Application of VEGF-B in psoriasis

Abstract

The invention discloses an application of VEGF-B (vascular endothelial growth factor-B) in psoriasis. Researches show that up-regulation of VEGF-B expression in a psoriasis animal model can significantly reduce levels of inflammatory factors such as IL-1beta, TNF-a and the like, and reduce dermal immune cell infiltration; meanwhile, excessive proliferation of keratinocytes is inhibited, normal differentiation of epidermis is promoted, and therefore scale formation and skin thickening are reversed. Different from the existing anti-VEGF-A strategy, the invention proves that the enhancement of the VEGF-B signal can resist inflammation and repair, and a new therapeutic target is provided for psoriasis. According to the invention, the limitation of the traditional anti-VEGF-A therapy is broken through, and the clinical value of VEGF family members is expanded.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of VEGF-B in psoriasis. Background Technology

[0002] Psoriasis is an immune-mediated skin disease characterized by abnormal proliferation of keratinocytes and chronic recurrent inflammation. Severe types often affect the scalp, extensor surfaces of the limbs, and trunk, and are closely associated with multi-system comorbidities such as psoriatic arthritis and cardiovascular metabolic syndrome.

[0003] Current clinical treatments for psoriasis typically include: topical corticosteroids and vitamin D3 analogs, calcineurin inhibitors, and keratolytic agents; phototherapy such as UVB and PUVA; traditional systemic drugs such as methotrexate, cyclosporine, and acitretin; and various biologics and small-molecule oral medications (such as PDE4 inhibitors) targeting cytokines such as TNF-α, IL-23, and IL-17. Traditional medications have short-lived effects, are prone to relapse, and have various adverse reactions. Although new-generation biologics have significantly improved the clearance rate of moderate to severe psoriasis, current treatments still have significant limitations: long-term use of topical corticosteroids can lead to adverse reactions such as skin atrophy; systemic immunosuppressants pose risks of liver and kidney toxicity and infection; biologics are expensive, require long-term use, and some patients experience initial or secondary treatment resistance, leading to a high risk of relapse upon discontinuation.

[0004] Currently, the pathogenesis and key targets of psoriasis remain unclear. Macrophages play a crucial regulatory role in the development and progression of psoriasis. Skin-resident Langerhans cells and dermal macrophages can sense antimicrobial peptides, their own DNA / RNA, and damage-associated molecular patterns (DAMPs) released by keratinocytes, and become polarized to a pro-inflammatory M1 phenotype, producing cytokines such as IL-23, TNF-α, and IL-1β. This drives the Th17 / Th1 response and amplifies the inflammatory cascade, promoting the formation of typical psoriasis-like lesions. Conversely, macrophages polarized to the M2 phenotype can secrete IL-10 and TGF-β, participating in the negative regulation of inflammation and tissue repair. In mouse psoriasis models such as IMQ, selective depletion or functional regulation of macrophages significantly affects the severity of lesions, suggesting that macrophages are not only key effector cells in psoriasis but also potential important therapeutic targets.

[0005] Currently approved treatments mainly focus on a few inflammatory cytokine targets, and specific regulatory methods for key cell populations such as macrophages remain scarce. Furthermore, some patients do not respond well to existing targeted drugs or experience unstable long-term efficacy. Therefore, there is an urgent need to explore new key therapeutic targets and develop safer, more effective, and long-lasting treatment strategies from a novel perspective of regulating the psoriatic skin lesion microenvironment and macrophage function.

[0006] In the pathological process of psoriasis, members of the vascular endothelial growth factor (VEGF) family and their receptor signaling pathways are believed to play an important regulatory role. However, different members have significantly different roles in psoriasis, and there are even functional conflicts, which poses a challenge to targeted therapy against specific VEGF family members.

