Epicardial-derived paracrine factors for repairing cardiac tissue

Abstract

Provided herein are epicardium-derived paracrine factors for repairing heart tissue. In particular, provided herein are compositions and kits comprising epicardium-derived paracrine factors, such as hypoglycosylated follicular- stimulating hormone-like 1 (FSTL1), for treating and repairing damage to heart tissue caused by cardiovascular disease, myocardial infarction (MI), other ischemic events, or heart growth defects, and methods of using the same.

Description

[0001] This application is a divisional application of the invention application filed on April 8, 2016, with Chinese application number 201680031325.3 and entitled "Epicardial-derived paracrine factor for repairing heart tissue".

[0002] Cross-citation of related applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 145,480, filed April 9, 2015, and U.S. Provisional Patent Application No. 62 / 196,766, filed July 24, 2015, the disclosures of which are incorporated herein by reference in their entirety. Invention Field

[0004] In particular, the present invention relates to compositions comprising epicardial-derived paracrine factors and their use in treating or preventing damage to cardiac (e.g., myocardial) tissue following ischemic events such as myocardial infarction. Background of the Invention

[0005] Acute myocardial infarction (AMI) is one of the leading causes of death in the Western world, and many risk factors (both environmental and genetic) contribute to its pathogenesis. The heart generally lacks sufficient endogenous regenerative capacity to repair itself after damage. Reconstruction of the corresponding left ventricle (LV) following myocardial infarction (MI) or other ischemic events leads to LV dilation and eventually heart failure (Holmes et al., 2005, Annu Rev Biomed Eng.; 7: 223-53). Following coronary artery occlusion, ischemic myocytes downstream of the occlusion immediately become necrotic and / or undergo apoptosis. Neutrophils immediately infiltrate the tissue, while leukocytes (mainly macrophages) arrive shortly thereafter and participate in the digestion of necrotic cell debris. Neutrophils in ischemic tissue can be toxic to surrounding myocytes because they release reactive oxygen species and proteolytic enzymes, which further damage surrounding myocytes (Nah & Rhee, Korean Circ J.; Oct; 39(10): 393-82009). Once damage occurs, it forms cytopenic scars, which cause systolic dysfunction and eventually heart failure.

[0006] To reduce the epidemiological and financial burden associated with ischemic events that invade the myocardium, it is imperative to develop novel compositions and strategies for preserving cardiomyocytes or stimulating their growth after damage caused by ischemic events such as myocardial infarction. Therapies are needed to address and / or treat cardiac (e.g., myocardial) tissue after damage. The inventions disclosed herein address these needs and provide additional benefits. Invention Overview

[0007] Specifically, this article provides compositions and kits containing epicardial-derived paracrine factors (such as hypoglycated follicle-like 1 (FSTL1)) for the treatment and repair of damage to cardiac (e.g., myocardial) tissue caused by cardiovascular disease, myocardial infarction (MI), or other ischemic events, as well as methods of using them.

[0008] Therefore, in some aspects, this document provides a method for repairing cardiac (e.g., myocardial) tissue after injury in a subject with a need, the method comprising contacting the cardiac (e.g., myocardial) tissue with an epicardial-derived paracrine factor. In some embodiments, the epicardial-derived paracrine factor is a hypoglycated follicle-staphylin-like 1 (FSTL1) polypeptide. In some embodiments of any of the embodiments disclosed herein, the injury is ischemia-reperfusion cardiac (e.g., myocardial) injury due to ischemic heart disease and / or due to cardiac dysplasia. In some embodiments of any of the embodiments disclosed herein, the injury is myocardial infarction and / or the heart contains scar tissue. In some embodiments of any of the embodiments disclosed herein, repairing cardiac (e.g., myocardial) tissue comprises increasing the number of cardiomyocytes in the cardiac (e.g., myocardial) tissue. In some embodiments, the number of cardiomyocytes is increased by at least two times compared to the number of cardiomyocytes in damaged scar tissue that has not been contacted with the epicardial-derived paracrine factor after injury. In some embodiments of any of the embodiments disclosed herein, repairing cardiac (e.g., myocardial) tissue comprises an improved percentage shortening of cardiac (e.g., myocardial) tissue compared to the percentage shortening fraction in cardiac (e.g., myocardial) tissue that has not been exposed to epicardial-derived paracrine factors after injury. In some embodiments of any of the embodiments disclosed herein, repairing cardiac (e.g., myocardial) tissue comprises improved wall motion compared to the same subject before treatment. In some embodiments of any of the embodiments disclosed herein, repairing cardiac (e.g., myocardial) tissue comprises improved blood perfusion area compared to the same subject before treatment. In some embodiments of any of the embodiments disclosed herein, repairing cardiac (e.g., myocardial) tissue comprises reduced cardiac (e.g., myocardial) accidents and hospitalizations compared to similar subjects who were not treated. In some embodiments of any of the embodiments disclosed herein, repairing cardiac (e.g., myocardial) tissue comprises an increased amount of cardiomyocyte cytoplasmic division in the cardiac (e.g., myocardial) tissue compared to the amount of cardiomyocyte cytoplasmic division in cardiac (e.g., myocardial) tissue that has not been exposed to epicardial-derived paracrine factors after injury. In some embodiments, the increased amount of cardiomyocyte cytoplasmic division is determined by the expression of Aurora B kinase. In some embodiments of any of the embodiments disclosed herein, repair of cardiac (e.g., myocardial) tissue involves reduced cardiomyocyte apoptosis. In some embodiments of any of the embodiments disclosed herein, the method results in an increase in the level of transcripts encoding heart-specific (e.g., myocardial) contractile proteins in cardiomyocytes. In some embodiments, the method results in a two-fold increase in the level of transcripts encoding heart-specific (e.g., myocardial) contractile proteins in cardiomyocytes. In some embodiments of any of the embodiments disclosed herein, the heart-specific (e.g., myocardial) contractile protein is selected from the group consisting of myh6, mlc2v, and mlc2a.In some embodiments of any of the implementations disclosed herein, the method results in rhythmic contractile Ca in cardiomyocytes. 2+ Actin +Increased cell proliferation. In some embodiments of any of the embodiments disclosed herein, the cardiac (e.g., myocardial) tissue is immediately exposed to the epicardial-derived paracrine factor following the injury. In some embodiments of any of the embodiments disclosed herein, the method increases the survival of the subject following the injury. In some embodiments of any of the embodiments disclosed herein, the method attenuates fibrosis in the cardiac (e.g., myocardial) tissue following the injury. In some embodiments of any of the embodiments disclosed herein, the method results in increased angiogenesis in the damaged area of ​​the cardiac (e.g., myocardial) tissue. In some embodiments, the increased angiogenesis is measured by the expression of von Willebrand factor (vWF) or smooth muscle actin in vascular cells. In some embodiments of any of the embodiments disclosed herein, the method induces cardiomyocytes to enter the cell cycle. In some embodiments, the cardiomyocyte entry into the cell cycle is assessed by the expression of phospho-histone H3. In some embodiments of any of the embodiments disclosed herein, the method results in cardiomyocyte entry into the cell cycle being at least 2-fold higher than the amount of cardiomyocytes entering the cell cycle in cardiac (e.g., myocardial) tissue that has not been exposed to the epicardial-derived paracrine factor following the injury. In some embodiments, the hypoglycated FSTL1 peptide is synthesized in prokaryotic cells. In some embodiments, the prokaryotic cells are bacterial cells. In some embodiments, the hypoglycated FSTL1 peptide is synthesized in eukaryotic cells treated with a glycosylation inhibitor. In some embodiments, the glycosylation inhibitor is tunicamycin. In some embodiments, the hypoglycated FSTL1 peptide is generated by replacing one or more glycosylated amino acids with one or more glycosylated incapable amino acids. In some embodiments, the one or more glycosylated amino acids are selected from the group consisting of N144, N175, N180, and N223. In some embodiments of any of the embodiments disclosed herein, the hypoglycated FSTL1 peptide does not protect cardiomyocytes from apoptosis following damage. In some embodiments of any of the embodiments disclosed herein, the hypoglycated FSTL1 peptide is injected directly into damaged myocardial tissue. In some embodiments of any of the embodiments disclosed herein, the hypoglycated FSTL1 peptide is delivered systematically. In some embodiments of any of the embodiments disclosed herein, the hypoglycated FSTL1 peptide is delivered intracardiacly. In some embodiments of any of the embodiments disclosed herein, the low-glycosylated FSTL1 peptide is embedded or seeded into a three-dimensional collagen patch. In some embodiments of any of the embodiments disclosed herein, the low-glycosylated FSTL1 peptide is embedded or seeded into a hydrogel.In some embodiments of any of the embodiments disclosed herein, the heart (e.g., myocardium) tissue is contacted from the epicardial site, the intracardiac site, and / or via one or more direct injections into the myocardium.

[0009] In another aspect, this document provides a pharmaceutical composition comprising a hypoglycosylated follicle-associated 1-like peptide (FSTL1) and one or more pharmaceutically acceptable excipients. In some embodiments, the hypoglycosylated FSTL1 peptide is synthesized in prokaryotic cells. In some embodiments, the prokaryotic cells are bacterial cells. In some embodiments, the hypoglycosylated FSTL1 peptide is synthesized in eukaryotic cells treated with a glycosylation inhibitor. In some embodiments, the glycosylation inhibitor is tunicamycin. In some embodiments, the hypoglycosylated FSTL1 peptide is obtained through genome editing. In some embodiments, the hypoglycosylated FSTL1 peptide is obtained by inserting modified RNA. In some embodiments, the hypoglycosylated FSTL1 peptide is obtained by treating a subject with a drug (e.g., causing treatment to inhibit the glycosylation of endogenous FSTL1 peptides). In some embodiments of any of the embodiments disclosed herein, the composition is formulated for direct injection into damaged cardiac (e.g., myocardial) tissue. In some embodiments of any of the embodiments disclosed herein, the composition is formulated for systemic administration. In some embodiments of any of the embodiments disclosed herein, the low-glycosylated FSTL1 peptide is embedded or seeded into a three-dimensional (3D) collagen patch. In some embodiments, the 3D collagen patch has an elastic modulus of 12 ± 4 kPa.

[0010] In other aspects, this document provides a kit comprising (i) a low-glycosylated follicle-staphylin-like 1 (FSTL1) peptide; and (ii) one or more pharmaceutically acceptable excipients. In some embodiments, the kit further comprises (iii) a three-dimensional (3D) collagen patch. In some embodiments of any of the embodiments disclosed herein, the low-glycosylated FSTL1 peptide is embedded or seeded into a three-dimensional (3D) collagen patch. In some embodiments of any of the embodiments disclosed herein, the 3D collagen patch has an elastic modulus of 12 ± 4 kPa. In some embodiments of any of the embodiments disclosed herein, the kit further comprises (iv) an adhesion means for adhering the 3D collagen patch to the epicardium or myocardium of a damaged heart. In some embodiments, said adhesion means is a suture.

[0011] In other respects, this document provides a method for repairing cardiac (e.g., myocardial) tissue following injury in subjects with a need, the method comprising contacting the cardiac (e.g., myocardial) tissue with a three-dimensional (3D) collagen patch inoculated or perfused with a recombinant low-glycosylated follicle-staphylin-like 1 (FSTL1) polypeptide. In some embodiments, the injury is ischemia-reperfusion injury. In some embodiments, the injury is myocardial infarction. In some embodiments of any of the embodiments disclosed herein, the 3D collagen patch is sutured to the cardiac (e.g., myocardial) tissue.

[0012] In another aspect, this document provides a three-dimensional (3D) collagen patch infused or seeded with a recombinant low-glycosylated follicle-staphylin-like 1 (FSTL1) peptide. In some embodiments, the recombinant low-glycosylated FSTL1 peptide is synthesized in prokaryotic cells. In some embodiments, the prokaryotic cells are bacterial cells. In some embodiments, the recombinant low-glycosylated FSTL1 peptide is synthesized in eukaryotic cells treated with a glycosylation inhibitor. In some embodiments, the glycosylation inhibitor is tunicamycin. In some embodiments of any of the embodiments disclosed herein, the 3D collagen patch has an elastic modulus of 12 ± 4 kPa.

[0013] Unless explicitly or clearly excluded from the context of the implementation or aspect, each aspect and implementation described herein can be used together.

[0014] Numerous patents, patent applications, and other types of publications (e.g., journal articles, electronic database entries, etc.) are referenced throughout this specification. All disclosures of the patents, patent applications, and other publications cited herein are incorporated herein by reference for all purposes. Brief description of the attached diagram

[0015] Figure 1 Depicting cardiac developmental activity within the epicardial secretomic group. (ad) Co-culturing ESC-derived cardiomyocytes (mCM) ESC Cardiogenic effects of epicardial (EMC) cell lines. a, b) mCM only. ESC (a) mCM co-cultured with EMC for 4 days ESC (b) Visualization of cardiomyocytes by α-actin immunofluorescence (green) and EMC by H2B-mCherry fluorescence (red). (c) Quantification of cardiomyocyte numbers expressed as fold changes (as in a and b) (n = 3). (d) mCM ESC The cardiac response was positive relative to the number of EMCs added to the co-culture (+: 1 x 10⁻⁶). 5 EMC per hole, ++: 5 x 10 5EMC per well, quantified using myocyte markers Myh6, Myh7, Mlc2a, and Mlc2v (n=3), normalized for Gapdh gene expression. *: compared with control (p<0.05), ■: statistically significant difference compared with "+" condition (p<0.05). e1) Effect of epicardial EMC-conditioned medium on cardiogenesis. e, f) mCM after 8 days of treatment with control (e) or EMC-conditioned medium (f). ESC α-actin staining (green). g) Quantification of cardiomyocyte number. h) mCM ESC Quantification of heart-specific markers, normalized for Gapdh expression. i) Quantification of the number of cardiomyocytes with rhythmic calcium transients, automated using a KineticImage Cytometer (Vala Sciences). Quantifications in gi are presented as fold changes. n = 3 in all experiments. *Statistically significant difference compared to controls (p < 0.05). jm) Conditioned culture medium from adult epicardial-derived cells (EPDCs) promotes cytokinesis in embryonic cardiomyocytes. j) From E12.5 GFP-positive hearts (Tnnt2-Cre; Rosa26) mTmG / + Embryonic cardiomyocytes were isolated. EPDC-conditioned medium promoted cardiomyocyte proliferation, and boiling the conditioned medium eliminated the growth-promoting effect. Cytokinesis analysis by immunostaining of Aurora B and the cardiac marker Tnnt2 showed increased cardiomyocyte cytokinesis after treatment with adult EPDC medium (medium group: 38 positive cells out of 19668; conditioned medium group: 74 / 22143); *P<0.05; n=5.

[0016] Figure 2 Depicting the improvement of cardiac function by an embryonic epicardial patch after permanent LAD ligation. a) Schematic diagram of collagen patch formation (plastic compression procedure, from...) 30 (Reconstruction). b) Evaluation of the mechanical properties of the engineered patch, measured by atomic force microscopy. A bar graph of the measured microstiffness distribution of the patch is shown in red. These values ​​are compared to the elastic range reported for common scaffold biomaterials. 31 Drawing. The gray area depicts the previously described... 32The optimal elastic range that maximizes myocyte contractility. c, d) Epicardial patch implantation. After permanent LAD ligation to induce myocardial infarction (c), the epicardial patch was sutured to the surface of the ischemic myocardium at two points (d). The inset in the small figure shows the prepared patch being immersed in culture medium before implantation. eg) Physiological effect of patch loaded with epicardial conditioned medium after myocardial infarction (MI). All samples were collected at week 2 after patch implantation. e) Summary of echocardiography analysis performed 2 weeks after infarction, including: sham (sham control), untreated infarcted mice (MI only), MI treated with patch only (MI + patch), and infarcted animals treated with patch loaded with epicardial conditioned medium (MI + patch + CM). All data were normalized to individual preoperative baseline values. f) Absolute value of shortening fraction (FS%) from e. g) Overall histological analysis of the heart stained with Masson's trichrome; samples as labeled. A minimum number of 8 mice (n) was used for each experimental group. *: p < 0.05 compared to the sham control, ●: p < 0.05 compared to MI only, and ■: p < 0.05 compared to MI + patch.

[0017] Figure 3 FSTL1, a dynamically expressed epicardial cardiogenic factor, was depicted after injury. a) MS / MS profile of R. GLCVDALIELSDENADWK.L. identified as FSTL1. Peptide probability = 1.0, Xcorr = 6.276, ΔCn = 0.471. bf) Effects of recombinant FSTL1 on mouse embryonic stem cell-derived cardiomyocytes (mCMs) after 8 days of treatment with either control medium alone (control) or medium containing human FSTL1 (FSTL1, 10 ng / ml). ESCThe effects of FSTL1 on the culture medium were changed every 2 days in all experiments and conditions. b, c) Identification of cardiomyocytes by α-actin immunostaining (green). d) Quantification of the number of cardiomyocytes (n=8) in b, c, expressed as fold change. e) Expression of cardiac-specific markers in b, c, normalized for Gapdh expression (n=3), expressed as fold change. f) Quantification of the number of cardiomyocytes with rhythmic calcium transients (fold change relative to control) with or without FSTL1 treatment, analyzed using Kinetic Image Cytometer (KIC) (n=6). g) Measurement of individual cardiomyocyte cell size (in pixels) after 2 days of culture in labeled concentrations of FSTL1 (n=5). *: Statistically significant difference from control (p<0.05). h) Epicardial expression of FSTL1 after mid-pregnancy. Direct protein visualization using FSTL1 antibody (red) demonstrated FSTL1 expression in the mouse epicardium at embryonic days E12.5, E15.5, and E17.5. Some interstitial expression was also detected. Image E12.5 shows colocalization of FSTL1 with the epicardial transcription factor Wilm's Tumor 1 (Wt1, green nuclear staining, white arrow). Images E15.5 and E17.5 are costained with the myocyte marker α-actin (green) and show no overlap with FSTL1 (white arrow) and the myocyte marker (yellow arrow). i) Dynamic expression of FSTL1 in the damaged adult epicardium. i) Top inset: Histological (Masson's trichrome staining) assessment of fibrotic tissue induced at sequential time following myocardial infarction (MI). Bottom inset: Immunohistochemistry of FSTL1 at sequential time following MI (brown). Inset shows FSTL1 expression in the epicardium of a sham-operated heart and the reduction of FSTL1 in the epicardium of a damaged heart. FSTL1 was also undetectable in fibrotic tissue, but it became upregulated in the myocardium after MI (observation of brown FSTL1 immunostaining in the myocardium after MI). High-resolution immunofluorescence images: Colocalization of FSTL1 (red) and epicardial marker Wt1 (green) in an undamaged (pseudo) adult heart (j). Selective epicardial localization of FSTL1 (red) in an adult pseudoheart (k). FSTL1 loss in epicardial cells and their derivatives after MI (l). Wt1-CreER; Rosa26 RFP / + Epicardial lineage markers (green) in mice following oral administration of tamoxifen (6 administrations over 3 weeks, with a 1-week break before MI). Hearts were collected 2 weeks after MI. Immunostaining of RFP (gray), FSTL1 (red), and Tnni3 (green) in Wt1 lineage cells showed that FSTL1 was absent in epicardial cells and their derivatives (gray) after MI, but abundant in myocardium (green).

