J Mol Med DOI 10.1007/s00109-015-1305-z

REVIEW

G-protein-mediated signaling in vascular smooth muscle cells — implications for vascular disease Till F. Althoff 1 & Stefan Offermanns 2,3

Received: 31 March 2015 / Revised: 14 May 2015 / Accepted: 2 June 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Differentiated vascular smooth muscle cells (VSMCs) are critical determinants of vascular tone and blood pressure. However, during vascular remodeling processes, which occur in response to changing hemodynamics or vascular injury, VSMCs lose most of their contractile functions in a dedifferentiation process, which goes along with cell proliferation and cell migration. VSMCs are under the constant control of a variety of mediators with vasocontractile or vasodilatory properties. Most of these mediators act through G-protein-coupled receptors, which, via different downstream signaling pathways, regulate the phosphorylation of myosin light chain and thereby control vascular tone. Recent work indicates that procontractile G-protein-mediated signal transduction pathways are also critical regulators of vascular smooth muscle dedifferentiation and redifferentiation. This review describes some of the key G-protein-mediated signal transduction pathways regulating vascular tone and VSMC differentiation and their involvement in cardiovascular diseases.

* Stefan Offermanns [email protected] 1

Department of Cardiology & Vascular Medicine and Center for Cardiovascular Research (CCR), Charité Campus Mitte, Charité Universitätsmedizin Berlin, 10117 Berlin, Germany

2

Department of Pharmacology, Max-Planck-Institute for Heart and Lung Research, Ludwigstr 43, 61231 Bad Nauheim, Germany

3

Medical Faculty, Johann Wolfgang Goethe University Frankfurt, 60590, Frankfurt am Main, Germany

Keywords Vascular smooth muscle cells . G-protein-coupled receptors . G-protein signaling

Introduction Differentiated vascular smooth muscle cells (VSMCs) are highly specialized to establish and regulate vascular tone, a key determinant of blood pressure and organ perfusion. VSMC tone is under the constant control of a variety of vasocontractile and vasodilatory mediators, most of which act through G-protein-coupled receptors (GPCRs), which then initiate downstream signaling processes that eventually regulate the phosphorylation state of the myosin light chain (MLC) [1]. However, the functional repertoire of VSMCs is not limited to the providing of contractile forces. In fact, VSMCs are highly plastic — a property that discriminates them from terminally differentiated cardiac or skeletal myocytes. VSMCs can switch between a quiescent, contractile state and phenotypes of increased proliferation, migration, and synthetic as well as inflammatory capacity [2]. This ability to dedifferentiate and redifferentiate is considered a prerequisite for vascular remodeling processes that enable vascular development and repair, as well as adaptation to chronically altered hemodynamics. However, dysregulation of VSMC plasticity plays a key role in pathological vascular remodeling and diseases as well. Whereas it is well established that VSMC contraction is under the control of G-protein-mediated signaling pathways, the central role of G-proteins in the regulation of smooth muscle cell plasticity and vascular remodeling has only recently been appreciated. Here, we review the current knowledge on G-protein-mediated signaling in VSMCs and its role in the regulation of vascular tone and

