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OBJECTIVE: Rarefaction of blood and lymphatic vessels in the skin has been reported in systemic sclerosis (SSc) (scleroderma). E26 transformation-specific-related factor (ERG) and Friend leukemia virus-induced erythroleukemia 1 (FLI-1) are important regulators of angiogenesis, but their role in lymphatic vasculature is lesser known. The goal of this study was to determine the role of ERG and FLI-1 in postnatal lymphangiogenesis and SSc lymphatic system defects. METHODS: Immunofluorescence was used to detect ERG and FLI-1 in skin biopsy samples from patients with SSc and healthy controls. Transcriptional analysis of ERG or FLI-1-silenced human dermal lymphatic endothelial cells (LECs) was performed using microarrays. Effects of ERG and FLI-1 deficiency on in vitro tubulogenesis in human dermal LECs were examined using a Matrigel assay. ERG and FLI-1 endothelial-specific knockouts and ERG lymphatic-specific knockouts were generated to examine vessel regeneration in mice. RESULTS: ERG and FLI-1 protein levels were reduced in the blood and lymphatic vasculature in SSc skin biopsy samples. ERG levels were shown to regulate genes involved in lymphatic vessel specification, including vascular endothelial growth factor receptor 3/FLT-4, lymphatic vessel endothelial hyaluronan receptor 1, SOX-18, and prospero homeobox 1 (PROX-1), whereas FLI-1 enhanced the function of ERG. The ERG-FLT-4 pathway regulated in vitro tubulogenesis in human LECs. Deficiency of ERG or FLI-1 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 FLI-1 are essential regulators of blood and lymphatic vessel regeneration. Deficiency of ERG and FLI-1 in SSc endothelial cells may contribute to the impairment of blood and lymphatic vasculature in patients with SSc.
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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.
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Envelhecimento , Bleomicina , Células Endoteliais , Lesão Pulmonar , Pulmão , Fibrose Pulmonar , Animais , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Envelhecimento/patologia , Bleomicina/toxicidade , Humanos , Camundongos , Fibrose Pulmonar/patologia , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/genética , Pulmão/patologia , Pulmão/metabolismo , Lesão Pulmonar/patologia , Lesão Pulmonar/metabolismo , Lesão Pulmonar/etiologia , Receptor trkB/metabolismo , Receptor trkB/genética , Camundongos Endogâmicos C57BL , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/genética , Proteínas de Sinalização YAP/metabolismo , Masculino , Análise de Célula Única , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Feminino , Modelos Animais de DoençasRESUMO
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.
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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.
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Proteínas Morfogenéticas Ósseas , Fator de Transcrição GATA6 , Estresse Oxidativo , Hipertensão Arterial Pulmonar , Animais , Camundongos , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Proliferação de Células , Células Cultivadas , Hipertensão Pulmonar Primária Familiar/patologia , Fator de Transcrição GATA6/genética , Fator de Transcrição GATA6/metabolismo , Miócitos de Músculo Liso/metabolismo , Hipertensão Arterial Pulmonar/genética , Hipertensão Arterial Pulmonar/metabolismo , Hipertensão Arterial Pulmonar/patologia , Artéria Pulmonar/patologia , Remodelação VascularRESUMO
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.
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OBJECTIVE: To evaluate the levels of periostin in patients with systemic sclerosis (SSc) and their association with features of systemic sclerosis. METHODS: The levels of periostin were assessed in the serum of 106 SSc patients and 22 healthy controls and by immunofluorescence staining in cardiac tissue from 4 SSc patients and 4 controls. Serum periostin was measured via enzyme-linked immunosorbent assay. The results were analyzed using Mann-Whitney test or Kruskal-Wallis test followed by Dunn's multiple comparisons tests and Spearman's test for correlations. Cardiac tissue from SSc patients and controls was stained for periostin and co-stained for periostin and collagen type I using immunofluorescence. RESULTS: Periostin levels were higher in patients with SSc compared to controls and directly correlated to modified Rodnan skin score and echocardiography parameters of left ventricular measurements. Immunofluorescence staining in SSc cardiac tissue showed patchy periostin expression in all SSc patients, but not in controls. Furthermore, there was extensive periostin expression even in areas without collagen deposition, while all established fibrotic areas showed colocalization of collagen and periostin. There was no association between periostin levels and interstitial lung disease, pulmonary hypertension or other vascular complications. CONCLUSION: Periostin is elevated in SSc cardiac tissue in vivo and circulating levels of periostin are increased in SSc, correlating with the extent of disease duration, degree of skin fibrosis, and left ventricular structural assessments. Periostin may be a potential biomarker that can provide further pathogenic insight into cardiac fibrosis in SSc.
