1.3.4 Shear stress response
1.3.4.2 Atheroprone shear stress
1.3.4.2 Atheroprone shear stress
In contrast to the ECs in atheroprotective regions, ECs in atheroprone regions exposed to low and/or oscillatory shear stress display a ‘cobblestone’
morphology [52]. Furthermore, these ECs display an impaired endothelial barrier function and higher rates of cell turnover, cellular senescence and show increased permeability to lipoproteins [48, 52, 175]. Furthermore, these dysfunctional ECs have increased expression of adhesion molecules leading to the adhesion and transmigration of monocytes over the endothelial barrier to the intima [53]. Another important aspect is the chronic endoplasmic reticulum (ER) stress that causes endothelial apoptosis [176]. Low and/or oscillatory shear stress is reducing the NO-‐dependent protection against atherosclerosis through reduction of eNOS synthesis [149]. Furthermore, increased reactive oxygen species (ROS) production through induced nicotinamide adenine dinucleotide phosphatase (NADPH) activity increases NO degradation and further activates the ECs [177, 178]. Twist related protein-‐1 (TWIST1) is preferentially expressed in low shear stress regions of adult arteries. TWIST1 was found to promote atherosclerosis by enhancing EC proliferation and inflammation through expression of VCAM-‐1 and ICAM-‐1 inflammatory adhesion molecules [179]. In contrast to high protective shear, low shear stress increases the thrombogenicity
through reduced expression of tissue plasminogen activator and prostacyclin [180].
Finally, different miRNAs are regulated by atheroprone shear stress. Indeed, the miR17 and miR92 cluster, miR34a, miR663 and the miR712-‐205 family have all been found to be up-‐regulated in these regions and to promote atherogenesis [157, 181, 182]. Interestingly, several groups of shear stress-‐regulated miRNAs have a dual role in atherogenesis. miR-‐21 expression was found to be upregulated in HUVECs after exposition to unidirectional shear stress. Here, miR-‐21 was found to decrease apoptosis and increase eNOS phosphorylation and NO production in ECs [183]. In contrast, another study reported that oscillatory shear stress-‐induced expression of miR-‐21 may inhibits the expression of peroxisome proliferator-‐activated receptor-‐alpha (PPARα), resulting in enhanced expression of VCAM-‐1 and monocyte chemotactic protein-‐1 (MCP-‐1) [184]. In addition, miR-‐126-‐carrying endothelial apoptotic bodies have been shown to be atheroprotective in mice through promoting a C-‐X-‐C motif chemokine 12 (CXCL12)-‐dependent recruitment of progenitor cells to the endothelial lining [185]. In contrast, the effects of miR-‐126 on VSMCs includes repression of genes (e.g. FOXO3, BCL2, and IRS1) known to hold these cells in the atheroprotective contractile phenotype.[186] Conferring to miR-‐126 a atheroprone role in VSMCs despite its atheroprotective effects on endothelial cells. Finally, miR-‐155 is increased in the thoracic aorta exposed to laminar shear stress compared to the lower curvature of the aortic arch associated with oscillatory and low shear stress [187]. Both pro-‐ and anti-‐atherosclerotic effects have been assigned to miR-‐155 depending on the context. Increased atherosclerosis with reduced plaque stability was present in LDLR-‐/-‐ mice
harboring a bone marrow deficiency of miR-‐155 [188]. Furthermore, the delivery of miR-‐155 in vivo reduced atherosclerotic plaque formation through targeting MAP3K10 [189]. In contrast, miR-‐155 expression was induced in atherosclerotic plaques and pro-‐inflammatory macrophages through treatment with oxidized LDL and IFN-‐γ. Here, leukocyte-‐specific mIR-‐155 deletion decreased the number of lesional macrophages and plaque size after partial carotid ligation in ApoE-‐/-‐-‐mice. The loss of miR-‐155 reduced expression of CC-‐
chemokine ligand 2 (CCL2) that enhances the recruitment of monocytes to atherosclerotic lesions [190]. Demonstrating the pro-‐inflammatory role of miR-‐
155 in macrophages.
Together, these data show the complexity of miRNAs and showing their versatility depending on cellular context and environment [172]. In addition to laminar flow, disturbed flow also modulates DNA methylation mainly through the methyltransferase activity of DNMT1 [52, 162, 165]. Thus, disturbed flow increases the methylation of the promotor of the atheroprotective transcription factor KLF4, thereby inhibiting its expression [52, 191]. Finally, NF-‐κB has a central role in the pro-‐inflammatory activation of the endothelium during atherogenesis and in function of shear stress [48, 52, 145, 192]. Many stimuli leading to endothelial dysfunction have been ascribed to NF-‐κB signaling in ECs and will be separately discussed in the next paragraph.
