Fli1 Promotes Vascular Morphogenesis by Regulating Endothelial Potential of Multipotent Myogenic Progenitors.

[Figure: see text].

FLI1 and ERG protein degradation is regulated via Cathepsin B lysosomal pathway in human dermal microvascular endothelial cells.

OBJECTIVES: Friend leukemia integration 1 and erythroblast transformation-specific, important regulators of endothelial cell homeostasis, are reduced in microvascular endothelial cells in scleroderma patients, and their deficiency has been implicated in disease pathogenesis. The goal of this study was to identify the mechanisms involved in the protein turnover of friend leukemia integration 1 and erythroblast transformation-specific in microvascular endothelial cells. METHODS: The effects of lysosome and proteosome inhibitors on friend leukemia integration 1 and erythroblast transformation-specific levels were assessed by Western blotting and capillary morphogenesis. The effect of scleroderma and control sera on the levels of friend leukemia integration 1 and erythroblast transformation-specific was examined. RESULTS: The reduction in the protein levels of friend leukemia integration 1 and erythroblast transformation-specific in response to interferon α or Poly:(IC) was reversed by blocking either lysosomal (leupeptin and Cathepsin B inhibitor) or proteosomal degradation (MG132). MG132, leupeptin or CTSB-(i) also counteracted the anti-angiogenic effects of Poly:(IC) or interferon α. Scleroderma sera reduced protein levels of friend leukemia integration 1 and erythroblast transformation-specific in comparison to control sera. Treatment with CTSB(i) increased the levels of friend leukemia integration 1 and erythroblast transformation-specific in a majority of serum-treated samples. CONCLUSIONS: Inhibition of cathepsin B was effective in reversing the reduction of friend leukemia integration 1 and erythroblast transformation-specific protein levels after treatment with interferon α or scleroderma sera, suggesting that targeting cathepsin B may have a beneficial effect in SSc vascular disease.

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.

Active roles of dysfunctional vascular endothelium in fibrosis and cancer.

Chronic inflammation is the underlying pathological condition that results in fibrotic diseases. More recently, many forms of cancer have also been linked to chronic tissue inflammation. While stromal immune cells and myofibroblasts have been recognized as major contributors of cytokines and growth factors that foster the formation of fibrotic tissue, the endothelium has traditionally been regarded as a passive player in the pathogenic process, or even as a barrier since it provides a physical divide between the circulating immune cells and the inflamed tissues. Recent findings, however, have indicated that endothelial cells in fact play a crucial role in the inflammatory response. Endothelial cells can be activated by cytokine signaling and express inflammatory markers, which can sustain or exacerbate the inflammatory process. For example, the activated endothelium can recruit and activate leukocytes, thus perpetuating tissue inflammation, while sustained stimulation of endothelial cells may lead to endothelial-to-mesenchymal transition that contributes to fibrosis. Since chronic inflammation has now been recognized as a significant contributing factor to tumorigenesis, it has also emerged that activation of endothelium also occurs in the tumor microenvironment. This review summarizes recent findings characterizing the molecular and cellular changes in the vascular endothelium that contribute to tissue fibrosis, and potentially to cancer formation.

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.

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.

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.).

A Novel In Vivo Protocol for Molecular Study of Radiation-Induced Fibrosis in Head and Neck Cancer Patients.

OBJECTIVES: Radiation-induced fibrosis is a common complication for patients following head and neck cancer treatment. This study presents a novel minimally invasive protocol for molecular study of fibrosis in the stromal tissues. METHODS: Subjects with radiation-induced fibrosis in the head and neck who were at least 6 months post treatment received submental core needle biopsies, followed by molecular processing and quantification of gene expression for 14 select pro-inflammatory and pro-fibrotic genes. Control biopsies from the upper arm were obtained from the same subjects. Patients were followed up at 1 and 2 weeks to monitor for safety and adverse outcomes. RESULTS: Six subjects were enrolled and completed the study. No subjects experienced adverse outcomes or complication. An 18 gauge core biopsy needle with a 10 mm notch inserted for up to 60 seconds was needed. Subcutaneous tissue yielded 3 ng of RNA, amplified to 6 µg of cDNA, allowing for adequately sensitive quantitative polymerase chain reaction (qPCR) analysis of approximately 28 genes. CONCLUSIONS: This study demonstrates the safety and utility of a novel technique for the molecular study of fibrosis in head and neck cancer patients. Longitudinal studies of patients undergoing radiation therapy will allow for identification of molecular targets that contribute to the process of fibrosis in the head and neck.

