Adenovirus-mediated overexpression of endothelial nitric oxide synthase (eNOS) induces collateral artery development and substantially increases blood flow after induction of experimental acute hindlimb ischemia. However, the optimal technique of gene delivery for this or any other form of gene therapy in limb ischemia is still unknown. The purpose of this study was to determine the effect of the two most commonly used techniques, intra-arterial and intramuscular injection, on blood flow recovery, collateral artery development, and preservation of muscle mass. We compared intra-arterial injection under vascular isolation, intra-arterial injection under transient vascular occlusion, and intramuscular injection of phosphate buffered saline (PBS) or adenovirus encoding either the eNOS (AdeNOS) or LacZ (AdlacZ) gene after induction of acute hindlimb ischemia. Delivery of AdeNOS by both intra-arterial injection techniques increased eNOS activity (22.30 versus 10.56, P<0.01), blood flow (0.90±0.02 versus 0.69±0.07, P<0.001) and collateral artery development (17.56484 versus 13.74259, P<0.05) more than by intramuscular delivery. Intra-arterial injection under transient vascular occlusion led to better preservation of muscle mass, muscle architecture, and clinical ischemic index, but led to greater transgene expression in distant organs and contralateral limb muscles. Intra-arterial injection of AdeNOS under transient vascular occlusion is the optimal technique to reverse severe hindlimb ischemia in the rat. This is the first systematic comparison of different delivery techniques used in gene therapy of experimental hindlimb ischemia.
This is a preview of subscription content, access via your institution
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Get just this article for as long as you need it
Prices may be subject to local taxes which are calculated during checkout
Schainfeld RM, Isner JM . Critical limb ischemia: nothing to give at the office? Ann Intern Med 1999; 130: 442–444.
Katusic ZS . Therapeutic angiogenesis: new indication for endothelial NO synthase gene transfer. Arterioscler Thromb Vasc Biol 2002; 22: 1254–1255.
Tsutsui M et al. Adventitial expression of recombinant eNOS gene restores NO production in arteries without endothelium. Arterioscler Thromb Vasc Biol 1998; 18: 1231–1241.
Murohara T et al. Nitric oxide synthase modulates angiogenesis in response to tissue ischemia. J Clin Invest 1998; 101: 2567–2578.
Gurjar MV, Sharma RV, Bhalla RC . eNOS gene transfer inhibits smooth muscle cell migration and MMP-2 and MMP-9 activity. Arterioscler Thromb Vasc Biol 1999; 19: 2871–2877.
Radomski MW, Palmer RM, Moncada S . An L-arginine/nitric oxide pathway present in human platelets regulates aggregation. Proc Natl Acad Sci USA 1990; 87: 5193–5197.
Kubes P, Suzuki M, Granger DN . Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci USA 1991; 88: 4651–4655.
Huang PL et al. Hypertension in mice lacking the gene for endothelial nitric oxide synthase. Nature 1995; 377: 239–242.
Kullo IJ et al. Expression and function of recombinant endothelial NO synthase in coronary artery smooth muscle cells. Arterioscler Thromb Vasc Biol 1997; 17: 2405–2412.
Yogo K et al. Different vasculoprotective roles of NO synthase isoforms in vascular lesion formation in mice. Arterioscler Thromb Vasc Biol 2000; 20: E96–E100.
Arnal JF et al. Endothelium-derived nitric oxide and vascular physiology and pathology. Cell Mol Life Sci 1999; 55: 1078–1087.
van Haperen R et al. Reduction of blood pressure, plasma cholesterol, and atherosclerosis by elevated endothelial nitric oxide. J Biol Chem 2002; 277: 48803–48807.
Dimmeler S et al. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 1999; 399: 601–605.
Breslin JW et al. VEGF increases endothelial permeability by separate signaling pathways involving ERK-1/2 and nitric oxide. Am J Physiol Heart Circ Physiol 2003; 284: H92–H100.
