Targeting Gbeta gamma signaling in arterial vascular smooth muscle proliferation: a novel strategy to limit restenosis

Abstract

Restenosis continues to be a major problem limiting the effectiveness of revascularization procedures. To date, the roles of heterotrimeric G proteins in the triggering of pathological vascular smooth muscle (VSM) cell proliferation have not been elucidated. betagamma subunits of heterotrimeric G proteins (Gbetagamma) are known to activate mitogen-activated protein (MAP) kinases after stimulation of certain G protein-coupled receptors; however, their relevance in VSM mitogenesis in vitro or in vivo is not known. Using adenoviral-mediated transfer of a transgene encoding a peptide inhibitor of Gbetagamma signaling (betaARKct), we evaluated the role of Gbetagamma in MAP kinase activation and proliferation in response to several mitogens, including serum, in cultured rat VSM cells. Our results include the striking finding that serum-induced proliferation of VSM cells in vitro is mediated largely via Gbetagamma. Furthermore, we studied the effects of in vivo adenoviral-mediated betaARKct gene transfer on VSM intimal hyperplasia in a rat carotid artery restenosis model. Our in vivo results demonstrated that the presence of the betaARKct in injured rat carotid arteries significantly reduced VSM intimal hyperplasia by 70%. Thus, Gbetagamma plays a critical role in physiological VSM proliferation, and targeted Gbetagamma inhibition represents a novel approach for the treatment of pathological conditions such as restenosis.

Figure 1

Effect of βARKct on MAP kinase activation in rat aorta VSM cells. (A) Representative Western blot demonstrating that the βARKct peptide is expressed in cellular extracts only after βARKct adenovirus infection. Shown in the right-hand lane of this blot is a βARKct-positive standard that is a COS cell extract overexpressing the ∼30-kDa βARKct peptide. (B) Histograms displaying MAP kinase activity expressed as the fold over basal activity in rat aorta VSM cells infected with either the βARKct adenovirus or an empty virus as a negative control. MAP kinase activity was induced by LPA (10⁻⁵ M), EGF (10⁻⁵ M), or serum (5% FBS). Data shown are the means ± SEM of five separate experiments done in triplicate. ∗, P < 0.05 βARKct versus empty virus (paired t test).

Figure 2

Effect of βARKct expression on VSM cell proliferation. Cultured VSM cells cultured from rat aorta were infected with either the βARKct adenovirus or an empty virus, and [³H]thymidine incorporation was measured after replacement of serum (5% FBS) to quiescent cells. Data shown represent the mean ± SEM of five experiments done in triplicate. ∗, P < 0.05 βARKct vs. empty virus (repeated-measurements ANOVA with a grouping factor).

Figure 3

Adenoviral transgene expression in balloon-injured rat carotid arteries. (A) X-gal staining of a representative section of an injured rat carotid artery 5 days after infection with a β-gal adenovirus. (B) Demonstration of βARKct mRNA expression by RT-PCR of rat carotid arteries 5 days after balloon injury and infection with the βARKct adenovirus (lanes 2–4 and 9). The ∼600-bp-amplified βARKct mRNA fragment is absent from empty virus-infected carotid arteries (lane 1) or a noninjured, nontreated control carotid artery (lane 5). Lane 6 shows the amplified product from a βARKct plasmid, and lanes 7 and 8 contain DNA markers. Lanes 10 and 11 represent negative controls (H₂O) for RT and the PCR, respectively.

Figure 4

In vivo effects of βARKct expression on balloon-injured rat carotid arteries. (A) Histologic staining of a normal, untreated rat carotid artery. (B– D) Sections of representative rat carotid segments 28 days after balloon injury. Restenosis and intimal hyperplasia are evident in injured arteries not treated (B) or treated with the empty adenovirus (C). This proliferative response was attenuated significantly and not as prominent in injured arteries treated with the βARKct adenovirus (D).

Figure 5

Effect of adenoviral-mediated gene transfer of the βARKct on intimal hyperplasia in balloon-injured rat carotid arteries. (A) Histogram displaying measurements of the areas of the medial and intimal layers of carotid arteries 28 days after balloon injury. The three groups are arteries not treated (control, n = 8), treated with an empty adenovirus (n = 7), or treated with the βARKct adenovirus (n = 9). (B) Data expressed as the intima-to-media ratio. Data in both panels represent the mean ± SEM. ∗, P < 0.05 vs. both control and empty virus (ANOVA).