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remodeling. Moreover, we draw attention to potential drug targets and innovative therapeutic approaches, based on the current understanding of the role of these processes in cardiovascular disease. G-protein-mediated signaling pathways regulating vascular smooth muscle contraction A central process in the regulation of smooth muscle cell contraction is the phosphorylation of MLC [3, 4]. MLC is phosphorylated by the Ca2+-calmodulin-activated MLC kinase (MLCK), resulting in increased interaction of myosin and actin and an increase in vascular tone, whereas MLC is dephosphorylated by the MLC phosphatase (MLCP), resulting in decreased vascular tone. In contrast to the regulation of MLCK, which is Ca2+-dependent, MLCP is regulated in a Ca2+-independent manner. The classic vasoconstrictors, such as endothelin-1, angiotensin II, or vasopressin act through GPCRs coupled to G-proteins of the Gq/G11, G12/G13, and in some cases also the Gi family. Gq/G11 link these receptors to the activation of β-isoforms of phospholipase C, leading to the formation of inositol-1,4,5-trisphosphate and the release of intracellularly stored Ca2+. MLCK is then activated by Ca2+ together with calmodulin, resulting in an increased phosphorylation of MLC and an increase in vascular smooth muscle tone (Fig. 1). There is good evidence that the Ca2+ which regulates MLCK activity is derived to a considerable extent from a transmembrane influx through particular cation channels, such as voltage-dependent L-type Ca2+ channels and transient receptor potential (TRP) channels [5]. Most receptors of vasoconstrictors are also able to regulate myosin light chain phosphorylation in a Ca2+-independent manner, a mechanism involving the regulation of the myosin phosphatase. This pathway is mediated by the small GTPbinding protein RhoA. After activation, RhoA stimulates Rho-kinase which, in turn, phosphorylates and inhibits myosin phosphatase [6] (Fig. 1). In most cases the G-proteins G12/ G13 link receptors to the Rho/Rho-kinase pathway in VSMCs [4, 7, 8]. Activation of G12/G13 by receptors of vasoconstrictors results in the activation of particular Rho guanine nucleotide exchange factors (RhoGEFs), which then promote the formation of active RhoA [9], and the RhoGEF protein LARG appears to play an important role in VSMCs under in vivo conditions [8]. There is also good evidence that activation of Gq/G11 can induce RhoA activation through the RhoGEF proteins p63RhoGEF [10] or LARG [11]. In addition, Gq/G11 can activate RhoA indirectly through Ca2+- and Jak2-dependent activation of the RhoGEF protein p115RhoGEF. The latter mechanism has been shown to link the angiotensin II (AT1) receptor to RhoA-dependent VSMC contraction [12]. Some

Fig. 1 Regulation of VSMC tone by G-protein-mediated pathways. Shown are two of the major procontractile G-protein-mediated signaling transduction pathways. Most vasoconstrictor receptors are coupled to both Gq/G11 and G12/G13. Activation of β-isoforms of phospholipase C (PLC) through Gq/G11 results in the release of intracellularly stored Ca2+ by inositol-1,4,5-trisphosphate (IP3), which, together with calmodulin, activates the myosin light chain kinase (MLCK). The Rho guanine nucleotide exchange factor LARG couples G12/G13 to the activation of RhoA, which then activates Rho-kinase (ROCK). ROCK in turn can phosphorylate and inhibit the myosin light chain phosphatase (MLCP). The angiotensin II (AT1) receptor has been shown to activate Rho and ROCK through a different pathway involving the RhoGEF protein p115-RhoGEF, which is phosphorylated by JAK2

GPCRs of vasoconstrictors also couple to G-proteins of the Gi family, which may contribute to the procontractile activity of their ligands by releasing βγ-subunits resulting in the activation of phospholipase C β-isoforms or by inhibiting adenylyl cyclase leading to a decrease in intracellular cAMP levels [13, 14]. The relevance of Gq/G11- and G12/G13-mediated signaling in vascular smooth muscle tone regulation has been studied in mice with induced smooth muscle-specific Gαq/Gα11 and Gα12/Gα13 deficiency. Induction of smooth muscle-specific Gα deficiency has been chosen as an approach to minimize compensatory effects resulting from loss of G-proteinmediated signaling functions; however, such effects cannot be completely excluded and need to be considered when interpreting the data obtained with these mouse models. Ex vivo experiments on isolated vessels, however, clearly showed that several procontractile receptors use both Gq/ G11- and G12/G13-mediated signaling transduction pathways to increase vascular smooth muscle tone. In contrast, the α1adrenergic receptor signals exclusively through Gq/G11 to contract vascular smooth muscle. Studies using the same approach under in vivo conditions revealed that the basal vascular tone and the basal blood pressure require Gq/G11-mediated signaling in VSMCs, whereas an increase of vascular tone

