*3.3.3 Ca2+-independent muscle relaxation regulated by NO*

Independent from NO-Ca2+ pathway, in SMCs NO also increases MLCP activity and limits MLCK activity, resulting in a dephosphorylation shift of myosin light chain phosphorylation balance [15]. Thus, myosin cross-bridge cycling is inhibited, causing smooth muscle relaxation.

## **4. Vascular structural integrity mediated by NO**

Anatomic alterations in the cardiovascular structure directly deteriorate cardiovascular functions. NO is a multifunctional regulator for homeostasis in the cardiovascular system. An intact endothelial layer is the hub for NO generation. Pathological changes in NO generation can trigger various local flaws that may progress to be systematic cardiovascular issues with time.

#### **4.1 NO-induced alterations in endothelial permeability**

Deviant NO level causes change of endothelial permeability, a key characteristic for mass transfer and extravasation. Interestingly, increase, decrease, and no change of vascular permeability due to the presence of NO have been reported. Using

high concentration (millimolar level) of exogenous NO donor spermine NONOate decreased endothelial permeability in the *in vitro* human umbilical vein endothelial cell (HUVEC) model [57]. And this effect was amplified by vitamin C, a chemical that increases the apparent half-life of NO. However, in the frog mesenteric capillary model, inhibition of NO synthesis by L-NMMA decreased capillary permeability [58]. Moreover, although NO effectively regulated basal vascular tone in the blood-brain barrier, it demonstrated no effect on its basal permeability [59]. Again, these results demonstrate that NO's biological effect is sensitive to NO concentration, duration, and environment.

Vascular permeability is mainly determined by tightness of cell-cell junctions [60]. Tight junctions (TJs) and adherens junctions (AJs) are the most abundant interendothelial junctions. And both junctions are closely related to actin cytoskeleton dynamics [61] (**Figure 2**). TJs are composed of series of transmembrane proteins that anchor to the actin cytoskeleton to hold cells together. They seal the cells to maintain cell polarity and prevent the molecules from traveling through the space between cells. AJs consist of clusters of transmembrane protein cadherin, which is connected to actin cytoskeleton on its cytoplasmic side and binds strongly with cadherins residing on the neighboring cell membrane. These junctions are important for transmitting mechanical force between cells and reinforcing tissues. Since both junctions directly connect cytoskeletons, the cytoskeleton's behavior will influence cell-cell junctions and thus control vascular permeability. When actin and myosin filaments undergo relative sliding to cause cell contraction, the cytoskeleton-associated membrane proteins will be pulled into the cells, and cell-cell junctions are disrupted. NO mediates cell contraction by adjusting [Ca2+]i. Therefore, deviated NO level may cause the change of cell-cell junctions [60].

Another important downstream molecule of NO is vascular endothelial growth factor (VEGF) which has been extensively studied in cancer research due to its angiogenic effect. VEGF was initially considered as a vascular permeability factor, because it caused the formation of leaky capillaries [62], which is an important characteristic in tumor and retinopathy. Low NO level induces VEGF synthesis under normoxia through the transcription factor hypoxia-inducible factor 1 (HIF-1) [25, 63]. VEGF activates Src kinases, which further phosphorylate cadherin and elicit its internalization [64]. Once cells lose cadherin interactions, gaps between cells form and endothelial permeability is increased.

#### **4.2 Inhibition of SMCs proliferation by NO**

One distinctive characteristic of vascular SMCs is the phenotypic plasticity. Two most important phenotypes are contractile and synthetic. Contractile SMCs guarantee the good performance of muscle contraction/relaxation, while synthetic SMCs are highly proliferative and migratory, crucial for vascular remodeling during pregnancy and injury healing. Dysregulation of the phenotype transition causes neointima formation [65]. NO plays important roles in suppressing SMCs' contractile to synthetic transition.

