-butanediol distearate, polyoxyethylene polyol fatty acid esters, dehydrated sorbitol fatty acid esters, and polyoxyethylene dehydrated sorbitol fatty acid esters. Suitable emulsifiers for topical formulations include anionic, cationic, nonionic, and amphoteric surfactants. Preferred ionic emulsifiers include phospholipids, such as lecithin and its derivatives. Lecithin and other phospholipids can be used to prepare liposomes containing compositions as described herein. When phospholipids such as lecithin are placed in water and thus form a bilayer or a series of bilayers, lipid vesicles form, and each bilayer is separated by water molecules once sufficient energy is provided. Liposomes can be produced by sonicating phospholipids in water. Low shear rates produce multilayered liposomes.Sustained high-shear sonication tends to form smaller monolayer liposomes. Hydrophobic chemicals can dissolve in the phospholipid bilayer. The lipid bilayer of the liposomes delivers the composition described herein to keratinocytes through fusion with the cell membrane of keratinocytes. Sterols, including, for example, cholesterol and cholesterol fatty acid esters; amides such as fatty acid amides, ethoxylated fatty acid amides, and fatty acid alkanolamides can also be used as emollients and / or penetration enhancers. Suitable viscosity enhancers or thickeners that can be used in the preparation of viscous gels or creams with aqueous matrices include sodium polyacrylate, xanthan gum, polyvinylpyrrolidone, acrylic polymers, carrageenan, hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, propyl cellulose, hydroxypropyl methyl cellulose, polyethoxylated polyacrylamide, polyethoxylated acrylates, and polyethoxylated alkanols. Suitable preservatives and / or antioxidants for topical formulations include benzalkonium chloride, benzyl alcohol, phenol, urea, parabens, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), tocopherol, and mixtures thereof. Suitable chelating agents for topical formulations include ethylenediaminetetraacetic acid (EDTA), its alkali metal salts, its alkaline earth metal salts, its ammonium salts, and its tetraalkylammonium salts. The pH of the carrier is preferably from about 4.0 to 10.0, more preferably from about 6.8 to about 7.8. The pH can be controlled using buffer solutions or other pH adjusters. Suitable pH adjusters include phosphoric acid and / or phosphates, citric acid and / or citrates, hydroxide salts (i.e., calcium hydroxide, sodium hydroxide, potassium hydroxide), and amines, such as triethanolamine. Suitable buffer solutions include buffer solutions containing solutions of potassium dihydrogen phosphate and dipotassium hydrogen phosphate, maintaining a pH between 5.8 and 8; and buffer solutions containing solutions of sodium dihydrogen phosphate and disodium hydrogen phosphate, maintaining a pH between 6 and 7.5. Other buffers include citric acid / sodium citrate and disodium hydrogen phosphate / citric acid.

[0066] The pharmaceutical compositions disclosed herein may additionally include conventional adjuvants, such as propionic acid, propylene glycol, conventional buffers, preservatives, hydrophilic emulsifiers, lipophilic emulsifiers, fragrances, emollients, deodorants, humectants, etc. Colorants may also optionally be added to the compositions disclosed herein. Adjuvants that are harmful to wounds or surrounding skin, as well as those that may react with the pharmaceutical composition and / or adversely reduce the effectiveness of the pharmaceutical composition, should be avoided.

[0067] The pharmaceutical compositions disclosed herein can be formulated into a wide variety of articles for topical application, including but not limited to lotions, creams, gels, sticks, sprays, ointments, emulsions, pastes, foams, powders, and film-forming products. Such pharmaceutical compositions can be formulated for time-controlled release. If the pharmaceutical composition is formulated as an emulsion, the emulsion may have a continuous aqueous phase and a discontinuous non-aqueous or oil phase (oil-in-water emulsion), or a continuous non-aqueous or oil phase and a discontinuous aqueous phase (water-in-oil emulsion).

[0068] In some instances, pharmaceutical compositions such as those disclosed herein also contain one or more preservatives. Examples of preservatives include, but are not limited to, chelating agents such as EDTA, diethylenetriaminepentaacetic acid (DTPA), and catechins; sodium benzoate; potassium sorbate; and sodium nitrate. The composition may contain about 0.01% to about 5%, or about 0.1% to about 3%, or about 0.015% to about 1%, or about 0.015% to about 0.5%, or about 0.01% to about 0.1%, or about 0.0225% to about 0.1% w / v, or about 0.015%, 0.225%, or 0.1% w / v of a preservative.

[0069] The compositions provided herein may also contain one or more antimicrobial agents. Antimicrobial agents can counteract the activity of any bacterial proteases that may hinder the healing environment, allowing the wound to progress toward optimal healing. Examples of antimicrobial agents include, but are not limited to, aloe vera, ashitaba, bacteriophages, β-defensins, quaternary ammonium compounds, chlorhexidine, copper, dispersin B, essential oils, gentamicin, lactoferrin, lysostaphylococcus, N-haloamines, nitric oxide, oleic acid, PLUNC, polyhexanide biguanide (PHMB), bacteriocins, selenium, silver compounds, triclosan, zinc, and combinations thereof. Aloe vera contains many photochemical compounds, including but not limited to tannins, saponins, flavonoids, and fumaric acid. As used herein, the term "PLUNC" refers to a gene or clone encoding a protein associated with palatine, lung, or nasal epithelial cancer, as well as the protein itself. Examples of quaternary ammonium compounds include benzyl chloride and benzalkonium chloride. An example of a β-defensin is the antimicrobial peptide cathelicidin (LL-37). Examples of silver compounds may include colloidal silver, ionic silver, nonionic silver, silver chloride, silver nanoparticles, and silver sulfadiazine. Examples of essential oils include, but are not limited to, cinnamon oil, clove oil, eucalyptus oil, and tea tree oil. An example of chlorhexidine is chlorhexidine gluconate. The composition may contain about 0.01% to about 1%, or about 0.05% to about 1%, or about 0.05% to about 0.5% w / v of an antimicrobial agent.

[0070] The pharmaceutical compositions disclosed herein may also contain other agents, such as growth factors, cytokines, and protease inhibitors. Examples of growth factors include, but are not limited to, epidermal growth factor (EGF), transforming growth factor-α (TGF-α), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) including acidic fibroblast growth factor (α-FGF) and basic fibroblast growth factor (β-FGF), transforming growth factor-β (TGF-β), and insulin-like growth factor (IGF-1 and IGF-2), and combinations thereof.

[0071] In some instances, pharmaceutical compositions such as those disclosed herein may be infused within, injected into, absorbed by, layered onto, encapsulated within, or coated onto a carrier material, such as bandages, gauze, wound dressings, adhesive bandages, scaffolds, or hydrogels. The carrier material may be bioabsorbable, including, for example, polyglycolic acid, polylactic acid, polydioxanone, polyhydroxybutyrate, polyhydroxyvalerate, polyamino acid polyorthoesters, polyvinyl alcohol, collagen, gelatin, chitosan, oxidized regenerated cellulose, hyaluronic acid, alginate, or derivatives thereof; or it may be non-bioabsorbable, including, for example, polyurethane, polyvinyl alcohol, or gauze. The carrier material differs from carriers used in pharmaceutical compositions and pharmaceutically acceptable carriers.

[0072] Examples of suitable carrier materials include, but are not limited to: bandages, gauze, wound dressings, adhesive bandages, scaffolds, hydrogels, particularly hydrogels containing cellulose derivatives, including hydroxyethyl cellulose, hydroxymethyl cellulose, carboxymethyl cellulose, hydroxypropyl methyl cellulose, and mixtures thereof; and hydrogels containing polyacrylic acid and gelatin. The aforementioned carrier materials may include alginate (as a thickener or stimulant), pH-controlling buffers (e.g., disodium hydrogen phosphate / sodium dihydrogen phosphate), osmotic pressure-regulating agents (e.g., sodium chloride), and stabilizers (e.g., EDTA).

[0073] In some specific instances, the carrier material is a hydrogel or a scaffold.

[0074] As used herein, the term "hydrogel" refers to a three-dimensional (3D) network of hydrophilic polymers. Hydrogels typically absorb large amounts of fluid while simultaneously maintaining their structure due to the chemical or physical cross-linking of the individual polymer chains. At equilibrium, a hydrogel is typically 60-90% fluid and only 10-30% polymer. In some instances, the water content of a hydrogel is approximately 70-80%. Hydrogels are particularly useful due to the inherent biocompatibility of cross-linked polymer networks. Hydrogels can be prepared by cross-linking hydrophilic biopolymers or synthetic polymers. Examples of hydrogels formed by the physical or chemical cross-linking of hydrophilic biopolymers include, but are not limited to, hyaluronic acid, chitosan, alginate, collagen, dextran, pectin, carrageenan, polylysine, gelatin, or agarose. These materials consist of high-molecular-weight framework chains made of linear or branched polysaccharides or peptides.

[0075] Hydrogels closely resemble the natural extracellular matrix of living cells. They can also be made biodegradable in vivo by incorporating polymers such as PLA, PLGA, or PGA. Furthermore, hydrogels can be modified with fibronectin, laminin, vitrin, or surface-modified with RGD, which can promote cell adhesion and proliferation. In addition, changes in molecular weight, block structure, degradable bonds, and cross-linking patterns can affect the strength, elasticity, and degradation properties of hydrogels.

[0076] Hydrogels can also be modified with functional groups to covalently link various proteins (e.g., collagen) or compounds (e.g., therapeutic agents). Therapeutic agents that can bind to the matrix include, but are not limited to, analgesics, anesthetics, antifungals, antibiotics, anti-inflammatory agents, anthelmintics, antitoxins, antiemetics, antihistamines, antihypertensives, antimalarials, antimicrobials, antipsychotics, antipyretics, antiseptics, antiarrhythmics, antituberculosis agents, antitussives, antiviral agents, cardiostimulants, laxatives, chemotherapeutic agents, color or fluorescent imaging agents, corticosteroids (e.g., steroids), antidepressants, sedatives, diagnostic aids, diuretics, enzymes, expectorants, hormones, hypnotics, minerals, nutritional supplements, parasympathomimetic agents, potassium supplements, radiation sensitizers, radioisotopes, sedatives, sulfonamides, stimulants, sympathomimetic agents, tranquilizers, anti-urinary tract infection drugs, vasoconstrictors, vasodilators, vitamins, xanthine derivatives, etc. Therapeutic agents can also be other small organic molecules, naturally isolated entities or their analogues, organometallic reagents, chelated metals or metal salts, peptide-based drugs, or peptide or non-peptide receptor binders or targets. Molecules that can be incorporated into the hydrogel matrix include, but are not limited to, vitamins and other nutritional supplements; glycoproteins (e.g., collagen); fibronectin; peptides and proteins, carbohydrates (simple and complex); proteoglycans; antigens; oligonucleotides (sense and antisense DNA and / or RNA); antibodies (e.g., antibodies against infectious agents, tumors, drugs, or hormones); and gene therapy agents.

[0077] Many classifications of hydrogels have been reported. For example, hydrogels can be divided into those formed from natural polymers and those formed from synthetic polymers. Depending on the ionic charge on the binding groups, hydrogels can be cationic, anionic, or neutral. Hydrogels can also be classified as inert hydrogels, physical hydrogels, chemical hydrogels, or biochemical hydrogels. Inert hydrogels are inactive to normal chemical or biological processes, and they are resistant to degradation and not absorbed by the body. Physical hydrogels can undergo a transition from liquid to gel in response to changes in environmental conditions such as temperature, ion concentration, pH, or other conditions such as the mixing of two components. Chemical hydrogels use covalent bonding, which introduces mechanical integrity and resistance to degradation compared to other weak materials. In biochemical hydrogels, biological agents such as enzymes or amino acids participate in the gelation process. Hydrogels can also be classified into different groups based on their structure: amorphous, semi-crystalline, crystalline, and hydrocolloid aggregates.

[0078] In some instances, the hydrogel is an inert hydrogel. In other instances, the hydrogel is a physical hydrogel, particularly a thermosensitive hydrogel. Thermosensitive hydrogels use temperature as an external stimulus to exhibit a solution-gel transition, and most thermosensitive polymers can form hydrogels near body temperature. Many inert and thermosensitive hydrogels are commercially available. For example, petrolatum is an inert hydrogel, and Pluronic F-127 is a thermosensitive hydrogel.

[0079] Scaffolds can be infused, coated, or contained with cells, growth factors, extracellular matrix components, nutrients, integrins, or other substances that promote cell growth. Scaffolds can also be used as carrier materials for the pharmaceutical compositions disclosed herein. Scaffolds can be formed from biological or synthetic scaffold materials and are used in the field of tissue engineering to support protein adhesion and inward cell growth for tissue repair and regeneration. Existing scaffold technologies rely on the inherent characteristics of the surrounding tissue space for protein adsorption and cell migration. Non-limiting examples of suitable scaffold materials include extracellular matrix proteins such as fibroin, collagen, or fibronectin, and synthetic or naturally occurring polymers, including bioabsorbable or nonabsorbable polymers such as polylactic acid (PLA), polyglycolic acid (PGA), polylactide-co-glycolic acid (PLGA), polyvinylpyrrolidone, polycaprolactone, polycarbonate, polyfumarate, caprolactone, polyamide, polysaccharides (including alginates (e.g., calcium alginate) and chitosan), hyaluronic acid, polyhydroxybutyrate, polyhydroxyvalerate, polydioxanone, polyorthoester, polyethylene glycol, poloxamer, polyphosphazene, polyanhydride, polyamino acids, polyacetal, polycyanoacrylate, polyurethane (e.g., Polyacrylates, ethylene-vinyl acetate polymers and other acyl-substituted cellulose acetate and their derivatives, polystyrene, polyvinyl chloride, polyvinyl fluoride, polyvinyl imidazole, chlorosulfonated polyolefins, polyethylene oxide, polyvinyl alcohol, And nylon. The scaffold may also contain ceramics, such as hydroxyapatite, coral apatite, calcium phosphate, calcium sulfate, calcium carbonate or other carbonates, bioglass, allogeneic grafts, autologous grafts, xenografts, decellularized tissue or any of the above complexes. In some instances, the scaffold may contain collagen (e.g., or The scaffold may contain any combination of two, three, or more materials, such as polylactic acid (PLA), polyglycolic acid (PGA), polylactide-co-glycolic acid (PLGA), polyurethane, polysaccharides, hydroxyapatite, or polyethylene glycol. Additionally, the scaffold may contain any combination of two, three, or more materials in separate or multiple regions of the scaffold, said materials being non-covalent or covalently combined (e.g., copolymers such as polyethylene oxide-polypropylene glycol block copolymers or terpolymers), or combinations thereof.

[0080] On the other hand, pharmaceutical compositions as disclosed herein are provided for use in treatment.

[0081] In one aspect, a method for treating a wound is provided, comprising administering to a subject in need a pharmaceutically effective amount of a pharmaceutical composition as disclosed herein.

[0082] This disclosure also provides methods for promoting epithelial tissue regeneration in subjects. In some instances, epithelial tissue regeneration at the wound site of a subject is promoted, and thus helps to promote wound healing in the subject.

[0083] This disclosure also provides pharmaceutical compositions as disclosed herein for treating wounds in a subject. This disclosure also provides pharmaceutical compositions as disclosed herein for promoting the regeneration of epithelial tissue in a subject. Use of the pharmaceutical compositions as disclosed herein in the preparation of a medicament for treating wounds in a subject is also provided. Use of the pharmaceutical compositions as disclosed herein in the preparation of a medicament for promoting the regeneration of epithelial tissue in a subject is also provided.

[0084] As used herein, the term "treatment" includes reducing or alleviating at least one adverse reaction or symptom of a disease or condition. As used herein, "pharmaceuticalally effective amount" means an amount of pharmaceutical composition sufficient to produce a beneficial or desired clinical effect. This amount may be administered in one or more doses. However, factors considered to be effective amounts can be precisely determined based on individual patient factors, including but not limited to the patient's age, wound size, wound type, wound severity, and route of administration of the pharmaceutical composition. This amount can be readily determined by a person skilled in the art based on known procedures (including clinical trials) and the methods disclosed herein.

