The 3 main connexins expressed in arteries are Cx37, Cx40 and Cx43, and their expression patterns vary with disease states. From studies on knockout mice, it has become the past 2 decades increasingly clear that these proteins play a crucial role in vascular physiology and disease. In this paragraph, I will summarize atherosclerosis studies involving connexin knockout mice.
1.4.2.1 Cx37
As previously described, Cx37 is lost in ECs in areas prone to atherosclerosis and on ECs overlaying atherosclerotic plaques. Indeed, Cx37-‐/-‐ApoE-‐/-‐ mice showed enhanced atherosclerotic lesion development in the thoracic-‐abdominal aorta and in the aortic sinus [244]. Adoptive transfer studies revealed that the deletion of Cx37 in monocytes/macrophages increased the amount of these cells in the plaques. This was due to an effect on monocyte adhesion rather than the migratory properties of the monocytes per se. In fact, it appeared that Cx37 regulates the ATP-‐dependent monocyte adhesion. Finally, ATP release and cell adhesion was different in a monocytic cell line transfected with different polymorphic isoforms of the connexin (Cx37-‐319S or Cx37-‐319P), leading to a possible explanation for the differential risk of myocardial infarction associated with these polymorphisms [244]. Using the afore-‐mentioned shear stress modifying cast model, it appeared that the extent of atherosclerosis in oscillatory regions of Cx37-‐/-‐ApoE-‐/-‐ mice was increased and that the stable plaque phenotype normally occurring in these regions was abrogated [75]. Finally, using a phage display approach Pfenniger and colleagues showed that Cx37 is a direct binding partner of eNOS. Binding of the two proteins did not only modulate Cx37
channel function but also eNOS enzyme activity, suggesting that endothelial expression of the protein may also affect early atherogenesis [261].
Thrombus formation after plaque rupture may lead to myocardial infarction [262]. Interestingly, Angelillo-‐Scherrer et al. showed that platelets expressing Cx37 form functional gap junction channels during platelet aggregation.
Moreover, this Cx37-‐mediated direct intercellular communication between platelets was limiting the thrombus propensity [239].
In conclusion, Cx37 has multiple functions in various cells that are related to atherosclerotic plaque formation and the complications of this disease.
1.4.2.2 Cx40
Similar to Cx37-‐deficient mice, Cx40-‐deficient mice showed affected eNOS function and a decreased endothelium-‐dependent vasorelaxation [263]. Further investigations revealed however that this effect was likely due to the concomitant down-‐regulation of Cx37 expression observed in Cx40-‐deficient mice [264]. Moreover, these mice showed decreased activity and expression of CD73 and increased VCAM-‐1 expression in ECs [217]. CD73 in ECs is known to up-‐regulate anti-‐adhesion signals via adenosine production [265]. In line with these data, mice with EC-‐specific deletion of Cx40 in ECs developed larger atherosclerotic lesions than control mice after 5 or 10 weeks of high cholesterol diet. Strikingly, even without high cholesterol diet these mice developed spontaneously atherosclerotic plaques in the aortic sinuses, demonstrating the importance of this protein in the initiation of the disease [217]. Using siRNA or antisense to decrease Cx40 in vitro in ECs, the expression and activity of CD73 was decreased and adhesion of neutrophils and monocytes increased.
Additionally, using an adenosine receptor agonist reversed this effect.
Altogether, these results pointed in the direction that Cx40 gap junctional communication propagates adenosine-‐evoked anti-‐inflammatory signals between ECs [217].
As for Cx37, Cx40 is expressed in platelets [240]. Vaiyapuri et al. showed that inhibition of Cx40 in human platelets or its deletion in mice reduced the platelet fibrinogen binding, aggregation, clot retraction and granule secretion. They concluded that Cx40, in an independent manner from Cx37, is involved in the regulation of platelet function thus contributing to haemostasis and thrombosis [240].
In conclusion, endothelial Cx40 protects against atherosclerotic plaque formation and platelet Cx40 fine-‐tunes platelet aggregation and clot retraction.
