Shear stress-induced atherosclerotic plaque composition in ApoE−/− mice is modulated by connexin37
Introduction
Atherosclerosis is a progressive lipid-driven immuno-inflammatory disorder of arteries that evolves over decades. It starts with a localized blood flow related “priming” of endothelial cells (ECs), which is followed by accumulation of lipids and by migration of inflammatory cells and smooth muscle cells (SMCs) towards the intimal space [1]. Monocytes/macrophages are the predominant inflammatory cells present in atherosclerotic plaques, but polymorphonuclear cells (PMNs), T and B lymphocytes as well as dendritic cells have also been described [2], [3], [4]. The composition of each plaque will determine its stability and its propensity to rupture [5]. As the vast majority of clinical consequences of atherosclerosis (such as myocardial infarction and stroke) are caused by acute thrombus formation at the site of a plaque rupture, recent research has aimed its focus at better understanding the mechanisms underlying formation of stable versus unstable plaque phenotypes.
Plaque vulnerability is enhanced when macrophages secreting matrix metalloproteinases predominate, whereas a high content of SMCs and collagen fibers denotes a more stable plaque phenotype [5], [6]. In addition, it is now accepted that different subsets of macrophages (M1, M2, Mox) could either favor plaque rupture or promote a stabilization of the lesion [7], [8], [9], [10]. Interestingly, macrophages are remarkably plastic and can switch from one phenotype to another depending on environmental stimuli encountered [11].
Disturbed blood flow is thought to promote atherogenesis [12], [13], [14], [15] by acting either through shear stress – a friction force deforming ECs and affecting endothelial phenotype – or through mass transport dependent diffusion of lipids. A recent meta-analysis has shown that the majority of studies on shear stress and atherosclerosis favors this association, though some studies failed to detect it [16]. Moreover, the pattern of shear stress disturbance seems to be crucial for plaque phenotype; i.e. low laminar shear stress (LLSS) promotes formation of unstable plaques, whereas oscillatory shear stress (OSS) induces plaques with a stable phenotype [17], [18], [19]. Accumulation of inflammatory cells and an increase in elastase activity can be detected in plaques arising in regions of the arterial tree that were subjected to LLSS [20].
Connexins are a family of transmembrane proteins that can form gap junction channels, thus allowing direct cell–cell communication. They can also form hemichannels connecting the cytoplasm to the extracellular space, thus participating in autocrine or paracrine signaling [21]. Connexin37 (Cx37) is expressed in ECs, monocyte/macrophages, platelets and some vascular SMCs [22]. Its expression pattern is altered in advanced atherosclerotic plaques, with loss of Cx37 in ECs overlying plaques, and increased expression in macrophage foam cells [23]. It has also been shown to play a protective role in atherosclerosis: Cx37 deletion increased the development of atherosclerosis in Apolipoprotein E-deficient (ApoE−/−) mice [24]. Expression of Cx37 by monocytes has been shown to be atheroprotective since it inhibits their adhesion by an autocrine ATP-dependent mechanism [24]. We recently showed that endothelial Cx37 expression is regulated by shear stress patterns [25], which may contribute in the specific localization of the disease in the arterial tree. However, it remains unknown whether shear stress-dependent plaque composition is also affected by Cx37. Thus we have investigated the effect of Cx37 on the phenotype of plaques induced by LLSS or OSS.
Section snippets
Animals
ApoE−/− mice on a C57BL/6 background were purchased at Jackson Laboratory (Bar Harbor, ME) and were further crossed in our animal facility. Male ApoE−/−Cx37−/− and female ApoE−/−Cx37+/− mice, both on a C57BL/6 background, were interbred. Cx37 wild-type (WT) and knock-out (KO) alleles were detected by polymerase chain reaction genotyping, as previously described [24]. All mice were kept in conventional housing. They were fed a normal chow diet. Animals of 15-to-16 weeks of age were used. Animal
Effect of a constrictive cast on wall shear stress
Time averaged wall shear stress (TAWSS) was 11.2 ± 0.8 Pa at baseline, which reduced to 9.5 ± 0.5 Pa upstream and 9.9 ± 2.7 Pa downstream of the cast at 6 weeks after cast placement. The cast-induced changes in TAWSS remained unchanged at 9 weeks after cast placement (Fig. 1B, left panel). The oscillatory shear index (OSI) was zero at baseline, which increased to 0.09 ± 0.03 at 6 weeks and 0.06 ± 0.02 at 9 weeks (p = NS) downstream of the cast (Fig. 1B, right panel), and remained at zero
Placement of a cast creates different shear stress regions
The cast-based technique has been introduced previously [37] and has been used extensively since then [18], [19], [25]. It has been tested for its reproducible reduction in blood flow [17], and for shear stress-dependent effects on the vessel wall [17], [18], [19], [25]. In this study, we demonstrated the hemodynamic effects of this cast, including reduction of TAWSS, and appearance of oscillations in the downstream region. The cast defines three flow regions: one region with low TAWSS and low
Acknowledgments
This work was supported by grants from the Swiss National Science Foundation (310030_143343 to BRK; CRSII3-141811 to BRK, TPV and RK), a joint grant from the SNF, the Swiss Academy of Medical Sciences and the Velux Foundation (323630-123735 to AP), the Fondation Gustave et Simone Prévot (to BRK), the Fondation Carlos et Elsie de Reuter (to BRK) and the British Heart Foundation (RG/11/13/209550 to RK).
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