New Indication for Endothelial NO Synthase Gene Transfer
Evidence continues to accumulate on the importance of NO in angiogenesis.1–7⇓⇓⇓⇓⇓⇓ A number of angiogenic substances, including vascular endothelial growth factor (VEGF), stimulate production of NO in endothelial cells.8,9⇓ In vivo biosynthesis of NO is essential for angiogenesis induced by tissue ischemia.3 Angiogenesis is severely impaired in ischemic hindlimb of endothelial NO synthase (eNOS)-deficient mice.3,10⇓ This impairment is not corrected by administration of VEGF, strongly suggesting that NO is downstream signal for angiogenic effect of VEGF. Indeed, in vascular endothelial cells, VEGF increases eNOS enzymatic activity via activation of protein kinase Akt and subsequent phosphorylation of eNOS.11,12⇓ More recent study demonstrated that 3-hydroxyl-3-methyl coenzyme A (HMG-CoA) reductase inhibitor simvastatin also promotes angiogenesis by activation of protein kinase Akt.13 This effect seems to be mediated by stimulation of eNOS enzymatic activity. The importance of protein kinase Akt in regulation of NO production in vivo was demonstrated by adenovirus-mediated delivery of active Akt into the vascular wall. Overexpression of Akt increased resting blood flow, whereas expression of dominant negative Akt inhibited endothelium-dependent relaxation mediated by NO.14 Thus, phosphorylation of eNOS by protein kinase Akt seems to be a major molecular mechanism underlying the angiogenic effect of VGEF and statins.
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Structural adaptation of the vascular tree in response to increased shear stress is of fundamental importance for normal function of cardiovascular system. For instance, exercise training increases cross-sectional area of coronary arteries and stimulates angiogenesis in the heart.15 This adaptive response appears to be designed to maintain normal shear stress in coronary circulation. Increasing capillary shear stress by vasodilatation of resistance arteries due to chronic administration of the α1-adrenergic antagonist prazosin stimulates expression of VGEF and angiogenesis in skeletal muscle capillaries.16 It is unclear whether this angiogenic effect is specific for α1-adrenergic blockade; however, this observation suggests that pharmacologically induced vasodilatation and subsequent increase in blood flow may stimulate angiogenesis. It is of major interest that shear stress activates protein kinase Akt leading to phosphorylation of eNOS and production of NO in endothelial cells.11,12⇓ Protein kinase Akt and NO are apparently key molecules responsible for adaptation to high shear stress and structural remodeling of vascular tree.
Loss of NO biological activity and/or biosynthesis is a central mechanism responsible for pathogenesis of vascular endothelial dysfunction.17 Restoration of normal NO levels in diseased arteries is a major therapeutic goal and could be achieved by supplementation with exogenous NO or by strategies designed to increase concentration of endogenous NO, including exercise, l-arginine, tetrahydrobiopterin, antioxidants, statins, angiotensin-converting enzyme inhibitors, and estrogen replacement. During the past decade, gene transfer technology emerged as a new approach in restoration of NO production. All three isoforms of NOS, endothelial (eNOS), neuronal (nNOS), and inducible (iNOS), have been patented and tested for potential therapeutic value in animal models of vascular disaeses.18–20⇓⇓ In 2001, the first Phase I NOS clinical trial was initiated. Plasmid based formulation of iNOS is being tested for treatment of coronary artery restenosis after balloon angioplasty.
Numerous preclinical studies of NOS isoforms suggest that wide variety of vascular diseases (eg, atherosclerosis, hypertension, vasospasm, diabetic vascular disease, impotence) could be prevented or treated with NOS gene therapy.19 The study by Smith and colleagues21 published in this issue of Arteriosclerosis Thrombosis and Vascular Biology provided the first experimental evidence that recombinant eNOS may stimulate therapeutic angiogenesis. The investigators used rat model of hindlimb ischemia and delivered eNOS or luciferase adenovirus into adductor skeletal muscle. Four weeks later, blood flow in ischemic limb was almost normalized in rats treated with recombinant eNOS but remained significantly lower in rats treated with vehicle or adenovirus encoding luciferase. eNOS gene delivery increased the number of capillaries, strongly suggesting that high local concentration of NO may stimulate angiogenesis in skeletal muscle.
The mechanisms by which NO stimulates angiogenesis are not completely understood. Overexpression of eNOS in the arterial wall causes vasodilatation and increase in local blood flow.19 These hemodynamic changes may lead to adaptive angiogenic response and explain findings reported in the study by Smith and colleagues.21 It would be of interest to determine whether adenovirus-mediated gene delivery of a vasodilator gene other than eNOS (eg, prostacyclin, natriuretic peptides, calcitonin gene-related protein) can also stimulate angiogenesis. These experiments will certainly help determine if the effect of recombinant eNOS is specific or could it be mimicked by other vasodilators. Besides vasodilatation, increased local concentration of NO may stimulate proliferation and migration of endothelial cells.1,2,22,23⇓⇓⇓ Both proliferation and migration of endothelial cells are essential for formation of new microvessels or larger arteries. Obviously, further investigation is needed to completely characterize molecular mechanisms underlying angiogenesis in response to overexpression of eNOS.
If indeed increased local concentration of NO is sufficient stimulus for angiogenesis, recombinant eNOS may have therapeutic advantage over VGEF. NO is essential for angiogenic effect of VGEF; however, biological availability or biosynthesis of NO is impaired in most of the patients with vascular disease and advanced age. This impairment may significantly dampen angiogenic effect of VGEF and could explain failures of VGEF gene–based therapy. In a clinical setting, the decision to deliver VGEF instead of eNOS may depend on assessment of endothelial function and endogenous NO production in ischemic tissue. Alternatively, one can try to deliver both VGEF and eNOS. Whether therapeutic angiogenesis achieved with this combination is superior to a single gene delivery remains to be determined.
