**9. Vasodilator-stimulated phosphoprotein**

physiological/pathophysiological importance in VSM and/or during CVD is not well under‐

As mentioned, several molecules exist in this wide family of cyclic nucleotides that are considered to be true second messengers. A second messenger is defined as being generated by a first-messenger-regulated enzyme, being activated by targeted effector proteins, exerting defined biological effects, being degraded by specific inactivation mechanisms, and whose effects can be duplicated by membrane-permeable analogues and/or bacterial nucleotidyl cyclase toxins. [17] Accordingly, both cyclic AMP and cyclic GMP are considered the tradi‐ tional second messengers, being generated by membrane-bound and/or soluble cyclase enzymes, exerting many effects via intracellular kinases and/or nonkinase targets such as ion channels, have broad biological effects, are inactivated by a family of phosphodiesterases (PDEs), and whose effects can be mimicked by specific analogues or toxins. Also, new theories suggest that the pyrimidines cyclic CMP and cyclic UMP are in fact emerging second messen‐

The most recognized cyclic nucleotide second messengers are cyclic AMP and cyclic GMP which are synthesized and operate in similar mechanistic fashion. Cyclic AMP is generated through several different avenues including adenylate cyclase (AC) stimulation by direct agonists or following beta-stimulation or through G protein-coupled receptor activation. Following stimulation of AC, adenosine triphosphate (ATP) is dephosphorylated to produce cyclic AMP and PPi. Similarly, following activation of GC through natriuretic peptides (activating particulate GC) or from the gaseous ligands (NO) or carbon monoxide (CO) which activate soluble GC, GTP is dephosphorylated to yield cyclic GMP and PPi. Cyclic AMP and cyclic GMP operate largely through downstream phosphorylation events on Ser/Thr or tyrosine (Tyr) residues on target proteins and serve diverse roles in normal vascular physiol‐ ogy and homeostasis and during the pathogenesis of CVD. The preferred target proteins for cyclic AMP and cyclic GMP are protein kinase A (PKA) and PKG, respectively [19]. Much of the work from our lab over the past few years has focused on cyclic AMP and cyclic GMP and their abilities to target downstream phosphorylatable substrates mainly through PKA and PKG to elicit functional control over a variety of physiological and pathophysiological parameters elemental to CVD. Some of our latest studies have identified the Ser/Thr kinases PKA, PKG, protein kinase C (PKC), and the metabolic gauge AMP-activated protein kinase (AMPK) as biologically important regulators of VSM proliferation, migration, and chemotaxis; ECM and MMP balance; and cellular apoptosis or necrosis using a variety of experimental approaches and commercially available rodent VSM cells, primary rodent VSM cells, and human primary VSM cells. We have observed capacity of Ser/Thr-specific protein phospha‐ tases (PPs) in moderating these kinase activities and in maintaining proper phosphorylative balance. We have also witnessed abilities of the Ser/Thr PPs to elicit control over VSM growth independent of direct kinase involvement. Additionally, many of these studies performed in cultured cells have been validated in whole animal models of injury-induced VSM growth, and these findings have verified biological ability of these cyclic nucleotide systems to operate in a whole body setting. Lastly, we have solidified these observations obtained in rodent models by recapitulating them in human primary coronary artery VSM preparations, thus

stood.

16 Muscle Cell and Tissue

gers in that they also fulfill these criteria [17,18].

Following generation of cyclic AMP and cyclic GMP and activation of their respective kinases PKA and PKG (among others, as discussed below), these kinases then target a variety of proteins and other factors to elicit functional consequences. Among these numerous bioactive targets is vasodilator-stimulated phosphoprotein or VASP. VASP belongs to the Ena/VASP Homology (EVH) family of cytoskeletal proteins that play essential roles in cellular dynamics and function. These proteins are involved in intracellular signaling processes and regulate "outside-in" communication via integrin–ECM interactions. VASP is comprised of an Nterminal EVH1 domain (targets focal adhesion and membrane domains), a proline-rich midregion that binds to Src-homology 3 (SH3) domains, and WW domain-containing proteins that aid in Ser/Thr binding, and a C-terminal EVH2 domain that mediates tetramerization and actin/focal adhesion binding. Figure 5 shows a schematic of the primary VASP sequence including essential EVH1 and EVH2 domains. **FIGURE 5**

**Figure 5.** Schematic for the primary sequence of VASP complete with an N-terminal EVH1 domain (that targets focal adhesion/membrane domains) and a C-terminal EVH2 domain that targets focal adhesion/actin binding. (Adapted from [20])

