**4. Novel compounds for NO-independent sGC/cGMP activation**

Organic NO donors such as nitrite and nitroglycerin are successful examples in clinical practice for more than a century. However, formation of harmful intermediate, peroxynitrite, and the long-term treatment with NO donors resulting in drug resistance limit the clinical applications of NO donor compounds. To overcome these obstacles, the novel agents for triggering sGC/cGMP cascade in NO-independent manner have been developed.

A series of 1-(substituted benzyl)-3-(substituted aryl)-condensed pyrazole derivatives were synthesized and identified as class novel antiplatelet agents [47, 48]. As one of the most promising analogues, 1-benzyl-3-(5′-hydroxymethyl-2′-furyl)

*Vascular Biology - Selection of Mechanisms and Clinical Applications*

therapeutic agents for cardiovascular diseases.

endogenous and competitive inhibitor of NOS [9–11].

could produce NO and nitrite.

**3. Pathological role of NO**

effects by activating sGC to increase the cGMP level and vascular effects known to be mediated by cGMP such as vasodilation, inhibition of platelet aggregation, and inflammatory reaction. Cyclic GMP modulates a number of signaling processes downstream of NO. The NO-cGMP cascade can be regulated by pharmacological modulation of protein kinases, phosphodiesterases (PDE), and ion channels to alter vascular tones as well as endothelial and vascular smooth muscle cell growth. Pharmacological alteration of the NO level has been a major strategy to develop

Deguchi and his colleagues found that GC activity in the supernatant of neuroblastoma and brain preparations were activated by L-arginine which has been identified as an endogenous activator of sGC [3]. Hibbs et al. noted the latter that the cytotoxic properties of macrophages in co-cultures with tumor cells could be enhanced with L-arginine but suppressed by N-N-methyl-arginine (LNMA), an inhibitor of nitric oxide synthase (NOS) [4]. This cytotoxicity action was accompanied by accumulation of nitrite in the conditioned medium. These important studies provide the insight to identify a pathway of L-arginine metabolism that

NOS is a group of isozymes which convert L-arginine to L-hydroxyarginine and

NO is essential in the maintenance of vascular homeostasis including smooth muscle relaxation, inhibition of platelet aggregation, attenuation of vascular smooth muscle cell (VSMC) proliferation, neurotransmission, and immune defense [12]. Therefore, the impaired NO pathway has been implicated in endothelial dysfunction and pathogenesis of a number of diseases featuring inflammatory reaction. These include arthritis, myocarditis, colitis, and nephritis. Altered NO synthesis has been noted in selected pathologic conditions such as amyotrophic lateral sclerosis, cancer, diabetes, and neurodegenerative diseases [13, 14]. In general, physiological NO actions on target tissues are brief, reversible, and dependent on the downstream cGMP-dependent signaling events. Conversely, the pathological actions noted with excessively and sustained NO production involved NO interaction with superoxide to generate peroxynitrite, a highly reactive free radical which exhibits the toxic actions of potent oxidants. Peroxynitrite, independent of the cGMP signaling events, has been implicated in oxidative injury noted in a number of disease models [15–17]. In addition to its free radical actions, peroxynitrite inactivates prostacyclin synthase to reduce prostacyclin levels, leading to vascular

ADMA, a risk factor for cardiovascular diseases, inhibits NOS to reduce biosynthesis of NO, resulting in impaired blood flow, accelerated atherogenesis, and suppressed angiogenesis [19]. ADMA is involved in the development of endothelial dysfunction. In essential hypertension patients, the L-arginine and ADMA levels are elevated and inversely related to endothelial function [20, 21]. Endothelial function depends on the integrity of eNOS and the availability and vascular signaling of

subsequently to NO and L-citrulline through cofactors including reduced nicotinamide-adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and tetrahydrobiopterin (BH4) [5]. The first NOS isoform to be identified was the neuronal NOS (nNOS or named as NOS-1) [6]. This was followed shortly thereafter by inducible NOS (iNOS), also known as type II NOS (NOS-2) [7], and then by endothelial NOS (eNOS or named as NOS-3) [8]. NOS can also be inactivated by asymmetric dimethyl arginine (ADMA), an

**74**

dysfunction [18].

indazole (YC-1) was selected for further investigation. The physiological property of YC-1 in stimulation of sGC was demonstrated by Ko and colleagues [49]. Potential regulatory mechanisms of YC-1 on cardiovascular protections were summarized in **Figure 1**. Ko et al. showed that YC-1 is an antithrombotic agent. It inhibits platelet aggregation by increasing platelet cGMP levels in an NO-independent manner. YC-1 action was noted to exert its antiplatelet effect through the activation of NO-independent sGC/cGMP pathway [50]. Nearly, all the newer generations of sGC stimulator except acryl-acrylamide family have been derived based on YC-1 as the parent compound [51]. YC-1 and its successors all require the presence of a reduced haem moiety within sGC to stimulate sGC, but they also act in synergy with NO by binding NO or iron-free precursor of haem to structurally resemble the NO-haem complex and stabilize sGC in its active configuration [52–54]. Stasch et al. also reported that YC-1 and its derivate, BAY 41-2272, bind to regulatory sites (cys 238 and cyst 243 regions) in the α1-subunit of sGC, resulting in conformational change and subsequent activation of recombinant sGC by NO-independent but haem-dependent mechanism [55]. Mulsch et al. also noted that the combined effect of nitrovasodilators and YC-1 in cultured VSMCs and isolated rabbit aortic rings reflected the direct synergistic action of YC-1 and NO on the sGC [56]. Wohlfart et al. reported that YC-1 can stimulate synthesis and release NO in endothelial cells independent of raising the cGMP content in a calcium-dependent manner [57]. In addition, YC-1 inhibits the cGMP-specific phosphodiesterase type 5 (PDE-5)

