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Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

39232048
71350
10.1038/s41598-024-71350-1
Article
A water-soluble caveolin-1 peptide inhibits psoriasis-like skin inflammation by suppressing cytokine production and angiogenesis
Asai Chika 1
Takamura Naoko 1
Watanabe Tomoya 1
Asami Miho 1
Ikeda Noriko 1
Reese Charles F. 2
Hoffman Stanley 2
Yamaguchi Yukie yui1783@yokohama-cu.ac.jp

1
1 https://ror.org/0135d1r83 grid.268441.d 0000 0001 1033 6139 Department of Environmental Immuno-Dermatology, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-Ku, Yokohama, 236-0004 Japan
2 https://ror.org/012jban78 grid.259828.c 0000 0001 2189 3475 Division of Rheumatology, Department of Medicine, Medical University of South Carolina, Charleston, SC USA
4 9 2024
4 9 2024
2024
14 205536 4 2024
27 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
The plasma membrane protein caveolin-1 (CAV-1) regulates signaling by inhibiting a wide range of kinases and other enzymes. Our previous study demonstrated that the downregulation of CAV-1 in psoriatic epidermal cells contributes to inflammation by enhancing JAK/STAT signaling, cell proliferation, and chemokine production. Administration of the CAV-1 scaffolding domain (CSD) peptide suppressed imiquimod (IMQ)-induced psoriasis-like dermatitis. To identify an optimal therapeutic peptide derived from CAV-1, we have compared the efficacy of CSD and subregions of CSD that have been modified to make them water soluble. We refer to these modified peptides as sCSD, sA, sB, and sC. In IMQ-induced psoriasis-like dermatitis, while all four peptides showed major beneficial effects, sB caused the most significant improvements of skin phenotype and number of infiltrating cells, comparable or superior to the effects of sCSD. Phosphorylation of STAT3 was also inhibited by sB. Furthermore, sB suppressed angiogenesis both in vivo in the dermis of IMQ-induced psoriasis mice and in vitro by blocking the ability of conditioned media derived from CAV-1-silenced keratinocytes to inhibit tube formation by HUVEC. In conclusion, sB had similar or greater beneficial effects than sCSD not only by cytokine suppression but by angiogenesis inhibition adding to its ability to target psoriatic inflammation.

Keywords

Psoriasis
Skin inflammation
Caveolin-1
CSD peptide
water-soluble subregions
Subject terms

Drug discovery
Diseases
Medical research
Pulmonary Focused Foundations in Innovation and ScholarshipNIHBasic Medical Research Fund of the Japanese Dermatological Associationissue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Psoriasis vulgaris is a chronic immune-mediated inflammatory skin disease characterized by scaly skin plaques caused by keratinocyte hyperproliferation and immune cell infiltration1. Its pathogenesis is mediated by inflammatory cytokines (e.g. tumor necrosis factor (TNF)-α, interleukin (IL)-17, IL-22, IL-23) produced by immune cells that infiltrate the dermis, leading to epidermal hyperproliferation via activation of signal transducer and activator of transcription 3 (STAT3)2. Psoriatic lesions also exhibit angiogenesis, increased endothelial cell proliferation, capillary bed dilation, capillary loop meandering, and increased capillary permeability3. Angiogenic mediators (e.g. TNF-α, vascular endothelial growth factor (VEGF), hypoxia-inducible factor, IL-8, angiopoietins) are abundant in the psoriatic skin. These features suggest aberrant angiogenesis accompanies psoriasis pathogenesis4.