[0018] Figure 4 The in vivo regeneration effect of FSTL1 on conditioned epicardial medium in engineered epicardial patches was depicted. a) Physiological analysis. a) Survival time progression for each condition was analyzed using the Kaplan-Meier method. b) Kinetics of fractional shortening [FS(%)] during the first 3 months of treatment, as measured by echocardiography. Data are provided as absolute values ​​of FS: sham (sham control), untreated infarcted mice (MI only), MI treated with patch only (MI+patch), and infarcted animals treated with patches loaded with FSTL1 (MI+patch+FSTL1). c) FS% at 2 and 4 weeks post-MI in FSTL1-TG mice compared to MI+patch and MI+patch+FSTL1. *: p < 0.05 compared to sham control, ●: p < 0.05 compared to MI only, and ■: p < 0.05 compared to MI+patch. de) Morphometry analysis. d) Representative Masson's trichrome staining and quantification of the area of ​​fibrosis as a percentage of total LV wall at 4 weeks post-MI (n>4). e) Echocardiographic assessment of left ventricular morphology. Abbreviations: LVIDd (end-diastolic left ventricular diameter); LVIDs (end-systolic left ventricular diameter); LVPWd (end-diastolic left ventricular posterior wall size); LVPWs (systolic left ventricular posterior wall thickness). *: p<0.05 compared to sham, ●: p<0.05 compared to MI only, and ■: p<0.05 compared to MI+. fi) Analysis of the vascular system at 4 weeks post-MI. f) Immunostaining of endothelial markers (vWF). g) Vascular area quantified by measuring the mean lumen area of ​​individual vessels relative to the total area of ​​histological sections. h) Immunostaining of smooth muscle markers (αSMA). i) Quantification of the number of vessels per unit area. *: p < 0.05 compared to sham control, ●: p < 0.05 compared to MI only, and ■: p < 0.05 compared to MI + patch. j) Patch-boundary zone visibility at week 4 post-MI. Trichrome staining of the infarct and labeled boundary zones reveals patch integration with host tissue and abundant cellularization of the patch by native cardiac cells. Observe the abundant muscle (red) within the patch and boundary zone of animals treated with patch + FSTL1 (three small right images, green arrows).

[0019] Figure 5The restored epicardial FSTL1 expression was depicted as promoting cardiomyocyte proliferation. All experiments were performed after permanent LAD ligation. Samples were analyzed at week 4 of treatment unless otherwise specified. (ah) Immunofluorescence staining. Immunofluorescence staining of cardiomyocyte marker α-actin (red) in the infarcted region (bd) and co-immunofluorescence staining of DNA replication markers phosphate-histone 3Ser10 (pH3, green) and α-actin (red) in the boundary zone in the four treatment groups analyzed 4 weeks after MI, compared with sham-operated animals (a, e). Inset in (ad) shows a lower magnification image, with dashed lines delineating the boundary between the patch and host tissue. Arrows in (g, h) indicate the pH3-positive zone. + α-actin of the nucleus + Cardiomyocytes. io) Quantification of cardiomyocyte proliferation. i) Illustration of cross-sections used for quantitative analysis, each section covering the infarct, attached, and spaced 250 μm apart, 1–2 mm from the apex. j) High-magnification images of pH3 (green) and α-actin (red), 3D rendering showing colocalization of cardiomyocyte nuclei with pH3 staining. k) pH3 in the four experimental groups. + α-actin + Quantification of the incidence of double-positive cells. Data were collected from 5-7 hearts in each group, targeting the total pH3 in each heart. + α-actin + Cells were counted in three different cross sections. 1) Cytokinesis was determined. 1) Co-immunofluorescence of cardiomyocytes (α-actin, green) and the cytokinesis marker Aurora B kinase (red) in the FSTL1 cohort; 3D visualization showing α-actin along the Z-axis. + Aurora B kinase between cardiomyocytes + Cleavage groove. m) Aurora B in 4 experimental groups + / α-actin + Quantification of cell incidence. Data were collected from 5-7 hearts in each group, targeting total Aurora B in each heart. + / α-actin + Cells were counted in three different cross sections. no) Proliferation was determined using cardiomyocyte nuclear markers. n) Co-immunofluorescence of cardiomyocyte nuclear markers PCM1 (red) and pH3 (green) in the +FSTL1 cohort; 3D rendering showed co-localization of cardiomyocyte nuclei with pH3 staining. o) PCM1 in the four experimental groups. + pH3 + Quantification of cell incidence. Data were collected from 5-7 hearts in each group, targeting total PCM1 in each heart. + pH3+ Cells were counted in three different cross sections. *: Statistical difference from spurious, P<0.05. **: Statistical difference from all other groups, P<0.05. (pv) Lineage tracing of newly generated myocytes. 4-OH-tamoxifen (OH-Tam) treatment of α-MHC-mERCremER; Rosa26 Z / EG / + (MCM + / ZEG+ mice were induced to express eGFP in pre-existing cardiomyocytes (illustrated in p). Collagen patches loaded with FSTL1 were applied concurrently with coronary artery ligation (MI). The heart was dissected, fixed, and stained 4 weeks after MI. q) shows the LV region of the sham-operated heart with effective cardiomyocyte labeling (α-actin white; eGFP green). rt) shows eGFP in the entire region (r), the infarcted region (s), and the boundary zone (t, u) at 4 weeks post-operation. + (Pre-existing, green) LV region of the infarcted heart containing cardiomyocytes. White arrow indicates pH3. + Non-cardiac cells. The yellow arrow indicates pH 3. + eGFP + Double-positive cells indicate cardiomyocytes pre-existing during mid-cycle. Observe pH3 in the boundary zone and infarct region. + eGFP + Double-positive cell clusters.

[0020] Figure 6 The proliferative activity of FSTL1 in early cardiomyocytes was depicted as dependent on cell-selective post-transcriptional FSTL1 modification. af) FSTL1 promotes the proliferation of immature cardiomyocytes derived from mESCs. a, d) mESCs were inoculated with 10 μg / ml EdU. ESC Stimulation with labeled concentrations of FSTL1 for 24 hours, followed by staining for α-actin (red) and EdU (green). Quantification of all α-actin was performed. + EdU in cardiomyocytes + α-actin + Percentage of cardiomyocytes (d). b, e) mCM ESC Stimulation with 10 ng / ml FSTL1 for 48 hours and staining for α-actin (red) and phospho-histone 3 (green). Quantification of all α-actin. + pH3 in cardiomyocytes + α-actin + The percentage of cardiomyocytes (e). c, f) will be mCM ESCStimulation with labeled concentrations of FSTL1 for 48 hours, followed by staining for α-actin (red) and the cytokinesis marker Aurora B (green). All α-actin was quantified. + Aurora B in cardiomyocytes + α-actin + Percentage of cardiomyocytes (f). g, h) FSTL1 expressed in mammalian cells is glycosylated. g) Western blot of HEK293 cells in conditioned medium for FSTL1 in the case of + / - FSTL1 overexpression and + / - blockade of protein glycosylation by tunicamycin (Tuni.), showing that FSTL1 is glycosylated. h) Western blot of recombinant human FSTL1 expressed in mammalian cells or bacteria for FSTL1, showing differences in glycosylation (red arrow: glycosylated form; black arrow: unglycosylated form). i) mCM ESC Stimulation with 10 nM H2O2 and 10 ng / ml FSTL1 generated by bacteria and mammals for 24 hours, followed by staining for α-actin and TUNEL (cell death). Quantification of all α-actin. + TUNEL in cardiomyocytes + α-actin + The percentage of cardiomyocytes (i) showed that mammalian-derived FSTL1 attenuated H2O2-induced apoptosis, while bacterial-derived FSTL1 did not. Furthermore, neither protein had any effect on apoptosis in the absence of H2O2 stimulation. (j, k) EdU incorporation and Aurora B quantification (using the same methods as a, c, d, f) compared bacterial and mammalian-derived FSTL1, showing that bacterial-derived FSTL1 promoted mCM... ESC Proliferation, while mammalian-generated FSTL1 cannot. l) FSTL1 is differentially glycosylated in cardiomyocytes and epicardial cells. Western blots of FSTL1 against conditioned media of Adeno-FSTL1-infected NRVCs and EMCs, with or without tunicamycin treatment, showed that unglycosylated proteins had the same size, while glycosylated FSTL1s had different sizes, suggesting differential modification of FSTL1 in cardiomyocytes and epicardial cells. m, n) EdU incorporation assay to compare the effects of conditioned media of Adeno-FSTL1-infected NRVCs and EMCs. Using Western blots normalized to the same concentration of FSTL1, EMC conditioned media induced mCMs to a similar degree to bacterial-generated FSTL1. ESCProliferation was observed in conditioned medium of Adeno-FSTL1-infected NRVCs, but not in NRVCs, suggesting that glycosylation status determines FSTL1 function. All experiments n=5. *: Statistical difference from control, P<0.05. o) The role of FSTL1 during cardiac injury, working model. In ischemic heart disease, FSTL1 becomes highly expressed in the myocardium. Myocardial-secreted FSTL1 is highly glycosylated (glycoFSTL1) and protects against apoptosis, and does not exhibit proliferative activity. FSTL1 is not expressed in the epicardium of damaged hearts. Hypoglycosylated forms (either secreted by intact epicardium or delivered in the epicardial patch) activate proliferation in replicative cardiomyocyte precursor cells located in the subepicardial space.

[0021] Figure 7 This study depicts the activation of cardiac regeneration by epicardial FSTL1 delivery in a preclinical model of ischemic cardiac injury. (ad) Physiological effects of FSTL1 patch delivery into the epicardium in a porcine experimental model of ischemia-reperfusion (I / R). MRI measured ~50% of baseline ejection fraction (EF), which decreased to ~30% after 1 week (ab). Pigs treated with patch + FSTL1 1 week after I / R recovered contractility by 2 weeks of treatment, with EF at ~40%. EF remained stable over the following 2 weeks, the longest duration analyzed (a, b). This contrasts with the steady decline in cardiac function in untreated animals (I / R untreated) or animals treated with patch alone (I / R + patch) (b). (cd) Pigs treated with patch + FSTL1 (c) show the smallest scar size (area) across all study groups, including I / R + patch animals (d). Green lines and arrows highlight the scar perimeter. eo) Assessment of patch integration with host cardiac tissue, angiogenesis, cellularization, and regeneration at week 4 post-implantation. e) Masson's trichrome staining of porcine heart showing patch + FSTL1 attachment to ischemic tissue and limited fibrosis. fh) Immunostaining of smooth muscle markers (αSMA, red) and EdU (green) showing newly formed arterial smooth muscle. Porcine heart treated with patch + FSTL1 showing ischemic areas (f, g) and boundary zones with FSTL1-loaded patches (h) with evidence of new DNA formation in vascular smooth muscle cells. The white lines and arrows in inset h delineate the approximate boundary between patch and host tissue. im) EdU incorporation analysis of cardiomyocytes resident in infarcted and boundary zones of porcine heart treated with patch + FSTL1, showing striated cardiomyocytes (α-actin). +(red), some of which (arrows in i) are also positive for DNA synthesis (EdU, green, examples in jm at high magnification). n) Patch +FSTL1 co-immunofluorescence of cardiomyocytes in the heart (α-actin, green) and cytokinase marker Aurora B kinase (red), 3D rendering showing α-actin in the Z-axis. + Aurora B kinase between cardiomyocytes + Intermediate.

[0022] Figure 8 Describing the mCM used in this study ESC Cell characterization. a) Schematic timeline of cell preparation and processing. bd) mCM ESC Immunostaining with α-actin showed that most cells were composed of α-actin. + (b) Furthermore, α-actin lacks striated structures (c). (d) mCM ESC Immunostaining with α-smooth muscle actin (αSMA) showed that most cells were αSMA. + Unlike mature cardiomyocytes that do not express SMA. 42 . ef)mCM ESC Automated detection of EdU incorporation in mCM. Treatment of mCM with 10 μg / ml EdU for 24 hours. ESC Captured images, stained with EdU, α-actin, and DAPI, were obtained using an InCell 1000 (General Electric) (e). Overlay maps of masked EdU, α-actin, and DAPI channels, obtained using automated detection software (f). (g)mCM ESC EdU is incorporated into the profile over time. mCM will be included at times of 0, 24, 48, and 144 hours. ESC Treat with 10 μg / ml EdU for 24 hours. Calculate all α-actin for each time period. + EdU in cardiomyocytes + / α-actin + Percentage of cardiomyocytes. Note the decrease in EdU incorporation over time. (h, i)mCM ESC Fluo 4 calcium images, and baseline background image (h) and peak image (i). j)mCM ESC (Red) Comparison of representative calcium transients in neonatal rat ventricular cardiomyocytes (NRVC, blue). Note the difference between mCM and NRVC. ESC The reduced amplitude, slower up-and-down striking rate, and prolonged calcium transient duration suggest mCM. ESC Immature calcium processing. In all experiments, mCM was applied.ESC Add FSTL1 one day later (time 0-24 in this figure).

[0023] Figure 9 Atomic force microscopy (AFM) analysis of engineered epicardial patches. AFM with a custom flat tip for patch fabrication (a), fabricated using electron beam deposition, and used to probe the stiffness of the gel in a 90 μm × 90 μm area (b, c).

[0024] Figure 10 Myocardial overexpression of FSTL1 (FSTL1-TG) in mice following permanent LAD ligation. (ad) FSTL1 protein expression kinetics after myocardial infarction. FSTL1-TG mice (C57 / Bl6 background) and littermate wild-type (WT) mice underwent LAD ligation. Heart tissue and serum were collected at baseline, and on days 1, 3, 7, and 28 post-surgery. FSTL1 protein levels in the ischemic region (IA) and distal region (RM) of the heart were analyzed by Western blot (a). FSTL1 expression relative to tubulin levels is reported (b). Serum FSTL1 levels were analyzed by Western blot (c). Ponceau-S staining is also shown to indicate isotropic loading of serum. Quantification of serum FSTL1 levels is shown in (d). n>3 in all groups. *: P<0.05 compared to WT baseline, #: P<0.05 compared to FSTL1-TG baseline. ANOVA was used for statistical significance (P<0.05). (ej) Long-term morphometric and functional responses of FSTL1-TG mice to permanent LAD ligation. Representative Masson's trichrome staining of WT (e) and FSTL1-TG (f) at 4 weeks after MI. Quantification of contents in fibrotic tissue at 4 weeks after MI (g). Echocardiographic measurements of systolic left ventricular internal dimensions (LVIDs) (h) and diastolic left ventricular diameter (LIVDd) (i) at 2 and 4 weeks after MI. Echocardiographic measurements of shortening fraction (FS%) in genotypes at 2 and 4 weeks after MI (j). (kn) Dual immunofluorescence staining of α-actin (cardiomyocytes) and pH3 (mitosis) (k) and α-actin (cardiomyocytes) and von Willebrand factor (vascular endothelial cells) (m) in FSTL1-TG and WT mice, quantified in (l, n). n = 5, *, significant difference from WT (P < 0.05).

[0025] Figure 11 This indicates that FSTL1 is lost from the epicardium and expressed in the myocardium after myocardial infarction. (ac) lineage marker Wt1-CreER; Rosa26 RFP / +Detection of FSTL1 in mice after MI. Wt1-CreER; Rosa26 RFP / + Epicardial lineage markers (green) in mice following oral administration of tamoxifen (6 administrations over 3 weeks, with a 1-week break before MI). Hearts were collected 2 weeks after MI. Immunostaining of RFP (green), FSTL1 (red), and Tnni3 (white) in Wt1 lineage cells shows that FSTL1 is absent in epicardial cells and their derivatives (green) after MI, but abundant in myocardium (gray) (high magnification images of a and b are shown in c).

[0026] Figure 12 Depicting FSTL1 retention in in vitro and in vivo patches. a, b) Enzyme-linked immunosorbent assay (ELISA) used to measure the amount of FSTL1 retained in collagen scaffolds after in vitro exposure to PBS for different time intervals (0–21 days) (a). The table lists the initial and final FSTL1 concentrations, and the relative values ​​over the first 24 hours (b). cf) FSTL1 retention in in vivo patches. Representative images of FSTL1 immunostaining in the animal treatment group are labeled at week 4 post-surgery. Note that although FSTL1 is expressed in the undamaged epicardium (arrow in the inset of c), its expression becomes undetectable in the infarcted area after MI (d). Similarly, FSTL1 was not detected in MI+patted animals (e), although it remained persistent in the patch area of ​​the MI+pat+FSTL1 group (red staining) (f).

[0027] Figure 13 Depicting reduced fibrosis after MI with patch + FSTL1. Representative Masson's trichrome staining of a series of transverse sections of the heart at 4 weeks post-MI under four conditions (pseudo, MI only, MI + patch, and MI + patch + FSTL1). Note the severe fibrosis in the MI only condition, the reduced fibrosis in the MI + patch condition, and the further reduction in the MI + patch + FSTL1 condition. Figure 4 Quantization in small image d.

[0028] Figure 14 Depicting post-treatment MRI images. Representative MRI images from the MI-only, MI+patch, and MI+patch+FSTL1 treatment groups in mice are shown, displaying 3D-FSPGR (rapid damage gradient echo) images and delayed-enhanced images using gadolinium contrast agent, confirming the reduction (delineated in green) and preserved contractility of the infarct area.

[0029] Figure 15Analysis of patch + FSTL1 function in a mouse model of ischemia / reperfusion (I / R) with delayed patch transplantation. Cardiac function assessments of pseudo-I / R and I / R treated with patch + FSTL1 at end-diastole and end-systole (AC), pre-transplantation (A, 1 week post-lesion), 2 weeks post-patch implantation (B), and 4 weeks post-transplantation (C). Values ​​were normalized for each individual animal by dividing by pre-operative baseline. D) Absolute values ​​of the fractional shortening (FS, %) at different time points before and after I / R from AC mice, assessed by echocardiography. Abbreviations and Figure 4 Same as in. *: p < 0.05 compared to false and ●: p < 0.05 compared to I / R. E) Co-immunofluorescence staining of DNA replication markers phosphate-histone 3 Ser10 (pH 3, green) and α-actin (red) in the boundary zone of the heart treated with +FSTL1 4 weeks after MI. F) pH 3 in the three experimental groups. + α-actin + Quantification of the incidence of double-positive cells. Data were collected from 3 hearts in each group, targeting the total pH3 in each heart. + α-actin + Cells were counted in three different cross sections. *: Statistically different from all other groups, P < 0.05.