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Esclerodermia Localizada , Escleroderma Sistêmico , Humanos , Escleroderma Sistêmico/patologia , Esclerodermia Localizada/patologia , Fibrose , Pele/patologia , BiomarcadoresRESUMO
Vascular dysfunction is a hallmark of chronic diseases in elderly. The contribution of the vasculature to lung repair and fibrosis is not fully understood. Here, we performed an epigenetic and transcriptional analysis of lung endothelial cells (ECs) from young and aged mice during the resolution or progression of bleomycin-induced lung fibrosis. We identified the transcription factor ETS-related gene (ERG) as putative orchestrator of lung capillary homeostasis and repair, and whose function is dysregulated in aging. ERG dysregulation is associated with reduced chromatin accessibility and maladaptive transcriptional responses to injury. Loss of endothelial ERG enhances paracrine fibroblast activation in vitro, and impairs lung fibrosis resolution in young mice in vivo. scRNA-seq of ERG deficient mouse lungs reveales transcriptional and fibrogenic abnormalities resembling those associated with aging and human lung fibrosis, including reduced number of general capillary (gCap) ECs. Our findings demonstrate that lung endothelial chromatin remodeling deteriorates with aging leading to abnormal transcription, vascular dysrepair, and persistent fibrosis following injury.
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Fibrose Pulmonar , Idoso , Envelhecimento/genética , Animais , Bleomicina , Células Endoteliais/metabolismo , Fibrose , Humanos , Pulmão/patologia , Camundongos , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Transdução de Sinais , Regulador Transcricional ERG/genética , Regulador Transcricional ERG/metabolismoRESUMO
OBJECTIVE: The development of precision therapeutics for systemic sclerosis (SSc) has been hindered by the lack of models that accurately mimic the disease in vitro. This study was undertaken to design and test a self-assembled skin equivalent (saSE) system that recapitulates the cross-talk between macrophages and fibroblasts in cutaneous fibrosis. METHODS: SSc-derived dermal fibroblasts (SScDFs) and normal dermal fibroblasts (NDFs) were cultured with CD14+ monocytes from SSc patients or healthy controls to allow de novo stroma formation. Monocyte donor-matched plasma was introduced at week 3 prior to seeding keratinocytes to produce saSE with a stratified epithelium. Tissue was characterized by immunohistochemical staining, atomic force microscopy, enzyme-linked immunosorbent assay, and quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Stroma synthesized de novo from NDFs and SScDFs supported a fully stratified epithelium to form saSE. A thicker and stiffer dermis was generated by saSE with SScDFs, and more interleukin-6 and transforming growth factor ß (TGFß) was secreted by saSE with SScDFs compared to saSE with NDFs, regardless of the inclusion of monocytes. Tissue with SSc monocytes and plasma had amplified dermal thickness and stiffness relative to control tissue. Viable CD163+ macrophages were found within the stroma of saSE 5 weeks after seeding. Additionally, SSc saSE contained greater numbers of CD163+ and CD206+ macrophages compared to control saSE. TGFß blockade inhibited stromal stiffness to a greater extent in SSc saSE compared to control saSE. CONCLUSION: These data suggest reciprocal activation between macrophages and fibroblasts that increases tissue thickness and stiffness, which is dependent in part on TGFß activation. The saSE system may serve as a platform for preclinical therapeutic testing and for molecular characterization of SSc skin pathology through recapitulation of the interactions between macrophages and fibroblasts.
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Ativação de Macrófagos , Escleroderma Sistêmico , Células Cultivadas , Fibroblastos/metabolismo , Fibrose , Humanos , Escleroderma Sistêmico/patologia , Pele/patologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
[Figure: see text].