Caveolin-1 (CAV-1) is the major structural protein in caveolae (flask-shaped pits in the plasma membrane). CAV-1 regulates signal transduction by interacting with various signaling molecules5. We reported a significant reduction in CAV-1 levels in the epidermis of psoriasis patients6. CAV-1 reduction induces Janus kinase (JAK)/STAT pathway activation in psoriatic inflammation, leading to further keratinocyte proliferation and cytokine/chemokine production. Likewise, we observed CAV-1 reduction in a mouse model in the epidermis of imiquimod (IMQ)-induced psoriasis-like inflammation. Restoring CAV-1 function using the CAV-1 scaffolding domain (CSD) peptide improved skin phenotype and suppressed psoriasis-related cytokine expression in this model6. CSD acts by mimicking the inhibitory effect of full-length CAV-1 on signaling7. Subregions of CSD may be superior to CSD as therapeutic agents because they are easy to synthesize and likely to show improved tissue penetration and reduced side effects such as immunogenicity. Thus, a CSD subregion may be a more specific therapeutic agent than full-length CSD8. Indeed, CSD subregions improve skin and lung fibrosis in bleomycin-treated mice. Bone marrow monocytes isolated from bleomycin-treated mice show enhanced migration in vitro towards CXCL12, while in vivo treatment with CSD and its subregions suppressed this enhanced migration9,10. CSD subregions also inhibit vascular hyperpermeability in mice with angiotensin II-induced myocardial fibrosis10. However, CSD subregion’s effects on psoriatic inflammation remain unknown.

To understand the activities of CSD subregions on psoriasis, we evaluated their beneficial effects on tissue morphology in the IMQ model and the molecular mechanisms underlying these effects. Another key feature of this study is the use of full-length CSD and subregions modified to be water-soluble, thereby enhancing the likelihood they can be developed to treat human disease. Previously, CSD and subregions were dissolved in DMSO, then diluted in aqueous solutions before use.

Results

DMSO- and water-soluble CSD-B peptides improve the skin phenotype in IMQ-induced psoriasis-like inflammation

Previously6, we studied the beneficial effects of CSD bearing the antennapedia internalization sequence. This peptide required solubilization in DMSO. Here, we first evaluated the beneficial effects of CSD subregions without the antennapedia internalization sequence on IMQ-induced psoriasis-like inflammation. These peptides (Supplementary Table 1) still required DMSO for solubilization. Of these peptides, CSD and B were the most effective in improving skin phenotype (Supplementary Fig. 1a,b). These studies demonstrate the antennapedia internalization sequence is not required for versions of CSD to improve IMQ-induced psoriasis-like inflammation.

To avoid DMSO toxicity11,12, we designed water-soluble versions of CSD and its subregions (designated sCSD, sA, sB, sC, Table 1). Subcutaneous injection of each of the four peptides significantly improved skin phenotype in terms of the clinical skin scores for erythema and scale compared to the IMQ group. Only sB significantly improved induration (Fig. 1a,b). Importantly, we compared the beneficial effects of the four peptides and did not find a statistically significant difference among their beneficial effects. Histological analyses indicated that the epidermal thickness was significantly reduced by all peptide treatments and the number of infiltrating cells was inhibited most significantly by sB (Figs. 1c–e). Comparing the beneficial effects of the peptides, sB significantly reduced the epidermal thickness compared to sA and sC and the number of infiltrating cells was significantly inhibited by sB compared to sA.Table 1 Amino acid sequences of water-soluble CSD and subregions.

Red letters are not part of caveolin-1. D-amino acids are lower case. Peptides were purchased from Elim BioPharmaceuticals, Inc. (Hayward, CA).

