**3. Roles of cAMP in differentiated VSMCs**

#### **3.1. cAMP induces relaxation of differentiated VMCs**

Elevation of intracellular cAMP after activation of Gs coupled receptors by vasorelaxing hormones such as adrenaline, noradrenaline and the endothelium-derived prostaglandine I2 (PGI2) induces a rapid and efficient relaxation of mature differentiated SMCs [53]. Moreover, the cAMP elevating agent forskolin induces a relaxant effect in VSMCs *in vivo* which is potentiated by inhibitors of PDE3 and PDE4, the two main PDE isoforms expressed in VSMCs [25,26,30] [54]. In SMCs, cAMP contributes to muscle relaxation through two different mechanisms; one through the stimulation of the Ca pump at the sarcolemmal membrane (Ca extrusion) and sarcoplasmic reticulum (Ca accumulation), and the other through the dephosphorylation of myosin light chain kinase (MLCK). De-phosphorylation of MLCK is accomplished by the myosin light chain phosphatase (MLCP) which is well known to be activated upon phoshorylation by the cAMP target PKA or the cGMP dependent protein kinase G (PKG) [55,56]. Conversely, when phosphorylated by Rho-associated kinase (ROCK) or PKC, MLCP activity is inhibited, resulting in contraction. A new mechanism of cAMP-mediated relaxation has been recently described in airway and aortic smooth muscle cells involving Epac, the last cAMP effector identified. Activation of Epac by an Epac selective cAMP analog in pre-contracted aortic smooth muscle cells and airway smooth muscle cells results in the down regulation of RhoA activity and in the increase of Rap1 or Rac1 activities, leading to cell relaxation [57,58]. cAMP pools involved in SMC relaxation may be mainly generated by the type 6 adenylyl cyclase (AC6). Indeed, overexpression of only AC6 (and not AC5, AC2, or AC1) in primary aortic VSMCs enhances smooth muscle relaxation [59]. Furthemore, a recent study using selective short interfering RNA sequences reveals that AC6 is the predominant isoenzyme involved in vasodilator-mediated cAMP accumulation in aortic VSMCs, account‐ ing for 60% of the total response to β-adrenoceptor (β-AR) stimulation [60].

#### **3.2. cAMP maintains a low rate of proliferation in differentiated VSMC**

A cause to effect relationship between the decreased expression of some specific components of the cAMP signalling and proliferative capacity of VSMC has been demonstrated. Inversely, emergence of PDEs in trans-differentiated VSMC allows them to proliferate.

#### *3.2.1. Role of CREB*

the availability of cAMP/cGMP to their effectors –AKAPs dynamically assemble the three different cAMP effectors to control the cellular actions of cAMP [37]. As their name implies, AKAPs were originally described to target PKA to distinct subcellular locations and confine activation to only a subset of potential targets. In reality, these proteins have the ability to form complexes with other signalling molecules including Epac proteins, protein kinases, phos‐ phatases, phosphodiesterases, AC, as well as GPCR and ion channels. AKAPs are localized to numerous cellular sites, including the plasma membrane, Golgi, centrosome, nucleus, mitchondria and cytosol. The first AKAP to be characterized was microtubule associated protein-2 (MAP2), initially identified because of it co-purified with RII from brain extract [52]. The AKAP family has grown and includes more than 50 structurally diverse, but functionally similar members. Despite their diversity, AKAP orthologues have been identified in a range of species, including yeast, nematodes, mice and humans. All AKAPs share common proper‐ ties: 1) they contain a PKA-anchoring domain 2) compartmentalization of individual AKAP-PKA units occur through specialized targeting domains that are present on each anchoring protein 3) they have the ability to form complexes with other signalling molecules including protein kinases, phosphatases, phosphodiesterases, AC, as well as GPCR and ion channels 4) AKAPs are recruited into much larger multiprotein complexes through the interactions with other adaptator molecules such as PDZ and SH3 domain containing proteins. These four properties of AKAPs allow these proteins to integrate multiple signalling pathways, allowing

