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1.
Arterioscler Thromb Vasc Biol ; 34(7): 1548-55, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24855063

ABSTRACT

OBJECTIVE: Collateral vessel formation can functionally compensate for obstructive vascular lesions in patients with atherosclerosis. Neovascularization processes are triggered by fluid shear stress, hypoxia, growth factors, chemokines, proteases, and inflammation, as well as reactive oxygen species, in response to ischemia. Polymerase δ-interacting protein 2 (Poldip2) is a multifunctional protein that regulates focal adhesion turnover and vascular smooth muscle cell migration and modifies extracellular matrix composition. We, therefore, tested the hypothesis that loss of Poldip2 impairs collateral formation. APPROACH AND RESULTS: The mouse hindlimb ischemia model has been used to understand mechanisms involved in postnatal blood vessel formation. Poldip2(+/-) mice were subjected to femoral artery excision, and functional and morphological analysis of blood vessel formation was performed after injury. Heterozygous deletion of Poldip2 decreased the blood flow recovery and spontaneous running activity at 21 days after injury. H2O2 production, as well as the activity of matrix metalloproteinases-2 and -9, was reduced in these animals compared with Poldip2(+/+) mice. Infiltration of macrophages in the peri-injury muscle was also decreased; however, macrophage phenotype was similar between genotypes. In addition, the formation of capillaries and arterioles was impaired, as was angiogenesis, in agreement with a decrease in proliferation observed in endothelial cells treated with small interfering RNA against Poldip2. Finally, regression of newly formed vessels and apoptosis was more pronounced in Poldip2(+/-) mice. CONCLUSIONS: Together, these results suggest that Poldip2 promotes ischemia-induced collateral vessel formation via multiple mechanisms that likely involve reactive oxygen species-dependent activation of matrix metalloproteinase activity, as well as enhanced vascular cell growth and survival.


Subject(s)
Ischemia/metabolism , Mitochondrial Proteins/metabolism , Muscle, Skeletal/blood supply , Neovascularization, Physiologic , Nuclear Proteins/metabolism , Animals , Apoptosis , Cell Proliferation , Cells, Cultured , Collateral Circulation , Disease Models, Animal , Heterozygote , Hindlimb , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/pathology , Humans , Hydrogen Peroxide/metabolism , Ischemia/genetics , Ischemia/pathology , Ischemia/physiopathology , Macrophages/metabolism , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Mice , Mice, Knockout , Mitochondrial Proteins/deficiency , Mitochondrial Proteins/genetics , Nuclear Proteins/deficiency , Nuclear Proteins/genetics , RNA Interference , Recovery of Function , Regional Blood Flow , Time Factors , Transfection
2.
PLoS One ; 9(5): e96657, 2014.
Article in English | MEDLINE | ID: mdl-24797518

ABSTRACT

Polymerase-δ interacting protein 2 (Poldip2) is an understudied protein, originally described as a binding partner of polymerase delta and proliferating cell nuclear antigen (PCNA). Numerous roles for Poldip2 have been proposed, including mitochondrial elongation, DNA replication/repair and ROS production via Nox4. In this study, we have identified a novel role for Poldip2 in regulating the cell cycle. We used a Poldip2 gene-trap mouse and found that homozygous animals die around the time of birth. Poldip2-/- embryos are significantly smaller than wild type or heterozygous embryos. We found that Poldip2-/- mouse embryonic fibroblasts (MEFs) exhibit reduced growth as measured by population doubling and growth curves. This effect is not due to apoptosis or senescence; however, Poldip2-/- MEFs have higher levels of the autophagy marker LC3b. Measurement of DNA content by flow cytometry revealed an increase in the percentage of Poldip2-/- cells in the G1 and G2/M phases of the cell cycle, accompanied by a decrease in the percentage of S-phase cells. Increases in p53 S20 and Sirt1 were observed in passage 2 Poldip2-/- MEFs. In passage 4/5 MEFs, Cdk1 and CyclinA2 are downregulated in Poldip2-/- cells, and these changes are reversed by transfection with SV40 large T-antigen, suggesting that Poldip2 may target the E2F pathway. In contrast, p21CIP1 is increased in passage 4/5 Poldip2-/- MEFs and its expression is unaffected by SV40 transfection. Overall, these results reveal that Poldip2 is an essential protein in development, and underline its importance in cell viability and proliferation. Because it affects the cell cycle, Poldip2 is a potential novel target for treating proliferative conditions such as cancer, atherosclerosis and restenosis.