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under hypertensive conditions requires activation of both the Gq/G11- and G12/G13-mediated signaling pathway [8]. The major mediators of vascular smooth muscle relaxation are cAMP and cGMP [15]. Of these two mediators, the formation of cAMP is under the control of GPCRs coupled to the G-protein Gs, resulting in the stimulation of adenylyl cyclase. The major mediators of cAMP-induced relaxation of VSMCs are protein kinase A (PKA) and protein kinase G (PKG), which are both activated by cAMP [16]. Multiple mechanisms have been described as to how PKA and PKG inhibit GPCRmediated smooth muscle contraction by interfering with the Gq/G11-mediated, Ca2+-dependent pathway as well as with the Ca2+-independent Rho/Rho-kinase-mediated signaling pathway [16]. Transcriptional control of VSMC differentiation Differentiated VSMCs display a quiescent, contractile phenotype as reflected by the abundant expression of smooth muscle-selective differentiation marker genes that predominantly code for proteins of the contractile apparatus, such as smooth muscle myosin heavy chain (SMMHC), SM α-actin, SM22α, (h1-) calponin and smoothelin [17]. However, VSMCs retain a considerable plasticity and are able to dedifferentiate towards distinct proliferative, migratory, synthetic, and/or inflammatory phenotypes. In this respect, it has to be stated that differentiation and proliferation are not mutually exclusive, and that, vice versa, VSMC dedifferentiation is not necessarily accompanied by a proliferative phenotype. The marked down-regulation of the above-mentioned smooth muscle differentiation marker genes though is a common feature of VSMC dedifferentiation. The majority of smooth muscle differentiation marker genes is regulated by serum response factor (SRF), a widely expressed transcription factor that binds to so-called serum response elements (SRE) which are characterized by CarG box [CC(A/T)6GG] DNA sequences [17]. However, the spectrum of SRF targets is not limited to SM differentiation marker genes but is in fact dominated by growth-related genes involved in smooth muscle proliferation and dedifferentiation. It is now well established that two families of transcriptional co-factors, the myocardin family and the ternary complex factor (TCF) family of Etsdomain proteins, differentially modulate the transcription of these distinct SRF target genes through their mutually exclusive binding to SRF [18] (Fig. 2). While transcriptional cofactors of the myocardin family, consisting of myocardin itself and myocardin related transcription factors (MRTFs) A and B, promote VSMC differentiation, competitive binding of TCFs induces transcription of early response growth genes as well as VSMC dedifferentiation and proliferation (Fig. 2). TCFs are phosphorylated and activated through an extracellular signal-regulated kinase 1/2 (Erk1/2) pathway, whereas RhoA-mediated signaling promotes nuclear translocation of

Fig. 2 Transcriptional control of VSMC plasticity. TCFs ternary complex factors, MRTF myocardin related transcription factor A, Myocd myocardin, Myo Fam myocardin family of transcriptional cofactors (incl. MRTF und Myocd)

MRTFs and induces smooth muscle differentiation (Fig. 2). However, it has to be considered that these data are almost exclusively based on in vitro studies. As VSMCs instantly change their phenotype upon isolation and further dedifferentiate in cell culture conditions, it is generally acknowledged that cell culture-based experiments on VSMCs have to be interpreted with great caution and necessitate validation in vivo [17].