[0007] VEGF-A is a key promoter of angiogenesis and inflammatory response in psoriatic lesions. Studies have shown that VEGF-A expression is significantly upregulated in psoriatic lesions. By activating the VEGFR-2 signaling pathway, it not only mediates abnormal proliferation of dermal microvessels and increased vascular permeability but also participates in inflammatory cell infiltration and excessive proliferation of keratinocytes, thereby exacerbating the pathological progression of psoriasis. Animal experiments have further confirmed that overexpression of VEGF-A can induce psoriatic dermatitis in mice. Based on this mechanism, existing treatments targeting VEGF signaling are mainly designed around the VEGF-A / VEGFR-2 pathway, such as VEGF Trap or anti-VEGF-A antibodies, aiming to inhibit angiogenesis and alleviate epidermal hyperplasia (see EP1947118B1). However, the improvement in lesions caused by VEGF-A inhibitors often relapses upon treatment discontinuation, suggesting that simply blocking angiogenesis is insufficient for long-term control of inflammation.

[0008] Unlike VEGF-A, VEGF-B, while belonging to the same family, exhibits fundamentally different biological characteristics and functional orientations. VEGF-B selectively binds to VEGFR-1 and neurociliary protein-1 (NRP-1) but does not activate VEGFR-2, thus lacking the strong angiogenic capacity of VEGF-A. Existing research suggests that VEGF-B is more inclined to participate in processes such as metabolic regulation and cell protection, for example, demonstrating anti-apoptotic and tissue-protective potential in myocardial ischemia-reperfusion injury models. However, the effects of VEGF-B differ under different pathological conditions: in autoimmune arthritis models, Vegfb Gene knockout can alleviate joint inflammation and pathological angiogenesis, suggesting that it may promote pathological angiogenesis and inflammation; however, in terms of T cell regulation, autocrine VEGF-B can support T cell activation by maintaining mitochondrial homeostasis, thereby enhancing anti-infection and anti-tumor immune responses. This dual effect makes the function of VEGF-B in complex inflammatory diseases such as psoriasis difficult to predict. Currently, no studies have revealed a direct link between VEGF-B and psoriasis, nor have any technical solutions for intervening in psoriasis by specifically regulating VEGF-B been proposed. Summary of the Invention

[0009] This invention is the first to discover that upregulating VEGF-B can alleviate the inflammatory response of psoriasis and improve epidermal thickening and scaling caused by abnormal proliferation of keratinocytes. These results suggest that VEGF-B may serve as a potential target for psoriasis intervention.

[0010] Therefore, the first object of the present invention is to provide the use of VEGF-B in the preparation of medicaments for psoriasis.

[0011] Preferably, the drug is used to reduce skin inflammation and / or improve epidermal thickening and scaling caused by abnormal proliferation of keratinocytes.

[0012] Preferably, the VEGF-B is selected from: (a) VEGF-B protein or its functional fragments, variants or derivatives; (b) The nucleic acid molecule encoding the protein described in (a); and (c) An expression vector containing the nucleic acid molecule described in (b).

[0013] Preferably, the expression vector is a viral vector or a non-viral vector.

[0014] Preferably, the viral vector includes an adeno-associated virus vector.

[0015] Preferably, the drug is an injectable dosage form, a topical dosage form, or an oral dosage form.

[0016] Preferably, the injectable dosage form is selected from subcutaneous injection, intramuscular injection, intravenous injection, or intradermal injection.

[0017] Preferably, the topical dosage form is selected from gels, sprays, patches, or lotions.

[0018] Preferably, the oral dosage form is selected from tablets, capsules, granules, powders, or solutions.

[0019] Preferably, the psoriasis includes plaque psoriasis, guttate psoriasis, pustular psoriasis, or erythrodermic psoriasis.