Morbidelli L et al. Nitric oxide mediates mitogenic effect of VEGF on coronary venular endothelium. Am J Physiol 1996; 270: H411–H415.
Ziche M et al. Nitric oxide synthase lies downstream from vascular endothelial growth factor-induced but not basic fibroblast growth factor-induced angiogenesis. J Clin Invest 1997; 99: 2625–2634.
Wilcox JN et al. Expression of multiple isoforms of nitric oxide synthase in normal and atherosclerotic vessels. Arterioscler Thromb Vasc Biol 1997; 17: 2479–2488.
Da Silva-Azevedo L, Baum O, Zakrzewicz A, Pries AR . Vascular endothelial growth factor is expressed in endothelial cells isolated from skeletal muscles of nitric oxide synthase knockout mice during prazosin-induced angiogenesis. Biochem Biophys Res Commun 2002; 297: 1270–1276.
Feng Y et al. VEGF induces nuclear translocation of Flk-1/KDR, endothelial nitric oxide synthase, and caveolin-1 in vascular endothelial cells. Biochem Biophys Res Commun 1999; 256: 192–197.
Papapetropoulos A et al. Nitric oxide synthase inhibitors attenuate transforming-growth-factor-beta 1-stimulated capillary organization in vitro. Am J Pathol 1997; 150: 1835–1844.
Silvestre JS et al. Vascular endothelial growth factor-B promotes in vivo angiogenesis. Circ Res 2003; 93: 114–123.
Zhang R et al. Nitric oxide enhances angiogenesis via the synthesis of vascular endothelial growth factor and cGMP after stroke in the rat. Circ Res 2003; 92: 308–313.
Cernadas MR et al. Expression of constitutive and inducible nitric oxide synthases in the vascular wall of young and aging rats. Circ Res 1998; 83: 279–286.
Tamarat R et al. Endothelial nitric oxide synthase lies downstream from angiotensin II-induced angiogenesis in ischemic hindlimb. Hypertension 2002; 39: 830–835.
Fukumura D et al. Predominant role of endothelial nitric oxide synthase in vascular endothelial growth factor-induced angiogenesis and vascular permeability. Proc Natl Acad Sci USA 2001; 98: 2604–2609.
Leibovich SJ et al. Production of angiogenic activity by human monocytes requires an L-arginine/nitric oxide-synthase-dependent effector mechanism. Proc Natl Acad Sci USA 1994; 91: 4190–4194.
Montrucchio G et al. Nitric oxide mediates angiogenesis induced in vivo by platelet-activating factor and tumor necrosis factor-alpha. Am J Pathol 1997; 151: 557–563.
Lloyd PG, Yang HT, Terjung RL . Arteriogenesis and angiogenesis in rat ischemic hindlimb: role of nitric oxide. Am J Physiol Heart Circ Physiol 2001; 281: H2528–H2538.
Smith RS et al. Human endothelial nitric oxide synthase gene delivery promotes angiogenesis in a rat model of hindlimb ischemia. Arterioscler Thromb Vasc Biol 2002; 22: 1279–1285.
Brevetti LS et al. Overexpression of endothelial nitric oxide synthase increases skeletal muscle blood flow and oxygenation in severe rat hind limb ischemia. J Vasc Surg 2003; 38: 820–826.
Lee PC et al. Impaired wound healing and angiogenesis in eNOS-deficient mice. Am J Physiol 1999; 277: H1600–H1608.
Kubis N et al. Decreased arteriolar density in endothelial nitric oxide synthase knockout mice is due to hypertension, not to the constitutive defect in endothelial nitric oxide synthase enzyme. J Hypertens 2002; 20: 273–280.
Aicher A et al. Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells. Nat Med 2003; 9: 1370–1376.
Murohara T et al. Role of endothelial nitric oxide synthase in endothelial cell migration. Arterioscler Thromb Vasc Biol 1999; 19: 1156–1161.