Antagonistic regulation of VSMC differentiation through Gq/G11 and G12/G13 Despite the large body of evidence on transcriptional control of VSMC plasticity, the extracellular cues and upstream signaling mechanisms regulating SRF-dependent VSMC differentiation under in vivo conditions have remained poorly understood. Besides extracellular stimuli like TGFβ and PDGFBB as well as PDGF-DD [17], several contractile GPCR ligands including angiotensin II, endothelin-1, thromboxane A2, thrombin, lysophosphatidic acid (LPA), and sphingosine-1phosphate (S1P) have been implicated in VSMC plasticity [17, 19–25]. All of these contractile ligands activate GPCRs that are dually coupled to Gq/G11 and G12/G13, albeit some receptors show a certain preference. VSMCs lacking the Gprotein α-subunits Gα12 and Gα13 or the RhoGEF protein LARG showed decreased expression levels of smooth muscle marker genes and had an exaggerated response to dedifferentiation stimuli in vitro as well as in a neointima model in vivo [26]. Consistent with this, LARG has been shown to be able to mediate differentiation of VSMCs via RhoA [27]. Evidence from cell-based studies suggest that RhoA can induce SRFdependent transcription of smooth muscle differentiation marker genes through two distinct mechanisms — up-regulation of myocardin expression [25, 28] and facilitation of MRTF-A nuclear translocation [18, 29] (Fig. 2). Both mechanisms have been found to operate in vivo in a G12/G13- and LARG-dependent manner [26] (Fig. 3). However, it remains unclear which of its downstream effectors link RhoA to the activation of the myocardin family of transcriptional co-factors. It has been suggested that the actin nucleating proteins

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Fig. 3 Gq/G11- and G12/G13-mediated signaling: synergistic regulation of vascular tone and antagonistic regulation of VSMC plasticity. PLC phospholipase C, ROCK Rho-kinase, IP3 inositol-1,4,5,-trisphosphate, MLCK myosin light chain kinase, MLCP myosin light chain phosphatase, MRTF myocardin-related transcription factor A, Myocd myocardin, Myo Fam myocardin family of transcriptional co-factors (incl. MRTF and Myocd)

mammalian homologue of diaphanous 1 and 2 (mDia1 and 2) as well as the formin homology domain-containing protein 1 (FHOD1) connect G12/G13-RhoA to the SRF-mediated transcriptional events as they are all RhoA-regulated and have been shown to promote actin polymerization, followed by MRTF-A nuclear translocation and VSMC differentiation marker gene expression [18, 29–31]. Pharmacological inhibition of another RhoA effector, Rho-kinase, has been shown to result in reduced neointima formation after vascular injury [32]. Subsequent studies using genetic mouse models, however, suggested that this effect was primarily due to the loss of Rho-kinase in leukocytes but not necessarily in vascular smooth muscle cells [33]. Interestingly, interference with G12/G13-LARG-RhoA-mediated VSMC differentiation resulted in drastic exacerbation of atherosclerosis with enhanced plaque progression and vulnerability [26]. These data provide strong evidence for a causal relationship between dysregulated VSMC differentiation and atherosclerosis, even though the exact mechanisms and specific VSMC phenotypes that account for this observation remain to be elucidated. In contrast, Gq/G11-mediated signaling appears to counteract differentiation and to promote proliferation of VSMCs. In vivo studies using mice with smooth muscle-specific deficiency in Gαq/Gα11 indicated that induction of early response genes and repression of smooth muscle differentiation marker genes involves Gq/G11-mediated activation of ERK1/2 and subsequent phosphorylation of its effector, the TCF Elk-1, in a mouse model for neointima formation [26] (Fig. 3). This is consistent with earlier studies providing evidence for a central

