Selective inhibition of integrin αvß6 leads to rapid induction of urinary bladder tumors in cynomolgus macaques.

Administration of a novel and selective small molecule integrin αvß6 inhibitor, MORF-627, to young cynomolgus monkeys for 28 days resulted in the rapid induction of epithelial proliferative changes in the urinary bladder of 2 animals, in the absence of test agent genotoxicity. Microscopic findings included suburothelial infiltration by irregular nests and/or trabeculae of epithelial cells, variable cytologic atypia, and high mitotic rate, without invasion into the tunica muscularis. Morphologic features and patterns of tumor growth were consistent with a diagnosis of early-stage invasive urothelial carcinoma. Ki67 immunohistochemistry demonstrated diffusely increased epithelial proliferation in the urinary bladder of several monkeys, including those with tumors, and αvß6 was expressed in some epithelial tissues, including urinary bladder, in monkeys and humans. Spontaneous urothelial carcinomas are extremely unusual in young healthy monkeys, suggesting a direct link of the finding to the test agent. Inhibition of integrin αvß6 is intended to locally and selectively block transforming growth factor beta (TGF-ß) signaling, which is implicated in epithelial proliferative disorders. Subsequent in vitro studies using a panel of integrin αvß6 inhibitors in human bladder epithelial cells replicated the increased urothelial proliferation observed in monkeys and was reversed through exogenous application of TGF-ß. Moreover, analysis of in vivo models of liver and lung fibrosis revealed evidence of epithelial hyperplasia and cell cycle dysregulation in mice treated with integrin αvß6 or TGF-ß receptor I inhibitors. The cumulative evidence suggests a direct link between integrin αvß6 inhibition and decreased TGF-ß signaling in the local bladder environment, with implications for epithelial proliferation and carcinogenesis.

Systemic Sclerosis Dermal Fibroblasts Induce Cutaneous Fibrosis Through Lysyl Oxidase-like 4: New Evidence From Three-Dimensional Skin-like Tissues.

OBJECTIVE: Systemic sclerosis (SSc) is a clinically heterogeneous disease characterized by increased collagen accumulation and skin stiffness. Our previous work has demonstrated that transforming growth factor ß (TGFß) induces extracellular matrix (ECM) modifications through lysyl oxidase-like 4 (LOXL-4), a collagen crosslinking enzyme, in bioengineered human skin equivalents (HSEs) and self-assembled stromal tissues (SAS). We undertook this study to investigate cutaneous fibrosis and the role of LOXL-4 in SSc pathogenesis using HSEs and SAS. METHODS: SSc-derived dermal fibroblasts (SScDFs; n = 8) and normal dermal fibroblasts (NDFs; n = 6) were incorporated into HSEs and SAS. These 3-dimensional skin-like microenvironments were used to study the effects of dysregulated LOXL-4 on ECM remodeling, fibroblast activation, and response to TGFß stimulation. RESULTS: SScDF-containing SAS showed increased stromal thickness, collagen deposition, and interleukin-6 secretion compared to NDF-containing SAS (P < 0.05). In HSE, SScDFs altered collagen as seen by a more mature and aligned fibrillar structure (P < 0.05). With SScDFs, enhanced stromal rigidity with increased collagen crosslinking (P < 0.05), up-regulation of LOXL4 expression (P < 0.01), and innate immune signaling genes were observed in both tissue models. Conversely, knockdown of LOXL4 suppressed rigidity, contraction, and α-smooth muscle actin expression in SScDFs in HSE, and TGFß-induced ECM aggregation and collagen crosslinking in SAS. CONCLUSION: A limitation to the development of effective therapeutics in SSc is the lack of in vitro human model systems that replicate human skin. Our findings demonstrate that SAS and HSE can serve as complementary in vitro skin-like models for investigation of the mechanisms and mediators that drive fibrosis in SSc and implicate a pivotal role for LOXL-4 in SSc pathogenesis.

Targeting MC1R depalmitoylation to prevent melanomagenesis in redheads.