[0030] Figure 16 All pH3 detected in a section of a heart treated with Patch +FSTL1 + A representative of cardiomyocytes. Masson's trichrome staining of the heart treated with patch + FSTL1 for 4 weeks after MI (a). In (b), adjacent sections are stained for α-actin, corresponding to the black boxed area with infarction and patch in (a). The dotted line in (b) indicates the boundary between the heart and patch. Adjacent sections are stained for α-actin and pH3, and all α-actin is found. + pH3 + Double-positive cardiomyocytes are shown in (c) (white arrow), with each image corresponding to the area in the numbered white box in (b).

[0031] Figure 17Depicting the effects of implanted patch + FSTL1 on apoptosis and inflammation. a) Representative TTC staining on day 2 after MI / patch treatment in all four groups (sham, MI, MI + patch, MI + patch + FSTL1). b) Quantification of area at risk, compared across all four groups. Data were collected from four hearts in each group, four cross sections, each approximately 2 mm after each heart. *: Statistical difference with sham, P < 0.05. c, d) Representative images of TUNEL assays (TUNEL, green; α-actin, red), comparing hearts 2 days after MI with patch alone and patch + FSTL1. e) TUNEL in the infarct region. + α-actin + The quantification was performed as a percentage of the total number of cardiomyocytes. No difference was observed between the MI+ patch and MI+ patch+FSTL1 conditions. Data were collected from 3 hearts in each group, 3 different cross sections (with...). Figure 5 (Same as in small image i). Ten 0.09mm images were obtained from the infarct region of each section. 2 Image and targeting TUNEL + α-actin + and total α-actin + Cell counting. (ae) On days 4 and 8 after MI, hearts treated with patch only and patch + FSTL1 were subjected to TUNEL staining for cell death and α-actin-associated staining for cardiomyocytes (ad). Minimal TUNEL staining was detected. + α-actin + Cells, with significant amounts of TUNEL + α-actin-secreting cells. All TUNEL + Nuclear quantification showed no significant difference between the patched and patch+FSTL1-treated hearts at both time points (e). (fj) Immunostaining with F4 / 80 (for macrophages) and α-actin (for cardiomyocytes) was performed on the same hearts in small figure ad (fi). F4 / 80 + Cell quantification showed no significant difference between the patch and patch +FSTL1 treated hearts at both time points (j).

[0032] Figure 18 The results show that FSTL1 does not induce proliferation in adult and neonatal cardiomyocytes, or cardiac progenitor cells. af) Adult cardiomyocytes derived from mouse primary isolates. a) MCMs treated with 4-OH-tamoxifen (OH-Tam) in a 3D collagen patch. + / ZEG + GFP isolated from mice +Demonstration of cardiomyocytes. bd) Changes in gene expression in adult cardiomyocytes treated with FSTL1, including proliferation (b), heart-specific (c), and hypertrophy (d) markers. Note that there were no changes in the expression of heart-specific genes, no increase in cell cycle markers (consistent with undetectable Ki67 immunostaining), and a decrease in hypertrophy markers. Cardiomyocytes embedded in 3D patches were treated with FSTL1 (10 ng / ml) for 7 days, with the medium changed every 2 days. e, f) FUCCI assay in 3D-cultured adult cardiomyocytes, performed 1 week after 3D culture. e) FSTL1 treatment for 7 days, with the medium changed every 2 days. f) Control of 3D-cultured adult cardiomyocytes in FSTL1-deficient conditions. Note that no cells in the S / G2 / M phase (GFP) were present under either condition. + Detectable signs of cardiomyocytes. Purple arrows point to purple nuclei, derived from co-localization of Hoechst (blue) and G1 phase FUCCI (red) markers. (gj) Primary neonatal rat ventricular cardiomyocytes (NRVC). (g, h) Freshly isolated NRVCs with 10 μg / ml EdU were stimulated with FSTL1 for 48 h and stained for α-actin (red) and EdU (green). Quantification of all α-actin + EdU in cardiomyocytes + / α-actin + Percentage of cardiomyocytes (h). i, j) NRVCs were stimulated with FSTL1 for 48 hours and stained for α-actin (red) and pH3 (green). Quantification of all α-actin. + pH3 in cardiomyocytes + / α-actin + Percentage of cardiomyocytes (j). No increase in proliferation was observed after FSTL1 treatment. (n=4)*: Statistical difference from control, P<0.05. km) Sca1 + Progenitor cells 19 Starvation-synchronization for 48 hours and stimulation with FSTL1 or control growth medium in the presence of EdU for 72 hours. (The text abruptly ends here, likely due to an incomplete translation or a missing section.) + Clones 3 were obtained from the clonal growth of SP-grade lin-Sca1 cells that had not yet undergone clonal growth. + Obtain the Sca1 set. k) Process Sca1 after 72 hours. + EdU and DAPI staining of cells. l) EdU staining after 72 hours of treatment. + Sca1 + Cell percentage. FSTL1 concentrations: 0, 1, 10, 100 ng / ml. Abbreviations: SS, serum starvation; CGM, control growth medium. m) Sca1 after 72 hours of FSTL1 treatment. +Cell number (n=5). No significant changes were observed after FSTL1 treatment.

[0033] Figure 19 FSTL1 detection was depicted in conditioned media of NRVC and EMC. Western blots of FSTL1 in conditioned media of NRVC and EMC with or without tunicamycin treatment showed the secretion of glycosylated FSTL1 in EMC but not NRVC.

[0034] Figure 20 Depicting mCM after FSTL1 processing ESC Detection of phosphate-Akt and PCNA in cells. Western blot analysis of phosphate-Akt (Ser473 and Thr308, both indicators of survival response in cardiomyocytes) and PCNA (proliferation markers) after treatment with FSTL1 at 10 ng / ml and 50 ng / ml for 1 h and 24 h showed no change in phosphate-Akt and PCNA after FSTL1 treatment. Invention Details

[0035] The invention disclosed herein is based on the inventors' observations that conditioned media obtained from epicardial-like cell cultures enhance cardiomyogenesis in vitro and in adult damaged hearts. The epicardium of the heart provides progenitor cells... 1,2 And mitogens (including FGF, IGF2, and PDGF) 3-5 The epicardium is the outer epithelial layer that contributes to myocardial growth during development. Recent studies suggest that the epicardium may retain the function of adult myocardium even after damage, potentially serving as a source of myogenic ancestors. 6,7 However, to date, no epicardial-derived paracrine factors have been shown to support myocardial regeneration in mammals after injury, although the identification of such factors and their mechanisms of action would provide insights into this poorly understood and inherently inefficient process. 8 .

[0036] As further detailed below, after submitting the conditioned medium to mass spectrometry, followed by subsequent analysis, follicle-stimulating hormone-like 1 (FSTL1) was identified as a component of the observed cardiogenic activity. FSTL1 was observed to be expressed in the adult epicardium but showed a remarkable decrease after myocardial infarction (MI), where it was then replaced by myocardial expression. As illustrated below in non-limiting examples, although endogenous myocardial or transgenic overexpression in the myocardium has no regenerative effect, applying FSTL1 to the epicardial surface of the heart via a compressed collagen patch recapitulates the activity of the epicardial conditioned medium. In some embodiments, engineered FSTL1 epicardial treatment reduces pathological regeneration after MI, restores angiogenesis, and induces pre-existing αMHC. +Cells enter the cell cycle, thereby improving cardiac function. As further shown in the non-limiting embodiments described below, in vitro studies have indicated that FSTL1 stimulates the proliferation of immature myocytes rather than progenitor cells. In some embodiments, the pro-proliferative properties of FSTL1 are related to tissue-specific posttranscriptional modifications of the protein, such as its glycosylation state. In other embodiments of the invention, administration of hypoglycosylated FSTL1 does not activate Akt-1 signaling activity. In yet another non-limiting embodiment described below, epicardial patch delivery of hypoglycosylated FSTL1 was also effective in a preclinical porcine model of myocardial infarction, highlighting the evolutionary conservation of this regenerative mechanism in mammals. Accordingly, without being bound by theory, engineered epicardial delivery of FSTL1 has the potential to be an attractive option for achieving therapeutic regeneration of cardiomyocytes following ischemic injury.

[0037] I. Definition

[0038] As used in this article, the phrase "cardiac tissue" refers to any tissue of the heart. Cardiac tissue includes cardiac muscle tissue, epicardial tissue, and endocardial tissue. Cardiac tissue encompasses any cell type found within the heart.

[0039] As used herein, the phrase "epicardial-derived paracrine factor" refers to any protein, polypeptide, or fragment thereof produced by cells of the outer epithelial layer of the heart and capable of eliciting one or more physiological, protective, proliferative, and / or repair responses in cardiac (e.g., myocardial) tissue following damage caused by cardiovascular disease, myocardial infarction, or other ischemic events. In one embodiment, the epicardial-derived paracrine factor is a component of a conditioned medium obtained from epicardial cell cultures.

[0040] As used in the context of this invention, the term "low-glycosylation" refers to a protein with a minimal number of carbohydrate modules post-translationally modified or completely lacking carbohydrate modules. In some embodiments, low-glycosylation refers to a protein completely lacking any carbohydrate modification, whatever it may be (e.g., N-linked glycans, O-linked glycans, or phospho-glycans). In another embodiment, this term refers to a protein having a reduced amount of carbohydrate modification relative to the amount of glycosylation that occurs in vivo under normal physiological conditions in mammalian cells (such as at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or any 100%). In another embodiment, this term refers to a protein having a reduced amount of carbohydrate modification relative to the amount of glycosylation that occurs in vivo under normal physiological conditions in mammalian cells (such as any one of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%). In another embodiment, this term refers to a protein having a reduced amount of carbohydrate modification relative to the amount of glycosylation that occurs in vivo under normal physiological conditions in mammalian cells (such as any one of about 1-100%, 5-95%, 10-90%, 20-80%, 30-70%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%). In another embodiment, this term refers to a protein having a reduced amount of carbohydrate modification relative to the amount of glycosylation that occurs in vivo under normal physiological conditions in mammalian cells (such as any reduced carbohydrate modification of about 1-100%, 5-95%, 10-90%, 20-80%, 30-70%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%). In still other embodiments, the low-glycosylated protein is engineered such that all glycosylation-capable amino acid residues (such as amino acid residues with N-linked, O-linked, or phospho-glycan capabilities) are replaced with non-glycosylation-capable amino acid residues.

[0041] As used herein, the phrase “repairing cardiac tissue after injury” refers to any type of action or treatment that reduces, minimizes, or even maintains the level of damage resulting from cardiovascular disease, myocardial infarction, or other ischemic events. Therefore, repairing damage indicates that the subject’s condition has not worsened and may be improved in relation to the damage of concern compared to the level of damage under conditions where the treatments or actions described herein for reducing damage are absent.

[0042] As used herein, “cardiovascular disease” or “heart disease” is a term used to describe a range of diseases or events that affect the heart and / or vascular system. Types of heart disease include, but are not limited to, coronary artery disease, cardiomyopathy, ischemic heart disease, heart failure, inflammatory heart disease, valvular heart disease, and aneurysm. Heart disease can be assessed using clinical parameters and / or assessments known to a person skilled in the art of diagnosing and / or treating heart disease, such as physical examination, detection of signs and symptoms of cardiovascular disease, electrocardiogram, echocardiography, chest X-ray, blood tests to detect cardiac biomarkers, etc. Biomarkers typically used in clinical settings include, but are not limited to, cardiac troponin (C, T, and I), CK and CK-MB, and myoglobin.

[0043] As used in this article, "myocardial infarction" or "MI" refers to the necrosis of the myocardium, which can be caused by an interruption of blood supply to the heart, resulting in a critical imbalance between the myocardium's oxygen supply and demand. This can originate from plaque rupture and thrombus formation in the coronary arteries, leading to an acute reduction in blood supply to a portion of the myocardium; that is, occlusion or blockage of the coronary artery following the rupture of a susceptible atherosclerotic plaque. If left untreated for a sufficient period, the resulting ischemia or restriction of blood supply and oxygen deficiency can cause damage or death, i.e., myocardial infarction. Generally, this damage is largely irreversible, and current clinical treatments primarily aim to delay the progression of heart failure to prolong survival. Myocardial infarction can be assessed using clinical parameters and / or assessments known to those skilled in the art of diagnosing and / or treating myocardial infarction (e.g., physical examination, detection of signs and symptoms of myocardial infarction, electrocardiogram, echocardiography, chest X-ray, blood tests to detect cardiac biomarkers (including troponin, CK, and CK-MB, etc.).

[0044] As used herein, “reperfusion” refers to the restoration of blood flow or supply to the heart or cardiac (e.g., myocardium) tissue that has become ischemic or hypoxic. Modalities of reperfusion include, but are not limited to, chemical dissolution of occluded thrombi (i.e., thrombolysis), administration of vasodilators, angioplasty, percutaneous coronary intervention (PCI), catheter insertion, and coronary artery bypass graft (CABG) procedures.

[0045] The “subject” or “individual” can be a vertebrate, a mammal, or a human. Mammals include, but are not limited to, farm animals, sporting animals, pets, primates, mice, and rats. In one respect, the subject is a human.

[0046] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0047] As used herein, the singular terms “a,” “an,” and “the / said” include plural references unless the context clearly indicates otherwise.

[0048] II. The Composition of the Invention

[0049] This article provides pharmaceutical compositions containing an epicardial-derived paracrine factor (e.g., FSTL1, such as hypoglycosylated FSTL1) and one or more pharmaceutically acceptable excipients or carriers.

[0050] In some embodiments, the epicardial-derived paracrine factor is follicle-like protein 1 (FSTL1; also known as follicle-associated protein 1). FSTL1 is a protein encoded by the FSTL1 gene in humans. This gene encodes a protein similar to follicle-associated protein (follicle-associated protein), an activin-binding protein. FSTL1 contains an FS module (a follicle-associated protein sequence containing 10 conserved cysteine ​​residues), a Kazal-type serine protease inhibitory domain, two EF hand domains, and a Von Willebrand factor-type C domain (Entrez gene: FSTL1 follicle-associated protein 1). In other embodiments, FSTL1 comprises the amino acid sequence of SEQ ID NO: 1 (NCBI reference sequence: NP_009016.1):

[0051] MWKRWLALALALVAVAWVRAEEELRSKSKICANVFCGAGRECAVTEKGEPTCLCIEQCKPHKRPVCGSNGKTYLNHCELHRDACLTGSKIQVDYDGHCKEKKSVSPSASPVVCYQSNRDELRRRIIQWLEAEIIPDGWFSKGSNYSEILDKYFKNF DNGDSRLDSSEFLKFVEQNETAINITTYPDQENNKLLRGLCVDALIELSDENADWKLSFQEFLKCLNPSFNPPEKKCALEDETYADGAETEVDCNRCVCACGNWVCTAMTCDGKNQKGAQTQTEEEMTRYVQELQKHQETAEKTKRVSTKEI(SEQ ID NO:1)

[0052] The present invention provides and covers nucleic acids encoding FSTL1. In various embodiments, the nucleic acid is a recombinant nucleic acid. In some embodiments, FSTL1 is encoded by the nucleic acid of SEQ ID NO: 2 (NCBI reference sequence: NM_007085.4):

[0053]

[0054]

[0055]

[0056] FSTL1 nucleic acid can be incorporated into vectors, such as expression vectors, using standard techniques known to those skilled in the art. Methods for linking DNA constructs containing nucleic acids of interest, such as FSTL1, promoters, terminators, and other sequences, and for inserting them into suitable vectors are well known in the art. Alternatively, vectors can be constructed using known recombination techniques (e.g., InvitrogenLife Technologies, Gateway Technology).

[0057] In some implementations, it may be desirable to overexpress FSTL1 nucleic acid at levels far higher than currently found in spontaneously generated cells. This can be achieved by selectively cloning the nucleic acid encoding those peptides into a multi-copy plasmid or by placing those nucleic acids under a strongly inducible or constitutive promoter. Methods for overexpressing the desired peptide are routine and well-known in molecular biology, and examples can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, 2001.

[0058] A variety of host cells can be used to generate recombinant host cells that express FSTL1. These host cells can be naturally FSTL1-producing cells or cells that do not naturally produce FSTL1. For example, mammalian cells, such as, but not limited to, Chinese hamster ovary (CHO) cells or epicardial-derived cell cultures, can be used to generate FSTL1. However, in other embodiments, recombinant FSTL1 is generated using cells derived from organisms that do not produce post-translationally glycosylated proteins (i.e., cells that do not post-translationally modify proteins with one or more carbohydrate modules).

[0059] Non-limiting examples of cells that do not produce post-translational glycosylated proteins include bacterial cells. Therefore, in one embodiment, the host cell is a bacterial cell. In another embodiment, the bacterial cell is a Gram-positive or Gram-negative bacterial cell. In yet another embodiment, the bacterial cell is selected from *Escherichia coli*, *Lactobacillus acidophilus*, *P. citrea*, *Bacillus subtilis*, *B. licheniformis*, *B. lentus*, *B. brevis*, *B. stearothermophilus*, *B. alkalophilus*, and *B. amyloliquefaciens*. The group consists of *Bacillus clausii*, *Bacillus halodurans*, *Bacillus megaterium*, *Bacillus coagulans*, *Bacillus circulans*, *Bacillus lautus*, *Bacillus thuringiensis*, *Streptomyces albus*, *Streptomyces lividans*, *Streptomyces coelicolor*, *Streptomyces griseus*, *Pseudomonas* sp., *Pseudomonas alcaligenes*, *Clostridium* sp., *Corynebacterium* sp., and *Corynebacterium glutamicum* cells.

[0060] The encoded FSTL1 polypeptide can be expressed by inserting FSTL1-encoding nucleic acids or vectors containing them into host cells (e.g., bacterial cells) using standard techniques. The introduction of DNA constructs or vectors into host cells can be performed using techniques such as transformation, electroporation, nuclear microinjection, transduction, transfection (e.g., Lipofection-mediated or DEAE-Dextrin-mediated transfection or transfection using recombinant phage viruses), incubation with calcium phosphate DNA precipitates, high-velocity bombardment of DNA-coated microprojectiles, and protoplast fusion. Universal transformation techniques are well known in the art (see, for example, Current Protocols in Molecular Biology (FMAusubel et al. (eds) Chapter 9, 1987; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor, 2001; and Campbell et al., Curr Genet, 16:53-56, 1989, each of which is cited in its entirety, particularly concerning transformation methods). The introduced nucleic acid can be integrated into the host cell's chromosomal DNA or maintained as an extrachromosomal replication sequence. In another embodiment, the FSTL1 peptide can be generated in the host cell via delivery of a chemically modified mRNA encoding a mutant FSTL1 glycosylation-deficient peptide (see Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction. Zangi L, et al. Nat Biotechnol. 2013). Oct; 31(10):898-907, which is incorporated herein in its entirety by reference. Chemically modified RNA (also referred to herein as modRNA) may include, for example, phosphate ester modifications to thiophosphate nucleotide linkages, modifications to the 2'-hydroxyl group of the ribose, or other modifications to the phosphate backbone or sugar modules of the mRNA.

