VEGF-A, VEGF-D, VEGF receptor-1, VEGF receptor-2, NF-kappaB, and RAGE in atherosclerotic lesions of diabetic Watanabe heritable hyperlipidemic rabbits.

Plaque angiogenesis may be associated with the development of unstable and vulnerable plaques. Vascular endothelial growth factors (VEGFs) are potent angiogenic factors that can affect plaque neovascularization. Our objective was to determine the effect of diabetes on atherosclerosis and on the expression of angiogenesis-related genes in atherosclerotic lesions. Alloxan was used to induce diabetes in male Watanabe heritable hyperlipidemic (WHHL) rabbits that were sacrificed 2 and 6 months after the induction of diabetes. Nondiabetic WHHL rabbits served as controls. Blood glucose (Glc), serum-free fatty acids (FFA), and serum triglyceride levels were significantly higher in diabetic rabbits. Accelerated atherogenesis was observed in the diabetic WHHL rabbits together with increased intramyocellular lipids (IMCL), as determined by 1H-NMR spectroscopy. Atherosclerotic lesions in the diabetic rabbits had an increased content of macrophages and showed significant increases in immunostainings for vascular endothelial growth factor (VEGF)-A, VEGF-D, VEGF receptor-1, VEGF receptor-2, RAGE, and NF-kappaB. VEGF-A165 and VEGFR-2 mRNA levels were significantly increased in aortas of the diabetic rabbits, where a trend toward increased plaque vascularization was also observed. These results suggest that diabetes accelerates atherogenesis, up-regulates VEGF-A, VEGF-D, and VEGF receptor-2 expression, and increases NF-kappaB, RAGE, and inflammatory responses in atherosclerotic lesions in WHHL rabbits.

Gene transfer into rabbit arteries with adeno-associated virus and adenovirus vectors.

BACKGROUND: Gene transfer offers considerable potential for altering vessel wall physiology and intervention in vascular disease. Therefore, there is great interest in developing optimal strategies and vectors for efficient, targeted gene delivery into a vessel wall. METHODS: We studied adeno-associated viruses (AAV; 9 x 10(8) to 4 x 10(9) TU/ml) for their usefulness to transduce rabbit arteries in vivo in comparison with adenoviruses (Adv; 1 x 10(9) to 1 x 10(10) pfu/ml). 100 microl of viruses or placebo solution were injected intraluminally into transiently isolated carotid segments. RESULTS: In normal arteries AAV transduced mainly medial smooth muscle cells (SMC) while Adv transduced exclusively endothelial cells (EC). Mechanical injury to EC layer and internal elastic lamina enabled Adv to penetrate and transduce medial SMC. Transgene expression in EC after the AAV-mediated gene transfer was very low. The use of the EC-specific Tie-1 promoter did not lead to specific transgene expression in EC. Transgene expression in SMC persisted for at least 100 days after the AAV treatment whereas the Adv-mediated effect diminished in 14 days. AAV caused only a modest increase in EC VCAM-1 expression and proliferation rate of vascular cells as compared with the mock-treated arteries while Adv caused an extensive inflammatory cell infiltration, VCAM-1 expression, vascular cell proliferation and morphological damages. CONCLUSIONS: Significant differences were observed between the AAV and the Adv vectors in their patterns of arterial transduction and consequent inflammatory responses. These distinct properties may be utilized for different applications in vascular biology research and gene therapy for cardiovascular diseases.

Angiogenic responses of vascular endothelial growth factors in periadventitial tissue.

Recent discovery of new members of the vascular endothelial growth factor (VEGF) family has generated much interest as to which members may be best suited for therapeutic angiogenesis in various tissues. In this study we evaluated angiogenic responses of the different members of the VEGF family in vivo using adenoviral gene transfer. Adenoviruses (1 x 10(9) plaque-forming units [pfu]) encoding for VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-C(deltaNdeltaC) and VEGF-D(deltaNdeltaC) (deltaNdeltaC are proteolytically cleaved forms) were transferred locally to the periadventitial space of the rabbit carotid arteries using a collar technique that allows efficient local transfection of the periadventitial tissue. Expression of the transfected VEGFs was confirmed by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). Seven days after the gene transfer maximum neovessel formation was observed in VEGF-A-, VEGF-D-, and VEGF-D(deltaNdeltaC)-transfected arteries. VEGF-C(deltaNdeltaC) also showed angiogenic activity whereas VEGF-B was not effective in inducing angiogenesis. Pericytes were detected around the neovessels, which also frequently showed the presence of intraluminal erythrocytes. Infiltration of inflammatory cells in response to VEGF-D and VEGF-D(deltaNdeltaC) was less prominent than that caused by other VEGFs. In line with the absence of lymphatics in the normal carotid arteries no significant evidence of lymphatic vessel formation was seen in response to any of the studied VEGFs in the periadventitial space. The results help to define possibilities for local angiogenic therapy around blood vessels and support the concept that angiogenic effects may be tissue-specific and depend both on the growth factor ligands and the target tissues. It is concluded that VEGF-A, VEGF-D, and VEGF-D(deltaNdeltaC) are the best candidates for therapeutic angiogenesis when delivered around large arteries.