Some genetic melanocortin-1 receptor (MC1R) variants responsible for human red hair color (RHC-variants) are consequently associated with increased melanoma risk. Although MC1R signaling is critically dependent on its palmitoylation primarily mediated by the ZDHHC13 protein-acyl transferase, whether increasing MC1R palmitoylation represents a viable therapeutic target to limit melanomagenesis in redheads is unknown. Here we identify a specific and efficient in vivo strategy to induce MC1R palmitoylation for therapeutic benefit. We validate the importance of ZDHHC13 to MC1R signaling in vivo by targeted expression of ZDHHC13 in C57BL/6J-MC1RRHC mice and subsequently inhibit melanomagenesis. By identifying APT2 as the MC1R depalmitoylation enzyme, we are able to demonstrate that administration of the selective APT2 inhibitor ML349 treatment efficiently increases MC1R signaling and represses UVB-induced melanomagenesis in vitro and in vivo. Targeting APT2, therefore, represents a preventive/therapeutic strategy to reduce melanoma risk, especially in individuals with red hair.

Oncostatin M and its role in fibrosis.

Oncostain M, a member of the IL-6 family of cytokines, is produced by immune cells in response to infections and tissue injury. OSM has a broad, often context-dependent effect on various cellular processes including differentiation, hematopoiesis, cell proliferation, and cell survival. OSM signaling is initiated by binding to type I (LIFRß/gp130) or type II (OSMRß/gp130) receptor complexes and involves activation of Janus kinase/signal transducer and activator of transcription, mitogen-activated protein kinase, and phosphatidylinositol-3-kinase. High levels of OSM have been detected in many chronic inflammatory conditions characterized by fibrosis, giving a rationale to target OSM for the treatment of these diseases. Here we discuss the current knowledge on the role of OSM in various stages of the fibrotic process including inflammation, vascular dysfunction, and activation of fibroblasts.

Targeting the upstream transcriptional activator of PD-L1 as an alternative strategy in melanoma therapy.

Programmed cell death ligand 1 (PD-L1) interacts with programmed cell death protein-1 (PD-1) as an immune checkpoint. Reactivating the immune response by inhibiting PD-L1 using therapeutic antibodies provides substantial clinical benefits in many, though not all, melanoma patients. However, transcriptional suppression of PD-L1 expression as an alternative therapeutic anti-melanoma strategy has not been exploited. Here we provide biochemical evidence demonstrating that ultraviolet radiation (UVR) induction of PD-L1 in skin is directly controlled by nuclear factor E2-related transcription factor 2 (NRF2). Depletion of NRF2 significantly induces tumor infiltration by both CD8+ and CD4+ T cells to suppress melanoma progression, and combining NRF2 inhibition with anti-PD-1 treatment enhanced its anti-tumor function. Our studies identify a critical and targetable PD-L1 upstream regulator and provide an alternative strategy to inhibit the PD-1/PD-L1 signaling in melanoma treatment.

Therapeutic Targeting of TAZ and YAP by Dimethyl Fumarate in Systemic Sclerosis Fibrosis.

Systemic sclerosis (scleroderma, SSc) is a devastating fibrotic disease with few treatment options. Fumaric acid esters, including dimethyl fumarate (DMF, Tecfidera; Biogen, Cambridge, MA), have shown therapeutic effects in several disease models, prompting us to determine whether DMF is effective as a treatment for SSc dermal fibrosis. We found that DMF blocks the profibrotic effects of transforming growth factor-ß (TGFß) in SSc skin fibroblasts. Mechanistically, we found that DMF treatment reduced nuclear localization of transcriptional coactivator with PDZ binding motif (TAZ) and Yes-associated protein (YAP) proteins via inhibition of the phosphatidylinositol 3 kinase/protein kinase B (Akt) pathway. In addition, DMF abrogated TGFß/Akt1 mediated inhibitory phosphorylation of glycogen kinase 3ß (GSK3ß) and a subsequent ß-transducin repeat-containing proteins (ßTRCP) mediated proteasomal degradation of TAZ, as well as a corresponding decrease of TAZ/YAP transcriptional targets. Depletion of TAZ/YAP recapitulated the antifibrotic effects of DMF. We also confirmed the increase of TAZ/YAP in skin biopsies from patients with diffuse SSc. We further showed that DMF significantly diminished nuclear TAZ/YAP localization in fibroblasts cultured on a stiff surface. Importantly, DMF prevented bleomycin-induced skin fibrosis in mice. Together, our work demonstrates a mechanism of the antifibrotic effect of DMF via inhibition of Akt1/GSK3ß/TAZ/YAP signaling and confirms a critical role of TAZ/YAP in mediating the profibrotic responses in dermal fibroblasts. This study supports the use of DMF as a treatment for SSc dermal fibrosis.