Endoplasmic reticulum stress and endothelial dysfunction.

Prolonged perturbation of the endoplasmic reticulum (ER) leads to ER stress and unfolded protein response (UPR) and contributes to the pathogenesis of various chronic disorders. This review focuses on the role of ER stress and UPR in endothelial cells and the relevance of these processes to vascular diseases. Chronic activation of ER stress and UPR pathways in endothelial cells leads to increased oxidative stress and inflammation and often results in cell death. Because endothelial cells play a pivotal role in maintaining vascular homeostasis, various pathological conditions interfering with this homeostasis including homocysteinemia, hyperlipidemia, high glucose, insulin resistance, disturbed blood flow, and oxidative stress can lead to endothelial dysfunction in part through the activation of ER stress. We discuss recently discovered aspects of the role of ER stress/UPR in those pathological conditions. We also summarize recent findings implicating ER stress and UPR in systemic hypertension as well as pulmonary arterial hypertension. Finally, this review will highlight a novel role of UPR mediators in the process of angiogenesis.

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.

Increased expression of endoplasmic reticulum stress and unfolded protein response genes in peripheral blood mononuclear cells from patients with limited cutaneous systemic sclerosis and pulmonary arterial hypertension.

OBJECTIVE: Pulmonary arterial hypertension (PAH), a common complication of limited cutaneous systemic sclerosis (lcSSc), is associated with alterations of markers of inflammation and vascular damage in peripheral blood mononuclear cells (PBMCs). Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) have been implicated in autoimmune and inflammatory diseases. The goal of this study was to assess whether markers of ER stress and the UPR are present in PBMCs from lcSSc patients with PAH. METHODS: PBMCs were purified from 36 healthy controls, 32 lcSSc patients with PAH, and 34 lcSSc patients without PAH. Gene expression in healthy control PBMCs stimulated with thapsigargin was analyzed by DNA microarray. Genes were validated by quantitative real-time reverse transcription-polymerase chain reaction in PBMCs from healthy controls and lcSSc patients. RESULTS: Several ER stress/UPR genes, including BiP, activating transcription factor 4 (ATF-4), ATF-6, and a spliced form of X-box binding protein 1, were up-regulated in PBMCs from lcSSc patients, with the highest levels in patients with PAH. Thapsigargin up-regulated heat-shock proteins (HSPs) and interferon (IFN)-regulated genes in PBMCs from healthy controls. Selected HSP genes (particularly DnaJB1) and IFN-related genes were also found at significantly elevated levels in PBMCs from lcSSc patients, while IFN regulatory factor 4 expression was significantly decreased. There was a positive correlation between DnaJB1 and severity of PAH (measured by pulmonary artery pressure) (r = 0.56, P < 0.05) and between ER stress markers and interleukin-6 levels (r = 0.53, P < 0.0001) in PBMCs from lcSSc patients. CONCLUSION: This study demonstrates an association between select ER stress/UPR markers and lcSSc with PAH, suggesting that ER stress and the UPR may contribute to the altered function of circulating immune cells in lcSSc.

ERG regulates lymphatic vessel specification genes and its deficiency impairs wound healing-associated lymphangiogenesis.

OBJECTIVE: Rarefaction of blood and lymphatic vessels in the skin has been reported in SSc (systemic sclerosis, scleroderma). ERG and FLI1 are important regulators of angiogenesis, but their role in lymphatic vasculature is less known. The goal of this study was to determine the role of ERG and FLI1 in postnatal lymphangiogenesis and SSc lymphatic system defects. METHODS: Immunofluorescence was used to detect ERG and FLI1 in SSc and healthy control (HC) skin biopsies. Transcriptional analysis of ERG or FLI1 silenced human dermal lymphatic endothelial cells (LECs) was performed using microarrays. Effects of ERG/FLI1 deficiency on in vitro tubulogenesis in human dermal LECs was examined using a Matrigel assay. Erg and Fli1 endothelial specific knockouts and Erg lymphatic specific knockouts were generated to examine vessel regeneration in mice. RESULTS: ERG and FLI1 protein levels were reduced in the blood and lymphatic vasculature in SSc skin biopsies. ERG was shown to regulate genes involved in lymphatic vessel specification, including VEGFR3/FLT4, LYVE-1, SOX18, and PROX1, while FLI1 enhanced the function of ERG. ERG/FLT4 pathway regulated in vitro tubulogenesis in human LECs. Deficiency of Erg or Fli1 similarly impaired the function of blood vessels in mice. However, only Erg deficiency affected the regeneration of lymphatic vessels during wound healing. CONCLUSION: ERG and FLI1 are essential regulators of blood and lymphatic vessel regeneration. Deficiency of ERG and FLI1 in SSc endothelial cells, may contribute to impairment of blood and lymphatic vasculature in SSc patients.

