Abstract: Psoriatic skin lesions exhibit a clearly altered vascular network in the form of numerous enlarged, tortuous, and hyperpermeable blood vessels. Vascular proliferation facilitates inflammatory cytokine distribution, inflammatory cell recruitment, and resident cell proliferation in the psoriatic condition (Wang et al., 2019Wang X. Sun X. Qu X. Li C. Yang P. Jia J. et al.Overexpressed fibulin-3 contributes to the pathogenesis of psoriasis by promoting angiogenesis.Clin Exp Dermatol. 2019; 44: e64-e72Crossref PubMed Scopus (9) Google Scholar). Cutaneous angiogenesis reflects the disease severity of patients with psoriasis (Malecic and Young, 2017Malecic N. Young H.S. Excessive angiogenesis associated with psoriasis as a cause for cardiovascular ischaemia.Exp Dermatol. 2017; 26: 299-304Crossref PubMed Scopus (21) Google Scholar), and inhibiting angiogenesis can effectively attenuate psoriatic lesions (Zibert et al., 2011Zibert J.R. Wallbrecht K. Schön M. Mir L.M. Jacobsen G.K. Trochon-Joseph V. et al.Halting angiogenesis by non-viral somatic gene therapy alleviates psoriasis and murine psoriasiform skin lesions.J Clin Invest. 2011; 121: 410-421Crossref PubMed Scopus (30) Google Scholar). Therefore, angiogenesis plays a critical role in the psoriasis pathogenesis. Many factors regulate cutaneous angiogenesis. TNF-like weak inducer of apoptosis (TWEAK or TNFSF12) is a proinflammatory cytokine that acts by binding to the receptor fibroblast GF–inducible 14 (Fn14 or TNFRSF12A). Previously, we reported that TWEAK/Fn14 signaling is activated in psoriatic lesions, promoting keratinocyte (KC) proliferation (Cheng et al., 2016Cheng H. Xu M. Liu X. Zou X. Zhan N. Xia Y. TWEAK/Fn14 activation induces keratinocyte proliferation under psoriatic inflammation.Exp Dermatol. 2016; 25: 32-37Crossref PubMed Scopus (28) Google Scholar). Subcutaneously injecting TWEAK in naive mice induces skin inflammation with histological and molecular features of psoriasis (Sidler et al., 2017Sidler D. Wu P. Herro R. Claus M. Wolf D. Kawakami Y. et al.TWEAK mediates inflammation in experimental atopic dermatitis and psoriasis.Nat Commun. 2017; 8: 15395Crossref PubMed Scopus (35) Google Scholar). Deficiency of TWEAK or Fn14 ameliorates psoriasiform lesions in murine models (Peng et al., 2018Peng L. Li Q. Wang H. Wu J. Li C. Liu Y. et al.Fn14 deficiency ameliorates psoriasis-like skin disease in a murine model.Cell Death Dis. 2018; 9: 801Crossref PubMed Scopus (9) Google Scholar; Sidler et al., 2017Sidler D. Wu P. Herro R. Claus M. Wolf D. Kawakami Y. et al.TWEAK mediates inflammation in experimental atopic dermatitis and psoriasis.Nat Commun. 2017; 8: 15395Crossref PubMed Scopus (35) Google Scholar). TWEAK also improves vasculogenesis in mice with acute myocardial infarction (Sheng et al., 2018Sheng Z. Ju C. Li B. Chen Z. Pan X. Yan G. et al.TWEAK promotes endothelial progenitor cell vasculogenesis to alleviate acute myocardial infarction via the Fn14-NF-κB signaling pathway.Exp Ther Med. 2018; 16: 4019-4029PubMed Google Scholar). Recently, we demonstrated that TWEAK directly enhances the migration and cytokine production of dermal microvascular endothelial cells (DMECs) (Liu et al., 2019Liu J. Liu Y. Peng L. Li J. Wu K. Xia L. et al.TWEAK/Fn14 signals mediate burn wound repair.J Invest Dermatol. 2019; 139: 224-234Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). However, the exact effect of TWEAK on angiogenesis remains unclear, especially in psoriatic inflammation. In this study, 5 μg of TWEAK was subcutaneously injected into BALB/c mice (Supplementary Materials and Methods) twice weekly for 2 weeks. Histological evaluation showed that TWEAK induced vascular proliferation in skin, as indicated by the proliferation of CD34-positive endothelial cells (Figure 1a and b), and the mice showed localized infiltration of macrophages (Iba-1-positive) and T lymphocytes (CD3-positive) (Supplementary Figure S1). However, the enhancement effect of TWEAK was partially abrogated in Fn14-deficient mice (Figure 1a and b). Consistently, the cutaneous expression levels of proangiogenic factors, including