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 , VasodilationABSTRACT
Analogs of 1,5-diarylpyrazoles with a novel pharmacophore at N1 were designed, synthesized and evaluated for the in-vitro cyclooxygenase (COX-1/COX-2) inhibitory activity. The variations at/around position-4 of the C-5 phenyl ring in conjunction with a CF3 and CHF2 groups at C-3 exhibited a high degree of potency and selectivity index (SI) for COX-2 inhibition. The in-vivo evaluation of these potent compounds with a few earlier ones indicated the 4-OMe-phenyl analog and the 4-NHMe-phenyl analog with a CF3, and the 4-OEt-phenyl analog with a CHF2 group at C-3 to possess superior potency than celecoxib. In addition to its impressive anti-inflammatory, antipyretic, analgesic and anti-arthritic properties, compound (DRF-4367) was found to possess an excellent pharmacokinetic profile, gastrointestinal (GI) safety in the long-term arthritis study and COX-2 potency in human whole blood assay. Thus, compound was selected as an orally active anti-inflammatory candidate for pre-clinical evaluation.