**3. Nitric oxide**

Nitric oxide is a signaling molecule involved in a wide array of cellular pathways; mainly, NO contributes to the normal functions of a variety of organ systems [52]. NO is highly reactive, and readily diffuses across cellular membranes. As a result, NO is found in many paracrine signaling pathways. NO is mainly synthesized from l-arginine, oxygen, and NADPH in a redox reaction, catalyzed by nitric oxide synthase (NOS) [53]. NOS has three isoforms, but only endothelial NOS (eNOS) and neuronal NOS (nNOS) are constantly and consistently expressed in cells. Both eNOS and nNOS are calcium dependent, while the other NOS isoform, cytokine inducible NOS (iNOS), is expressed by pro-inflammatory cytokines on an as needed basis [54]. iNOS and nNOS are both soluble enzymes that exist within the cytosol. eNOS, however, is found to localize to the plasma or Golgi body membranes. Because of its unique and wide cellular and subcellular distribution, NOS has many diverse functions throughout the entirety of the body [1, 54, 55].

### **4. Cilia and nitric oxide interplay**

Although primary cilia and NO have various independent roles within the body, especially in the vasculature, their functions often intersect and cooperate with each other. Most research on the interaction between endothelial primary cilia and nitric oxide focuses on vascular homeostasis, but their interactions extend into other areas [1]. However, this chapter will focus on signaling cascades that lead

to NO biosynthesis or increased NO bioavailability. The following discussion of the interactions between primary cilia and nitric oxide will focus on vasodilation, wound healing, dopamine signaling, and cellular proliferation.

#### **4.1 Vasodilation**

Primary cilia and NO independently effect the vasculature in different ways, but recent studies suggest a direct relationship may exist between the two. Vascular endothelial cells are present in the blood vessel wall and are in continuous contact with blood flow-generated fluid shear stress. Endothelial cells are known mechanotransducers of fluid shear stress, which causes the biosynthesis of NO. This helps regulate vascular tone; NO will diffuse into the surrounding smooth muscle, producing vasorelaxation [56].

Evidence supports primary cilia as the main sensor in this mechanosensitive pathway. As stated previously, PC-1, a mechanosensory protein that malfunctions in polycystic kidney disease, localizes to vascular endothelial primary cilia. In an *in vitro* study performed by Nauli et al., which investigated PC-1's fluid shear mechanosensory properties, it was found that in contrast to wildtype endothelial cells, the PC-1 knockout cells failed to produce an increase in cytosolic calcium and the corresponding NO flux in response to fluid shear stress. The authors, to demonstrate that calcium and NO signals are induced in response to ciliary PC-1 activation, used *Tg737orpk/orpk* endothelial cells that lack ciliary ultrastructure but have functional PC-1. The results showed that neither calcium nor NO signals were present at flow rates up to 50 dyne/cm2 [3]. These results suggest that PC-1 is responsible for proper cilia mechanosensory function, and that ciliary PC-1 specifically elicits NO production.

Follow-up studies by AbouAlaiwi et al. Showed that PC-2, which, as stated previously, is a calcium permeable cation channel that forms a complex with PC-1, is also important for mechanotransduction. Studies using a PC-2 knockdown line of cells showed a reduction in calcium and NO flux under shear stress when compared to control cells. This was further validated in *ex vivo* studies, where endothelial cells isolated from *pkd2−/−* mice arteries failed to respond to fluid shear stress [4]. These results indicate both PC-1 and PC-2 are needed for cilia mechanosensation, and further suggest that activation of the PC-1/PC-2 complex will start the signaling cascade needed for calcium-dependent NO biosynthesis. In addition, the results show that the increase in intracellular calcium is caused by an increase in intra-ciliary calcium. However, other researchers have proposed that calcium moves bidirectionally between the cilia and the cytosol [57–59]. Regardless, the increase in intracellular calcium triggers the calcium/calmodulin complex, which activates constitutive NOS, such as eNOS, by binding to its target site on the enzyme [60].

