Myocardial angiogenesis induced by plasmid VEGF-A165 gene transfer : Experimental and clinical studies

Detta är en avhandling från Stockholm : Karolinska Institutet, Department of Medicine

Sammanfattning: New therapeutic options have emerged for patients with medically intractable angina who are not candidates for conventional revascularisation techniques, from increasing understanding of the biology of vessel formation and how different angiogenic growth factors participate in the process. Administration of vascular endothelial growth factor-A165 (VEGF-A165), a key regulator of vessel growth, has been shown to augment myocardial collateral growth. The research programme behind this thesis focuses on angiogenic gene therapy using plasmid encoding VEGF-A165 as a vector. In study I the bioactivity of phVEGF-A165 after intramyocardial injection in normoxic and hypoxic rats was examined morphologically. A dose-response correlation between the number of VEGF-A expressing cells as well as the angiogenic effect in terms of microvessel density was observed. Hypoxic conditions had a tendency to enhance both plasmid expression and angiogenesis in rat myocardium. In study II the morphological localization of gene expression following catheter-based transendocardial injection of plasmid encoding VEGF-A165 or a reporter protein in pig hearts was examined. After three days plasmid transfection and protein expression was macroscopically localised only in the target areas and microscopically localised at all levels of the left ventricular wall. VEGF-A165 and reporter protein were expressed to the same degree. In study III the behaviour of plasmids encoding VEGF-A165 or a reporter gene injected directly into rat hearts was explored. Transfection was localized only at the injection site, with minimally detected systemic spread. The time kinetics of different plasmids was similar, with a rapid though short gene expression independent of which promotor was used. Doseresponse expression of different genes was likewise similar, with an increase in protein levels detected only up to a certain dose injected plasmid after which protein expression plateaued In study IV plasmid uptake and protein expression was compared after injection of the same dose of the same reporter plasmid into skeletal and cardiac muscle in rats. Plasmid DNA levels one day after gene transfer was approximately 3-fold higher in skeletal than in cardiac muscle, but had by day three abruptly decreased to levels significantly lower than in cardiac muscle. Luciferase activity in cardiac muscle was 40-fold higher than in skeletal muscle one day and 10-fold higher three days posttransfection. The efficacy of plasmid gene transfer hence differs between tissues. In study V seven patients with end-stage angina pectoris were included in an open phase 1 trial testing the safety and efficacy of intramyocardial injection of phVEGF-A165 via a mini-thoracotomy as sole therapy. A transient increase in plasma VEGF-A levels was observed. Two months after gene transfer improved perfusion in the target region was detected in four patients and improved function in all patients. Furthermore, a significant symptomatic relief that was retained under the twelve-month follow-up period was documented. Conclusions: These results suggest that positive therapeutic effects can be achieved by angiogenic myocardial gene therapy using plasmid vectors. Catheter-based intramyocardial gene transfer is feasible to accomplish with high anatomical precision, making it possible to design randomized, blinded clinical trials. The results also emphasize the importance of continued research on the pharmacokinetics and pharmacodynamics of plasmid vectors as well as improvement and refinement of plasmid constructs in order to amplify their clinical utility.

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