VASP as a Ser/Thr-containing protein was originally characterized as a substrate for cyclic nucleotide-directed kinase-mediated phosphorylation signals, with cyclic AMP-driven PKA preferentially phosphorylating VASPSer157 and cyclic GMP-directed PKG primarily acting on VASPSer239 [21-24]. To date, at least four distinct Ser/Thr phosphorylation sites have been identified on VASP: Ser157 and Ser239 as well as Thr278 and Ser322 [25-27]. Interestingly, despite this original characterization of cyclic nucleotide-directed kinases "selectively" phosphory‐ lating discrete Ser/Thr residues on downstream substrates including VASP, emerging reports from our lab and others have documented crosstalk and promiscuity of these and other Ser/Thr kinases to phosphorylate discrete residues on VASP. Recent observations from our laboratory in commercial [28] and primary VSM cells [19,29-31] as well unpublished findings in human primary coronary VSM cells document capacities of cyclic AMP and cyclic GMP to not only phosphorylate their respective canonical targets PKA and PKG but to also have abilities to phosphorylate other kinases as well. In these studies, findings reveal that cyclic AMP, stimulated via direct or indirect means, phosphorylates its accepted target PKA but also phosphorylates the preferred PKG target VASPSer239. Similarly, cyclic GMP, also activated directly or indirectly, phosphorylates its accepted target VASPSer239 as well as the supposed PKA substrate VASPSer157. We have also documented abilities of both cyclic AMP and cyclic GMP to stimulate members of the diverse PKC family [30]. Recently we observed that AMP kinase, another Ser/Thr enzyme, not only phosphorylates its canonical target VASPThr278 [32] but also has capacity to phosphorylate the preferred PKA target VASPSer157, yet interestingly without observable effects on VASPSer239 [33,34]. Recently it was reported that PKD, a down‐ stream product of PKC and a Ser/Thr kinase involved in ECM receptor-mediated signal transduction pathways, phosphorylates both its reported VASPSer322 site as well as the PKA VASPSer157 site [35]. Considering this crosstalk among these kinases and the target VASP, it is important to note that while Ser/Thr and Tyr kinases can target a specific residue, these enzymes are also attracted to flanking residues alongside the known phosphoacceptor site. By this virtue, the catalytic cleft of the kinase interacts not only with its preferred Ser, Thr, and/or Tyr phosphoacceptor residue but also with their flanking regions, thereby binding to similar recognition sequences among similar substrate family members and in turn reducing their specificity for a particular residue. This kinase crosstalk then affords broad impact of upstream kinase signaling but also clouds precision of downstream targeting. Regardless of this observed promiscuity, these critical cyclic nucleotide/kinase/VASP signaling cascades elicit a vast array of significant biological effects in VSM and are of critical importance in vascular physiology and pathology.

Functionally, VASP operates as an anticapping protein and assists in the down-regulation of platelet adhesion molecules, promotes endothelial barrier protection, and assists in vessel structural integrity [36,37]. VASP was recently shown to protect against vascular inflammation and insulin resistance following exposure to high-fat diet [36]. In cancer research, VASP is implicated as a possible pharmacotherapeutic target aimed at controlling aberrant cancer cell migration [35,36]. In those studies, protection by VASP is attributed to its multiple phosphor‐ ylation residues (namely Ser157, Ser239, Thr278, and Ser322) that significantly and differentially impact its function as an actin-binding protein. Interestingly, like cancer cells, VSM cells rely on reorganization of their actin cytoskeleton as their primary mechanism of movement [38-40],

Vascular Smooth Muscle as a Therapeutic Target in Disease Pathology http://dx.doi.org/10.5772/60878 19

**FIGURE 6**

VASP as a Ser/Thr-containing protein was originally characterized as a substrate for cyclic nucleotide-directed kinase-mediated phosphorylation signals, with cyclic AMP-driven PKA preferentially phosphorylating VASPSer157 and cyclic GMP-directed PKG primarily acting on VASPSer239 [21-24]. To date, at least four distinct Ser/Thr phosphorylation sites have been identified on VASP: Ser157 and Ser239 as well as Thr278 and Ser322 [25-27]. Interestingly, despite this original characterization of cyclic nucleotide-directed kinases "selectively" phosphory‐ lating discrete Ser/Thr residues on downstream substrates including VASP, emerging reports from our lab and others have documented crosstalk and promiscuity of these and other Ser/Thr kinases to phosphorylate discrete residues on VASP. Recent observations from our laboratory in commercial [28] and primary VSM cells [19,29-31] as well unpublished findings in human primary coronary VSM cells document capacities of cyclic AMP and cyclic GMP to not only phosphorylate their respective canonical targets PKA and PKG but to also have abilities to phosphorylate other kinases as well. In these studies, findings reveal that cyclic AMP, stimulated via direct or indirect means, phosphorylates its accepted target PKA but also phosphorylates the preferred PKG target VASPSer239. Similarly, cyclic GMP, also activated directly or indirectly, phosphorylates its accepted target VASPSer239 as well as the supposed PKA substrate VASPSer157. We have also documented abilities of both cyclic AMP and cyclic GMP to stimulate members of the diverse PKC family [30]. Recently we observed that AMP kinase, another Ser/Thr enzyme, not only phosphorylates its canonical target VASPThr278 [32] but also has capacity to phosphorylate the preferred PKA target VASPSer157, yet interestingly without observable effects on VASPSer239 [33,34]. Recently it was reported that PKD, a down‐ stream product of PKC and a Ser/Thr kinase involved in ECM receptor-mediated signal transduction pathways, phosphorylates both its reported VASPSer322 site as well as the PKA VASPSer157 site [35]. Considering this crosstalk among these kinases and the target VASP, it is important to note that while Ser/Thr and Tyr kinases can target a specific residue, these enzymes are also attracted to flanking residues alongside the known phosphoacceptor site. By this virtue, the catalytic cleft of the kinase interacts not only with its preferred Ser, Thr, and/or Tyr phosphoacceptor residue but also with their flanking regions, thereby binding to similar recognition sequences among similar substrate family members and in turn reducing their specificity for a particular residue. This kinase crosstalk then affords broad impact of upstream kinase signaling but also clouds precision of downstream targeting. Regardless of this observed promiscuity, these critical cyclic nucleotide/kinase/VASP signaling cascades elicit a vast array of significant biological effects in VSM and are of critical importance in