#### **Figure 1.**

*Schematic overview of regulatory mechanisms of YC-1 on cardiovascular protections. cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; ECs, endothelial cells; FAK, focal adhesion kinase; GMP, guanosine monophosphate; GTP, guanosine triphosphate; HO-1, heme oxygenase-1; Hsp70, heat shock protein 70; iNOS, inducible nitric oxide synthase; MMP2, matrix metalloproteinase-2; MMP-9, matrix metalloproteinase-9; oxLDL, oxidized low-density lipoprotein; PDE5, phosphodiesterase 5; PKG, protein kinase G; sGC, soluble guanylyl cyclase; TGF-β, transforming growth factor-beta; VASP, vasodilator-stimulated phosphoprotein; VSMC, vascular smooth muscle cells.*

**77**

**5.2 Antitumor effects**

*Therapeutic Applications and Mechanisms of YC-1: A Soluble Guanylate Cyclase Stimulator*

in platelets and in aortic extracts to raise cGMP levels and prolong its duration of action [58, 59]. The vasodilator-stimulated phosphoprotein (VASP) has been reported to be involved in cGMP- and cAMP-mediated antiplatelet actions [60]. Becker et al. noted that VASP is the target of YC-1 since VASP phosphorylation can be directly increased through stimulation of the cGMP/protein kinase G/VASP

In addition to the effects in antiplatelet aggregation and antithrombosis, YC-1 has been demonstrated to provide several beneficial effects including cardiovascular protections; antitumor, neuroprotective, and anti-inflammatory effects; as well

YC-1 inhibits VSMC proliferation, similar to specific guanylate cyclase inhibitors, suggesting that the antiproliferative effect of YC-1 is mediated by cGMP [62]. A similar conclusion has also been drawn by other investigators [63, 64]. As shown in **Figure 1**, NO-/cGMP-dependent processes have been suggested to modulate VSMC phenotype and the arterial response to endovascular injury [65, 66]. It has been reported that YC-1 can upregulate expression of iNOS and inducible heme oxygenase-1 (HO-1) at the transcriptional and translational level as well as stimulate sGC and cGMP production in the balloon-injured artery [63]. These results support the proposal that YC-1 can be developed as a potent new therapeutic agent for reducing restenosis *via* endogenous carbon monoxide (CO)- and/ or NO-mediated cGMP-dependent processes. Wu et al. found that two important modulators, transforming growth factor (TGF)-β1 and focal adhesion kinase (FAK), responsible for VSMC proliferation and migration were reduced in content in the cultured VSMC treated with YC-1. The effect of YC-1 on preventing balloon injury-induced vascular stenosis has also been demonstrated in a rat carotid angioplasty model [64]. Liu et al. also found that YC-1 can inhibit neointima formation in balloon-injured rat carotid through suppressing the expression and actions of matrix metalloproteinase (MMP)-2 and MMP-9 [67]. YC-1 can also prevent oxLDL-mediated apoptosis by inducing heat shock protein 70 (Hsp70) expression in VSMCs suggesting its cytoprotective effect in vascular diseases [68]. Similarly, Hsp70 overexpression has also been involved in protective effect of YC-1 on heat stroke [69]. In vivo evidence shows that YC-1 and zaprinast, an inhibitor of cGMPselective PDE, inhibit injury-induced vascular remodeling through anti-mitogenic

and pro-apoptotic actions in a rat carotid artery balloon injury model [70]. Moreover, YC-1 has also been found to induce cell cycle arrest of HUVEC through upregulation of p21 and p27 protein *via* inhibition of the cyclin/cyclin-dependent kinase (CDK) system. This finding suggests that YC-1-induced antiproliferation effect in HUVEC is *via* a cGMP-independent manner [71]. Besides, the prevention effects of YC-1 on the development of hypoxia-induced pulmonary arterial hypertension (PAH), right ventricular hypertrophy (RVH), and pulmonary vascular

A growing body of evidence indicates that hypoxia-inducible factor-1 (HIF-1) contributes to tumor progression and metastasis. YC-1 inhibits HIF-1-mediated

remodeling has been clearly mentioned in animal model [72].

*DOI: http://dx.doi.org/10.5772/intechopen.84572*

**5. Additional pharmacological activities of YC-1**

pathway [61].

as optical protections.

**5.1 Cardiovascular protections**

*Therapeutic Applications and Mechanisms of YC-1: A Soluble Guanylate Cyclase Stimulator DOI: http://dx.doi.org/10.5772/intechopen.84572*

in platelets and in aortic extracts to raise cGMP levels and prolong its duration of action [58, 59]. The vasodilator-stimulated phosphoprotein (VASP) has been reported to be involved in cGMP- and cAMP-mediated antiplatelet actions [60]. Becker et al. noted that VASP is the target of YC-1 since VASP phosphorylation can be directly increased through stimulation of the cGMP/protein kinase G/VASP pathway [61].
