Polymerase δ-interacting protein 2 promotes postischemic neovascularization of the mouse hindlimb

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.

Keywords: NADPH oxidase; apoptosis; ischemia; metalloproteases; neovascularization.

Figure 1. Poldip2^+/- mice exhibit reduced blood flow recovery after ischemic injury

Femoral artery ligation was performed on Poldip2^+/+ and Poldip2^+/- mice and blood flow was assessed in the adductor muscles of the nonischemic (NIL) and ischemic limbs (IL) by LASER Doppler Perfusion Imaging (LDPI) at the indicated time points. A) Representative images from LDPI analysis immediately after the surgery and at the 21 day time point. B) Quantitative analysis presented as perfusion ratios (ischemic leg (IL) / nonischemic leg (NIL)). Data represent mean±SEM (n = 15-20 per genotype). * P<0.05 vs. Poldip2^+/+

Figure 2. Poldip2^+/- mice exhibit reduced angiogenesis and endothelial cell proliferation

A) Representative photomicrographs and quantification of capillary density in ischemic gastrocnemius muscle stained with antibody against I-isolectin B4 (n = 3-6 per genotype). Bars are means ± SEM. * P<0.05 vs. Poldip2 ^+/+. B) Representative photomicrographs and quantification of endothelial cell infiltration around SIS (small intestine submucosa) area at day 12 after implantation. Endothelial cells were assessed by staining with antibody against I-isolectin B4. C) Time course of serum-induced proliferation in HUVEC treated with siControl or siPoldip2 (n = 3 per condition). Bars are means ± SEM. * P<0.05 vs. siControl. Bar scale = 200 μm.

Figure 3. Poldip2 regulates arteriogenesis and apoptosis

A) Representative photomicrographs and quantification of arterioles in ischemic gastrocnemius muscle stained with antibody against smooth muscle α-actin. (n = 3-6 per genotype). Bars are means ± SEM. * P<0.05 vs. Poldip2^+/+. Scale bar = 200 μm. B) Representative photomicrographs visualized by autofluorescence (green) and quantification of apoptotic cells in ischemic gastrocnemius muscle stained by TUNEL (red) 21 days after surgery. (n = 3 per genotype). Bars are means ± SEM. * P<0.05 vs. Poldip2^+/+. Scale bar = 100 μm.

Figure 4. Poldip2 downregulation reduces macrophage infiltration, but does not alter macrophage phenotype

Inflammatory cell infiltration in ischemic tissue was assessed by staining of macrophage cells at 7 days with an antibody against Mac-3. Representative photomicrographs of total macrophage content are shown. Quantitative analyses are presented as ratios (ischemic leg (IL) / nonischemic leg (NIL)) after analysis of total fluorescence using ImageJ software. (n = 3-4 per genotype). Bars are means ± SEM. * P<0.05 vs. Poldip2^+/+. B) mRNA levels of IL-1β, IL-6, iNOS, Mannose Receptor (MRC), Arginase (ARG) and IL-10 were measured in ischemic muscle at 7 days after surgery. B2M (beta-2 microglobulin) rRNA was used to normalize each sample. (n = 3-5 per genotype). Bars are means ± SEM.

Figure 5. MMP2 and MMP9 activity is reduced in Poldip2^+/- mice

A) Representative gelatin zymography images and quantification of MMP2 and MMP9 activity from muscle immediately downstream of the femoral artery ligation in Poldip2^+/- and Poldip2^+/+ mice. (n = 3-5 per genotype). Bars are means ± SEM. * P<0.05 vs. Poldip2^+/+ B) Gelatinase activity from ischemic and nonischemic muscle was measured using the EnzChek gelatinase assay kit. (n = 3-4 per genotype). Bars are means ± SEM. * P<0.05 vs. Poldip2^+/+.

Figure 6. Reduced hydrogen peroxide production and running distance in Poldip2^+/- mice

A) H₂O₂ production from ischemic and nonischemic gastrocnemius muscle was assessed by Amplex Red assay. (n = 3 per genotype). Bars are means ± SEM. * P<0.05 vs. Poldip2 ^+/+. B) Spontaneous running activity recorded over a 7-day period and expressed as total distance run. (n = 4-10 per genotype). Bars are means ± SEM. * P<0.05 vs. Poldip2 ^+/+.