IL-17A, IL-18, IL-23A, and VEGFA, were significantly increased in the mice, whereas Fn14 deficiency mitigated the effect of TWEAK (Supplementary Figure S2). TWEAK was further topically applied to a psoriasiform mouse model. BALB/c mice were treated with imiquimod cream either combined or not with 0.01% TWEAK in the cream. Histological evaluation showed that TWEAK exacerbated proliferation of CD34-positive vascular endothelial cells (Figure 1c and d). Similar results were observed via macrophage (Iba-1–positive) and T lymphocyte (CD3-positive) infiltration (Supplementary Figure S3). Notably, Fn14 deficiency counteracted this effect of TWEAK (Figure 1c and d). These findings were consistent with those of a previous report (Peng et al., 2018Peng L. Li Q. Wang H. Wu J. Li C. Liu Y. et al.Fn14 deficiency ameliorates psoriasis-like skin disease in a murine model.Cell Death Dis. 2018; 9: 801Crossref PubMed Scopus (9) Google Scholar). Moreover, IL-17A, IL-18, IL-23, and VEGFA expression levels were attenuated in the Fn14-deficient mice (Supplementary Figure S4). KCs are the main source of VEGFA in inflamed skin (Johnson and Wilgus, 2014Johnson K.E. Wilgus T.A. Vascular endothelial growth factor and angiogenesis in the regulation of cutaneous wound repair.Adv Wound Care (New Rochelle). 2014; 3: 647-661Crossref PubMed Google Scholar). The in vitro effects of TWEAK on VEGFA expression were investigated in KCs, which were stimulated with the M5 cytokine cocktail containing IL-17A to recapitulate the features of psoriasis (Cheng et al., 2016Cheng H. Xu M. Liu X. Zou X. Zhan N. Xia Y. TWEAK/Fn14 activation induces keratinocyte proliferation under psoriatic inflammation.Exp Dermatol. 2016; 25: 32-37Crossref PubMed Scopus (28) Google Scholar). TWEAK promoted IL-17A expression in non–M5 cocktail–treated cells (Figure 1e). Furthermore, TWEAK induced protein expression of VEGFA, IL-18, and IL-23, which was amplified upon stimulation with the M5 cocktail (Figure 1f). Furthermore, the Fn14 antagonist L524-0366 reversed the stimulatory effect of TWEAK (Figure 1e and g). The proangiogenic GF, hypoxia inducible factor-1α (HIF-1α), is increased in psoriatic lesions (Abdou et al., 2018Abdou A.G. Farag A.G.A. Hammam M. Taie D.M. Abdelaziz R.A. Immunohistochemical expression HIF1α in chronic plaque psoriasis, an association with angiogenesis and proliferation.J Immunoassay Immunochem. 2018; 39: 249-262Crossref PubMed Scopus (2) Google Scholar). We observed that HIF-1α was highly expressed in both KCs and DMECs after M5 cocktail stimulation (Figure 1h and i). Notably, acriflavine, an HIF-1α inhibitor, reversed the M5 cocktail–induced Fn14 upregulation in these cells (Figure 1j and k). The mRNA expression levels of these molecules were altered consistently (Supplementary Figure S5). Thus, HIF-1α mediated Fn14 upregulation in psoriatic inflammation. The effect of TWEAK on DMEC proliferation was directly evaluated via the spheroid capillary sprouting assay, which showed that TWEAK significantly enhanced capillary sprouting of M5 cocktail–stimulated DMECs (Figure 2a and b). The capillary tube formation assay showed that DMECs developed more capillary networks when treated with TWEAK (Figure 2c and d). The antagonists targeting Fn14 (L524-0366) and HIF-1α (acriflavine) counteracted these effects (Figure 2a–d). Moreover, the specific inhibitors of the NF-κB (JSH-23) and phosphatidylinositol 3-kinase (wortmannin) signaling pathways suppressed the TWEAK-induced stimulation of the proangiogenic cytokines VEGFA, IL-18, and IL-23 in M5 cocktail–stimulated keratinocytes (Supplementary Figure S6). Both inhibitors attenuated the effect of TWEAK on spheroid sprouting and tube formation among DMECs (Figure 2e–g). Hence, the NF-κB and phosphatidylinositol 3-kinase signaling pathways mediated the proangiogenic effect of TWEAK. This study provided compelling evidence that TWEAK and Fn14 interaction contributes to proangiogenic cytokine production and angiogenesis in psoriatic inflammation. As one of the main proangiogenic factors in psoriasis, VEGFA enhances vascular growth by regulating vascular endothelial cell