8. Purinergic receptors and the primary cilium

several isoforms, including VEGF165, which has been named VEGF-A or VEGF, the isoform involved in many of the functions attributed to the VEGF family. All members of the VEGF family activate tyrosine kinase receptors known as VEGF receptors (VEGFRs), which include VEGFR-1 (also known as fms-like tyrosine kinase 1 or Flt-1), VEGFR-2 (or kinase insertdomain containing receptor, KDR) and VEGFR-3 [58, 59]. Activation of VEGFRs has been implicated in several vascular functions, including angiogenesis, vascular tone regulation and

Importantly, VEGF and VEGFRs have also been associated with sensing FSS. High expression of VEGF [62] and the activation of VEGFR2 [63, 64] have been linked to the FSS sensing. Moreover, the activation of VEGFRs generally leads to NO synthesis in many kinds of cells, including endothelial cells [58]. Therefore, it is not surprising that VEGFR2 triggers NOdependent flow regulation. Jin et al. [63] showed that FSS leads to VEGFR2 activation in a ligand-independent manner and leads to eNOS activation in cultured endothelial cells. Intracellular downstream pathway associated with NO synthesis due to FSS-stimulated VEGFR2 activation included phosphoinositide 3-kinase (PI3K) and PKB/Akt. Interestingly, contrary to PKB/Akt, the PI3K pathway has not been associated to endothelial primary cilium FSS sensory function [29]. Also, in vivo experiments confirmed that VEGFR2 is a key mechanotransducer that activates eNOS in response to blood flow [63]. Despite these evidences, as far as we known there is no information related to VEGFRs present in the primary cilium as potential

VEGF can also bind to neuropilins (NRP), a family of transmembrane glycoproteins that play key role in axonal guidance, angiogenesis, tumorigenesis and immunological response [65]. NRPs have been characterized as co-receptors for VEGFRs and plexins, the receptors of the extracellular secreted ligands, belonging to class III semaphorins [60]. In turn, semaphorins are a class of secreted and membrane proteins that were originally identified as axonal growth cone guidance molecules. At least two neuropilin genes, NRP1 and NRP2, have been identified [66]. Genetic studies in mice have confirmed that NRPs are key components of vasculogenesis, angiogenesis and lymphangiogenesis [65, 66]. Nevertheless, NRPs can bind to growth factors such as VEGF, placental growth factor (PlGF), hepatocyte growth factor (HGF) and fibroblast growth factor (FGF), among others. And due to VEGF binding, NRPs can also modulate blood flow [65].

In endothelial cells, NRPs are thought to increase signaling through the VEGFRs acting as a coreceptor of VEGF and by stabilizing the VEGF/VEGFR complexes and therefore enhancing VEGF activity. Thus, the interaction of VEGF-A165 with NRP1 is required for stable binding of VEGF-A165 to VEGFR-2, full activation of VEGFR-2 and downstream signaling and biological

Limited information about the localization of NRP in primary cilium is available. Before presenting those evidences, we should give some information about hedgehog (HH) signaling. Briefly, HH signaling is essential for tissue patterning and organ formation during embryonic

endothelial cell survival, among others [59–61].

98 Endothelial Dysfunction - Old Concepts and New Challenges

7. Neuropilins and the primary cilium

regulator of FSS sensing.

responses [65, 67].

Since early 1970s [70], adenosine triphosphate (ATP) has been recognized as an extracellular signaling molecule activating a pathway defined as "purinergic signaling" where ATP, ADP and adenosine are involved. The signaling pathway starts with the activation of a family of membrane receptors. At this moment, separate families for adenosine purinergic (P1) and ATP and ADP purinergic (P2) receptors have been characterized. Briefly, adenosine receptor or P1 family includes at least four members of G-protein-coupled receptor subtypes identified as A1, A2A, A2B and A3. In contrast, the P2 family encompasses seven members of purinergic receptor type X (P2X), a family of ion channels receptor subtypes (P2X1–7) and at least eight members of P2Y G-protein-coupled receptor subtypes (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 and P2Y14) [71]. P2Y1, P2Y2, P2Y4 and P2Y6 are associated with the intracellular calcium (iCa2+) signaling pathway, whereas P2Y2, P2Y13 and P2Y14 are associated with cyclic adenosine monophosphate (cAMP) signaling. In contrast, P2Y11 has been shown to be associated with both iCa2+ and cAMP signaling [71].