[0085] As used herein, the term "subject" includes warm-blooded animals, preferably mammals, including humans. In some specific instances, the subject is a primate. In one specific instance, the subject is a human.

[0086] In some instances, the subject is a patient with or is believed to have an underlying medical condition or disease. In some other instances, the subject has or is believed to have cancer. In still other instances, the subject has or is believed to have diabetes. In yet another few instances, the subject is undergoing or has undergone further treatment, which includes, but is not limited to, chemotherapy, chemoprevention, radiation therapy, immunosuppressive therapy, steroid therapy, etc.

[0087] As used in this article, the term "wound" refers to damage to the body, which typically involves a tear or rupture of a membrane (such as the skin). Trauma can also include damage to underlying tissues and, in most cases, is the result of external physical forces on the body.

[0088] In some cases, the wound is characterized by slow healing or failure to heal.

[0089] For example, a non-healing wound is one that does not heal in the manner of a set of ordered stages within a predictable amount of time, as is the case with most wounds; a wound that does not heal within three months is generally considered a non-healing wound. Wounds can exhibit a range of healing rates, with acute and non-healing wounds at opposite ends of this range.

[0090] Possible causes of non-healing or slow-healing wounds may include, for example, pre-existing and / or underlying conditions or diseases, or because the subject is undergoing further treatment that impairs wound healing. These conditions or diseases may be pathological or non-pathological and can worsen or exacerbate wound healing by their presence in the subject. Examples of such conditions and / or diseases are, but are not limited to, cancer, diabetes (type I and II), skin diseases, autoimmune diseases, epithelial lining, inflammatory conditions of the dermis and / or subdermis (internal and external), eczema, etc.

[0091] Examples of wounds include, but are not limited to, chronic wounds, acute wounds, traumatic wounds, subacute wounds and lacerations, wounds caused by burns, partial-thickness burns, ulcers (such as diabetic ulcers, pressure ulcers, or venous insufficiency ulcers), flaps, and grafts. In one instance, the wound is caused by a burn or a chronic wound.

[0092] The pharmaceutical compositions disclosed herein can be applied to wounds by direct topical application. Alternatively, the pharmaceutical composition can be applied to a carrier material and then applied to the wound. Such methods may include applying the pharmaceutical composition to a bandage, gauze, or dressing to be applied to a wound. The pharmaceutical compositions provided herein may also be added to other compositions known for treating wounds.

[0093] The term “topical” application refers to application to a site of skin, dermis, or tissue, and application to such a site may include application to an adjacent site or within a site of tissue.

[0094] As used herein, the term "tissue site" broadly refers to a wound or defect located on or within a tissue, including but not limited to bone tissue, adipose tissue, muscle tissue, nerve tissue, dermis, vascular tissue, connective tissue, cartilage, tendon, or ligament.

[0095] In addition to local application, the pharmaceutical compositions disclosed herein can also be administered to subjects in need via other routes. Examples of administration include, but are not limited to, intravenous, intravascular, intramuscular, subcutaneous, intracerebral, intraperitoneal, soft tissue injection, surgical site, arthroscopic site, and percutaneous insertion, such as by direct injection, intubation, or catheter insertion. Any administration can be a single or multiple administration of the pharmaceutical composition. Administration can be performed at a single site or more than one site on the subject to be treated. Multiple administrations can occur substantially simultaneously or separately at different times.

[0096] This disclosure also shows that Agrin fragments or derivatives as described herein promote collective keratinocyte migration. It is also shown that Agrin-promoted collective keratinocyte migration is achieved by engaging MMP12 as a downstream effector. Since wound re-epithelialization begins after keratinocytes at the wound margin are activated to migrate, this disclosure also provides a method for promoting wound re-epithelialization, comprising administering an Agrin fragment or derivative thereof as disclosed herein.

[0097] As used in this article, the term "reepithelialization" refers to the process by which a new barrier is created between the wound and the environment through the migration of epithelial cells. The cellular and molecular processes involved in the initiation, maintenance, and completion of reepithelialization are essential for successful wound closure.

[0098] The invention described herein can be suitably practiced without the presence of any one or more elements or limitations not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” and “containing” should be understood broadly and without limitation. Furthermore, the terminology and expressions used herein have been used as descriptive rather than limiting terms, and their use is not intended to exclude any equivalents or portions thereof of the shown and described features; however, it should be recognized that various modifications can be made within the scope of the claimed invention. Therefore, it should be understood that although the invention has been specifically disclosed by way of preferred embodiments and optional features, modifications and variations of the invention disclosed herein and embodied can be adopted by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention.

[0099] Those skilled in the art will understand that various methods and techniques known in the art can be used to implement certain embodiments of the present invention.

[0100] Throughout this disclosure, certain embodiments may be disclosed in range form. It should be understood that the range form description is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the disclosed range. Therefore, the range description should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0101] This invention has been described broadly and generally herein. Each narrower group of species and subgenus falling within the scope of this general disclosure also forms part of this invention. This includes inventions with a general description that includes conditions or negative limitations removing any subject matter from that genus, regardless of whether the removed material has been specifically described herein.

[0102] Other embodiments are described within the scope of the following claims and non-limiting embodiments. Furthermore, in the context of the description of features or aspects of the invention according to the Markush Group, those skilled in the art will recognize that the invention is also described according to any single member or subgroup of the Markush Group.

[0103] Experimental Section

[0104] Materials and methods

[0105] Antibodies, siRNA and reagents

[0106] The following antibodies were used in the study: mouse monoclonal anti-Agrin (D-2), Santa Cruz Biotechnology, Cat#sc-374117; RRID: AB_10947251; human Agrin clone PIF12 (internal generation); rabbit polyclonal Agrin antibody, Novus Biologicals, Cat#NBP1-90209; mouse monoclonal anti-integrin β1, Abcam, Cat#ab24693; RRID: AB_448230; anti-integrin β1, activated, clone HUTS-4, Cat#MAB2079Z, RRID: AB_2233964; mouse monoclonal anti-β actin (C4), Santa Cruz Biotechnology, Cat#sc-47778; RRID: AB_2714189; rabbit polyclonal anti-GAPDH (FL-335), Santa Cruz Biotechnology. Biotechnology, Cat#sc-25778; RRID: AB_10167668; Phosphomyosin light chain 2 (Ser19) antibody #3671, RRID: AB_330248; Anti-myosin light chain (phosphate-S20) antibody (ab2480), RRID: AB_303094; Myosin light chain 2 antibody #3672, RRID: AB_330278; MMP-12 antibody clone G-2, Cat#sc-390863; Anti-MMP12 antibody, Abcam, Cat#ab137444; MMP12 polyclonal antibody, Thermo Fisher Scientific, Cat#PA5-27254, RRID: AB_2544730; Rabbit polyclonal anti-CD31, Abcam, Cat#ab28364; RRID: AB_726362; Goat anti-rabbit IgG-HRP, Santa Cruz Biotechnology, Cat#sc-2030; RRID: AB_631747; Goat anti-mouse IgG-HRP, Santa Cruz Biotechnology, Cat#sc-2005; RRID: AB_631736; Goat anti-mouse IgM-HRP, Santa Cruz Biotechnology, Cat#sc-2973; RRID: AB_650513; Goat anti-rabbit IgG (H+L) Alexa Flour 488 Invitrogen, Cat#A11034; Goat anti-mouse IgM Alexa Flour 594, Invitrogen, Cat#A21044.The following siRNAs were used in this study: human Agrin recessive siRNAs (a set of three) HSS139721, HSS180123, and HSS180124; mouse Agrin recessive siRNAs (a set of three) MSS201833, MSS201834, and MSS201835 (Thermo Fisher Scientific); human Agrin smartpool from Dharmacon (Cat#L-031716-00-0050); and human MMP12 (Cat#L-005954-00-0050). Specific siRNA sequences are listed in Table 3. Lipofectamine RNAimax (Invitrogen) was used for siRNA transfection according to the manufacturer's recommended guidelines. Fibronectin was obtained from Gibco and Advanced Biomatrix. Mouse tail type I collagen was obtained from Corning. Alexa-488-conjugated F-actin phalloidin was obtained from Thermo Fisher Scientific. Brestatin and Pluronic F127 were obtained from Sigma, MMP408 from Merck Millipore, Aquaphore from Beirsdorf AG, and Vaseline from Unilever. Dimethyl sulfoxide was obtained from Kanto Chemical, co., Inc., Cat#10378-00. CellTiter. The AQueous One Solution Cell Proliferation Assay (MTS, Promega Cat#G3580) was used for proliferation assay. 5-Bromo-2'-deoxyuridine-BrdU (#Cat ab142567) and anti-BrdU antibody (Cat#ab6326) were purchased from Abcam.

[0107] mice

[0108] Six- to eight-week-old female experimental ICR (named IcrTac:ICR) mice purchased from InVivos were used for in vivo assays of wound healing from perforated biopsies. In explant assays of mouse skin, randomized proportions of male and female newborn ICR mouse pups were used. Experiments were conducted whenever possible, taking into account ethical considerations and minimal animal disposal practices. Animal studies were not blinded. Animals were randomly assigned to different experimental groups. The number of animals used in each experiment is represented in the legend as n = x mice / group. All animal experiments were conducted according to protocols reviewed by the Agency for Science, Technology and Research (A*STAR) Bioresource Centre (BRC) in Singapore, strictly adhering to the Institutional Animal Care and Use Committee (IACUC) guidelines on the ethical use of animal models in biological research.

[0109] Cell lines and human skin explants

[0110] The human epidermal keratinocyte line HaCaT was maintained in Dulbecco modified Eagle medium (DMEM) (Gibco) containing 10% fetal bovine serum (FBS) and antibiotics penicillin and streptomycin (Gibco Cat#15140148). Cells obtained from ATCC (… CRL-2522 TM Normal human foreskin fibroblasts (BJ) were passaged in antibiotic-containing DMEM (Gibco). Normal primary adult epidermal keratinocytes (HEK), purchased from CELL Applications, Inc. (Cat#C-12003), were maintained in the manufacturer's recommended keratinocyte growth medium as recommended. Primary keratinocytes from the C57BL mouse strain were purchased from Cell Biologics (Cat#C57-6066K) and maintained in the manufacturer's recommended epithelial cell growth medium (Cat#M6621, Cell Biologics). Primary mouse dermal fibroblasts were isolated from the C57BL mouse strain (Cat#m-GFP-6067) and cultured in complete fibroblast medium (Cell Biologics, Cat#M2267). Full-thickness epidermis (EPIDERM-FT) on the basement membrane with a 3 mm perforation wound was measured. TM Human skin explants were purchased from MatTek Lifesciences and maintained using the company's culture medium. All cells were proliferated under standard culture conditions of 37°C and 5% CO2.

[0111] In vitro and in vivo RNA interference and knockdown

[0112] Dissolve the desired siRNA in the manufacturer's recommended buffer. For in vitro studies, mix 50 nM control or targeting siRNA with 5 μl Lipofectamine RNAimax (Thermo Fisher Scientific) and incubate at room temperature for 30 minutes. Add the siRNA:lipofectamine mixture to 250 μl of reduced growth factor medium. Validate the knockdown by RT-PCR or Western blot after 72 hours. For in vivo and in vitro RNAi, mix 75 nM latent siRNA with 8 μl Lipofectamine RNAiMAX (Thermo Fisher Scientific) at room temperature for 30 minutes. Subsequently, slowly incorporate the siRNA mixture into a 1:1 mixture of Aquaphore gel and phosphate-buffered saline (PBS) for local delivery. For treatment with skin explants, add the siRNA:lipofectamine mixture to Aquaphore on day 0 post-injury to form a 1:1 mixture and incubate for the specified number of days. For effective knockdown, reapply the topical siRNA ointment on day 4 post-injury. For explants rescued with sAgrin, an Aquaphore mixture containing 20 μg of protein was added on days 2, 4, and 6 post-injury.

[0113] Recombinant Agrin Expression and Purification

[0114] The C-terminal fragment (AgrinZ8LG3, with sequence: ) containing an 8-amino acid 'Z8' insert and an LG3 domain is used.

[0115] The bacteria, DTLAFDGRTFVEYLNAVTESELANEIPVEKALQSNHFELSLRTEATQGLVLWSGKATERADYVALAIVDGHLQLSYNLGSQPVVLRSTVPVNTNRWLRVVAHREQREGSLQVGNEAPVTGSSPLGATQLDTDGALWLGGLPELPVGPALPKAYGTGFVGCLRDVVVGRHPLHLLEDAVTKPELRPCPTP (SEQ ID NO: 7)), were cloned into the pET28 vector containing His-ta and expressed in *E. coli* (strain BL21-DE3). The bacteria were transformed with the plasmid. Single colonies were inoculated into TB medium containing antibiotics and induced with IPTG at 15°C for 16 hours. Cells were harvested by centrifugation, and the cell pellet was lysed in lysis buffer and then sonicated. The supernatant containing Agrin protein fragments, obtained by purifying a Superdex 75 column in imidazole buffer using gel filtration chromatography, was sterilized by passing it through a 22 μm low protein binding filter and then dissolved in PBS solution.

[0116] Perforation-biopsy wound healing model

[0117] Mice were housed under standard conditions of 21°C and a 12-hour light-dark cycle with free access to food. Subsequently, mice were anesthetized intraperitoneally (ip) with ketamine (100 mg / kg) diluted in 100 ml saline solution and xylazine (10 mg / kg). Hair was shaved from the base of the neck to the back across the entire shoulder area. The skin was wiped with alcohol and 10% povidone-iodine (Betadine) antibacterial solution. A circular, symmetrical 4 mm wound was created on either side of the shoulder area using a sterile 4 mm perforated biopsy needle (Integra Miltex, Integra York, PA, Inc.). The wound skin was carefully removed using a scalpel and scissors to create a full-thickness wound, which was kept open for local application during the analysis period. For a wound healing model with fixation, a 10 mm clear annular nylon sheet (Grace Bio-Laboratories, Bend, OR) was placed around the 4 mm wound. The fixation was positioned so that the wound was centered, and the fixation was applied with an adhesive (Krazy). (Elmer's Inc.) Adhere to the skin and cover with a Tegaderm dressing. Treat the wound with different formulations based on petroleum jelly, thermosensitive hydrogel (Pluronic F127-Sigma), or commercially available skin ointment (Aquaphore-Beiersdorf, Inc.). For petroleum jelly-based ointment emulsions, use 10 mg / cm³.2 Vaseline ointment was mixed with a filtered PBS solution containing a specified amount of Agrin (100 μg, 200 μg, or 500 μg) and 100 μg BSA (w / w) and applied topically every two days. For siRNA-mediated knockdown experiments, 75 nM of the specified siRNA was incubated with lipofectamine in PBS. Subsequently, a 1:1 mixture of PBS (containing siRNA):Aquaphore was applied topically to the wound area every two days. A topical formulation containing 20% ​​(w / w) Pluronic F127 (Sigma) was dissolved in a sterile, 0.22 μL filtered solution containing a final concentration of 200 μg / ml of sAgrin or BSA. The mixture was dissolved overnight at 4°C to ensure a liquid phase. The mixture formed a homogeneous gel at a temperature above approximately 20°C and was topically dispersed to cover the wound. Wound closure was assessed every other day using calipers. For both models, animals were kept in solitary cages in institutional animal facilities throughout the observed healing timeline. All animal experiments on wound healing were conducted in accordance with protocols reviewed by the Agency for Science, Technology and Research (A*STAR) Bioresource Centre (BRC) and strictly adhered to the Institutional Animal Care and Use Committee (IACUC) guidelines on the ethical use of animal models in biological research.