[0020] The beneficial effects of this invention are: This invention reveals for the first time that vascular endothelial growth factor B (VEGF-B) can serve as a potential target for psoriasis intervention, breaking through the technical inertia of the field that has long focused on VEGF-A targeting strategies, filling the gap in the existing technology's understanding of the function of VEGF-B in the pathogenesis of psoriasis, and providing an innovative intervention method for psoriasis treatment that is different from the traditional anti-angiogenic pathway. Attached Figure Description

[0021] Figure 1 Phenotypic and histological changes of the skin on the back of a mouse model of psoriasis on day 6. Vegf-bExpression levels. (A) is a macroscopic photograph of the back skin of mice in the psoriasis model group and the normal control group on day 6 after modeling. (B) is a photomicrograph of H&E staining of the back skin tissue of the two groups of mice (scale bar 200 μm). (C) is the expression level of the psoriasis skin tissue of the two groups of mice. Vegf-b mRNA relative expression levels qRT-PCR detection graph. All data are presented as mean ± standard error (SE). ***: p < 0.001.

[0022] Figure 2 Myeloid cell specific Vegf-b Effects of knockout on the phenotype of psoriatic mice. Where (A) represents mice derived from... Vegf-b BMDMs of cKO mice and littermate WT mice Vegf-b (a) Graph showing relative mRNA expression levels detected by qRT-PCR. (b) Comparison of macroscopic images of the dorsal skin of the two groups of mice after modeling. (c) Microscopic images of F4 / 80 immunofluorescence staining of the dorsal skin tissue of the two groups of mice after modeling (scale bar 200 μm). (d) Statistical graph of the number of F4 / 80 positive cells. All data are presented as mean ± standard error (SE). *: p < 0.05, **: p < 0.01.

[0023] Figure 3 The effects of skin overexpression of VEGF-B on the phenotype of psoriatic mice are shown in the following figures: (A) Experimental protocol for the effect of skin overexpression of VEGF-B on psoriasis. (B) qRT-PCR detection of relative VEGF-B mRNA expression levels in skin tissues of mice in the VEGF-B overexpression group and the GFP control group. (C) Macroscopic photographs of the dorsal skin of the two groups of mice. (D) H&E staining micrographs of the dorsal skin tissues of the two groups of mice (scale bar 200 μm). (E) Quantitative statistical graph of epidermal thickness. (F) F4 / 80 immunofluorescence staining micrographs of the skin tissues of the two groups of mice (scale bar 200 μm). (G) Statistical graph of the number of F4 / 80 positive cells. (H) Representative Western blotting images of IL-1β, TNF-α, and internal reference β-actin in the skin tissues of the two groups of mice. (I) Statistical graph of relative IL-1β protein expression. (J) Statistical graph of relative TNF-α protein expression. All data are presented as mean ± standard error (SE). *: p < 0.05, **: p < 0.01, ***: p < 0.001. Detailed Implementation

[0024] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents and materials used are commercially available unless otherwise specified.

[0026] Example 1 I. Materials (1) Mice Wild-type C57BL / 6J and LysM-cre Vegf-b loxP / loxP All mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0027] Vegf-b loxP / loxP Crossing mice with LysM-cre transgenic mice produces mice that specifically knock out myeloid cells. Vegf-b ( Vegf-b cKO mice, the Vegf-b The principle behind constructing cKO mice is: ... Vegf-b flanking insertion of genes loxP The "floxed mouse" was crossed with the "transgenic mouse carrying the Lysozyme M (LysM) promoter to drive Cre recombinase"; since the LysM promoter only activates Cre in premyeloid and mature myeloid cells (macrophages, granulocytes, etc.), the Cre-loxP system specifically excises Cre in these cells. Vegf-b The coding sequence is obtained from Cre, while other tissues retain gene integrity due to the lack of Cre activity, thus acquiring conditional myeloid cell lineage. Vegf-b Knock out rats ( Vegf-b cKO).

[0028] (2) Antibody Table 1 Antibodies .

[0029] (3) qPCR primer sequences Table 2 Primers .

[0030] (4) AAV virus AAV-VEGF-B and its control AAV-GFP were purchased from Vigene Biotechnology (Shandong, China).