1.3.4.2.1 NF-‐κB
High levels of NF-‐κB expression are typically found in regions of low shear stress and regions of disturbed flow a nuclear localization of this transcription factor is generally observed [193-‐196]. The most abundant form of NF-‐κB in the
endothelium is the RelA/p50 heterodimer [194]. In normal physiological conditions, NF-‐κB is sequestered in the cytoplasm through the binding of inhibitory IκB. Signaling through various pathways converge on the IκB kinase (IKK) complex phosphorylating IκB and leading to ubiquitination and proteasome-‐dependent degradation IκB [197, 198]. This dissociation of IκB from NF-‐κB makes it possible to the transcription factor to translocate freely to the nucleus, where it binds to transcription factor binding sites (TFBS) initiating the transcription of various genes [198]. Many effector molecules involved in endothelial cell dysfunction are under the control of NF-‐κB. Moreover, other flow-‐sensitive endothelial genes have non-‐canonical NF-‐κB binding sites [53].
In response to atheroprone shear stress different NF-‐κB-‐dependent adhesion molecules and pro-‐inflammatory molecules are up-‐regulated, including VCAM-‐1, ICAM-‐1, E-‐selectin, MCP-‐1, tissue factor, vWF, plasminogen activator inhibitor (PA-‐1), tumor necrosis factor (TNF)-‐α, interleukin (IL)-‐1 and interferon (IFN)-‐γ [53, 180].
Interestingly, Cuhlmann and colleagues proposed a possible explanation related to NF-‐κB for the spatial distribution of the atherosclerotic lesions in the vasculature. Using the constructive perivascular cuff model they showed that in low or low/oscillatory pro-‐inflammatory shear stress regions transcription factor p65 (RelA) was up-‐regulated through JNK1 and the downstream transcription factor activating transcription factor (ATF)-‐2. They suggest that the crosstalk between the JNK and NF-‐κB pathways may explain non-‐uniform spatial distribution of the atherosclerotic lesions [194]. In addition, although the expression of RelA is similar in low and low/oscillatory shear stress regions only low/oscillatory shear stress induced nuclear localization of RelA and up-‐
regulation of VCAM-‐1 expression [194]. These data suggest that ECs exposed to the low/oscillatory shear environment have a particular pro-‐inflammatory phenotype distinct from ECs exposed to low shear stress [194]. Interestingly, Pfenniger et al. showed that shear stress through the modulation of the gap junctional protein connexin37 forms distinct communication compartments in arteries. In concordance with the results of Cuhlmann and colleagues, introducing distinct EC compartments that segregate atheroprone regions from protected regions limiting the spread of pro-‐inflammatory mediators to neighboring regions [126].
Figure 8: Summary of atheroprotective and atheroprone effects of laminar and disturbed flow, respectively, on EC biology. Adapted from [199]
1.4 Connexins
Connexins are transmembrane proteins that are expressed in almost all cells of the body. They form intercellular channels, called gap junction channels, facilitating cell-‐cell communication by connecting the cytoplasm of two neighboring cells. Gap junction channels are formed when two opposed connexons (each composed of 6 connexins) from two neighboring cells dock in the extracellular space [200]. Furthermore, connexons may under specific conditions function as hemichannels and form a communicating path between
the cytoplasm and the extracellular space. Twenty-‐one different connexins have been found in humans and 20 in mice [201]. Connexins consist of 4 α-‐helical transmembrane domains and 2 extracellular loops (ELs) that are rather conserved between the different connexins. In addition, a cytoplasmic loop (CL), a C-‐terminal (CT) and N-‐terminal (NT) tail are located in the cytoplasm (Figure 9).
Figure 9: From connexin to gap junction channel. Connexins are membrane-‐spanning proteins composed of four transmembrane helices (M1-‐M4), two extracellular loops (EL1 and EL2), one intracellular loop (CL) and one N-‐ and C-‐terminal end located in the cytoplasm (NH2 and COOH). Six connexin subunits oligomerize to form a connexon. Two connexons from neighboring cells dock to form a gap junction channel.
These parts are unique to each type of connexin and differ in length and composition [202]. The different connexins are named after their specific molecular weight. Furthermore, connexins are classified in 5 subfamilies according to their sequence homology [203]. Gap junction channels can adopt different configurations. First, connexons can be homomeric when the six connexins forming the connexon are identical or they are heteromeric when two or more different connexins are forming the connexon. Secondly, gap junction channels can be homotypic if both connexons are formed by identical connexins
or considered heterotypic if they are formed by two different connexons.
Additionally, heteromeric heterotypic channels are formed when two connexons containing each multiple different connexins dock together [204]. Due to the versatility in the composition of gap junction channels it is not surprising that they differ in their gating properties, unitary conductance and permeability to ions and different molecules. In general, small molecules with a molecular weight below 1 kDa like adenosine triphosphate (ATP), glutathione, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphqte (cGMP) and inositol triphosphate (IP3) are passing through gap junction channels, but depending on the structure and charge of the metabolite this may differ considerably for channels made of different connexins [205-‐207]. Interestingly, recent evidence shows that even siRNA and miRNA may pass through gap junction channels under certain conditions [208]. Next to their channel function, connexins have also been found to interact with other proteins. First, regulation of gap junctional communication can be modulated by the interaction with associated proteins, such as protein kinases and protein phosphatases. Secondly, connexins have been found to interact with structural proteins such as zona occludens-‐1 (ZO-‐1) and microtubules [209, 210]. Finally, a growing number of connexin-‐associated proteins have broadened the range of connexin roles to transcriptional and cytoskeletal regulation [210].