Fig. 1 Skin phenotype of mice with IMQ-induced psoriasis-like inflammation treated with water-soluble CSD and subregions. (a) Representative images of the skin phenotype of mice treated with water-soluble peptides. Mice were treated with IMQ and vehicle (IMQ + Veh), IMQ and sCSD (IMQ + sCSD), IMQ and water-soluble subregions (IMQ + sA, IMQ + sB, IMQ + sC), or Vaseline for 6 days. (b) Scores represent the erythema, induration, and scales scores in each group for water-soluble peptide-treated mice at each time point. (c) Representative hematoxylin and eosin staining of the skin of mice treated with IMQ and water-soluble peptides. Scale bar = 100 μm. (d) The epidermal thickness and (e) the number of infiltrating cells were analyzed. The data were obtained from 10 random fields of view for each mouse. (f) The relative gene expression levels of IL-23, TNF-α, IL-17A, IL-17C, and IL-17F in the skin of mice treated with water-soluble peptides were determined by quantitative PCR. All the graphs indicate the mean ± SD of each group (n = 5). One-way analysis of variance (post hoc Holm-Sidak’s test) indicated *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (IMQ + Veh vs. IMQ + sCSD, IMQ + sA, IMQ + sB, or IMQ + sC), ^^P < 0.01, ^^^P < 0.001, or ^^^^P < 0.0001 (Vase vs. IMQ + Veh), and +P < 0.05, ++P < 0.01 (IMQ + sB vs IMQ + sA or IMQ + sC), respectively.

We also evaluated the expression of psoriasis-related cytokines in mouse skin. All four peptides significantly decreased IL-23, IL-17A, and IL-17C expression compared to the IMQ treatment alone (Fig. 1f).

sCSD and sB suppress IL-22-induced STAT3 activation in CAV-1 silenced keratinocytes

Because STAT3 is involved in keratinocyte proliferation and affects the expression of several cytokines13 and CAV-1 downregulation induces STAT3 activation in keratinocytes6, we examined whether STAT3 activation is targeted by versions of CSD. IL-22-induced pSTAT3 levels were significantly enhanced in CAV-1-silenced keratinocytes compared with control keratinocyte and were suppressed by sCSD and sB treatment at 15 min (Fig. 2a,b). Only sB demonstrated this effect at 60 min.Fig. 2 Suppressed activation of the STAT3 pathway in CAV-1-silenced keratinocytes by sCSD and sB treatment. (a) Representative images of immunoblotting for pSTAT3. pSTAT3 was evaluated at the indicated time points after IL-22 stimulation in CAV-1-silenced (siC-treated) keratinocytes treated with Veh, sCSD or sB, or control (siN-treated) keratinocytes. Original blots are presented in Supplementary Figure S2. (b) Protein levels were quantified by densitometry and expressed as the ratio of pSTAT3 to total STAT3. Statistical analyses were performed comparing siC + sCSD and siC + sB to siC + Veh at each time point. Deta was obtained from five independent experiments. One-way analysis of variance (post hoc Holm-Sidak’s test) indicated **P < 0.01 or ***P < 0.001 (siC + Veh vs. siC + sCSD or siC + sB), and ^P < 0.05 or ^^P < 0.01 (siN + Veh vs. siC + Veh), respectively. (c) Transcriptional activity of STAT3 was detected in CAV-1-silenced (siC-treated) or control (siN-treated) keratinocyte treated with indicated peptides following IL-22 stimulation using a luciferase STAT3 promoter assay. Three independent experiments were performed. The values of siN + Veh were set at 1. **P < 0.01 vs. siC with Veh.

Consistent with the immunoblotting data, luciferase expression by a reporter responsive to STAT3 activation was enhanced in CAV-1-silenced keratinocytes and was suppressed by sCSD and sB (Fig. 2c). These results indicate that sCSD and sB can modulate STAT3 activation as reported previously for CSD6.