NO donors and 8-Br-cGMP showed similar effect in inhibiting SMC migration and proliferation, indicating NO's inhibitory effect might be through the cGMP-dependent pathway [66]. SMCs overgrow when stimulated by serum and epidermal growth factor (EGF). Many studies were based on these models, though divergent results were reported. EGF induces SMC proliferation through mitogen-activated protein kinases (MAPK) pathway, also called extracellular signal-regulated kinases (ERK) pathway. Ras (a small GTPase) and Raf (kinase of MAPK kinase, or MAPKKK) are the critical upstream protein kinases in this pathway. NO blocks MAPK pathway by prohibiting Raf from being activated by Ras-GTP in rat aortic SMCs. It is believed that PKG

**11**

*Nitric Oxide and Oxidative Stress-Mediated Cardiovascular Functionality: From Molecular…*

phosphorylates Raf, resulting in the conformation change. Thus, Ras-GTP cannot recognize Raf, causing the block of MAPK pathway and the accumulation of Ras-GTP [67]. Meanwhile, elevation of cGMP induced by IL-1β is correlated with the activation of PKA, and it can be prevented by blocking NO and cGMP pathways. Interestingly, this effect is cAMP independent, but PKA inhibitor, not PKG inhibitor, can prevent the inhibition of the proliferation, indicating that cGMP-PKA cross talk plays impor-

NO-cGMP pathway may inhibit SMC growth by impairing cytoskeleton reorganization. Vasodilator-stimulated phosphoprotein (VASP) is characterized as a substrate of both PKG and PKA [69]. It targets focal adhesions and is involved in actin filament formation. Cell morphology change during proliferation relies on VASP, and its activation relies on the phosphorylation of Ser157 primarily mediated by PKA [70]. However, PKG can phosphorylate Ser239 and Thr278 to impair VASP's

NO also directly mediates proteins associated with cell cycle and cell metabolism by cGMP-independent mechanisms. Cyclin A and cyclin-dependent kinase 2 expression levels can be blunted by exogenous NO donor DETA NONOate in an *in vitro* vascular SMC model [72]. Ornithine decarboxylase (ODC) catalyzes the ornithine decarboxylation to form polyamines, which are necessary for cell growth and proliferation. ODC's active center can be masked by nitrosylation. And NO biosynthesis's intermediate product N(omega)-hydroxyarginine can inhibit ODC

Thrombus formation is critical for hemostasis during injury. However, thrombus in blood vessels can cause stroke and heart attack. Stable thrombus reduces lumen size and stiffens blood vessels. Unstable thrombus may rupture with blood flow and block the vessel. Activation of platelet is a critical step for thrombus formation, which involves exocytosis processes to expose P-selectin on the platelet surface and activate glycoprotein IIb/IIIa. Both processes depend on the elevation of [Ca2+]i controlled by IP3 pathway. NO suppresses platelet activation through NO-cGMP pathway [74]. Although, the inhibition pathway has not been fully characterized, evidences have shown that cGMP-PKG blocks agonist-induced IP3 formation in platelet [75], and PKG can phosphorylate IP3 receptor to inhibit Ca2+ release from the ER [35]. When the endothelium loses its integrity, there will be a local shortage of thromboregulators such as NO, prostacyclin, and ectonucleotidase CD 39, resulting in thrombogenesis [76]. Collagen and tissue factors also trigger the coagulation reactions [76]. The use of blood contact implant is another common source of thrombus. Note that, all materials are thrombogenic to some degrees. To enhance implant biocompatibility, an efficient method is to use NO releasing polymers to fabricate or surface coat the blood contacting devices (such as vascular graft/stent, intravascular catheter, and sensor implants). Common strategies include: physically incorporating NO releasing chemicals into polymer matrices, chemically linking NO releasing agent to polymer backbones, and developing materials that can trigger NO generation using endogenous NO donors circulating in the blood. By using the first two strategies, successful trials have been reported to achieve long-term (over few weeks to months) NO releasing and antithrombotic applications [77–79]. Good NO donors include N-diazeniumdiolate and S-nitrosothiols. Both hydrophilic and hydrophobic polymers that are commonly used in medical device fabrication have been successfully modified for NO release including poly(vinyl chloride), polymethacrylates, various hydrogels, polyethylene terephthalate, polyurethane, and silicone rubbers [77]. The third strategy directly uses endogenous NO donors as

*DOI: http://dx.doi.org/10.5772/intechopen.82556*

tant roles in suppressing rat aortic SMCs' proliferation [68].

activity and inhibit actin cytoskeleton reorganization [70, 71].

enzyme activity [73] to disrupt cell proliferation.

