Polymerase delta-interacting protein 2 (Poldip2) is a multi-functional protein with numerous roles in the vasculature, including the regulation of cell apoptosis and migration, as well as extracellular matrix deposition; however, its role in VSMC proliferation and neointimal formation is unknown. In this study, we investigated the role of Poldip2 in intraluminal wire-injury induced neointima formation and proliferation of vascular smooth muscle cells in vitro and in vivo. Poldip2 expression was observed in the intima and media of human atherosclerotic arteries, where it colocalized with proliferating cell nuclear antigen (PCNA). Wire injury of femoral arteries of Poldip2 +/+ mice induced robust neointimal formation after 2 weeks, which was impaired in Poldip2 +/‒ mice. PCNA expression was significantly reduced and expression of the cell cycle inhibitor p21 was significantly increased in wire-injured arteries of Poldip2 +/‒ animals compared to wild-type controls. No difference was observed in apoptosis. Downregulation of Poldip2 in rat aortic smooth muscle cells significantly reduced serum-induced proliferation and PCNA expression, but upregulated p21 expression. Downregulation of p21 using siRNA reversed the inhibition of proliferation induced by knockdown of Poldip2. These results indicate that Poldip2 plays a critical role in the proliferation of VSMCs.

Objective-Actin cytoskeleton assembly and organization, as a result of focal adhesion (FA) formation during cell adhesion, are dependent on reactive oxygen species and the cellular redox environment. Poldip2 (polymerase δ-interacting protein 2), a novel regulator of NOX4 (NADPH oxidase 4), plays a significant role in reactive oxygen species production and cytoskeletal remodeling. Thus, we hypothesized that endogenous reactive oxygen species derived from Poldip2/NOX4 contribute to redox regulation of actin and cytoskeleton assembly during integrin-mediated cell adhesion. Approach and Results-Using vascular smooth muscle cells, we verified that hydrogen peroxide (H2O2) levels increase during integrin-mediated cell attachment as a result of activation of NOX4. Filamentous actin (F-actin) was oxidized by sulfenylation during cell attachment, with a peak at 3 hours (0.80±0.04 versus 0.08±0.13 arbitrary units at time zero), which was enhanced by overexpression of Poldip2. Depletion of Poldip2 or NOX4 using siRNA, or scavenging of endogenous H2O2 with catalase, inhibited F-actin oxidation by 78±26%, 99±1%, and 98±1%, respectively. To determine the consequence of F-actin oxidation, we examined the binding of F-actin to vinculin, a protein involved in FA complexes that regulates FA maturation. Vinculin binding during cell adhesion as well as migration capacity were inhibited after transfection with actin containing 2 oxidation-resistant point mutations (C272A and C374A). Silencing of Poldip2 or NOX4 also impaired actin-vinculin interaction, which disturbed maturation of FAs and inhibited cell migration. Conclusions-These results suggest that integrin engagement during cell attachment activates Poldip2/Nox4 to oxidize actin, which modulates FA assembly.

Objective Cellular senescence influences organismal aging and increases predisposition to age-related diseases, in particular cardiovascular disease, a leading cause of death and disability worldwide. PGC-1α is a master regulator of mitochondrial biogenesis and function, oxidative stress and insulin resistance. Senescence is associated with telomere and mitochondrial dysfunction and oxidative stress, inferring a potential causal role of PGC-1α in senescence pathogenesis. Methods and Results We generated a PGC-1α+/−/ApoE−/− mouse model and show that PGC-1α deficiency promotes a vascular senescence phenotype that is associated with increased oxidative stress, mitochondrial abnormalities, and reduced telomerase activity. PGC-1α disruption results in reduced expression of the longevity-related deacetylase sirtuin 1 (SIRT1) and the antioxidant catalase, and increased expression of the senescence marker p53 in aortas. Further, angiotensin II (Ang II), a major hormonal inducer of vascular senescence, induces prolonged lysine acetylation of PGC-1α and releases the PGC-1α·FoxO1 complex from the SIRT1 promoter, thus reducing SIRT1 expression. The phosphorylation defective mutant PGC-1α S570A is not acetylated, is constitutively active for FoxO1-dependent SIRT1 transcription and prevents Ang II-induced senescence. Acetylation of PGC-1α by Ang II interrupts the PGC-1α-FoxO1-SIRT1 feed-forward signaling circuit leading to SIRT1 and catalase downregulation and vascular senescence. Conclusions PGC-1α is a primary negative regulator of vascular senescence. Moreover, the central role of post-translational modification of PGC-1α in regulating Ang II-induced vascular senescence may inform development of novel therapeutic strategies for mitigating age-associated diseases such as atherosclerosis.

Melanoma is the cancer with the highest increase in incidence, and transformation of radial growth to vertical growth (i.e., noninvasive to invasive) melanoma is required for invasive disease and metastasis. We have previously shown that p42/p44 MAP kinase is activated in radial growth melanoma, suggesting that further signaling events are required for vertical growth melanoma. The molecular events that accompany this transformation are not well understood. Akt, a signaling molecule downstream of PI3K, was introduced into the radial growth WM35 melanoma in order to test whether Akt overexpression is sufficient to accomplish this transformation. Overexpression of Akt led to upregulation of VEGF, increased production of superoxide ROS, and the switch to a more pronounced glycolytic metabolism. Subcutaneous implantation of WM35 cells overexpressing Akt led to rapidly growing tumors in vivo, while vector control cells did not form tumors. We demonstrated that Akt was associated with malignant transformation of melanoma through at least 2 mechanisms. First, Akt may stabilize cells with extensive mitochondrial DNA mutation, which can generate ROS. Second, Akt can induce expression of the ROS-generating enzyme NOX4. Akt thus serves as a molecular switch that increases angiogenesis and the generation of superoxide, fostering more aggressive tumor behavior. Targeting Akt and ROS may be of therapeutic importance in treatment of advanced melanoma.