The Activation of Human Dermal Microvascular Cells by Poly(I:C), Lipopolysaccharide, Imiquimod, and ODN2395 Is Mediated by the Fli1/FOXO3A Pathway.

Endothelial cell (EC) dysfunction has been associated with inflammatory and autoimmune diseases; however, the factors contributing to this dysfunction have not been fully explored. Because activation of TLRs has been implicated in autoimmune diseases, the goal of this study was to determine the effects of TLR ligands on EC function. Human dermal microvascular ECs (HDMECs) treated with TLR3 [Poly(I:C)], TLR4 (LPS), and TLR7 (imiquimod) agonists showed decreased proliferation and a reduced total number of branching tubules in three-dimensional human dermal organoid ex vivo culture. In contrast, the TLR9 ligand class C, ODN2395, increased angiogenesis. The antiproliferative effects of TLR3, TLR4, and TLR7 ligands correlated with significant downregulation of a key regulator of vascular homeostasis, Fli1, whereas TLR9 increased Fli1 levels. Furthermore, Poly(I:C) and LPS induced endothelial to mesenchymal transition that was reversed by the pretreatment with TGF-ß neutralizing Ab or re-expression of Fli1. We showed that Fli1 was required for the HDMEC proliferation by transcriptionally repressing FOXO3A. In contrast to TLR9, which suppressed activation of the FOXO3A pathway, TLR3, TLR4, and TLR7 ligands activated FOXO3A as indicated by decreased phosphorylation and increased nuclear accumulation. The inverse correlation between Fli1 and FOXO3A was also observed in the vasculature of scleroderma patients. This work revealed opposing effects of TLR9 and TLR3, TLR4, and TLR7 on the key angiogenic pathways, Fli1 and FOXO3A. Our results provide a mechanistic insight into the regulation of angiogenesis by TLRs and confirm a central role of Fli1 in regulating vascular homeostasis.

Anti-connective tissue growth factor (CTGF/CCN2) monoclonal antibody attenuates skin fibrosis in mice models of systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is characterized by fibrosis of the skin and internal organs. Although the involvement of connective tissue growth factor (CTGF/CCN2) has been well-documented in SSc fibrosis, the therapeutic potential of targeting CTGF in SSc has not been fully investigated. Our aim was to examine the therapeutic potential of CTGF blockade in a preclinical model of SSc using two approaches: smooth muscle cell fibroblast-specific deletion of CTGF (CTGF knockout (KO)) or a human anti-CTGF monoclonal antibody, FG-3019. METHODS: Angiotensin II (Ang II) was administered for 14 days by subcutaneous osmotic pump to CTGF KO or C57BL/6 J mice. FG-3019 was administered intraperitoneally three times per week for 2 weeks. Skin fibrosis was evaluated by histology and hydroxyproline assay. Immunohistochemistry staining was used for alpha smooth muscle actin (αSMA), platelet-derived growth factor receptor ß (PDGFRß), pSmad2, CD45, von Willebrand factor (vWF), and immunofluorescence staining was utilized for procollagen and Fsp1. RESULTS: Ang II-induced skin fibrosis was mitigated in both CTGF KO and FG-3019-treated mice. The blockade of CTGF reduced the number of cells expressing PDGFRß, procollagen, αSMA, pSmad2, CD45, and Fsp1 in the dermis. In addition, inhibition of CTGF attenuated vascular injury as measured by the presence of vWF-positive cells. CONCLUSIONS: Our data indicate that inhibition of CTGF signaling presents an attractive therapeutic approach in SSc.

