Perlecan Heparan Sulfate Proteoglycan Is a Critical Determinant of Angiogenesis in Response to Mouse Hind-Limb Ischemia

Abstract

Background: Perlecan is a heparan sulfate proteoglycan (HSPG) constituent of the extracellular matrix with roles in cell growth, differentiation, and angiogenesis. The role of the HS side chains in regulating in vivo angiogenesis after hind-limb ischemia is unknown. Methods: Heparan sulfate (HS)-deficient perlecan (Hspg2(Delta 3/Delta 3)) mice (n = 35), containing normal perlecan core protein but deficient in HS side chains, and wild-type (n = 33) littermates underwent surgical induction of hind-limb ischemia. Laser Doppler perfusion imaging (LDPI) and contrast-enhanced ultrasonography (CEU) provided serial assessment of hind-limb perfusion. Harvested muscles underwent immunostaining for endothelial cell density (CD31), real-time reverse transcription polymerase chain reaction RT-PCR for vascular endothelial growth factor (VEGF) mRNA expression and western blot analysis for VEGF and fibroblast growth factor (FGF) 2 protein expression at days 2 and 28. Results: Serial LDPI showed significantly greater perfusion recovery in ischemic limbs of wild-type compared with Hspg2(Delta 3/Delta 3) mice. CEU showed that normalized microvascular perfusion was increased in wild-type compared with Hspg2(Delta 3/Delta 3) mice at day 28 (0.67 +/- 0.12 vs 0.26 +/- 0.08; P = 0.001). CD31-positive cell counts were significantly higher in wild-type compared with Hspg2(Delta 3/Delta 3) mice on day 28 (122 +/- 30 cells vs 84 +/- 34 cells per high-power field [HPF]; P < 0.05). Endogenous VEGF mRNA expression (P < 0.05) and VEGF protein expression (P < 0.002) were significantly decreased in the ischemic limbs of Hspg2(Delta 3/Delta 3) mice compared with wild-type mice at day 2 and day 28, respectively. FGF2 protein expression showed no significant differences. Conclusions: These results suggest that the HS side chains in perlecan are important mediators of the angiogenic response to ischemia through a mechanism that involves upregulation of VEGF expression.