role of ERK1/2 in the regulation of TCFs [18]. How Gq/G11 regulates ERK1/2 in VSMCs is not fully clear. In vitro studies and studies in other organ systems suggest that Gq/G11 can mediate Erk1/2 activation through transactivation of receptor tyrosine kinases [34] or by direct regulation of Erk1/2 through βγ-subunits released from Gq/G11 [35]. In VSMCs, several mechanisms have been described that link Gq/G11-mediated signaling to the activation of RhoA as well [34]. However, loss of Gq/G11 in VSMCs did not affect RhoA activation after vessel injury [26]. Thus, while the Gq/G11- and G12/G13-mediated signaling pathways synergistically regulate vascular tone, they have antagonistic functions in the regulation of the differentiation state of VSMCs (Fig. 3). After induction of vascular injury, both signaling pathways appear to be activated in smooth muscle cells; however, the net response of VSMCs directly after injury is a dedifferentiation, which relies on the Gq/G11mediated signal transduction pathway but also on other mitogenic signaling processes, such as growth factor-dependent signaling via receptor tyrosine kinases [17, 26]. The activation of G12/G13-mediated signaling at the same time prevents an overshooting response during vascular remodeling and may facilitate the redifferentiation of VSMCs once an appropriate response to proliferative stimuli has occurred. Eventually, a well-balanced parallel activation of Gq/G11- and G12/G13-mediated signaling in VSMCs appears to be necessary for an optimal response to vascular injury or to changing hemodynamic influences. Supposably, the relative intensities of signaling through the two pathways are continuously regulated in the course of vascular remodeling processes, an effect which may in part be mediated by regulators of G-protein signaling (RGS) proteins. Several RGS proteins have been shown to be present in VSMCs [36–38], and some of them are able to differentially regulate Gq/G11 and G12/G13. It is also conceivable that Gq/G11- and G12/G13-mediated signaling pathways in VSMCs are differentially regulated by ligands of receptors which preferentially couple to one of the pathways and whose expression may change during the remodeling process (see below). GPCRs involved in the regulation of VSMC differentiation during remodeling Many classic procontractile GPCR ligands such as angiotensin II, endothelin-1, or thromboxane A2 have been shown to be capable of inducing smooth muscle differentiation through GPCRs that are dually coupled to Gq/G11- as well as G12/G13proteins [17, 21, 22, 39]. However, promitogenic and dedifferentiation promoting effects have been reported as well [17, 21, 40]. This can probably be best explained by the antagonistic activity of Gq/G11- and G12/G13-mediated signaling, allowing the activated receptor to promote or to inhibit VSMC differentiation depending on the presence of other factors.

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While most of these data are based on in vitro studies or on effects of systemically acting GPCR ligands in models of VSMC differentiation and dedifferentiation in vivo, it is currently not clear which role classic vasoconstrictor receptors play in VSMC differentiation and dedifferentiation in vivo. A potential role of the angiotensin II (AT1) receptor in VSMCs has been challenged by a study showing that smooth musclespecific angiotensin II (AT1) receptor deficiency was without impact on atherosclerosis or aneurysm formation in LDL receptor-deficient mice [41]. Also, various blood-borne mediators, such as S1P, LPA, or thrombin, which are present after vascular injury and which act through Gq/G11- and G12/G13-coupled receptors, have been shown to regulate VSMC differentiation [20, 23–25, 42]. Both proproliferative as well as differentiation promoting effects of S1P have been observed in vitro [20, 43], which has been explained by differential expression of S1P receptor isoforms. Of the five S1P receptors, VSMCs primarily express S1P3 and S1P2 and, at lower levels, also S1P1 receptors [44]. While S1P1 couples exclusively to Gi, the other two receptor subtypes couple to Gq/G11 and G12/G13, with a preference of S1P2 for G12/G13 and of S1P3 for Gq/G11 [45]. Activation of S1P2 and subsequently of G12/G13, LARG, and RhoA has been shown to promote MRTF-A nuclear translocation and VSMC differentiation [27]. In line with a role of G12/G13mediated signaling in promoting VSMC differentiation, global deficiency of the G12/G13-coupled receptor S1P2 leads to enhanced neointima formation in mouse arteries through increased VSMC migration and proliferation, whereas mice devoid of the Gq/G11-coupled S1P3 are protected from neointima formation to some extent [46]. Similarly, a S1P1/S1P3 antagonist markedly decreased neointimal hyperplasia in response to acute balloon injury of the rat carotid artery and promoted S1P-induced expression of VSMC differentiation marker genes, whereas pharmacological inhibition of S1P2 had opposite effects [20]. Interestingly, S1P1 and S1P3 were transiently upregulated in response to injury. In contrast, S1P2 expression was initially decreased but increased at 7 to 10 days post injury [20]. This pattern of early expression of S1P receptors enhancing proliferation and migration, including the Gq/G11-coupled S1P3, followed by delayed reinduction of the G12/G13-coupled S1P2, which promotes VSMC differentiation and quiescence, may explain how VSMC plasticity and vascular remodeling in an environment of abundant contractile stimuli is fine-tuned by differential G-protein signaling via Gq/G11 and G12/G13. In contrast to S1P, the role of other blood-borne mediators acting through GPCRs, such as LPA and thrombin, in the regulation of VSMC differentiation is less clear. The serine protease thrombin was shown to induce VSMC differentiation via protease-activated receptor (PAR)-1, RhoA, and myocardin [23, 25]. However, earlier reports suggest that thrombin acts as a potent mitogen on VSMCs as well [47].