Buckwalter JB et al. Endogenous vascular remodeling in ischemic skeletal muscle: a role for nitric oxide. J Appl Physiol 2003; 94: 935–940.
Welling TH et al. Systemic delivery of the interleukin-1 receptor antagonist protein using a new strategy of direct adenoviral-mediated gene transfer to skeletal muscle capillary endothelium in the isolated rat hindlimb. Hum Gene Ther 1996; 7: 1795–1802.
Paek R et al. Correlation of a simple direct measurement of muscle pO(2) to a clinical ischemia index and histology in a rat model of chronic severe hindlimb ischemia. J Vasc Surg 2002; 36: 172–179.
Rios CD et al. Adenovirus-mediated gene transfer to normal and atherosclerotic arteries. A novel approach. Arterioscler Thromb Vasc Biol 1995; 15: 2241–2245.
Tsurumi Y et al. Direct intramuscular gene transfer of naked DNA encoding vascular endothelial growth factor augments collateral development and tissue perfusion. Circulation 1996; 94: 3281–3290.
Majesky MW . A little VEGF goes a long way. Therapeutic angiogenesis by direct injection of vascular endothelial growth factor-encoding plasmid DNA. Circulation 1996; 94: 3062–3064.
Asahara T et al. Synergistic effect of vascular endothelial growth factor and basic fibroblast growth factor on angiogenesis in vivo. Circulation 1995; 92: II365–II371.
Nabel EG . Gene therapy for cardiovascular disease. Circulation 1995; 91: 541–548.
Pu LQ et al. Enhanced revascularization of the ischemic limb by angiogenic therapy. Circulation 1993; 88: 208–215.
Vassalli G et al. A mouse model of arterial gene transfer: antigen-specific immunity is a minor determinant of the early loss of adenovirus-mediated transgene expression. Circ Res 1999; 85: e25–e32.
Taniyama Y et al. Therapeutic angiogenesis induced by human hepatocyte growth factor gene in rat and rabbit hindlimb ischemia models: preclinical study for treatment of peripheral arterial disease. Gene Therapy 2001; 8: 181–189.
Cho WK et al. Modulation of Starling forces and muscle fiber maturity permits adenovirus-mediated gene transfer to adult dystrophic (mdx) mice by the intravascular route. Hum Gene Ther 2000; 11: 701–714.
Vajanto I et al. Evaluation of angiogenesis and side effects in ischemic rabbit hindlimbs after intramuscular injection of adenoviral vectors encoding VEGF and LacZ. J Gene Med 2002; 4: 371–380.
Isner JM et al. Clinical evidence of angiogenesis after arterial gene transfer of phVEGF165 in patient with ischaemic limb. Lancet 1996; 348: 370–374.
Brevetti LS et al. Administration of adenoviral vectors induces gangrene in acutely ischemic rat hindlimbs: role of capsid protein-induced inflammation. J Vasc Surg 2001; 34: 489–496.
Ooboshi H et al. Altered vascular function after adenovirus-mediated overexpression of endothelial nitric oxide synthase. Am J Physiol 1997; 273: H265–H270.
We express our appreciation to Rickmer Braren, YungHae Kim, Yuankai Lin, Yuki Sakai, and Ari Hoffman for their excellent assistance. This work was supported by the National Institutes of Health Grant HL68042 (GLT), HL75353 (LMM) and Pacific Vascular Research Foundation (LMM).
Rights and permissions
About this article
Cite this article
Yan, J., Tang, G., Wang, R. et al. Optimization of adenovirus-mediated endothelial nitric oxide synthase delivery in rat hindlimb ischemia. Gene Ther 12, 1640–1650 (2005). https://doi.org/10.1038/sj.gt.3302563
- endothelial nitric oxide synthase
- intra-arterial injection with tourniquet
- intra-arterial injection without tourniquet
- intramuscular injection
- limb ischemia