Endothelial Erg Regulates Expression of Pulmonary Lymphatic Junctional and Inflammation Genes in Mouse Lungs Impacting Lymphatic Transport.

The ETS transcription factor ERG is a master regulator of endothelial gene specificity and highly enriched in the capillary, vein, and arterial endothelial cells. ERG expression is critical for endothelial barrier function, permeability, and vascular inflammation. A dysfunctional vascular endothelial ERG has been shown to impair lung capillary homeostasis, contributing to pulmonary fibrosis as previously observed in IPF lungs. Our preliminary observations indicate that lymphatic endothelial cells (LEC) in the human IPF lung also lack ERG. To understand the role of ERG in pulmonary LECs, we developed LEC-specific inducible Erg-CKO and Erg-GFP-CKO conditional knockout (CKO) mice under Prox1 promoter. Whole lung microarray analysis, flow cytometry, and qPCR confirmed an inflammatory and pro-lymphvasculogenic predisposition in Erg-CKO lung. FITC-Dextran tracing analysis showed an increased pulmonary interstitial lymphatic fluid transport from the lung to the axial lymph node. Single-cell transcriptomics confirmed that genes associated with cell junction integrity were downregulated in Erg-CKO pre-collector and collector LECs. Integrating Single-cell transcriptomics and CellChatDB helped identify LEC specific communication pathways contributing to pulmonary inflammation, trans-endothelial migration, inflammation, and Endo-MT in Erg-CKO lung. Our findings suggest that downregulation of lymphatic Erg crucially affects LEC function, LEC permeability, pulmonary LEC communication pathways and lymphatic transcriptomics.

Lung injury-induced activated endothelial cell states persist in aging-associated progressive fibrosis.

Progressive lung fibrosis is associated with poorly understood aging-related endothelial cell dysfunction. To gain insight into endothelial cell alterations in lung fibrosis we performed single cell RNA-sequencing of bleomycin-injured lungs from young and aged mice. Analysis reveals activated cell states enriched for hypoxia, glycolysis and YAP/TAZ activity in ACKR1+ venous and TrkB+ capillary endothelial cells. Endothelial cell activation is prevalent in lungs of aged mice and can also be detected in human fibrotic lungs. Longitudinal single cell RNA-sequencing combined with lineage tracing demonstrate that endothelial activation resolves in young mouse lungs but persists in aged ones, indicating a failure of the aged vasculature to return to quiescence. Genes associated with activated lung endothelial cells states in vivo can be induced in vitro by activating YAP/TAZ. YAP/TAZ also cooperate with BDNF, a TrkB ligand that is reduced in fibrotic lungs, to promote capillary morphogenesis. These findings offer insights into aging-related lung endothelial cell dysfunction that may contribute to defective lung injury repair and persistent fibrosis.

Endothelial cells recruit macrophages and contribute to a fibrotic milieu in bleomycin lung injury.

Systemic sclerosis (SSc) is a systemic autoimmune disease that causes inflammation, vasculopathy, and fibrosis of the skin and internal organs. One of the most severe complications of SSc involves the development of pulmonary fibrosis. Endothelial cell injury precedes the development of fibrosis, and is believed to be an initiating event. Therefore, we aimed to characterize the role of endothelial cells in the progression of pulmonary fibrosis, using a well-established bleomycin (BLM) model of pulmonary fibrosis. Endothelial cells were isolated by cell sorting, and the analysis of gene expression was performed with quantitative RT-PCR. Endothelial injury was induced between the first and second week, as shown by the elevated expression of the vascular injury markers matrix metallopeptidase-12 and von Willebrand factor. After injury, endothelial activation was indicated by the up-regulation of selectins, CCL chemokines, and inflammatory mediators, including complement anaphylatoxin receptors (C3aR and C5aR), oncostatin M, and leukemia inhibitory factor. The endothelial cell overexpression of fibrotic mediators, including connective tissue growth factor, plasminogen activator inhibitor-1, osteopontin, fibronectin, and fibroblast specific protein-1, was observed in the second and fourth weeks. This study suggests that endothelial cells actively contribute to the disease process via multiple mechanisms, including the recruitment of inflammatory cells and the establishment of a profibrotic environment during the development of BLM-induced pulmonary fibrosis.

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.

Single Cell Transcriptomics of Fibrotic Lungs Unveils Aging-associated Alterations in Endothelial and Epithelial Cell Regeneration.