[0118] Mouse skin explant assay

[0119] Three-day-old ICR strain mouse pups were euthanized. Posterior skin from either side of the shoulder region was washed in 70% ethanol and surgically removed. The excised skin was washed in 70% ethanol and placed (dermal side down) on a pre-prepared gel substrate with defined hardness, with or without 20 μg / ml sAgrin. The explants were allowed to adhere for 2 hours, and then... 31 Adding 300μM Ca 2+ DMEM (dimethylformaldehyde) culture medium for keratinocytes was used. The growth of keratinocytes and skin tissue were imaged periodically under an Axiovert-200 inverted microscope, and then fixed in 4% paraformaldehyde and immunostained with an antibody against keratin 17.

[0120] Substrate hardness manipulation and micropatterning

[0121] Following the manufacturer's recommendations, tissue culture plates were coated with Col-T-gel (Fischer Scientific) with a hardness ranging from 0.8 kPa (soft) to 30 kPa (hard) and cured in a tissue culture incubator at 37°C for 40 minutes. Some experiments were performed using cells seeded on hydrogel plates of specified hardness: 0.2 kPa and 16 kPa soft and hard hydrogels (CytoSoft, Advanced Biomatrix, Inc.). The hardness of the siloxane substrate was determined by the manufacturer according to previously published protocols. For some experiments, a specified amount of sAgrin was incorporated into a 0.8 kPa collagen gel prior to gelation. After gelation, trypsin-digested cells or excised mouse skin explants were seeded onto gels of different hardnesses, either alone or containing sAgrin. Cells / tissues were incubated for the specified number of days under standard culture conditions with 500 μl of the recommended culture medium. Cross-shaped micropatterns with fibronectin-coated islands were fabricated using photolithography on a 19.5 × 19.5 mm coverslip from Cytoo, Inc., France. The surface area of ​​the micropatterns was 800 μm. 2 Or 1600μ 2 50,000 cells initially treated with siRNA were placed on micropatterns for 4–6 hours, and then treated for immunofluorescence. For some experiments, 10 μg / ml sAgrin was added to a slide 18 hours before cell addition.

[0122] 3D-hardness-dependent keratinocyte-fibroblast co-culture migration assay

[0123] A 3D in vitro wound healing model was created using collagen constructs with defined stiffness, modified from previously disclosed protocols. Collagen constructs containing 300,000 primary mouse dermal fibroblasts (DF) were prepared using Col-T-gel (0.8 kPa - soft) or (30 kPa - hard) according to the manufacturer's recommended protocol. For some experiments, DF treated with siControl and siAgrin was incorporated into either the soft or hard collagen constructs. The constructs carrying fibroblasts were cured in an incubator for 45 minutes. A second layer of collagen containing 350,000 primary mouse keratinocytes (KRT) was added to match the stiffness of the underlying layer, and the constructs were cured for 3–4 hours. After removing excess culture medium, the constructs were gently washed twice with 1×PBS. An inverted 2 μm pipette tip was inserted and gently rotated at the center of the construct to create a circular wound. Collagen and cells were rapidly removed from the wound area and replaced with 40 μL of soft or hard Col-T gel matched to the consistency and setup of the construct. The resulting wound construct was placed in an incubator and imaged after 30 minutes for use at day 0 timepoint. Migrating keratinocytes were imaged until day 5 post-traumatic initiation.

[0124] Western blot analysis

[0125] Manipulated cells were washed twice with cold phosphate-buffered saline (PBS) and lysed for 15 min at 4°C with cold 1% NP-40 lysis buffer supplemented with 1× protease inhibitor mixture (Roche Applied Biosciences). Cell lysates were centrifuged at 13,000 rpm for 15 min. Protein estimation was then performed using Bradford reagent. Subsequently, 40–50 μL of total protein was mixed with an equal volume of 2× Laemmli sample buffer and heated at 95°C for 5 min. The mixture was then separated by SDS-PAGE gel electrophoresis. The separated proteins were transferred to nitrocellulose membranes and blocked in 5% skim milk reconstituted in 1× PBS containing 0.1% Tween-20, and probed overnight with the appropriate primary antibody. The membranes were then washed with 1× PBS supplemented with 0.1% Tween-20; washed three times at 15-minute intervals. The membranes were then incubated for 1 h with conjugated horseradish peroxidase HRP secondary antibody (SantaCruz Biotechnology). After incubation, the blots were washed three more times as described above, then covered with an enhanced chemiluminescence (ECL) substrate (Pierce / Bio-Rad) and visualized on X-ray film using an image processor or digitally visualized using a Chemidoc analyzer (Bio-Rad). The density of each band was quantified using Image-J software.

[0126] Immunofluorescence and confocal microscopy

[0127] Cells were cultured overnight on eight-well slides or coverslips. They were then washed twice with PBS and fixed with 4% paraformaldehyde for 15 minutes. Subsequently, they were incubated at room temperature with a solution of 1 mM Ca. +2 and 1mM Mg +2 Cells were permeabilized for 15 minutes with 0.1% Triton X in phosphate-buffered saline (PBSCM). The permeabilized cells were incubated with the designated antibody in fluorescent dilution buffer (FDB) for 1–2 hours at room temperature or overnight at 4°C, then washed five times with PBSCM and incubated with secondary antibody Alexa Fluor (Thermo Fisher Scientific) for 1 hour at room temperature. The slides were washed five more times with PBSCM and mounted on Vectashield medium containing DAPI. The stained cells were then imaged using a Zeiss confocal microscope. Images were processed and analyzed using Zen Blue software. Intensity measurements were obtained by selecting parameters using the image analysis tools within Zen Blue software.

[0128] Histology and Immunofluorescence - Histology

[0129] Human skin equivalents and mouse skin tissues were harvested, embedded, and subjected to routine histology using hematoxylin and eosin. For immunostaining, tissues were fixed in 10% neutral buffered formalin for 24 hours. Subsequently, tissue sections were prepared using an ultramicrotome and antigen-retrieval was performed at pH 9 or pH 6 (for the specified antibody). Primary antibodies were used at a 1:50 dilution, while the corresponding Alexa-488 and Alexa-594 secondary antibodies (ThermoFisher Scientific) were used at 1:100 and 1:500 concentrations in fluorescent dilution buffer, respectively. Slides were mounted on VECTASHIELD hardset containing DAPI. TM In an anti-fading sealing medium. Following a previously published protocol, collagen was quantified by staining paraffin-embedded mouse skin tissue with Sirius red to serve as a marker for ECM deposition.

[0130] In vitro wound healing assay

[0131] Unidirectional scraping was performed on the confluent monolayers of control and Agrin-depleted cells in 6-well plates using a 20 μL pipette tip. Cells were then washed with 1×PBS at room temperature and incubated in complete medium at 37°C and 5% CO2, with or without sAgrin, as indicated. Phase-contrast images of the wound area were taken periodically at specified time points. For some wound healing migration assays, keratinocytes were cultured for 18 hours on a soft or hard substrate within a barrier plate (Nalge Nunc., Inc.). After the barrier was removed, cells were allowed to migrate and imaged using an Axiovert 200 inverted microscope at specified time points.

[0132] Traction microscopy

[0133] Substrate Preparation – First, two portions of silicone elastomer DOWSIL CY52-276 (Dow Inc.) were thoroughly mixed at a 1:1 weight ratio. The mixture was then poured into a 35mm glass-bottomed culture dish (iwaki 3930-035, Asahi Techno Glass Corporation, Japan) placed on a horizontal surface, achieving a thickness of approximately 500 μm. After overnight pre-curing, the silicone-coated culture dish was further baked at 80°C for 2 hours for complete curing. To maximize subsequent microsphere adhesion, the silicone film was pretreated with (3-aminopropyl)triethoxysilane (A3648, Sigma-Aldrich). For silanization, 1 ml of a solution containing 96% v / v, 2% v / v APTES, and 1% v / v acetic acid (A6283, Sigma-Aldrich) was added to the glass-bottomed culture dish, covering the entire silicone film, and allowed to react for 10 minutes. The silicone membrane was then rinsed twice with 96% v / v ethanol and then quickly immersed in Milli-Q water to remove all solution: this method of removing solution from the membrane is superior to drying with nitrogen, as the latter may introduce dust into the surface, subsequently affecting the uniformity of the attached microsphere distribution. The culture dish was then baked at 80°C for 2 hours to promote siloxane bond formation. To physically adsorb fluorescent latex microspheres onto the silicone membrane, 6 μl of 100 nm orange fluorescent (540 / 560) carboxylate-modified microspheres (F8800, ThermoFisher Scientific) were first diluted in 10 ml of Milli-Q water and sonicated for 10 min. The diluted microspheres were then passed through a 0.22 μm filter directly into a 15 ml Falcon tube containing 500 μl of 500 mM MES buffer (pH 6.0) (M3671, Sigma Aldrich). The microspheres were then sonicated again for 10 minutes and then covered with an APTES silanized silicone membrane placed in the previously prepared culture dish. The solution was left to stand for 5 minutes to allow the microspheres to attach. To remove the solution, the culture dish was then carefully and quickly tilted to remove all solution in a single action, as the surface tension of the solution would cause the microspheres to separate if reflux was allowed. The microsphere-coupled culture dish was then baked at 80°C for 2 hours and is typically used within one week. To promote cell adhesion, the microsphere-coupled silicone membrane was coated with 50 μg / ml human plasma fibronectin (10838039001, from Sigma Aldrich) to a surface density of 5 μg / cm³. 2 The hardness of the silicone substrate used for TFM was determined by atomic force microscopy according to the previously established protocol described in the following section.

[0134] Traction imaging – Live-cell imaging of HACAT cell sheet migration and microsphere displacement was performed over 24 hours at 10-minute intervals using a Nikon Biostation IMQ at 37°C and 5% CO2. Phase contrast and epifluorescence images were acquired using an internal 20× objective (0.5NA) and a 1.3-megapixel monochrome camera. The light source for epifluorescence was an Intensilight Hg pre-centered fiber optic illuminator, and the orange microspheres were imaged using a Texas Red filter array. To account for z-drift, 2–3 planes spaced 1 μm apart on either side of the focal plane were typically taken in the z-stack. Three independent samples were imaged for each condition, and for each independent sample, 3–4 locations were randomly selected and tracked.

[0135] Cell stiffness via atomic force microscopy (AFM)

[0136] The stiffness of mouse keratinocytes was measured using the Nanowizard IV BioAFM system (JPK Instruments, Germany). Confluent mouse keratinocyte monolayers were cultured in PBS-containing dishes. The monolayer was scraped across the center of the dish using a 100 μl pipette tip and characterized after 4 hours. Indentations were made on randomly selected cells at the wound edge, with polystyrene beads (~4.5 μm in diameter) attached to the end of a cantilever using a 3 nN force at 1 Hz (k = 0.03 N / m, Novascan Technologies, Inc., Ames, IA). More than 60 cells from triplicate experiments were characterized and averaged to assess Young's modulus for each condition. Young's modulus values ​​were calculated using JPK data processing software (JPK Instruments, Germany), employing a Hertzian contact model with a spherical indenter (4.5 μm in diameter; Poisson's ratio 0.5) to fit the extension curve.

[0137] The fabrication of the polyacrylamide block for collective traction force measurement in a wound healing assay model was first performed by preparing a polyacrylamide precursor mixture as follows. To prepare 1 ml of such a precursor mixture, 200 μl of 40% w / v aqueous AC (1610140, Bio-Rad), 200 μl of 2% w / v aqueous BIS (1610142, Bio-Rad), 1.5 μl of TEMED (1610800, Bio-Rad), and 583 μl of Milli-Q water were thoroughly mixed together. Then, 500 μl of this mixture was combined with 8 μl of 10% ammonium persulfate (1610700, Bio-Rad) and rapidly piped into a 3D-printed mold with a cavity size of 1.2 × 1.2 × 0.5 cm. A slightly larger square piece of 3-(trimethoxysilyl)propyl methacrylate (440159, Sigma-Aldrich) silanized glass was then placed on top. Once the reaction is complete, the glass will covalently bond with the polyacrylamide and act as a solid support for the block; it will also contribute to adding weight to the block. The silanization procedure for this glass support is similar to the silanization procedure for the silicone membrane described in the main text, except that TMSPMA is used instead of APTES. After 30 minutes, the solid hydrogel is removed from the mold and immersed in Milli-Q water overnight to wash away any remaining toxic components. The mold and the finished block are as follows. Figure 2 As shown in (a), the intended placement of the block in the glass-bottomed petri dish is shown in Figure 02(b).

[0138] Wound assessment using microfabrication devices in collective traction microsurgery

[0139] Before seeding HaCaT cells, a 35 mm culture dish containing an ECM-coated silicone membrane was filled with a polyacrylamide hydrogel block (1.2 × 1.2 × 0.5 cm, see also). Figure 22 This will ultimately be used to create clean wounds in HaCaT cell sheets. Polyacrylamide hydrogel is used because it resists protein binding, thus preventing cell adhesion and also preventing ECM removal from the silicone surface. After equilibrating the hydrogel block in the culture dish with growth medium for 30 minutes, the dish was replaced with fresh medium, and approximately 7 × 10⁻⁶ cells were added. 5 Cells were evenly seeded around the hydrogel block. The device was incubated for 24 hours before removing the hydrogel block, and then the cells were imaged (see also...). Figure 2 ).

[0140] Force transmission via ligand-conjugated magnetic beads

[0141] The ligands were conjugated to 4.5 μm epoxy paramagnetic beads (Dynabeads M-450Epoxy, Thermo Fisher Scientific) according to the manufacturer's manual. In short, the beads were washed twice in 0.1 M sodium phosphate buffer (pH 7.4). Subsequently, at 4 °C, 8 × 10⁸ beads were subjected to... 7 20 μgs of Agrin, BSA, and FN were conjugated to each magnetic bead in 0.1 M sodium phosphate buffer (pH 7.4) and gently rotated over 16–18 hours. The degree of protein conjugation to the beads was verified using a subset of ligand-conjugated beads by reducing SDS-PAGE gel analysis. Residual beads were washed and then stored in 0.1% BSA-phosphate-buffered saline (PBS) (pH 7.4) at 4°C. They were then resuspended in culture medium and added to cells at 37°C for 30 min. After a brief wash with PBS to remove excess unattached beads, cells were placed 6 mm from a permanent neodymium magnet (KJ Magnetics, USA) for 30 min, allowing vertical tension on the beads to be on the order of approximately 200 pN. Cells were then fixed in 4% paraformaldehyde, permeabilized, and treated for immunofluorescence.

[0142] Mathematical calculation of traction force

[0143] The displacement fields associated with the fluorescent bead images were calculated using PIVLab, an open-source MATLAB package for particle image veocimetry based on cross-correlation. Images of the substrate in a stress-free state (i.e., sufficiently long after 1% v / v sodium dodecyl sulfate (L4509, Sigma Aldrich) was added to the culture to kill cells) were used as reference images to calculate these deformations. During cross-correlation, a four-window pass was used, with each (square) window measuring 64, 32, 16, and 16 pixels, and 50% window overlap between strides. These calculated displacement fields were then used in MATLAB code to perform Fourier Transform Traction Cytometry (FTTC) to infer the traction force. For all FTTC calculations, Young's modulus and Poisson's ratio were assumed to be 10 kPa and 0.5, respectively. The optimal L2 regularization parameter was selected using the L-curve criterion. More specifically, L-curves were constructed for frames 1, 10, 20, 30, 40, 50, and 60 of each sample movie, and then the optimal regularization parameter corresponding to each L-curve was selected using MATLAB's L-angle function. The median of all optimal regularizations (i.e., the parameter computed for each frame of each sample) was considered the optimal regularization parameter (found to be 1.26 × 10⁻⁶). -9 Then, the regularization parameter is fixed for all samples. Note that a larger regularization parameter will reduce the size of the force field we calculate. This is done to avoid introducing artificial differences in the magnitude of the calculated traction field by systematically selecting smaller or larger regularization parameters for different sample conditions.

[0144] Image stitching

[0145] An internal script based on image cross-correlation is used to stitch adjacent image fields together to generate a larger traction field.