The calcium/calmodulin complex can also indirectly activate eNOS through activation of the AKT/PKB pathway, which stimulates AMPK (**Figure 2**) [61]. eNOS activation is mainly calcium dependent, but some studies have shown that a calcium independent pathway exists, via heat shock protein 90 (HSP90). HSP90 is also known to localize to cilia axonemes, and may act as a signal transductor that interacts with eNOS in the vasculature [62, 63]. HSP90 activation can lead to an increase in eNOS activity while calcium levels are high, and can also lead to more eNOS activity at low calcium levels due to its ability to directly bind to eNOS and increase the binding affinity for calmodulin [64, 65].

#### **4.2 Wound healing**

An under researched aspect of NO and primary cilia is their interaction in the vascular smooth muscle cell (VSMC) layer. Depending on blood vessel type, all

**55**

**Figure 2.**

*Primary Cilia are Sensory Hubs for Nitric Oxide Signaling*

three isoforms of NOS may exist within the VSMC layers [66]. During normal function, the cilia on the VSMC layer extend towards the extracellular matrix. Under abnormal conditions, such as a scratch wound, VSMC primary cilia will migrate to the wound edge. A recent study showed that VSMC cilia express polycystins, as well as α3-and β1-integrins. When the researchers blocked integrin function, the percent of cilia migrating to the wound edge dropped from about 88% to around 30% [67]. This drop suggests that VSMC primary cilia may be

*Primary cilia activation via fluid shear stress and NO signaling in the vascular endothelia. The left panel shows primary cilia bending while under fluid shear stress, with the resultant production and release of nitric oxide (NO). The production and release of NO is dependent on the activation of endothelial primary cilia within the vasculature. The bending of cilia via fluid-shear stress activates the mechanosensory polycystins complex, which initiates the synthesis and the release of NO. This biochemical cascade, shown in the right panel, involves an extracellular calcium influx (Ca2+), followed by the activation of multiple calcium-dependent proteins, including calmodulin (CaM), protein kinase C (PKC) and Akt/PKB. Figure is adopted from Ref. [1].*

In further support of the purported role primary cilia play in wound healing, results from experiments using VSMC that lacked cilia showed a slower scratchwound healing time than the ciliated control cells displayed [67]. While the cilia in the wounded area are directly exposed to constant fluid shear stress from blood flow, activation of the mechanosensory ciliary polycystin complex could occur, resulting in an increase in intracellular calcium [1]. This could potentially trigger vasoconstriction in VSMC, resulting in the isolation of the wounded area to allow the platelets to begin clot formation. As soon as the calcium amount reached a certain level, the calcium/calmodulin complex would form and activate eNOS and nNOS, leading to vasodilation and the next step in the wound healing process.

Further studies by Schneider et al. reveled that, once cellular growth was halted, platelet derived growth factor receptor alpha (PDGFRα), a tyrosine kinase with a prominent function in cellular proliferation, was found to localize to fibroblast primary cilia. Furthermore, ligand activation of PDGFRα will lead to the activation of the AKT and MEK/ERK proliferative pathways. Because AKT and ERK1/2 regulate eNOS activity in endothelial cells, PDGFRα activation could indirectly lead to eNOS

involved in integrin-mediated wound healing.

*DOI: http://dx.doi.org/10.5772/intechopen.89680*

#### **Figure 2.**

*Basic and Clinical Understanding of Microcirculation*

**4.1 Vasodilation**

producing vasorelaxation [56].

to NO biosynthesis or increased NO bioavailability. The following discussion of the interactions between primary cilia and nitric oxide will focus on vasodilation,

Primary cilia and NO independently effect the vasculature in different ways, but recent studies suggest a direct relationship may exist between the two. Vascular endothelial cells are present in the blood vessel wall and are in continuous contact with blood flow-generated fluid shear stress. Endothelial cells are known mechanotransducers of fluid shear stress, which causes the biosynthesis of NO. This helps regulate vascular tone; NO will diffuse into the surrounding smooth muscle,

Evidence supports primary cilia as the main sensor in this mechanosensitive pathway. As stated previously, PC-1, a mechanosensory protein that malfunctions in polycystic kidney disease, localizes to vascular endothelial primary cilia. In an *in vitro* study performed by Nauli et al., which investigated PC-1's fluid shear mechanosensory properties, it was found that in contrast to wildtype endothelial cells, the PC-1 knockout cells failed to produce an increase in cytosolic calcium and the corresponding NO flux in response to fluid shear stress. The authors, to demonstrate that calcium and NO signals are induced in response to ciliary PC-1 activation, used *Tg737orpk/orpk* endothelial cells that lack ciliary ultrastructure but have functional PC-1. The results showed that neither calcium nor NO signals were present at flow rates up to 50 dyne/cm2 [3]. These results suggest that PC-1 is responsible for proper cilia mechanosensory

