No abstract

Cells from multiple origins contribute to vascular smooth muscle cell (VSMC) development. Phenotypic heterogeneity of VSMCs is associated with their point of developmental origin; however, the mechanisms driving such differences are unknown. We here examined the mechanisms controlling vascular bed-specific differences in Rgs5 expression during development. Rgs5 levels were similar across different regions of the vasculature in neonatal animals but were >15-fold higher in descending aortas compared with carotid arteries of adult mice. Thus, vessel bed-specific changes in regulation of Rgs5 expression occurred during vessel maturation. Examination of adult Rgs5-LacZ reporter mice revealed lower Rgs5 expression in VSMCs originating from the third (carotid artery) branchial arch compared with those originating in the fourth and sixth (aortic B segment, right subclavian, and ductus arteriosus) branchial arches. Indeed, a mosaic Rgs5 expression pattern, with discreet LacZ boundaries between VSMCs derived from different developmental origins, was observed. Furthermore, Rgs5-LacZ expression was correlated with the site of VSMC origin (splanchic mesoderm ≈ local mesenchyme > somites > proepicardium > mesothelium). Surprisingly, Rgs5 reporter activity in cultured carotid artery- and descending aorta-derived cells did not recapitulate the differences observed in vivo. Consistent with a developmental origin-specific epigenetic mechanism driving the observed expression differences in vivo, the Rgs5 promoter showed increased methylation on CpG dinucleotides in carotid arteries compared with that in descending aortas in adult but not in neonatal mice. In vitro methylation of the Rgs5 promoter confirmed that its activity is sensitive to transcriptional down-regulation by CpG methylation. These data suggest that an origin-dependent epigenetic program regulates vascular bed- and maturation state-dependent regulation of VSMC-specific gene transcription.

Hypertension is a leading risk factor for the development of cardiovascular disease. Data from human and animal studies suggest that RGS2, a potent inhibitor of G q signaling, is important for blood pressure regulation. Several RGS2 mutations in the Japanese population have been found to be associated with hypertension. The product of one of these alleles, R44H, is mutated within the amino terminal amphipathic ␣-helix domain, the region responsible for plasma membrane-targeting. The functional consequence of this mutation and its potential link to the development of hypertension, however, are not known. In this study, we showed that R44H was a weaker inhibitor of receptor-mediated G q signaling than wild-type RGS2. Confocal microscopy revealed that YFP-tagged R44H bound to the plasma membrane less efficiently than wild-type RGS2. R44 is one of the basic residues positioned to stabilize lipid bilayer interaction of the RGS2 amphipathic helix domain. Tryptophan fluorescence and circular dichroism studies of this domain showed that the R44H mutation prevented proper entrenchment of hydrophobic residues into the lipid bilayer without disrupting helix-forming capacity. Together, these data suggest that decreasing the side-chain length and flexibility at R44 prevented proper lipid bilayer association and function of RGS2. Finally, the R44H protein did not behave as a dominantnegative interfering mutant. Thus, our data are consistent with the notion that a R44H missense mutation in human RGS2 produces a hypomorphic allele that may lead to altered receptor-mediated G q inhibition and contribute to the development of hypertension in affected subjects.Heterotrimeric G-proteins are important mediators of cardiovascular cell signaling and physiology. G-protein-coupled receptors (GPCRs) mediate the biologic function of neurotransmitters and hormones including catecholamines, angiotensin II (Ang II), and endothelin-1 (ET-1) to control a number of cardiovascular parameters, such as vascular resistance, cardiac output, and plasma volume. It is therefore important to maintain proper regulation and coordination of G-protein signaling to ensure proper blood pressure homeostasis. Regulators of G-protein signaling (RGS) proteins are one family of more than 35 proteins that serve this function through their activity as GTPase activating proteins for G-protein ␣ subunits. Within the RGS superfamily, RGS2 is comparatively well suited to regulate signaling events required for blood pressure homeostasis. RGS2 is a selective and efficient inhibitor of G q , the primary mediator of vasoconstrictive stimuli including norepinephrine, Ang II, and ET-1. RGS2 also inhibits some types of adenylyl cyclase (Sinnarajah et al., 2001;Salim et al., 2003), a pathway important for dopamine-mediated regulation of blood pressure in the kidneys.