5. HGF Gene Therapy for Myocardial Ischemia

In addition to stimulating peripheral angiogenesis, overexpression of HGF in the myocardium was also reported to stimulate angiogenesis and collateral formation in a rat myocardial infarction model.33 Moreover, intramuscular injection of the HGF plasmid into the ischemic myocardium resulted in a significant increase in blood flow and prevention of cardiac dysfunction in a canine model.34 We also injected human HGF plasmid DNA at doses of 0.4 or 4 mg into ischemic myocardium of pigs induced by ameroid constrictor using the NOGA™ system. At one month after injection, the ischemic area was significantly reduced in the 4 mg HGF group, accompanied by a significant increase in the capillary density and regional myocardial perfusion in the ischemic area as compared to the control group. These favorable outcomes suggest potential utility of HGF gene transfer for treatment of patients with ischemic heart disease.

The molecular mechanisms of the angiogenic activity of HGF seem to be largely dependent on the Ets pathway. Members of Ets family of transcription factors share a DNA-binding domain that binds to a core GGA(A/T) DNA sequence. In situ hybridization studies have revealed that the proto-oncogene c-Ets-1 is expressed in endothelial cells at the start of blood vessel formation, under both normal and pathological conditions. Thus, the Ets family may activate the transcription of genes encoding collagenase-1, stromelysine-1 and urokinase plasminogen activator, which are proteases involved in extracellular matrix degradation. It is believed that activation of these proteases is a major role played by Ets transcription factors in the regulation of angiogenesis. Our previous study demonstrated that HGF upregulated Ets activity and Ets-1 protein expression in a myocardial infarction model.33 In addition, exogenously expressed HGF also stimulated endogenous HGF expression through induction of Ets activity (Fig. 5), since the promoter region of the HGF gene contains an Ets binding site.6

More recently, an anti-fibrotic action of HGF has been identified, as HGF inhibits collagen synthesis via downregulation of TGF-β and stimulates collagen degradation via upregulation of MMP-1 and uPA.35 Although the mechanisms of HGF inhibition of TGF-β synthesis are

Fig. 5. Molecular mechanisms of angiogenesis induced by HGF through Ets-1. HGF stimulated various actions on collateral formation through Ets-1, revealing that HGF plays a pivotal role as a master gene in the cascade of angiogenesis.

not clear, HGF stimulates, as already mentioned, various metalloproteases such as MMP-1 through induction of Ets-1 activity.36 Prevention of fibrosis by HGF was confirmed by studies in which administration of human rHGF protein or gene transfer of human HGF prevented and/or induced regression of fibrosis in liver and pulmonary injury models.37,38 Similar findings were obtained in the myocardium, where overexpression of HGF reduced fibrosis in a cardiomyopathic hamster model.36 In addition, our recent study demonstrated a significant decrease in a fibrotic area by HGF in porcine chronic angina model, associated with a significant decrease in collagen I, III and TGF-β synthesis as compared to the control (unpublished data). Thus, HGF may also provide a new therapeutic opportunity to treat fibrotic cardiovascular diseases such as cardiomyopathy. Of importance, decrease in the ventricular fibrillation (VF) frequency and increased VF threshold were reported after HGF gene therapy.39 These findings suggest that HGF gene therapy may have an anti-arrhythmic effect after myocardial ischemia. Overall, HGF is a very unique angiogenic growth factor which has a number of cardioprotective effects including inhibition of fibrosis and apoptosis and suppression of arrhythmias. Currently, a phase I study using human HGF plasmid DNA in USA is ongoing to test the validity of this concept. Overall, coronary artery disease may also be curable using therapeutic angiogenesis by gene therapy.

