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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 5, 2003 as doi:10.1096/fj.02-0754fje. |
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,2
* Division of Gene Therapy Science,
Department of Geriatric Medicine,
Division of Biochemistry, Department of Oncology, Biomedical Research Center, and
Department of Pharmacology, National Cardiovascular Research Center, Osaka University Medical School, Suita 565, Japan
2Correspondence: Division of Gene Therapy Science, Osaka University Medical School, 2–2 Yamada-Oka, Suita 565-0871, Japan. E-mail: morishit{at}geriat.med.osaka-u.ac.jp
SPECIFIC AIM
The current therapeutic angiogenesis strategy to treat ischemic disease using angiogenic growth factors has been limited to use of a single gene. To enhance the angiogenic activity, we examined the angiogenic activity of cotransfection of genes of an angiogenic growth factor, hepatocyte growth factor (HGF), and a vasodilator substance, prostacyclin synthase.
PRINCIPAL FINDINGS
1. Angiogenesis induced by intramuscular (i.m.) injection of HGF or prostacyclin synthase plasmid in mouse model
Accompanied by a significant increase in human immunoreactive HGF in the hind limb transfected with human HGF vector, injection of human HGF vector into the ischemic hind limb resulted in a significant increase in blood flow from 2 wk after transfection as assessed by laser Doppler imaging (Fig. 1
, P<0.01). Moreover, transfection of human HGF vector significantly increased capillary density in the mouse ischemic hind limb around the injection site compared with the hind limb transfected with control vector at 4 wk after transfection (P<0.01). These results clearly demonstrated that transfection of human HGF vector into the ischemic hind limb induced therapeutic angiogenesis, which could be applied for the treatment of peripheral arterial disease. In contrast, no human HGF could be detected in muscles transfected with control vector at 4 days after transfection (P<0.01). Similarly, human 6 keto PGF1
could be detected in the ischemic hind limb transfected with human prostacyclin synthase, but not control vector (P<0.01). Human HGF protein was increased by cotransfection of the HGF and prostacyclin synthase genes. Injection of human prostacyclin synthase vector resulted in a weak but significant increase in capillary density at 4 wk after transfection. The increase in blood flow induced by the prostacyclin synthase gene was similar to that induced by the HGF gene at 2 wk after transfection (Fig. 1
, P<0.01). Cotransfection of the HGF and prostacyclin synthase genes demonstrated a further increase in blood flow compared with single gene transfection of HGF or prostacyclin synthase alone at 2 wk after transfection (Fig. 1
, P<0.01). Similarly, capillary density was most increased in mice transfected with the HGF and prostacyclin synthase genes compared with the HGF vector (P<0.01).
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2. Angiogenesis induced by i.m. injection of HGF or prostacyclin synthase plasmid in rabbit model
Similar to the mouse model, human immunoreactive HGF was observed in rabbits transfected with human HGF vector, but not control vector at 4 days after transfection (HGF: 2.1±0.4 ng/g tissue vs. control; not detected; P<0.01). Injection (i.m.) of HGF plasmid into the ischemic limb on day 10 after surgery produced significant augmentation of collateral vessel development as assessed by angiography on day 30 in the ischemia model, as shown in Fig. 2
(P<0.01). Serial angiograms revealed progressive linear extension of collateral arteries from the origin stem artery to the distal point of the reconstituted parent vessel in HGF-treated animals. Consistent with induction of angiogenesis, a significant increase in blood flow as assessed by a Doppler flow wire under basal conditions was observed in rats transfected with HGF plasmid compared with rats transfected with control vector (P<0.01). A significant increase in the blood pressure ratio of ischemic limb to normal limb was also observed in rabbits transfected with HGF plasmid compared with control vector (P<0.01). Cotransfection of the HGF and prostacyclin synthase genes demonstrated a further increase in angiographic scores compared with single gene transfection of the HGF or prostacyclin synthase gene alone (Fig. 2
, P<0.01). Similarly, capillary density was most increased in rabbits transfected with the HGF and prostacyclin synthase genes compared with HGF vector (P<0.01).
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3. Attenuation of decreased in neuronal velocity by i.m. injection of HGF and prostacyclin synthase plasmids in mouse diabetic ischemic hind limb model
Using diabetes ischemic hind limb animal models, we have investigated the hypothesis that experimental diabetic ischemic neuropathy can be reversed by coadministration of HGF and prostacyclin synthase genes. To answer this question, we used a new diabetic neuropathy model produced by creating hind limb ischemia in diabetic mice. Different from a previous report using a simple diabetes model, the diabetic ischemia model was characterized by severe impairment of nerve conduction velocity. In these ischemic mice with streptozotocin-induced diabetes, consistent with a profound reduction in the number of vessels, severe peripheral neuropathy developed in parallel, characterized by significant slowing of motor and sensory nerve conduction velocity, compared with nondiabetic control animals (P<0.01). In contrast, 4 wk after i.m. gene transfer of the HGF and prostacyclin synthase genes, vascularity and blood flow in the ischemic hind limb of treated animals were similar to those of nondiabetic control mice. In addition, constitutive overexpression of the HGF and prostacyclin synthase genes resulted in restoration of large and small fiber peripheral nerve function. Improvement of nerve function was not accompanied by a change in blood glucose level or body weight, since transfection of the HGF and prostacyclin synthase genes did not alter these parameters (data not shown). These findings suggest the feasibility of a novel treatment strategy for peripheral neuropathy.