Follow-up studies by AbouAlaiwi et al. Showed that PC-2, which, as stated previously, is a calcium permeable cation channel that forms a complex with PC-1, is also important for mechanotransduction. Studies using a PC-2 knockdown line of cells showed a reduction in calcium and NO flux under shear stress when compared to control cells. This was further validated in *ex vivo* studies, where endothelial cells isolated from *pkd2−/−* mice arteries failed to respond to fluid shear stress [4]. These results indicate both PC-1 and PC-2 are needed for cilia mechanosensation, and further suggest that activation of the PC-1/PC-2 complex will start the signaling cascade needed for calcium-dependent NO biosynthesis. In addition, the results show that the increase in intracellular calcium is caused by an increase in intra-ciliary calcium. However, other researchers have proposed that calcium moves bidirectionally between the cilia and the cytosol [57–59]. Regardless, the increase in intracellular calcium triggers the calcium/calmodulin complex, which activates constitutive NOS, such as eNOS, by binding to its target site on the enzyme [60]. The calcium/calmodulin complex can also indirectly activate eNOS through activation of the AKT/PKB pathway, which stimulates AMPK (**Figure 2**) [61]. eNOS activation is mainly calcium dependent, but some studies have shown that a calcium independent pathway exists, via heat shock protein 90 (HSP90). HSP90 is also known to localize to cilia axonemes, and may act as a signal transductor that interacts with eNOS in the vasculature [62, 63]. HSP90 activation can lead to an increase in eNOS activity while calcium levels are high, and can also lead to more eNOS activity at low calcium levels due to its ability to directly bind to eNOS and

An under researched aspect of NO and primary cilia is their interaction in the vascular smooth muscle cell (VSMC) layer. Depending on blood vessel type, all

wound healing, dopamine signaling, and cellular proliferation.

function, and that ciliary PC-1 specifically elicits NO production.

increase the binding affinity for calmodulin [64, 65].

**54**

**4.2 Wound healing**

*Primary cilia activation via fluid shear stress and NO signaling in the vascular endothelia. The left panel shows primary cilia bending while under fluid shear stress, with the resultant production and release of nitric oxide (NO). The production and release of NO is dependent on the activation of endothelial primary cilia within the vasculature. The bending of cilia via fluid-shear stress activates the mechanosensory polycystins complex, which initiates the synthesis and the release of NO. This biochemical cascade, shown in the right panel, involves an extracellular calcium influx (Ca2+), followed by the activation of multiple calcium-dependent proteins, including calmodulin (CaM), protein kinase C (PKC) and Akt/PKB. Figure is adopted from Ref. [1].*

three isoforms of NOS may exist within the VSMC layers [66]. During normal function, the cilia on the VSMC layer extend towards the extracellular matrix. Under abnormal conditions, such as a scratch wound, VSMC primary cilia will migrate to the wound edge. A recent study showed that VSMC cilia express polycystins, as well as α3-and β1-integrins. When the researchers blocked integrin function, the percent of cilia migrating to the wound edge dropped from about 88% to around 30% [67]. This drop suggests that VSMC primary cilia may be involved in integrin-mediated wound healing.

In further support of the purported role primary cilia play in wound healing, results from experiments using VSMC that lacked cilia showed a slower scratchwound healing time than the ciliated control cells displayed [67]. While the cilia in the wounded area are directly exposed to constant fluid shear stress from blood flow, activation of the mechanosensory ciliary polycystin complex could occur, resulting in an increase in intracellular calcium [1]. This could potentially trigger vasoconstriction in VSMC, resulting in the isolation of the wounded area to allow the platelets to begin clot formation. As soon as the calcium amount reached a certain level, the calcium/calmodulin complex would form and activate eNOS and nNOS, leading to vasodilation and the next step in the wound healing process.