The latter appears to be mediated primarily by PAR-1 with a possible contribution of PAR-4. Thrombin-induced proliferation of VSMCs in vitro and in vivo seems to involve multiple signal transduction pathways, including ERK1/2-mediated signaling, and is accompanied by Ca2+- and PKC-dependent up-regulation of the early response genes egr-1, c-fos, and cjun [47]. Interestingly, while physiological expression levels of PAR1 and PAR4 in VSMCs are low, in particular, PAR1 expression has been shown to increase in the context of vascular injury or within human atherosclerotic lesions [47–49]. Like S1P and thrombin, LPA is formed locally at sites of vascular injury and can be found in atherosclerotic lesions [24]. While LPA has been shown to be capable of inducing smooth muscle differentiation marker gene expression in vitro [23], there are several reports of mitogenic effects on VSMCs as well [24]. Moreover, local infusion of LPA induced neointimal hyperplasia in murine as well as rat common carotid arteries. However, G-protein-coupled LPA receptors were apparently not involved in this response [24, 50]. Gs- and Gi-mediated signaling As mentioned above, the increase of cAMP levels resulting from the activation of Gs-coupled receptors leads to a relaxation of vascular smooth muscle by interfering both with the Ca2+-dependent and Ca2+-independent Rho/Rho-kinase-mediated signaling pathway [16] (Fig. 4). These effects are mediated to a considerable degree by the protein kinases PKA and PKG, which are both activated by cAMP [16]. A couple of

Fig. 4 Some of the Gs- and Gi-mediated signaling in vascular smooth muscle cell regulation. PLC phospholipase C, ROCK Rho-kinase, IP3 inositol-1,4,5-trisphosphate, MLCK myosin light chain kinase, MLCP myosin light chain phosphatase, PKA protein kinase A, PKG protein kinase G. See also text for additional mechanisms downstream of PKG/PKA

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molecular mechanism by which PKA and PKG inhibit procontractile signaling pathways have been described, including an inhibition of Gq/G11-mediated signaling by phosphorylation and activation of the Bregulator of G-protein signaling 2^ (RGS2) [51, 52], a phosphorylation and inhibition of myosin phosphatase [53], an inhibition of IP3-induced release of intracellularly stored Ca2+ by phosphorylation of the inositoltrisphosphate receptor-accociated cGMP kinase substrate (IRAG) [54], as well as an inhibition of RhoA activity by cAMP through PKA-dependent and PKA-independent mechanisms [55–57]. While Gs-mediated increases in intracellular cAMP levels can affect both Gq/G11- and G12/G13-mediated signaling, the net effect of an activation of Gs-coupled receptors, including the β2 adrenergic receptor, the G-protein-coupled estrogen receptor (GPER/GPR30), or the prostaglandin IP and EP2 receptors on VSMC proliferation and migration is an inhibition [58–62]. These effects could even be increased in transgenic models of Gs-overexpression in airway smooth muscle cells [63]. The Gs-induced antimitogenic effects appear to be mediated by cyclic AMP-dependent signaling via PKA — mostly independent of Gq/G11 and G12/G13 [60, 64]. In various in vitro studies, experimental enhancement of intracellular cyclic AMP levels consistently prevented VSMC entry into G1 from the G0 phase of the cell cycle—possibly via PKA- and CREB-mediated control of cell cycle regulatory proteins including p70 s6 kinase, Cdk4, Cyclin D1, cdk2, p53, and p21 [64]. Moreover, VSMC migration as well as DNA and protein synthesis has been shown to be reduced upon induction of cyclic AMP formation [59, 65, 66]. Interestingly, Gs-mediated induction of cyclic AMP and its effector PKA were consistently found to disrupt PDGF-induced activation of ERK1/2 in VSMCs in this context, indicating a possible impact on VSMC dedifferentiation [60, 61, 64, 67]. Finally, in vivo evidence based on local application of various cyclic AMPelevating agents in the balloon catheter injury model of the rat demonstrated that cAMP via PKA inhibits VSMC growth and proliferation as well as neointima formation [68]. In fact, these results prompted clinical studies that found beneficial effects of the phosphodiesterase inhibitor cilostazol in the prevention of human restenosis [69, 70]. In line with these data, activation of different Gs-coupled receptors in vivo prevented VSMC proliferation and vascular remodeling [60, 71]. Together, these observations clearly indicate that G-proteins mediating the receptor-dependent stimulation (Gs) or inhibition (Gi) of adenylyl cyclases are involved in the regulation of VSMC plasticity and vascular remodeling. In accordance with the antimitogenic role of Gs-mediated signaling via cyclic AMP in VSMCs, many Gi-coupled receptors, including S1P1 [20, 46], EP3 [59], or the apelin receptor [72, 73] have been implicated in VSMC hyperproliferation, migration, and pathological vascular remodeling. Those receptors were all shown to promote VSMC proliferation

