Studies have demonstrated that nuclear factor of activated T cells 5 (NFAT5) is not only a tonicity-responsive transcription factor but also activated by other stimuli, so we aim to investigate whether NFAT5 participates in collateral arteries formation in rats. We performed femoral artery ligature (FAL) in rats for hindlimb ischaemia model and found that NFAT5 was up-regulated in rat adductors with FAL compared with sham group. Knockdown of NFAT5 with locally injection of adenovirus-mediated NFAT5-shRNA in rats significantly inhibited hindlimb blood perfusion recovery and arteriogenesis. Moreover, NFAT5 knockdown decreased macrophages infiltration and monocyte chemotactic protein-1 (MCP-1) expression in rats adductors. In vitro, with interleukin-1β (IL-1β) stimulation and loss-of-function studies, we demonstrated that NFAT5 knockdown inhibits MCP-1 expression in endothelial cells and chemotaxis of THP-1 cells regulated by ERK1/2 pathway. More importantly, exogenous MCP-1 delivery could recover hindlimb blood perfusion, promote arteriogenesis and macrophages infiltration in rats after FAL, which were depressed by NFAT5 knockdown.Besides, NFAT5 knockdown also inhibited angiogenesis in gastrocnemius muscles in rats. Our results indicate that NFAT5 is a critical regulator of arteriogenesis and angiogenesis via MCP-1-dependent monocyte recruitment, suggesting that NFAT5 may represent an alternative therapeutic target for ischaemic diseases. K E Y W O R D S arteriogenesis, hindlimb ischaemia, macrophage, monocyte chemoattractant protein 1, nuclear factor of activated T cells 5 | 2053 LIN et aL. S U PP O RTI N G I N FO R M ATI O N Additional supporting information may be found online in the Supporting Information section. How to cite this article: Lin X-C, Pan M, Zhu L-P, et al. NFAT5 promotes arteriogenesis via MCP-1-dependent monocyte recruitment.

BackgroundProphylactic exercise improves clinical outcomes in patients experiencing severe ischemic diseases. Previous studies have shown that exercise could alter the amount or content of circulating exosomes. However, little is known about the role of precursory exercise-derived circulating exosomes (Exe-Exo) in ischemic diseases. We therefore aimed to explore the function and mechanism of Exe-Exo in endogenous revascularization and perfusion recovery in peripheral arterial disease.Methods and ResultsWe first determined that 4 weeks of precursory treadmill exercise improved perfusion recovery on days 7, 14 and 21 after unilateral femoral artery ligation (FAL) but had no effect immediately after ligation. Then, local muscle delivery of Exe-Exo promotes arteriogenesis, angiogenesis and perfusion recovery, which could be abolished by GW4869, a well-recognized pharmacological agent inhibiting exosome release. This suggests that Exe-Exo mediated exercise-induced revascularization. In vitro, Exe-Exo enhanced endothelial cell proliferation, migration and tube formation. In addition, we identified miR-125a-5p as a novel exerkine through exosomal miRNA sequencing and RT-qPCR validation. Inhibition of miR-125a-5p abrogated the beneficial effects of Exe-Exo both in vivo and in vitro. Mechanistically, these exercise-afforded benefits were attributed to the exosomal miR-125a-5p downregulation of ECE1 expression and the subsequent activation of the AKT/eNOS downstream signaling pathway. Specifically, skeletal muscle may be a major tissue source of exercise-induced exosomal miR-125a-5p via fluorescence in situ hybridization.ConclusionsEndogenous circulating exosomal miR-125a-5p promotes exercise-induced revascularization via targeting ECE1 and activating AKT/eNOS downstream signaling pathway. Identify exosomal miR-125a-5p as a novel exerkine, and highlight its potential therapeutic role in the prevention and treatment of peripheral arterial disease.

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Background Endothelial progenitor cells (EPCs) play an important role in vascular repair. However, the functions of EPCs are obviously weakened in inflammatory microenvironment during restenosis. Therefore, we investigated whether omentin-1, an anti-inflammatory factor, can reduce neointima formation after carotid artery injury (CAI) in rats via improving EPCs functions damaged by inflammation and the underlying mechanisms. Methods Rats bone marrow-derived EPCs were isolated and cultured. EPCs were transfected with adenovirus vectors expressing human omentin-1 or green fluorescent protein (GFP). Rats received 2×106 EPCs with expressing omentin-1 or GFP by tail vein injection directly after CAI and again 24 h later. Hematoxylin-eosin staining and immunohistochemistry were used for analyzing neointimal hyperplasia. Besides, EPCs were treated with omentin-1 and tumor necrosis factor-α (TNF-α) in order to explore the underlying mechanism. Results Omentin-1 could significantly promote EPCs proliferation and tube formation, as well as inhibit apoptosis. EPCs overexpressed with omentin-1 using adenovirus vectors could extremely reduce the neointimal hyperplasia after CAI in rats. Besides, TNF-α could notably induce EPCs dysfunction including reduced proliferation, migration tube formation and increased apoptosis, which can be remarkably attenuated by omentin-1 via inhibiting of p38/CREB pathway. Besides, p38 agonist (anisomycin) could significantly reverse the protective effects of omentin-1 which attenuated the injury effects of TNF-α on EPCs. Conclusions EPCs overexpressed with omentin-1 can significantly reduce neointima formation after arterial injury by enhancing the functions of EPCs via inhibiting the p38/CREB pathway. Our results indicate that gene modified EPCs with omentin-1 may be an alternative strategy for the treatment of restenosis.