Polymerase delta interacting protein 2 sustains vascular structure and function

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.

Keywords: Nox4; Poldip2; blood vessel; extracellular matrix; hydrogen peroxide.

Figure 1. Poldip2 expression is specifically reduced in Poldip2+/− mice

A. mRNAs of Poldip2, p22phox and indicated catalytic subunits of Nox enzymes were measured by quantitative RT-PCR in whole aortas from wild-type (black bars) and Poldip2+/− (gray bars) mice. Data represent average ± SEM from 4 animals; *** P < 0.001. B. Representative Western blots of Poldip2 and CDK4 (loading control) performed using indicated amounts of whole mouse aorta protein (left). Densitometric quantification of Western blot results (right). Data represent averages ± SEM from 7 mice in each group; * P < 0.05.

Figure 2. NADPH oxidase activity is inhibited in Poldip2+/− mice

Superoxide (O₂^•−, left) and hydrogen peroxide (H₂O₂, right) were measured in kidney slices from wild-type (black bars) and Poldip2+/− (gray bars) mice by ESR. Data represent average ± SEM from 5–10 mice in each group; ** P < 0.01.

Figure 3. Aortic contraction and compliance are reduced in Poldip2+/− mice

Isometric force normalized to cross-sectional area (CSA) was measured in isolated aortic rings from wild-type (black symbols) and Poldip2+/− mice (gray symbols) exposed to indicated concentrations of phenylephrine (A) or potassium chloride (B). To evaluate stiffness, aortic segments were incrementally elongated (C) or inflated (D, E). Data represent average ± SEM from 5–9 vessels. *** P < 0.001, * P < 0.05 +/+ vs. +/−.

Figure 4. Disrupted elastic lamellae and increased extracellular matrix in Poldip2+/− aorta

Transmission electron micrographs of transverse aortic sections from wild-type (left) and Poldip2+/− (right) mice at increasing magnifications (top to bottom). The vascular lumen is visible at the top of images A–D. Elastic lamellae (EL) appear dark after post-staining with tannic acid in A and B. Breaks in elastic lamellae in Poldip2+/− are marked with arrows in B and D. Morphometric measurements of interlamellar amorphous (C and D) or fibrillar (F) extracellular matrix (ECM) areas in 195 images from 3 wild-type and 3 Poldip2+/− mice are expressed as average ± SEM in E; * P < 0.05.

Figure 5. The increase in extracellular collagen I produced by Poldip2+/− VSMC is abrogated by H₂O₂ supplementation

Cultured VSMC from wild-type and Poldip2+/− mice were incubated without (Control) or with 2 ng glucose oxidase for 3 days, before collection of media for Western blot analysis. Equal amounts of protein were loaded in each lane. Representative blot (top) and average densitometric data ± SEM from 9–11 independent experiments (bottom); * P < 0.05 vs. +/+.

Figure 6. Aortic dilatation is inhibited in Poldip2+/− mice

Dilatation was induced by surgical application of CaCl₂ to the abdominal aorta of 9–10 week-old male wild-type (black bar) or Poldip2+/− (gray bar) mice in a model of experimental aneurysm. Aortic diameters were measured before and 8 weeks after treatment and expressed as % change. Data represent average ± SEM from 9–10 mice in each group, ** P < 0.01.