through inhibition of adenylyl cyclase-dependent cAMP production which resulted in enhanced transactivation of growth factor receptors and induction of MAP kinase (MAPK) signaling via ERK1/2 [46, 59]. Because the expression levels of Gi are relatively high, their receptor-dependent activation results in the release of relatively high amounts of βγ-complexes. Activation of Gi is therefore believed to be the major coupling mechanism that results in the activation of βγmediated signaling processes, and serum-induced MAPK activation and proliferation of VSMCs in vitro was shown to be primarily mediated by G-protein βγ-subunits [74]. Moreover, inhibition of Gβγ-mediated signaling in vivo through adenoviral bARKct (β-adrenergic receptor kinase C-terminus) gene transfer markedly reduced neointimal hyperplasia in response to balloon injury in rat carotid arteries [74]. Summary/outlook Besides particular myogenic regulatory mechanisms, VSMC tone in vivo is under the control of various stimuli, which, in most cases, act through GPCRs. Work of the last years has shown that both Ca2+-dependent and Ca2+-independent contraction of VSMCs is regulated by heterotrimeric G-proteins. Whereas Gq/G11 control Ca2+-dependent and, in some cases, also Ca2+-independent tone regulation, G12/G13 through Rho/ Rho-kinase control Ca2+-independent processes. More recent data also indicate that the response of VSMCs to vascular injury, which results in the dedifferentiation and eventually redifferentiation of VSMCs, is controlled by mediators acting through GPCRs as well. Here, the procontractile G-proteinmediated signal transduction pathways mediated by Gq/G11 and G12/G13 appear to play antagonistic roles by promoting dedifferentiation and redifferentiation, respectively. In the future, it will be important to better understand the mechanisms that regulate these G-protein-mediated signaling pathways governing VSMC function. In particular, the spectrum of GPCRs and their G-protein coupling properties as well as their expression levels under specific physiological and pathophysiological conditions will be important to be explored. It may also be beneficial to understand the role of RGS proteins in this context in more detail, as they may be able to direct the signaling flow through distinct G-protein-mediated signaling pathways. Eventually, exploiting this knowledge will help to develop new pharmacological strategies allowing to interfere with or to modulate smooth muscle functions, taking into consideration that different signaling pathways have synergistic or antagonistic functions depending on the physiological or pathophysiological context.

Acknowledgments Dr. Althoff is a participant in the Charité Clinical Scientist Program funded by the Charité-Universitätsmedizin Berlin and the Berlin Institute of Health.

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G-protein-mediated signaling in vascular smooth muscle cells - implications for vascular disease.

Differentiated vascular smooth muscle cells (VSMCs) are critical determinants of vascular tone and blood pressure. However, during vascular remodeling...
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