[0146] 3D-angiogenesis assay of dermal endothelial cells and fibroblasts

[0147] One hundred thousand human dermal microvascular endothelial cells (HDMECs) were pre-labeled overnight with Cell Tracking Blue CMAC (7-amino-4-chloromethylcoumarin-Invitrogen) and mixed with an equal volume of GFP-expressing BJ cells. Both cell types were treated with relevant siRNA and induced to form co-culture spheroids according to a previously established protocol. Images of sprouting ECs were captured 24 hours after embedding and quantified using the Sprout morphology plugin (Image J) from Fiji, as previously described.

[0148] MMP12 activity assay

[0149] MMP12 activity was monitored using gelatin and casein proteomic profiling, following a previously published protocol. In short, control or Agrin-depleted HaCaT cells were grown to 80-90% confluence in FBS-free medium. The supernatant collected from each culture dish was centrifuged at 10,000 rpm for 5 minutes and concentrated using an Amicon ultra column. 20 μL of medium was mixed with 2×SDS loading buffer and loaded onto a 10% gelatin or casein gel. The gel was washed with incubation buffer and then stained with Coomassie Brilliant Blue for 1 hour. The gel was destained for 30 minutes and then imaged using a Chemidoc imaging system.

[0150] Quantitative reverse transcription PCR (RT-PCR) and RNA sequencing

[0151] Total RNA was extracted from the designated cells using the Qiagen RNeasy mini kit, following the manufacturer's recommended protocol. The total RNA was then reverse transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems). The resulting cDNA (200 ng) was used as a template for RT-PCR, which employed a SYBR-based PCR method. TM Green or Taqman RPremixed solutions and probes from ThermoFisher Scientific were used. Data were normalized relative to GAPDH as an endogenous control. RT-PCR primers used for each target gene in the study are listed in Table 1. For RNA sequencing, RNA quality was analyzed on a Bioanalyzer instrument (Agilent) using the Agilent RNA 6000 Pico kit. A total of 4 μg of RNA was used for RNA sequencing library preparation using the TruSeq Stranded mRNA Library Prep kit (Illumina) according to the manufacturer's instructions. Library amplification was limited to 7 PCR cycles. The purified libraries were analyzed by qPCR (KAPA Library Quantification Kit for Illumina, Roche). The Agilent High Sensitivity DNA Kit was used to assess fragment lengths of a subset of the libraries, and the remaining libraries were then pooled and further sequenced on a NextSeq500 (Illumina). Sequencing conditions were represented by high-output single-read runs with 75 bp read lengths. Raw reads from the fastq file were aligned to the hg38 genome using STAR 2.6.1d. Bam files were sorted and indexed using samtools. Based on features from the gencode.v29.annotation.gtf file, the relative number of reads mapped to each gene was quantified using htseqcount. The raw read counts were then input into EdgeR in R for differential expression analysis using Gene-ontology, GSEA, or other analyses. Gene set enrichment analysis was performed using the GSEA pre-sorting tool of Gene Set Enrichment Analysis (GSEA) software 3.0, using a gene set from the Molecular Signature Database (MSigDB) v6.2. Metascape (…) was used to perform the gene set enrichment analysis. https: / / metascape.org / Identify enriched gene ontology terms and KEGG pathways. Only consider genes with a false discovery rate (FDR) of less than 1% and expression changes of more than 2-fold between conditions.

[0152] Table 1. List of RT-PCR primers used

[0153]

[0154]

[0155] Statistical analysis

[0156] The number of biological and technical replicates for each experiment is shown in the legend. For most in vitro experiments, three biological replicates were performed unless otherwise indicated in the legend. For in vivo experiments, no animals were excluded from the analysis, and no power analysis was used to predetermine sample size. Animal age and sex are mentioned in the Methods section. Randomization was not applied to experiments using cell lines. Data are presented as mean + / - sd. GraphPad Prism software was used, with Student's t test employed to detect paired comparisons, and ANOVA or multiple t tests used to compare multiple groups. When *p < 0.05, ** p<0.005 *** Data were considered statistically significant when p < 0.0005. Data considered insignificant were denoted as 'ns'. No prior statistical tests or hypotheses were used to determine sample sizes for in vitro, ex vivo, and in vivo experiments. Three biological replicates were selected for in vitro experiments, as this follows common practice in biomedical research.

[0157] result

[0158] Skin trauma triggers the agrin-rich microenvironment

[0159] Reconstruction of lost ECM components significantly impacts epithelial wound healing to generate a new matrix that supports keratinocyte reepithelialization, thereby promoting wound closure. Focusing on the early stages of wound healing (where keratinocytes expressing keratin 17 (KRT17) are a marker of reepithelialization), we identified wound markers of ECM proteins, including Agrin (AGRN), basement membrane proteoglycan (Perlecan) (HSPG2), and phosphatidylinositol proteoglycans (Glypican) 1–3 (GPC1–3), which enhanced within 1–10 days after perforated wound biopsy in mouse skin. Figure 1 a) Following this trend, the expression levels of these ECM proteins were validated in vitro using a scratch wound model that phenotypically replicates a mechanical injury environment across several skin cell types, including immortalized and primary human keratinocytes (HaCaT and HEK) and human skin fibroblasts (BJ). Figure 1 As shown in b, the inventors analyzed the mRNA and protein levels in migrating cells initiating wound coverage over an early timeframe of 24 hours. Proteoglycans AGRN, GPC1, and HSPG2 were chosen because they were significantly upregulated during the early healing phase in the mouse model. Figure 1a). Increased mRNA levels of selected ECM wound tag genes were observed in migrating skin cells following the initial mechanical trauma; among them, AGRN expression was consistently increased in all analyzed skin cell types during the 24-hour post-traumatic period observed. Figure 1 b). Notably, a significant increase in Agrin expression was observed in keratinocytes (HaCaT and HEK) within 3 to 24 hours post-injury, while a significant enhancement was observed in fibroblasts (BJ) 24 hours post-injury. Figure 1 b). Conversely, within 3 to 24 hours post-injury, GPC1 mRNA increased 2 to 5-fold only in HaCaT cells, while no significant increase was observed in HEK and BJ cells. Figure 1 b). HSPG2 did not show any significant increase in any of the cell lines analyzed. Figure 1 b). With Figure 1 Consistent with the data analyzed in a, depleted GPC1 was observed to have a greater inhibitory effect on the migration rate of HaCaT cells after trauma compared with Agrin knockdown. Figure 2 Although Agrin expression is more robustly induced in damaged keratinocytes and fibroblasts (ac). Figure 1 (b) However, GPC1 can also serve as an important ECM proteoglycan for promoting skin wound healing. In this study, the inventors focused on Agrin because its role in skin injury-related models had never been documented. Furthermore, Agrin protein levels showed a consistent approximately 2 to 4-fold increase within 24 hours post-injury in human and mouse keratinocyte and fibroblast groups. Figure 1 c). Even in the uninjured state, human and mouse keratinocytes expressed higher levels of Agrin compared to their respective dermal fibroblasts, suggesting that keratin-derived Agrin may play a crucial role in skin injury repair. Figure 1 d). In a mouse model of 4mm perforated wound healing, robust agrin expression was detected as early as day 2 at the wound margin and around the wound bed, peaking on day 4 and then normalizing by day 8 post-injury. Figure 1 e). Immunohistochemical analysis further revealed that, compared with the dermis on day 2, the expression of Agrin in the keratinocyte layer of the epidermis significantly increased, reaching its maximum on day 4 after wound injury. Figure 1 f). Although increased agrin expression was also observed in the damaged dermis between days 2 and 4, no significant changes were detected in the subcutaneous tissue and dermal white adipose tissue (D-WAT) at any stage after injury. Figure 1 f). In summary, these results reveal that Agrin expression is significantly triggered in both the epidermis and dermis of the skin following mechanical injury.

[0160] Agrin depletion impairs skin wound healing.

[0161] To test the functional relevance of the agrin-enriched microenvironment in promoting wound healing in vivo, the inventors used three independent recessive siRNAs to knock down agrin in mouse skin to observe the effect on the healing rate of perforated biopsy wounds under "fixation-free" and "fixation-in" conditions, respectively. Figure 3 a). Latent siRNAs offer enhanced stability, minimal off-target effects, and accessibility to skin tissue, and are therefore increasingly used for effective knockdown in animal models. In both models, local injection of siRNA at the intended injury site three days prior to injury effectively reduced basal Agrin levels at the skin lesion site on the day of injury. Figure 3 b, left figure). Disordered or Agrin siRNA was mixed into a topical ointment formulation and applied to the open wound site every two days to robustly deplete Agrin expression throughout the early healing phase (including 9 days post-injury) in a fixative-free model. On day 4, in mice ectopically treated with Agrin siRNA, a significant reduction in the strong Agrin expression detected at the wound margin and around the wound bed in control skin validated the efficacy of Agrin knockdown. Figure 3 c). Inhibition of Agrin expression significantly delayed skin wound healing in vivo, indicating that Agrin is important for skin wound repair. Figure 4 a). Furthermore, effective wound healing depends on the ability of keratinocytes to express several wound-responsive keratins (e.g., keratin 6, 14, and 17), which drive the former's migration behavior at the wound site. Wound closure is initiated by a segment of migrating keratinocytes called the "epithelial tongue." The prominent epithelial tongue largely covers the wound site in skin treated with disordered siRNA, leaving the vast majority of the wound uncovered in Agrin-depleted skin sections. Figure 3 d). In this regard, a significant loss (>50%) of keratinocyte migration at the site of injury was observed in mouse skin treated with Agrin siRNA (as shown by the relative K17 occupancy). Figure 4 b).

[0162] Furthermore, the 4mm perforated wound was surrounded by a 10mm fixation firmly adhered to the skin, thus representing a "closed" fixation condition for wound healing. An ointment containing the corresponding siRNA was applied, followed by Tegaderm covering the wound area. Figure 4c). Therefore, the use of fixation minimizes purse-string mediated wound contraction and significantly promotes wound closure through reepithelialization. Compared with wound dressings without fixation, wound dressings with fixation reduced the wound healing rate on day 7. Figure 4 c). By using this in vivo model as an indicator of keratinocyte migration, skin agrin levels were effectively suppressed ( Figure 3 b, right figure). Agrin depletion in a fixed mouse model delays skin wound healing by attenuating keratinocyte reepithelialization, as indicated by reduced K17 occupancy. Figure 4 Combined with impaired reepithelialization, Agrin depletion severely inhibits the deposition of maturing and intermediate collagen fibers in the wound bed, indicating that impaired ECM replenishment in Agrin-depleted skin leads to a delayed healing response. Figure 4 f).

[0163] In addition, siRNA targeting human agrin was used to inhibit its expression in keratinocyte and human skin explant models. Figure 3 Notably, keratin-expressing keratinocytes efficiently migrated to close wounds in skin explants treated with control siRNA, a migration significantly inhibited by Agrin siRNA#1. Figure 3 fg). Agrin depletion (via Agrin siRNA#1) robustly attenuated the migration of K17-expressing epithelial tongues, thereby significantly delaying ex vivo wound closure rates in these human skin explants. Figure 4 gh, 3g). Interestingly, supplementation with the C-terminal recombinant protein fragment of Agrin (sAgrin), which has binding sites for its receptor lipoprotein-associated receptor-4 (Lrp4) and integrin, significantly rescued wound healing and keratinocyte migration in a human skin explant model. Figure 4 Based on these in vivo and in vitro functional studies, it was observed that Agrin knockdown severely impaired transpore migration and scratch wound-responsive migration rates in dermal fibroblasts and keratinocytes (human and mouse origin), which were again partially salvaged by Agrin. Figure 5 ad).

[0164] Since keratin reepithelialization is closely coordinated with its proliferative state, the question then arose whether Agrin supported keratin cell proliferation during the migration phase following wound injury. During the early in vitro migration within 4 hours post-wound injury, Agrin depletion did not affect the proliferation rate of leading cells, as measured by a 5-bromo-2'-deoxyuridine (BrdU) incorporation assay. Figure 6a). However, Agrin knockdown significantly reduced the proliferation of follower cells far from the wound edge, while supplementation with sAgrin salvaged this result ( Figure 6 a). Therefore, Agrin depletion differentially affects the proliferative state of leader and follower cells in vitro. In vivo, effective wound reepithelialization is achieved when highly proliferating keratinocytes (followers) at the wound margin push migrating cells (leaders) across the wound. Figure 6 b). As shown in the wound sections treated with control siRNA, during reepithelialization, proliferating keratinocytes at the epidermal base (solid arrow) were actively pushed by the hyperproliferating wound margin region (arrow) on day 7. Figure 6 b). Combined with damaged proliferation at the epidermal base, Agrin depletion severely inhibited keratinocyte proliferation in the dermis at the wound margin and wound bed. Figure 6 (bc). Therefore, this evidence suggests that Agrin enrichment in the wound environment supports keratinocyte proliferation, which triggers an efficient collective migration phase and wound closure.

[0165] Agrin sensitizes keratinocytes to ECM stiffness and fluid collective migration.

[0166] The role of Agrin in wound healing response has been demonstrated. The inventors investigated whether Agrin generates a mechanosensitive environment conducive to mass keratinocyte migration and wound closure. Since bulk stiffness from the ECM stimulates migration in multiple cell types, the inventors reasonably hypothesize that Agrin can integrate ECM stiffness signals with mass keratinocyte migration within the traumatic skin environment. The mass migration of HaCaT cells cultured on a hard (30 kPa) substrate was significantly higher than that on a compliant substrate (0.8 kPa). Figure 7 a). Interestingly, incorporation of sAgrin into the compliant substrate significantly improved the migration rate, comparable to that in the hard ECM alone. Figure 7 a). Collective keratinocyte migration is induced by the expression of keratin 17 (K17), particularly by leader cells located at the wound edge. Under these conditions, supplementation of the soft substrate with sAgrin stimulates collective migration by activating K17-expressing leader cells. Figure 7 b). Conversely, compared to control cells, Agrin-depleted keratinocytes on a hard matrix exhibited slower migration potential, which was significantly rescued when Agrin was integrated into the hard matrix. Figure 7 c). Agrin depletion in cells undergoing hard substrate formation also leads to a significant loss of K17-expressing migration leader cells. Figure 7d). K17-expressing leader cells were significantly restored in Agrin-depleted cells, which then exhibited enhanced migration upon sensing supplemental sAgrin in the hard ECM. Figure 7 cd).

[0167] As a complement to the 2D scratch wound assay, the inventors further designed an in vitro 3D substrate stiffness-dependent migration assay to summarize the role of Agrin in mediating keratinocyte migration under the simultaneous influence of potential dermal culture and bulk substrate stiffness experienced in natural skin tissue. In this strategy, primary mouse dermal fibroblasts (DF) were first embedded in a compliant collagen matrix (0.8 kPa), and then covered with mouse keratinocytes (KRT). Figure 7 e). A wound is created at the center, and the resulting area is immediately replenished with a soft matrix, allowing keratinocytes to migrate through an accommodative environment. (e.g.) Figure 7 As shown in e, supplementation with sAgrin in the soft matrix strongly enhanced keratinocyte migration on day 5 post-injury. In cases regenerated from the hard matrix, agrin depletion in both KRT and DF robustly eliminated migration rate ( Figure 7 f, Figure 2). Interestingly, supplementation with sAgrin in the hard matrix strongly restored keratinocyte migration (f). Figure 7 f, Figure 3). Notably, Agrin expressed by DF is insufficient to rescue Agrin-depleted keratinocytes from migrating onto the hard matrix. Figure 7 f, Figure 4). Furthermore, Agrin-depleted DF had the least effect on blocking keratinocyte migration on the hard matrix (f, Figure 4). Figure 7 (f, Figure 5). In summary, these data indicate that the increase in Agrin expression within keratinocytes is primarily a response to ECM stiffness sensing, which is both necessary and sufficient to drive migration within the wound microenvironment.