proliferation, migration, and sprouting (Sankar et al., 2017Sankar L. Arumugam D. Boj S. Pradeep P. Expression of angiogenic factors in psoriasis vulgaris.J Clin Diagn Res. 2017; 11: EC23-EC27PubMed Google Scholar; Uccelli et al., 2019Uccelli A. Wolff T. Valente P. Di Maggio N. Pellegrino M. Gürke L. et al.Vascular endothelial growth factor biology for regenerative angiogenesis.Swiss Med Wkly. 2019; 149: w20011PubMed Google Scholar). TWEAK expression correlates positively with VEGF in angioproliferative diseases and upregulates VEGF expression in various cells (Lei et al., 2015Lei M. Qin L. Wang A. Jin Y. Zhao X. Qi X. Fn14 receptor appears as a modulator of ovarian steroid-related regulation of goat endometrial epithelial cell IL-18 expression.Am J Reprod Immunol. 2015; 73: 428-436Crossref PubMed Scopus (6) Google Scholar; Ruffolo et al., 2019Ruffolo C. Toffolatti L. Massani M. Pozza A. Campo Dell'Orto M. Saadeh L.M. et al.Interferon-gamma and tumor necrosis factor-related weak inducer of apoptosis expression in neoangiogenesis in colorectal polypoid lesions.Eur Surg Res. 2019; 60: 186-195Crossref PubMed Scopus (2) Google Scholar; Shimada et al., 2012Shimada K. Fujii T. Tsujikawa K. Anai S. Fujimoto K. Konishi N. ALKBH3 contributes to survival and angiogenesis of human urothelial carcinoma cells through NADPH oxidase and tweak/Fn14/VEGF signals.Clin Cancer Res. 2012; 18: 5247-5255Crossref PubMed Scopus (39) Google Scholar). In this study, TWEAK upregulated IL-18,IL-23, and VEGFA expression in M5 cocktail–stimulated psoriatic KCs. TWEAK also increased IL-17A expression in normal KCs. Importantly, hypoxia induces VEGFA expression during skin injury (Johnson and Wilgus, 2014Johnson K.E. Wilgus T.A. Vascular endothelial growth factor and angiogenesis in the regulation of cutaneous wound repair.Adv Wound Care (New Rochelle). 2014; 3: 647-661Crossref PubMed Google Scholar). We determined that psoriatic inflammation induced HIF-1α in skin cells, further upregulating Fn14 expression and thereby amplifying the TWEAK-induced downstream cytokine release and angiogenesis. Fn14 was lowly expressed in normal cells and unaffected by the HIF-1α inhibitor, thus confirming that HIF-1α–induced mediation of the TWEAK effect was restricted to psoriatic environments. In conclusion, TWEAK promotes angiogenesis, thereby exacerbating cutaneous psoriatic disease. This study had some limitations. The in vitro data were generated in cells stimulated with the M5 cytokine cocktail, which only recapitulated some features of psoriasis. The TWEAK-induced cytokines in the KCs were not confirmed to have proangiogenic functions in full skin structures. Additionally, no direct evidence was found that blocking angiogenesis attenuates the effect of TWEAK on psoriatic inflammation. These issues require further study. No datasets were generated or analyzed during the current study. All animal protocols used in this study (No. 2016028) were approved by the University Research Ethics Committee. Wei Liu: http://orcid.org/0000-0002-2463-1427 Dingwei Zhang: http://orcid.org/0000-0002-0918-8818 Mai Luo: http://orcid.org/0000-0003-3424-3716 Fangyan Jia: http://orcid.org/0000-0001-6388-9087 Lingling Peng: http://orcid.org/0000-0002-5717-5325 Xiaoli Li: http://orcid.org/0000-0001-8106-2358 Yumin Xia: http://orcid.org/0000-0002-3493-7198 The authors state no conflict of interest. This study was supported by the National Natural Science Foundation of China, China (Project No. 81630081 ) and the Innovation Capability Support Plan (Project No. 2019TD-034 ) and the Key Research and Development Plan (Project No. 2020ZDLSF02-08 ) of Shaanxi Province, China. Conceptualization: XL, YX; Formal Analysis: ML; Funding Acquisition: XL, YX; Investigation: WL, DZ, LP; Methodology: WL, DZ, FJ; Supervision: YX; Writing - Original Draft Preparation: YX Fibroblast GF-inducible 14–deficient BALB/c mice were generated by CRISPR/Cas9-mediated genome editing (Peng et al., 2018Peng L. Li Q. Wang H. Wu J. Li C. Liu Y. et al.Fn14 deficiency ameliorates psoriasis-like skin disease in a murine model.Cell Death Dis. 2018; 9: 801Crossref PubMed