Blockade of PDGF Receptors by Crenolanib Has Therapeutic Effect in Patient Fibroblasts and in Preclinical Models of Systemic Sclerosis.

Systemic sclerosis (SSc) is a multi-organ fibrotic disease with few treatment options. Activated fibroblasts are the key effector cells in SSc responsible for the excessive production of collagen and the development of fibrosis. Platelet-derived growth factor (PDGF), a potent mitogen for cells of mesenchymal origin, has been implicated in the activation of SSc fibroblasts. Our aim was to examine the therapeutic potential of crenolanib, an inhibitor of PDGF receptor signaling, in cultured fibroblasts and in angiotensin II-induced skin and heart fibrosis. Crenolanib effectively inhibited proliferation and migration of SSc and healthy control fibroblasts and attenuated basal and transforming growth factor-ß-induced expression of CCN2/CTGF and periostin. In contrast to healthy control fibroblasts, SSc fibroblasts proliferated in response to PDGFAA, whereas a combination of PDGFAA and CCN2 was required to elicit a similar response in healthy control fibroblasts. PDGF receptor α mRNA correlated with CCN2 and other fibrotic markers in the skin of SSc patients. In mice challenged with angiotensin II, PDGF receptor α-positive cells were increased in the skin and heart. These PDGF receptor α-positive cells co-localized with PDGF receptor ß, procollagen, and periostin. Treatment with crenolanib attenuated the skin and heart fibrosis. Our data indicate that inhibition of PDGF signaling presents an attractive therapeutic approach for SSc.

Synergistic Role of Endothelial ERG and FLI1 in Mediating Pulmonary Vascular Homeostasis.

Endothelial cell (EC) activation underlies many vascular diseases, including pulmonary arterial hypertension (PAH). Several members of the E-twenty six (ETS) family of transcription factors are important regulators of the gene network governing endothelial homeostasis, and their aberrant expression is associated with pathological angiogenesis. The goal of this study was to determine whether deficiencies of the ETS family member, Friend leukemia integration 1 transcription factor (FLI1), and its closest homolog, ETS-related gene (ERG), are associated with PAH. We found that endothelial ERG was significantly reduced in the lung samples from patients with PAH, as well as in chronically hypoxic mice. Functional studies revealed that depletion of ERG or FLI1 in human pulmonary ECs led to increased expression of inflammatory genes, including IFN genes, whereas genes regulating endothelial homeostasis and cell-cell adhesion were down-regulated. Simultaneous knockdown of both ERG and FLI1 had synergistic or additive effects on the expression of these genes, suggesting that ERG and FLI1 coregulate at least a subset of their target genes. Functionally, knockdown of ERG and FLI1 induced cell monolayer permeability with a potency similar to that of vascular endothelial growth factor. Notably, stimulation of ECs with Toll-like receptor 3 ligand poly(I:C) suppressed ERG expression and induced ERG dissociation from the IFNB1 promoter, while promoting signal transducers and activators of transcription 1 (STAT1) recruitment. Consistent with the up-regulation of inflammatory genes seen in vitro, Erg and Fli1 double-heterozygote mice showed increased immune cell infiltration and expression of cytokines in the lung. In conclusion, loss of ERG and FLI1 might contribute to the pathogenesis of vascular lung complications through the induction of inflammation.

Dimethyl Fumarate ameliorates pulmonary arterial hypertension and lung fibrosis by targeting multiple pathways.

Pulmonary arterial hypertension (PAH) is a fatal condition for which there is no cure. Dimethyl Fumarate (DMF) is an FDA approved anti-oxidative and anti-inflammatory agent with a favorable safety record. The goal of this study was to assess the effectiveness of DMF as a therapy for PAH using patient-derived cells and murine models. We show that DMF treatment is effective in reversing hemodynamic changes, reducing inflammation, oxidative damage, and fibrosis in the experimental models of PAH and lung fibrosis. Our findings indicate that effects of DMF are facilitated by inhibiting pro-inflammatory NFκB, STAT3 and cJUN signaling, as well as ßTRCP-dependent degradation of the pro-fibrogenic mediators Sp1, TAZ and ß-catenin. These results provide a novel insight into the mechanism of its action. Collectively, preclinical results demonstrate beneficial effects of DMF on key molecular pathways contributing to PAH, and support its testing in PAH treatment in patients.