sB peptide suppressed angiogenesis and angiogenesis-related factors in vivo

Aberrant angiogenesis is associated with psoriasis4,14. CAV-1 plays an important role in pathological angiogenesis15. Therefore, we investigated its role in angiogenesis in IMQ-induced psoriasis. First, we validated that subcutaneous vascularity is increased in IMQ-treated mice and that versions of CSD decrease vascularity. Excessive vessel formation occurred in the subcutaneous tissues of mice treated with IMQ (Fig. 3a). In IMQ-treated mice, vessel content was significantly reduced by sA and even more strongly by sB (Fig. 3b). Comparing the effects of the peptides, sB significantly reduced vessel content compared to sC. When the experiment was quantified in terms of CD31-positive vessels, similar results were obtained. CD31-positive vessels were increased in number by IMQ and reduced by only sB (Fig. 3c,d).Fig. 3 Decreased subcutaneous vascularity in mice with IMQ-induced psoriasis-like inflammation treated by sB peptide. Mice were treated with IMQ and Veh (IMQ + Veh) alone, IMQ and sCSD peptide (IMQ + sCSD), IMQ with a water-soluble CSD subregion (IMQ + sA, IMQ + sB, IMQ + sC), or Vaseline (Vase) for 6 days. (a) Representative images of the subcutaneous vasculature in the back skin of the mice. (b) The length of the blood vessels in the white square was measured using ImageJ software. (c) Representative immunohistochemical images of CD31 expression in mouse skin with IMQ-induced psoriasis-like inflammation. Arrows indicate blood vessels. (d) The number of CD31 positive blood vessels. All the graphs indicate the mean ± SD of each group (n = 4 for IMQ + Veh, n = 5 for other groups). One-way analysis of variance (post hoc Holm-Sidak’s test) indicated *P < 0.05, **P < 0.01, or ***P < 0.001 (IMQ + Veh vs. IMQ + sCSD, IMQ + sA, IMQ + sB, or IMQ + sC), ^^P < 0.01or ^^^^P < 0.0001 (Vase vs. IMQ + Veh), and +P < 0.05 (IMQ + sB vs IMQ + sC), respectively.

Because sB is the most effective version of CSD for suppressing blood vessel formation, we used a protein array system to further analyze angiogenesis-related factor expression, focusing on the ability of sB to reverse IMQ’s angiogenic effects. Proteins of interest were identified by an alteration in mean spot pixel density of > 30% in IMQ + sB compared to IMQ alone. These proteins (CXCL12, osteopontin, TIMP-1, VEGF, MMP-3) are highlighted with squares (Fig. 4a) and their expression quantified (Fig. 4b). In all cases except VEGF their expression was induced by IMQ; in all cases their expression was inhibited by sB, but not by sCSD. The mRNA expression of these angiogenesis-related factors was evaluated using quantitative reverse transcription PCR. sB, but not sCSD, decreased IMQ-induced mRNA expression of all these factors except VEGF compared to IMQ alone (Fig. 4c). Comparing these peptides , sB significantly reduced mRNA expression of MMP3, CXCL12, and TIMP-1 compared to the effects of sCSD.Fig. 4 Angiogenesis-related factors in mice with IMQ-induced psoriasis-like inflammation. Mice were treated with IMQ and Veh (IMQ + Veh), IMQ and sCSD (IMQ + sCSD), IMQ and sB (IMQ + sB), or Vaseline for 6 days. (a) Images of Proteome Profiler Mouse Angiogenesis Array membranes. A mixture of skin tissue extracts from three mice in each group was used. The mean spot pixel densities that changed by > 30% of the IMQ level are boxed. (b) Graph indicates the relative mean spot pixel densities quantified using ImageJ software. (c) Relative gene expression levels of MMP-3, SPP1, CXCL12, TIMP-1, and VEGF-A in mouse skin were determined using quantitative PCR. Graphs indicate the mean ± SD of each group (n = 5). One-way analysis of variance (post hoc Holm-Sidak’s test) indicated *P < 0.05 or **P < 0.01 (IMQ + Veh vs. IMQ + sCSD or IMQ + sB), ^P < 0.05, ^^^P < 0.001, or ^^^^P < 0.0001 (Vase vs. IMQ + Veh), and +P < 0.05, ++P < 0.01, +++P < 0.001 (IMQ + sB vs. IMQ + sCSD), respectively.