[0061] In some implementations, FSTL1 is a hypoglycosylated FSTL1. Hypoglycosylated FSTL1 can be obtained by generating recombinant FSTL1 in host cells that are naturally not post-translationally modified with carbohydrate modules (such as bacteria, e.g., *E. coli*) or engineered to prevent post-translational modification with carbohydrate modules. Alternatively, hypoglycosylated FSTL1 can be generated in mammalian or other eukaryotic cells that are normally post-translationally modified with carbohydrate modules but have been treated with one or more glycosylation inhibitors. Suitable glycosylation inhibitors include, but are not limited to, tunicamycin (which blocks all protein N-glycosylation), streptotoxin, mycotoxin, bimycin / amycin, tebufenozide, antibiotic 24010, and antibiotic MM. 19290, Bacitracin, Corynebacterium toxin, Pyrooxytetracycline, Duimycin, 1-Deoxymannosinoside, Deoxymannosinoside, N-Methyl-1-Deoxymannosinoside, Frefield's amycin A, Glucose analogs, Mannose analogs, 2-Deoxy-D-glucose, 2-Deoxyglucose, D-(+)-mannose, D-(+)-galactose, 2-Deoxy-2-fluoro-D-glucose, 1,4-Dideoxy-1,4-imino-D-mannitol (DIM), Fluorinated glucose, Fluorinated mannitol, UDP-2-Deoxyglucose, GDP-2-Deoxyglucose, Hydroxymethylglutaryl-CoA reductase inhibitor, 25-hydroxycholesterol, Hydroxycholesterol, Stromalin, Cycloheximide, Purinemycin Actinomycin D, Monensin, m-chlorocarbonyl-cyanide phenylhydrazone (CCCP), monensin, polyterpenol-phosphoryl-2-deoxyglucose, N-acetyl-D-glucosamine, hypoxanthine, thymidine, cholesterol, glucosamine, mannosamine, succinamine, glutamine, brominated monensinol, monensinol epoxide, monensinol derivatives, glycosylmethyl-p-nitrophenyltriazine, β-hydroxyvaline, threo-β-fluoroasparagine, D-(+)-gluconic acid δ-lactone, di(2-ethylhexyl) phosphate, tributyl phosphate, dodecyl phosphate, 2-dimethylaminoethyl phosphate of (diphenylmethyl)-phosphate, [2-(diphenyloxyphosphinooxy)ethyl]trimethylammonium iodide, iodoacetate / salt, 2-deoxy-D-glucose, and fluoroacetate / salt.

[0062] Alternatively, in other implementations, the recombinant FSTL1 is engineered to prevent glycosylation when generated using eukaryotic or other glycosylation-capable host cells. In most biological contexts, glycosylation is either N-linked or O-linked. N-linked glycosylation occurs extensively in eukaryotes and archaea, but very rarely in eubacteria. In N-linked glycosylation, a glycan (i.e., a carbohydrate-containing module) attaches to the nitrogen atom of the asparagine or arginine amino acid side chain. N-linked glycans almost always attach to the nitrogen atom of the asparagine (Asn) side chain, which is part of the Asn-X-Ser / Thr concordance sequence, where X is any amino acid other than proline (Pro), serine (Ser), and threonine (Thr). O-linked glycosylation is a form of glycosylation that occurs in the Golgi apparatus of eukaryotes. In O-linked glycosylation, a glycan attaches to the hydroxyl oxygen atom of the serine, threonine, tyrosine, hydroxylysine, or hydroxyproline amino acid side chain.

[0063] Therefore, in some embodiments, recombinant FSTL1 is engineered to prevent N-linked glycosylation. In this case, some or all of the glycosylation-capable arginine or asparagine amino acids in the polypeptide sequence can be replaced with glycosylation-free amino acids (e.g., glutamine). In other embodiments, recombinant FSTL1 is engineered to prevent O-linked glycosylation. In this case, all glycosylation-capable serine, threonine, tyrosine, hydroxylysine, or hydroxyproline residues in the polypeptide sequence can be replaced with glycosylation-free amino acids (e.g., alanine). In still other embodiments, recombinant FSTL1 is engineered to prevent either O-linked or N-linked glycosylation by replacing all glycosylation-capable amino acids with glycosylation-free amino acids. In yet another embodiment, one or more asparagine (N) residues located at positions X144, X180, X175, and / or X223 in the FSTL1 amino acid sequence are replaced with glycosylation-free amino acids (such as, but not limited to, glutamine (Q)). Engineered glycosylation-free FSTL1 can be generated in host cells via transfection with a viral vector, plasmid, or chemically synthesized mRNA or mRNA mimic carrying a gene encoding glycosylation-free FSTL1. Alternatively, a gene encoding glycosylation-free FSTL1 can be integrated into the chromosome of a host cell under the control of an inducible or constitutive expression promoter. In yet another embodiment, a glycosylation-free FSTL1 polypeptide can be generated in host cells via delivery of modified mRNA encoding a glycosylation-free FSTL1 polypeptide.

[0064] The invention described herein covers FSTL1 incorporated into pharmaceutical compositions (e.g., sterile pharmaceutical compositions) containing one or more pharmaceutically acceptable carriers. As used herein, a "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" according to the invention is a component such as a carrier, diluent, or excipient that is compatible with other components in the composition (i.e., it can be combined with the pharmaceutical agents and / or compositions of the invention without eliminating the biological activity of the pharmaceutical agent or composition (e.g., FSTL1, such as hypoglycosylated FSTL1)) and is suitable for use in subjects as provided herein, without excessive adverse side effects (such as toxicity, irritation, allergic reactions, and death). Side effects are "excessive" when the risk outweighs the benefits provided by the pharmaceutical composition. Non-limiting examples of pharmaceutically acceptable ingredients include, but are not limited to, any standard drug carriers such as phosphate-buffered saline solutions, water, sterile water, polyethylene glycol, polyvinylpyrrolidone, lecithin, peanut oil, sesame oil, emulsions such as oil / water emulsions or water / oil emulsions, microemulsions, nanocarriers, and various types of wetting agents. Additives such as water, alcohols, oils, glycols, preservatives, fragrances, colorants, suspending agents, etc., may also be included in the composition along with the carrier, diluent, or excipient. In one embodiment, the pharmaceutically acceptable carrier suitable for use in the compositions disclosed herein is sterile, pathogen-free, and / or otherwise safe for administration to subjects without the risk of associated infection and other excessive adverse side effects.

[0065] Any pharmaceutical composition containing FSTL1 (such as FSTL1 containing low-glycosylated components) disclosed herein can be formulated for administration using any number of administration methods available in the art. Administration can be performed via a variety of routes, including patches, catheters, stents, oral, rectal, percutaneous, subcutaneous, intravenous, intramuscular, intranasal, and so on. In some embodiments, the above-described administration methods can be used to deliver suspensions containing FSTL1 (e.g., low-glycosylated FSTL1 mixed with gelatin sponge particles). These compositions are effective as both injectable and oral compositions. Such compositions are prepared in a manner well known in the pharmaceutical industry and contain at least one active compound. When used as an oral composition, the polypeptide composition is protected from acid digestion in the stomach by a pharmaceutically acceptable protectant.

[0066] In some embodiments, any pharmaceutical composition containing FSTL1 (such as FSTL1 containing low-glycosylation) disclosed herein can be incorporated into engineered patches for direct application to the epicardium or damaged myocardial tissue. In one embodiment, a compressed collagen gel is used to generate a three-dimensional (3D) collagen patch to deliver low-glycosylation FSTL1 directly to the epicardium. In some embodiments, a highly hydrated collagen gel can be compressed to remove excess water and generate a dense biomaterial with improved biological and mechanical properties.

[0067] As described in Example 2 below, a highly hydrated collagen gel undergoes unrestricted compression by applying static compressive stress of ~1,400 Pa for 5 minutes, resulting in a ~98-99% volume reduction. The elastic modulus of this compressed collagen is close to that of the embryonic epicardium, which is optimal for the contractility of immature cardiomyocytes. The elasticity of the compressed collagen patch can be evaluated by atomic force microscopy (AFM) in a nanoindentation mode, using forced triggering with indentations of ~100 nm that result in minimal local strain of less than about 10% (such as less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5%, encompassing all values ​​falling between these percentages) to minimize the effects of substrate-related artifacts. This 3D collagen patch can be seeded with recombinant FSTL1 (such as low-glycosylated FSTL1) and then applied directly to the epicardium or damaged or compromised areas of myocardium, for example, by suturing. In some embodiments, the 3D collagen patch has an elastic modulus comparable to that reported for embryonic epicardium (E ~ 12 ± 4 kPa, such as about 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, 15 kDa, or any one of 16 kDa). In other embodiments, the 3D collagen patch has a lower elastic modulus than that of mature epicardium (E > 30-40 kPa). In still other embodiments, the 3D collagen patch has a lower elastic modulus than that of fibrotic cardiac tissue (E > 100 kPa), but higher than that of most currently used scaffold biomaterials (E ≤ 1 kPa). In another embodiment, the 3D collagen patch has an elastic modulus of approximately 1 kPa, 2 kPa, 3 kPa, 4 kPa, 5 kPa, 6 kPa, 7 kPa, 8 kPa, 9 kPa, 10 kPa, 11 kPa, 12 kPa, 13 kPa, 14 kPa, 15 kPa, 16 kPa, 17 kPa, 18 kPa, 19 kPa, 20 kPa, 21 kPa, 22 kPa, 23 kPa, 24 kPa, 25 kPa, 26 kPa, 27 kPa, 28 kPa, or 29 kPa.

[0068] Further information on the construction and use of 3D collagen-based patches to deliver substances directly to the heart can be found in Serpooshan, V. et al., Acta Biomater, 2010; 6, 3978-3987; Serpooshan, V. et al., J Biomed Mater Res A, 2011; 96, 609-620; and Abou Neel et al., Soft Matter, 2006; 2, 986-992, which are incorporated herein by reference.

[0069] Another option for delivering low-glycosylated FSTL1 peptides to cardiac tissue is as a component of self-polymerizing hydrogels delivered via catheter technology. Further information on this type of delivery can be found in Koudstaal et al., J. of Cardiovasc. Trans. Res. (2014) 7:232-241, the contents of which are incorporated herein by reference. Catheter delivery can also be used for suspensions containing FSTL1, such as low-glycosylated FSTL1 mixed with gelatin sponge particles.

[0070] III. The Method of the Invention

[0071] This document describes a method for repairing cardiac (e.g., myocardial) tissue after injury in subjects with a need, comprising contacting the cardiac (e.g., myocardial) tissue with an epicardial-derived paracrine factor. In some embodiments, the epicardial-derived paracrine factor is a hypoglycated follicle-like 1 (FSTL1) peptide. In other embodiments of the invention, administration of hypoglycated FSTL1 does not activate Akt-1 signaling activity (see...). Figure 20 In other embodiments of the invention, administration of low-glycosylated FSTL1 does not result in a decrease in cardiomyocyte apoptosis (see [link to invention]). Figure 6 ).

[0072] Damage to cardiac (e.g., myocardial) tissue can be associated with any number of known diseases or conditions that invade the heart or circulatory system, and includes, but is not limited to, coronary artery disease, cardiomyopathy, ischemic heart disease, heart failure, inflammatory heart disease, valvular heart disease, and aneurysm. In one embodiment, the damage is caused by myocardial infarction (MI; such as acute myocardial infarction (AMI)). In another embodiment, the damage is caused by an ischemic event followed by reperfusion.

[0073] Repairing damaged cardiac (e.g., myocardial) tissue can involve increasing the number of cardiomyocytes, which can be indirectly measured in living subjects using several imaging methods, such as delayed enhancement-modulated MRI (DE-MRI), as well as the reduction in the size of myocardial infarction. See, for example, Hendel RC et al, JACC 48(7):1475-97 and Sardella G et al, JACC 2009;53(4):309-15, which are incorporated herein by reference in their entirety. In some implementations, contacting cardiac (e.g., myocardial) tissue with epicardial-derived paracrine factors (such as hypoglycated FSTL1) results in loss of muscle recovery and infarct size reduction of approximately 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 90%, 100%, or approximately 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%. In other embodiments, contacting cardiac (e.g., myocardial) tissue with epicardial-derived paracrine factors (such as hypoglycated FSTL1) results in loss of muscle recovery and infarct size reduction of at least about 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 90%, 100%, or about 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%. In other embodiments, contacting cardiac (e.g., myocardial) tissue with epicardial-derived paracrine factors (such as hypoglycated FSTL1) results in loss of muscle recovery and infarct size reduction of up to about 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 90%, 100%, or about 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%.

[0074] In some embodiments of any of the methods disclosed herein, the epicardial-derived paracrine factor (such as hypoglycated FSTL1) is infused, seeded, or embedded in a 3D collagen-based patch (such as any of those described herein). The collagen-based patch can then be brought into direct contact with the epicardium or a damaged area of ​​myocardium (such as a myocardial area exposed to ischemic events, such as myocardial infarction). The 3D collagen patch can be applied to the epicardium or myocardium to bring the patch into contact with the damaged tissue via suturing or by any other means known in the art.

[0075] In other embodiments, the epicardial-derived paracrine factor (such as hypoglycated FSTL1) is a component of a hydrogel delivered to the epicardium, endocardium, or damaged myocardial region (via, for example, catheter technology; Koudstaal et al., J. of Cardiovasc. Trans. Res. (2014) 7:232-241, incorporated herein by reference in its entirety).

[0076] In some embodiments of any of the methods disclosed herein, an increase in the number of cardiomyocytes is achieved by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more times compared to the number of cardiomyocytes in cardiac (e.g., myocardial) tissue that has not been exposed to epicardial-derived paracrine factors after injury. Assessment of cardiomyocyte replication is routine in the art and can be measured, for example, by determining the number of α-actin-positive cells in a cardiac (e.g., myocardial) tissue sample from a subject. In some other embodiments, the effect on cardiac (e.g., myocardial) tissue can be achieved by placing low-glycosylated FSTL1 in the proximal segment of the cardiac compartment (i.e., the endocardium), for example by delivery via catheter technology. In some embodiments, a suitable catheter may be a NOGA catheter (Johnson & Johnson).

[0077] In some implementations, the effects on cardiac (e.g., myocardial) tissue can be achieved by placing low-glycosylated FSTL1 intracardiacly into the heart via a percutaneous catheter delivery system, such as those developed and available by BioCardia (www.biocardia.com).

[0078] In some implementations, the effects on cardiac (e.g., myocardial) tissue can be achieved by extracardiac placement of low-glycosylated FSTL1, similar to those used in other applications (e.g., Epicardial Catheter Systems). TM Similar catheter devices to those used in St. Jude Medical Center.

[0079] In some implementations, the effect on cardiac (e.g., myocardial) tissue can be achieved by placing low-glycosylated FSTL1 when it is impregnated in a drug-dilutable stent (e.g., those available from Abbott Laboratories or Biosensors International, etc.).

[0080] In some implementations, the effect on cardiac (e.g., myocardial) tissue can be achieved by systematically placing low-glycosylated FSTL1, using an approved formulation.

[0081] In some implementations, the effect on cardiac (e.g., myocardial) tissue can be achieved by placing a low-glycosylated FSTL1, i.e., by using compounds or drugs that inhibit endogenous glycosylation of the FSTL1 protein, which are readily available and known to those skilled in the art.

[0082] In some implementations, the effect on cardiac (e.g., myocardial) tissue can be achieved by placing a hypoglycosylated FSTL1, i.e., by introducing a modRNA that encodes a specific mutagenic site targeting the N-glycosylation site in the FSTL1 mRNA sequence.

[0083] In some implementations, the effects on cardiac (e.g., myocardial) tissue can be achieved by placing a hypoglycosylated FSTL1, i.e., genome editing using CRISPR / Cas9 technology or similar technologies (see, for example, Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Hao Yin, et al. Nature Biotechnology 32, 551-553 (2014) doi:10.1038 / nbt.2884, incorporated herein by reference in its entirety).

[0084] In some other implementations, the effects on cardiac (e.g., myocardial) tissue can be achieved by delivering a small molecule mimic of low-glycosylated FSTL1.

[0085] In other embodiments, repair of damaged cardiac (e.g., myocardial) tissue includes an improvement in the percentage reduction of cardiac (e.g., myocardial) tissue compared to the percentage reduction in cardiac (e.g., myocardial) tissue that has not been exposed to epicardial-derived paracrine factors after injury. In some embodiments, exposing cardiac (e.g., myocardial) tissue to epicardial-derived paracrine factors (such as hypoglycated Fstl1) results in an improvement in the percentage reduction of cardiac (e.g., myocardial) tissue compared to the same subject before treatment of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% or greater, including all values ​​falling between these percentages. In some implementations, contacting cardiac (e.g., myocardial) tissue with epicardial-derived paracrine factors (such as hypoglycated Fstl1) results in a reduction of the percentage of cardiac (e.g., myocardial) tissue compared to the same subject before treatment, with improvements of up to about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, including all values ​​falling between these percentages. In some implementations, contacting cardiac (e.g., myocardial) tissue with epicardial-derived paracrine factors (such as hypoglycated Fstl1) results in an improvement of at least about 1-100%, 5-95%, 10-90%, 20-80%, 30-70%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100% in any of these percentages, including all values ​​falling between these percentages. In some implementations, exposing cardiac (e.g., myocardial) tissue to epicardial-derived paracrine factors (such as hypoglycated Fstl1) results in an improvement of up to approximately 1-100%, 5-95%, 10-90%, 20-80%, 30-70%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100% in any of these percentages compared to the same subject before treatment. Repair of damaged cardiac (e.g., myocardial) tissue may also include an increase in the amount of cardiomyocyte cytokinesis compared to the amount of cardiomyocyte cytokinesis in cardiac (e.g., myocardial) tissue that has not been exposed to epicardial-derived paracrine factors after injury.In some embodiments, contacting cardiac (e.g., myocardial) tissue with epicardial-derived paracrine factors (such as low-glycosylated FSTL1) results in an improvement in the amount of cardiomyocyte cytokinesis of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% or greater. In some embodiments, contacting cardiac (e.g., myocardial) tissue with epicardial-derived paracrine factors (such as low-glycosylated FSTL1) results in an improvement in the amount of cardiomyocyte cytokinesis of at most about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% or greater. In some embodiments, contacting cardiac (e.g., myocardial) tissue with epicardial-derived paracrine factors (such as hypoglycosylated FSTL1) results in an improvement in the amount of cardiomyocyte cytokinesis by at least about 1-100%, 5-95%, 10-90%, 20-80%, 30-70%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%. In some implementations, exposing cardiac (e.g., myocardial) tissue to epicardial-derived paracrine factors (such as hypoglycosylated FSTL1) results in an improvement in the amount of cardiomyocyte cytokinesis by up to approximately 1-100%, 5-95%, 10-90%, 20-80%, 30-70%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%. Assessment of cardiomyocyte cytokinesis is routine in the art and can be measured, for example, by determining the expression level of Aurora B kinase in a cardiac (e.g., myocardial) tissue sample from a subject. Current methods only allow these studies to be performed post-mortem, after biopsy, or after transplantation.