Scopus (16) Google Scholar). Mice were housed in the specific pathogen-free animal facility at the university. Ten-week-old male mice were used in the experiments. All animal protocols used in this study (No. 2016028) were approved by the University Research Ethics Committee. The wild-type mice were shaved on the back, and then received a subcutaneous injection of recombinant murine TNF-like weak inducer of apoptosis (TWEAK) (R&D Systems, Minneapolis, MN) or BSA (Sigma-Aldrich, St. Louis, MO). TWEAK and BSA were dissolved in PBS at 50 μg/ml each. Mice were injected twice weekly over a 14-day period at a volume of 100 μl each time. Mice were killed on day 14, and skin tissue was harvested (Supplementary Figure S7). Psoriasiform skin disease was induced in BALB/c mice with 5% imiquimod cream (Sichuan Mingxin Pharmaceuticals Co, Ltd, Chengdu, China) as previously described (Peng et al., 2018Peng L. Li Q. Wang H. Wu J. Li C. Liu Y. et al.Fn14 deficiency ameliorates psoriasis-like skin disease in a murine model.Cell Death Dis. 2018; 9: 801Crossref PubMed Scopus (16) Google Scholar). In some experiments, lyophilized TWEAK or BSA was prepared by mixing with the cream (0.01%), without altering the concentration of other ingredients. The topical dose of the cream was 25 mg daily. Mice were evaluated for PASI scores (An et al., 2017An J. Zhang D. Wu J. Li J. Teng X. Gao X. et al.The acitretin and methotrexate combination therapy for psoriasis vulgaris achieves higher effectiveness and less liver fibrosis.Pharmacol Res. 2017; 121: 158-168Crossref PubMed Scopus (14) Google Scholar). Skin tissues were collected on day 7 (Supplementary Figure S7). H&E staining solution was used to stain the paraffin-embedded tissue sections. Immunohistochemistry analysis was performed as described previously (Liu et al., 2017Liu Y. Peng L. Li L. Liu C. Hu X. Xiao S. et al.TWEAK/Fn14 activation contributes to the pathogenesis of bullous pemphigoid.J Invest Dermatol. 2017; 137: 1512-1522Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Briefly, deparaffinized sections were blocked with Dual Endogenous Enzyme Block (DAKO, Glostrup, Denmark). Rabbit IgG against CD34, CD3, or Iba-1 (2 μg/ml; Abcam, Cambridge, United Kingdom) and polymer-horseradish peroxidase–labeled goat anti-rabbit IgG (2 μg/ml; DAKO) were also applied. Then, a 3,3′-diaminobenzine-chromogen substrate (DAKO) was used for color development. Histological evaluations were performed by two pathologists blinded to the grouping. Cutaneous angiogenesis was analyzed using a computer-assisted morphometric method (Výbohová et al., 2015Výbohová D. Mellová Y. Adamicová K. Adamkov M. Hešková G. Quantitative comparison of angiogenesis and lymphangiogenesis in cutaneous lichen planus and psoriasis: immunohistochemical assessment.Acta Histochem. 2015; 117: 20-28Crossref PubMed Scopus (11) Google Scholar). Primary human keratinocytes and dermal microvascular endothelial cells were purchased from ScienCell Laboratories (Carlsbad, CA) and cultured according to the protocol provided by the vendor. Primary human keratinocytes and dermal microvascular endothelial cells were cultured in Keratinocyte Medium and Endothelial Cell Medium (ScienCell Laboratories), respectively. Before stimulation assays, the cells were incubated in Keratinocyte Growth Supplement-free Medium (ScienCell Laboratories) for 24 hours. The M5 cocktail (IL-1α, IL-17A, IL-22, oncostatin M, and TNF-α, each at 10 ng/ml) was added to the cell cultures as previously described (Cheng et al., 2016Cheng H. Xu M. Liu X. Zou X. Zhan N. Xia Y. TWEAK/Fn14 activation induces keratinocyte proliferation under psoriatic inflammation.Exp Dermatol. 