3.2. Polycystin-1, polycystin-2 and polaris

vertebrate cells [24]. Motile cilia contain microtubules (MT) arranged in a (9 + 2) manner consisting of a nine doublets MT ring surrounding a central pair of MT and presenting protein spokes and dynein inner and outer arms necessary for movement. In contrast the primary cilium shows (9 + 0) organization with nine pairs of MT at the periphery lacking the central pair of MTs, as well as the protein spoke and the dynein arms (Figure 1). In both cases, MT extend from a basal body originating from "mother" centriole of the centrosome [25]. The structure and maintenance of the primary cilium are regulated by intraflagellar transport (IFT)

In physiological conditions, nearly all quiescent differentiated mammalian cells exhibit a primary cilium, which emanates from the surface as a single long hair-shaped projection [27]. Therefore, primary cilia are found in a large number of mammalian cells including stem cells, epithelial and endothelial cells [19]. Their presence was demonstrated in adult vascular system (reviewed in [2]), developing chicken endocardium [4], embryonic mouse aortic endothelium [9], cultured human umbilical vein endothelial cells (HUVECs) [28, 29] and epithelial cells including macula densa [30] or tubular epithelial cells [20]. Nevertheless, alteration in the number, length and structural features has been implicated in pathological conditions such as

polycystic kidney disease, atherosclerosis and hypertension, among others [18, 23, 31].

Depending on structural and functional features, five distinct domains were described in the

1. The ciliary membrane housing many sensory receptors and channels supporting sensory

2. The soluble compartment or cilioplasm constituting the fluid between the ciliary membrane and the axoneme and where IFT machinery is located to assemble and maintain the

3. The axoneme composed of tubulin that supports ciliary transport. It is composed of nine

4. The ciliary tip is the distal part of the axoneme where specialized proteins localize whose

IFT is required for assembly and maintenance of cilia. Briefly, ciliogenesis is initiated in the apical cytoplasm at the basal body. Proteins involved in cilium formation concentrate and assemble into complexes that migrate along the cilia axonemal microtubules through a process called IFT. The anterograde movement of particles from the cell body to the tip of the flagella/cilia is driven by

5. The basal body, the network foundation from which the primary cilium emanates.

3. Primary cilium sensing fluid-shear depends on mechanoproteins

particles [26].

primary cilium [2] (Figure 1):

92 Endothelial Dysfunction - Old Concepts and New Challenges

function of cilia.

pairs of MTs.

function is still unclear.

3.1. Intraflagellar transport

polycystins and structural polaris

cilia.

Among sensory molecules housing into the primary cilium, both polycystin-1 (PKD1) and polycystin-2 (PKD2) have been described. These are membrane integral proteins. Experimental data show that they are highly expressed in human endothelial and epithelial cells and are required for normal physiological cilia function (reviewed in [2]). The importance of these proteins has been highlighted due to the finding that mutations in pkd1 or pkd2 genes result in polycystic kidney disease, hence their name [9].

PKD1 is a 3327 amino acids long transmembrane protein with 11 membrane-spanning domains. Its long extracellular N-terminus has a mechanosensory function, while its short intracellular C-terminus is involved in intracellular signaling and interaction with PKD2 [34, 35]. PKD1 has been shown to mediate fluid-shear sensing in epithelial and endothelial cells [9, 36].

PKD2, a 968 amino acids long protein, is a non-selective Ca2+ permeable transient receptor potential (TRP) channel consisting of six membrane-spanning domains and intracellular Cand N-terminal domains [37]. The sensory function of PKD2 depends on PKD1 and has to be localized to endothelial primary cilia [38]. Accordingly, PKD2 functioning as a Ca2+ channel [29] allows extracellular Ca2+ influx into the cilioplasm in response to FSS [39]. Thus, mechanistically, PKD1 and PKD2 interact through their C-terminus [29, 34, 35] and localized to the ciliary membrane; they are able to detect extracellular FSS and to increase cytosolic Ca2+. This turns on a signaling cascade leading to the production of NO [9, 38, 40].