[0086] In some embodiments, repairing damaged cardiac (e.g., myocardial) tissue may include a reduction in cardiomyocyte apoptosis compared to the amount of cardiomyocyte apoptosis in cardiac (e.g., myocardial) tissue that has not been exposed to epicardial-derived paracrine factors after injury. In some embodiments, exposing cardiac (e.g., myocardial) tissue to epicardial-derived paracrine factors (such as hypoglycated FSTL1) results in a reduction of cardiomyocyte apoptosis in cardiac (e.g., myocardial) tissue by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or greater, including all values ​​falling between these percentages. In some implementations, contacting cardiac (e.g., myocardial) tissue with epicardial-derived paracrine factors (such as hypoglycated FSTL1) results in a reduction of cardiomyocyte apoptosis in cardiac (e.g., myocardial) tissue by up to about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, including all values ​​falling between these percentages. In some embodiments, contacting cardiac (e.g., myocardial) tissue with epicardial-derived paracrine factors (such as hypoglycosylated FSTL1) results in a reduction of cardiomyocyte apoptosis in cardiac (e.g., myocardial) tissue by at least about 1-100%, 5-95%, 10-90%, 20-80%, 30-70%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, or greater, including all values ​​falling between these percentages. In some embodiments, contacting cardiac (e.g., myocardial) tissue with epicardial-derived paracrine factors (such as hypoglycosylated FSTL1) results in a reduction of cardiomyocyte apoptosis in the cardiac (e.g., myocardial) tissue by up to approximately 1-100%, 5-95%, 10-90%, 20-80%, 30-70%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%, encompassing all values ​​falling between these percentages. Assessment of cardiomyocyte apoptosis is routine in the art (postmortem or ex vivo, after heart separation) and can be measured, for example, by TUNEL staining of a cardiac (e.g., myocardial) tissue sample from a subject.

[0087] Repairing damaged cardiac (e.g., myocardial) tissue may also include elevated levels of transcripts encoding one or more heart-specific contractile proteins in cardiomyocytes compared to the levels of these contractile proteins in cardiac (e.g., myocardial) tissue that has not been exposed to epicardial-derived paracrine factors after injury. In some embodiments, exposing cardiac (e.g., myocardial) tissue to epicardial-derived paracrine factors (such as hypoglycosylated Fstl1) results in an increase in the level of one or more transcripts encoding heart-specific contractile proteins of up to about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or greater. In some embodiments, contacting cardiac (e.g., myocardial) tissue with epicardial-derived paracrine factors (such as hypoglycosylated Fstl1) results in an increase in the level of one or more transcripts encoding heart-specific contractile proteins by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, contacting cardiac (e.g., myocardial) tissue with epicardial-derived paracrine factors (such as hypoglycosylated Fstl1) results in an increase in the level of one or more transcripts encoding heart-specific contractile proteins by at least about 1-100%, 5-95%, 10-90%, 20-80%, 30-70%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%. In some embodiments, contacting cardiac (e.g., myocardial) tissue with epicardial-derived paracrine factors (such as hypoglycosylated Fstl1) results in an increase in the level of one or more transcripts encoding heart-specific contractile proteins to up to about 1-100%, 5-95%, 10-90%, 20-80%, 30-70%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%. In some embodiments, the heart-specific contractile protein is selected from the group consisting of myh6, mlc2v, and mlc2a. Evaluation of heart-specific contractile protein transcripts is routine in the art and can be measured by, for example, Northern blotting, Western blotting, reverse transcriptase (RT) PCR, FACS analysis, immunohistochemistry, or in situ hybridization.

[0088] Repairing damaged cardiac (e.g., myocardial) tissue can involve rhythmic, contractile calcium cells within cardiomyocytes. 2+ Actin + Cell proliferation. In some implementations, contact between cardiac (e.g., myocardial) tissue and epicardial-derived paracrine factors (such as hypoglycated Fstl1) leads to rhythmic contractile Ca2+ in cardiomyocytes. 2+ Actin + The amount of cells and the rhythmic contractile Ca in cardiac (e.g., myocardial) tissue that has not been exposed to epicardial-derived paracrine factors after damage 2+ Actin + The number of cells increases by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more times. Cardiac cardiomyocytes possess rhythmic, contractile calcium... 2+ Actin + Cell evaluation is routine in the art (see Example 1 below).

[0089] Damaged cardiac (e.g., myocardial) tissue may be exposed to any epicardial-derived paracrine factor (such as low-glycosylated FSTL1) composition (such as pharmaceutical compositions) disclosed herein before, during, or after damage to the cardiac (e.g., myocardial) tissue. In some embodiments, exposing cardiac (e.g., myocardial) tissue to an epicardial-derived paracrine factor composition in subjects considered to be at risk of cardiovascular disease, MI, or another ischemic myocardial event thereby mitigating or preventing damage to the myocardium from such event. In other embodiments, exposing cardiac (e.g., myocardial) tissue to an epicardial-derived paracrine factor composition immediately after the onset of an ischemic event caused by cardiovascular disease or MI, such as about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, or 24 minutes. 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 10.5 hours, 11 hours, 11.5 hours, or 12 hours or more (including all time periods falling between these values). In some embodiments, the composition is applied less than 1 minute after the cardiac injury. Alternatively, in other embodiments, the cardiac (e.g., myocardial) tissue is exposed to an epicardial-derived paracrine factor composition after injury, such as at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, or 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 1 month, 2 months, 3 months, 4 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or one or more years (including all time periods falling between these values) after an ischemic event caused by cardiovascular disease or MI.

[0090] Any method disclosed herein for treating damage to cardiac (e.g., myocardial) tissue may result in prolonged survival in subjects following the damage. As used herein, prolonged survival includes, for example, a survival increase of at least about 5% (e.g., at least about 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, or more than 200% or greater) compared to the relative survival of subjects not subjected to the methods currently described. Survival time may be measured, for example, in days, weeks, months, or years. In some embodiments, exposing the damaged cardiac (e.g., myocardial) tissue to epicardial-derived paracrine factors according to any method described herein may prolong the survival of subjects by at least 6 months, 7 months, 8 months, 9 months, 10 months, 12 months, 18 months, 24 months, 36 months, or more.

[0091] In some embodiments, repairing damaged cardiac (e.g., myocardial) tissue may include a reduction or attenuation of fibrosis in the cardiac (e.g., myocardial) tissue compared to the amount of fibrosis in cardiac (e.g., myocardial) tissue that has not been exposed to epicardial-derived paracrine factors after injury. In some embodiments, exposing cardiac (e.g., myocardial) tissue to epicardial-derived paracrine factors (such as hypoglycated FSTL1) results in a reduction of fibrosis in the cardiac (e.g., myocardial) tissue by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any greater, including all values ​​falling between these percentages. In some implementations, contacting cardiac (e.g., myocardial) tissue with epicardial-derived paracrine factors (such as hypoglycated FSTL1) results in a reduction of fibrosis in cardiac (e.g., myocardial) tissue by up to about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, including all values ​​falling between these percentages. In some embodiments, contacting cardiac (e.g., myocardial) tissue with epicardial-derived paracrine factors (such as hypoglycated FSTL1) results in a reduction of fibrosis in the cardiac (e.g., myocardial) tissue by at least about 1-100%, 5-95%, 10-90%, 20-80%, 30-70%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%, encompassing all values ​​falling between these percentages. Assessment of cardiomyocyte fibrosis is routine in the art and can be measured by DE-MRI or by histological examination (postmortem or biopsy) of cardiac (e.g., myocardial) tissue. In some embodiments, contacting cardiac (e.g., myocardial) tissue with epicardial-derived paracrine factors (such as hypoglycated FSTL1) results in a reduction of fibrosis in the cardiac (e.g., myocardial) tissue by up to approximately 1-100%, 5-95%, 10-90%, 20-80%, 30-70%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%, encompassing all values ​​falling between these percentages. Assessment of cardiomyocyte fibrosis is routine in the art and can be measured by DE-MRI or by histological examination (postmortem or biopsy) of cardiac (e.g., myocardial) tissue.

[0092] Repairing damaged cardiac (e.g., myocardial) tissue may additionally include increased angiogenesis in the damaged areas of the cardiac (e.g., myocardial) tissue. In some embodiments, contacting cardiac (e.g., myocardial) tissue with epicardial-derived paracrine factors (such as hypoglycated FSTL1) results in an increase in the amount of angiogenesis in the cardiac (e.g., myocardial) tissue compared to the relative amount of angiogenesis in cardiac (e.g., myocardial) tissue that was not contacted with epicardial-derived paracrine factors after injury, or an increase in blood perfusion of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any greater. In some embodiments, contacting cardiac (e.g., myocardial) tissue with epicardial-derived paracrine factors (such as hypoglycated FSTL1) results in an increase in the amount of angiogenesis in the cardiac (e.g., myocardial) tissue compared to the relative amount of angiogenesis in cardiac (e.g., myocardial) tissue that was not contacted with epicardial-derived paracrine factors after injury, or an increase in blood perfusion of up to about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of any one of these. In some embodiments, contacting cardiac (e.g., myocardial) tissue with epicardial-derived paracrine factors (such as hypoglycated FSTL1) results in an increase in the amount of angiogenesis in the cardiac (e.g., myocardial) tissue that is restored or blood perfusion is increased by at least about 1-100%, 5-95%, 10-90%, 20-80%, 30-70%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% after injury. In some embodiments, contacting cardiac (e.g., myocardial) tissue with epicardial-derived paracrine factors (such as hypoglycated FSTL1) results in an increase in the amount of angiogenesis in the cardiac (e.g., myocardial) tissue compared to the relative amount of angiogenesis in cardiac (e.g., myocardial) tissue that was not contacted with epicardial-derived paracrine factors after injury, or an increase in blood perfusion of up to about 1-100%, 5-95%, 10-90%, 20-80%, 30-70%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% of any one.Assessing angiogenesis in cardiac (e.g., myocardial) tissue is routine in the art and can be assessed by measuring the expression of proteins such as von Willebrand factor (vWF) or smooth muscle actin in vascular cells (see Example 4 below).

[0093] In some further embodiments, repair of damaged cardiac (e.g., myocardial) tissue includes an increase in the number of cardiomyocytes entering the cell cycle. In some embodiments, exposing cardiac (e.g., myocardial) tissue to epicardial-derived paracrine factors (such as hypoglycated Fstl1) results in an increase in the number of cardiomyocytes entering the cell cycle in cardiac (e.g., myocardial) tissue by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more times compared to the number of cardiomyocytes entering the cell cycle in cardiac (e.g., myocardial) tissue that has not been exposed to epicardial-derived paracrine factors after injury. Assessment of the number of cardiomyocytes entering the cell cycle in cardiac (e.g., myocardial) tissue is routine in the art and can be assessed by measuring, for example, the expression of phosphate-histone H3 (see Example 5 below).

[0094] In some embodiments of any of the methods disclosed herein, the epicardial-derived paracrine factor (such as hypoglycated Fstl1) is infused, seeded, or embedded in a 3D collagen-based patch (such as any of those described herein). The collagen-based patch can then be brought into direct contact with the epicardium or a damaged area of ​​myocardium (such as a myocardial area exposed to ischemic events, such as myocardial infarction). The 3D collagen patch can be applied to the epicardium or myocardium to bring the patch into contact with the damaged tissue via suturing or by any other means known in the art.

[0095] In other embodiments, the epicardial-derived paracrine factor (such as hypoglycated Fstl1) is a component of a hydrogel delivered to the epicardium, endocardium, or damaged myocardial region (via, for example, catheter technology; Koudstaal et al., J. of Cardiovasc. Trans. Res. (2014) 7: 232-241).

[0096] IV. Reagent Kit

[0097] This document also provides kits comprising (i) epicardial-derived paracrine factors (such as low-glycosylated FSTL1 peptides); and (ii) one or more pharmaceutically acceptable excipients. One or both of these kit components may be made sterile so that they can be administered to individuals in need (e.g., individuals with cardiac impairment, such as MI). The kit may optionally contain a 3D collagen patch (such as any of those disclosed herein) that can inoculate or perfuse the epicardial-derived paracrine factor prior to administration to a subject. Alternatively, the pre-inoculated or pre-perfused 3D collagen patch may be included in the kit along with written instructions regarding its use and application to the damaged cardiac (e.g., myocardial) tissue or epicardium of the subject in need. The kit may further include means or tools, such as, but not limited to, suture materials, for adhering the 3D collagen patch to the epicardium or damaged cardiac (e.g., myocardial) tissue.

[0098] Any kit disclosed herein may also include a hydrogel (such as a self-polymerizing hydrogel) as a carrier of epicardial-derived paracrine factors (such as a hypoglycosylated FSTL1 peptide). In one embodiment, the kit further includes one or more catheters for delivering the hydrogel (such as a hydrogel infused with a hypoglycosylated FSTL1 peptide) to the endocardium, epicardium, and / or one or more damaged myocardial regions.

[0099] The kit may also include written instructions on using the kit, such as instructions on infusing epicardial-derived paracrine factors into a 3D collagen patch, suturing the patch to the myocardium or epicardium, infusing epicardial-derived paracrine factors into a hydrogel (such as a self-polymerizing hydrogel), and delivering the hydrogel to the epicardium or one or more damaged myocardial regions via catheter technology.

[0100] The intention is that every maximum numerical limit given in this specification includes every lower numerical limit, as explicitly stated herein. Every minimum numerical limit given in this specification includes every higher numerical limit, as explicitly stated herein. Every numerical range given in this specification includes every narrower numerical range falling within such a wider range, as explicitly stated herein.

[0101] The invention can be further understood by submitting the following embodiments, which are provided by way of example and are not intended to be limiting. Example

[0102] Example 1: Epicardial paracrine signal transduction activates cardiomyocyte proliferation

[0103] The epicardium of the heart provides progenitor cells. 1,2And mitogens (including FGF, IGF2, and PDGF) 3-5 The epicardium is the outer epithelial layer that contributes to myocardial growth during development. Recent studies suggest that the epicardium may retain the function of adult myocardium even after damage, potentially serving as a source of myogenic ancestors. 6 , 7 However, no epicardial-derived paracrine factors have been shown to support myocardial regeneration in mammals, although their identity and mechanisms of action would provide insights into this poorly understood and inherently inefficient process. 8 This embodiment describes the identification of such an epicardial-derived paracrine factor.

[0104] Materials and methods

[0105] Progenitor cells Sca1 + Myh6 - The progenitor of cardiomyocytes is as described 19 Obtained by Schneider Labs.

[0106] epicardial mesothelial cells (EMC) as described 33 Maintained in DMEM containing 10% FBS and antibiotics / antifungal agents. Nuclear labeling was performed using EMC stably transduced with H2B-mCherry lentivirus.

[0107] Mouse embryonic stem cell-derived cardiomyocytes (mCM) ESC ): A stable mouse ESC line (Myh6-Puror; Rex-Blastr) for drug resistance selection in cardiomyocytes was generated through lentiviral transduction and selection with blastomycin, which is consistent with our previously reported human line. 34 resemblance.

[0108] mCM ESC Differentiation of Myh6-Puror; Rex-Blastr mESCs into embryoids (EBs) was achieved up to day 4 on differentiation medium containing: Iscove's modified Dulbecco medium (IMDM) supplemented with 10% FBS, 2 mM glutamine, and 4.5 x 10⁻⁶ mg / L. -4 M monothioglycerol, 0.5 mM ascorbic acid, 200 μg / mL transferrin (Roche), 5% protein-free hybridoma medium (PFHM-II, Invitrogen), and antibiotic / antifungal agent were added, and the mixture was spread onto adherent cell culture plates one day after spontaneous cell agitation began. Myh6 was then purified. +Cardiomyocytes were treated with puromycin for 24 hours on day 9 of differentiation. Subsequently, the cells were treated with trypsin and plated as monolayers of cardiomyocytes. Conditioned medium and FSTL1 treatment were typically performed 24 hours after monolayer plating. The length of treatment is indicated in the legend of each figure.

[0109] Embryonic cardiomyocytes. Tnt-Cre;Rosa26 cells from e12.5 embryos were sorted using fluorescence-activated cell sorting (FACS). mTmG / + Heart-based purification of cardiomyocytes. The heart was dissociated and GFP isolated via collagenase IV digestion. + Cells were used for FACS purification. GFP was cultured. + The cells were identified as cardiomyocytes by the expression of their cardiomyocyte-specific markers, α-actin-co-2 (ACTN2) and cardiac troponin T (TNNT2). They beat rhythmically during in vitro culture.

[0110] Conditioned culture medium for rat epicardial mesothelial cells (EMC). EMC 33 cells were cultured in 10% FBS DMEM containing penicillin / streptomycin until confluence (~1 x 10⁻⁶). 6 / cm 2 The cells were then washed three times with PBS, and the culture medium was replaced with phenol red-free, penicillin / streptomycin-containing serum-free DMEM. After culturing for another two days, the culture medium was collected as conditioned medium (20 ml of medium was added for conditioned medium, and 18 ml was collected after two days). The collected medium was filtered through a 0.22 μm pore membrane (Millipore). A control conditioned medium was prepared in the same manner but without EMC cells.

[0111] Adult mouse EPDC conditioned medium in Zhou's laboratory 9 Genetically generated. In short, intensive feeding was used to train 8-week-old adult Wt1. CreERT2 / + Rosa26 mTmG / + Heart-bearing mice were orally injected with 4 mg of tamoxifen 4-5 times over a 2-week period. Myocardial infarction was then induced in 11-week-old adult mice by ligation of the left anterior descending coronary artery. One week after the injury, Wt1 samples were collected. CreERT2 / + Rosa26 mTmG / +Hearts were then digested into single cells using collagenase IV. The digestion solution was prepared by adding 4 ml of 1% collagenase IV and 1 ml of 2.5% trypsin to 44.5 ml of Hank's balanced salt solution, supplemented with 0.5 ml of chicken serum and 0.5 ml of horse serum. Cells were resuspended in Hank's balanced salt solution, and 4 ml of the digestion solution was added to each tube and gently shaken at 37°C for 6 minutes. After removing the supernatant containing dissociated cells, another 4 ml of digestion solution was added to repeat the digestion 6 times. After the final digestion, the cells were filtered through a 70 μm filter and centrifuged at 200 g for 5 minutes at 4°C to form cell clumps. The cells were then resuspended in Hank's balanced salt solution for FACS separation. Dissociated cells from GFP hearts were used as a control for gate setting in FACS. + Wt1 CreERT2 / + Rosa26 mTmG / + Cardiac GFP+ cells (epicardial-derived cells, EPDCs) were isolated, and these GFP+ cells... + The purified population was confirmed to be GFP under a fluorescence microscope. + Cells. FSTL1 expression (as determined by PCR) was restored in cultured GFP+EDPCs. Then, fully conditioned medium from EPDC was added to the myocyte assay. Dilutions are indicated in the legend.

