KLF4-Induced Connexin40 Expression Contributes to Arterial Endothelial Quiescence

Abstract

Shear stress, a blood flow-induced frictional force, is essential in the control of endothelial cell (EC) homeostasis. High laminar shear stress (HLSS), as observed in straight parts of arteries, assures a quiescent non-activated endothelium through the induction of Krüppel-like transcription factors (KLFs). Connexin40 (Cx40)-mediated gap junctional communication is known to contribute to a healthy endothelium by propagating anti-inflammatory signals between ECs, however, the molecular basis of the transcriptional regulation of Cx40 as well as its downstream effectors remain poorly understood. Here, we show that flow-induced KLF4 regulated Cx40 expression in a mouse EC line. Chromatin immunoprecipitation in ECs revealed that KLF4 bound to three predicted KLF consensus binding sites in the Cx40 promoter. HLSS-dependent induction of Cx40 expression was confirmed in primary human ECs. The downstream effects of Cx40 modulation in ECs exposed to HLSS were elucidated by an unbiased transcriptomics approach. Cell cycle progression was identified as an important downstream target of Cx40 under HLSS. In agreement, an increase in the proportion of proliferating cell nuclear antigen (PCNA)-positive ECs and a decrease in the proportion of ECs in the G0/G1 phase were observed under HLSS after Cx40 silencing. Transfection of communication-incompetent HeLa cells with Cx40 demonstrated that the regulation of proliferation by Cx40 was not limited to ECs. Using a zebrafish model, we finally showed faster intersegmental vessel growth and branching into the dorsal longitudinal anastomotic vessel in embryos knock-out for the Cx40 orthologs Cx41.8 and Cx45.6 . Most significant effects were observed in embryos with a mutant Cx41.8 encoding for a channel with reduced gap junctional function. Faster intersegmental vessel growth in Cx41.8 mutant embryos was associated with increased EC proliferation as assessed by PH3 immunostaining. Our data shows a novel evolutionary-conserved role of flow-driven KLF4-dependent Cx40 expression in endothelial quiescence that may be relevant for the control of atherosclerosis and diseases involving sprouting angiogenesis.

A connexin/ifi30 pathway bridges HSCs with their niche to dampen oxidative stress

“…First, we examined the status of HSPCs in connexin41.8 ( cx41.8 ) mutant embryos. This connexin has been previously described in zebrafish 32 , 33 and we recently reported that cx41.8 plays an important role on ECs proliferation 34 . In zebrafish embryos, cx41.8 is enriched in caudal ECs, as well as in HSPCs and macrophages, to a lesser extent, as scored by qPCR on sorted kdrl:GFP + , ikaros:GFP + , and mpeg1:GFP + cells (Fig.…”

Evolutionary Protection of Krüppel-Like Factors 2 and 4 in the Development of the Mature Hemovascular System

“…Also induced by laminar sheer stress, KLF4 promotes anti-inflammatory, and anti-thrombotic gene expression (25)(26)(27). Further, KLF4 promotes endothelial quiescence throughout a vascular bed via modulating gap junctional proteins and through the maintenance of proper proteostasis (28,29). These mechanisms protect against age-associated vascular disease, atherosclerosis, and ischemic stroke.…”

Endothelial connexins in vascular function

“…Targeting Cx40 in vivo reduces angiogenesis in the developing mouse retina (56) and inhibits tumor growth by reducing angiogenesis (57). In an unbiased transcriptomics approach, cell cycle progression was identified as an important downstream target of Cx40 under arterial level of wall shear stress (58), illustrating that the regulation of proliferation by Cx40 may well extend beyond capillary ECs. Moreover, zebrafish embryos knockout for the Cx40 orthologues Cx41.8 and Cx45.6 displayed faster intersegmental vessel growth and branching into the dorsal longitudinal anastomotic vessels, an effect that was associated with increased EC proliferation (58).…”

“…In an unbiased transcriptomics approach, cell cycle progression was identified as an important downstream target of Cx40 under arterial level of wall shear stress (58), illustrating that the regulation of proliferation by Cx40 may well extend beyond capillary ECs. Moreover, zebrafish embryos knockout for the Cx40 orthologues Cx41.8 and Cx45.6 displayed faster intersegmental vessel growth and branching into the dorsal longitudinal anastomotic vessels, an effect that was associated with increased EC proliferation (58). This suggests that the close relation between Cx40 and EC proliferation in sprouting angiogenesis is probably well conserved during evolution.…”