Background/Purpose: Since vascular manifestations such as Raynaud’s phenomenon and morphological changes on nailfold capillaroscopy often precede the onset of other clinical manifestations of systemic sclerosis (SSc), the identification of pathways linking vasculopathy to organ fibrosis might thus provide important insights into early disease mechanisms and allow early targeted intervention for both fibrotic and vascular events. Recently, activating transcription factor 3 (ATF3), a member of the ATF/cAMP-responsive element binding (CREB) family of transcription factors, which regulates cellular response to stress, was characterized as a downstream mediator of canonical TGF-β signaling in fibroblast activation and tissue fibrosis. In this study we aimed to investigate the role of ATF3 linking vasculopathy to organ fibrosis.

Methods: ATF3 expression in vessels of either human skin samples of SSc patients and healthy volunteers, or murine skin and lung tissue of Fra2 transgenic (tg) mice was analyzed by multi-chanel immunofluorescence (IF) and confocal laser scanning microscopy. Human umbilical vein (HUVEC), human microvascular endothelial cells (HMEC) and smooth muscle cells (SMC) as well as immortalized blood (BEC-TI) and lymphatic (LEC-TI) endothelial cells were transfected with pCMV-ATF3 and pCMV plasmid constructs and cell proliferation and survival were measured by microtiter tetrazolium (MTT) and apoptosis assays, migration by chemotaxis-induced migration assay, and tube formation in a matrigel basement membrane matrix. Additionally, ex vivo mouse fetal metatarsal assays were performed using ATF3 knockout and wildtype littermates to study the angiogenetic process.

Results: ATF3 was significantly upregulated in fibroblasts of skin biopsies of SSc patients and of various organs of fibrosis models. ATF3 deficiency ameliorated fibrosis in various mouse models including Fra2 tg mice, a genetic model resembling both fibrosis and vasculopathy. Notably, ATF3 was significantly upregulated in vascular cells of fibrotic tissues of SSc patients and Fra2 tg mice. Multi-color IF and confocal laser scanning microscopy of skin and lung biopsies of SSc patients and Fra2 tg mice revealed an increased expression of ATF3 especially in microvascular endothelial cells and smooth muscle cells. ATF3 overexpression in smooth muscle cells led to an extensively enhanced proliferation and increased migratory capacity whereas endothelial cells showed a SSc-like phenotype with reduced proliferation and migration. After ATF3 overexpression, tube formation capacity was completely altered as assessed by cumulative tube length, tube numbers and capillary sprouting. To investigate vessel outgrowth from a different perspective, we used the ex vivo fetal mouse metatarsal assay. ATF3 knockout mice showed a completely altered angiogenic response as assessed by tube length, number of branches and number junctions compared to wildtype controls.

Conclusion: We identified ATF3 as a new profibrotic factor in endogenous activated fibroblasts as wells as in disturbed vasculature suggesting ATF3 as a potential therapeutic target intervening both fibrotic and vascular manifestations.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/activating-transcription-factor-3-a-new-linkage-between-vasculopathy-and-organ-fibrosis-in-systemic-sclerosis/