Rad GTPase Attenuates Vascular Lesion Formation by Inhibition of Vascular Smooth Muscle Cell Migration

Harvard University, Cambridge, Massachusetts, United States
Circulation (Impact Factor: 14.43). 04/2005; 111(8):1071-7. DOI: 10.1161/01.CIR.0000156439.55349.AD
Source: PubMed


Rad (Ras associated with diabetes) GTPase is a prototypic member of a new subfamily of Ras-related GTPases with unique structural features, although its physiological role remains largely unknown. In the present study, we characterized the Rad function in vascular smooth muscle cells (VSMCs) and the influence of adenovirus-mediated Rad (Ad-Rad) gene delivery on vascular remodeling after experimental angioplasty.
We documented for the first time that neointimal formation using balloon-injured rat carotid arteries was associated with a significant increase in Rad expression as determined by immunohistochemistry and quantitative real-time reverse-transcriptase polymerase chain reaction. The levels of Rad expression in VSMCs were highly induced by platelet-derived growth factor and tumor necrosis factor-alpha. Morphometric analyses 14 days after injury revealed significantly diminished neointimal formation in the Ad-Rad-treated carotid arteries compared with Ad-GFP or PBS controls, whereas the mutated form of Rad GTPase, which can bind GDP but not GTP, increased neointimal formation. Overexpression of Rad significantly inhibited the attachment and migration of VSMCs. In addition, Rad expression dramatically reduced the formation of focal contacts and stress fibers in VSMCs by blocking the Rho/ROK signaling pathway.
Our data clearly identified Rad GTPase as a novel and critical mediator that inhibits vascular lesion formation. Manipulation of the Rad signaling pathway may provide new therapeutic approaches that will limit vascular pathological remodeling.

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    • "These characteristic structures allow Rad to interact with a variety of cellular effectors to participate in many biological functions . Rad can inhibit insulin-stimulated glucose uptake in myocyte and adipocyte cell lines [6] and regulate neurite extension via Rho/RhoA kinase (ROK) signaling [7], and Rad expression is upregulated in vascular smooth muscle cells (VSMCs) during vascular lesion formation [8]. Regarding the role of Rad in the heart, we previously reported that Rad could function as a potent inhibitor of voltage-dependent calcium channels by directly binding to their b-subunit and that dominant-negative suppression of Rad induced ventricular arrhythmias [9]. "
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    ABSTRACT: Ras-related small G-protein Rad plays a critical role in generating arrhythmias via regulation of the L-type Ca2+ channel (LTCC). The aim was to demonstrate the role of Rad in intracellular calcium homeostasis by cardiac-specific dominant-negative suppression of Rad. Transgenic (TG) mice overexpressing dominant-negative mutant Rad (S105N Rad TG) were generated. To measure intracellular Ca2+ concentration ([Ca2+]i), we recorded [Ca2+]i transients and Ca2+ sparks from isolated cardiomyocytes using confocal microscopy. The mean [Ca2+]i transient amplitude was significantly increased in S105N Rad TG cardiomyocytes, compared with control littermate mouse cells. The frequency of Ca2+ sparks was also significantly higher in TG cells than in control cells, although there were no significant differences in amplitude. The sarcoplasmic reticulum Ca2+ content was not altered in the S105N Rad TG cells, as assessed by measuring caffeine-induced [Ca2+]i transient. In contrast, phosphorylation of Ser2809 on the cardiac ryanodine receptor (RyR2) was significantly enhanced in TG mouse hearts compared with controls. Additionally, the Rad-mediated RyR2 phosphorylation was regulated via a direct interaction of Rad with protein kinase A (PKA).
    Full-text · Article · Sep 2014 · Biochemical and Biophysical Research Communications
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    • "Total RNA was extracted from cultured RASMCs with Trizol (Invitrogen) and 1 µg was used for cDNA synthesis using a first strand cDNA synthesis kit according to the manufacturer's instructions (Invitrogen). Rad mRNA level was assessed by real-time quantitative RT-PCR as previously described [7] and 18S level was measured for normalization of variations in RNA input and cDNA synthesis. "
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    ABSTRACT: Ras associated with diabetes (Rad) inhibits vascular lesion formation by reducing the attachment and migration of vascular smooth muscle cells (VSMCs). However, the transcriptional regulation of Rad in VSMCs is unclear. We found that Platelet-Derived Growth Factor (PDGF)induced Rad expression in a time- and dose-dependent manner in rat aortic smooth muscle cells (RASMCs) using quantitative real-time PCR. By serial deletion analysis of the Rad promoter, we identified that two GC-rich early growth response-1 (Egr-1) binding sites are essential for PDGF-induced Rad promoter activation. Overexpression of Egr-1 in RASMCs strongly stimulated Rad expression while the Egr-1 corepressor, NGFI-A binding protein 2 (NAB2), repressed PDGF-induced Rad up-regulation in a dose-dependent manner. Direct binding of Egr-1 to the Rad promoter region was further confirmed by chromatin immunoprecipitation assays. Our results demonstrate that Rad is regulated by PDGF through the transcriptional factor Egr-1 in RASMCs.
    Full-text · Article · Apr 2011 · PLoS ONE
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    • "These genes may be components of cytokine signaling pathways, a common injury response pathway. Rad expression has been directly linked to cytokine signaling in vascular smooth muscle cells where it is stimulated by tumor necrosis factor-α (Fu et al., 2005). Tumor necrosis factor-α is induced in injured nerves and may be the signal for Rad induction (Murphy et al., 1995; Ohtori et al., 2004; Schafers et al., 2003). "
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    ABSTRACT: Unlike mammals, teleost fish are able to mount an efficient and robust regenerative response following optic nerve injury. Although it is clear that changes in gene expression accompany axonal regeneration, the extent of this genomic response is not known. To identify genes involved in successful nerve regeneration, we analyzed gene expression in zebrafish retinal ganglion cells (RGCs) regenerating their axons following optic nerve injury. Microarray analysis of RNA isolated by laser capture microdissection from uninjured and 3-day post-optic nerve injured RGCs identified 347 up-regulated and 29 down-regulated genes. Quantitative RT-PCR and in situ hybridization were used to verify the change in expression of 19 genes in this set. Gene ontological analysis of the data set suggests regenerating neurons up-regulate genes associated with RGC development. However, not all regeneration-associated genes are expressed in differentiating RGCs indicating the regeneration is not simply a recapitulation of development. Knockdown of six highly induced regeneration-associated genes identified two, KLF6a and KLF7a, that together were necessary for robust RGC axon re-growth. These results implicate KLF6a and KLF7a as important mediators of optic nerve regeneration and suggest that not all induced genes are essential to mount a regenerative response.
    Full-text · Article · Jan 2008 · Developmental Biology
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