Conditioned media derived from sB-treated keratinocytes inhibited HUVEC tube formation

We performed a tube formation assay to determine whether angiogenesis-related factors released from CAV-1-downregulated epidermis promote angiogenesis (Fig. 5a). Conditioned medium from CAV-1-silenced keratinocytes increased network formation. This effect was reversed in sB-treated keratinocytes (Fig. 5a). Conditioned medium from sB-treated, CAV-1-silenced keratinocytes also decreased branches, branching points, and tube lengths compared to medium from CAV-1-silenced keratinocytes (Fig. 5b). These results indicate that sB inhibits angiogenesis by suppressing angiogenesis-related factor release from CAV-1-silenced keratinocytes.Fig. 5 sB peptide treatment of CAV-1 silenced keratinocyte suppressed tube formation in HUVECs. HUVECs were cultured on Matrigel. Conditioned media derived from CAV-1 silenced keratinocytes (siC + Veh), CAV-1 silenced keratinocytes treated with sCSD peptide (siC + sCSD), CAV-1 silenced keratinocytes treated with sB peptide (siC + sB), or control, siN-treated keratinocytes (siN + Veh) was added 4 h prior to observation of tube formation. (a) Representative images of the tube formation assay. (b) Quantitative analysis of branch count, junction count, and skeleton length. Data were obtained from the entire plate using ImageJ software. Three independent experiments were performed. One-way analysis of variance (post hoc Holm-Sidak’s test) indicated *P < 0.05 vs. siC + Veh.

Discussion

We previously showed that injection of full-length CSD improves the skin phenotype of mice with IMQ-induced psoriasis-like inflammation6. Here, we have extended our studies to CSD subregions modified to be water-soluble. Water-solubility is an important feature aiding in the development of novel therapeutic agents for clinical use. We found all three water-soluble CSD subregions (as well as water-soluble full-length CSD) improve skin phenotype and reduce epidermal thickness in IMQ-treated mice. It is noteworthy that all three subregions are beneficial. Some reports indicate amino acids 90–92 of CAV-1 are essential for regulating eNOS/CAV-1 interactions16,17. In another study, a CSD subregion including 90–92 inhibited fibrosis, whereas other subregions were ineffective18. A careful examination of ref. 18 raises the possibility that this group failed to observe beneficial effects with peptides lacking 90–92 because their peptides were not capped on either terminus.

In contrast, Reese et al.10 reported that all CSD peptide subregions, whether or not they include 90–92, inhibit lung and skin fibrosis in vivo. Additionally, they found that bone marrow-derived monocytes from bleomycin-treated mice exhibit enhanced migration in vitro compared to control mouse monocytes and that in vivo treatment of the mice with all three CSD subregions reversed this enhanced migration. Finally, they found that all three subregions inhibited the in vitro migration of TGFβ-activated human monocytes.

While all four peptides reduced IMQ-induced epidermal thickening, we find sB (the subregion including 90–92), was best in improving skin phenotype (erythema, induration, scale), and that only sB reduced IMQ-induced inflammatory cell infiltration and vessel formation. Therefore, we speculate that versions of CSD act, at least in part, by regulating eNOS/CAV-1 interactions.