2016; 25: 32-37Crossref PubMed Scopus (34) Google Scholar). The cells were also stimulated with recombinant human TWEAK (R&D Systems) or BSA (0–250 ng/ml, 0–48 hours). Some cells were simultaneously treated with a specific inhibitor of the NF-κB (JSH-23, 20 μM) or phosphatidylinositol 3-kinase (wortmannin, 25 μg/ml) signaling pathways (Sigma-Aldrich). The fibroblast GF–inducible 14 and hypoxia inducible factor–1α antagonists L524-0366 (10 μM) and acriflavine (5 μM), respectively, were purchased from Sigma-Aldrich and used in some experiments as previously described (Dhruv et al., 2013Dhruv H. Loftus J.C. Narang P. Petit J.L. Fameree M. Burton J. et al.Structural basis and targeting of the interaction between fibroblast growth factor-inducible 14 and tumor necrosis factor-like weak inducer of apoptosis.J Biol Chem. 2013; 288: 32261-32276Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar; Wigerup et al., 2016Wigerup C. Påhlman S. Bexell D. Therapeutic targeting of hypoxia and hypoxia-inducible factors in cancer.Pharmacol Ther. 2016; 164: 152-169Crossref PubMed Scopus (325) Google Scholar). The sprouting assay was performed as previously described (Rosano et al., 2020Rosano S. Corà D. Parab S. Zaffuto S. Isella C. Porporato R. et al.A regulatory microRNA network controls endothelial cell phenotypic switch during sprouting angiogenesis.Elife. 2020; 9: e48095Crossref PubMed Scopus (20) Google Scholar). Briefly, dermal microvascular endothelial cells were trypsinized and cultured in hanging drops in endothelial cell basal medium (Thermo Fisher Scientific, Waltham, MA) containing methylcellulose (Sigma-Aldrich). After overnight incubation, spheroids were embedded in a solution containing rat tail collagen I solution (Sigma-Aldrich). Sprouting was observed under stimulation of TWEAK (100 ng/ml, 24 hours). Capillary tube formation was performed by culturing dermal microvascular endothelial cells on Matrigel (Becton-Dickinson, Franklin Lakes, NJ) as described previously (Machnicka et al., 2020Machnicka B. Ponceau A. Picot J. Colin Y. Lecomte M.C. Deficiency of αII-spectrin affects endothelial cell-matrix contact and migration leading to impairment of angiogenesis in vitro.Cell Mol Biol Lett. 2020; 25: 3Crossref PubMed Scopus (1) Google Scholar). Spheroids were imaged by a digital microscope (Leica, Wetzlar, Germany). The ImageJ software (National Institutes of Health, Bethesda, MD) was used to measure the capillary length. Total RNA was extracted from fresh tissue and cell cultures using the Trizol reagent (Ambion, Carlsbad, CA). RNA was reverse transcribed into cDNA using a commercial kit (Takara Bio, Kusatsu, Japan). Then, qPCR was performed on a 7900HT Fast PCR System (Applied Biosystems, Waltham, MA). SYBR Green Master Mix (Takara Bio) was used as fluorescent dye. The expression levels of the target genes were calculated using the 2−ΔCt method. The PCR primers were synthesized by AuGCT Biotech Co, Ltd, (Beijing, China) and are listed in Supplementary Table S1. Protein lysates were prepared from cell cultures using RIPA buffer supplemented with a protease inhibitor cocktail (Beyotime Biotech, Beijing, China). The samples were separated on SDS-PAGE gels, followed by transfer onto polyvinylidene difluoride membranes (Millipore, Burlington, MA). The membranes were incubated with rabbit IgG against fibroblast GF–inducible 14, hypoxia inducible factor–1α, or β-actin (Abcam; 2 μg/ml). Biotinylated goat anti-rabbit IgG (2 μg/ml; Beyotime Biotech) and horseradish peroxidase–streptavidin (Beyotime Biotech) were sequentially used. An enhanced chemiluminescence kit (Thermo Fisher Scientific) was used to detect signals of reactive proteins. The protein band intensities were measured using the ImageJ software. The levels of IL-17A, IL-18, IL-23, and VEGFA were determined in culture supernatants using the commercial Quantikine ELISA kits (R&D Systems). The assays were performed following the procedures provided by the manufacturer. Data are expressed as the mean ± SEM. Statistical analysis was performed using GraphPad Prism version 8.0 (GraphPad Software Inc, La Jolla, CA). One-way ANOVA was used for the comparison of more than two groups of variables. Two-tailed Student's t-test was used for the comparison of the two groups. Differences were considered statistically significant if the P-value was < 0.05. Abbreviations: F, forward; Fn14, fibroblast growth factor-inducible 14; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HIF, hypoxia-inducible factor; IL, interleukin; R, reverse; VEGFA, vascular endothelial growth factor A.