However, the regulation of endothelial Cx40 expression, potential protein partners for Cx40 as well as its role in the progression of atherosclerosis remain to be investigated.
1.4.2.3 Cx43
As Cx43 is widely expressed in many atheroma-‐associated cell types, targeting Cx43 in atherosclerosis may have a multitude effects, both beneficial and detrimental. Importantly, Cx43 gap junctional communication between ECs may be rather specifically inhibited in vivo by mimetic peptides and this decreased the adhesion of neutrophils cells in a model of lung inflammation.[216] Although it remains controversial whether neutrophils express Cx43 in all conditions, it has been shown that neutrophil adhesion is decreased when these leukocytes
were obtained from Cx43+/-‐ mice or from mice with a cell-‐specific deletion of Cx43 [243].
Studies performed on LDLR-‐/-‐ mice with reduced levels of Cx43 (Cx43+/-‐) on high cholesterol diet showed a 50% reduction in atherosclerotic plaque formation in thoracic-‐abdominal aortas and in aortic sinuses compared to the control LDLR-‐/-‐
mice. Furthermore, the plaques contained less inflammatory cells and had a thicker fibrous cap with more collagen and VSMCs, typical for a stable plaque phenotype. In addition, treatment of LDLR-‐/-‐ -‐mice with Pravastatin (statins are lipid lowering drugs well known to reduce the cardiovascular risk (reviewed in:
[266] and [267]) increased Cx43 expression, which was associated with decreased the amount of inflammatory cells and increased the thickness of the fibrous cap compared to control mice [268]. Atherosclerosis studies on Cx43+/-‐
mice with a ubiquitous reduction of Cx43 are hard to interpret due to expression of this protein in ECs, VSMCs, macrophages, T cells, B cells and possibly neutrophils. Tie2-‐Cre+Cx43fl/flApoE-‐/-‐ mice were first supposed to have endothelial-‐specific deletion of Cx43 but it was later shown that the Cre-‐
recombinase was also active in monocytes. These mice showed reduced atherosclerotic plaque development with reduced lipid content and calcium deposition compared to control mice, illustrating a crucial role for endothelial and monocytic Cx43 in atherogenesis [269]. Subsequently, Morel et al.
reconstituted LDLR-‐/-‐ mice with Cx43+/+, Cx43+/-‐ or Cx43-‐/-‐ hematopoietic fetal liver cells to examine specifically the role of Cx43 in immune cells. Here, the progression of atherosclerosis and the amount of neutrophils was lower in aortic roots in Cx43+/-‐ but not in Cx43-‐/-‐ compared to the control Cx43+/+ mice. Such a critical role for the exact amount of Cx43 has been described previously for
VSMCs in the context of restenosis [270, 271] and for neural crest cell during heart development [272-‐274]. Interestingly, no difference in terms of polarization (M1/M2) was found between Cx43+/+, Cx43+/-‐ or Cx43-‐/-‐
macrophages but detailed microarray analysis revealed modified gene expression for the chemokines Ccl2, Ccl3, and Cxcl12 and the complement component C3a. This brought the authors to the tempting conclusion that the levels of Cx43 expression in macrophages determines their chemo-‐attractant secretion [246].
Finally, given the important role of VSMCs in plaque stability it is surprising that the mechanism regulating Cx43 expression in these cells is not fully understood.
Blackburn and colleagues performed the first study shedding light on this mechanism. They showed that the transcription factor NF-‐κB was involved in the regulation of Cx43 and that the phosphorylation state of Cx43 was important for VSMC proliferation [275]. Interestingly, Rama and colleagues added to that, that transforming growth factor beta (TGF-‐β)-‐treated human aortic SMCs increased their Cx43 expression level which correlated with an increased synthetic activity.
Altogether, this leads to the conclusion that Cx43 levels and phosphorylation states in VSMCs may be important for the stability of atherosclerotic lesions [276, 277].
In conclusion, the different data from various research groups are pointing in the direction that Cx37 and Cx40 are atheroprotective and that Cx43 is atheroprone in ECs of large arteries.