[0031] II. Methods 1. Establishment of an imiquimod (IMQ)-induced mouse model of psoriasis 1) The hair on the back (area 2 cm × 1.5 cm) of 8-week-old C57BL / 6J or transgenic mice was removed with an electric shaver and depilatory cream one day before modeling.

[0032] 2) On the second day, gently wipe and clean the skin at the modeling site to ensure no skin damage. The model was treated with 62.5mg of 5% IMQ applied to the hairless skin area for 5 consecutive days, while the control group was treated with the same amount of Vaseline. After application, mice were placed individually in cages for 6 hours to ensure full absorption of the drug and to prevent mutual licking.

[0033] 3) On day 6 of modeling, assess the pathological phenotype of the mice and collect samples.

[0034] 2. Intradermal injection in mice The fur on the back of the mice was removed using an electric shaver and depilatory cream. Before injection, the mice were fully anesthetized, and the skin was disinfected with 75% ethanol. Using an insulin injector, the needle was inserted at a depth of 30 mm. 。 Gently insert the horn between the epidermis and dermis, and slowly inject 50 μL of the drug solution. Successful injection is indicated by the formation of a noticeable bulge on the skin surface with a diameter of 3-5 mm after injection, and a prolonged duration of this bulge. After the drug is fully absorbed, place the mouse on a heating pad to awaken it.

[0035] 3. Extraction and culture of mouse bone marrow-derived macrophages (BMDMs) Mice were euthanized by dislocation after anesthesia. They were then immersed in a beaker containing sufficient 75% ethanol for 5 minutes for sterilization. The tibia and femur were isolated on a sterile operating table. Bone marrow was flushed from the tibia and femur using pre-cooled culture medium, repeatedly rinsing three times until no obvious red color was visible inside the leg bones. The culture medium containing bone marrow cells was repeatedly pipetted to disperse cell clumps, then filtered through a 70 μm cell filter and transferred to 15 mL centrifuge tubes. The tubes were centrifuged at 1500 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in erythrocyte lysis buffer and allowed to stand for 5 minutes. The cells were then centrifuged again at 1500 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in cold, prepared bone marrow macrophage induction medium and plated (2 × 10⁻⁶ cells / mL). 6 (Live cells / plate). Replace half of the bone marrow macrophage induction medium on the third day of culture, replace all the medium on the fifth day, and it can be used for subsequent experiments on the seventh day.

[0036] 4. RNA isolation and extraction, cDNA synthesis and quantitative real-time PCR (qRT-PCR) BMDMs or skin powder ground in liquid nitrogen were collected. Total RNA was isolated from cells using TRIzol reagent (TIANGEN) and synthesized into cDNA using the DNase-containing FastKing RT kit (TIANGEN). qRT-PCR was performed using a SYBR Green (ROCHE) and ABI QuantStudio 6 Flex instrument (Life Technologies). Results were normalized to β-actin transcripts. The relative folding changes of gene expression were calculated using the delta-delta Ct method.

[0037] 5. Western blot experiment 1) Preparing liquids: ①10×Running Buffer: Add 144 g glycine, 10 g SDS powder, and 30.3 g Tris powder sequentially, using ddH2O as the solvent, and bring the volume to 1 L to prepare a storage solution. Dilute to 1×Running Buffer to prepare the electrophoresis working solution for use. ②5×SDS Loading Buffer: Add 4 g of SDS powder, 20 mg of bromophenol blue, 3.085 g of DTT, 10 mL of Tris-HCl (1M pH 6.8), and 20 mL of glycerol in sequence, and use ddH2O as a solvent to make up to 40 mL. ③ 10× Transfer Buffer: Add 30.3 g Tris powder and 144 g glycine sequentially, using ddH2O as the solvent, and bring the volume to 1 L to prepare a storage solution. Dilute with 10× Transfer Buffer 100 mL + methanol 200 mL + ddH2O 700 mL to prepare 1× Transfer Buffer as the working solution for use. 2) Preparation of separating gel and stacking gel: ① Prepare 10% separating gel (10 mL): Table 3 shows the preparation of 10% separating gel. .