Further studies by Schneider et al. reveled that, once cellular growth was halted, platelet derived growth factor receptor alpha (PDGFRα), a tyrosine kinase with a prominent function in cellular proliferation, was found to localize to fibroblast primary cilia. Furthermore, ligand activation of PDGFRα will lead to the activation of the AKT and MEK/ERK proliferative pathways. Because AKT and ERK1/2 regulate eNOS activity in endothelial cells, PDGFRα activation could indirectly lead to eNOS activation [68]. Moreover, data from recent studies on endothelial progenitor cells indicate that platelet-derived growth factor AA (PDGF-AA) might contribute a vital role in wound healing, possibly by its effects on angiogenesis through the PI3K/ Akt/eNOS signaling pathway [69]. Bone morphological protein (BMP) receptor II (BMPRII), which is highly expressed on endothelial cells in lung vasculature, as well as moderately expressed in smooth muscle, is also involved in cell wound migration. In this pathway, migration is trigged by the ligands BMP2 and BMP4, which result in eNOS being phosphorylated [70]. While not conclusive, this evidence, when taken all together, suggests that primary cilia may have a significant part to play in the wound healing process.

When tissues begin to repair themselves after a wound, clots must be dissolved to maintain proper blood flow. This is known as clot retraction and platelet inhibition. NO is known to inhibit platelet aggregation, secretion, adhesion, and fibrinogen binding; all through activation of guanylyl cyclase and cGMP, alongside the inhibition of thromboxane A2. By this mechanism, platelet aggregation and accumulation are reduced, enabling the clot to dissolve, and the wound to heal fully [71–73]. Given the evidence, it is possible that an interaction between primary cilia and NO could be important in the wound healing and repair processes.

#### **4.3 Dopamine signaling**

Hypertension present in polycystic kidney disease (PKD) patients in the later stages of the disease is made worse by increased kidney volume. However, hypertension can also be seen in children, as well as the early stages of PKD, long before renal function starts to deteriorate. Some evidence suggests that an increase in sympathetic activation occurs in these patients, independently of their kidney function. Dopamine, an endogenous neuronal hormone that acts within the sympathetic nervous system, is confirmed to be involved in the regulation of blood pressure. Abnormal dopamine signaling can lead to hypertensive states in humans. Dopamine receptor 1 (D1) and dopamine receptor 5 (D5) receptors have been found to localize to primary cilia [39, 74–76]. While there are no current therapies that target D1 or D5, some studies using dopamine 1-like receptor subtypes showed vasodilatory effects in peripheral arteries [77].

The D5 receptor is thought to have both a chemosensory and mechanosensory role within primary cilia. Subjecting endothelial ciliary knockout cells *pkd1−/−* (lacking PC-1), and *Tg737orpk/orpk* cells that have no cilia, to dopamine under static conditions revealed a significantly subdued calcium influx when compared to the control cells. The researchers contributed this to the presence of underdeveloped cilia in the knockout cells, which would have less D5 receptors on them due to their smaller size. Under flow conditions with added dopamine, the mechanosensory function of the cilia knockout cells was restored, in comparison to the untreated knockout cells. Because calcium influx in these cell lines is associated with eNOS activation, the results of this study suggest a potential restoration of lost vasodilatory responses caused by a failed ciliary induction of NO biosynthesis [74]. There is additional evidence that suggests dopamine receptor 2 (D2) may also localize, or possibly get transported to, the primary cilia [78]. In one study, cerebral vasospasms were reversed with dopamine treatment; but when haloperidol, a D2 selective antagonist drug, was administered, the vasorelaxation failed to occur. It was also reported that, after administration of dopamine, a large increase in eNOS and iNOS expression was seen, and administration of haloperidol also blocked this effect [79].

D2 is also possibly transported to the primary cilia under specific conditions to mediate NOS activity within cells. Evidence supporting the role of ciliary dopamine receptors in the mediation of NO can be found in Autosomal Dominant Polycystic

**57**

*Primary Cilia are Sensory Hubs for Nitric Oxide Signaling*

Kidney Disease (ADPKD) patient clinical trials. ADPKD patients experience extrarenal maladies that mainly affect their cardiovascular system, such as hypertension. The hypertensive state could be brought on, in part, by the inability of primary endothelial cilia to respond to alterations in blood pressure. This would cause a failure to synthesize NO. In a study conducted by Lorthioir et al., flow-mediated dilation of normotensive ADPKD patients was compared to that of adults without ADPKD. It was shown that ADPKD patients had significantly less vasodilation during sustained flow increases, as well as a total loss of NO release when compared to those without ADPKD. When ADPKD patients were administered brachial infusions of 0.25–0.5 μg/kg/min of dopamine, there was an increase in flow-mediated dilation, and a statistically significant increase in dilatory response at the highest dose [80]. According to these results, dopamine receptors may facilitate a connection between

primary cilia, NO, and blood pressure regulation in ADPKD patients [1].

