Sarco/Endoplasmic Reticulum Ca ²⁺ -ATPase Gene Transfer Reduces Vascular Smooth Muscle Cell Proliferation and Neointima Formation in the Rat

Abstract

Abstract-Proliferation of vascular smooth muscle cells (VSMC) is a primary cause of vascular disorders and is associated with major alterations in Ca 2ϩ handling supported by loss of the sarco/endoplasmic reticulum calcium ATPase, SERCA2a. To determine the importance of SERCA2a in neointima formation, we have prevented loss of its expression by adenoviral gene transfer in a model of balloon injury of the rat carotid artery. Two weeks after injury, the intima/media ratio was significantly lower in SERCA2a-infected than in injured noninfected or injured ␤-galactosidaseinfected carotids (0.29Ϯ0.04 versus 0.89Ϯ0.19 and 0.72Ϯ0.14, respectively; PϽ0.05), and was comparable to that observed in control carotids (0.21Ϯ0.03). The pathways leading to proliferation were analyzed in serum-stimulated VSMC. Forced expression of SERCA2a arrested cell cycle at the G1 phase and prevented apoptosis. SERCA2a inhibits proliferation through inactivation of calcineurin (PP2B) and its target transcription factor NFAT (nuclear factor of activated T-cells) resulting in lowering of cyclin D1 and pRb levels. By using NFAT-competing peptide VIVIT, we showed that NFAT activity is strongly required to promote VSMC proliferation. In conclusion, we provide the first evidence that increasing SERCA2a activity inhibits VSMC proliferation and balloon injury-induced neointima formation. Key Words: vascular smooth muscle cell proliferation Ⅲ gene transfer Ⅲ SERCA2a Ⅲ calcium signaling Ⅲ nuclear factor of activated T-cells C ell hyper-proliferation is an important etiology factor of cardiovascular diseases such as primary atherosclerosis, restenosis, and vein-graft disease. 1,2 The neointimal vascular smooth muscle cell (VSMC) proliferation constitutes a primary cause of vascular disorders 1 but, despite increasing knowledge about the cell-cycle regulation of VSMC, the molecular mechanism governing VSMC proliferation remains elusive. Thus, identifying genetic modifiers of VSMC proliferation is a major focus in cardiovascular biology and medicine. 3 VSMCs have the ability to transition between quiescent differentiated and proliferating phenotypes. Acquisition of proliferating phenotype by VSMC is associated with alterations in Ca 2ϩ handling supported by modification of Ca 2ϩ transporter expression. 4 In quiescent VSMC, the Ca 2ϩ signal consists mainly of localized elementary calcium events. The global increase in Ca 2ϩ concentration is rapidly reduced by calcium pumps, keeping the cytoplasmic Ca 2ϩ concentration low. Spontaneous spark frequency decreases after activation of phosphoinositol-3 kinase (PI3K) and G protein-coupled receptors, probably attributable to inhibition of the ryanodine receptor (RyR). 6,7,14 -16 Several previous studies have suggested that SERCA2a is involved in the control of proliferation and growth: transgenic mice with only one allele of the ATP2a2 gene (SERCA2) develop numerous cancers of the upper digestive tract and skin, and cardiac hypertrophy 17,18 ; low levels of SERCA2a are associated with cardiac hypertrophy bot...

The non-excitable smooth muscle: Calcium signaling and phenotypic switching during vascular disease

“…The authors concluded that the delivery of SERCA2a to the injured media reversed the alterations in Ca 2+ handling observed in proliferating VSMC, suggesting SERCA2a expression and activity are important in maintaining the non-proliferative phenotype of smooth muscle [82,83]. In addition, overexpression of SERCA2a in proliferating VSMC in culture reduced the amplitude and propagation of IP 3 -evoked Ca 2+ signals [82], supporting a role of IP 3 -mediated Ca 2+ signaling in VSMC proliferation.…”

“…Interestingly, the same authors provided evidence that adenoviral overexpression of SERCA2a inhibits VSMC proliferation and subsequent neointima formation following balloon catheter-induced injury in the rat carotid artery [82]. Specifically, overexpression of SERCA2a in serum-stimulated VSMC inhibited proliferation through inactivation of calcineurin and its target transcription factor nuclear factor of activated T-cells (NFAT) resulting in lowering of Cyclin D1 levels and cell cycle arrest at the G1 phase [82]. The authors concluded that the delivery of SERCA2a to the injured media reversed the alterations in Ca 2+ handling observed in proliferating VSMC, suggesting SERCA2a expression and activity are important in maintaining the non-proliferative phenotype of smooth muscle [82,83].…”

“…Work by Lipskaia et al showed that in proliferating VSMC, SERCA2a expression is downregulated, while SERCA2b expression remains unchanged [82]. When VSMC are growth arrested or in confluent cultures SERCA2a expression is regained, pointing to an important link between alteration in the expression of Ca 2+ -handling proteins and cell proliferation.…”

“…When VSMC are growth arrested or in confluent cultures SERCA2a expression is regained, pointing to an important link between alteration in the expression of Ca 2+ -handling proteins and cell proliferation. Interestingly, the same authors provided evidence that adenoviral overexpression of SERCA2a inhibits VSMC proliferation and subsequent neointima formation following balloon catheter-induced injury in the rat carotid artery [82]. Specifically, overexpression of SERCA2a in serum-stimulated VSMC inhibited proliferation through inactivation of calcineurin and its target transcription factor nuclear factor of activated T-cells (NFAT) resulting in lowering of Cyclin D1 levels and cell cycle arrest at the G1 phase [82].…”

“…Specifically, overexpression of SERCA2a in serum-stimulated VSMC inhibited proliferation through inactivation of calcineurin and its target transcription factor nuclear factor of activated T-cells (NFAT) resulting in lowering of Cyclin D1 levels and cell cycle arrest at the G1 phase [82]. The authors concluded that the delivery of SERCA2a to the injured media reversed the alterations in Ca 2+ handling observed in proliferating VSMC, suggesting SERCA2a expression and activity are important in maintaining the non-proliferative phenotype of smooth muscle [82,83]. In addition, overexpression of SERCA2a in proliferating VSMC in culture reduced the amplitude and propagation of IP 3 -evoked Ca 2+ signals [82], supporting a role of IP 3 -mediated Ca 2+ signaling in VSMC proliferation.…”