A series of mutation and deletion experiments demonstrated that besides PKD1 and PKD2, the protein polaris also orchestrates FSS sensing. The physiological Ca2+ and NO increase in response to FSS is abolished when the pkd1, pkd2 and polaris genes are mutated or knocked out [29]. Interestingly, mutations or deletion of polaris seem to affect the structural integrity of cilia through the PKD1 and PKD2 mislocalization, which remain concentrated at the basal body [9, 29, 32, 41]. Together these findings evidence that polaris mediates the PKD1 and PKD2 primary cilium localization, implying a polaris cilium sensory function regulation. In order to achieve a proper fluid-shear sensing by endothelial cells and an adequate response, all three components, PKD1, PKD2 and polaris, are thus indispensable.

#### 3.3. Molecular cascade involved in shear stress-induced calcium and NO signaling

FSS leads to cilia bending leading to PKD2-mediated increase of intracellular Ca2+ that leads to activation of ryanodine receptors (RyR) and inositol 1,4,5-triphosphate receptor (InsP3R) present in the endoplasmic reticulum, which then releases its stores of Ca2+ enhancing the intracellular levels of Ca2+ [42, 43]. Subsequently, Ca2+ activates several intracellular signaling pathways, including the activation of the eNOS-bound calmodulin, thus increasing the production of NO that diffuses from endothelial cells to neighboring VSMC inducing vasodilatation [2, 29]. This particular pathway is summarized in Figure 2.

The works of AbouAlaiwi et al. [29] have helped to elucidate this last mechanism. In order to prove that FSS-dependent primary cilia bending induces extracellular Ca2+ influx, they used Ca2+ chelator EGTA (ethylene glycol-bis (β-aminoethyl ether)-N,N,N<sup>0</sup> ,N<sup>0</sup> -tetraacetic acid). In these experiments, EGTA abolished both Ca2+ and NO increases. In addition, the inhibitor of the eNOS, NG-nitro-L-arginine methyl ester (L-NAME) blocked the FSS-induced NO release without affecting Ca2+ increase. The same effect was shown after blocking calcium-dependent mechanisms of NO production using calphostin C as an inhibitor of protein kinase C (PKC) or W7 as antagonist of calmodulin. Similarly, inhibiting protein kinase B (PKB)/Akt abolished NO release without altering Ca2+ increase. Inhibiting IP3 kinase using LY-294002 did not alter neither Ca2+ nor NO increase. These findings indicate that calmodulin, PKC and Akt/PKB are downstream of the calcium pathway and that they are necessary for NO release during primary cilium-mediated FSS signaling [29] (Figure 2).

4. The regulation of ciliary function

shear stress [2].

Changes in fluid patterns generate differential biomechanical forces, which lead to alteration of cilia function or structure [2]. Indeed, almost all blood vessels possess cilia [4, 23]. Particularly, arteries with low FSS or high fluid turbulence have cilia [2]. A prolonged exposure of endothelial cells to high FSS induces the disassembly of cilia [28] and inactivation of PKD1 by proteolytic cleavage [9], suggesting that primary cilia may not be required only to sense high

Figure 2. Mechanotransduction of FSS at the endothelial primary cilium. Extracellular FSS leads to cilia bending and activation of polycycstin-1/-2 complex, conducing to extracellular calcium influx. Calcium binds to ryanodine receptors and an efflux of intracellular organelle calcium. This is followed by activation of calmodulin (CaM), protein kinase C

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

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(PKC), protein kinase B (PKB/Akt), eNOs and NO production. Figure reproduced with permission [38].

Sensing Fluid-Shear Stress in the Endothelial System with a Special Emphasis on the Primary Cilium http://dx.doi.org/10.5772/intechopen.73134 95

Figure 2. Mechanotransduction of FSS at the endothelial primary cilium. Extracellular FSS leads to cilia bending and activation of polycycstin-1/-2 complex, conducing to extracellular calcium influx. Calcium binds to ryanodine receptors and an efflux of intracellular organelle calcium. This is followed by activation of calmodulin (CaM), protein kinase C (PKC), protein kinase B (PKB/Akt), eNOs and NO production. Figure reproduced with permission [38].