[0038] ②Prepare a 5% concentrate (5 mL): Table 4 shows the preparation of 5% concentrated gel. .

[0039] 3) Protein gel electrophoresis: Assemble the prepared gel and add the newly prepared electrophoresis solution to the tank. Check for leakage. Remove the comb and add the marker and sample to the gel wells in sequence. Add electrophoresis solution to the electrophoresis tank and set the program to 80 V for 30 min constant voltage electrophoresis. Then switch to 120 V for 1 h constant voltage electrophoresis. 4) Transfer: First, activate the PVDF membrane with methanol, and remove the gel from step 3). Assemble the transfer clamps in a "sandwich" structure, taking care to avoid leaving air bubbles during assembly. Assemble the transfer apparatus, add the transfer working solution, set the program to a constant current of 250 mA for 2 h, and place it on ice for transfer. 5) Blocking: Take out the membrane after the transfer is completed, add 1×TBST and place it on a shaker at room temperature for 5 min to clean it, discard it, add 5% skim milk as the blocking solution and place it on a shaker at room temperature for blocking for 1 h. 6) Primary antibody incubation: Discard the blocking solution, add 1×TBST and place on a shaker at room temperature to wash until the blocking solution is clean. Discard the liquid, add the primary antibody prepared with 5% BSA as antibody dilution solution, and incubate overnight on a shaker in a 4°C cold storage. 7) Membrane washing: Wash the membrane three times with 1×TBST, 10 min each time; 8) Secondary antibody incubation: Discard the solution, add secondary antibody prepared with 5% skim milk at a ratio of 1:5000, and incubate on a shaker at room temperature for 1 h; 9) Same as step 7). 10) Exposure and development: Prepare the developer (A solution: B solution = 1:1 ratio, prepare and use immediately). Place the film on the plate, add developer to the film, shake well, expose and develop on the machine, and save the results.

[0040] 6. Immunofluorescence staining Frozen sections of skin were incubated for 15 minutes in 0.5% Triton X-100 (Sigma, X100) prepared in 1 × PBS, then blocked with 5% normal goat serum for 1 hour, and incubated overnight at 4°C with primary antibody. After washing three times with 1 × PBS, sections were incubated with secondary antibody at room temperature for 1 hour, followed by DAPI incubation for 5 minutes for nuclear staining (Sigma, D9542). Imaging was performed using an AX10 imager Z2 (Zeiss) microscope, and analysis was conducted using ZEN 2012 (Zeiss) and ImageJ.

[0041] 7. H&E staining Mouse skin sections were dewaxed as follows: xylene I (SCRC, 10023418) for 20 minutes, xylene II (SCRC, 10023418) for 20 minutes, 100% ethanol I (SCRC, 100092683) for 5 minutes, 100% ethanol II (SCRC, 100092683) for 5 minutes, and 75% ethanol for 5 minutes. Sections were stained with hematoxylin solution (Servicebio, G1003) for 3-5 minutes, rinsed with tap water, then stained with hematoxylin differentiation solution (Servicebio, G1003), and rinsed with tap water. Sections were treated with hematoxylin-Scott Tap blue (Servicebio, G1003) and rinsed with tap water. Sections were treated with 85% ethanol for 5 minutes, 95% ethanol for 5 minutes, and then eosin dye (Servicebio, G1003) for 5 minutes. After dehydration, the sections were sealed with neutral adhesive (SCRC, 10004160). Images were taken using an AX10 imager Z2 (Zeiss) microscope.