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Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Morfogênese , Mioblastos/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Células Endoteliais/citologia , Endotélio Vascular/embriologia , Endotélio Vascular/metabolismo , Mesoderma/citologia , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína MyoD/genética , Proteína MyoD/metabolismo , Mioblastos/citologia , Fator Regulador Miogênico 5/genética , Fator Regulador Miogênico 5/metabolismo , Proteína Proto-Oncogênica c-fli-1/genéticaRESUMO
Microvascular injury is considered an initial event in the pathogenesis of scleroderma and endothelial cells are suspected of being the target of the autoimmune process seen in the disease. EBV has long been proposed as a trigger for autoimmune diseases, including scleroderma. Nevertheless, its contribution to the pathogenic process remains poorly understood. In this study, we report that EBV lytic antigens are detected in scleroderma dermal vessels, suggesting that endothelial cells might represent a target for EBV infection in scleroderma skin. We show that EBV DNA load is remarkably increased in peripheral blood, plasma and circulating monocytes from scleroderma patients compared to healthy EBV carriers, and that monocytes represent the prominent subsets of EBV-infected cells in scleroderma. Given that monocytes have the capacity to adhere to the endothelium, we then investigated whether monocyte-associated EBV could infect primary human endothelial cells. We demonstrated that endothelial cells are infectable by EBV, using human monocytes bound to recombinant EBV as a shuttle, even though cell-free virus failed to infect them. We show that EBV induces activation of TLR9 innate immune response and markers of vascular injury in infected endothelial cells and that up-regulation is associated with the expression of EBV lytic genes in infected cells. EBV innate immune modulation suggests a novel mechanism mediating inflammation, by which EBV triggers endothelial cell and vascular injury in scleroderma. In addition, our data point to up-regulation of EBV DNA loads as potential biomarker in developing vasculopathy in scleroderma. These findings provide the framework for the development of novel therapeutic interventions to shift the scleroderma treatment paradigm towards antiviral therapies.
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Endotélio Vascular/patologia , Infecções por Vírus Epstein-Barr/complicações , Imunidade Inata , Escleroderma Sistêmico/imunologia , Pele/patologia , Adulto , Idoso , Biópsia , DNA Viral/isolamento & purificação , Células Endoteliais/imunologia , Células Endoteliais/patologia , Endotélio Vascular/citologia , Endotélio Vascular/imunologia , Infecções por Vírus Epstein-Barr/sangue , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/virologia , Feminino , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/imunologia , Herpesvirus Humano 4/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/metabolismo , Escleroderma Sistêmico/sangue , Escleroderma Sistêmico/patologia , Escleroderma Sistêmico/virologia , Pele/irrigação sanguínea , Pele/imunologia , Receptor Toll-Like 9/metabolismo , Carga Viral , Adulto JovemRESUMO
BACKGROUND: Aldehyde dehydrogenase 1 family member A1 (RALDH1)-producing dermal dendritic cells (DCs), a conventional DC subset regulating skin fibrosis, are decreased in the involved skin of patients with systemic sclerosis (SSc). In this study, we investigated the contribution of Fli1 deficiency, a potential predisposing factor of SSc, to the phenotypical alteration of RALDH1-producing dermal DCs by using SSc model mice and SSc skin samples. METHODS: Bleomycin (BLM)-induced skin fibrosis was generated with Fli1+/- and wild-type mice. The proportions of DC and CD4+ T cell subsets were determined by flow cytometry in the dermis of BLM-treated mice. Fli1 expression in dermal DCs was evaluated by immunofluorescence with skin samples of SSc and healthy control subjects. RESULTS: RALDH activity of dermal DCs was significantly decreased in BLM-treated Fli1+/- mice compared with BLM-treated wild-type mice, whereas the proportion of CD103-CD11b- dermal DCs, a major DC subset producing RALDH1 in response to BLM injection, was comparable between groups. Relevant to this finding, the proportion of regulatory T cells (Tregs) in the dermis was decreased in BLM-treated Fli1+/- mice relative to BLM-treated wild-type mice, while the proportions of Th1, Th2 and Th17 cells were unaltered. In the involved skin of SSc patients, Fli1 was downregulated in CD11c+ cells, including dermal DCs. CONCLUSIONS: Fli1 deficiency inhibits RALDH1 activity of CD103-CD11b- dermal DCs and related induction of Tregs in BLM-treated mice. Considering Fli1 reduction in SSc dermal DCs, Fli1deficiency may impair the dermal DC-Treg system, contributing to the development of skin fibrosis in SSc.
