Himadri Roy

Biology of vascular endothelial growth factors

Angiogenesis is the process by which new blood vessels are formed from existing vessels. The vascular endothelial growth factors (VEGFs) are considered as key molecules in the process of angiogenesis. The VEGF family currently includes VEGF‐A, ‐B, ‐C, ‐D, ‐E, ‐F and placenta growth factor (PlGF), that bind in a distinct pattern to three structurally related receptor tyrosine kinases, denoted VEGF receptor‐1, ‐2, and ‐3. VEGF‐C and VEGF‐D also play a crucial role in the process of lymphangiogenesis. Here, we review the biology of VEGFs and evaluate their role in pathological angiogenesis and lymphangiogenesis.

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.

VEGF-A,VEGF-D and VEGF-DΔNΔC induced intimal hyperplasia in carotid arteries

Background  The role of vascular endothelial growth factors (VEGFs) in intimal hyperplasia and atherogenesis remains unknown. Several studies have suggested that some members of the VEGF family reduce intimal hyperplasia, but others have proposed that VEGFs accelerate restenosis and atherosclerosis. This investigation conducted a comparative study with adenoviruses encoding different VEGFs in a rabbit carotid artery collar model of intimal hyperplasia in order to analyze the role of VEGFs in the formation of intimal hyperplasia.

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

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.

Molecular genetics of atherosclerosis

Atherosclerosis is a complex multifocal arterial disease involving interactions of multiple genetic and environmental factors. Advances in techniques of molecular genetics have revealed that genetic polymorphisms significantly influence susceptibility to atherosclerotic vascular diseases. A large number of candidate genes, genetic polymorphisms and susceptibility loci associated with atherosclerotic diseases have been identified in recent years and their number is rapidly increasing. In this review we focus on some of the major candidate genes and genetic polymorphisms associated with human atherosclerotic vascular diseases.

Gene Therapy for Cardiovascular Diseases

Gene therapy offers a promising alternative for the treatment of various cardiovascular diseases. Blood vessels are among the easiest targets for gene therapy and in some pathological conditions only a temporary expression of the transfected gene will be required to achieve a beneficial biological effect. These conditions include the prevention of postangioplasty restenosis, postbypass atherosclerosis, peripheral atherosclerotic vascular disease and thrombus formation. Further developments in gene transfer vectors and gene delivery techniques are required before a full therapeutic potential or gene therapy in cardiovascular diseases can be evaluated.

VEGF-A, VEGF-D, VEGF receptor-1, VEGF receptor-2, NF-κB, 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‐B. 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‐B, RAGE, and inflammatory responses in atherosclerotic lesions in WHHL rabbits.—Roy, H., Bhardwaj, S., Babu, M., