**4. Roles of cAMP in trans-differentiated VSMCs**

#### **4.1. cAMP inhibits proliferation of trans-differentiated VMCs**

cAMP is well known to diminish cell growth and to promote cell-differentiation in general, it can even be antagonistic to the effect of growth factors [81]. The first clue that cAMP might have a role in controlling growth of cultured cells emerged from two studies. Burk observed that two drug inhibitors of cAMP phosphodiesterase activity, caffeine and theophylline, slowed the growth of normal and transformed baby hamster kidney (BHK) cells [82]. At the same time, Ryan and Heidrick reported that cAMP itself inhibited the growth of Hela cells [83]. The first demonstration that cAMP inhibits proliferation of VSMCs was done by Southgate and Newby showing the inhibitory effect of 8-Br-CAMP on serum-induced proliferation 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

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

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

cAMP is well known to diminish cell growth and to promote cell-differentiation in general, it can even be antagonistic to the effect of growth factors [81]. The first clue that cAMP might have a role in controlling growth of cultured cells emerged from two studies. Burk observed that two drug inhibitors of cAMP phosphodiesterase activity, caffeine and theophylline, slowed the growth of normal and transformed baby hamster kidney (BHK) cells [82]. At the same time, Ryan and Heidrick reported that cAMP itself inhibited the growth of Hela cells [83]. The first demonstration that cAMP inhibits proliferation of VSMCs was done by Southgate and Newby showing the inhibitory effect of 8-Br-CAMP on serum-induced proliferation of

proliferation [72].

130 Current Trends in Atherogenesis

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

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

inhibitor, promotes VSMC differentiation through CREB [73].

**4. Roles of cAMP in trans-differentiated VSMCs**

**4.1. cAMP inhibits proliferation of trans-differentiated VMCs**

signal-regulated kinase activity [80].

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

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 in VSMCs [100,101].

#### **4.2. cAMP has dual opposite effects on migration of trans- differentiated VMCs**

over, the forced expression of AC8 in primary rat VSMC cultures triggered the recolonization of a wounded zone, whereas blocking it in IL-1β−cells stopped the IL-1βinduced migration [21] This finding was extending *in vivo*, on human samples, where only the neo-intimal VSMCs a high level of AC8. Of note, AC8 is well known for its role in stress adaptation, mood disorders and opiate dependence [115]. The involvement of this enzyme in VSMC trans-differentiation was therefore unexpected. Molecular mecha‐ nisms underlying AC8-mediated VSMC migration does not involve PKA but could in‐ volve Epac1 since it has been shown that Epac 1 expression is upregulated in the neointima after vascular injury of mouse arteries and induces VSMC migration. More‐ over Rap1, one of the described targets of Epac, is well known for its involvement in cell

The Role of Cyclic 3'-5' Adenosine Monophosphate (cAMP) in Differentiated and Trans-Differentiated...

http://dx.doi.org/10.5772/54726

133

**4.3. cAMP has dual opposite effects on inflammation of trans-differentiated VMCs**

is the result of the emergence of AC8 in IL1β−treated VSMC.

**4.4. cAMP inhibits collagen synthesis of trans-differentiated VSMCs**

A study from Adkins and coll. demonstrate that the elevation of intracellular cAMP by rapamycin inhibits the secretion of the pro-inflammatory molecule Tumor Necrosis Factor alpha (TNF-α) in lipopolyssacharide treated VSMCs from human saphenous vein segments [110]. Adversely, Clement and collaborators suggest that the production of cAMP specifically by AC8 is involved in the potentiator effect of prostaglandin E2 (PGE2) on the secretion of phospholipase A2 (sPLA2), a marker of inflammation, in response to interleukine 1 β (IL1β) in primary cultures of rat aortic smooth muscle cells [20]. In details, authors show that PGE2 i) induces the transition of CMLV towards a trans-differentiated/ inflammatory state through the activation of the subtype 4 Gs-linked PGE2 receptor EP4, ii) acts in synergy with IL1β to potentiate the secretion of phospholipase A2 and the disorganization of the alpha actin cytoskeleton. This potentiator effect is the result of a simultaneous activation of PGE2 receptors EP4 and EP3: in differentiated VSMC, EP3 receptors inhibit cyclase activity induced by EP4 and become activator of this activity in trans-differentiated VSMC. This switch of regulation

Synthetic VSMCs, in the atherosclerotic and neointimal lesions, produce an abundant exracellular matrix (ECM), rich in type I collagen (collagen I). This ECM plays an important role in vessel wall thickening and in the occlusion of the vessel lumen. In addition, collagen I in vascular lesions may also regulate VSMC proliferation/migration, platelet circulation, mono‐ cyte activation, lipid accumulation, calcification, and plaque stability [74]. cAMP elevating agents have been shown to inhibit collagen I synthesis induced by fetal calf serum- and TGFβ [75]. Emergence of PDE1-c in trans-differentiated SMCs from rat aortic and human saphe‐ nous vein explants opposes the inhibitory effect of cAMP on collagen 1 synthesis, and accounts, at least in part, for the increase of collagen 1 expression in trans-differentiated VSMCs. The use of specific pharmacological inhibitors and si-RNA reveal that the cAMP-mediated inhibitory effect on collagen 1 synthesis involves cyclic nucleotide gated channels but not PKA,

migration [116].

nor Epac [76].

#### *4.2.1. cAMP inhibit migration of trans-differentiated VSMCs*

A growing body of evidence emerged in the beginning of the 1990's implicating cAMP in the inhibition of trans-differentiated VSMC migration. These studies, using analogs of cAMP, activators of ACs and cAMP raising agents in VSMCs, have demonstrated that an increase in cAMP positively correlates with the inhibition of VSMC migration. Indeed, raising the intracellular concentration of cAMP either with dopamine, acting throught D1 receptors, adrenomedullin, or forskolin, inhibited migration of VSMCs stimulated with PDGF or serum [102-104]. Studies in rat aortic SMCs suggest that vasoactive agents that elevate intracellular cAMP inhibit cell movement by disassembling actin stress fibers of the cytoskeleton [105,106]. Furthermore, downregulation of PKA abrogates inhibition of VSMC chemotaxis by forskolin [89]. The inhibitory effect of cAMP on VSMC migration is re-inforced by the fact that inhibiting all together PDE3 and PDE4D, the two main PDE isoforms expressed in VSMCs that account for cAMP hydrolysis in VSMC [26,30,107] markedly potentiated both the anti-migratory effect and the increase in cAMP caused by forskolin and significantly inhibited PDGF-induced VSMC proliferation and migration [90,108,109]. In addition, Newman et al demonstrated that forskolin inhibits TNFα-induced interleukin 6 expression and migration in human vascular smooth muscle cells [110]. This effect could involve the transcription factor CREB since PDGFinduced migration was decreased by active CREB and augmented with dominant negative CREB [66,95] ; In addition, a negative correlation has been described between the CREB level and the PDGF-activated SMC migration [64]. Nonetheless, the role of CREB in SMC migration remains unclear since CREB has been demonstrated to be involved in UTP, arachidonic acid and TNF alpha-induced SMC migration of VSMCs [111,112]. Moreover, recent studies show that oxidized and non-oxidized fatty acids induce SMC motility through this transcription factor [113,114].

#### *4.2.2. A specific endogenous pool of cAMP induces migration of trans- differentiated VSMCs*

By demonstrating that differential expression of ACs isoforms in two VSMC models ac‐ count for opposite effects of isoprenaline on cAMP production in VSMC, Webb and coworkers suggested for the first time that changes of AC isoform(s) expression in VSMCs could account for the manifestation of vascular diseases [18]. In line with this study, Li‐ mon's group recently demonstrated that the emergence of the calcium/calmodulin posi‐ tively regulated AC isoform 8 (AC8) in trans-differentiated VSMCs is involved in VSMC migration. Type 8 AC is barely undetectable in differentiated VSMCs and is strongly in‐ duced in trans-differentiated VSMCs. A causal relationship between AC8 apparition and the migratory capacities of VSMCs has been established. Indeed, authors show that 10 days after balloon angioplasty2 , rat carotid artery displayed high AC8 immuno-labelling only in the neo-intima and was no longer detectable when it was analyzed after the reendothelization phenomenon during which VSMC migration/proliferation halted. More‐

<sup>2</sup> Balloon angioplasty in rat carotid artery serves as an in vivo model of VSMC migration and proliferation.

over, the forced expression of AC8 in primary rat VSMC cultures triggered the recolonization of a wounded zone, whereas blocking it in IL-1β−cells stopped the IL-1βinduced migration [21] This finding was extending *in vivo*, on human samples, where only the neo-intimal VSMCs a high level of AC8. Of note, AC8 is well known for its role in stress adaptation, mood disorders and opiate dependence [115]. The involvement of this enzyme in VSMC trans-differentiation was therefore unexpected. Molecular mecha‐ nisms underlying AC8-mediated VSMC migration does not involve PKA but could in‐ volve Epac1 since it has been shown that Epac 1 expression is upregulated in the neointima after vascular injury of mouse arteries and induces VSMC migration. More‐ over Rap1, one of the described targets of Epac, is well known for its involvement in cell migration [116].

**4.2. cAMP has dual opposite effects on migration of trans- differentiated VMCs**

*4.2.2. A specific endogenous pool of cAMP induces migration of trans- differentiated VSMCs*

By demonstrating that differential expression of ACs isoforms in two VSMC models ac‐ count for opposite effects of isoprenaline on cAMP production in VSMC, Webb and coworkers suggested for the first time that changes of AC isoform(s) expression in VSMCs could account for the manifestation of vascular diseases [18]. In line with this study, Li‐ mon's group recently demonstrated that the emergence of the calcium/calmodulin posi‐ tively regulated AC isoform 8 (AC8) in trans-differentiated VSMCs is involved in VSMC migration. Type 8 AC is barely undetectable in differentiated VSMCs and is strongly in‐ duced in trans-differentiated VSMCs. A causal relationship between AC8 apparition and the migratory capacities of VSMCs has been established. Indeed, authors show that 10

only in the neo-intima and was no longer detectable when it was analyzed after the reendothelization phenomenon during which VSMC migration/proliferation halted. More‐

2 Balloon angioplasty in rat carotid artery serves as an in vivo model of VSMC migration and proliferation.

, rat carotid artery displayed high AC8 immuno-labelling

A growing body of evidence emerged in the beginning of the 1990's implicating cAMP in the inhibition of trans-differentiated VSMC migration. These studies, using analogs of cAMP, activators of ACs and cAMP raising agents in VSMCs, have demonstrated that an increase in cAMP positively correlates with the inhibition of VSMC migration. Indeed, raising the intracellular concentration of cAMP either with dopamine, acting throught D1 receptors, adrenomedullin, or forskolin, inhibited migration of VSMCs stimulated with PDGF or serum [102-104]. Studies in rat aortic SMCs suggest that vasoactive agents that elevate intracellular cAMP inhibit cell movement by disassembling actin stress fibers of the cytoskeleton [105,106]. Furthermore, downregulation of PKA abrogates inhibition of VSMC chemotaxis by forskolin [89]. The inhibitory effect of cAMP on VSMC migration is re-inforced by the fact that inhibiting all together PDE3 and PDE4D, the two main PDE isoforms expressed in VSMCs that account for cAMP hydrolysis in VSMC [26,30,107] markedly potentiated both the anti-migratory effect and the increase in cAMP caused by forskolin and significantly inhibited PDGF-induced VSMC proliferation and migration [90,108,109]. In addition, Newman et al demonstrated that forskolin inhibits TNFα-induced interleukin 6 expression and migration in human vascular smooth muscle cells [110]. This effect could involve the transcription factor CREB since PDGFinduced migration was decreased by active CREB and augmented with dominant negative CREB [66,95] ; In addition, a negative correlation has been described between the CREB level and the PDGF-activated SMC migration [64]. Nonetheless, the role of CREB in SMC migration remains unclear since CREB has been demonstrated to be involved in UTP, arachidonic acid and TNF alpha-induced SMC migration of VSMCs [111,112]. Moreover, recent studies show that oxidized and non-oxidized fatty acids induce SMC motility through this transcription

*4.2.1. cAMP inhibit migration of trans-differentiated VSMCs*

factor [113,114].

132 Current Trends in Atherogenesis

days after balloon angioplasty2

#### **4.3. cAMP has dual opposite effects on inflammation of trans-differentiated VMCs**

A study from Adkins and coll. demonstrate that the elevation of intracellular cAMP by rapamycin inhibits the secretion of the pro-inflammatory molecule Tumor Necrosis Factor alpha (TNF-α) in lipopolyssacharide treated VSMCs from human saphenous vein segments [110]. Adversely, Clement and collaborators suggest that the production of cAMP specifically by AC8 is involved in the potentiator effect of prostaglandin E2 (PGE2) on the secretion of phospholipase A2 (sPLA2), a marker of inflammation, in response to interleukine 1 β (IL1β) in primary cultures of rat aortic smooth muscle cells [20]. In details, authors show that PGE2 i) induces the transition of CMLV towards a trans-differentiated/ inflammatory state through the activation of the subtype 4 Gs-linked PGE2 receptor EP4, ii) acts in synergy with IL1β to potentiate the secretion of phospholipase A2 and the disorganization of the alpha actin cytoskeleton. This potentiator effect is the result of a simultaneous activation of PGE2 receptors EP4 and EP3: in differentiated VSMC, EP3 receptors inhibit cyclase activity induced by EP4 and become activator of this activity in trans-differentiated VSMC. This switch of regulation is the result of the emergence of AC8 in IL1β−treated VSMC.

#### **4.4. cAMP inhibits collagen synthesis of trans-differentiated VSMCs**

Synthetic VSMCs, in the atherosclerotic and neointimal lesions, produce an abundant exracellular matrix (ECM), rich in type I collagen (collagen I). This ECM plays an important role in vessel wall thickening and in the occlusion of the vessel lumen. In addition, collagen I in vascular lesions may also regulate VSMC proliferation/migration, platelet circulation, mono‐ cyte activation, lipid accumulation, calcification, and plaque stability [74]. cAMP elevating agents have been shown to inhibit collagen I synthesis induced by fetal calf serum- and TGFβ [75]. Emergence of PDE1-c in trans-differentiated SMCs from rat aortic and human saphe‐ nous vein explants opposes the inhibitory effect of cAMP on collagen 1 synthesis, and accounts, at least in part, for the increase of collagen 1 expression in trans-differentiated VSMCs. The use of specific pharmacological inhibitors and si-RNA reveal that the cAMP-mediated inhibitory effect on collagen 1 synthesis involves cyclic nucleotide gated channels but not PKA, nor Epac [76].