PDGFRß Is a Novel Marker of Stromal Activation in Oral Squamous Cell Carcinomas.

Carcinoma associated fibroblasts (CAFs) form the main constituents of tumor stroma and play an important role in tumor growth and invasion. The presence of CAFs is a strong predictor of poor prognosis of head and neck squamous cell carcinoma. Despite significant progress in determining the role of CAFs in tumor progression, the mechanisms contributing to their activation remain poorly characterized, in part due to fibroblast heterogeneity and the scarcity of reliable fibroblast surface markers. To search for such markers in oral squamous cell carcinoma (OSCC), we applied a novel approach that uses RNA-sequencing data derived from the cancer genome atlas (TCGA). Specifically, our strategy allowed for an unbiased identification of genes whose expression was closely associated with a set of bona fide stroma-specific transcripts, namely the interstitial collagens COL1A1, COL1A2, and COL3A1. Among the top hits were genes involved in cellular matrix remodeling and tumor invasion and migration, including platelet-derived growth factor receptor beta (PDGFRß), which was found to be the highest-ranking receptor protein genome-wide. Similar analyses performed on ten additional TCGA cancer datasets revealed that other tumor types shared CAF markers with OSCC, including PDGFRß, which was found to significantly correlate with the reference collagen expression in ten of the 11 cancer types tested. Subsequent immunostaining of OSCC specimens demonstrated that PDGFRß was abundantly expressed in stromal fibroblasts of all tested cases (12/12), while it was absent in tumor cells, with greater specificity than other known markers such as alpha smooth muscle actin or podoplanin (3/11). Overall, this study identified PDGFRß as a novel marker of stromal activation in OSCC, and further characterized a list of promising candidate CAF markers that may be relevant to other carcinomas. Our novel approach provides for a fast and accurate method to identify CAF markers without the need for large-scale immunostaining experiments.

MMP-12 deficiency attenuates angiotensin II-induced vascular injury, M2 macrophage accumulation, and skin and heart fibrosis.

MMP-12, a macrophage-secreted elastase, is elevated in fibrotic diseases, including systemic sclerosis (SSc) and correlates with vasculopathy and fibrosis. The goal of this study was to investigate the role of MMP-12 in cardiac and cutaneous fibrosis induced by angiotensin II infusion. Ang II-induced heart and skin fibrosis was accompanied by a marked increase of vascular injury markers, including vWF, Thrombospondin-1 (TSP-1) and MMP-12, as well as increased number of PDGFRß+ cells. Furthermore Ang II infusion led to an accumulation of macrophages (Mac3+) in the skin and in the perivascular and interstitial fibrotic regions of the heart. However, alternatively activated (Arg 1+) macrophages were mainly present in the Ang II infused mice and were localized to the perivascular heart regions and to the skin, but were not detected in the interstitial heart regions. Elevated expression of MMP-12 was primarily found in macrophages and endothelial cells (CD31+) cells, but MMP-12 was not expressed in the collagen producing cells. MMP-12 deficient mice (MMP12KO) showed markedly reduced expression of vWF, TSP1, and PDGFRß around vessels and attenuation of dermal fibrosis, as well as the perivascular fibrosis in the heart. However, MMP-12 deficiency did not affect interstitial heart fibrosis, suggesting a heterogeneous nature of the fibrotic response in the heart. Furthermore, MMP-12 deficiency almost completely prevented accumulation of Arg 1+ cells, whereas the number of Mac3+ cells was partially reduced. Moreover production of profibrotic mediators such as PDGFBB, TGFß1 and pSMAD2 in the skin and perivascular regions of the heart was also inhibited. Together, the results of this study show a close correlation between vascular injury markers, Arg 1+ macrophage accumulation and fibrosis and suggest an important role of MMP-12 in regulating these processes.

Proteome-wide analysis and CXCL4 as a biomarker in systemic sclerosis.

