Lucy S. Brevetti

Adeno-associated viral vector-mediated gene transfer of VEGF normalizes skeletal muscle oxygen tension and induces arteriogenesis in ischemic rat hindlimb

Critical limb ischemia is an important clinical problem that often leads to disability and limb loss. Vascular endothelial growth factor (VEGF), delivered either as recombinant protein or as gene therapy, has been shown to promote both collateral artery formation (arteriogenesis) and capillary angiogenesis in animal models of hindlimb ischemia. However, none of the previous studies has demonstrated an improvement in tissue hypoxia, the condition that drives the molecular response to ischemia. Furthermore, the optimal vector and route of gene delivery have not been determined. Recently, adeno-associated viral (AAV) vectors, which efficiently transduce skeletal muscle and produce sustained transgene expression, have been used as gene therapy vectors. We asked whether an intra-arterial injection of AAV-VEGF(165) normalizes muscle oxygen tension by increasing skeletal muscle oxygen tension, and promotes arteriogenesis and angiogenesis in a rat model of severe hindlimb ischemia. We found that AAV-VEGF treatment normalized muscle oxygen tension in the ischemic limb. In contrast, vehicle and AAV-lacZ-treated limbs remained ischemic. Collateral arteries were more numerous in AAV-VEGF-treated rats, but, surprisingly, capillaries were not. We conclude that intra-arterial AAV-mediated gene transfer of AAV-VEGF(165) normalizes muscle oxygen tension and leads to arteriogenesis in rats with severe hindlimb ischemia.

Overexpression of endothelial nitric oxide synthase increases skeletal muscle blood flow and oxygenation in severe rat hind limb ischemia

OBJECTIVE Although nitric oxide (NO) has a critical role in angiogenesis, the therapeutic potential of NO synthase overexpression in severe ischemia remains undefined. We tested the hypothesis that overexpression of endothelial NO synthase (eNOS) would improve tissue perfusion in severe hind limb ischemia. METHODS Severe hind limb ischemia was induced in 122 adult male Sprague-Dawley rats. Ten days after the induction of hind limb ischemia, vascular isolation and intraarterial delivery of an adenoviral vector encoding eNOS (AdeNOS), a control adenoviral vector (AdE1), or phosphate-buffered saline solution (PBS) was performed. Skeletal muscle blood flow, muscle oxygen tension, angiography, and immunohistochemistry for capillary counts were measured. RESULTS Gene transfer of AdeNOS increased eNOS protein expression and enzyme activity. Two weeks after gene transfer, skeletal muscle blood flow was fourfold higher in eNOS-transduced than in AdE1-transduced or PBS treated rats and was similar to exercise-induced maximal flow in nonischemic muscle. eNOS overexpression increased muscle oxygen tension in a titer-dependent fashion. This increase persisted 1 month after transduction, even though eNOS enzyme activity had declined to normal levels. Angiography and capillary counts showed that eNOS overexpression increased the size and number of collateral arteries, but did not significantly increase the capillary-muscle fiber ratio. CONCLUSIONS eNOS overexpression in an ischemic rat hind limb significantly increased skeletal muscle blood flow, muscle oxygen tension, and collateral arteries (arteriogenesis). Furthermore, eNOS overexpression did not result in capillary angiogenesis above control levels. These studies demonstrate the potential for eNOS overexpression as treatment for severe limb ischemia in human beings.

Therapeutic angiogenesis for critical limb ischemia: invited commentary.

Lower extremity arterial occlusive disease results in tissue ischemia of the legs and is relatively common in the elderly. Clinically, it may be asymptomatic, cause muscle pain during exercise, or progress to a severe degree of ischemia that may result in limb loss. Although bypass surgery and angioplasty have increased the rate of limb salvage in these patients, amputation of the affected limb remains a common outcome for many patients. Therapeutic angiogenesis is the administration of angiogenic factors, or genes encoding these factors, to promote neovascularization and thereby increase blood flow to the ischemic leg. We have developed an animal model of hindlimb ischemia in which to study therapeutic angiogenesis. We chose nitric oxide as the angiogenic factor for our experiments because of its ability to induce angiogenesis, vasodilation, and inhibit inflammation. In this review, we will discuss our experience with our model of hindlimb ischemia, as well as discuss our results of gene therapy for therapeutic angiogenesis using nitric oxide.

Oral administration of L-Arginine decreases necrosis of the epigastric skin flap in the rat

Background:We investigated the effect of prolonged oral arginine administration on tissue necrosis and perfusion in the rat skin flap. Methods:Twenty-five Sprague-Dawley rats had an 8 × 8-cm epigastric skin flap elevated and were divided in 2 groups, l-Arginine and Control, which respectively received oral 6% l-arginine solution or water for 8 days postoperatively. On postoperative day 8, area of flap necrosis was measured, and the animals were perfused systemically with 15-μm colored fluorescent microspheres before (blue) and after (yellow-green) ligation of the flap pedicle. Results:l-Arginine reduced total flap necrosis (6.53 ± 3.76 cm2 versus 11.91 ± 4.12 cm2; P < 0.01). After pedicle ligation, total flap perfusion remained unchanged in Control but diminished in the l-Arginine group (Control: 0.47 ± 0.23 and 0.42 ± 0.06; P = nonsignificant versus l-Arginine: 0.58 ± 0.29 and 0.27 ± 0.19; P < 0.01). Serum levels of l-arginine were higher in the l-arginine-treated animals (504 ± 154 versus 152 ± 34 μmol/l; P < 0.0001). Conclusions:Postoperative oral administration of l-arginine decreased flap necrosis in the rat epigastric skin flap. Flap perfusion following oral l-arginine was more dependent on the main vascular pedicle.