386. Intra-Arterial Adenoviral Mediated β2Adrenergic Receptor (AR) Gene Delivery Induces Neoangiogenesis in Rat Ischemic Hindlimb

Abstract

Top of pageAbstract We have shown that β2AR gene delivery to the endothelium corrects impaired endothelial vasodilation in hypertensive rats. Endothelium mediates other vascular biology responses, including neoangiogenesis; furthermore, during chronic ischemia there is local adrenergic activation. Based on this background, we tested the hypothesis that increasing the number of endothelial β2AR in ischemic districts would result in neoangiogenesis. We used the rat ischemic hind limb (HL) model in 15 WKY rats, by legating the right common femoral artery. Through means of an incision made distal to the ligature, we injected intraarterially a solution containing 5∗109PFU of recombinant adenovirus encoding for the human β2 AR (ADβ2AR, n=8), the naturally occurring, signaling challenged ILE164 variant of the human β2AR (ADβ2-164AR, n=5) or an empty virus (ADEV, n=7) and incubate for 15 minutes while venous blood return from the HL was blocked. After 15 days, we performed selective HL digital angiographies in live anaesthetized rats, with a computer controlled injection during i.a. nitroglycerin (20 μg i.a.) to induce maximal vasodilation. We estimated blood flow (BF) measuring the number of angiography frames needed by the contrast medium to reach the origin of the dorsal paw artery from the injection (TFC). HL perfusion was also assessed by dyed beads dilution, injecting in the abdominal aorta 108 dyed beads, and measuring the release of dye in the digested gastrocnemious samples of the ischemic HL. As reference we used the contralateral non ischemic muscle. We also counted the capillary (<10μ diameter) per muscle fiber density (C/F) on tibial muscle sections stained with isolectin B4. The non ischemic HL of ADβ2AR, ADβ2-164AR and ADEV rats showed similar TFCs (4.9±0.18, 5.3±0.65 and 5.1±0.2 TFC, ns). ADβ2AR, ADβ2-164AR and ADEV ischemic HL presented greater TFCs compared to the non ischemic HL but the ADβ2AR TFCs were significantly reduced to ADEV and ADβ2-164AR (11±2.5; 23±6.8 and 30±0.1 respectively, p<0.001). BF estimated by dyed microspheres dilution was higher in the ADβ2AR vs the ADEV and ADβ2-164AR ischemic HL (fold of non ischemic control: 1.9±0.6, 0.5±0.14, and 0.45±1 respectively, p<0.05). Finally, capillary density was higher in ADβ2AR compared to ADβ2-164AR and ADEV (0.82±0.04, 0.56±0.03 and vs 0.53±0.03 C/F respectively, p<0.001). In conclusion, our data show that ADβ2AR increases blood flow in ischemic HL, suggest a role for endothelial β2AR in neoangiogenesi, which might be impaired in patients harbouring the ILE164 variant of the b2AR gene.