Type-2 diabetic Lep^db/db mice show a defective microvascular phenotype under basal conditions and an impaired response to angiogenesis gene therapy in the setting of limb ischemia

Diabetes mellitus is associated with macro- and micro-angiopathy, leading to increased risk of peripheral ischemia. In the present study, we have characterized the microvascular phenotype at the level of limb muscles and the spontaneous angiogenesis response to surgically-induced unilateral limb ischemia in a murine model of type-2 diabetes, the obese C57BL/KsOlaHsd-Lepr^db/db mice (Lepr^db/db), and in non-diabetic heterozygous Lepr^db/+. Wild type C57BL mice (WT) were used as controls. The basal microvascular phenotype was determined in mice aged 3 or 5 months, while the response to limb ischemia was studied only in 5-month old mice. Moreover, in 5-month old ischemic Lepr^db/db and Lepr^db/+, we have tested the therapeutic potential of local angiogenesis gene therapy with human tissue kallikrein (hTK) or constitutively-activated Akt kinase (Myr-Akt). We found that in the muscles of 3- or 5-month old Lepr^db/db, apoptosis of endothelial cells was enhanced and the densities of capillary and arteriole were reduced. Arterioles of Lepr^db/db showed hypertrophic remodelling and, occasionally, lumen occlusion. Following ischemia, Lepr^db/db showed a defective reparative angiogenesis in ischemic muscle, delayed blood flow recovery, and worsened clinical outcome as compared with controls. Five-month old Lepr^db/+ displayed an increase in endothelial cell apoptosis under basal conditions, while capillary and arteriole densities were normal. Lepr^db/+ mounted a proper reparative angiogenesis response to limb ischemia and regained blood flow to the ischemic limb, regularly. Local gene therapy with hTK or Myr-Akt induced angiogenesis in ischemic muscles of Lepr^db/+ and Lepr^db/db. However, in the Lepr^db/db neither gene therapy approach improved the blood flow recovery and the clinical outcome from ischemia. In contrast, either hTK or Myr-Akt gene transfer improved the post-ischemic recovery of Lepr^db/+. Type-2 diabetes has a negative impact on the basal microvascular phenotype and severely impairs post-ischemic recovery of limb muscles. Gene therapy-induced stimulation of neovascularization might not suffice as a sole therapeutic strategy to combat type-2 diabetes-related vascular complications. In type-2 diabetic patients, therapeutic angiogenesis may need to be further optimized before being recommended for clinical applications.