**4.3 Prevention of thrombogenesis by NO**

*Nitric Oxide and Oxidative Stress-Mediated Cardiovascular Functionality: From Molecular… DOI: http://dx.doi.org/10.5772/intechopen.82556*

phosphorylates Raf, resulting in the conformation change. Thus, Ras-GTP cannot recognize Raf, causing the block of MAPK pathway and the accumulation of Ras-GTP [67]. Meanwhile, elevation of cGMP induced by IL-1β is correlated with the activation of PKA, and it can be prevented by blocking NO and cGMP pathways. Interestingly, this effect is cAMP independent, but PKA inhibitor, not PKG inhibitor, can prevent the inhibition of the proliferation, indicating that cGMP-PKA cross talk plays important roles in suppressing rat aortic SMCs' proliferation [68].

NO-cGMP pathway may inhibit SMC growth by impairing cytoskeleton reorganization. Vasodilator-stimulated phosphoprotein (VASP) is characterized as a substrate of both PKG and PKA [69]. It targets focal adhesions and is involved in actin filament formation. Cell morphology change during proliferation relies on VASP, and its activation relies on the phosphorylation of Ser157 primarily mediated by PKA [70]. However, PKG can phosphorylate Ser239 and Thr278 to impair VASP's activity and inhibit actin cytoskeleton reorganization [70, 71].

NO also directly mediates proteins associated with cell cycle and cell metabolism by cGMP-independent mechanisms. Cyclin A and cyclin-dependent kinase 2 expression levels can be blunted by exogenous NO donor DETA NONOate in an *in vitro* vascular SMC model [72]. Ornithine decarboxylase (ODC) catalyzes the ornithine decarboxylation to form polyamines, which are necessary for cell growth and proliferation. ODC's active center can be masked by nitrosylation. And NO biosynthesis's intermediate product N(omega)-hydroxyarginine can inhibit ODC enzyme activity [73] to disrupt cell proliferation.

#### **4.3 Prevention of thrombogenesis by NO**

Thrombus formation is critical for hemostasis during injury. However, thrombus in blood vessels can cause stroke and heart attack. Stable thrombus reduces lumen size and stiffens blood vessels. Unstable thrombus may rupture with blood flow and block the vessel. Activation of platelet is a critical step for thrombus formation, which involves exocytosis processes to expose P-selectin on the platelet surface and activate glycoprotein IIb/IIIa. Both processes depend on the elevation of [Ca2+]i controlled by IP3 pathway. NO suppresses platelet activation through NO-cGMP pathway [74]. Although, the inhibition pathway has not been fully characterized, evidences have shown that cGMP-PKG blocks agonist-induced IP3 formation in platelet [75], and PKG can phosphorylate IP3 receptor to inhibit Ca2+ release from the ER [35].

When the endothelium loses its integrity, there will be a local shortage of thromboregulators such as NO, prostacyclin, and ectonucleotidase CD 39, resulting in thrombogenesis [76]. Collagen and tissue factors also trigger the coagulation reactions [76]. The use of blood contact implant is another common source of thrombus. Note that, all materials are thrombogenic to some degrees. To enhance implant biocompatibility, an efficient method is to use NO releasing polymers to fabricate or surface coat the blood contacting devices (such as vascular graft/stent, intravascular catheter, and sensor implants). Common strategies include: physically incorporating NO releasing chemicals into polymer matrices, chemically linking NO releasing agent to polymer backbones, and developing materials that can trigger NO generation using endogenous NO donors circulating in the blood. By using the first two strategies, successful trials have been reported to achieve long-term (over few weeks to months) NO releasing and antithrombotic applications [77–79]. Good NO donors include N-diazeniumdiolate and S-nitrosothiols. Both hydrophilic and hydrophobic polymers that are commonly used in medical device fabrication have been successfully modified for NO release including poly(vinyl chloride), polymethacrylates, various hydrogels, polyethylene terephthalate, polyurethane, and silicone rubbers [77]. The third strategy directly uses endogenous NO donors as