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Família Aldeído Desidrogenase 1/metabolismo , Retinal Desidrogenase/metabolismo , Escleroderma Sistêmico , Linfócitos T Reguladores , Animais , Células Dendríticas , Modelos Animais de Doenças , Fibrose , Humanos , Células de Langerhans , Camundongos , Proteína Proto-Oncogênica c-fli-1/genética , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/patologia , Pele/patologiaRESUMO
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.
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Catepsina B/metabolismo , Derme/metabolismo , Células Endoteliais/metabolismo , Lisossomos/metabolismo , Microvasos/metabolismo , Proteólise , Proteína Proto-Oncogênica c-fli-1/metabolismo , Adulto , Idoso , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Regulador Transcricional ERG/metabolismoRESUMO
OBJECTIVE: In prevous studies, we established a new animal model, KLF5+/- ;Fli-1+/- mice, in which fundamental pathologic features of systemic sclerosis (SSc) are broadly recapitulated. SSc vasculopathy is believed to occur as a result of impaired vascular remodeling, but its detailed mechanism of action remains unknown. To address this, the present study investigated the properties of dermal microvascular endothelial cells (DMECs), bone marrow-derived endothelial progenitor cells (BM-EPCs), and bone marrow-derived mesenchymal stem cells (BM-MSCs), a precursor of pericytes, in KLF5+/- ;Fli-1+/- mice. METHODS: Neovascularization and angiogenesis were assessed in KLF5+/- ;Fli-1+/- mice by in vivo Matrigel plug assay and in vitro tube formation assay, respectively. The properties of mouse BM-EPCs and BM-MSCs were assessed with in vitro studies. Dermal vasculature was visualized in vivo by injecting the mice with fluorescein isothiocyanate-conjugated dextran. RESULTS: Neovascularization was diminished in skin-embedded Matrigel plugs from KLF5+/- ;Fli-1+/- mice. DMECs from KLF5+/- ;Fli-1+/- mice showed defective tubulogenic activity, decreased expression of VE-cadherin and CD31, and an imbalance in the expression of Notch1/Dll4, suggesting that angiogenesis and anastomosis are disturbed. KLF5+/- ;Fli-1+/- mouse BM-MSCs exhibited enhanced proliferation and migration and increased collagen production following stimulation with transforming growth factor ß1, indicating that these cells differentiate preferentially into myofibroblasts rather than pericytes. KLF5+/- ;Fli-1+/- mouse BM-EPCs displayed a transition toward mesenchymal cells, suggesting that vasculogenesis is impaired. Wound healing was delayed in KLF5+/- ;Fli-1+/- mice (mean ± SD healing time 15.67 ± 0.82 days versus 13.50 ± 0.84 days; P = 0.0017), and the vascular network was poorly developed in wound scar tissue. CONCLUSION: The characteristics observed in the KLF5+/- ;Fli-1+/- mouse model - specifically, impaired neovascularization and vascular maturation - are similar to those observed in human SSc, and could be at least partially attributable to the induction of SSc-like properties in DMECs, BM-EPCs, and BM-MSCs. These findings indicate the critical contribution of Klf5 and Fli1 deficiency in vascular cells and related cell precursors to the development of SSc vasculopathy.