Epidermal keratinocytes in psoriatic lesions exhibit STAT3 activation that is promoted by increased cytokine and growth factor levels19. For example, IL-22, an effector cytokine expressed by Th17 and Th22 cells, triggers epidermal activation and hyperproliferation in psoriasis patients via STAT3 activation13. Various keratinocyte-derived cytokines and chemokines further mobilize infiltration of activated immune cells into the dermis20. Previously, we demonstrated that CAV-1 reduction increased STAT3 phosphorylation in human keratinocytes and that CSD reduced the number of pSTAT3-positive cells in the epidermis of mice with IMQ-induced psoriasis-like inflammation6. Thus we speculated that CSD suppresses inflammation in psoriasis by regulating STAT3 activation. Supporting our speculation, Okada et al.21 reported that pSTAT3 was decreased in CAV-1-overexpressing HeLa cells. Overexpression of the CSD region alone in HeLa cells suppressed the expression of pSTAT3. The mechanism through which CAV-1 regulates STAT3 has not yet been elucidated. One report indicated that CAV-1 is homologous to the SOCS pseudosubstrate and functions as a negative regulator of STAT signaling, and that CAV-1 prevents the binding of JAKs and STAT proteins to their receptors by binding phosphotyrosine residues of cytokine receptors22. Another report demonstrated that CAV-1 suppressed STAT3 activity via CSD by downregulating cadherin-11, an activator of STAT323,24. Herein we found that both full-length sCSD and also the sB subregion suppressed both the phosphorylation of STAT3 and its ability to act as a transcriptional activator in CAV-1-silenced keratinocytes. Inflammatory signals decrease eNOS expression via STAT3 phosphorylation and activate STAT3 binding to the eNOS promoter25. Since CSD and its subregion containing 90–92 are speculated to regulate eNOS expression16,17, sCSD and sB may suppress STAT3 phosphorylation via eNOS. Taken together, versions of CSD may be effective receptor inhibitors in psoriasis, similar to the mechanism by which JAK inhibitors are effective. These results demonstrate that restoring CAV-1 function using sCSD and sB suppresses STAT3 activation in CAV-1-silenced keratinocytes and that CSD’s effects involve STAT3 activation.

Increased dermal vascularity is a characteristic of psoriasis16. CAV-1 deficiency in mice results in aberrant angiogenesis15. Therefore, we explored whether using our peptides to mimic CAV-1 suppresses angiogenesis. Indeed, we observed that several angiogenesis-related factors were upregulated in IMQ-treated mice, and that sB suppressed their expression while sCSD had significantly less of a beneficial effect.

Some angiogenesis-related factors, namely VEGF, CXCL8 and TNF-α are upregulated in psoriatic keratinocytes4,26,27. Thus, we speculate that keratinocytes are the source of angiogenesis-related factors upregulated in IMQ-treated mice and that the reduction of CAV-1 in keratinocytes results in their upregulation. As predicted, our experiments showed that conditioned medium derived from CAV-1-silenced keratinocytes promotes HUVEC tube formation (although the effect did not reach statistical significance) and that the restoration of CAV-1 activity in keratinocytes significantly reduced the HUVEC tube formation induced by the conditioned medium. Our results are also consistent with publications showing that CAV-1 reduction upregulates angiogenesis-related factors as evidenced by an inverse correlation between CAV-1 and VEGF in osteosarcoma28, increased CXCR12 expression in CAV-1 silenced fibroblast29, and a significant negative correlation between osteopontin and caveolin-1 levels in human osteoarthritic cartilage30.

The contribution of CAV-1 to angiogenesis is controversial. Consistent with our data, restoration of CAV-1 function using CSD inhibited VEGF-stimulated angiogenic signaling31 and VEGF-induced tumor vascular permeability in mice32. In contrast to our data, CAV-1 upregulation was reported to enhance HUVEC tube formation and CSD treatment of endothelial cells enhanced capillary-like tubule formation33. These discrepancies may be explained in part by the fact that while in most systems versions of CSD mimic the effects of endogenous CAV-1, in some systems they act as competitors of the activity of endogenous CAV-131,32,34–36.

In summary, our study suggests that water-soluble versions of CSD and its three subregions have beneficial effects on psoriasis. Among the four peptides, in general sB had the greatest beneficial effect. Anti-angiogenic effects may be involved in sB’s efficacy. While both sCSD and sB contain the 90–92 amino acid sequence reported to regulate eNOS activity, sB may be more effective than sCSD and a better candidate for drug development because of its smaller size resulting in easier synthesis, improved tissue penetration, and a decreased potential for causing side effects including an immune response. Based on the total picture provided by our data, we have tentatively selected sB as our Lead Compound in pursuing our goal of developing a treatment for psoriasis. Nevertheless, we cannot rule out the possibility that when dose, route, and frequency of administration are altered, another one of our peptides may become more beneficial than sB.

