Age and Sex Impact the Role of Thrombospondin‐2 and Thrombospondin‐5 in Response to Hindlimb Ischemia

Peripheral arterial disease progression to critical limb ischemia remains a significant burden in the aged, necessitating revascularization. Therapies to enhance neovascularization, such as angiogenesis or arteriogenesis, may provide an option for patients not suitable for surgery. The thrombospondins (TSPs) are secreted matricellular glycoproteins, with TSP-1, TSP-2, and TSP-5 implicated in neovascularization. Currently, there is little data regarding the effects of TSP-2, TSP-5, sex, and aging on angiogenesis and arteriogenesis. In vitro, the effect of TSP-2 or TSP-5 on early or late passage endothelial cell (EC) tubule formation or disruption was assessed. In vivo, angiogenesis, and arteriogenesis in male and female, young (14-16 weeks) and old (105-110 weeks) wild-type, TSP-2 and TSP-5 knockout mice were compared. To assess the effect of sex hormones, neovascularization was assessed in ovariectomized young female mice. After 14 days, angiogenesis and arteriogenesis were quantified using immunohistochemistry. Laser Doppler was used to confirm > 50% decrease in blood flow. In vitro: TSP-2 inhibited angiogenesis in early passage ECs and disrupted tubules in late passage ECs. TSP-5 prevented early passage tubule disruption. Late passage cells showed greater disruption than early passage. In vivo: TSP-2 was anti-angiogenic in young mice and anti-arteriogenic in old mice; TSP-5 had no effect on angiogenesis but largely negatively impacted arteriogenesis. Females showed reduced angiogenesis/arteriogenesis versus males, aging blunted both responses, and TSP-2/TSP-5 knockouts altered sex- and age-specific ischemic responses. In conclusion, we broadly elucidate the differential effects of TSP-2, TSP-5, sex and aging on neovascularization after inducing limb ischemia.