ATP is released by almost all cell types after gentle mechanical stimulation and acts in an autocrine or paracrine manner [72]. Living cells under stressful conditions (i.e., hypoxia) or dying cells release ATP [72]. Interestingly, purinergic signaling parallel to flow sensor activity of the primary cilium [73]. Purinergic signaling associated with flow sensing was detected in several structures such as kidney tubules [20], intrahepatic bile ducts [74, 75], endothelial cells [31], among other lining cells. Therefore, deflection of the primary cilium has been related with ATP release leading to autocrine or paracrine activation of purinergic receptors.

The relationship between ATP, purinergic signaling and primary cilium has been studied in the kidney tubular system [73]. The main physiological area that established a relationship between these three elements is related to urinary flow sensor, where a flow-stimulated increase of iCa2+ has been characterized. Initial investigation suggested that deflecting the cilium releases a paracrine factor, such as ATP that can activate a G-protein-coupled receptor and generate inositol triphosphate (IP3) leading to iCa2+ increase throughout the cytosol due to the release of Ca2+ from the intracellular stores. Also, increasing the tubular flow triggered an increase in iCa2+. The same experiments were also performed in renal tubules from mice lacking P2Y2 receptors or cells lacking the primary cilium. In those experiments, the response to tubular flow was markedly reduced only in those cells lacking the P2Y2 [76]. These results strongly suggested that tubular flow triggers ATP release, followed by auto- and paracrine activation of epithelial P2 receptors. However, a direct link to the primary cilium could not be established in these experiments. Despite that, information regarding how purinergic signaling can be associated or not to function of primary cilia is missing.

depends on its localization at the primary cilium and on PKD1. Thus, impaired function and expression of PKD2 are associated with endothelial dysfunction. Interestingly, prolonged exposure of endothelial cells to high FSS induces the disassembly of cilia [28] and inactivation of PKD1 by proteolytic cleavage [9], reducing the ability of endothelial cells to properly sense

Sensing Fluid-Shear Stress in the Endothelial System with a Special Emphasis on the Primary Cilium

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Initial evidence showed that primary cilia were present in the vascular beds where flow is disturbed, and related to atherosclerosis [89]. Particularly, the primary cilia were found in the endothelial cells of human aortic fatty dots and streaks, but not in those of the normal intima [89]. Moreover, recent evidences also found the primary cilium in cells exposed to laminar blood flow [27]. Regarding atherosclerotic plaque, primary cilia have been shown to be located at the atherosclerotic predilection sites, where flow is disturbed and around atherosclerotic lesions in the aortic arch in wild-type mice and apolipoprotein E-deficient mice, respectively [23]. In addition, experimentally induced pathologic turbulent flow in mice leads to induction of primary cilia, and subsequently to atherogenesis, suggesting a role of primary cilia in

Contrary, another evidence found an inverse correlation between the presence of endothelial primary cilia and vascular calcified areas, although the signaling mechanisms involved remain unknown [22]. In order to analyze this phenomenon, Sanchez-Duffhues et al. [22] used the Tg737 cilium-defective mouse model and they found that non-ciliated aortic endothelial cells acquire the ability to trans-differentiate into mineralizing osteogenic cells. The mechanism for this trans-differentiation requires the presence of bone morphogenetic proteins (BMP). Therefore, these data emphasize the role of the endothelial cells in vascular calcification and generation of atherosclerosis. Whether these findings are associated or not to iCa2+, eNOS activation

Apparently, differences in blood flow patterns along the endothelium trigger abnormal vascular responses that have been associated with pathophysiological consequences, such as atherosclerosis. While endothelial cells exposed to laminar blood flow are protected from atherosclerosis formation, turbulent blood flow, which occurs at bends and bifurcations of blood vessels, facilitates the process of atherosclerosis. Primary cilia presence and function have barely been studied in both endothelial activation and dysfunction. Hence, more studies are required to better

Phenotypic cell alterations resulting from flow-induced mechanical strains and their implication in diseases are a growing field of research in many cell types such as vascular endothelial,

In the chapter, we presented the role of the primary cilium as one of the multiple physiological mechanosensors for FSS in endothelial and renal cells, where it regulates vascular homeostasis

alterations in blood pressure.

endothelial activation and dysfunction [23].

and NO synthesis remains unclear.

understand these issues.

10. Concluding remarks

smooth muscle, kidney epithelial cells and chondrocytes.

9.2. Atherosclerosis