Akt/protein kinase B (PKB) activation/phosphorylation by angiotensin II (Ang II) is a critical signaling event in hypertrophy of vascular smooth muscle cells (VSMCs). Conventional wisdom asserts that Akt activation occurs mainly in plasma membrane domains. Recent evidence that Akt activation may take place within intracellular compartments challenges this dogma. The spatial identity and mechanistic features of these putative signaling domains have not been defined. Using cell fractionation and fluorescence methods, we demonstrate that the early endosomal antigen-1 (EEA1)-positive endosomes are a major site of Ang II-induced Akt activation. Akt moves to and is activated in EEA1 endosomes. The expression of EEA1 is required for phosphorylation of Akt at both Thr-308 and Ser-473 as well as for phosphorylation of its downstream targets mTOR and S6 kinase, but not for Erk1/2 activation. Both Akt and phosphorylated Akt (p-Akt) interact with EEA1. We also found that PKC-α is required for organizing Ang II-induced, EEA1-dependent Akt phosphorylation in VSMC early endosomes. EEA1 expression enables PKC-α phosphorylation, which in turn regulates Akt upstream signaling kinases, PDK1 and p38 MAPK. Our results indicate that PKC-α is a necessary regulator of EEA1-dependent Akt signaling in early endosomes. Finally, EEA1 down-regulation or expression of a dominant negative mutant of PKC-α blunts Ang II-induced leucine incorporation in VSMCs. Thus, EEA1 serves a novel function as an obligate scaffold for Ang II-induced Akt activation in early endosomes.

Angiotensin II (Ang II) is a pleuripotential hormone that is important in the pathophysiology of multiple conditions including aging, cardiovascular and renal diseases, and insulin resistance. Reactive oxygen species (ROS) are important mediators of Ang II-induced signaling generally and have a well defined role in vascular hypertrophy, which is inhibited by overexpression of catalase, inferring a specific role of H2O2. The molecular mechanisms are understood incompletely. The transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is a key regulator of energy metabolism and ROS-scavenging enzymes including catalase. We show that Ang II stimulates Akt-dependent PGC-1α serine 570 phosphorylation, which is required for the binding of the histone acetyltransferase GCN5 (general control nonderepressible 5) to PGC-1α and for its lysine acetylation. These sequential post-translational modifications suppress PGC-1α activity and prevent its binding to the catalase promoter through the forkhead box O1 transcription factor, thus decreasing catalase expression. We demonstrate that overexpression of the phosphorylation-defective mutant PGC-1α (S570A) prevents Ang II-induced increases in H2O2 levels and hypertrophy ([3H]leucine incorporation). Knockdown of PGC-1α by small interfering RNA promotes basal and Ang II-stimulated ROS and hypertrophy, which is reversed by polyethylene glycol-conjugated catalase. Thus, endogenous PGC-1α is a negative regulator of vascular hypertrophy by up-regulating catalase expression and thus reducing ROS levels. We provide novel mechanistic insights by which Ang II may mediate its ROS-dependent pathophysiologic effects on multiple cardiometabolic diseases.

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.

Clinical studies show that metformin attenuates all‐cause mortality and myocardial infarction compared with other medications for type 2 diabetes, even at similar glycemic levels. However, there is paucity of data in the euglycemic state on the vasculoprotective effects of metformin. The objectives of this study are to evaluate the effects of metformin on ameliorating atherosclerosis.

Background: Polymerase δ-interacting protein 2 (Poldip2) is a multifunctional protein that regulates vascular extracellular matrix composition and matrix metalloproteinase (MMP) activity. The blood-brain barrier (BBB) is a dynamic system assembled by endothelial cells, basal lamina, and perivascular astrocytes, raising the possibility that Poldip2 may be involved in maintaining its structure. We investigated the role of Poldip2 in the late BBB permeability induced by cerebral ischemia. Methods: Transient middle cerebral artery occlusion (tMCAO) was induced in Poldip2 +/+ and Poldip2 +/- mice. The volume of the ischemic lesion was measured in triphenyltetrazolium chloride-stained sections. BBB breakdown was evaluated by Evans blue dye extravasation. Poldip2 protein expression was evaluated by western blotting. RT-PCR, zymography, and ELISAs were used to measure mRNA levels, activity, and protein levels of cytokines and MMPs. Cultured astrocytes were transfected with Poldip2 siRNA, and mRNA levels of cytokines were evaluated as well as IΚBα protein degradation. Results: Cerebral ischemia induced the expression of Poldip2. Compared to Poldip2 +/+ mice, Poldip2 +/- animals exhibited decreased Evans blue dye extravasation and improved survival 24 h following stroke. Poldip2 expression was upregulated in astrocytes exposed to oxygen and glucose deprivation (OGD) and siRNA-mediated downregulation of Poldip2 abrogated OGD-induced IL-6 and TNF-α expression. In addition, siRNA against Poldip2 inhibited TNF-α-induced IΚBα degradation. TNF-α, IL-6, MCP-1, VEGF, and MMP expression induced by cerebral ischemia was abrogated in Poldip2 +/- mice. The protective effect of Poldip2 depletion on the increased permeability of the BBB was partially reversed by systemic administration of TNF-α. Conclusions: Poldip2 is upregulated following ischemic stroke and mediates the breakdown of the BBB by increasing cerebral cytokine production and MMP activation. Therefore, Poldip2 appears to be a promising novel target for the development of therapeutic strategies to prevent the development of cerebral edema in the ischemic brain.