Elevation of intracellular cAMP after activation of Gs coupled receptors by vasorelaxing hormones such as adrenaline, noradrenaline and the endothelium-derived prostaglandine I2 (PGI2) induces a rapid and efficient relaxation of mature differentiated SMCs [53]. Moreover, the cAMP elevating agent forskolin induces a relaxant effect in VSMCs *in vivo* which is potentiated by inhibitors of PDE3 and PDE4, the two main PDE isoforms expressed in VSMCs [25,26,30] [54]. In SMCs, cAMP contributes to muscle relaxation through two different mechanisms; one through the stimulation of the Ca pump at the sarcolemmal membrane (Ca extrusion) and sarcoplasmic reticulum (Ca accumulation), and the other through the dephosphorylation of myosin light chain kinase (MLCK). De-phosphorylation of MLCK is accomplished by the myosin light chain phosphatase (MLCP) which is well known to be activated upon phoshorylation by the cAMP target PKA or the cGMP dependent protein kinase G (PKG) [55,56]. Conversely, when phosphorylated by Rho-associated kinase (ROCK) or PKC, MLCP activity is inhibited, resulting in contraction. A new mechanism of cAMP-mediated relaxation has been recently described in airway and aortic smooth muscle cells involving Epac, the last cAMP effector identified. Activation of Epac by an Epac selective cAMP analog in pre-contracted aortic smooth muscle cells and airway smooth muscle cells results in the down regulation of RhoA activity and in the increase of Rap1 or Rac1 activities, leading to cell relaxation [57,58]. cAMP pools involved in SMC relaxation may be mainly generated by the

the convergence of signals to a common target [36,37].

128 Current Trends in Atherogenesis

**3. Roles of cAMP in differentiated VSMCs**

**3.1. cAMP induces relaxation of differentiated VMCs**

The cAMP Response Element Binding Protein (CREB) is a transcription factor, well known to be phosphorylated and activated by PKA. CREB expression has been shown to be dramatically decreased in cultured trans-differentiated VSMCs and in the media of numerous rodent and porcine models of vascular diseases. Depletion of this transcription factor *in vivo* elicits changes consistent with those observed in SMCs from pathologically remodelled arteries whereas forced depletion of CREB with small interfering RNA in aortic SMCs is sufficient to induce their proliferation, hypertrophy, migration, de-differentiation, and ECM production. Furthe‐ more, CREB is inactivated in VSMCs by several proliferative stimuli and overexpression of wild type or constitutively active CREB, in primary cultures of SMC arrests cell cycle progres‐ sion induced by these stimuli [61-66]. Additionally, Transforming growth factor beta and thiazolidinediones activate CREB to oppose to aortic SMC proliferation induced by growth factors [62,67]. Nevertheless, some apparent contradictory studies show that CREB is involved in VSMC proliferation induced by ATP and thrombin [68,69].

#### *3.2.2. Role of CREB AKAP12β and AKAP5*

AKAP12β, a member of the AKAP family, is markedly decreased in human and rodent vascular lesions. Overexpression of AKAP12 β attenuates serum-induced SMC growth in *vitro* and a causal relationship exists between the induction of the expression of this protein and the inhibition of serum-induced VSMC proliferation by all trans retinoic acid [70]. An other AKAP shown to repress VSMC growth is AKAP5 (AKAP79/AKAP75/AKAP150 in human, bovine, rat respectively) since over-expression of this protein inhibits serum-induced VSMC prolifer‐ ation and local delivery of AKAP5 to balloon-injured vessels wall reduced the extent of neointimal burden [71].

#### *3.2.3. Role of PDE1-C*

PDE1C, a PDE isoform hydrolyzing both cAMP and cGMP, is expressed in proliferating human VSMCs but is absent in quiescent cells. In *vivo*, PDE1C is expressed in human foetal aortas containing proliferating SMCs, but not in newborn aortas in which SMC proliferation has ceased. Moreover, a causal relationship has been established between the emergence of PDE1-c in VSMCs and their capacity to proliferate, since specific inhibition of PDE1C in SMCs isolated from normal aorta or from lesions of atherosclerosis results in suppression of SMC proliferation [72].