BACKGROUND: Plasmacytoid dendritic cells have been implicated in the pathogenesis of systemic sclerosis through mechanisms beyond the previously suggested production of type I interferon. METHODS: We isolated plasmacytoid dendritic cells from healthy persons and from patients with systemic sclerosis who had distinct clinical phenotypes. We then performed proteome-wide analysis and validated these observations in five large cohorts of patients with systemic sclerosis. Next, we compared the results with those in patients with systemic lupus erythematosus, ankylosing spondylitis, and hepatic fibrosis. We correlated plasma levels of CXCL4 protein with features of systemic sclerosis and studied the direct effects of CXCL4 in vitro and in vivo. RESULTS: Proteome-wide analysis and validation showed that CXCL4 is the predominant protein secreted by plasmacytoid dendritic cells in systemic sclerosis, both in circulation and in skin. The mean (±SD) level of CXCL4 in patients with systemic sclerosis was 25,624±2652 pg per milliliter, which was significantly higher than the level in controls (92.5±77.9 pg per milliliter) and than the level in patients with systemic lupus erythematosus (1346±1011 pg per milliliter), ankylosing spondylitis (1368±1162 pg per milliliter), or liver fibrosis (1668±1263 pg per milliliter). CXCL4 levels correlated with skin and lung fibrosis and with pulmonary arterial hypertension. Among chemokines, only CXCL4 predicted the risk and progression of systemic sclerosis. In vitro, CXCL4 down-regulated expression of transcription factor FLI1, induced markers of endothelial-cell activation, and potentiated responses of toll-like receptors. In vivo, CXCL4 induced the influx of inflammatory cells and skin transcriptome changes, as in systemic sclerosis. CONCLUSIONS: Levels of CXCL4 were elevated in patients with systemic sclerosis and correlated with the presence and progression of complications, such as lung fibrosis and pulmonary arterial hypertension. (Funded by the Dutch Arthritis Association and others.).

Endothelial GATA-6 deficiency promotes pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a chronic and progressive disease characterized by pulmonary vasculopathy with elevation of pulmonary artery pressure, often culminating in right ventricular failure. GATA-6, a member of the GATA family of zinc-finger transcription factors, is highly expressed in quiescent vasculature and is frequently lost during vascular injury. We hypothesized that endothelial GATA-6 may play a critical role in the molecular mechanisms underlying endothelial cell (EC) dysfunction in PAH. Here we report that GATA-6 is markedly reduced in pulmonary ECs lining both occluded and nonoccluded vessels in patients with idiopathic and systemic sclerosis-associated PAH. GATA-6 transcripts are also rapidly decreased in rodent PAH models. Endothelial GATA-6 is a direct transcriptional regulator of genes controlling vascular tone [endothelin-1, endothelin-1 receptor type A, and endothelial nitric oxide synthase (eNOS)], pro-inflammatory genes, CX3CL1 (fractalkine), 5-lipoxygenease-activating protein, and markers of vascular remodeling, including PAI-1 and RhoB. Mice with the genetic deletion of GATA-6 in ECs (Gata6-KO) spontaneously develop elevated pulmonary artery pressure and increased vessel muscularization, and these features are further exacerbated in response to hypoxia. Furthermore, innate immune cells including macrophages (CD11b(+)/F4/80(+)), granulocytes (Ly6G(+)/CD45(+)), and dendritic cells (CD11b(+)/CD11c(+)) are significantly increased in normoxic Gata6-KO mice. Together, our findings suggest a critical role of endothelial GATA-6 deficiency in development and disease progression in PAH.

Interferon-γ promotes vascular remodeling in human microvascular endothelial cells by upregulating endothelin (ET)-1 and transforming growth factor (TGF) ß2.

