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

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 invivo angiogenesis after hind-limb ischemia is unknown. Methods: Heparan sulfate (HS)-deficient perlecan (Hspg2^δ3/δ3) 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 and28. Results: Serial LDPI showed significantly greater perfusion recovery in ischemic limbs of wild-type compared with Hspg2^δ3/δ3 mice. CEU showed that normalized microvascular perfusion was increased in wild-type compared with Hspg2^δ3/δ3 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^δ3/δ3 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^δ3/δ3 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.