rabbit aortic smooth muscle cells [84]. This inhibitory effect of cAMP on VSMC growth was confirmed *in vitro* [85,86] and *in vivo* by Indolfi et al., demonstrating that local or oral admin‐ istration of cell-permeable, cyclic AMP analog, 8-Br-cAMP and non-selective phosphodiester‐ ase-inhibitor drugs to rats markedly inhibits neo-intimal formation after balloon injury *in vivo* and/or *in vitro* in SMC [87,88]. Selective inhibitors of PDE3A and PDE4D, the two main PDE isoforms expressed in VSMCs that account for cAMP hydrolysis [25,26,30] were also shown to inhibit proliferation of trans-differentiated VSMCs. PDE3 and PDE4 inhibitors markedly potentiate both the anti-proliferative effect and the increase in cAMP caused by forskolin and PGI2 and significantly inhibit PDGF-induced VSMC proliferation and migration [89,90]. [Of note, PDE4D is the first gene that has been linked to common forms of stroke such as cardiogenic and carotid strokes [91]. Moreover, PDE3 inhibitors administred orally are able to inhibit VSMC proliferation in a model of photochemically-induced vascular injury (Kondo et al., Atherosclerosis, 1999), and a recent publication clearly demonstrates that PDE3A depletion *in vitro* and *in vivo* inhibits mitogen-induced VSMC proliferation [61]. The AC isoform that could play a role in cAMP-mediated inhibition of VSMC growth is the type 3 adenylyl cyclase (AC3) since Wong et al. demonstrated that this protein mediates the inhibitory effect of prostaglandin E2 (PGE2) on basal and PDGF-BB-induced proliferation in murine and human arterial VSMC [51]. Various molecular mechanisms have been proposed to explain AMP-mediated inhibition of VSMCs. Such mechanisms include subsequent suppression of growth factor-mediated activation of mitogenic protein kinases in VSMCs. Indeed, cAMP can oppose to the mitogen-activated protein (MAP) kinases ERK1/2 [61,92], to JNK1 [93] as well as to the phosphatidylinositol 3-kinase effector S6K1 [92]. In addition, cAMP can regulate gene/ protein expression which may contribute to its anti-proliferative action. For example, cAMP elevating agents restore expression of p53-p21 in response to PDGF [61,94], prevents seruminduced expression of cyclin-dependent kinases [95], inhibits basal and glucose-induced VSMC growth by a down-regulation of the transcription factor E2F [25] and can reduce the serum-induced expression of the S-Phase kinase-Associated Protein 2 (Skp2), an important factor for cell cycle progression in VSMCs [96]. Furthermore, prostacyclin-induced cAMP intracellular elevation inhibits the proliferation of arterial smooth muscle cells by inhibiting the smad1/5 driven expression of Id1 (inhibitor of DNA binding protein) gene [97]. Some of the genic effects of cAMP in VSMCs may be mediated by CREB since this transcription factor has been demonstrated to inhibit the expression of a number of cell-cycle and mitogenic genes in trans-differentiated VSMCs as well as genes encoding growth factors, growth factor receptors, and cytokines [61,64,98]. The cAMP effectors PKA and Epac both are involved in cAMP VSMC growth inhibition. Indeed, PKA inhibitors have been shown to reverse or, at least, inhibit the effect of cAMP elevating agents on VSMC proliferation [71,77,87,88,99]. Concerning the involvement of Epac in VSMC proliferation, Mayer and collaborators and Hewer and collaborators respectively demonstrated that Epac is involved in the adenosinemediated decrease of cell proliferation in human VSMCs and acts synergically with PKA to mediate cAMP-dependent cell-cycle arrest and associated induction of a stellate- morphology

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in VSMCs [100,101].

#### **3.3. Others roles of cAMP in differentiated VSMC**

#### *3.3.1. cAMP maintains the contractile phenotype of differentiated VSMCs*

As mentioned above, CREB depletion elicits changes consistent with those observed in SMCs from pathologically remodelled arteries *in vivo.* These changes include modifications in the expression of SMC markers and contractile factors such as SM myosin, and strongly suggest that cAMP is important in maintaining the contractile phenotype of differentiated VSMCs [64]. The role of CREB in the maintenance of the contractile phenotype is reinforced by a recent publication showing that cAMP elevation by cilostazol, a potent type 3 phosphodiesterase inhibitor, promotes VSMC differentiation through CREB [73].

#### *3.3.2. cAMP has dual opposite effects on apoptosis of differentiated VSMCs*

Some studies demonstrate that cAMP is pro-apoptotic in SMCs whereas others present cAMP as an anti-apoptotic factor in these cells. The opposite effect of cAMP on apoptosis in the same type cell can be explained by the compartmentalization of cAMP signalling since these studies use different ways to elevate intracellular cAMP. Some studies use cAMP elevating agents, whereas others use hormones such as prostacyclin. In aortic VSMC, Torella et al. show that cAMP analogs inhibits apoptosis through Ser83 phosphorylation of p85αPI3K [77]. Addition‐ ally, in the same model, the AC activator forskolin reduces apoptosis in serum-deprived rat aortic VSMC at a site upstream of caspase 3 via activation of PKA [78]. In line with these studies, inhibition of CREB function in aortic VSMC induces apoptosis of rat aortic VSMC, possibly through downregulation of bcl2 expression [79]. Adversely, cAMP elevation in response to prostacyclin induces apoptosis in rat aortic VSMC through the inhibition of extracellular signal-regulated kinase activity [80].
