Endothelial cell-derived pro-fibrotic factors increase TGF-β1 expression by smooth muscle cells in response to cycles of hypoxia-hyperoxia

Background: The vascular pathology of peripheral artery disease (PAD) encompasses abnormal microvascular architecture and fibrosis in response to ischemia-reperfusion (I/R) cycles. We aimed to investigate the mechanisms by which pathological changes in the microvasculature direct fibrosis in the context of I/R. Methods: Primary human aortic endothelial cells (ECs) were cultured under cycles of normoxia-hypoxia (NH) or normoxia-hypoxia-hyperoxia (NHH) to mimic I/R. Primary human aortic smooth muscle cells (SMCs) were cultured and treated with media from the ECs. Findings: The mRNA and protein expression of the pro-fibrotic factors platelet derived growth factor (PDGF)-BB and connective tissue growth factor (CTGF) were significantly upregulated in ECs undergoing NH or NHH cycles. Treatment of SMCs with media from ECs undergoing NH or NHH cycles led to significant increases in TGF-β1, TGF-β pathway signaling intermediates, and collagen expression. Addition of neutralizing antibodies against PDGF-BB and CTGF to the media blunted the increases in TGF-β1 and collagen expression. Treatment of SMCs with PAD patient-derived serum also led to increased TGF-β1 levels. Interpretation: In an in-vitro model of I/R, which recapitulates the pathophysiology of PAD, increased secretion of PDGF-BB and CTGF by ECs was shown to be predominantly driving TGF-β1-mediated expression by SMCs. These cell culture experiments help elucidate the mechanism and interaction between ECs and SMCs in microvascular fibrosis associated with I/R. Thus, targeting these pro-fibrotic factors may be an effective strategy to combat fibrosis in response to cycles of I/R. Funding: National Institute on Aging at the National Institutes of Health grant number R01AG064420. Research in context: Evidence before this study: Previous studies in gastrocnemius biopsies from peripheral artery disease (PAD) patients showed that transforming growth factor beta 1 (TGF-β1), the most potent inducer of pathological fibrosis, is increased in the vasculature of PAD patients and correlated with collagen deposition. However, the exact cellular source of TGF-β1 remained unclear. Added value of this study: Exposing cells to cycles of normoxia-hypoxia-hyperoxia (NHH) resulted in pathological changes that are consistent with human PAD. This supports the idea that the use of NHH may be a reliable, novel in vitro model of PAD useful for studying associated pathophysiological mechanisms. Furthermore, pro-fibrotic factors (PDGF-BB and CTGF) released from endothelial cells were shown to induce a fibrotic phenotype in smooth muscle cells. This suggests a potential interaction between these cell types in the microvasculature that drives increased TGF-β1 expression and collagen deposition. Thus, targeting these pro-fibrotic factors may be an effective strategy to combat fibrosis in response to cycles of ischemia-reperfusion.