[0042] III. Results 1. Vegf-b Downregulated expression in the skin of psoriatic mice A mouse model of psoriasis was established by applying 5% imiquimod (IMQ) to the skin of C57BL / 6J mice. Continuous application of IMQ for 5 days induced severe skin lesions in mice, including erythema and silvery-white flaky scales. Figure 1 A in the text). H&E staining results showed that compared with normal mice (CTL), the epidermis of the model mice was significantly thickened, indicating acanthosis caused by excessive proliferation of keratinocytes, accompanied by parakeratosis and extension of epidermal ridges, indicating the successful establishment of the psoriasis model. Figure 1 (B) qRT-PCR results showed that compared with normal mice (CTL), Vegf-b The expression was significantly downregulated in the skin of psoriasis model mice. Figure 1 (C in the middle).

[0043] 2. Myeloid cell-specific knockout Vegf-b This exacerbated the psoriasis phenotype. A specific knockout assay was established in myeloid cells using the Cre-loxP system. Vegf-b ( Vegf-b cKO mice, and in littermates wild-type (WT) and Vegf-b A psoriasis model was established in cKO mice. qRT-PCR results showed... Vegf-b In origin Vegf-bThe expression of deletion was found in the macrophages (BMDMs) of cKO mice. Figure 2 (A) Compared to littermate WT mice, Vegf-b cKO mice exhibit more severe pathological features of psoriasis, primarily characterized by more silvery-white, layered scales, presenting a cracked appearance. Figure 2 (B in the text). Immunofluorescence staining results showed that, compared to littermate WT mice, Vegf-b cKO mice had more F4 / 80 subcutaneous infiltration. + macrophages ( Figure 2 (C and D in the text).

[0044] 3. Overexpression of VEGF-B in mouse skin can significantly alleviate the psoriasis phenotype. VEGF-B was overexpressed in the skin of a mouse model of psoriasis via intradermal injection of AAV. Figure 3 The qRT-PCR results showed that VEGF-B was successfully overexpressed in the skin of mice in the AAV-VEGF-B group. Figure 3 (B in the text). Six days after modeling, back skin samples were collected for relevant indicator testing. Macroscopic observation showed that, compared to the AAV-GFP control group, VEGF-B overexpression significantly alleviated the pathological phenotype of psoriasis, including significantly inhibiting the appearance of erythema and scaling. Figure 3 C in the text). H&E staining results showed that VEGF-B overexpression alleviated acanthosis caused by excessive proliferation of keratinocytes (C). Figure 3 (D and E in the text). Immunofluorescence staining results showed that VEGF-B overexpression significantly reduced F4 / 80. + Macrophage infiltration in subcutaneous tissue ( Figure 3 F and G in the figure). Western blotting results showed that VEGF-B overexpression significantly downregulated the expression of inflammatory factors IL-1β and TNF-α (F and G in the figure). Figure 3 (H, I, and J in the text).

[0045] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of VEGF-B in the preparation of drugs for psoriasis.

2. The use according to claim 1, characterized in that, The drug is used to reduce skin inflammation and / or improve epidermal thickening and scaling caused by abnormal proliferation of keratinocytes.

3. The use according to claim 1, characterized in that, The VEGF-B is selected from: (a) VEGF-B protein or its functional fragments, variants or derivatives; (b) The nucleic acid molecule encoding the protein described in (a); and (c) An expression vector containing the nucleic acid molecule described in (b).

4. The use according to claim 3, characterized in that, The expression vector can be a viral vector or a non-viral vector.

5. The use according to claim 4, characterized in that, The viral vector includes adeno-associated virus vectors.

6. The use according to claim 1, characterized in that, The drug is in the form of an injectable dosage form, a topical dosage form, or an oral dosage form.

7. The use according to claim 6, characterized in that, The injectable dosage form is selected from subcutaneous injection, intramuscular injection, intravenous injection, or intradermal injection.

8. The use according to claim 6, characterized in that, The topical dosage form is selected from gels, sprays, patches, or lotions.

9. The use according to claim 6, characterized in that, The oral dosage form is selected from tablets, capsules, granules, powders, or solutions.

10. The use according to claim 1, characterized in that, The psoriasis includes plaque psoriasis, guttate psoriasis, pustular psoriasis, or erythrodermic psoriasis.

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