[0112] Cardiomyocyte proliferation (treated with conditioned medium) is as previously described. 9 The MTT assay was performed using Celltiter 96 Aqueous One solution (Promega). After adding Celltiter 96 Aqueous One reagent to the cell culture medium, the plate was incubated at 37°C for 3–4 hours, and then the absorbance at 490 nm was recorded using a 96-well plate reader. The absorbance at 490 nm is closely related to the cell number. Thus, the MTT readout on the y-axis labeled with the MTT assay (A490) reflects the relative cell number from each well between treatment groups.

[0113] Calcium imaging: Contractile calcium transients were recorded using a Kinetic Image Cytometer (KIC, Vala Sciences) with a Fluo4 NW calcium indicator (Life Science). As described. 38 Data was processed using Cyteseer software (Vala Sciences) containing the KIC analysis package.

[0114] RNA extraction and Q-RT-PCR: Total RNA was extracted using TRIzol (Invitrogen) and reverse transcribed into cDNA using the QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer's instructions. The cDNA sample synthesized from 100 ng of total RNA was submitted for RT-QPCR performed using the LightCycler 480 SYBR Green I Master Kit (Roche) on a LightCycler 480 Real-Time PCR System (Roche). Primer sequences used in this example and other examples disclosed herein are listed below:

[0115]

[0116] result

[0117] To search for epicardial signals that promote cardiogenesis, epicardial mesothelial cells (EMC) were compared with Myh6. + Mouse embryonic stem cell (ESC) derived cardiomyocytes (called mCM) ESC Co-culture. Self-differentiated Myh6-Puro r Puromycin-selective preparation of mCM in mouse ESCs ESC (For details and cell phenotype, see [link]) Figure 8 (Materials and Methods). Co-culture with EMC consistently increased α-actin. + The number of muscle cells ( Figure 1 The expression of small figures (ac) and cardiomyocyte markers (including Myh6, Myh7, Mlc2a, and Mlc2v) Figure 1 (Inset d). Undiluted EMC conditioned medium increased the number of myocytes (2.4-fold increase in α-actin was detected). + cell, Figure 1 Small image (eg) and Myh6 (1.8 times), Mlc2v (1.9 times), and Mlc2a (1.3 times) expression ( Figure 1 (See small figure h) to reproduce the effect of co-culture. Furthermore, the EMC-conditioned medium, compared to the standard medium, exhibits a rhythmic contractile Ca2+. 2+ Transient α-actin + The number of cells (8.6 times) Figure 1 (Figure i). Thus, factors secreted from the epicardial-like culture increased the number of contractile cells in ESC-derived cardiomyocyte cultures. The co-culture did not promote cardioogenesis of undifferentiated (Myh6-) ESCs (not shown).

[0118] To assess whether adult epicardium also contains such activity, samples were prepared from 3-4 month old Wt1 cells.CreERT2 / + Rosa26 mTmG / + mice 9 Conditioned culture medium for FACS-isolated epicardial-derived cells (EPDCs) Figure 1 Figure j shows materials and methods. When added to E12.5 embryonic cardiomyocytes (also previously based on eGFP fluorescence from TNT-Cre; Rosa26), mTmG / + When isolated from mouse FACS, adult EPCD conditioned medium significantly enhanced the proliferation of cardiomyocytes in serum-free medium (p<0.05). Figure 1 (Small figure k). Boiling the EPDC medium before incubation eliminates this effect, consistent with the fact that the intrinsic activity of the protein is... EPDC conditioned medium allows for the connection of adjacent Tnnt2... + The incidence of Aurora B kinase in the cleavage fissures of cells nearly doubled (0.19 to 0.33%, P < 0.05). Figure 1 The small figures (l, m) indicate the activity in the adult epicardium that promotes the cytokinesis of embryonic cardiomyocytes.

[0119] Example 2: Engineered epicardial weakening and regeneration with improved cardiac function

[0120] This example describes the role of factors secreted by the epicardium in the adult heart.

[0121] Materials and methods

[0122] Adult ventricular myocytes as previously disclosed 35 Isolate from 3-month-old FVB mice. In short, mice were anesthetized with sodium pentobarbital (100 mg / kg intravenously). The heart was removed and treated at 37°C with calcium-free... 2+ The solution (in mM: 120 NaCl, 14.7 KCl, 0.6 KH₂PO₄, 0.6 Na₂HPO₄, 1.2 MgSO₄⁻ 7H₂O, 4.6 NaHCO₃, 10 Na-HEPES, 30 taurine, 10 BDM, 5.5 glucose) was used for retrograde perfusion, followed by enzymatic digestion with collagenase. The ventricles were cut into small pieces and further digested. A stop buffer (Ca-free) was added. 2+The cells were prepared by centrifuging the solution (12.5 μM CaCl2 + 10% fetal bovine serum) at 40 g for 3 minutes. Myocytes were resuspended in stop buffer, increasing the CaCl2 concentration to 1 mM. Cells were then resuspended in MEM + 5% fetal bovine serum + 10 mM DM + 2 mM L-glutamine and added to collagen solution for prepolymerization (250,000 cells per ml or per patch). After collagen gelation and plastic compression, cells were plated in the above (spread) medium and cultured overnight, then transferred to culture medium: MEM + 1 mg / ml fetal bovine serum albumin + 25 μM blebbistatin + 2 mM L-glutamine, with or without recombinant FSTL1 (AVISCERA BIOSCIENCE, 10 ng / ml). On day 7, as previously described... 36 3D culture specimens were subjected to fluorescence-based cell cycle indicators (FUCCI, Premo) based on ubiquitous protein morphology. TM The FUCCI Cell Cycle Sensor (Life Technologies, US) assay is used. In this assay, G1 and S / G2 / M cells emit red and green fluorescence, respectively. Premo is calculated using the following equation. TM geminin-GFP and Premo TM Volume of Cdt1-RFP:

[0123] The cell count is the estimated total cell count at the time of cell labeling (equal to the CM inoculation density), PPC (particles per cell) is the number of virus particles per cell (in this assay, = 40), and 1 × 10 8 This represents the number of virus particles per mL of reagent. Add the calculated reagent volume directly to the cell patch in complete cell culture medium, mix gently, and incubate overnight (≥16 hours) in an incubator. Image the patch sample using a standard fluorescence microscope with a GFP and RFP filter kit.

[0124] Compressed collagen gel for engineered epicardial patches: A highly hydrated collagen gel – used as a cardiac patch in this study – was generated as follows: 1.1 ml of 1X DMEM (Sigma, MO, US) was added to 0.9 ml of sterile rat tail type I collagen solution (3.84 mg / ml, Millipore, MA, US) in acetic acid. The resulting 2 ml collagen-DMEM mixture was thoroughly mixed and neutralized with 0.1 M NaOH (~50 μl). The entire process was performed on ice to avoid premature gelation of the collagen. In the case of patches containing epicardial factors, EMC culture medium was collected as above and 0.6 ml was mixed with 0.5 ml of DMEM. The collagen solution (0.9 ml) was then dispensed into the wells of 24-well plates (15.6 mm in diameter) and incubated at 37 °C for 30 min in a tissue culture incubator to allow polymerization. As previously described. 39,40 Plastic compression was performed to remove excess water and generate a dense biomaterial with improved biological and mechanical properties. In short, as a casting, a highly hydrated collagen gel (at a volume of ~0.9 ml) underwent unrestricted compression by applying static compressive stress of ~1,400 Pa for 5 minutes (see details). 39,41 This results in a 98-99% volume reduction. The elastic modulus of the compressed collagen was assessed using atomic force microscopy (AFM) in nanoindentation mode (the target was an elastic modulus approximating that of the embryonic epicardium, which is optimal for the contractility of immature cardiomyocytes). 32 Forced triggering, resulting in minimal local strain (indentation ~100 nm) of less than 10%, was used to minimize the effects of substrate-related artifacts. Custom-designed flat AFM tips were fabricated using focused ion beam milling, and the stiffness of the gel was probed by scanning a 90 μm × 90 μm area. Figure 9 Small image (ac).

[0125] Permanent LAD occlusion (MI): Male 10-12 week old C57BL / 6J mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Procedures involving animal use and surgery were approved by the Stanford Research Animal Care and Use Committee (IACUC). Animal care and interventions were provided in accordance with the Laboratory Animal Welfare Act. Mice were anesthetized in an isoflurane inhalation chamber, endotracheally intubated using a 22-gauge catheter (Becton, Dickinson Inc., Sandy, Utah), and connected to a small animal volume-controlled ventilator (Harvard Apparatus, Holliston, MA). A left thoracotomy was performed via the fourth intercostal space, and the lungs were retracted to expose the heart. After opening the pericardium, a 7-0 suture was placed to occlude the left anterior descending artery (LAD) approximately 2 mm below the edge of the left atrium. Ligation was considered successful when the LV wall became pale. In the experimental group treated with patches, the prepared collagen patches were sutured (at two points) to the surface of the ischemic myocardium immediately after ligation. The animals were held on a heating pad until they recovered. Another group of mice underwent sham ligation; they had a similar surgical procedure but without LAD ligation. The minimum number of mice, n=8, was used in each study group.

[0126] TTC staining: On day 2 after MI / patching treatment, mouse hearts from all four groups were harvested and sectioned perpendicularly along the long axis into four sections (approximately 2 mm thick). The sections were placed in the wells of a 12-well cell culture plate and incubated at 37°C for 15 minutes with 1% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma-Aldrich) solution. The sections were then washed with PBS, visualized under a stereomicroscope, and photographed with a digital camera.

[0127] Echocardiography: In vivo cardiac function was assessed by echocardiography at 2 and 4 weeks after LAD ligation. Two-dimensional (2D) analysis was performed on mice using a GE Vivid 7 ultrasound platform (GE Health Care, Milwaukee, WI) equipped with a 13 MHz transducer. Mice were sedated with isoflurane (100 mg / kg, inhalation) and their chests were shaved. Mice were placed in a supine or left lateral decubitus position on the heated platform for echocardiography. 2D clipping and M-mode images were recorded from the mid-left ventricle in short-axis views at the papillary muscle apex. LV diameter (LVID) and posterior wall thickness (LVPW) were measured at both terminal diastole and systole. Fractional shortening (FS, %) and ejection fraction (EF, %) were calculated from the LV dimensions in the 2D short-axis views via extrapolation from the 2D data. Echocardiographic assessment was performed using a minimum number (n) of 8 mice per experimental group. Measurements were performed blinded by two independent groups.

[0128] result

[0129] Next, the roles of factors secreted by the epicardium in the adult heart were assessed using conditioned medium delivered with an epicardial 3D collagen patch. Figure 2 Figure a) is designed to have an elastic modulus (E ~ 12 ± 4 kPa) similar to that reported by the embryonic epicardium. 10 It has a comparable elastic modulus, lower than that of mature epicardium (E>30-40 kPa) and fibrotic heart tissue (E>100 kPa), but higher than that of most currently used scaffold biomaterials (E≤1 kPa). Figure 2 Small image b and Figure 9 The engineered patch was inoculated with EMC-conditioned medium and sutured onto the epicardium of an infarcted adult rat heart. Figure 2 (Insets c, d). Patches were implanted immediately after permanent ligation of the left anterior descending (LAD) coronary artery (myocardial infarction (MI)). Two weeks later, hearts treated with epicardium-culture medium-patches (MI+patches+CM groups) and empty patches (MI+patches, unconditioned culture medium) showed significantly better morphological parameters compared to MI-only animals, including left ventricular diameters at terminal diastole and systole (LVIDd and LVIDs, respectively) and left ventricular posterior wall dimensions at terminal diastole and systole (LVPWd and LVPWs, respectively). Figure 2 Figure e and Table 1) are consistent with the model in which collagen patches provide mechanical support to inhibit pathological modification. 10 It is worth noting that MI + patch + CM treatment provides additional benefits compared to all other conditions, with significantly better ventricular systolic parameters ( Figure 2 Figures e, f and Table 1 thus indicate the activity of epicardial secretion involved in maintaining function.

[0130] Example 3: FSTL1 is an epicardial factor that can induce cardiomyocyte proliferation.

[0131] This embodiment provides data suggesting that FSTL1 plays a role in epicardial-myocardial communication to promote cardiomyogenesis.

[0132] Materials and methods

[0133] LC-MS / MS analysis of conditioned medium: First, tris(2-carboxyethyl)phosphine (TCEP) was added to 1 mL of conditioned medium to a final concentration of 10 mM, and the protein sample was reduced at 37 °C for 30 min. Then, iodoacetamide was added to a final concentration of 20 mM, and the solution was alkylated at 37 °C in the dark for 40 min. Mass spectrometry-grade trypsin (Promega) was then added to the solution at a 1:100 ratio. After digestion overnight at 37 °C, the sample was desalted using a SepPack cartridge, dried using a SpeedVac, and resuspended in 100 μL of 5% formic acid. The obtained peptides were analyzed online using an LC-MS / MS system consisting of a Michrom HPLC, a 15 cm Michrom Magic C18 column, a low-flow advanced Michrom MS source, and an LTQ-Orbitrap XL (ThermoScientific, Waltham, MA). Peptides were separated using a 120-minute gradient of 0–30% B (0.1% formic acid, 100% acetonitrile), with a total LC time of 141 minutes. The LTQ-Orbitrap XL was set to scan precursors in Orbitrap at a resolution of 60,000, followed by data-dependent MS / MS of the top four precursors. The raw LC-MS / MS data were then submitted to Sorcerer Enterprise (Sage-N Research Inc.) for protein identification against the IPI rat protein database, which contains cysteine ​​peptide sequences, allowing up to two missed cleavages and a precursor quality tolerance of 50.0 ppm. A molecular weight of 57 Da was added to all cysteine ​​residues to account for carboxamide methylation. Differential searches included 16 Da for methionine oxidation. Search results were viewed, sorted, filtered, and statically analyzed using PeptideProphet and ProteinProphet (ISB). The minimum transproteome pipeline (TPP) probability scores for proteins and peptides were set to 0.95 to ensure that the TPP error rate was below 0.01.

[0134] Recombinant FSTL1 was purchased from AVISCERA BIOSCIENCE (00347-02-100, generated in E. coli) and R&Dsystem (1694-FN-050, generated in mouse myeloma cell lines, NSO-derived).

[0135] Histology and Immunohistochemistry: Histological analyses of this and other examples were performed according to standard paraffin embedding protocols. For immunohistochemistry, unless otherwise described, embedded embryo sections were sectioned to a thickness of 7 μm. The antibodies used in this example and other examples disclosed herein are as follows: 1:200 α-actin (Sigma, A7811), 1:300 α-smooth muscle actin (Sigma A2547), 1:100 phosphate-histone 3 (rabbit Millipore 06-570), 1:300 phosphate-histone 3 (mouse Abcam ab14955), 1:100 WT1 (Abcam, ab15249), 1:250 AuroraB (Millipore 04-1036, batch number 221196), 1:200 PCM1 (Sigma-Aldrich HPA023370), and 1:200 FSTL1 (R&DMAB17381). At least five sections were stained for histological studies and three for immunohistochemical studies. The inclusion criterion for patch implantation was patch coverage of >70% of the infarct (histologically controlled). TUNEL assay (Roche 11684795910) was performed on frozen sections as instructed.

[0136] result

[0137] To identify bioactive epicardial secretory proteins, EMC-conditioned medium was analyzed by mass spectrometry. Comparison of the spectra with the IPI rat database identified 1596 peptide readouts corresponding to 311 unique proteins, of which 95 readouts were due to 16 discrete secretory proteins. The 10 proteins with the highest spectra counts were selected for analysis in mCM. ESC The test was conducted during the assay. Of these, only follicle-stimulating hormone-like 1 (also known as FSTL1, FRP, or TSC36) was recorded to have cardiac activity. Figure 3 Figure a) shows a secretory glycoprotein of the BM-40 / SPARC / Osteonectin family, which shares a single cysteine-rich domain with follicle-stimulating hormone (FSTL). Unlike FSTL, FSTL1 does not block activin, and its biochemical and biological functions are poorly characterized. 11 Delivery of FSTL1 in the heart leads to a short-term anti-apoptotic effect. 12,13 However, no myocardial repair function is attributed to FSTL1; indeed, FSTL1 levels are elevated in blood flow after acute myocardial infarction, and for this reason, it is considered a biomarker for acute coronary syndrome. 14 .

[0138] mCM was treated with bacterially synthesized recombinant human FSTL1 (10 ng / ml). ESCThe number of cardiomyocytes increases threefold in 8 days. Figure 3 (small image bd), and increased the level of transcripts encoding heart-specific contractile proteins by 2-fold (myh6, mlc2v, and mlc2a, Figure 3 Small figure e) and rhythmic contractile Ca 2+ Transient α-actin + The number of cells increased 7 times ( Figure 3 (Figure f) without inducing hypertrophy. Indeed, FSTL1 reduces cardiomyocyte size in a dose-dependent manner (Figure f). Figure 3 (Figure g). In summary, these data suggest that FSTL1 plays a role in epicardial-myocardial communication to promote cardiomyogenesis.

[0139] Direct visualization via immunostaining revealed that FSTL1 was restricted to the epicardium as early as mid-pregnancy. Figure 3 Figure h), although it existed earlier in the myocardium of the primitive heart tube. 15 Epicardial expression was not previously documented, although it persisted throughout adulthood. Figure 3 (Small figure ik). Notably, FSTL1 is significantly altered following ischemic injury, resulting in its increased abundance in the myocardium. Figure 3 (Figure i-1), and surprisingly absent in the epicardium and infarct area ( Figure 3 Small figures i, l and Figure 10 ).

[0140] Example 4: Localized FSTL1 delivery improves cardiac function after MI

[0141] Previous studies have shown that transient overexpression of FSTL1 in cardiomyocytes or direct systemic infusion of recombinant human FSTL1 is anti-apoptotic after acute ischemia / reperfusion. 12,13 However, in this embodiment, we examine whether it imparts any long-term benefits.