Ability of “uncoupled eNOS” (in the presence of suboptimal concentrations of l-arginine or tetrahydrobiopterin) to generate superoxide anions instead of NO is a potentially major drawback in gene therapy application of recombinant eNOS.17 In the present study by Smith and colleagues,21 eNOS was delivered to ischemic skeletal muscle of otherwise healthy rats. Levels of cyclic GMP, an indirect measure of NO production, were increased three days after eNOS gene delivery. Based on presented findings, it appears that l-arginine or tetrahydrobiopterin levels in skeletal muscle are not limiting for NO production. Whether the same holds true four weeks after eNOS gene delivery is unknown. Successful clinical application of eNOS gene therapy will depend on the presence of optimal conditions required for proper eNOS enzymatic activity in target tissue. Studies on aged animals24 or animal models of vascular diseases may provide additional useful information before eNOS gene–based therapeutic angiogenesis could be tested in clinical arena.
This work was supported in part by National Heart, Lung, and Blood Institute grants HL-53524, HL-58080, and HL-066958, by National Institute for Neurological Disorders and Stroke grant NS-37491, by the American Heart Association Bugher Foundation Award for the Investigation of Stroke, and by the Mayo Foundation. The secretarial assistance of Janet Beckman is gratefully acknowledged.
- ↵Ziche M, Morbidelli L, Masini E, Amerini S, Granger HJ, Maggi CA, Geppetti P, Ledda F. Nitric oxide mediated angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P. J Clin Invest. 1994; 94: 2036–2044.
- ↵Murohara T, Horowitz JR, Silver M, Tsurumi Y, Chen D, Sullivan A, Isner JM. Vascular endothelial growth factor/vascular permeability factor enhances vascular permeability via nitric oxide and prostacyclin. Circulation. 1998; 97: 99–107.
- ↵Matsunaga T, Warltier DC, Weihrauch DW, Moniz M, Tessmer J, Chilian WM. Ischemia-induced coronary collateral growth is dependent on vascular endothelial growth factor and nitric oxide. Circulation. 2000; 102: 3098–3103.
- ↵Fukumura D, Gohongi T, Kadambi A, Izumi Y, Ang J, Yun CO, Buerk DG, Huang PL, Jain RK. Predominant role of endothelial nitric oxide synthase in vascular endothelial growth factor-induced angiogenesis and vascular permeability. Proc Nat Acad Sci U S A. 2001; 98: 2604–2609.
- ↵Parenti A, Morbidelli L, Ledda F, Granger HJ, Ziche M. The bradykinin/B1 receptor promotes angiogenesis by up-regulation of endogenous FGF-2 in endothelium via the nitric oxide synthase pathway. FASEB J. 2001; 15: 1487–1489.
- ↵Rikitake Y, Hirata K, Kawashima S, Ozaki M, Takahashi T, Ogawa W, Inoue N, Yokoyama M. Involvement of endothelial nitric oxide in sphingosine-1-phosphate-induced angiogenesis. Arterioscler Thromb Vasc Biol. 2002; 22: 108–114.
- ↵van der Zee R, Murohara T, Luo Z, Zollmann F, Passeri J, Lekutat C, Isner JM. Vascular endothelial growth factor/vascular permeability factor augments nitric oxide release from quiescent rabbit and human vascular endothelium. Circulation. 1997; 95: 1030–1037.
- ↵Hood JD, Meininger CJ, Ziche M, Granger HJ. VEGF upregulates ecNOS message, protein, and NO production in human endothelial cells. Am J Physiol. 1998; 274: H1054–H1058.
- ↵Qian HS, Liu P, Kauser K, Rubanyi GM. Nitric oxide deficiency leads to impaired angiogenesis and severe dysfunction of microcirculation in a mouse hind limb ischemia model.In: Proceedings of the 7th World Congress of Microcirculation. Sydney, Australia: Monduzzi Editore; 2001: 525–529.
- ↵Laughlin MH, McAllister RM. Exercise training-induced coronary vascular adaptation. J Appl Physiol. 1992; 73: 2209–2225.
- ↵Katusic ZS. Vascular endothelial dysfunction: does tetrahydrobiopterin play a role? Am J Physiol Heart Circ Physiol. 2001; 281: H981–H986.
- ↵Chen AFY, O’Brien T, Katusic ZS. Functional influence of gene transfer of recombinant nitric oxide synthase to cardiovascular system.In: Ignarro L, ed. Nitric Oxide Biology and Pathobiology. San Diego, Calif: Academic Press; 2000: 525–545.
- ↵von der Leyen HE, Dzau VJ. Therapeutic potential of nitric oxide synthase gene manipulation. Circulation. 2001; 103: 2760–2765.
- ↵Smith RS, Lin K-F, Agata J, Chao L, Chao J. Human endothelial NO synthase gene delivery promotes angiogenesis in rat model of hindlimb ischemia. Arterioscler Thromb Vasc Biol. 2002: 22; 1279–1285
- ↵Morbidelli L, Chang CH, Douglas JG, Granger HJ, Ledda F, Ziche M. Nitric oxide mediates mitogenic effect of VEGF on coronary venular endothelium. Am J Physiol. 1996; 270: H411–H415.
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- ↵Rivard A, Fabre JE, Silver M, Chen D, Murohara T, Kearney M, Magner M, Asahara T, Isner JM. Age-dependent impairment of angiogenesis. Circulation. 1999; 99: 111–120.