Polymerase delta interacting protein 2 sustains vascular structure and function.

OBJECTIVE: On the basis of previous evidence that polymerase delta interacting protein 2 (Poldip2) increases reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (Nox4) activity in vascular smooth muscle cells, we hypothesized that in vivo knockdown of Poldip2 would inhibit reactive oxygen species production and alter vascular function. APPROACH AND RESULTS: Because homozygous Poldip2 deletion is lethal, Poldip2(+/-) mice were used. Poldip2 mRNA and protein levels were reduced by ≈50% in Poldip2(+/-) aorta, with no change in p22phox, Nox1, Nox2, and Nox4 mRNAs. NADPH oxidase activity was also inhibited in Poldip2(+/-) tissue. Isolated aortas from Poldip2(+/-) mice demonstrated impaired phenylephrine and potassium chloride-induced contractions, increased stiffness, and reduced compliance associated with disruption of elastic lamellae and excessive extracellular matrix deposition. Collagen I secretion was elevated in cultured vascular smooth muscle cells from Poldip2(+/-) mice and restored by H2O2 supplementation, suggesting that this novel function of Poldip2 is mediated by reactive oxygen species. Furthermore, Poldip2(+/-) mice were protected against aortic dilatation in a model of experimental aneurysm, an effect consistent with increased collagen secretion. CONCLUSIONS: Poldip2 knockdown reduces H2O2 production in vivo, leading to increases in extracellular matrix, greater vascular stiffness, and impaired agonist-mediated contraction. Thus, unaltered expression of Poldip2 is necessary for vascular integrity and function.

Microtubules regulate angiotensin II type 1 receptor and Rac1 localization in caveolae/lipid rafts: role in redox signaling.

OBJECTIVE: Microtubules are important in signal transduction temporal-spatial organization. Full expression of angiotensin II (Ang II) signaling in vascular smooth muscle cells (VSMCs) is dependent on the reactive oxygen species (ROS) derived from nicotinamide-adenine dinucleotide phosphate (NAD(P)H) oxidase and the dynamic association of the Ang II type 1 receptor (AT1R) with caveolae/lipid rafts. Translocation of the small GTPase Rac1 to the plasma membrane is an essential step for activation of NAD(P)H oxidase; however, its precise localization in the plasma membrane after agonist stimulation and how it is targeted are unknown. We hypothesized that microtubules are involved in regulating multiphasic Ang II signaling events in VSMC. METHODS AND RESULTS: We show that Ang II promotes Rac1 and AT1R trafficking into caveolae/lipid rafts, which is blocked by disruption of microtubules with nocodazole. As a consequence, nocodazole significantly inhibits Ang II-stimulated H2O2 production, its downstream ROS-dependent epidermal growth factor receptor transactivation, Akt phosphorylation, and vascular hypertrophy without affecting Rac1 activation or ROS-independent extracellular signal-regulated kinase 1/2 phosphorylation. CONCLUSIONS: These results suggest that proper Rac1 and AT1R trafficking into caveolae/lipid rafts requires the integrity of microtubules and provide insight into the essential role of microtubules for the spatial-temporal organization of ROS-dependent and caveolae/lipid rafts-dependent AT(1)R signaling linked to vascular hypertrophy.

Upregulation of Nox-based NAD(P)H oxidases in restenosis after carotid injury.

Restenosis, a frequent complication of coronary angioplasty, is associated with increased superoxide (O2*(-)) production. Although the molecular identity of the responsible oxidase is unclear, an NAD(P)H oxidase appears to be involved. In smooth muscle, p22phox and 2 homologues of gp91phox, nox1 and nox4, are expressed, whereas fibroblasts contain gp91phox. To begin investigating the possibility that these oxidase components might contribute to the increased O2*(-) that accompanies neointimal formation, we measured their expression after balloon injury of the rat carotid artery. The increase in O2*(-) production 3 to 15 days after surgery was not due to inflammatory cell infiltration but appeared to be derived from medial and neointimal smooth muscle cells and adventitial fibroblasts. Nox1 and p22phox mRNAs were increased 2.7- and 3.6-fold, respectively, at day 3 after injury and remained elevated for 15 days. gp91Phox was increased 7 to 15 days after injury, and nox4 expression was increased 2-fold, but only at day 15 after surgery. These results confirm and extend our previous in vitro data and suggest that in the vasculature, the nox-based NAD(P)H oxidases serve different functions. This dynamic regulation of oxidase components may be critical to smooth muscle phenotypic modulation in restenosis and atherosclerosis.