Abstract-Atherosclerosis and arterial injury-induced neointimal hyperplasia involve medial smooth muscle cell (SMC) proliferation and migration into the arterial intima. Because many 7-transmembrane and growth factor receptors promote atherosclerosis, we hypothesized that the multifunctional adaptor proteins ␤-arrestin1 and -2 might regulate this pathological process. Deficiency of ␤-arrestin2 in ldlr Ϫ/Ϫ mice reduced aortic atherosclerosis by 40% and decreased the prevalence of atheroma SMCs by 35%, suggesting that ␤-arrestin2 promotes atherosclerosis through effects on SMCs. To test this potential atherogenic mechanism more specifically, we performed carotid endothelial denudation in congenic wild-type, ␤-arrestin1 Ϫ/Ϫ , and ␤-arrestin2 Ϫ/Ϫ mice. Neointimal hyperplasia was enhanced in ␤-arrestin1mice, and diminished in ␤-arrestin2 Ϫ/Ϫ mice. Neointimal cells expressed SMC markers and did not derive from bone marrow progenitors, as demonstrated by bone marrow transplantation with green fluorescent protein-transgenic cells. Moreover, the reduction in neointimal hyperplasia seen in ␤-arrestin2 Ϫ/Ϫ mice was not altered by transplantation with either wild-type or ␤-arrestin2 Ϫ/Ϫ bone marrow cells. After carotid injury, medial SMC extracellular signal-regulated kinase activation and proliferation were increased in ␤-arrestin1Ϫ/Ϫ and decreased in ␤-arrestin2 Ϫ/Ϫ mice. Concordantly, thymidine incorporation and extracellular signal-regulated kinase activation and migration evoked by 7-transmembrane receptors were greater than wild type in ␤-arrestin1 Ϫ/Ϫ SMCs and less in ␤-arrestin2 Ϫ/Ϫ SMCs. Proliferation was less than wild type in ␤-arrestin2 Ϫ/Ϫ SMCs but not in ␤-arrestin2 Ϫ/Ϫ endothelial cells. We conclude that ␤-arrestin2 aggravates atherosclerosis through mechanisms involving SMC proliferation and migration and that these SMC activities are regulated reciprocally by ␤-arrestin2 and ␤-arrestin1. These findings identify inhibition of ␤-arrestin2 as a novel therapeutic strategy for combating atherosclerosis and arterial restenosis after angioplasty.

Accumulating evidence suggests that receptor protein-tyrosine kinases, like the platelet-derived growth factor receptor-␤ (PDGFR␤) and epidermal growth factor receptor (EGFR), may be desensitized by serine/threonine kinases. One such kinase, G protein-coupled receptor kinase-2 (GRK2), is known to mediate agonistdependent phosphorylation and desensitization of multiple heptahelical receptors. In testing whether GRK2 could phosphorylate and desensitize the PDGFR␤, we first found by phosphoamino acid analysis that cells expressing GRK2 could serine-phosphorylate the PDGFR␤ in an agonist-dependent manner. Augmentation or inhibition of GRK2 activity in cells, respectively, reduced or enhanced tyrosine phosphorylation of the PDGFR␤ but not the EGFR. Either overexpressed in cells or as a purified protein, GRK2 demonstrated agonist-promoted serine phosphorylation of the PDGFR␤ and, unexpectedly, the EGFR as well. Because GRK2 did not phosphorylate a kinase-dead (K634R) PDGFR␤ mutant, GRK2-mediated PDGFR␤ phosphorylation required receptor tyrosine kinase activity, as does PDGFR␤ ubiquitination. Agonist-induced ubiquitination of the PDGFR␤, but not the EGFR, was enhanced in cells overexpressing GRK2. Nevertheless, GRK2 overexpression did not augment PDGFR␤ down-regulation. Like the vast majority of GRK2 substrates, the PDGFR␤, but not the EGFR, activated heterotrimeric G proteins allosterically in membranes from cells expressing physiologic protein levels. We conclude that GRK2 can phosphorylate and desensitize the PDGFR␤, perhaps through mechanisms related to receptor ubiquitination. Specificity of GRK2 for receptor protein-tyrosine kinases, expressed at physiologic levels, may be determined by the ability of these receptors to activate heterotrimeric G proteins, among other factors.

Activation of cells intrinsic to the vessel wall is central to the initiation and progression of vascular inflammation. As the dominant cellular constituent of the vessel wall, vascular smooth muscle cells (VSMCs) and their functions are critical determinants of vascular disease. While factors that regulate VSMC proliferation and migration have been identified, the endogenous regulators of VSMC proinflammatory activation remain incompletely defined. The Kruppel-like family of transcription factors (KLFs) are important regulators of inflammation. In this study, we identified Kruppel-like factor 15 (KLF15) as an essential regulator of VSMC proinflammatory activation. KLF15 levels were markedly reduced in human atherosclerotic tissues. Mice with systemic and smooth muscle-specific deficiency of KLF15 exhibited an aggressive inflammatory vasculopathy in two distinct models of vascular disease: orthotopic carotid artery transplantation and diet-induced atherosclerosis. We demonstrated that KLF15 alters the acetylation status and activity of the proinflammatory factor NF-κB through direct interaction with the histone acetyltransferase p300. These studies identify a previously unrecognized KLF15-dependent pathway that regulates VSMC proinflammatory activation.