Lung regeneration deteriorates with aging leading to increased susceptibility to pathologic conditions, including fibrosis. Here, we investigated bleomycin-induced lung injury responses in young and aged mice at single-cell resolution to gain insights into the cellular and molecular contributions of aging to fibrosis. Analysis of 52,542 cells in young (8 weeks) and aged (72 weeks) mice identified 15 cellular clusters, many of which exhibited distinct injury responses that associated with age. We identified Pdgfra + alveolar fibroblasts as a major source of collagen expression following bleomycin challenge, with those from aged lungs exhibiting a more persistent activation compared to young ones. We also observed age-associated transcriptional abnormalities affecting lung progenitor cells, including ATII pneumocytes and general capillary (gCap) endothelial cells (ECs). Transcriptional analysis combined with lineage tracing identified a sub-population of gCap ECs marked by the expression of Tropomyosin Receptor Kinase B (TrkB) that appeared in bleomycin-injured lungs and accumulated with aging. This newly emerged TrkB + EC population expressed common gCap EC markers but also exhibited a distinct gene expression signature associated with aberrant YAP/TAZ signaling, mitochondrial dysfunction, and hypoxia. Finally, we defined ACKR1 + venous ECs that exclusively emerged in injured lungs of aged animals and were closely associated with areas of collagen deposition and inflammation. Immunostaining and FACS analysis of human IPF lungs demonstrated that ACKR1 + venous ECs were dominant cells within the fibrotic regions and accumulated in areas of myofibroblast aggregation. Together, these data provide high-resolution insights into the impact of aging on lung cell adaptability to injury responses.

GATA6 coordinates cross-talk between BMP10 and oxidative stress axis in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a life-threatening condition characterized by a progressive increase in pulmonary vascular resistance leading to right ventricular failure and often death. Here we report that deficiency of transcription factor GATA6 is a shared pathological feature of PA endothelial (PAEC) and smooth muscle cells (PASMC) in human PAH and experimental PH, which is responsible for maintenance of hyper-proliferative cellular phenotypes, pulmonary vascular remodeling and pulmonary hypertension. We further show that GATA6 acts as a transcription factor and direct positive regulator of anti-oxidant enzymes, and its deficiency in PAH/PH pulmonary vascular cells induces oxidative stress and mitochondrial dysfunction. We demonstrate that GATA6 is regulated by the BMP10/BMP receptors axis and its loss in PAECs and PASMC in PAH supports BMPR deficiency. In addition, we have established that GATA6-deficient PAEC, acting in a paracrine manner, increase proliferation and induce other pathological changes in PASMC, supporting the importance of GATA6 in pulmonary vascular cell communication. Treatment with dimethyl fumarate resolved oxidative stress and BMPR deficiency, reversed hemodynamic changes caused by endothelial Gata6 loss in mice, and inhibited proliferation and induced apoptosis in human PAH PASMC, strongly suggesting that targeting GATA6 deficiency may provide a therapeutic advance for patients with PAH.

Elevated expression of cav-1 in a subset of SSc fibroblasts contributes to constitutive Alk1/Smad1 activation.

Previous studies have shown that the transforming growth factor (TGF)ß/Alk1/Smad1 signaling pathway is constitutively activated in a subset of systemic sclerosis (SSc) fibroblasts and this pathway is a critical regulator of CCN2 gene expression. Caveolin-1 (cav-1), an integral membrane protein and the main component of caveolae, has also been implicated in SSc pathogenesis. This study was undertaken to evaluate the role of caveolin-1 in Smad1 signaling and CCN2 expression in healthy and SSc dermal fibroblasts. We show that a significant subset of SSc dermal fibroblasts has up-regulated cav-1 expression in vitro, and that cav-1 up-regulation correlates with constitutive Smad1 phosphorylation. In addition, basal levels of phospho-Smad1 were down-regulated after inhibition of cav-1 in SSc dermal fibroblasts. Caveolin-1 formed a protein complex with Alk1 in dermal fibroblasts, and this association was enhanced by TGFß. By using siRNA against cav-1 and adenoviral cav-1 overexpression we demonstrate that activation of Smad1 in response to TGFß requires cav-1 and that cav-1 is sufficient for Smad-1 phosphorylation. We also show that cav-1 is a positive regulator of CCN2 gene expression, and that it is required for the basal and TGFß-induced CCN2 levels. In conclusion, this study has revealed an important role of cav-1 in mediating TGFß/Smad1 signaling and CCN2 gene expression in healthy and SSc dermal fibroblasts.