Subject(s)
Autophagy , Fibroblasts/cytology , Gene Expression Regulation, Developmental , Mitochondrial Proteins/metabolism , Nuclear Proteins/metabolism , Animals , Apoptosis , Cell Cycle , Cell Proliferation , DNA Repair , DNA Replication , Female , Gene Silencing , Genotype , Homozygote , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphorylation , Proliferating Cell Nuclear Antigen/metabolism , Signal Transduction
3.
Arterioscler Thromb Vasc Biol ; 33(9): 2154-61, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23825363

ABSTRACT

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.


Subject(s)
Aorta/metabolism , Aortic Aneurysm/prevention & control , Mitochondrial Proteins/metabolism , Nuclear Proteins/metabolism , Animals , Aorta/drug effects , Aorta/pathology , Aorta/physiopathology , Aortic Aneurysm/genetics , Aortic Aneurysm/metabolism , Aortic Aneurysm/pathology , Aortic Aneurysm/physiopathology , Blood Pressure , Cells, Cultured , Collagen Type I/metabolism , Cytochrome b Group/metabolism , Dilatation, Pathologic , Disease Models, Animal , Dose-Response Relationship, Drug , Elastic Tissue/metabolism , Extracellular Matrix/metabolism , Gene Expression Regulation , Genotype , Male , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitochondrial Proteins/deficiency , Mitochondrial Proteins/genetics , Myocytes, Smooth Muscle/metabolism , NADH, NADPH Oxidoreductases/metabolism , NADPH Oxidase 1 , NADPH Oxidase 2 , NADPH Oxidase 4 , NADPH Oxidases/metabolism , Nuclear Proteins/deficiency , Nuclear Proteins/genetics , Oxidants/pharmacology , Phenotype , RNA, Messenger/metabolism , Vascular Stiffness , Vasoconstrictor Agents/pharmacology , Vasodilation
4.
Am J Physiol Heart Circ Physiol ; 305(5): H747-55, 2013 Sep 01.
Article in English | MEDLINE | ID: mdl-23812391

ABSTRACT

Vascular diseases frequently accompany diabetes mellitus. Based on the current understanding of atherosclerosis as an inflammatory disorder of the vascular wall, it has been speculated that diabetes may accelerate atherosclerosis by inducing a proinflammatory milieu in the vasculature. ANG II and bone morphogenic proteins (BMPs) have been implicated in vascular inflammation. We evaluated the effect of angiotensin receptor blockade by valsartan and BMP inhibition by noggin on markers of vascular inflammation in a mouse model of diabetes. Noggin had no effect on blood pressure but decreased serum glucose levels, whereas valsartan significantly decreased blood pressure, but not serum glucose. Both inhibitors reduced reactive oxygen species production in the aorta. Additionally, noggin and valsartan diminish gene transcription and protein expression of various inflammatory molecules in the vascular wall. These observations indicate that although both inhibitors block superoxide production and have similar effects on inflammatory gene expression, glycemia and blood pressure may represent a secondary target differentially affected by noggin and valsartan. Our data clearly identify the BMP pathway as a potentially potent therapeutic target in diabetic inflammatory vascular disease.


Subject(s)
Bone Morphogenetic Proteins/antagonists & inhibitors , Carrier Proteins/therapeutic use , Diabetes Mellitus, Type 2/complications , Hyperglycemia/prevention & control , Vasculitis/prevention & control , Angiotensin II Type 1 Receptor Blockers/pharmacology , Angiotensin II Type 1 Receptor Blockers/therapeutic use , Animals , Blood Glucose/drug effects , Blood Pressure/drug effects , Blood Pressure/physiology , Carrier Proteins/pharmacology , Diabetes Mellitus, Type 2/physiopathology , Disease Models, Animal , Hyperglycemia/physiopathology , Male , Mice , Mice, Mutant Strains , Reactive Oxygen Species/metabolism , Tetrazoles/pharmacology , Tetrazoles/therapeutic use , Valine/analogs & derivatives , Valine/pharmacology , Valine/therapeutic use , Valsartan , Vasculitis/physiopathology
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