Material and Methods

Murine model of psoriasis-like skin inflammation

In vivo experiments involved 6-week-old C57BL/6 J female mice (Japan SLC, Shizuoka, Japan). To induce skin inflammation, 62.5 mg of 5% IMQ cream (Beselna cream; Mochida Pharmaceutical, Tokyo, Japan) was applied to the back of mice daily for 6 days as described6. Vaseline was the control. Subcutaneous vessels were measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Animal experiments in this study were performed in accordance with Yokohama City University Institutional Animal Care and Use Committee guidelines and with ARRIVE guidelines (https://arriveguidelines.org). All animal experiments were approved by the Institutional Committee of Laboratory Animal Experimentation (Animal Research Center of Yokohama City University, Yokohama, Japan). (Approval No: F-A-21-029).

CSD treatment

Our four water-soluble peptides (sCSD, sA, sB, sC) are defined in Table 1. Peptides were dissolved in sterile water. 100 μl of a 0.2 mM peptide solution was injected intradermally into the back skin of mice following each IMQ treatment. On day 7, mice were euthanized by CO2 asphyxiation, and skin surrounding the injection site harvested.

Measurement of skin scores, epidermal thickness, and cellular infiltration

Clinical skin scores were determined on days 1 through 7 using the modified psoriasis severity index score. Degrees of skin erythema, induration, and scales were classified: 0, no symptoms; 1, mild; 2, moderate; 3, severe; and 4, very severe. Sections from paraffin-embedded mouse skin were stained with hematoxylin and eosin. Images were captured using a BZ-9000 microscope (Keyence, Osaka, Japan). Epidermal thickness was measured from the stratum basale to the stratum granulosum as described6. The average value from 10 random fields was calculated for each mouse. Infiltrated cells were also counted from 10 random 10,000 μm2 fields per mouse.

Immunohistochemistry

The endothelial cell marker CD31 was used to enable the evaluation of angiogenesis. Briefly, samples fixed with 10% formaldehyde were paraffin embedded. Slides were cut and deparaffinized. Endogenous peroxidase activity was blocked with 3% H2O2. After heat-mediated antigen retrieval, slides were incubated with 1% bovine serum albumin to block nonspecific binding. Anti-CD31 (ab28364, Abcam) antibodies were added and incubated at 4℃ overnight. After incubation with peroxidase-conjugated secondary antibodies (ab205718, Abcam), slides were stained with DAB and counterstained with hematoxylin. Images were captured using a BZ-9000 microscope (Keyence, Osaka, Japan).

Expression profiling of angiogenesis-related factors in mouse skin

Mixed skin tissue extracts from three mice per group (IMQ + Veh, IMQ + sCSD, IMQ + sB, Vaseline) were analyzed using a proteome profiler array (mouse angiogenesis array kit, R&D Systems, Minneapolis, MN, ARY015), allowing the simultaneous assessment of the relative levels of 53 angiogenesis-related proteins. The mean spot pixel densities of Vaseline and IMQ + Veh were compared. Factors that differed by > 30% are marked with colored squares. Array procedures and data analyses were performed following the manufacturer's instructions.

Cell culture and stimulation

Normal human epidermal keratinocytes (NHEKs) were purchased from Takara Bio (Shiga, Japan) and cultured in Keratinocyte Basal Medium (KBM) overnight prior to human recombinant rIL-22 stimulation (200 ng ml−1, R&D Systems, Minneapolis, MN). Cells were harvested 24 or 48 h after stimulation.