Systemic sclerosis (SSc) is a complex disease characterized by vascular alterations, activation of the immune system and tissue fibrosis. Previous studies have implicated activation of the interferon pathways in the pathogenesis of SSc. The goal of this study was to determine whether interferon type I and/or type II could play a pathogenic role in SSc vasculopathy. Human dermal microvascular endothelial cells (HDMVECs) and fibroblasts were obtained from foreskins of healthy newborns. The RT Profiler PCR Array System was utilized to screen for EndoMT genes. Treatment with IFN-α or IFN-γ downregulated Fli1 and VE-cadherin. In contrast, IFN-α and IFN-γ exerted opposite effects on the expression of α-SMA, CTGF, ET-1, and TGFß2, with IFN-α downregulating and IFN-γ upregulating this set of genes. Blockade of TGFß signaling normalized IFN-γ-mediated changes in Fli1, VE-cadherin, CTGF, and ET-1 levels, whereas upregulation of α-SMA and TGFß2 was not affected. Bosentan treatment was more effective than TGFß blockade in reversing the actions of IFN-γ, including downregulation of α-SMA and TGFß2, suggesting that activation of the ET-1 pathway plays a main role in the IFN-γ responses in HDMECs. IFN-γ induced expression of selected genes related to endothelial-to-mesenchymal transition (EndoMT), including Snail1, FN1, PAI1, TWIST1, STAT3, RGS2, and components of the WNT pathway. The effect of IFN-γ on EndoMT was mediated via TGFß2 and ET-1 signaling pathways. This study demonstrates distinct effects of IFN-α and IFN-γ on the biology of vascular endothelial cells. IFN-γ may contribute to abnormal vascular remodeling and fibrogenesis in SSc, partially via induction of EndoMT.

A role for estrogen receptor-α and estrogen receptor-ß in collagen biosynthesis in mouse skin.

Hormonal regulation of the dermal collagenous extracellular matrix has a key role in maintaining proper tissue homeostasis. However, the factors and pathways involved in this process are not fully defined. This study investigated the role of estrogen receptors (ERs) in the regulation of collagen biosynthesis in mice lacking either ERα or ERß. Collagen content was significantly increased in the skin of ERα(-/-) mice, as measured by acetic acid extraction and the hydroxyproline assay, and correlated with the decreased levels of matrix metalloproteinase (MMP)-15 and elevated collagen production by ERα(-/-) fibroblasts. In contrast, collagen content was decreased in the skin of ERß(-/-) mice, despite markedly increased collagen production by ERß(-/-) fibroblasts. However, expression of several MMPs, including MMP-8 and -15, was significantly elevated, suggesting increased degradation of dermal collagen. Furthermore, ERß(-/-) mice were characterized by significantly reduced levels of small leucine proteoglycans, lumican (Lum), and decorin (Dcn), leading to defects in collagen fibrillogenesis and possibly less stable collagen fibrils. ERα(-/-) mice also exhibited fibrils with irregular structure and size, which correlated with increased levels of Lum and Dcn. Together, these results demonstrate distinct functions of ERs in the regulation of collagen biosynthesis in mouse skin in vivo.

Angiotensin II induces skin fibrosis: a novel mouse model of dermal fibrosis.

INTRODUCTION: Systemic sclerosis (SSc) is an autoimmune inflammatory disorder of unknown etiology characterized by fibrosis of the skin and internal organs. Ang II (angiotensin II), a vasoconstrictive peptide, is a well-known inducer of kidney, heart, and liver fibrosis. The goal of this study was to investigate the profibrotic potential of Ang II in the mouse skin. METHODS: Ang II was administered by subcutaneous osmotic mini pumps to C57BL/6 male mice. Collagen-content measurements were performed with Gomori Trichrome staining and hydroxyproline assay. The mRNA expression level of collagens, TGF-ß1, TGF-ß2, TGF-ß3, CTGF, αSMA, CD3, Emr1, CD45/B220, MCP1, and FSP1 were quantified with real-time polymerase chain reaction (PCR). Immunostaining was performed for markers of inflammation and fibrosis, including, phospho-Smad2, αSMA, CD3, Mac3, CD45/B220, and CD163B. Fibrocytes were identified by double staining with CD45/FSP1 and CD45/PH4. Endothelial cells undergoing endothelial-to-mesenchymal transition (EndoMT) were identified by double staining with VE-cadherin/FSP1. RESULTS: Ang II-infused mice develop prominent dermal fibrosis in the area proximal to the pump, as shown by increased collagen and CTGF mRNA levels, increased hydroxyproline content, and more tightly packed collagen fibers. In addition, elevated mRNA levels of TGF-ß2 and TGF-ß3 along with increased expression of pSmad2 were observed in the skin of Ang II-treated mice. Dermal fibrosis was accompanied by an increased number of infiltrating fibrocytes, and an increased number of αSMA-positive cells, as well as CD163B⁺ macrophages in the upper dermis. This correlated with significantly increased mRNA levels of αSMA, Emr1, and MCP1. Infiltration of CD3-, CD45/B220-, and Mac3-positive cells was observed mainly in the hypodermis. Furthermore, an increased number of double-positive VE-cadherin/FSP1 cells were detected in the hypodermis only. CONCLUSIONS: This work demonstrates that Ang II induces both inflammation and fibrosis in the skin via MCP1 upregulation and accumulation of activated fibroblasts. Additionally, our data suggest that populations of these fibroblasts originate from circulating blood cells. Ang II infusion via osmotic minipumps could serve as a useful mouse model of skin fibrosis to gain new insights into pathogenic mechanisms and to test new antifibrotic therapies.