Functionally, VASP operates as an anticapping protein and assists in the down-regulation of platelet adhesion molecules, promotes endothelial barrier protection, and assists in vessel structural integrity [36,37]. VASP was recently shown to protect against vascular inflammation and insulin resistance following exposure to high-fat diet [36]. In cancer research, VASP is implicated as a possible pharmacotherapeutic target aimed at controlling aberrant cancer cell migration [35,36]. In those studies, protection by VASP is attributed to its multiple phosphor‐ ylation residues (namely Ser157, Ser239, Thr278, and Ser322) that significantly and differentially impact its function as an actin-binding protein. Interestingly, like cancer cells, VSM cells rely on reorganization of their actin cytoskeleton as their primary mechanism of movement [38-40],

vascular physiology and pathology.

18 Muscle Cell and Tissue

**Figure 6.** Exercise increases VASPSer239 in rat thoracic aorta (TA) segments following a 10 day treadmill running proto‐ col. **A**: Tissue homogenates of TA sections were pooled from 4 animals per group (sedentary (Sed.) or exercised (Ex.)) and immunoprecipitation (IP) for VASPSer239 was performed. IP fractions were then subjected to gel electrophoresis fol‐ lowed by ECL Western blotting for VASPSer239. **B** represents results from densitometry for VASPSer239 from TA homoge‐ nates in Sed. or Ex. cohorts. Preliminary results show that exercise-induced shear stress approximately doubles VASPSer239 expression in rat TA sections compared to sedentary controls. These early results suggest that shear stress acts to reduce VSM migration via VASPSer239, which may serve as a mechanism underlying beneficial effects of exercise against CVD.

and so VASP serves as a plausible target for control of migration in the context of CVD. In our lab, we have recently examined site-specific phosphorylated VASP as a controller of VSM migration and proliferation in commercial and primary VSM cells [19,28-31]. In summary, results suggest that VASPSer239 acts primarily to control cell migration, yet VASPSer157 serves to regulate cell proliferation, both key components in CVD pathogenesis. Also, by virtue of its name, VASP is inherently involved in regulation of hemodynamics via its upstream activators (cyclic AMP/cyclic GMP/kinases) and downstream effectors. In the context of an endothelialcompromised (diseased) blood vessel with fenestration of the intimal–medial barrier and basement membrane, underlying VSM cells become exposed to elevated levels of blood flow and associated mechanical forces. In this regard, recent guidance from the American Heart Association [1] states physical activity as the first-line homeopathic therapy at ameliorating the symptoms associated with PAD, a main form of CVD. While it is known that exercise is beneficial for the circulatory system via formation of collaterals and arterialization of existing capillaries, our question of how hemodynamic forces alleviate PAD remains unanswered [41, 42]. Since exercise was recently shown to stimulate phosphorylated VASP [43], VASP is likely linked to beneficial outcomes observed following exercise. With this in mind, using a 10 day treadmill exercise regimen, preliminary observations show that hyperemic fluid shear stress has capacity to increase VASPSer239 in rat aorta segments compared to sedentary controls (Figure 6). This dilatory response can increase circumferential hoop stress and in the presence of an increased pressure head, consequently increase fluid shear stress. This effect can drastically alter the actin cytoskeleton within the cells of the exposed neointima [44,45] and perhaps then serve to mitigate abnormal cellular migration and growth associated with CVD. Indeed, these early findings offer support for VASP and phosphorylated VASP species, as crucial cytoske‐ letal and signal transduction proteins, as capable of controlling deleterious vascular growth integral for CVD.

Indeed, VASP holds great promise regarding its ability to control VSM growth underlying vascular disorders but represents just one of the many end-targets of cyclic nucleotide-driven cellular signals. Vascular cyclic AMP and cyclic GMP and their Ser/Thr kinases also have capacity to exert regulatory control over synthetic TGF-beta signaling [19] as well as other cellto-cell and focal adhesion/cytoskeletal proteins including gap junctional connexins [46,47], paxillin [34,48], G-actin and F-actin rations [34], and FAK [28,34]. We have also reported on capacities of these signals to regulate ECM-degrading MMPs as a potential route of action in VSM [34,49]. The broad influence of cyclic nucleotide signaling and its many end-target proteins including VASP and associated cytoskeletal components in VSM presents an attrac‐ tive and biologically feasible therapeutic strategy aimed against basic elements of CVD and many forms of vascular dysfunction.
