Shear stress modulates the expression of the atheroprotective protein Cx37 in endothelial cells

Abstract

High laminar shear stress (HLSS) is vasculoprotective partly through induction of Kruppel-like factor 2 (KLF2). Connexin37 (Cx37) is highly expressed in endothelial cells (ECs) of healthy arteries, but not in ECs overlying atherosclerotic lesions. Moreover, Cx37 deletion in apolipoprotein E-deficient (ApoE^−/−) mice increases susceptibility to atherosclerosis. We hypothesized that shear stress, through KLF2 modulation, may affect Cx37 expression in ECs. Cx37 expression and gap-junctional intercellular (GJIC) dye transfer are prominent in the straight portion of carotid arteries of ApoE^−/− mice, but are reduced at the carotid bifurcation, a region subjected to oscillatory flow. Shear stress-modifying vascular casts were placed around the common carotid artery of ApoE^−/− mice. Whereas Cx37 expression was conserved in HLSS regions, it was downregulated to ~ 50% in low laminar or oscillatory flow regions. To study the mechanisms involved, HUVECs or bEnd.3 cells were exposed to flow in vitro. Cx37 and KLF2 expression were increased after 24 h of HLSS. Interestingly, shear-dependent Cx37 expression was significantly reduced after silencing of KLF2. Moreover after exposure to simvastatin, a well-known KLF2 inducer, KLF2 binds to the Cx37 promoter region as shown by ChIP. Finally, GJIC dye transfer was highly reduced after KLF2 silencing and was increased after exposure to simvastatin. HLSS upregulates the expression of Cx37 in ECs by inducing its transcription factor KLF2, which increases intercellular communication. Therefore, this effect of shear stress on Cx37 expression may contribute to the synchronization of ECs and participate in the protective effect of HLSS.

Introduction

Atherosclerosis is characterized by specific intimal lesions, called atheromas, whereby lipids and leukocytes accumulate in the wall of arteries over time [1], [2]. Although most risk factors for atherosclerosis are systemic (such as smoking, diabetes mellitus, hypercholesterolemia, hypertension), the lesions do not distribute evenly over the arterial tree [3], [4]. They rather develop at specific sites such as around the origin of branching arteries, where blood flow is turbulent (oscillatory shear stress, OSS), or at the inner curvature of vessels, where low laminar shear stress (LLSS) is present. In fact, those two patterns of wall shear stress have been shown to induce endothelial dysfunction and ultimately to promote atherosclerosis [5], [6]. In contrast, high laminar shear stress (HLSS) is atheroprotective through the upregulation of protective anti-inflammatory mediators in endothelial cells (ECs). The transcription factor KLF2 has recently been shown to be a key mediator in this signal transduction: its induction represses pro-inflammatory pathways such as the NF-κB and MAP-K cascades, and activates the transcription of protective factors such as endothelial nitric oxide synthase (eNOS) and thrombomodulin [7].

Connexins (Cx) are a family of transmembrane proteins that form gap junctions. Six connexins oligomerize to form a hemichannel, and the assembly of two hemichannels from adjacent cells forms a gap junction channel. The accretion of a large number of gap junction channels forms a full gap junction, which enables the passage of ions and small metabolites and thus provides a direct communication pathway between cells in contact [8]. Synchronization of cells through gap junctions has been shown to play a crucial role for tissue homeostasis in various organs. Under some specific conditions, unapposed hemichannels have also been shown to provide a communication pathway between the cytoplasm and the extracellular space, enabling the release of paracrine mediators [9]. Three connexins can be found in ECs: Cx37, Cx40 and Cx43 [10]. As they each form channels with different electrical properties and permeability, it is believed that they each play a unique role in vascular physiology and disease. This study is focused on Cx37, which is known to form gap junction channels with a relatively large single channel conductance but quite specific and restricted permeability to large molecules depending on their ionic charge [11]. The discovery of a single nucleotide polymorphism in the human Cx37 gene, which has been correlated to atherosclerosis-related diseases (carotid and coronary artery stenosis, myocardial infarction [12]) led to further investigations of this protein in relation to atherosclerosis. In a healthy artery, Cx37 is highly expressed in ECs. However, this expression is lost in the endothelium overlying advanced atheromas, both in humans and in mouse models for the disease [13]. Moreover, Cx37 deletion increased the development of atherosclerosis in ApoE^−/− mice fed a high cholesterol diet. A role for this protein in monocyte adhesion has already been demonstrated, as Cx37 hemichannels appeared to allow the release of ATP, which reduces monocyte adhesion, and thus progression of atherosclerotic plaque development [14]. However, a role for this connexin in the initiation of atherosclerosis is also likely: given the strong inverse relationship between atherosclerosis and endothelial Cx37 expression, it appeared very likely that a local risk factor for atherosclerosis would affect Cx37 and that reduced endothelial synchronization by gap junctions would participate in endothelial dysfunction, the first step of atherosclerosis development. Shear stress patterns being one of the strongest local determinants for atherosclerosis, we hypothesized that blood flow sensed by the endothelium would regulate Cx37 expression. This hypothesis is supported by the fact that Cx37 is markedly less expressed in the venous circulation, which is exposed to LLSS, than in the arterial tree [15]. Moreover, the promoter region of this gene contains numerous CACCC elements, which are known as KLF-consensus binding sites.