Fli1 is a negative regulator of estrogen receptor α in dermal fibroblasts.

Estrogen is an important regulator of dermal fibroblast functions, including extracellular matrix (ECM) synthesis. Estrogen mediates its effects through estrogen receptors (ERs), ERα and ERß; however, regulation of ERs in dermal fibroblasts remains poorly understood. Friend leukemia integration factor 1 (Fli1), a member of the Ets transcription factor family, has been shown to play a pivotal role in regulation of the ECM genes in dermal fibroblasts. The aim of this study was to examine a possible interaction between Fli1 and estrogen pathways, focusing on ERα. We show that treatment of human dermal fibroblasts with transforming growth factor-ß (TGF-ß) increases ERα protein and mRNA levels. Similarly, ERα expression was increased in response to small interfering RNA (siRNA)-mediated depletion of Fli1, suggesting that Fli1 is a mediator of the TGF-ß effects on ERα expression. Accordingly, we showed that Fli1 binds to the most proximal region of the ERα promoter, and dissociates from the promoter upon TGF-ß treatment. An inverse correlation between Fli1 and ERα expression levels was confirmed in cultured skin fibroblasts obtained from Fli1(+/-) mice and in the skin of Fli1(+/-) mice in vivo. This study supports a role of Fli1 as a negative regulator of the ERα gene in dermal fibroblasts.

Endothelial Fli1 deficiency impairs vascular homeostasis: a role in scleroderma vasculopathy.

Systemic sclerosis or scleroderma (SSc) is a complex autoimmune connective tissue disease characterized by obliterative vasculopathy and tissue fibrosis. The molecular mechanisms underlying SSc vasculopathy are largely unknown. Friend leukemia integration factor 1 (Fli1), an important regulator of immune function and collagen fibrillogenesis, is expressed at reduced levels in endothelial cells in affected skin of patients with SSc. To develop a disease model and to investigate the function of Fli1 in the vasculature, we generated mice with a conditional deletion of Fli1 in endothelial cells (Fli1 CKO). Fli1 CKO mice showed a disorganized dermal vascular network with greatly compromised vessel integrity and markedly increased vessel permeability. We show that Fli1 regulates expression of genes involved in maintaining vascular homeostasis including VE-cadherin, platelet endothelial cell adhesion molecule 1, type IV collagen, matrix metalloproteinase 9, platelet-derived growth factor B, and S1P(1) receptor. Accordingly, Fli1 CKO mice are characterized by down-regulation of VE-cadherin and platelet endothelial cell adhesion molecule 1, impaired development of basement membrane, and a decreased presence of alpha-smooth muscle actin-positive cells in dermal microvessels. This phenotype is consistent with a role of Fli1 as a regulator of vessel maturation and stabilization. Importantly, vascular characteristics of Fli1 CKO mice are recapitulated by SSc microvasculature. Thus, persistently reduced levels of Fli1 in endothelial cells may play a critical role in the development of SSc vasculopathy.