[0168] In the next example, the ability of sAgrin to sensitize the growth of collective ex vivo keratinocytes from mouse skin explants that had undergone growth on different substrate stiffnesses was tested. Accordingly, collective keratinocyte growth was monitored when full-thickness posterior skin from 2-day-old mouse pups was placed on collagen matrices corresponding to soft or hard substrates, with or without sAgrin. Figure 7 g). Compared to explants on a soft collagen matrix alone, sAgrin-supplemented compliant substrates significantly enhanced keratinocyte growth ( Figure 7h). In these skin explants that underwent hard matrix treatment without any exogenous sAgrin and were Agrin-depleted through mouse-specific siRNA treatment, no keratinocytes grew after 5 days of culture. Figure 7 i, middle figure). The difference is that Agrin-depleted skin explants sensitized to a hard substrate scaffolded with sAgrin exhibited higher keratinocyte growth (i, middle figure). Figure 7 i, Figure 3). In summary, these data suggest that Agrin enhances ECM stiffness sensing in keratinocytes, which guides collective migration after injury.

[0169] Cells introduce subtle changes in their intrinsic material properties by enhancing the tension of their motility-enhancing cytoskeleton, thereby responding to bulk ECM stiffness. To examine the mechanosensory effect of Agrin-enriched environments on keratinocytes, the stiffness of damaged migrating keratinocytes was first measured using atomic force microscopy as a marker of the cells' intrinsic material properties. The stiffness of migrating mouse keratinocytes, which was recorded as ~2.1 kPa 4 h after wound scraping, significantly decreased to ~0.9 kPa after Agrin knockdown. Figure 7 (j and 8a-b). Notably, sAgrin is the cause of a significant increase in cell stiffness (~1.5 kPa), thereby enhancing the mechanosensory perception of these migrating keratinocytes. Figure 7 (j and 8b). Therefore, Agrin knockdown “softens” migrating cells, preventing them from crossing the wound bed. Next, traction force microscopy (TFM) was performed to measure the cumulative force exerted by migrating cells on the basal layer in a mechanical stress environment simulating wound injury. In this collective migration model, the maximum traction force was exerted by cells at the ECM leading edge. Collective migration of Agrin-depleted cells was severely impaired, indicating a lower mean traction stress compared to control cells. Figure 7 k). While control cells exhibited higher collective traction at the leading edge, agrin-depleted cells showed significantly lower traction within both leader and follower cells, resulting in lower migration potential. Figure 7 The mean traction stress at the leading edge of cells was ~4.67 Pa 6 h after migration, decreasing by ~32% after Agrin depletion. Furthermore, velocity fields were plotted using particle image velocimetry (PIV) to characterize the hydrodynamics of collective cell migration involved in epithelial wounds. A uniform velocity distribution was observed in control cells, attributed to large velocity vectors and incidental vortex motions and eddies exhibiting hydroelastic-like migration behavior. Figure 7 This fluid migration is largely disrupted by the inhibition of Agrin (l). Figure 7Notably, supplementing Agrin-depleted cells with sAgrin significantly enhanced collective traction stress, coordinated velocity field, and collective fluid migration at the front edge. Figure 7 These results indicate that Agrin endows ECMs with intrinsic cellular stiffness and enhanced traction, leading to collective keratinocyte migration through coordinated hydrodynamics.

[0170] Agrin mechanotransduction regulates post-injury cell mechanics

[0171] Regarding this result of enhanced keratinocyte fluid mobility, an important question is whether Agrin modulates cellular mechanics during wound injury by coordinating cytoskeletal structures. The formation of actomyosin cables at the leading edge of embryonic and adult wound healing demonstrates an organized cytoskeletal structure that determines the integrity of collective migration in keratinocytes. First, the inventors examined whether Agrin coordinates actomyosin dynamics at the leading edge during wound stress. In control keratinocytes, trauma resulted in robust actomyosin cables within 4 hours (…). Figure 9 a). Cells lacking Agrin lack these actomyosin cables, while exogenous Agrin treatment restores these actomyosin cables. Figure 9 a). This actomyosin cable network was highlighted by significant induction of phosphorylated myosin light chains (pMLC) within 2 hours of wound injury in control cells, which conferred enhanced contractility and migration speed. Figure 9 ab). Agrin-depleted cells consistently showed severe inhibition of pMLC activation within 2 hours post-traumatic stress, and this was restored by sAgrin pretreatment. Figure 9 b). Recombinant sAgrin treatment resulted in strong recruitment of pMLCs to the wound margin, forming stable actomyosin cables, which were eliminated by the MLC inhibitor brestatin. Figure 9 c). Secondly, the formation of robust actomyosin cables is essential for collective cell migration, as brestatin treatment eliminates sAgrin-induced HaCaT migration after wound injury ( Figure 9 d). This evidence supports the actomyosin structure of Agrin at the post-traumatic tissue front to maintain cell migration potential.

[0172] To gain a deeper understanding of how cellular mechanosensory is calibrated through actomyosin involvement resulting from an agrin-induced force recognition mechanism, the inventors used permanent magnets to apply mechanical forces to injured keratinocytes via ligand-coated magnetic beads that mimic the effects of aberrant mechanical forces experienced by the wound microenvironment. In this setup, the magnetic beads were conjugated to control proteins (bovine serum albumin-BSA or fibronectin-FN) and sAgrin, respectively, and subsequently bound to the cell surface. The permanent magnets, positioned 6 mm above the cells in the culture plate, ensured a continuous application of a 200 pN force for 30 minutes after wound scraping via the ligand-coated beads. Figure 10 a). Effective ligand conjugation with beads via covalent bonding is demonstrated by the abrupt reduction in residual free protein after conjugation. Figure 10 b). Similar to the integrin ligand FN, sAgrin beads significantly increased the localization of activated integrin β1-Agrin in control migrating keratinocytes, indicating that local application of force induced enhanced mechanical tension. Figure 10 c). In control cells without any additional magnetic force, actomyosin cables were detected 30 minutes after trauma. Figure 9 e, without force applied); however, the application of external force further surged pMLC recruitment, which localized near sAgrin beads that form robust actomyosin cables (e, without force applied); Figure 9 e, Shi Litu). In stark contrast, Agrin knockdown cells in such... Figure 9 The pMLC and actomyosin chord localizations reported in a were poor under normal post-traumatic conditions. Figure 9 e). It is expected that transient force transfer via sAgrin should partially manifest as increased actomyosin recruitment, thus reflecting an enhanced mechanical response to injured cells. Therefore, applying force with sAgrin for 30 minutes resulted in an approximately 2-fold increase in pMLCs near the bead, which partially salvaged actomyosin cable activity in Agrin-depleted cells in culture without any prior sAgrin exposure. Figure 9 ef). After applying force to injured cells, sAgrin induced more pMLC recruitment in control cells compared to FN, but was similar to pMLC recruitment induced by multiligand glycan-4 (Syndecan-4, SDC4). Figure 10 (de, siControl diagram). Although FN acts as a global mechanosensor to stimulate integrins and multiligand glycan-4 regulates cellular mechanics, these are completely insufficient to rescue myosin mechanotropic tension in Agrin-deficient cells. Essentially, the reduced pMLC in Agrin-depleted cells is only restored by forces transduced by sAgrin (not by FN and SDC4). Figure 10(de, siAgrin figure). Similarly, compared to FN or BSA beads, sAgrin-coated magnetic beads enhanced pMLC recruitment in Agrin-depleted cells at 30 minutes post-traumatic stress. Figure 10 fh). Additionally, the specificity of Agrin as a mechanotransducer was tested using the following strategy: First, increased sAgrin-coated beads resulted in greater pMLC recruitment in a dose-dependent manner 30 minutes after wound scraping. Figure 10 i). Secondly, increasing the application of force from continuous stimulation via sAgrin-coated beads to up to 60 minutes resulted in enhanced pMLC mechanosensory after wound injury (i). Figure 10 (j) These results suggest that Agrin functions as a mechanotransducer of external forces, sufficient to comprehensively modify the cytoskeleton dynamics required for collective migration following wound injury.

[0173] In addition to the loss of ECM components, keratinocytes typically traverse wound areas under varying geometrical tensions, adapting to large-scale changes in morphology and cytoskeleton. To simulate whether Agrin affects the ability of keratinocytes to shift their cytoskeletal tension upwards when exposed to geometrical constraints, normal HEK cells were cultured in cross-shaped FN patterns with different surface areas. The FN-coated cross-shaped micropatterns forced cells into a polarized orientation, where F-actin stress fibers originated from the dorsal arc, while numerous actomyosin stress fibers bundled together at the lateral arcs towards the base. Smaller (800 μ) 2 Fibronectin patterns compress cells, and even further lead to severe loss of F-actin stress fibers. Figure 9 g). Although the sAgrin cross pattern was only supplemented within a small area, it partially rescued F-actin stress fibers and recruited more pMLCs to the transverse arc ( Figure 9 g). sAgrin's role in inducing stress fiber formation in geometrically constrained HEK cells at large cross-shaped (1600 μm) areas. 2 The pattern is further highlighted, in which cells exhibit prominent tension features, including, respectively, the tension of actomyosin at elongated F-actin stress fibers and transverse arcs. Figure 9 g, see figure below). Notably, sAgrin conferred a significantly higher tension curvature, enriching activated MLCs at the transverse arcs of cells within the larger cross-shaped pattern. Figure 9 Importantly, F-actin stress fibers and pMLC-enriched transverse arc tension bundles were completely eliminated in Agrin-depleted cells. Figure 9i, representing the first and second figures respectively. Interestingly, sAgrin incorporated into the FN cross matrix restored the tension characteristics of the F-actin stress fiber network and the transverse arcs enriched in pMLC ( Figure 9 (i, Figure 3). In summary, these results indicate that the tension-sensing mechanism is conferred by Agrin in keratinocytes under geometric constraints, thereby modulating it towards the ECM matrix, which is conducive to the healing process.

[0174] MMP12 acts as a mediator of agrin-mechanistic transduction after wound injury.

[0175] To identify downstream effectors of Agrin-mediated mechanical stress in keratinocytes following wound injury, the inventors conducted a whole-transcriptome comparison of control cells cultured in simulated high-rigidity ECM plastics and Agrin-depleted cells using RNA-seq analysis. Figure 11 a). Multidimensional scaling (MDS) revealed distinct gene profiles between the control and the large populations of Agrin-depleted RNA. Figure 11 a). After stratifying the astonishing number of genes differentially regulated by Agrin in the hard ECM, several highly significant clusters were selected using gene ontology (GO) analysis. Figure 12 a, Figure 11 b. Tables 2 and 3). GO, network, and gene set enrichment analysis (GSEA) revealed that most of the genes sharply downregulated upon Agrin knockdown belong to ECM regulation, including matrix-related and structural components of the ECM. Figure 11 bd). During agrin depletion, several matrix histones, including matrix metalloproteinases (MMPs), are consistently reduced among the top 10 significantly regulated genes by agrin, while other proteins such as Serpine2 and interleukin 1a-b are upregulated. Figure 12 a and 11d). Consistent with its effects on cell migration, the GSEA analysis also revealed an overall reduction in gene clusters that positively regulate wound healing (a and 11d). Figure 12 b). We validated the mRNA expression of a selected set of upregulated and downregulated genes ( Figure 11 e). It was found that the expression of several MMP mRNAs, including MMP 12, MMP 10 and MMP 1, was significantly reduced after Agrin knockdown.

[0176] Table 2. Primers used for pro-inflammatory cytokines and chemokines in the study

[0177]

[0178] Table 3. Small interfering RNA (siRNA) sequences

[0179]

[0180] The next set of experiments focused on identifying key MMPs that mediate the mechanotransmission of Agrin in the wound environment. First, we assessed whether MMP 1, MMP 10, and MMP 12 independently affect keratinocyte migration after wound scraping. Accordingly, knockdown of MMP 1 and MMP 10 reduced keratinocyte migration, and sAgrin treatment did not restore keratinocyte migration. Figure 13 a). In contrast, MMP12 knockdown showed the greatest suppression of post-damage migration velocity ( Figure 13 b). Notably, no consistent upregulation of MMP1 and MMP10 mRNA levels was observed in HaCaT cells after wound injury. Figure 13 c). Most importantly, MMP1 or MMP10 depletion had no effect on reducing actomyosin tension at the wound edge. Figure 13 d). When cultured on a large cross pattern, MMP1 or MMP10 depleted cells did not show any reduction in the tension characteristics of the F-actin stress fiber network and the transverse arc enriched by pMLC. Figure 13 e). Although regulated by Agrin, these observations rule out any significant role of MMP1 or MMP10 as downstream mediators of wound healing mechanisms.

[0181] MMP12 has emerged as the most important potential candidate to mediate the mechanical tension of Agrin in wound healing. In a cohort of immortalized and primary keratinocyte and dermal fibroblast cultures, similar to its mRNA level, MMP12 protein levels significantly decreased after Agrin depletion. Figure 14 a). Due to the decrease in both mRNA and protein levels after Agrin depletion, the loss of MMP12's gelatin and casein degradation catalytic activity was significant in Agrin-silenced keratinocytes, and it was restored in a dose-dependent manner by supplementation of sAgrin. Figure 14 b).

[0182] Next, it was determined whether MMP12 activation was a result of Agrin sensitization to ECM stiffness in a wound-related Agrin-enriched environment. First, HaCaT cells were cultured in a single soft substrate (0.8 kPa) or in a soft substrate with increased concentrations of supplemental sAgrin. Importantly, the soft substrate incorporating sAgrin strongly stabilized MMP12 protein levels in a dose-dependent manner. Figure 12c, Figure 1). Enhanced exposure time of cultured keratinocytes to a soft substrate regulated by sAgrin also increased MMP12 levels, indicating that Agrin rigorously mimics the ECM stiffness inducer to positively regulate MMP12 levels in keratinocytes. Figure 12 c, right figure). Conversely, the depletion of Agrin in cells undergoing hard ECM led to a significant decrease in MMP12 levels ( Figure 12 d). Agrin knockdown cells recovered their MMP12 levels in a dose-dependent manner after sensing sAgrin inducers from the hard ECM. Figure 12 d). Secondly, unlike MMP1 or MMP10, wound injury strongly activates MMP12 mRNA (~4-fold) and protein levels, which closely mirrors the mRNA and protein levels observed during Agrin overexpression in several skin cell lines. Figure 14 c, 13c and Figure 1 b). As early as 6 hours after wound scraping, MMP12 and Agrin were detected in cells at the migration front; supplementation with sAgrin further enhanced this effect, thereby mimicking a favorable platform for collective keratinocyte migration after wound injury. Figure 14 d). In mouse skin treated with control siRNA, the migrating epithelial tongue mediating intralesional wound closure efficiently expressed MMP12 ( Figure 14 e). Local agrin depletion significantly deprived the weakened epithelial tongue of MMP12 levels, which subsequently failed to close the wound ( Figure 14 e). In an in vivo fixation wound healing model, Agrin depletion also strongly suppressed MMP12 expression on day 4 post-injury, leading to a poor healing rate. Figure 14 f). This was reproduced in vitro, where MMP12 knockdown of mimicking human immortalized and primary mouse keratinocytes following wound scraping resulted in migration loss similar to that observed in Agrin-depleted cells. Figure 12 e). In summary, this evidence suggests that the Agrin microenvironment of wound stress activates MMP12 for collective keratinocyte migration.