6. HGF Gene Therapy for Restenosis After Angioplasty

Another important cardiovascular disease potentially amenable to gene therapy is post-angioplasty restenosis. Intimal hyperplasia develops in a large part as a result of vascular smooth muscle cell (VSMC) proliferation and migration induced by a complex interaction of multiple growth factors that are activated by vascular “injury”. It has been hypothesized that rapid regeneration of endothelial cells without replication of VSMC may also modulate vascular growth, because multiple anti-proliferative endothelium-derived substances (PGI2, NO, CNP) are secreted from endothelial cells. This concept was first tested by overexpression of the VEGF-165 gene.40 Using a similar strategy, we also reported preclinical experiments in which overexpression of the HGF gene in balloon-injured arteries can accelerate re-endothelialization, thereby attenuating intimal hyperplasia.41 In this study, we found that re-endothelialized balloon-injured arteries showed improvement in endothelial dysfunction induced by balloon angioplasty.41 HGF also has a strong anti-apoptotic effect in endothelial cells.42−47 Of interest, HGF can abrogate the decrease in DNA synthesis and cell death of endothelial cells mediated by serum-free treatment.42 An additive effect of HGF and FGF2 was observed in the prevention of endothelial cell death, equivalent to the effect of serum treatment.47 Thus HGF can be classified as a new member of a class of growth factors, such as FGFs and VEGFs, with anti-cell death activity. Interestingly, high D-glucose- induced aortic endothelial cell death was also attenuated by recombinant HGF.43 The mechanisms by which HGF prevented endothelial cell death mediated by these conditions are still unclear. One possibility is HGF upregulation of an anti-apoptotic factor, bcl-2, and inhibition of the translocation of bax from the cytosol to the mitochondria membrane in human endothelial cells.44,46 HGF is known to stimulate phosphatidylinositol-3 -kinase (PI3K), protein tyrosine phosphatase-2, phospholipase C-r, pp60c-src and grb2/hSos1.48−50 Moreover, HGF also stimulates the Rhoand Ras-mediated signal transduction pathways51 as well as ERK and Akt signaling that play pivotal roles in the mitogenic and anti-apoptosis actions of HGF in endothelial cells.52 A unique feature of the HGF signal transduction system is re-phosphorylation

378 R. Morishita & T. Ogihara

of ERK by HGF.52 This re-phosphorylation of ERK may be due to auto-induction of endogenous HGF, given previously demonstrated ability of exogenous HGF to induce endogenous HGF expression.53 Indeed, addition of neutralizing anti-HGF antibody after HGF stimulation attenuated the re-phosphorylation of ERK.52 This unique property of the HGF signal transduction system is involved in the potent mitogenic activity and anti-apoptotic action of HGF. Further studies are needed to clarify the utility of gene therapy to treat restenosis after angioplasty.

7.Next Five Years Perspective — Future Direction of HGF Therapy

To potentially improve the efficiency of angiogenic therapy with HGF we tested a new strategy, combining transfection of HGF and prostacyclin synthase genes.54 Prostacyclin synthase was chosen because of the utility of vasodilator agents such as prostaglandins and phosphodiesterase type III inhibitors to treat patients with peripheral artery disease. A combination of angiogenesis induced by HGF and vasodilation of newly generated blood vessels induced by prostacyclin may enhance blood flow recovery and maintain new vessel formation. The combined therapy resulted in an improvement in peripheral neuropathy, characterized by significant slowing of nerve conduction velocity and was more effective than a single-gene transfection.

Peripheral neuropathy is common and ultimately accounts for significant morbidity in diabetes. However, there are currently no therapeutic options for patients with diabetic neuropathy. Earlier work using animal models of hindlimb ischemia also documented favorable effects of VEGF gene transfer on ischemic peripheral neuropathy.55 It is intriguing to note that the neurological and neurophysiological findings in a prospective study of patients undergoing phVEGF165 gene transfer for critical limb ischemia showed clinical improvement in electrophysiological measurements in diabetic patients. Although the model used in this study was more severe compared with the previous work, co-transfection of HGF and prostacyclin synthase genes was able to improve the electrophysiological measures. As HGF has been reported