*Vascular Biology - Selection of Mechanisms and Clinical Applications*

tion, duration, and environment.

high concentration (millimolar level) of exogenous NO donor spermine NONOate decreased endothelial permeability in the *in vitro* human umbilical vein endothelial cell (HUVEC) model [57]. And this effect was amplified by vitamin C, a chemical that increases the apparent half-life of NO. However, in the frog mesenteric capillary model, inhibition of NO synthesis by L-NMMA decreased capillary permeability [58]. Moreover, although NO effectively regulated basal vascular tone in the blood-brain barrier, it demonstrated no effect on its basal permeability [59]. Again, these results demonstrate that NO's biological effect is sensitive to NO concentra-

Vascular permeability is mainly determined by tightness of cell-cell junctions [60]. Tight junctions (TJs) and adherens junctions (AJs) are the most abundant interendothelial junctions. And both junctions are closely related to actin cytoskeleton dynamics [61] (**Figure 2**). TJs are composed of series of transmembrane proteins that anchor to the actin cytoskeleton to hold cells together. They seal the cells to maintain cell polarity and prevent the molecules from traveling through the space between cells. AJs consist of clusters of transmembrane protein cadherin, which is connected to actin cytoskeleton on its cytoplasmic side and binds strongly with cadherins residing on the neighboring cell membrane. These junctions are important for transmitting mechanical force between cells and reinforcing tissues. Since both junctions directly connect cytoskeletons, the cytoskeleton's behavior will influence cell-cell junctions and thus control vascular permeability. When actin and myosin filaments undergo relative sliding to cause cell contraction, the cytoskeleton-associated membrane proteins will be pulled into the cells, and cell-cell junctions are disrupted. NO mediates cell contraction by adjusting [Ca2+]i. Therefore,

Another important downstream molecule of NO is vascular endothelial growth factor (VEGF) which has been extensively studied in cancer research due to its angiogenic effect. VEGF was initially considered as a vascular permeability factor, because it caused the formation of leaky capillaries [62], which is an important characteristic in tumor and retinopathy. Low NO level induces VEGF synthesis under normoxia through the transcription factor hypoxia-inducible factor 1 (HIF-1) [25, 63]. VEGF activates Src kinases, which further phosphorylate cadherin and elicit its internalization [64]. Once cells lose cadherin interactions, gaps between

One distinctive characteristic of vascular SMCs is the phenotypic plasticity. Two most important phenotypes are contractile and synthetic. Contractile SMCs guarantee the good performance of muscle contraction/relaxation, while synthetic SMCs are highly proliferative and migratory, crucial for vascular remodeling during pregnancy and injury healing. Dysregulation of the phenotype transition causes neointima formation [65]. NO plays important roles in suppressing SMCs' contrac-

NO donors and 8-Br-cGMP showed similar effect in inhibiting SMC migration and proliferation, indicating NO's inhibitory effect might be through the cGMP-dependent pathway [66]. SMCs overgrow when stimulated by serum and epidermal growth factor (EGF). Many studies were based on these models, though divergent results were reported. EGF induces SMC proliferation through mitogen-activated protein kinases (MAPK) pathway, also called extracellular signal-regulated kinases (ERK) pathway. Ras (a small GTPase) and Raf (kinase of MAPK kinase, or MAPKKK) are the critical upstream protein kinases in this pathway. NO blocks MAPK pathway by prohibiting Raf from being activated by Ras-GTP in rat aortic SMCs. It is believed that PKG

deviated NO level may cause the change of cell-cell junctions [60].

cells form and endothelial permeability is increased.

**4.2 Inhibition of SMCs proliferation by NO**

tile to synthetic transition.

**10**

the NO reservoir to catalyze NO generation from S-nitrosoglutathione or nitrite in the body. Currently, its main challenge is to adjust the NO releasing rate to be more biologically relevant.