CONCLUSION AND SIGNIFICANCE
A novel therapeutic strategy using angiogenic growth factors to expedite and/or augment collateral artery development has recently entered the realm of treatment of ischemic diseases. Although the clinical utility of gene therapy using VEGF (VEGF165, VEGF121, VEGF-2), HGF, FGF, and HIF-1 has been demonstrated, the current clinical trials and animal experiments are limited to use of a single angiogenic growth factor to stimulate angiogenesis. Here, we introduce a new strategy, therapeutic angiogenesis using cotransfection of the HGF and prostacyclin synthase genes, as gene therapy for treatment of patients with critical limb ischemia. The reason we chose prostacyclin synthase was to consider the utility of vasodilator agents such as prostaglandins and phosphodiesterase type III inhibitors to treat human patients with PAD. A combination of angiogenesis induced by HGF and vasodilation of newly generated blood vessels induced by prostacyclin would enhance blood flow recovery and maintain new vessel formation (see Fig. 3
). As expected, blood flow and capillary density in mice cotransfected with the HGF and prostacyclin synthase genes was much greater than that with single transfection of "naked" plasmid DNA.
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As diabetic patients often present with advanced coronary and peripheral artery disease, in developing human gene therapy it is important to know whether compensatory mechanisms for vascular ischemia are affected in this condition. Accordingly, we and others have reported a delay of angiogenesis in a diabetic state. Nevertheless, our previous studies demonstrated the utility of HGF gene transfer to stimulate neovascularization of ischemic tissues in a diabetes model. 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 hind limb ischemia also documented favorable effects of VEGF gene transfer on ischemic peripheral neuropathy. Numerous reports have shown direct effects of reduced blood flow or ischemia on nerve conduction velocity and integrity of the peripheral nervous system. Diabetic neuropathy has been causally related to microangiopathy and endoneurial ischemia. 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, cotransfection of the HGF and PGIS genes was able to improve the electrophysiological findings. As HGF has been reported to have direct effects on nerve cells, the results of these experiments do not exclude the possible contribution of direct effects of HGF on nerve integrity.
What is the clinical relevance of the second-generation therapeutic angiogenesis strategy presented in this study? First, although a single i.m. injection of HGF plasmid was sufficient to prevent necrosis, enhancement of therapeutic angiogenesis by increasing the dose or injection time or transfection efficiency using viral and nonviral vector systems would be important. Indeed, the clinical efficacy using the VEGF gene did not reach 100%. To treat a wider range of patients such as those with critical limb ischemia with diabetes or on hemodialysis, it is necessary to achieve higher efficiency. However, when increasing the dose or injection time, cost and toxicity issues might be a problem. Alternatively, using highly efficient vector systems may cause side effect issues and toxicity due to the vector itself. Indeed, adenoviral vectors often used clinically cause deleterious side effects. Stimulation of collateral formation induced by HGF plasmid DNA with prostacyclin is relatively safe, since prostacyclin is widely used to treat patients for a long time, without severe side effects. Second, it is important to achieve therapeutic effects at an earlier time point such as 2 wk after transfection. Using single gene transfection, therapeutic efficacy appears from 3 or 4 wk after transfection, since the increase in blood vessels induced by the expression of growth factors from a transgene requires a long period. Thus, to consider the clinical setting, this second generation of therapeutic angiogenesis may be useful to treat PAD patients. Alternatively, pharmaceutical drugs such as oral prostacyclin analogs are widely used to treat PAD patients, and the combination of gene therapy using a single gene with oral drugs is more likely in the clinical setting. A recent report using VEGF121 is attractive. The authors reported that the vasodilator response to nitroglycerine was significantly restored in patients transfected with the VEGF121 gene. Thus, it is likely to stimulate collateral formation after gene therapy combined with oral vasodilator drugs in clinical practice. These experimental findings are thus encouraging for the treatment of diabetic neuropathy and PAD, although these areas clearly require further investigation and monitoring with regard to safety issues.
Here we have demonstrated that cotransfection of the prostacyclin synthase and HGF genes was more effective than single gene transfection to stimulate angiogenesis and improve diabetic neuropathy in a mouse hind limb ischemia model. These data provide important information for the clinical application of therapeutic angiogenesis to treat peripheral arterial disease.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0754fje; to cite this article, use FASEB J. (February 5, 2003) 10.1096/fj.02-0754fje 
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