CAV-1 silencing

CAV-1-specific small interfering RNA (stealth RNAi) was purchased from Invitrogen (Carlsbad, CA, USA). A random RNAi was the negative control. For reverse transfection, 100 pM of each RNAi in 500 μl OptiMEM, plus 5 μl Lipofectamine RNAiMAX (Invitrogen) was added to wells. After 20 min, 8 × 104 NHEKs in KBM containing growth factors without antibiotics were seeded into each well. Cells were cultured for 48 h. For the final 16 h, the medium was replaced with KBM without growth factors. Finally, cells were treated 1 h with sCSD, then stimulated with IL-22.

Quantitative polymerase chain reaction

Total RNA was extracted using TRIzol reagent and an Illustra RNAspin Mini RNA Isolation Kit (GE Healthcare, Uppsala, Sweden). First-strand cDNA was synthesized using a High-Capacity RNA-to-cDNA Kit according to the manufacturer’s instructions (Applied Biosystems, Carlsbad, CA, USA). Quantitative polymerase chain reaction (PCR) was performed in duplicates using TaqMan gene expression assays on a CFX96 Touch Real-Time PCR machine (Bio-Rad, Tokyo, Japan). Gene expression levels were normalized to β-actin or GAPDH as appropriate and compared using the 2 − ΔΔCt method.

Western blotting analysis

Samples were prepared using RIPA buffer containing protease inhibitor cocktail as described37. Equal protein loads were analyzed by western blotting using: pSTAT3 (Tyr705), STAT3, or GAPDH loading control (Cell Signaling). Signals were detected using horseradish peroxidase-conjugated secondary antibodies and ECL Prime Western Blotting Detection Reagent (GE Healthcare) on a Doc™ XRS+ (Bio-Rad). Signals were quantified using Image Lab (Bio-Rad).

Luciferase reporter gene assay

STAT3 transcription was measured using pGL4.47[luc2P/SIE/Hygro] (Promega, Madison, WI, USA) according to the manufacturer’s instructions. NHEKs (2 × 105 cells/well) were co-transfected with a STAT3-responsive reporter and CAV-1 RNAi or control RNAi. After 48 h, the cells were treated with 0.2 mM sCSD 1 h prior to treatment with 200 ng ml−1 of IL-22. Three hours after IL-22 stimulation, cells were harvested. Cell lysates were analyzed for luciferase activity using the Dual-Luciferase Reporter Assay System (Promega).

Tube formation assay

75 µl Matrigel (356,234, Corning, NY, USA) was added to wells of 96-well culture plates and incubated at 37 °C for 30 min to solidify the gel. 1 × 104 HUVECs were seeded into each Matrigel-coated well. Conditioned media derived from CAV-1-silenced keratinocytes stimulated with either IL22, IL22 plus sCSD, or IL22 plus sB was added (1:3 with vascular cell basal medium) and cultured 4 h. Conditioned media from siN-treated (non-silenced) keratinocytes stimulated with IL22 served as the control. Tube formation was observed by microscopy. Branch number (branch count), branch point number (junction count), and tube length (skeleton length) were measured (ImageJ software, National Institutes of Health).

Statistical analysis

Statistical comparisons were performed using one-way analysis of variance (post-hoc Holm-Sidak’s tests) as indicated. Tests were performed using GraphPad Prism version 7.0 software (GraphPad Software, San Diego, CA, USA). Statistical significance was set at p < 0.05.

Supplementary Information

Supplementary Information.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-71350-1.

Author contributions

Y.Y. designed the study, supervised, wrote and revised the main manuscript. C.A. and N.T. conducted the study and wrote the draft of the manuscript. T.W., M.A. and N.I. conducted the study. CF.R. and S.H. supervised and wrote and revised the main manuscript. All authors reviewed the manuscript.

Funding

This work was supported by the 2020 Basic Medical Research Fund of the Japanese Dermatological Association (donated by Shiseido) (YY). SH was supported by R41 grants from NIH (HL164283, AR081740, HL167327). CFR was supported by the Pulmonary Focused Foundations in Innovation and Scholarship (5T32HL144470-04).

Data availability

All data generated or analyzed during this study are provided in the supplementary Table S2.

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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