[0142] Materials and methods

[0143] In vivo delayed-enhancement magnetic resonance imaging (DEMRI): To prepare for the scan, anesthesia was induced with 2% isoflurane and maintained with 1.25–1.5% isoflurane while respiratory rate was monitored. Subcutaneous ECG leads were inserted to monitor heart rate, and body temperature was maintained at 37°C. Functional parameters were recorded at 1 and 4 weeks post-treatment using a 3T GESigna Excite clinical scanner with a dedicated mouse coil (Rapid MR International, Germany). The following sequence was performed for MRI acquisition: (1) DEMRI was performed after an intraperitoneal injection of 0.2 mmol / kg gadopentetate dimeglumine (Magnevist, Berlex Laboratories) with a gated fGRE-IR sequence at FOV 3.4 cm, slice thickness 0.9 mm, matrix 128x128, TE 5 ms, TI 150–240 ms, and FA 60°; and (2) multi-volume cardiac MRI was performed with fSPGR at FOV 7 cm, slice thickness 0.9 mm, matrix 256x256, TE 5.5 ms, and FA 30. Two-dimensional imaging planes were placed along the short axis of the left ventricle (LV) using coronal and axial reconnaissance images. This qualitative study used a minimum number (n) of 2 mice per experimental group.

[0144] Vessel counting: Vessel density parameters were measured from histological sections of heart samples stained for von Willebrand factor (vWF), a marker of endothelial cells in the vessel wall. Up to 60 sections were analyzed for each treatment group (4 mice per group). Analysis was performed using ImageJ to calculate: 1) the total luminal area of ​​the vessels, and 2) the number of vessels stained with vWF+. In each case, histograms of the vessel parameters were obtained as a fraction of the total surface area analyzed, and medians were plotted for each treatment group. Statistical significance of differences from the sham group was determined by one-tailed ANOVA (p < 0.05).

[0145] Enzyme-linked immunosorbent assay (ELISA): To evaluate FSTL1 retention in the in vitro engineered patch system, collagen scaffolds loaded with FSTL1 (5 μg / ml) were immersed in PBS at 37°C with agitation for different times (0, 12 hours, 1 day, and 21 days), and FSTL1 concentration was determined using an ELISA kit (USCN Life Science, Inc., Houston, USA). The limit of detection for this technique was 0.50 ng / ml. The scaffolds were pretreated for 5 min with 1 mg / ml type I collagenase (Sigma Aldrich, MO, US) and 5 mg / ml hyaluronidase (Sigma Aldrich, MO, US) dissolved in phosphate-buffered saline, followed by centrifugation at 5,000 x g for 20 min. 100 μl aliquots of the collected samples were added to 96-well plates and incubated at 37°C for 2 h. Then, 100 μL of prepared assay reagent A was added to the wells, followed by incubation at the same temperature for 1 h. After three aspiration and washing cycles, 100 μL of prepared reagent B was added to each well and incubated at 37 °C for 30 min. After five aspiration and washing cycles, 90 μL of substrate solution was added to each well, followed by incubation at 37 °C for 25 min. 50 μL of stop solution was added to each well, and the absorbance of each well was immediately read at 450 nm. The concentration of FSTL1 was defined using a standard curve of standard solutions. The test was performed four times.

[0146] Ischemia-reperfusion (I / R): 10-11 week old male C57 / BL6 mice were anesthetized and cannulated as described above. A left thoracotomy was then performed. The pericardium was gently opened and the left anterior descending coronary artery (LAD) was ligated against the PE10 conduit using 8-0 nylon sutures (Ethicon, Inc. Johnson & Johnson Co., USA). The PE10 conduit was removed 30 minutes after occlusion. Successful coronary artery occlusion was verified by visual inspection (by noticing the formation of pale white tissue in the distal myocardium after ligation). The chest was then closed around the adjacent ribs using 7-0 sutures and the skin was closed with 6-0 sutures. Buprenorphine was administered subcutaneously at a BID dose for at least 1 day. For the patch-treated group, a second thoracotomy was performed one week after I / R, and the prepared collagen patch was sutured (at two points) to the surface of the ischemic myocardium. The sham control consisted of age-matched mice that underwent the same surgical procedure (two thoracotomies) except for LAD ligation. In the ischemia-reperfusion study, cardiac function was assessed in vivo before surgery (baseline), 1 week after I / R, and 2 and 4 weeks after implantation.

[0147] FSTL1-TG mice (used in the MI experiment) were 12-15 week old female and male mice with a C57BL6 background. Furthermore, the research protocol was approved by the Institute for Research Animal Care and Use (IACUC) at Boston University.

[0148] result

[0149] In transgenic mice (which express FSTL1 under the control of the striated muscle-restricted MCK promoter) (FSTL1-TG16, Figure 11 Cardiac function was assessed in inset (Figures a and b). FSTL1-TG mice showed a small but significant improvement in contractility after permanent LAD ligation; however, they did not show long-term improvements in morphometric parameters or scar size, despite abundant FSTL1 overexpression (Figures a and b). Figure 11 (Small figure ac). Thus, myocardial overexpression of FSTL1 was insufficient to recapitulate the cardioprotective effect of epicardial conditioned medium delivered to the epicardial surface. Next, the effect of epicardial hFSTL1 delivery on cardiac function was evaluated. Collagen patches were prepared as before, but this time 10 μg of recombinant bacterially synthesized hFSTL1 / patch was pre-loaded onto the epicardial surface of the infarcted heart before polymerization and application (see Materials and Methods for details). The patches retained immunodetectable hFSTL1 for up to 21 days in vitro and 28 days in vivo, the longest duration of which was tested (…). Figure 12 Immediately after MI, a freshly prepared hFSTL1 patch (patch + FSTL1) was applied to the epicardial surface of the heart. Patch + FSTL1 resulted in significantly improved animal survival compared to animals with MI alone and those with patch alone. Figure 4 Small figure a).

[0150] Contractile echocardiography time-process measurements (% fractional shortening, FS%) demonstrated that Patch +FSTL1 induced a stable recovery of cardiac function between 2 weeks and 3 months after MI, at which point the FS% was close to that in sham-operated animals. Figure 4 (See Figure b and Table 1). Conversely, untreated animals showed a severe decrease in FS% after 4 weeks, with no subsequent improvement. Treatment with patches alone mitigated the decrease in cardiac function relative to untreated, but unlike FSTL1, there was no subsequent improvement in cardiac function. Figure 4 (See Figure b and Table 1).

[0151] Table 1. Raw echocardiographic values ​​(mean ± SEM) obtained at days 0 (baseline), 14, and 28 post-treatment in a mouse model with permanent LAD ligation. Patch was implanted concurrently with the lesion.

[0152]

[0153] *: Statistically significant difference compared to the sham comparison (P<0.05).

[0154] ● The difference was statistically significant compared with MI alone (P<0.05).

[0155] ▲ The difference was statistically significant compared with MI+ patches (P<0.05).

[0156] ■ The difference between MI+patent+CM was statistically significant (P<0.05).

[0157] Given that FSTL1 is upregulated in the myocardium after MI 16 Subsequently, it was tested whether epicardial delivery of FSTL1 was essential for inducing beneficial effects. This was performed by implanting FSTL1 alone or with FSTL1 plus FSTL1 in FSTL1-TG mice with myocardial infarction. 16 Compared to patch treatment alone, contractility parameters showed a dramatic and specific increase in transgenic animals inoculated with patch + FSTL1, with notable changes at week 2 of treatment and improvements reaching up to 50% by week 4. Figure 4 Figure c). Thus, epicardial delivery of recombinant FSTL1 is effective, even in the context of myocardial transgene overexpression of FSTL1, and further points to specific benefits of epicardial FSTL1 delivery beyond that of either patch or myocardial FSTL1 overexpression alone.

[0158] Improved cardiac function and survival following FSTL1 implantation were accompanied by a significant reduction in fibrosis. Figure 4 Small image d, Figure 13 The thinning of LVs in the +FSTL1 and only FSTL1 conditions is similar; both treatments significantly reduce LV thinning compared to the MI-only condition. Figure 4 (Figures d, e and Table 1). In an independent experimental group, delayed enhancement magnetic resonance imaging (DEMRI) analysis at 4 weeks post-lesion confirmed that MI+patch+FSTL1 treatment reduced scar size ( Figure 14 ).

[0159] The study also investigated whether patch + FSTL1 would have a similar beneficial effect if applied after cardiac function decline. For this purpose, an ischemia-reperfusion (I / R) model was used, and the patch was implanted 1 week after injury. All animals showed reduced contractility (from 37% FS before injury to 22% FS 1 week after I / R before patch placement). Cardiac function in untreated animals progressively declined (22%, 20%, and 16% FS at 1, 3, and 5 weeks after I / R, respectively). In contrast, the patch + FSTL1 cohort recovered to 34% FS and stabilized at 3 weeks after I / R, corresponding to complete FS recovery (…). Figure 15 (and Table 2). Similar to the permanent ligation model ( Figure 4Functional recovery is accompanied by recovery of morphometric parameters. Figure 15 (and Table 2). These data indicate that epicardial delivery of FSTL1 induces recovery from ischemic injury.

[0160] Table 2. Raw echocardiographic values ​​(mean ± SEM) at long-term (2nd and 3rd months) after treatment in a mouse model with permanent LAD ligation. Patch was implanted simultaneously with the lesion.

[0161]

[0162]

[0163] *: Statistically significant difference compared to the sham comparison (P<0.05).

[0164] ● The difference was statistically significant compared with MI alone (P<0.05).

[0165] ■ The difference was statistically significant compared with MI+ patches (P<0.05).

[0166] ▲ The difference between MI+patent+CM was statistically significant (P<0.05).

[0167] The FSTL1 in the patch enhances angiogenesis in both the underlying myocardium and the collagen patch at the infarct boundary, as assessed by immunostaining of von Willebrand factor (vWF) and smooth muscle actin (αSMA). Figure 4 (Inset fi). Compared to 0.9% of the area in the MI+ patch group and 0.4% of the area in the MI-only group, approximately 1.5% of the patch area and the underlying myocardium in the MI+ patch+FSTL1 group were occupied by blood vessels. Figure 4 (Small figure g). This value indicates that the vascular system (3.1% area) observed in a considerable region of the distal LV wall of the sham-operated animal was recovered by nearly half. MI+pattern +FSTL1 group (82 vessels / mm) 2 The number of vessels per unit surface area (of any size) in each histological section relative to MI+ patch (35 vessels / mm²). 2 ) and only MI (15 veins / mm) 2 The processing group also improved ( Figure 4 (Inset i). In contrast, the sham-manipulated animal showed 136 blood vessels / mm. 2 The results again indicated that the patch + FSTL1 restored angiogenesis to approximately half the level in mice without infarction. Furthermore, smooth muscle cells surrounded numerous blood vessels, particularly in the MI + patch + FSTL1 group. Figure 4(Small figure h). Masson's trichrome staining shows serial transplantation of patch + FSTL1 into the host myocardium and demonstrates host cell migration into the patch, including evidence of streaked cells at MI and 4 weeks after patch placement (green arrow). Figure 4 (The last two columns in small image j).

[0168] Example 5: In vivo FSTL1-induced cardiomyocytes to enter the cell cycle

[0169] This example demonstrates that epicardial-delivered FSTL1 may have a different function than FSTL1 produced in the myocardium.

[0170] Materials and methods

[0171] The method used in Example 5 is as described herein.

[0172] result

[0173] The +FSTL1 cohort showed intra-patent α-actin + Evidence of striated muscle cells ( Figure 5 (small image ad). Striped cells were rarely observed in the patch under FSTL1 deficiency. Importantly, FSTL1 induces α-actin, which is also positive for phosphate-histone H3 (Ser10) (pH3). + The incidence of cardiomyocytes was 6.2-fold higher than that seen in MI-only animals (compared to 2.5 times per cross section in MI-only). Figure 5 For each of the 15.6 sections in the MI+Patch+FSTL1 processing group (small image i), p<0.05; Figure 5 Little Tuek and Figure 16 This suggests that FSTL1 promotes entry into S phase and DNA replication. The immunoreactivity of Aurora B kinase between α-actin-stained cells and the non-overlap of nuclear DAPI staining in the three-dimensional reconstruction of confocal optical sections were also observed. Figure 5 Figure 1 shows the incidence of cardiomyocytes with intermediate-localized Aurora B kinase significantly elevated in MI++FSTL1-containing hearts compared to other conditions. Figure 5 Figure m) confirms the localization of the intermediate (which is a transient bridge in the cell during cell division), suggesting α-actin. + Cells are induced to undergo cytokinesis. PCM1 is used as a marker of the cardiomyocyte nucleus. 17 A significant increase in the incidence of pH3-positive PCM1+ nuclei was observed. Figure 5 Figure n, o). Increased cardiomyocyte proliferation was also observed 4 weeks post-transplantation in hearts treated with +FSTL1 following I / R injury. Figure 15FSTL1 had no effect on cardiomyocyte apoptosis or risk area immediately after MI, or on apoptosis and inflammation on days 4 and 8 after MI. Figure 17 Although FSTL1 has been shown to prevent apoptosis and may regulate acute inflammation following ischemic injury, 12,13,16 .

[0174] Compared with FSTL1 delivery, pH3 in the border zone myocardium of FSTL1-TG mice was higher. + The number of cardiomyocytes was not increased compared to the wild-type control. Figure 10 Small figure k, l), despite increased angiogenesis ( Figure 10 Small graphs m, n and Figure 16 FSTL1, which indicates epicardial delivery, may have a different function than FSTL1 produced in the myocardium.

[0175] Example 6: Origin of proliferating FSTL1-responsive cardiomyocytes

[0176] This example demonstrates that the glycosylation state of FSTL1 is related to changes in its functional state.

[0177] Materials and methods

[0178] Neonatal rat ventricular cardiomyocytes (NRVCs) were isolated using a neonatal rat cardiomyocyte isolation kit (Cellutron) and cultured at 37°C and 5% CO2. In short, the ventricles of 1-2 day old Hsd:SD rats (Sprague Dawley) were dissected, and the cells were digested five times at 37°C for 15 minutes each time with an enzyme mixture. The cells were then pooled, pre-spread on uncoated cell culture dishes for 90 minutes to remove fibroblasts, and cultured at 3 × 10⁻⁶ cells / mL. 5 cells / cm 2 Cells were spread onto 1% gelatin-coated cell culture dishes in high-serum medium (DME / F12 [1:1], 0.2% BSA, 3mM sodium pyruvate, 0.1mM ascorbic acid, 4 mg / L transferrin, 2mM L-glutamine, and 5 mg / L ciprofloxacin, supplemented with 10% horse serum and 5% fetal bovine serum (FBS)). After 24 hours, the medium was replaced with low-serum medium (the same but with 0.25% FCS) and the cells were cultured until use.

[0179] Automated in vitro cell proliferation and cell death assay: Cells (mCM) were plated in 384 format. ESCCells were incubated with EdU (and NRVC) (details of the exposure dose and duration are specified in the illustration), fixed in 4% PFA for 2 hours, washed in PBS, and stained for EdU using the Click-it EdU Assay Kit (Life Technologies). Cells were then washed in PBS, immunostained with α-coactin antibody (Sigma, A7811) to identify cardiomyocytes, and stained with DAPI (4',6-diamidinyl-2-phenylindole) to identify nuclei. Then, as described... 37 Plate imaging was performed using the InCell 1000 System (GE Healthcare) and automated analysis was conducted in the Developer Toolbox (GE Healthcare). EdU was generated for the percentage of cardiomyocytes incorporating EdU into chromosomal DNA. + / α-actin + Nucleus and α-actin + The ratio of nuclei. Similarly, cells (mCM) are arranged in a 384 plate format. ESC Cells (including NRVC) were fixed in 4% PFA for 2 hours, washed in PBS, and immunostained with pH3 antibody (Millipore 06-570) (for nuclei in mitosis), or Aurora B (Millipore 04-1036) (for cytokinesis), or TUNEL (Roche) (for cell death), and α-actin antibody (Sigma, A7811) (for cardiomyocytes) and DAPI (for nuclei). Imaging and analysis were performed in the same manner as with the EdU assay. pH3 was calculated. + α-actin + Double positive nucleus, Aurora B + α-actin + Double positive cells, and TUNEL + α-actin + Double positive nuclei relative to α-actin + The percentage of total cell nuclei is used to determine the percentage of cardiomyocytes undergoing mitosis, cytokinesis, and apoptosis, respectively.

[0180] FSTL1 Overexpression and Western Blotting: Hek293 cells were transiently transfected with the human FSTL1 plasmid (GEDharmacon, ID: ccsbBroad304_02639pLX304-Blast-V5-FSTL1) using Lipofectamine 2000 (simulated transfection was performed using Lipofectamine and plasmid-free transfection). 48 hours post-transfection, the serum-containing medium was replaced with serum-free DMEM and incubated with the cells for 24 hours. Chlamydiae was administered at 2 μg / ml. Conditioned medium was collected from chlamydiae samples at 16 hours (when cells appeared healthy). The conditioned medium was rotated at 400g for 7 minutes and then concentrated approximately 20-fold using a Microcon-10kDa cutoff column (Millipore). Samples were combined 1:1 with 2x SDS sample buffer containing protease inhibitor, DTT, and 5 mM EDTA, boiled at 95°C for 10 min, and run on a 4-15% acrylamide Mini-Protean TGX gel. The samples were then transferred to a nitrocellulose membrane and incubated with a 1:1,000 dilution of anti-V5 primary antibody MAB15253 (Pierce) and a 1:10,000 dilution of 800 nm conjugated anti-mouse secondary antibody (Odyssey). Neonatal rat ventricular cardiomyocytes were infected with an adenovirus expressing untagged mouse FSTL1 at MOI 50. The culture medium was replaced with serum-free medium 24 hours post-infection. Infected NRVC and EMC cells were conditioned in serum-free DMEM / F12 penicillin / streptomycin medium for 24 hours. The medium was then conditioned with 1 μg / ml tunicamycin for 16 hours. Conditioned medium was vortexed at 400g for 7 minutes and then concentrated using a Microcon-10kDa cutoff column (Millipore). Samples were combined at a 1:1 ratio with 2x SDS sample buffer containing protease inhibitors, DTT, and 5mM EDTA, boiled at 95°C for 10 minutes, and run on an AnyKD Mini-Protean TGX gel. The gel was then transferred to a nitrocellulose membrane and incubated with a 1:500 dilution of anti-FSTL1 MAB1694 (R&D) primary antibody and a 1:10,000 dilution of an 800nm ​​conjugated anti-rat secondary antibody (Odyssey). Blocking and antibody incubation were performed in an Odyssey Blocker. Western blotting against recombinant FSTL1 (100ng each) was performed in the same manner.