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Células Endoteliais/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Células-Tronco Mesenquimais/metabolismo , Neovascularização Patológica/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , Escleroderma Sistêmico/metabolismo , Vasculite/metabolismo , Animais , Modelos Animais de Doenças , Células Endoteliais/patologia , Fatores de Transcrição Kruppel-Like/genética , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Knockout , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Proteína Proto-Oncogênica c-fli-1/genética , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/patologia , Vasculite/genética , Vasculite/patologiaRESUMO
Scleroderma (SSc) is an autoimmune connective tissue disease characterized by immune dysregulation, vasculopathy, and fibrosis. We have previously demonstrated that low Fli1 expression in SSc fibroblasts and endothelial cells plays an important role in SSc pathogenesis. Cells of myeloid and lymphoid origin also express Fli1 and are dysregulated in patients with SSc, playing key roles in disease pathogenesis. However, the role for immune Fli1 in SSc is not yet clear. Our aim was to elucidate whether Fli1 contributes to the immune dysregulation seen in SSc. Comparison of the expression of Fli1 in monocytes, B- and T-cell fractions of PBMCs isolated from SSc patients and healthy controls (HC), showed an increase in Fli1 levels in monocytes. We used siRNA transfected human myeloid cells and mouse peritoneal macrophages obtained from Fli1 flox/flox LysMCre+/+ mice, and found that markers of alternative macrophage activation were increased with Fli1 deletion. Coculture of Fli1-deficient myeloid cells and primary human or mouse fibroblasts resulted in a potent induction of collagen type I, independent of TGFß upregulation. We next analyzed global gene expression profile in response to Fli1 downregulation, to gain further insight into the molecular mechanisms of this process and to identify differentially expressed genes in myeloid cells. Of relevance to SSc, the top most upregulated pathways were hallmark IFN-γ and IFN-α response. Additionally, several genes previously linked to SSc pathogenesis and fibrosis in general were also induced, including CCL2, CCL7, MMP12, and CXCL10. ANKRD1, a profibrotic transcription co-regulator was the top upregulated gene in our array. Our results show that Fli1-deficient myeloid cells share key features with cells from SSc patients, with higher expression of profibrotic markers and activation of interferon responsive genes, thus suggesting that dysregulation of Fli1 in myeloid cells may contribute to SSc pathogenesis.
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Células Mieloides/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/imunologia , Escleroderma Sistêmico/metabolismo , Animais , Doenças Autoimunes , Células Cultivadas , Técnicas de Cocultura , Modelos Animais de Doenças , Regulação para Baixo , Fibroblastos/metabolismo , Fibrose/metabolismo , Fibrose/patologia , Expressão Gênica , Voluntários Saudáveis , Humanos , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Monócitos/metabolismo , RNA Interferente Pequeno , Pele/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
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.
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Fibroblastos/fisiologia , Proteína-Lisina 6-Oxidase/fisiologia , Escleroderma Sistêmico/etiologia , Escleroderma Sistêmico/patologia , Pele/patologia , Adulto , Bioengenharia , Feminino , Fibrose/etiologia , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Cultura de Tecidos , Adulto JovemRESUMO
Systemic sclerosis is an autoimmune disease characterized by fibrosis of skin and internal organs, vasculopathy, and dysregulation of immune system. A diagnostically important feature of immunological abnormalities in systemic sclerosis is the presence of circulating antinuclear antibodies, which may be detected in 90-95% of patients with either of the four main laboratory methods: immunofluorescence, enzyme-linked immunosorbent assay, immunodiffusion, and immunoblotting. There are several antinuclear antibodies specific for systemic sclerosis. These include antibodies against topoisomerase (anti-TOPO I), kinetochore proteins (ACA), RNA polymerase enzyme (anti-RNAP III), ribonuclear proteins (anti-U11/U12 RNP, anti-U1 RNP, anti-U3 RNP) and nucleolar antigens (anti-Th/To, anti-NOR 90, anti-Ku, antiRuvBL1/2, and anti-PM/Scl). Autoantibodies specific for systemic sclerosis have been linked to distinct clinical features. Therefore, detecting a particular antibody type is important in predicting a possible organ involvement and prognosis and may have an impact on monitoring and treatment.
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Anticorpos Antinucleares/imunologia , Autoimunidade , Suscetibilidade a Doenças , Escleroderma Sistêmico/etiologia , Autoanticorpos/imunologia , Autoantígenos/imunologia , Biomarcadores , Diagnóstico Diferencial , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Prevalência , Escleroderma Sistêmico/diagnóstico , Escleroderma Sistêmico/epidemiologia , Escleroderma Sistêmico/metabolismoRESUMO
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.