The relation between the location of atherosclerotic plaques in the vasculature and the presence of low and/or oscillatory wall shear stress has been recognized for several decades [311]. It is increasingly recognized that ECs located in
athero-‐susceptible sites have specific gene expression patterns in vivo, which may be reproduced in in vitro studies [41, 312]. These specific expression patterns include connexins. Elegant detailed studies by Gabriels and Paul onto the expression and distribution of vascular connexins in rat aorta in situ revealed that Cx40 and Cx37 were present in nearly all ECs of the thoracic and abdominal aorta at cell-‐cell junctions. In contrast, Cx43 was undetectable in most of the arterial endothelium but was specifically localized at the downstream edge of the ostia of branching vessels and at bifurcations known to experience disturbed blood flow [214]. The link between shear stress patterns and connexin expression was further strengthened by the in vivo observation of Inai et al. that the endothelium on the upstream and downstream surfaces of cardiac valves have different connexin expression patterns. ECs on upstream surfaces were found to have 70-‐ to 200-‐fold higher Cx43 levels compared to downstream surfaces. However, the expression of Cx37 was almost equal and Cx40 was not detected at all [313]. However, the exact mechanisms inducing this specific expression patterns and their contribution to vascular (patho)physiology have been only partially resolved.
1.4.5.1 Cx43
Cowan and colleagues performed the first in vitro experiments assessing the levels of Cx43 in response shear stress. They subjected cultured ECs to a laminar shear stress of 15 dynes/cm2 resulting in an 4-‐fold increase of Cx43 mRNA levels after 1 hour, which remained elevated during the 16 hour experiment [314]. In vitro experiments using a parallel plate chamber demonstrated that Cx43 mRNA expression levels were increased 6-‐ to 8-‐fold after 5 hours in regions of flow
disturbance compared to no-‐flow and remained increased after 16h.
Interestingly, ECs exposed to 30 hours of disturbed flow showed reduced dye transfer (lucifer yellow) compared to cells exposed to undisturbed flow. They concluded that shear stress gradients in regions of disturbed flow regulate EC intercellular communication through both the functionality and the expression of Cx43 [315]. Another study used the bEnd.3 endothelial cell line (PymT-‐
transformed mouse ECs) grown in elastic tubes and exposed to different levels of hydrostatic pressure, shear stress and circumferential stretch and the investigated the expression of Cx43. They showed that Cx43 was sensitive to a change in hemodynamic environment and in particular to OSS and circumferential stretch (4% greater diameter) but not to pressure [137].
Although it became clear that Cx43 is up-‐regulated in ECs exposed to disturbed flow no clear regulatory mechanism was demonstrated. Using different flow profiles and a specific inhibitor of mitogen-‐activated protein kinase kinase (PD-‐
98059) Bao et al. demonstrated that the temporal gradients in shear activates ERK1 and ERK2 and induces the expression of c-‐fos and Cx43 [316]. Altogether, we can conclude that Cx43 is up-‐regulated in ECs exposed to disturbed flow patterns and that mitogen-‐activated proteins kinase pathways are possibly involved in its regulation [305, 317, 318].
1.4.5.2 Cx37
It has been shown that Cx37 is highly expressed in ECs of straight regions of the common carotid artery and virtually absent in ECs at carotid bifurcations in young ApoE-‐/-‐ mice. Accordingly, the carotid bifurcation regions with decreased Cx37 expression showed almost no cell-‐cell communication in a scrape loading
assay with propidium iodide, a Cx37 permeable dye [126]. Using shear stress-‐
modifying vascular casts imposing specific flow patterns in the straight parts of carotid arteries of mice it was shown that Cx37 was down-‐regulated in regions of low laminar and oscillatory shear stress but conserved in regions of high laminar shear stress, unambiguously demonstrating that Cx37 is a shear stress regulated gene. In vitro exposure of cultures of bEnd.3 cells, HUVECs or human coronary artery ECs (HCAECs) to HLSS also induced an up-‐regulation of Cx37 expression [126, 319]. The promoter of Cx37 contains multiple CACCC elements, which are binding sites for KLF2 and KLF4. Indeed, KLF2 silencing in EC cultures under HLSS decreased the expression of Cx37, whereas silencing KLF4 did not affect Cx37 expression. Moreover, binding of KLF2 to the KLF consensus binding motifs in the Cx37 promotor was demonstrated by chromatin immunoprecipitation (ChIP). Thus, through KLF2 HLSS modulates the expression of Cx37 in the endothelium, thereby contributing to the formation of distinct communication compartments in arteries [126]. The responsiveness of endothelial Cx37 expression to changes in hydrostatic pressure or circumferential stretch are currently not known.
1.4.5.3 Cx40
The expression of Cx40 is lost in ECs overlying the atherosclerotic lesion [320].
However, preliminary immunofluorescent stainings comparing Cx40 expression in the straight part and the bifurcation region of the mouse carotid artery revealed no gross differences at both locations [126, 280]. This suggests that either Cx40 expression in ECs covering the atherosclerotic lesion is regulated by factors secreted by atheroma-‐associated cells or that the immunostaining
methodology is not sufficiently sensitive to detect small changes in Cx40 expression at the bifurcation.
A strong expression of Cx40 was observed during flow-‐driven collateral arterial network formation in the developing chick embryo [321]. In adult life, similar increases in flow drive arteriogenesis and the formation of collateral arterial networks in occlusive peripheral artery disease for instance. Interestingly, mice with ubiquitous genetic deletion of Cx40 and mice with endothelial-‐specific tamoxifen-‐inducible ablation of Cx40 showed reduced arteriogenesis after femoral or mesenteric artery occlusion [321, 322]. In HUVEC cultures, an unidirectional shear stress of 6-‐10 dyne/cm2 caused a long-‐term induction of Cx40 protein expression, with two short-‐term mRNA peaks at 4 and 16 h, indicating the dynamic nature of the adaptation process [323]. Furthermore, experiments inhibiting PI 3-‐kinase (PI3K) or protein kinase B (Akt) revealed a reduced expression of Cx40 with PI3K being involved in basal Cx40 expression and Akt taking part in the shear stress-‐dependent induction of Cx40 [323].
Finally, Yao et al. showed that normal laminar shear stress (15 dyne/cm2) inhibited EC proliferation via sirtuin-‐1 (SIRT1) and Cx40 [324]. Although these limited and fragmented data suggest a potential relation between shear stress patterns, Cx40 expression and atherosclerosis, no direct evidence has been obtained so far.