[0183] Several observations further suggest that MMP12 mediates agrin-induced collective migration after trauma. First, sAgrin failed to stimulate migration in MMP12-depleted human and mouse keratinocytes ( Figure 12 e). Secondly, knockdown of MMP12 in keratinocytes undergoing hard substrate (30 kPa) severely impaired the migration of K17-expressing cells, and this could not be restored by providing sAgrin. Figure 14g). Third, short-term treatment of keratinocytes with MMP408 (a specific MMP12 inhibitor) impaired the migration of sAgrin-induced K17-expressing keratinocytes in soft substrates and under normal cell culture conditions. Figure 14 Fourth, as previously reported in the 3D hardness-dependent migration assay, sAgrin-infused soft matrix stimulated the migration of mouse keratinocytes onto dermal fibroblasts after injury. Figure 12 f, Figure 1 and Figure 2). Conversely, MMP12 depletion in mouse keratinocytes and the underlying dermal fibroblasts completely eliminated keratinocyte migration, which could not even be rescued by supplementing with a compliance matrix containing sAgrin. Figure 12 f, Figures 3 and 4). In the in vitro setting, the enhanced K17-expressing migration and growth of mouse skin explants induced by soft matrix supplementation with sAgrin was abruptly eliminated in the presence of an MMP408 inhibitor. Figure 12 g and 14j).

[0184] Recalling that MMP12 regulates Agrin-driven migration, it is reasonable to identify MMP12 as a mediator of Agrin-sensing mechanotropic tension in trauma-stressed keratinocytes. Therefore, the next set of experiments focused on determining the role of MMP12 in comprehensively modulating cytoskeletal tension in response to Agrin-rich environments, as prepared during the early wound healing phase. The robust actomyosin cables generated in Agrin-rich keratinocytes post-traumatically are disrupted with MMP12 depletion, accompanied by significant inhibition of pMLC at the wound margin. Figure 12 h). Even sAgrin treatment failed to restore the actomyosin cable network, revealing a significant defect in these MMP12-mediated inhibition of cytoskeleton orientation in cells. Figure 12 Similarly, within 2 hours post-traumatic stress, sAgrin treatment failed to salvage severely reduced pMLC levels in MMP12-depleted keratinocytes. Figure 12 Furthermore, when extended in a large cross-shaped geometric pattern, MMP12 knockdown cells and cells provided with sAgrin did not show pMLC enrichment in F-actin stress fibers and transverse arcs compared to control cells. Figure 14 k) indicates that Agrin's comprehensive regulation of cytoskeleton tension during collective cell migration following wound injury requires functional MMP12.

[0185] Since MMP12 inhibition resulted in impaired collective migration, the researchers investigated whether the decreased cytoskeletal tension was a consequence of the gradual weakening of Agrin's mechanotransduction capacity caused by MMP12 inhibition. To this end, the inventors first plotted the cell stiffness of mouse keratinocytes under MMP12 inhibition 4 hours post-injury. Notably, MMP12 knockdown significantly reduced the stiffness of migrating keratinocytes, which could not be restored by sAgrin treatment. Figure 14 In addition to reduced cell stiffness, the traction stress of MMP12 knockdown keratinocytes migrating on TFM substrates was partially reduced at the migration front. Figure 12 Interestingly, sAgrin treatment failed to induce sufficient traction at the migration front of MMP12-depleted keratinocytes (j). Figure 12 j). Compared to control cells, MMP12 knockdown suppressed uniform velocity distribution at the leading edge, which further reduced hydroelastic-like migration in cells supplemented with sAgrin. Figure 14 These results indicate that Agrin binds to MMP12 to provide fluid migration behavior to keratinocytes. Furthermore, Agrin-induced mechanotransmission-mediated pMLC activation in injured keratinocytes was also inhibited after MMP12 depletion. Neither FN nor sAgrin-mediated mechanotransmission could rescue the reduced pMLC strength in MMP12-depleted cells at 30 minutes post-traumatic injury. Figure 12 In summary, these results reveal that macroscopic defects in actomyosin tension in MMP12-depleted cells eliminate the mechanosensory capacity conferred by Agrin after skin injury. Furthermore, Agrin mechanotransduction in the wound environment, which facilitates accelerated keratinocyte migration and wound closure, is largely dependent on MMP12.

[0186] Agrin cannot heal wounds in the skin of MMP12-deficient mice.

[0187] To test whether sAgrin confers a mechanosensitive wound healing environment by engaging MMP12 in vivo, MMP12 was depleted via a latent siRNA on days 0 and 10 post-traumatic injury. This latent siRNA effectively suppressed skin MMP12 levels. Figure 15 ab). When compared with those treated with control siRNA, inhibition of MMP12 expression in mouse skin delayed wound healing rate ( Figure 16 More importantly, the accelerated healing rate and degree of reepithelialization observed in sAgrin-treated control animals within 10 days post-injury were significantly reduced after MMP12 depletion. Figure 16ab). It was further noted that the increased pMLC expression in the wound margins and wound beds of the control and sAgrin-treated areas was significantly attenuated in MMP12-depleted skin sections. Figure 16 c). Furthermore, sAgrin-induced maturation and mixed collagen fiber deposition was significantly reduced after MMP12 depletion at the wound bed. Figure 16 d). Therefore, MMP12 depletion results in a mechanosensitive environment, primarily due to the loss of ECM replenishment and actomyosin mechanotropic properties, even when treated with sAgrin. Reduced angiogenesis (fewer blood vessels) was also observed in the wound bed of MMP12-depleted mouse skin. Figure 16 Therefore, sAgrin treatment failed to induce robust angiogenesis of CD31 when MMP12 was inhibited in mouse skin. Figure 16 e). In summary, these observations reveal that sAgrin requires MMP12 to generate a mechanoreceptive pro-angiogenic wound-healing niche in vivo.

[0188] Agrin, as a bio-additive in hydrogel materials, promotes wound healing.

[0189] The environment induced by Agrin during wound closure prompted the inventors to test whether sAgrin itself accelerates wound healing in vitro and in vivo, making it a clinically relevant biomaterial for wound healing. The inventors produced a recombinant sAgrin protein containing a 'z' insert via gel filtration, yielding a purified protein corresponding to a size of 23 kDa. Figure 17 a). Besides, for example, BrDu doping determination ( Figure 17 In addition to the fact shown in b) that sAgrin supports the proliferation of primary mouse keratinocytes, an accelerated migration rate was observed when keratinocytes were treated with increased concentrations of sAgrin. Figure 17 c). Furthermore, sAgrin treatment also enhanced the in vitro migration of K17-expressing leading keratinocytes and the growth of keratinocytes from mouse skin explants in vitro. Figure 17 In Agrin-depleted cells, mechanotransmission via sAgrin (rather than FN) effectively enhanced MMP12 recruitment in the pMLC activation region. Figure 17 f). These results reveal that Agrin specifically stimulates keratinocyte migration by supporting its proliferative phase.

[0190] Finally, using two independent full-thickness mouse perforated biopsy wound healing models with and without fixation, the wound healing properties of sAgrin versus the control protein were evaluated in two different hydrogels for topical application. In the first example, either sAgrin or the control protein (BSA) was incorporated into petrolatum as an inert hydrogel material for topical delivery to injured skin according to the fixation-free healing model. The healing-promoting efficacy of sAgrin incorporated into petrolatum at different concentrations was determined. As shown, in the fixation-free wound healing model, sAgrin significantly accelerated the healing rate of mouse perforated wounds in a dose-dependent manner. Figure 17 g). Subsequently, a single concentration of sAgrin (200 μg) dissolved in Pluronic F-127 (as a thermosensitive hydrogel, which is widely used for local delivery) was tested to monitor the healing process. Similarly, sAgrin-promoted hydrogel treatment significantly improved the rate of wound healing in vivo compared to the control local hydrogel formulation. Figure 18 a). Enhanced proliferation of Ki67+ ve cells at the base of the migrating epidermis and at the wound margin in sAgrin-treated mouse skin sections supported an improved healing rate. Figure 17 h).

[0191] Furthermore, the efficacy of sAgrin compared to rat tail collagen-I and BSA as biological additives was tested using a mouse model of wound healing with fixation. Within 12 days post-injury, the incorporation of 200 μg of sAgrin into Pluronic F127 significantly accelerated wound healing compared to similar concentrations of collagen or BSA. Figure 18 b). These results demonstrate that Agrin, as a bio-additive, can accelerate the wound healing response, independent of locally delivered hydrogel materials used for wound healing in two independent mouse models. Furthermore, following sAgrin treatment, migrating epithelial tongues were observed to significantly extend from the wound margins of wounds without fixation. Figure 18 c). Under fixation conditions on day 10 post-traumatic period, compared to the BSA or collagen treatment groups, animals treated with sAgrin primarily exhibited healed skin and regenerated hair follicles near the wound area. Figure 18 c). During the early stages of wound healing in the fixative-free model (day 2), it was also accompanied by robust K17-expressing keratinocyte growth, MMP12 expression, and pMLC activation observed at the wound margins and wound beds of mouse skin treated with sAgrin-based hydrogel. Figure 18df). Because wound healing under immobilization conditions requires a longer time frame, similar increases in K17 occupancy, MMP12 expression, and pMLC activity were identified in sAgrin-treated animals on day 7 post-injury, compared to their BSA or collagen-treated counterparts. Figure 18 df). Because ECM supplementation within the wound bed guides the entire healing process, sAgrin-treated wound beds showed higher levels of mature and mixed collagen fiber deposition compared to the BSA and collagen-treated groups. Figure 18 g). Under conditions without fixation, a similar enhancement of collagen deposition by sAgrin was observed in the wound bed on day 7. Figure 17 Therefore, these data also indicate that sAgin promotes optimal collagen deposition in the wound bed to maintain the firmness of the wound healing microenvironment. Figure 18 g and 17i).

[0192] It is known that the rapid expression of inflammatory cytokines in the early stages of wound healing initiates an efficient skin healing process. Therefore, this study investigated whether sAgrin selectively activates key cytokines and chemokines to regulate the inflammatory response associated with wound healing. The inventors analyzed the mRNA expression of several cytokines and chemokines known to be induced within 48 hours of skin injury in mice. Specifically, compared to the BSA or collagen treatment groups, mouse skin wounds treated with sAgrin significantly induced the expression of TGF-β1, VEGF-A, MCP-1, MIP-1β, and MIP-2 within 48 hours of injury. Figure 19 a). In fact, enhanced protein expression of TGF-β1, VEGF-A, and MCP-1 was demonstrated in the sAgrin-treated group compared to mouse skin wound tissue treated with BSA and collagen. Figure 19 b). The timely involvement of a group of highly selective pro-inflammatory proteins by sAgrin may lay the foundation for accelerating the wound healing process.

[0193] Determining whether the improved healing rate induced by sAgrin occurs via sustained angiogenesis in the wound bed has potential implications for the field of regenerative medicine. Therefore, on day 6, in a fixation-free model, a stable enhancement of angiogenesis was observed in the wound bed of Agrin-treated mouse skin, with an increase in both the number of blood vessels and their respective diameters. Figure 18 h). Compared to BSA or collagen, in the fixation model, an increased number and diameter of blood vessels were also observed in the sAgrin-treated mouse skin wound bed on day 12 post-injury. Figure 18Therefore, in vivo data suggest that Agrin can enhance the interaction between dermal fibroblasts and endothelial cells, thereby enhancing angiogenic activity within the wound bed. To test this in vitro, Agrin was depleted in human dermal fibroblasts (GFP-labeled BJ) and human dermal microvascular endothelial cells (cell-tracker blue-labeled HDMEC) and subjected to a 3D co-culture budding angiogenesis assay. Figure 20 a). Notably, the control spheroid co-culture produced strong shoot generation, which was significantly reduced by inhibiting Agrin in endothelial cells and fibroblasts, respectively. Figure 20 b, as shown in Figures 2 and 3 respectively. Interestingly, simultaneous depletion of Agrin in both fibroblasts and endothelial cells leads to a stronger elimination of budding angiogenesis. Figure 20 (b) In summary, these results indicate that Agrin supports a pro-angiogenic environment in the wound bed by promoting interactions between dermal fibroblasts and endothelial cells. Therefore, in addition to laying a solid foundation for use as a biomaterial to promote skin tissue repair, the evidence in this study suggests that local delivery of sAgrin accelerates in vivo wound healing by enhancing the mechanotransduction environment that maintains optimal ECM deposition, angiogenesis, and thus promotes collective keratinocyte migration.

[0194] discuss

[0195] Wound healing represents a complex but highly coordinated biological process that restores damaged tissue structures to a normal state. The sequence of skin wound healing is characterized by a homeostatic phase where injury signals trigger clot formation to restrict blood flow, followed by an inflammatory phase of debridement of injured cells. Next, a proliferative phase controls the proliferation, survival, and migration of keratinocytes through the wound area in a process known as reepithelialization. The timely execution of all of the above ultimately leads to wound closure and restoration of tissue integrity. Therefore, the primary criterion for effective keratinocyte migration after injury depends on the deposition rate of new extracellular matrix (ECM) and its components, which subsequently trigger angiogenesis conducive to the healing process.

[0196] Beyond its scaffolding function, the ECM acts as a "dynamic communication layer" with its surrounding tissues, typically shielding them from mechanical stresses and / or instructing them to maintain tissue balance against adverse external stresses, thus preserving tissue integrity. Furthermore, the skin acts as a superior organ of mechanoreceptors, analyzing how mechanical forces are integrated within structured tissue structures to maintain critical biological functions. To emphasize this dynamic mechanical feedback between skin cells and their surrounding ECM, a large number of soluble and matrix-binding proteins are spatiotemporally regulated by the ECM. During injury, a significant portion of the ECM is lost, rendering the underlying tissue unable to respond to substantial external mechanical stresses—a phenomenon known as mechanosensing. This lack of mechanosensing capacity during injury largely contributes to large-scale deficiencies in actomyosin tension, which influences cell survival and migration. As an attractive hypothesis for explaining the early stages of wound healing, de novo expression of key ECM proteins within the wound healing microenvironment could repair the underlying skin tissue by improving mechanosensing and potentially restoring the mechanoreciprocity between the ECM and skin tissue. Under this premise, a primary interest lies in identifying key ECM components that integrate mechanical stimuli and establish mechanosensing within injured cells, thereby adapting them to the wound stress environment.

[0197] In this study, the inventors investigated whether Agrin could enhance skin tissue repair by modulating the mechanoreceptive wound microenvironment by improving mechanosensory perception in keratinocytes.

[0198] In tissue injury, the integrated alterations to the disordered extracellular matrix (ECM) and surrounding tissues, encompassing mechanical forces, tissue compression, proprioceptive stress from the ECM, and collective cell migration, are essential for initiating the healing process. Notably, this coordinated effort to replenish the injured ECM, thereby sensitizing the wound environment to adverse physical stresses, leading to an effective healing response and limiting hypertrophic scar formation, is crucial in regenerative medicine. The comprehensive evidence presented in this article reveals several important findings that advance our understanding of the mechanobiology of wound healing and provide significant implications for tissue repair strategies. First, Agrin, representing an important ECM proteoglycan, is triggered in injured skin tissue, and this Agrin-enriched microenvironment modulates the mechanical landscape for efficient wound healing. Figure 12(g) Secondly, Agrin guides keratinocytes to migrate collectively at the wound site by sensitizing them to various physical parameters, such as bulk ECM stiffness, mechanical forces, and geometric constraints. Thirdly, the mechanosensory capacity conferred by Agrin powerfully and comprehensively repairs the cytoskeleton structure after wound injury and is largely dependent on MMP12 activation. Therefore, Agrin mechanotransduction via MMP12 activation provides an integrated mechanism that enables injured skin cells to counteract the adverse mechanical stresses caused by wound injury and accelerate their migration rate. Fourthly, recognizing the importance of the mechanosensory state of Agrin-enriched wound environments, the results of this study further reveal the significant clinical value that appropriate use of sAgrin as a biological additive wound healing material can provide for trauma.