Primary cilia also help regulate cell proliferation. As stated in the ciliogenesis section, the cilia extends from the basal body, which is composed of mother and daughter centrioles, and cilia are reabsorbed after cell cycle re-entry [12]. In cancerous cell clusters, cilia are missing from the more prolific dividers, which suggests that despite not playing a major role in cell division, primary cilia are important for

NO possibly plays a role in cell proliferation as well, in conjunction with primary cilia. NO has been proven to halt the cell cycle by preventing the transition from G1 to S phase, in a dose dependent manner. The spike in NO is caused by an increase in free l-arginine, which is mediated by various cytokines. PC-1 is a known mediator of the JAK/STAT pathway by activating STAT3; when the cytosolic tail of PC-1 is cleaved upon once luminal flow halts, it can coactivate STAT-1, −3, and −6, along with JAK2. The PC-1 tail causes the cells to sensitize to cytokines and growth factor signaling, which then causes an exaggerated cellular response, which could potentially lead to an increase in l-arginine [85, 86]. Through this mechanism, overly

The superfamily of TGF-β signaling provides a fascinating system of cellular crosstalk, in which the effects of the same ligand can be unique depending on the cell type and the physiological conditions. This family is composed of more than 30 different ligand types of the TGF-β-activin-Nodal BMP subfamilies that can activate receptor serine/threonine kinases of types I and II (TGFβRI/II and BMP-RI/II, respectively). Ciliopathies widely overlap with phenotypes associated with aberrant TGF-β/BMP signaling. Prominent examples include structural heart defects associated with congenital heart disease (CHD) [87], suggesting that cardiac primary cilia may contribute to cellular events regulated by TGF-β/BMP signaling events during heart development. Moreover, different components of the TGF-β signalosome, including TGF-βRI, TGF-βRII, SMAD2/3, SMAD4, and SMAD7 are present at the cilia-centrosome axis. In a recent study, Feng et al. concluded that high salt (HS)-induced endothelial dysfunction and the development of salt-dependent increases in blood pressure (BP) were related to endothelial TGF-β signaling. Specifically, TGF-β-dependent ALK5 signaling increases endothelial NADPH oxidase-4 (NOX4), an enzyme that produces hydrogen peroxide, which limits NO bioavailability and ultimately promotes increased BP [88]. BMPRII contributes to cell proliferation through its interactions with primary cilia, eNOS, and NO. Using pulmonary artery endothelial cells, studies have shown that stimulation of BMPRII results in eNOS activation. BMPRII ligands BMP2 and BMP4 stimulate eNOS phosphorylation at a regulatory site via activation by protein kinase A. This eNOS

*DOI: http://dx.doi.org/10.5772/intechopen.89680*

starting and stopping cell mitosis [81–84].

prolific cell division would be arrested.

**4.4 Cell proliferation**

Kidney Disease (ADPKD) patient clinical trials. ADPKD patients experience extrarenal maladies that mainly affect their cardiovascular system, such as hypertension. The hypertensive state could be brought on, in part, by the inability of primary endothelial cilia to respond to alterations in blood pressure. This would cause a failure to synthesize NO. In a study conducted by Lorthioir et al., flow-mediated dilation of normotensive ADPKD patients was compared to that of adults without ADPKD. It was shown that ADPKD patients had significantly less vasodilation during sustained flow increases, as well as a total loss of NO release when compared to those without ADPKD. When ADPKD patients were administered brachial infusions of 0.25–0.5 μg/kg/min of dopamine, there was an increase in flow-mediated dilation, and a statistically significant increase in dilatory response at the highest dose [80]. According to these results, dopamine receptors may facilitate a connection between primary cilia, NO, and blood pressure regulation in ADPKD patients [1].