[0181] Cardiac cell lineage markers: Cardiac cell lineage markers were achieved by intraperitoneal injection of 4-OH tamoxifen at a dose of 20 mg / kg / day into Myh6 cells with a C57BL6 background in 8-week-old infants. mERCremER :Rosa26 Z / EG mice18 The process lasted for two weeks, followed by a one-week break before harvesting cardiomyocytes. Figure 5 Small image p), or perform MI surgery and grafting. Four weeks after MI, collect animals for immunostaining ( Figure 5 Small image (qu).

[0182] result

[0183] In vivo, cardiomyocytes induced to enter the cell cycle by FSTL1 may originate from pre-existing myocytes (Myh6). + (The cells themselves) or are derived de novo from an ancestral population. To differentiate these possibilities, the cardiomyocyte-specific Myh6 promoter was used. 18 Tamoxifen-induced Cre can genetically mark pre-existing Myh6 under controlled conditions. + Cardiomyocytes and their fate was tracked after MI and FSTL1 transplantation. Figure 5 (small image p). Injection of Myh6 mERCremER :Rosa26 Z / EG 4-OH tamoxifen in mice effectively labels pre-existing cardiomyocytes with eGFP before MI (mitosis). Figure 5 (Small image q). Four weeks after grafting, eGFP... + pH3 + Cells are clearly visible in the infarcted area and the boundary zone. Figure 5 The small image (ru) indicates that the patch +FSTL1 acts on cells expressing Myh6 before LAD ligation and patch transplantation.

[0184] α-actin in the cycle + What is the origin of these cells? Adult cardiomyocytes generally have difficulty entering the cell cycle, and FSTL1 does not promote DNA replication or cell division in adult or neonatal rat ventricular cardiomyocytes in vitro. Figure 18 (Small image aj). Similarly, FSTL1 does not stimulate the proliferation or differentiation of primary cardiogenetic progenitor cells (Lin-, Sca1) from adult rat hearts into clonal expansions that can form cardiomyocytes upon reimplantation into adult hearts. + SP + ) 19 ( Figure 18 In contrast, increasing the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into α-actin... + mCM ESC ( Figure 6 In small figures a and d), increasing pH 3 + α-actin + The number of cells ( Figure 6Figures b and e) and localized Aurora B kinase in cleavage / intermediate regions ( Figure 6 Small image c, f), mCM ESC Cells responded to FSTL1 in a dose-dependent manner. This was observed in Myh6. + Lineage tracing results of cell proliferation in vivo after treatment with FSTL1 ( Figure 5 The small image (pu) combination suggests that Myh6, located proximal to the epicardium, has the ability to proliferate in response to epicardial FSTL1. + / α-actin + The presence of cells.

[0185] However, unusually, neither endogenous myocardial induction by FSTL1 expression nor direct transgenic overexpression of FSTL1 could activate regeneration. Figure 4 , Figure 10 Therefore, given that our previous experiments were all conducted using bacterially synthesized human FSTL1, it is particularly important to test whether cell-specific modifications of FSTL1 are involved. Figure 4 , Figure 5 , Figure 6 (Small figure af). FSTL1 is highly glycosylated in mammalian cells. Figure 6 Small image g), while the recombinant FSTL1 generated in bacteria is not like this ( Figure 6 Small figure h). Therefore, in apoptosis and proliferation assays, in mCM ESC The function of recombinant FSTL1 generated in bacterial (naked) and mammalian (glycosylated) cells was tested on cells. Human FSTL1 expressed in mammals protects mCM. ESC They are immune to H2O2-induced apoptosis, unlike bacterial-expressed FSTL1. Figure 6 (Figure i). Conversely, bacterial expression of human FSTL1 promotes mCM. ESC Proliferation, unlike human FSTL1 expressed in mammals ( Figure 6 Figures j and k show that these key functional differences are therefore associated with FSTL1 glycosylation status. The study also compared the levels of FSTL1 overexpression in neonatal rat ventricular cardiomyocytes (NRVCs) with those of NRVCs that did not produce detectable amounts of endogenous FSTL1. Figure 19 – Endogenous FSTL1 expressed in EMC-epicardial cells. Western blot analysis indicated a significant size difference between myocardial and epicardial forms of FSTL1, with the difference “disappearing” with tunicamycin treatment (an inhibitor of glycosylation). Figure 6 Figure i shows that FSTL1 undergoes post-transcriptional modification (glycosylation) in a cell-specific manner. Subsequently, in the proliferation assay, in mCM... ESCThe above methods were used to functionally test the production of these Fstl forms in cardiomyocytes and epicardial cells. Conditioned medium from myocardial cells derived from NRVCs infected with unlabeled FSTL1 adenovirus showed efficacy against mCM. ESC Proliferation was not affected. In contrast, EMC-conditioned medium significantly promoted mCM proliferation in a low-glycosylation FSTL1-dependent manner. ESC proliferation( Figure 6 The small figures (m, n) show a degree comparable to that of bacterially synthesized hFSTL1, demonstrating a link between cell-specific posttranscriptional modifications and changes in the functional state of FSTL1.

[0186] Example 7: Epicardial FSTL1 delivery activates cardiac regeneration in a preclinical pig model.

[0187] This embodiment shows that the recovery effect of the patch +FSTL1 delivery in the epicardium appears to be evolutionarily conserved.

[0188] Materials and methods

[0189] Application of the patch in a porcine model of ischemia-reperfusion: The porcine study was conducted as follows in Yorkshire pigs (45 days old) by occlusion of the left anterior descending artery (LAD) via percutaneous coronary angioplasty to dilate the catheter. Complete reperfusion was performed 90 minutes after occlusion to simulate a clinical MI disease model. One week after MI, a left thoracotomy was performed and the patch was sutured to the infarct. Animal groups included: sham control, untreated I / R treatment (n=3), I / R treated with the patch alone (I / R + patch, n=1), and I / R treated with a patch loaded with FSTL1 (I / R + patch + FSTL1, n=2). EdU delivery: 250 mg / wk EdU was perfused into the circulation using an osmotic mini-pump during the 4-week study period (weeks 1 to 5 after I / R).

[0190] Statistical Analysis: The number of samples (n) used in this and all other examples is documented in the text and shown in the figures. All in vitro experiments were performed independently at least twice. Gene expression experiments were performed independently three times, while EdU proliferation assays and cell size measurements were performed independently more than ten times. No sample size was predetermined, and retrospective analysis using Gpower 3.1 for significantly different results in most in vitro studies yielded a power >0.8. Sample size was assessed for animal studies. Animals that did not survive for at least 4 weeks after surgery were excluded from functional and histological studies. No randomization was applied. Group assignments were not known between animal surgery and outcome analysis in mouse myocardial infarction experiments. Values ​​presented are expressed as mean ± SEM. The principle of using mean ± SEM instead of SD is that SEM quantifies the uncertainty of mean assessment, while SD indicates the deviation of data from the mean. In other words, SEM provides an assessment of the reported mean value, while SD gives a concept of the variability of a single observation. Statistical significance was tested using one-way ANOVA and the Student's T test (P < 0.05). Survival curves were generated using PRISM (GraphPad) and the time series (Mantel-Cox) test was used to examine significant differences in mouse survival under different conditions.

[0191] result

[0192] Engineered epicardial delivery of FSTL1 was evaluated in a porcine model of myocardial ischemia-reperfusion injury. Prior to infarction, ejection fraction (EF) was ~50%, as determined by magnetic resonance imaging (MRI). One week after I / R, EF% decreased to ~30%, after which patch + FSTL1 was applied to the epicardium of the damaged tissue. Pigs treated with patch + FSTL1 recovered contractility by week 2 of treatment (week 3 of the experiment), achieving approximately 40% EF, and remained stable for up to two weeks. The longest duration analyzed ( Figure 7 (Insets a, b). This contrasts with the steady decline in cardiac function in untreated animals and animals treated with individual patches. Figure 7 Inset b). Pigs treated with patch +FSTL1 showed the lowest fibrotic tissue formation (scar size) among all treatments, including patch-only animals (see representative MRI images). Figure 7 (Insets c and d)). Analysis at week 4 post-graft transplantation (week 5 of the experiment) showed that the graft had integrated into the host tissue and exhibited limited fibrosis. Figure 7 (small figure e) and vascular smooth muscle cells in the boundary zone of the ischemic area ( Figure 7 Small image fh) and cardiomyocytes ( Figure 7EdU markers were also detected in the border zone of the heart treated with +FSTL1, indicating the presence of localized Aurora B kinase (an indicator of cytokinesis). Figure 7 (Small figure n). Thus, the restorative effect of FSTL1 delivery in the epicardium appears to be evolutionarily conserved.

[0193] Example 8: Application of hypoglycosylated FSTL1 does not activate Akt-1 signaling activity.

[0194] This example demonstrates processing mCM using FSTL1. ESC It does not lead to the activation of Akt-1.

[0195] As described above, implement mCM processing using FSTL1. ESC And the Western blot of phosphate-Akt.

[0196] The results are shown in Figure 20 It describes the mCM after FSTL1 processing. ESC Detection of phosphate-Akt and PCNA in cells. Western blots of phosphate-Akt (Ser473 and Thr308, both involved in survival responses in cardiomyocytes) and PCNA (proliferation markers) after treatment with FSTL1 at 10 ng / ml and 50 ng / ml for 1 hour and 24 hours showed no significant changes in phosphate-Akt or PCNA after FSTL1 treatment.

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Claims

1. Use of a hypoglycosylated follicle-like 1 (FSTL1) peptide in the preparation of a medicament for use in a method for repairing cardiac tissue after injury in a subject with a need, the method comprising contacting the cardiac tissue with the hypoglycosylated follicle-like 1 (FSTL1) peptide, wherein the hypoglycosylated FSTL1 peptide comprises one or more asparagine residues at positions 144, 175, 180, and 223 of the FSTL1 amino acid sequence SEQ ID NO: 1, with one or more substitutions of one or more glutamine residues.

2. Use according to claim 1, wherein, This low-glycosylated FSTL1 polypeptide contains an asparagine residue at position 180 of the FSTL1 amino acid sequence SEQ ID NO: 1 replaced with a glutamine residue.

3. Use according to claim 1, wherein, (A) The damage is ischemia-reperfusion cardiac injury, due to ischemic heart disease, and / or due to cardiac dysplasia, or (B) The damage is myocardial infarction and / or the heart contains scar tissue.

4. Use according to claim 1, wherein, Repairing heart tissue involves increasing the number of cardiomyocytes within that heart tissue.

5. The use according to claim 4, wherein, The number of cardiomyocytes was at least three times higher than that in cardiac tissue that had not been exposed to low-glycosylated follicle-staphylin-like 1 (FSTL1) peptide after injury.

6. Use according to claim 1, wherein, Repairing cardiac tissue involves the restoration of elevated levels of damaged cardiac tissue, including cardiomyocytes and / or the cardiac vascular system.

7. Use according to claim 6, wherein, The number of cardiomyocytes was at least three times higher than that in cardiac tissue that had not been exposed to low-glycosylated follicle-staphylin-like 1 (FSTL1) peptide after injury.

8. Use according to claim 1, wherein, The repair of cardiac tissue includes an improved percentage of shortening in cardiac tissue compared to the percentage of shortening in cardiac tissue that was not exposed to low-glycosylated follicle-like 1 (FSTL1) peptide after damage.

9. Use according to claim 1, wherein, Repairing cardiac tissue involves reducing the amount of fibrosis by at least 2% in the same heart before damage by contact with a hypoglycosylated follicle-stimulating hormone-like 1 (FSTL1) peptide, delivery, or genome editing treatment.

10. The use according to claim 1, wherein, Repairing cardiac tissue involves increasing the perfusion area by at least 2% compared to the amount of perfusion area in the same heart before damage, following contact with a hypoglycated follicle-stimulating hormone-like 1 (FSTL1) peptide or genome editing treatment.

11. Use according to claim 1, wherein, Repairing cardiac tissue involves an increased amount of cardiomyocyte cytoplasmic division compared to cardiac tissue that has not been exposed to low-glycosylated follicle-staphylin-like 1 (FSTL1) peptide after injury.

12. Use according to claim 11, wherein, The increase in the amount of cytoplasmic division of cardiomyocytes was measured by the expression of Aurora B kinase.

13. Use according to claim 1, wherein, Repairing heart tissue involves reducing cardiomyocyte apoptosis.

14. The use according to claim 1, wherein, The method resulted in an increase in the level of transcripts encoding heart-specific contractile proteins in cardiomyocytes.

15. The use according to claim 14, wherein (i) the method results in a 2-fold increase in the level of a transcript encoding a heart-specific contractile protein in cardiomyocytes, and / or (ii) the heart-specific contractile protein is selected from the group consisting of myh6, mlc2v, and mlc2a.

16. The use according to claim 1, wherein, The method results in rhythmic contractile Ca 2+ tropomyosin + cell multiplication.

17. Use according to claim 1, wherein, Immediately after the damage, the cardiac tissue was contacted with the hypoglycosylated follicle-like 1 (FSTL1) peptide.

18. The use according to claim 1, wherein, Contact the heart tissue with the hypoglycosylated follicle-like 1 (FSTL1) peptide at any time following the damage.

19. Use according to claim 1, wherein, The method reduces cardiac events and hospitalizations.

20. The use according to claim 1, wherein, The method reduces fibrosis in the heart tissue following the damage.

21. The use according to claim 3, wherein, The method resulted in increased angiogenesis in the damaged areas of the heart tissue.

22. The use according to claim 21, wherein, The increased angiogenesis was measured by the expression of von Willebrand factor (vWF) or smooth muscle actin in vascular cells.

23. The use according to claim 1, wherein, The method induces cardiomyocytes to enter the cell cycle.

24. The use according to claim 23, wherein, (A) The cardiomyocytes entering the cell cycle are assessed by the expression of phospho-histone H3, and / or (B) the method results in a cardiomyocytes entering the cell cycle at least twice as much as the amount of cardiomyocytes entering the cell cycle in cardiac tissue that has not been exposed to low-glycosylated follicle-staphylin-like 1 (FSTL1) peptide after injury.

25. The use according to claim 1, wherein, (A) The hypoglycated FSTL1 polypeptide is synthesized in prokaryotic cells, or (B) The hypoglycated FSTL1 polypeptide is synthesized in eukaryotic cells treated with an inhibitor of glycosylation.

26. The use according to claim 1, wherein, The low-glycosylated FSTL1 peptide is delivered into the damaged myocardial tissue via a collagen patch.

27. The use according to claim 1, wherein, The hypoglycosylated FSTL1 peptide was directly injected into the damaged myocardial tissue.

28. The use according to claim 1, wherein, The system delivers the low-glycosylated FSTL1 peptide.

29. The use according to claim 1, wherein, The low-glycosylated FSTL1 peptide is delivered into the heart.

30. The use according to claim 29, wherein, The contents are delivered into the heart via a catheter.

31. The use according to claim 1, wherein, The low-glycosylated FSTL1 peptide is delivered to the epicardium via catheter.

32. The use according to claim 1, wherein, The low-glycosylated FSTL1 peptide was delivered using a drug-eluting stent catheter.

33. The use according to claim 1, wherein, The low-glycosylated FSTL1 peptide is embedded or seeded into a three-dimensional collagen patch.

34. The use according to claim 1, wherein, Contact with the heart tissue via one or more of the following methods: from the epicardium, the interior of the heart, and / or by direct injection into the myocardium.

35. The use according to claim 1, wherein, The low-glycosylated FSTL1 peptide was expressed in the heart using a modified dRNA (modRNA).

36. The use according to claim 1, wherein, This hypoglycosylated FSTL1 peptide was expressed through genome editing.

37. A sterile pharmaceutical composition comprising a hypoglycosylated follicle-like 1 (FSTL1) polypeptide and one or more pharmaceutically acceptable excipients, wherein the hypoglycosylated FSTL1 polypeptide comprises one or more asparagine residues at positions 144, 175, 180 and 223 of the FSTL1 amino acid sequence SEQ ID NO: 1, replaced by one or more glutamine residues.

38. The sterile pharmaceutical composition according to claim 37, wherein, This low-glycosylated FSTL1 polypeptide contains an asparagine residue at position 180 of the FSTL1 amino acid sequence SEQ ID NO: 1 replaced with a glutamine residue.

39. The sterile pharmaceutical composition of claim 37, further comprising an inhibitor of FSTL1 glycosylation.

40. The sterile composition according to claim 39, wherein, The inhibitor of FSTL1 glycosylation includes tunicamycin.

41. The sterile pharmaceutical composition according to claim 37, wherein, The hypoglycosylated FSTL1 polypeptide is synthesized in prokaryotic cells.

42. The sterile pharmaceutical composition according to claim 41, wherein, The prokaryotic cells mentioned are bacterial cells.

43. The sterile pharmaceutical composition according to claim 37, wherein, The hypoglycosylated FSTL1 peptide was synthesized in eukaryotic cells treated with an inhibitor of glycosylation.

44. The sterile pharmaceutical composition according to claim 43, wherein, The inhibitor of glycosylation is tunicamycin.

45. The sterile pharmaceutical composition according to claim 37, wherein, (i) the composition is formulated for direct injection into damaged cardiac tissue, or (ii) the composition is formulated for systemic administration.

46. The sterile pharmaceutical composition according to claim 37, wherein, The hypoglycosylated FSTL1 peptide is synthesized in cells containing modified RNA (modRNA), or the hypoglycosylated FSTL1 peptide is expressed through genome editing.

47. The sterile pharmaceutical composition according to claim 37, wherein, The low-glycosylated FSTL1 peptide is embedded or seeded into a three-dimensional (3D) collagen patch.

48. The sterile pharmaceutical composition according to claim 47, wherein, The 3D collagen patch has an elastic modulus of 12 ± 4 kPa.

49. A kit comprising (i) a hypoglycosylated follicle-staphylin-like 1 (FSTL1) polypeptide, wherein the hypoglycosylated FSTL1 polypeptide comprises one or more asparagine residues at positions 144, 175, 180 and 223 of the FSTL1 amino acid sequence SEQ ID NO: 1, with one or more substitutions of one or more glutamine residues; and (ii) one or more pharmaceutically acceptable excipients.

50. The kit according to claim 49, wherein, This low-glycosylated FSTL1 polypeptide contains an asparagine residue at position 180 of the FSTL1 amino acid sequence SEQ ID NO: 1 replaced with a glutamine residue.

51. A three-dimensional (3D) collagen patch infused or seeded with a recombinant low-glycosylated follicle-staphylin-like 1 (FSTL1) polypeptide, wherein the low-glycosylated FSTL1 polypeptide comprises one or more asparagine residues at positions 144, 175, 180 and 223 of the FSTL1 amino acid sequence SEQ ID NO: 1, replaced by one or more glutamine residues.

52. The three-dimensional (3D) collagen patch according to claim 51, wherein, This low-glycosylated FSTL1 polypeptide contains an asparagine residue at position 180 of the FSTL1 amino acid sequence SEQ ID NO: 1 replaced with a glutamine residue.

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