[0199] Epithelial cells experience a wide range of physical stresses, including morphological changes in ECM stiffness, cell shape and geometry, lack of adhesion, and the application of mechanical forces. Each of these parameters determines cell behavior in the wound environment; however, little is known about the nature of the ECM proteins that enable injured cells to respond to these physical parameters. Driven by this belief, Agrin partially sensitizes keratinocytes to perform collective migration in response to a number of physical parameters, including bulk ECM stiffness, uplifting cytoskeletal tension within geometrically constrained structures, and acting as a local mechanotransducer when external forces are applied to injured cells. Figure 21 Nevertheless, since the integrin FN binding under external force cannot restore actomyosin cables in the absence of Agrin, the magnitude of cytoskeletal repair and enhanced mechanosensory response to the wound microenvironment appear to be uniquely targeted at Agrin mechanotransduction. Consistent with the idea that collective migration of cells to the hardened ECM generates higher traction, the potential of Agrin as an ECM participant is crucial, as it confers considerable elasticity on keratinocytes post-injury, helping them to possess higher traction, cytoskeletal tension, and improved motility. While Agrin depletion independently inhibited 2D scratch wound healing in both keratinocytes and dermal fibroblasts, a 3D hardness-dependent keratinocyte-fibroblast co-culture migration assay showed that Agrin derived from dermal fibroblasts was insufficient to restore the migration of Agrin-depleted keratinocytes on a hard substrate. Furthermore, knockdown of Agrin in dermal fibroblasts did not affect the migration rate of stratified keratinocytes with intact Agrin on a hard substrate. Given that uninjured and injured keratinocytes express higher levels of agrin than dermal fibroblasts, these research series argue that agrin expressed by keratinocytes acts as a major regulator to coordinate migration occurring in the trauma area when sensing proprioceptive ECM stiffness.

[0200] Dynamic ECM remodeling induced by the Agrin-enriched environment is not fully established shortly after trauma. Transcriptomic data from this study revealed an overall loss of collagen and ECM structural components in Agrin-depleted keratinocytes. Given that Agrin depletion delays wound healing, it is clear that Agrin provides a comprehensive ECM remodeling signal through MMP12 to sustain wound healing. This is supported by several observations showing that ECM-integrated sAgrin restores intracellular migration responses in keratinocytes both in vitro and in vitro, responses attenuated by MMP12 depletion or inhibition. For example, keratinocyte growth from mouse skin explants grown on a collagen gel that closely mimics the underlying dermal rigidity was strongly increased when sAgrin was incorporated with subsequently activated MMP12. Furthermore, in addition to manipulating tissue mechanics to provide better resilience to mechanical damage, the accelerated healing effect elucidated in vivo by sAgrin hydrogel treatment through MMP12 activity may involve dynamic ECM remodeling, as indicated by increased collagen deposition.

[0201] In summary, this study reveals that damage-triggered Agrin enhances mechanosensory through MMP12 activity in keratinocytes, thereby integrating a wide range of mechanical stimuli and ultimately promoting wound healing. sequence list <110> Singapore Agency for Science, Technology and Research <120> Wound healing compositions and their uses <130> 73177PCT <160> 119 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic sequence <400> 1 Glu Leu Ala Asn Glu Ile Pro Val 1 5 <210> 2 <211> 184 <212> PRT <213> Artificial sequence <220> <223> synthetic sequence <400> 2 Pro Phe Leu Ala Asp Phe Asn Gly Phe Ser His Leu Glu Leu Arg Gly 1 5 10 15 Leu His Thr Phe Ala Arg Asp Leu Gly Glu Lys Met Ala Leu Glu Val 20 25 30 Val Phe Leu Ala Arg Gly Pro Ser Gly Leu Leu Leu Tyr Asn Gly Gln 35 40 45 Lys Thr Asp Gly Lys Gly Asp Phe Val Ser Leu Ala Leu Arg Asp Arg 50 55 60 Arg Leu Glu Phe Arg Tyr Asp Leu Gly Lys Gly Ala Ala Val Ile Arg 65 70 75 80 Ser Arg Glu Pro Val Thr Leu Gly Ala Trp Thr Arg Val Ser Leu Glu 85 90 95 Arg Asn Gly Arg Lys Gly Ala Leu Arg Val Gly Asp Gly Pro Arg Val 100 105 110 Leu Gly Glu Ser Pro Val Pro His Thr Val Leu Asn Leu Lys Glu Pro 115 120 125 Leu Tyr Val Gly Gly Ala Pro Asp Phe Ser Lys Leu Ala Arg Ala Ala 130 135 140 Ala Val Ser Ser Gly Phe Asp Gly Ala Ile Gln Leu Val Ser Leu Gly 145 150 155 160 Gly Arg Gln Leu Leu Thr Pro Glu His Val Leu Arg Gln Val Asp Val 165 170 175 Thr Ser Phe Ala Gly His Pro Cys 180 <210> 3 <211> 188 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence <400> 3 Pro Phe Leu Ala Asp Phe Asn Gly Phe Ser His Leu Glu Leu Arg Gly 1 5 10 15 Leu His Thr Phe Ala Arg Asp Leu Gly Glu Lys Met Ala Leu Glu Val 20 25 30 Val Phe Leu Ala Arg Gly Pro Ser Gly Leu Leu Leu Tyr Asn Gly Gln 35 40 45 Lys Thr Asp Gly Lys Gly Asp Phe Val Ser Leu Ala Leu Arg Asp Arg 50 55 60 Arg Leu Glu Phe Arg Tyr Asp Leu Gly Lys Gly Ala Ala Val Ile Arg 65 70 75 80 Ser Arg Glu Pro Val Thr Leu Gly Ala Trp Thr Arg Val Ser Leu Glu 85 90 95 Arg Asn Gly Arg Lys Gly Ala Leu Arg Val Gly Asp Gly Pro Arg Val 100 105 110 Leu Gly Glu Ser Pro Val Pro Lys Ser Arg Lys His Thr Val Leu Asn 115 120 125 Leu Lys Glu Pro Leu Tyr Val Gly Gly Ala Pro Asp Phe Ser Lys Leu 130 135 140 Ala Arg Ala Ala Ala Val Ser Ser Gly Phe Asp Gly Ala Ile Gln Leu 145 150 155 160 Val Ser Leu Gly Gly Arg Gln Leu Leu Thr Pro Glu His Val Leu Arg 165 170 175 Gln Val Asp Val Thr Ser Phe Ala Gly His Pro Cys 180 185 <210> 4 <211> 4 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 4 Light Looks Angry Light 1 <210> 5 <211> 167 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 5 Glu Tyr Leu Asn Ala Val Thr Glu Ser Glu Lys Ala Leu Gln Ser Asn 1 5 10 15 His Phe Glu Leu Ser Leu Arg Thr Glu Ala Thr Gln Gly Leu Val Leu 20 25 30 Trp Ser Gly Lys Ala Thr Glu Arg Ala Asp Tyr Val Ala Leu Ala Ile 35 40 45 Val Asp Gly His Leu Gln Leu Ser Tyr Asn Leu Gly Ser Gln Pro Val 50 55 60 Val Leu Arg Ser Thr Val Pro Val Asn Thr Asn Arg Trp Leu Arg Val 65 70 75 80 Val Ala His Arg Glu Gln Arg Glu Gly Ser Leu Gln Val Gly Asn Glu 85 90 95 Ala Pro Val Thr Gly Ser Ser Pro Leu Gly Ala Thr Gln Leu Asp Thr 100 105 110 Asp Gly Ala Leu Trp Leu Gly Gly Leu Pro Glu Leu Pro Val Gly Pro 115 120 125 Ala Leu Pro Lys Ala Tyr Gly Thr Gly Phe Val Gly Cys Leu Arg Asp 130 135 140 Val Val Val Gly Arg His Pro Leu His Leu Leu Glu Asp Ala Val Thr 145 150 155 160 Lys Pro Glu Leu Arg Pro Cys 165 <210> 6 <211> 175 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence <400> 6 Glu Tyr Leu Asn Ala Val Thr Glu Ser Glu Leu Ala Asn Glu Ile Pro 1 5 10 15 Val Glu Lys Ala Leu Gln Ser Asn His Phe Glu Leu Ser Leu Arg Thr 20 25 30 Glu Ala Thr Gln Gly Leu Val Leu Trp Ser Gly Lys Ala Thr Glu Arg 35 40 45 Ala Asp Tyr Val Ala Leu Ala Ile Val Asp Gly His Leu Gln Leu Ser 50 55 60 Tyr Asn Leu Gly Ser Gln Pro Val Val Leu Arg Ser Thr Val Pro Val 65 70 75 80 Asn Thr Asn Arg Trp Leu Arg Val Val Ala His Arg Glu Gln Arg Glu 85 90 95 Gly Ser Leu Gln Val Gly Asn Glu Ala Pro Val Thr Gly Ser Ser Pro 100 105 110 Leu Gly Ala Thr Gln Leu Asp Thr Asp Gly Ala Leu Trp Leu Gly Gly 115 120 125 Leu Pro Glu Leu Pro Val Gly Pro Ala Leu Pro Lys Ala Tyr Gly Thr 130 135 140 Gly Phe Val Gly Cys Leu Arg Asp Val Val Val Gly Arg His Pro Leu 145 150 155 160 His Leu Leu Glu Asp Ala Val Thr Lys Pro Glu Leu Arg Pro Cys 165 170 175 <210> 7 <211> 189 <212> PRT <213> artificial sequence <220> <223> composite sequence <400> 7 Asp Thr Leu Ala Phe Asp Gly Arg Thr Phe Val Glu Tyr Leu Asn Ala 1 5 10 15 Val Thr Glu Ser Glu Leu Ala Asn Glu Ile Pro Val Glu Lys Ala Leu 20 25 30 Gln Ser Asn His Phe Glu Leu Ser Leu Arg Thr Glu Ala Thr Gln Gly 35 40 45 Leu Val Leu Trp Ser Gly Lys Ala Thr Glu Arg Ala Asp Tyr Val Ala 50 55 60 Leu Ala Ile Val Asp Gly His Leu Gln Leu Ser Tyr Asn Leu Gly Ser 65 70 75 80 Gln Pro Val Val Leu Arg Ser Thr Val Pro Val Asn Thr Asn Arg Trp 85 90 95 Leu Arg Val Val Ala His Arg Glu Gln Arg Glu Gly Ser Leu Gln Val 100 105 110 Gly Asn Glu Ala Pro Val Thr Gly Ser Ser Pro Leu Gly Ala Thr Gln 115 120 125 Leu Asp Thr Asp Gly Ala Leu Trp Leu Gly Gly Leu Pro Glu Leu Pro 130 135 140 Val Gly Pro Ala Leu Pro Lys Ala Tyr Gly Thr Gly Phe Val Gly Cys 145 150 155 160 Leu Arg Asp Val Val Val Gly Arg His Pro Leu His Leu Leu Glu Asp 165 170 175 Ala Val Thr Lys Pro Glu Leu Arg Pro Cys Pro Thr Pro 180 185 <210> 8 <211> 437 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence <400> 8 Leu Gly Arg Glu Gly Thr Phe Cys Gln Thr Ala Ser Gly Gln Asp Gly 1 5 10 15 Ser Gly Pro Phe Leu Ala Asp Phe Asn Gly Phe Ser His Leu Glu Leu 20 25 30 Arg Gly Leu His Thr Phe Ala Arg Asp Leu Gly Glu Lys Met Ala Leu 35 40 45 Glu Val Val Phe Leu Ala Arg Gly Pro Ser Gly Leu Leu Leu Tyr Asn 50 55 60 Gly Gln Lys Thr Asp Gly Lys Gly Asp Phe Val Ser Leu Ala Leu Arg 65 70 75 80 Asp Arg Arg Leu Glu Phe Arg Tyr Asp Leu Gly Lys Gly Ala Ala Val 85 90 95 Ile Arg Ser Arg Glu Pro Val Thr Leu Gly Ala Trp Thr Arg Val Ser 100 105 110 Leu Glu Arg Asn Gly Arg Lys Gly Ala Leu Arg Val Gly Asp Gly Pro 115 120 125 Arg Val Leu Gly Glu Ser Pro Val Pro His Thr Val Leu Asn Leu Lys 130 135 140 Glu Pro Leu Tyr Val Gly Gly Ala Pro Asp Phe Ser Lys Leu Ala Arg 145 150 155 160 Ala Ala Ala Val Ser Ser Gly Phe Asp Gly Ala Ile Gln Leu Val Ser 165 170 175 Leu Gly Gly Arg Gln Leu Leu Thr Pro Glu His Val Leu Arg Gln Val 180 185 190 Asp Val Thr Ser Phe Ala Gly His Pro Cys Thr Arg Ala Ser Gly His 195 200 205 Pro Cys Leu Asn Gly Ala Ser Cys Val Pro Arg Glu Ala Ala Tyr Val 210 215 220 Cys Leu Cys Pro Gly Gly Phe Ser Gly Pro His Cys Glu Lys Gly Leu 225 230 235 240 Val Glu Lys Ser Ala Gly Asp Val Asp Thr Leu Ala Phe Asp Gly Arg 245 250 255 Thr Phe Val Glu Tyr Leu Asn Ala Val Thr Glu Ser Glu Leu Ala Asn 260 265 270 Glu Ile Pro Val Glu Lys Ala Leu Gln Ser Asn His Phe Glu Leu Ser 275 280 285 Leu Arg Thr Glu Ala Thr Gln Gly Leu Val Leu Trp Ser Gly Lys Ala 290 295 300 Thr Glu Arg Ala Asp Tyr Val Ala Leu Ala Ile Val Asp Gly His Leu 305 310 315 320 Gln Leu Ser Tyr Asn Leu Gly Ser Gln Pro Val Val Leu Arg Ser Thr 325 330 335 Val Pro Val Asn Thr Asn Arg Trp Leu Arg Val Val Ala His Arg Glu 340 345 350 Gln Arg Glu Gly Ser Leu Gln Val Gly Asn Glu Ala Pro Val Thr Gly 355 360 365 Ser Ser Pro Leu Gly Ala Thr Gln Leu Asp Thr Asp Gly Ala Leu Trp 370 375 380 Leu Gly Gly Leu Pro Glu Leu Pro Val Gly Pro Ala Leu Pro Lys Ala 385 390 395 400 Tyr Gly Thr Gly Phe Val Gly Cys Leu Arg Asp Val Val Val Gly Arg 405 410 415 His Pro Leu His Leu Leu Glu Asp Ala Val Thr Lys Pro Glu Leu Arg 420 425 430 Pro Cys Pro Thr Pro 435 <210> 9 <211> 177 <212> PRT <213> artificial sequence <220> <223> composite sequence <400> 9 Ala Pro Val Pro Ala Phe Glu Gly Arg Ser Phe Leu Ala Phe Pro Thr 1 5 10 15 Leu Arg Ala Tyr His Thr Leu Arg Leu Ala Leu Glu Phe Arg Ala Leu 20 25 30 Glu Pro Gln Gly Leu Leu Leu Tyr Asn Gly Asn Ala Arg Gly Lys Asp 35 40 45 Phe Leu Ala Leu Ala Leu Leu Asp Gly Arg Val Gln Leu Arg Phe Asp 50 55 60 Thr Gly Ser Gly Pro Ala Val Leu Thr Ser Ala Val Pro Val Glu Pro 65 70 75 80 Gly Gln Trp His Arg Leu Glu Leu Ser Arg His Trp Arg Arg Gly Thr 85 90 95 Leu Ser Val Asp Gly Glu Thr Pro Val Leu Gly Glu Ser Pro Ser Gly 100 105 110 Thr Asp Gly Leu Asn Leu Asp Thr Asp Leu Phe Val Gly Gly Val Pro 115 120 125 Glu Asp Gln Ala Ala Val Ala Leu Glu Arg Thr Phe Val Gly Ala Gly 130 135 140 Leu Arg Gly Cys Ile Arg Leu Leu Asp Val Asn Asn Gln Arg Leu Glu 145 150 155 160 Leu Gly Ile Gly Pro Gly Ala Ala Thr Arg Gly Ser Gly Val Gly Glu 165 170 175 Cys <210> 10 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence <400> 10 Pro Pro Lys Pro Cys Asp Ser Gln Pro Cys Phe His Gly Gly Thr Cys 1 5 10 15 Gln Asp Trp Ala Leu Gly Gly Gly Phe Thr Cys Ser Cys Pro Ala Gly 20 25 30 Arg Gly Gly Ala Val Cys Glu 35 <210> 11 <211> 38 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence <400> 11 Gly Asp His Pro Cys Leu Pro Asn Pro Cys His Gly Gly Ala Pro Cys 1 5 10 15 Gln Asn Leu Glu Ala Gly Arg Phe His Cys Gln Cys Pro Pro Gly Arg 20 25 30 Val Gly Pro Thr Cys Ala 35 <210> 12 <211> 38 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence <400> 12 Glu Lys Ser Pro Cys Gln Pro Asn Pro Cys His Gly Ala Ala Pro Cys 1 5 10 15 Arg Val Leu Pro Glu Gly Gly Ala Gln Cys Glu Cys Pro Leu Gly Arg 20 25 30 Glu Gly Thr Phe Cys Gln 35 <210> 13 <211> 40 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence <400> 13 Ala Gly His Pro Cys Thr Arg Ala Ser Gly His Pro Cys Leu Asn Gly 1 5 10 15 Ala Ser Cys Val Pro Arg Glu Ala Ala Tyr Val Cys Leu Cys Pro Gly 20 25 30 Gly Phe Ser Gly Pro His Cys Glu 35 40 <210> 14 <211> 582 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 14 catatggaca ccctggcgtt cgatggtcgt acctttgttg agtacctgaa cgcggtgacc 60 gagagcgaac tggcgaacga gatcccggtt gaaaaggcgc tgcagagcaa ccacttcgag 120 ctgagcctgc gtaccgaagc gacccaaggt ctggtgctgt ggagcggcaa agcgaccgaa 180 cgtgcggact acgttgcgct ggcgattgtg gatggtcacc tgcagctgag ctataacctg 240 ggcagccaac cggtggttct gcgtagcacc gttccggtga acaccaaccg ttggctgcgt 300 gtggttgcgc accgtgagca gcgtgaaggt agcctgcaag ttggcaacga agcgccggtg 360 accggtagca gcccgctggg tgcgacccag ctggacaccg atggtgcgct gtggctgggt 420 ggcctgccgg aactgccggt tggtccggcg ctgccgaagg cgtatggtac cggctttgtg 480 ggttgcctgc gtgacgtggt tgttggtcgt cacccgctgc acctgctgga ggatgcggtt 540 accaaaccgg aactgcgtcc gtgcccgacc ccgtaaggat cc 582 <210> 15 <211> 1326 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 15 catatgctgg gtcgtgaggg caccttctgc caaaccgcga gcggtcaaga tggtagcggt 60 ccgtttctgg cggatttcaa cggttttagc cacctggaac tgcgtggcct gcacaccttc 120 gcgcgtgacc tgggcgagaa gatggcgctg gaagtggttt ttctggcgcg tggtccgagc 180 ggtctgctgc tgtacaacgg ccagaagacc gacggtaaag gcgatttcgt tagcctggcg 240 ctgcgtgacc gtcgtctgga gtttcgttat gatctgggta aaggtgctgc ggttattcgt 300 agccgtgagc cggtgaccct gggtgcgtgg acccgtgtga gcctggaacg taacggtcgt 360 aagggtgcgc tgcgtgttgg tgatggtccg cgtgtgctgg gcgagagccc ggttccgcac 420 accgtgctga acctgaagga accgctgtac gttggtggcg cgccggactt cagcaagctg 480 gcgcgtgcgg cggcggttag cagcggtttt gatggcgcga ttcagctggt tagcctgggt 540 ggccgtcaac tgctgacccc ggagcacgtg ctgcgtcaag ttgacgttac cagctttgcg 600 ggtcacccgt gcacccgtgc gagcggtcat ccgtgcctga acggtgcgag ctgcgttccg 660 cgtgaagcgg cgtacgtgtg cctgtgcccg ggtggcttta gcggtccgca ctgcgagaag 720 ggtctggtgg agaagagcgc gggtgacgtg gataccctgg cgttcgatgg ccgtaccttt 780 gttgagtatc tgaacgcggt gaccgagagc gaactggcga acgagatccc ggttgaaaag 840 gcgctgcaga gcaaccactt cgagctgagc ctgcgtaccg aagcgaccca aggtctggtg 900 ctgtggagcg gcaaagcgac cgaacgtgcg gactacgttg cgctggcgat tgtggatggt 960 cacctgcagc tgagctataa cctgggcagc caaccggtgg ttctgcgtag caccgttccg 1020 gtgaacacca accgttggct gcgtgtggtt gcgcaccgtg agcagcgtga aggtagcctg 1080 caagttggca acgaagcgcc ggtgaccggt agcagcccgc tgggtgcgac ccagctggac 1140 accgatggtg cgctgtggct gggtggcctg ccggaactgc cggttggtcc ggcgctgccg 1200 aaggcgtatg gtaccggctt tgtgggttgc ctgcgtgacg tggttgttgg tcgtcacccg 1260 ctgcacctgc tggaggatgc ggttaccaaa ccggaactgc gtccgtgccc gaccccgtaa 1320 ggatcc 1326 <210> 16 <211> 19 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 16 gatgcacgca cctcgatgt 19 <210> 17 <211> 15 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 17 ggcccccctg gcatt 15 <210> 18 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 18 gaccccagac aaatgtgatc ct 22 <210> 19 <211> 18 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 19 ttcaggctcg ggattcca 18 <210> 20 <211> 26 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 20 cctagtctat tcatagctaa tcaaga 26 <210> twenty one <211> twenty two <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> twenty one agtggaggaa agctgtgcat ac 22 <210> twenty two <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> twenty two aaagcgaccg gcaatataaa 20 <210> twenty three <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> twenty three atagctcagg acccgatttg 20 <210> twenty four <211> twenty one <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> twenty four acgcagctct gtgtcaaagg t 21 <210> 25 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 25 tgatgaacga gactcgacag c 21 <210> 26 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 26 gcgggagcga gcagatccag 20 <210> 27 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 27 ggaagctggc aaactggcag 20 <210> 28 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 28 tctcattgca agatcatcgc c 21 <210> 29 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 29 ccccatgaat aacacagcac c 21 <210> 30 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 30 ttagaggaga aatgccaagg aa 22 <210> 31 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 31 cactgatgaa ggaggaagga ag 22 <210> 32 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 32 tggactctcc ggattgacca agtt 24 <210> 33 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 33 atttgggata gttggctcct cgct 24 <210> 34 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 34 cccatctatc agcaggctcc 20 <210> 35 <211> 19 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 35 aaagcgatac cgccttctg 19 <210> 36 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 36 taagaatgct tccaggacca ctc 23 <210> 37 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 37 gctgtaacat ctgccgattt ttc 23 <210> 38 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 38 ctcggcatga atctgatgaa tg 22 <210> 39 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 39 tggcagttcc cacgttattg 20 <210> 40 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 40 tggactatca gctccgctac tatg 24 <210> 41 <211> 19 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 41 gtggcagtgt tggtctcgc 19 <210> 42 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 42 ctacacgcta caccctcgac 20 <210> 43 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 43 acgtcctcac accaggaaac 20 <210> 44 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 44 gcgcccaaac cgaagtcata 20 <210> 45 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 45 atgggggatg caggattgag 20 <210> 46 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 46 ccgtatttct actcggatgc tg 22 <210> 47 <211> 18 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 47 tggcttgggt ttcctgtc 18 <210> 48 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 48 ggactcagct ccatgtctat cc 22 <210> 49 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 49 actgaggtct cccctaagat cc 22 <210> 50 <211> 18 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 50 gagaaagtgg ttgagagg 18 <210> 51 <211> 18 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 51 gtaatcaacg acggcaag 18 <210> 52 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 52 tgacaggatg cagaaggaga tta 23 <210> 53 <211> 19 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 53 agccaccgat ccacacaga 19 <210> 54 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 54 ctataaattg agcccgcagc 20 <210> 55 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 55 gaccaaatcc gttgactccg 20 <210> 56 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 56 acaccgtcct caacctgaag 20 <210> 57 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 57 ccaggttgta gctcagttgc 20 <210> 58 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 58 cggccccgcc atggagctcc 20 <210> 59 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 59 ggcagttacc gccaccgggg 20 <210> 60 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 60 agcatggacg tggctgtgcc 20 <210> 61 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 61 ggcgtgcgtg tgtagcctgt 20 <210> 62 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 62 aatggcacgg actaatttgc 20 <210> 63 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 63 tatgagggtt tgtggggtgt 20 <210> 64 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 64 aatgctgcag ccccaaggaa t 21 <210> 65 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 65 ctgggcaact ggacaactca actc 24 <210> 66 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 66 catcactgcc acccagaaga ctg 23 <210> 67 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 67 atgccagtga gcttcccgtt cag 23 <210> 68 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 68 gcctcaaacc ttccaaatca 20 <210> 69 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 69 tggatccatt tcctcaaagg 20 <210> 70 <211> 17 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 70 cctgtccaaa ctaaggc 17 <210> 71 <211> 19 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 71 ggttttctca tagatggcg 19 <210> 72 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 72 gtacctccac catgccaagt 20 <210> 73 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 73 tcacatctgc aagtacgttc g 21 <210> 74 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 74 ccttccagga tgaggacatg a 21 <210> 75 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 75 tgagtcacag aggatgggct c 21 <210> 76 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 76 cctccaactg tctaccagtt cc 22 <210> 77 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 77 gcctggctaa gttattgtgc 20 <210> 78 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 78 tgaatgcctg agagtcttgg 20 <210> 79 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 79 ttggcagcaa acagcttatc 20 <210> 80 <211> 18 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 80 tgctcgtggc tgccttct 18 <210> 81 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 81 caggaagtgg gagggtcaga 20 <210> 82 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 82 cgcccagaca gaagtcatag 20 <210> 83 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 83 tcctcctttc caggtcagtt a 21 <210> 84 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 84 tgcacccaaa ccgaagtcat 20 <210> 85 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 85 ttgtcagaag ccagcgttca c 21 <210> 86 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 86 caagctcatc tgtgcagacc 20 <210> 87 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 87 cgcctgtagt gcataagagt cc 22 <210> 88 <211> 18 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 88 caccactccc tgctgctt 18 <210> 89 <211> 18 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 89 acacttggcg gttccttc 18 <210> 90 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 90 taggctggag atctacaaga gg 22 <210> 91 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 91 agtgcttgag gtggttgtgg 20 <210> 92 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 92 agccttccgc taccttctta 20 <210> 93 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 93 gctgttgcct ttgttcttgg 20 <210> 94 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 94 cgtgtggata caggatgttg acag 24 <210> 95 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 95 aggaggagcc catctttctg taac 24 <210> 96 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 96 ggttcacgat cctgctcctg ac 22 <210> 97 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 97 gctgggcctg agaacactca ag 22 <210> 98 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 98 tgagcaagag aggccctatc 20 <210> 99 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 99 aggcccctcc tgttattatg 20 <210> 100 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 100 gcctcttctc attcctgctt g 21 <210> 101 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 101 ctgatgagag ggaggccatt 20 <210> 102 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 102 acggccttcc ctacttcaca 20 <210> 103 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 103 catttccacg atttcccaga 20 <210> 104 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 104 cgtgcgtgac atcaaagaga a 21 <210> 105 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 105 tggatgccac aggattccat 20 <210> 106 <211> 25 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 106 ccuuugucga guaccucaac gcugu 25 <210> 107 <211> 25 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 107 acagcguuga gguacucgac aaagg 25 <210> 108 <211> 25 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 108 cauacggcaa cgagugucag cugaa 25 <210> 109 <211> 25 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 109 uucagcugacacucguugccguaug 25 <210> 110 <211> 25 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 110 gcgauuuaug gacuuugacu gguuu 25 <210> 111 <211> 25 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 111 aaaccaguca aaguccauaa aucgc 25 <210> 112 <211> 25 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 112 ggagaccugc caguuuaacucugua 25 <210> 113 <211> 25 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 113 uacagaguua aacuggcagg ucucc 25 <210> 114 <211> 25 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 114 agguucccuu caggugggca augaa 25 <210> 115 <211> 25 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 115 uucauugccc accugaaggg aaccu 25 <210> 116 <211> 25 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 116 ccaaaguccu gugauucca gccuu 25 <210> 117 <211> 25 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 117 aaggcuggga aucacaggac uuugg 25 <210> 118 <211> 25 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 118 ggagcucacg gagacuucaa cuauu 25 <210> 119 <211> 25 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 119 aauaguugaagucuccgugagcucc 25

