Gene Therapy to Treat Myocardial Ischemic Disease Using Therapeutic Angiogenesis

Similar ideas have been applied to treat coronary artery disease. A human gene therapy trial to treat coronary artery disease using VEGF165 gene has been started by Professor Isner and colleagues (21,22). His group performed intramuscular injection of naked plasmid encoding VEGF gene into ischemic myocardium through mini-operation. Similar to human trials in PAD, transfection of VEGF gene resulted in a marked increase in blood flow and improved clinical symptoms without apparent toxicity (21). More recently, the results from 13 consecutive patients with chronic stable angina have been reported (22). Although all of them had failed conventional therapy (drugs, percutaneous transluminal coronary angioplasty, and/or coronary artery bypass graft), reduction in the size of the defects documented by serial single-photon emission computed tomography imaging was observed after direct myocardial injection of phVEGF165 via a minithoraco-tomy (22).

Gene Therapy And Myocardial Ischemia

Fig. 2. Model of collateral formation induced by vascular endothelial growth factor (VEGF) (A) and hepatocyte growth factor (HGF) (B). HGF stimulated the growth and migration of endothelial cells together with the migration, but not proliferation, of vascular smooth muscle cells (VSMC) through c-met. In contrast, VEGF only stimulated the growth and migration of endothelial cells without the migration or proliferation of VSMC, because of the lack of receptors in VSMC.

Fig. 2. Model of collateral formation induced by vascular endothelial growth factor (VEGF) (A) and hepatocyte growth factor (HGF) (B). HGF stimulated the growth and migration of endothelial cells together with the migration, but not proliferation, of vascular smooth muscle cells (VSMC) through c-met. In contrast, VEGF only stimulated the growth and migration of endothelial cells without the migration or proliferation of VSMC, because of the lack of receptors in VSMC.

These data clearly suggest thatphVEGF165 gene therapy may successfully rescue foci of hibernating myocardium. In addition, the recent report summarized the anesthetic management of 30 patients with class 3 or 4 angina, enrolled in a phase 1 clinical trial of direct myocardial gene transfer of naked DNA-encoding VEGF165, as sole therapy for refractory angina. Twenty-nine of 30 patients experienced reduced angina (56.2 ± 4.1 episodes/ week preoperatively vs 3.8 ± 1.6 postoperatively) and reduced sublingual nitroglycerin consumption (60.1 ± 4.4 tablets/week preoperatively vs 2.9 ± 1.1 postoperatively) (23). Even at 1-yr follow-up, the average number of angina episodes per week and average number of nitroglycerin tablets used per week significantly improved at all measured time points after gene transfer (24). This observation persisted at a 12-mo follow-up, when the average number of anginal episodes was 10 ± 19 and average weekly nitroglycerin tablet usage was 3 ± 8 (p < 0.05 vs baseline for both). Following this success, gene therapy using VEGF121 gene was performed by intramuscular injection of adenoviral vector (25). A phase I study using adenovirus-mediated transfection of VEGF121 gene demonstrated clinical safety (25). It is noteworthy that no evidence of systemic or cardiac-related adverse events related to vector administration was observed up to 6 mo after therapy (26). Intracoronary gene transfer of VEGF165 resulted in a significant increase in myocardial perfusion, although no differences in clinical restenosis rate or minimal lumen diameter were present after the 6-mo follow-up (27). More recently, intracoronary infusion of adenovirus encoding FGF gene was performed in a multicenter trial as phase I/IIa. The report documented that intracoronary infusion of FGF gene improved cardiac dysfunction without severe toxicity (28). Seventy-nine patients with chronic stable angina Canadian Cardiovascular Society class 2 or 3 underwent double-blind randomization (1:3) to placebo (n = 19) or Ad5-FGF4 (n = 60). Excitingly, a protocol-specified, subgroup analysis showed the greatest improvement in patients with baseline exercise tolerance test < 10 min (1.6 vs 0.6 min, p = 0.01, n = 50). In addition, the report documented that treatment of 52 patients with stable angina and reversible ischemia with FGF4 adenoviral injection resulted in a significant reduction of ischemic defect size (29). Currently, the phase IIb/ III trials using adenoviral delivery of FGF4 are now underway.

In addition to these angiogenic growth factors, overexpression of HGF was also reported to stimulate angiogenesis and collateral formation in a rat myocardial infarction model (30). Moreover, it was reported that intramuscular injection of HGF gene into the ischemic myocardium resulted in a significant increase in blood flow and prevention of cardiac dysfunction in a canine model (31). The molecular mechanisms of the angiogenic activity of HGF seem to be largely dependent on the ets pathway (an essential transcription factor for angiogenesis), because members of the ets family play important roles in regulating gene expression in response to the multiple developmental and mitogenic signals. The ets family of transcription factors has a DNA-binding domain in common 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 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 the ets family takes part in regulating angiogenesis by controlling the transcription of these genes, whose activity is necessary for the migration of endothelial cells from pre-existing capillaries. Our previous study demonstrated that HGF upregulated ets activity and ets-1 protein in a myocardial infarction model (30). In addition, exogenously expressed HGF also stimulated endogenous HGF expression through induction of ets activity (32) (Fig. 3), because the promoter region of the HGF gene contains a number of putative regulatory elements, such as a B cell- and a macrophage-specific transcription factor binding site (PU.1/ETS),

Fig. 3. Molecular mechanisms of angiogenesis induced by hepatocyte growth factor (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.

as well as an interleukin (IL)-6 response element (IL-6 RE), a transforming growth factor (TGF)-P inhibitory element, and a cAMP response element (33). Severe ischemic heart disease may also be curable using therapeutic angiogenesis by HGF, as a result of the autoinduction of the endogenous HGF system.

Recently, an antifibrotic action of HGF has been identified, as HGF inhibited collagen synthesis through TGF-P and stimulated collagen degradation through upregulation of matrix metalloproteinase (MMP-1) and urinary plasminogen activator (uPA) (34). Although the mechanisms through which HGF inhibited TGF-P synthesis are not clear, HGF stimulated various metallo-proteases, such as MMP-1 through induction of ets-1 activity (35). Prevention of fibrosis by HGF was confirmed by previous studies in which administration of human rHGF or gene transfer of human HGF prevented and/or regressed fibrosis in liver and pulmonary injury models (36,37). Thus, HGF may also provide a new therapeutic strategy to treat fibrotic cardiovascular disease, e.g., cardiomyopathy. Our group has also applied to start a human gene therapy protocol using intracardiac muscular injection of HGF plasmid DNA through surgical operation. Overall, the treatment for coronary artery disease may also be curable using therapeutic angiogenesis by gene therapy.

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