Claims

1. Use of a pharmaceutical composition in the preparation of a medicament for treating skin wounds, wherein the pharmaceutical composition comprises a pharmaceutically effective amount of an Agrin fragment, wherein the Agrin fragment comprises an LG3 domain of Agrin and an eight-amino acid insert ELANEIPV (SEQ ID NO: 1) at the z-site of the LG3 domain, wherein the Agrin fragment comprises the sequence DTLAFDGRTFVEYLNAVTESELANEIPVEKALQSNHFELSLRTEATQGLVLWSGKATERADYVALAIVDGHLQLSYNLGSQPVVLRSTVPVNTNRWLRVVAHREQREGSLQVGNEAPVTGSSPLGATQLDTDGALWLGGLPELPVGPALPKAYGTGFVGCLRDVVVGRHPLHLLEDAVTKPELRPCPTP (SEQ ID NO: 7).

2. Use of a hydrogel in the preparation of a medicament for treating skin wounds, wherein the hydrogel comprises a pharmaceutical composition comprising a pharmaceutically effective amount of an Agrin fragment, wherein the Agrin fragment comprises an LG3 domain of Agrin and an eight-amino acid insert ELANEIPV (SEQ ID NO: 1) at the z-site of the LG3 domain, wherein the Agrin fragment comprises the sequence DTLAFDGRTFVEYLNAVTESELANEIPVEKALQSNHFELSLR TEATQGLVLWSGKATERADYVALAIVDGHLQLSYNLGSQPVVLRSTVPVNTNRWLRVVAHREQREGSLQVGNEAPVTGSSPLGATQLDTDGALWLGGLPELPVGPALPKAYGTGFVGCLRDVVVGRHPLHLLEDAVTKPELRPCPTP (SEQ ID NO: 7).

3. The use according to claim 2, wherein the hydrogel is an inert hydrogel or a thermosensitive hydrogel.

4. Use of a scaffold in the preparation of a medicament for treating skin wounds, wherein the scaffold comprises a pharmaceutical composition comprising a pharmaceutically effective amount of an Agrin fragment, wherein the Agrin fragment comprises an LG3 domain of Agrin and an eight-amino acid insert at the z-site of the LG3 domain. (SEQ ID NO: 1), wherein the Agrin fragment contains the sequence DTLAFDGRTFVEYLNAVTESELANEIPVEKALQSNHFELSLRTEATQGLVLWSGKATERADYVALAIVDGHLQLSYNLGSQPVVLRSTVPVNTNRWLRVVAHREQREGSLQVGNEAPVTGSSPLGATQLDTDGALWLGGLPELPVGPALPKAYGTGFVGCLRDVVVGRHPLHLLEDAVTKPELRPCPTP (SEQ ID NO: 7).

5. The use according to any one of claims 1-2 and 4, wherein the LG3 domain of Agrin, which contains no inserts at the z-site, comprises a sequence. (SEQ ID NO: 5).

6. The use according to any one of claims 1-2 and 4, wherein the Agrin fragment further comprises the LG2 domain of Agrin.

7. The use according to claim 6, wherein the LG2 domain of Agrin, which has no inserts at the y-site, contains a sequence. (SEQ ID NO: 2).

8. The use according to claim 6, wherein the Agrin fragment comprises a sequence (SEQ ID NO: 8).

9. The use according to any one of claims 1-2 and 4, wherein the Agrin fragment is soluble.

10. The use according to any one of claims 1-2 and 4, wherein the wound is a slow-healing wound or a non-healing wound.

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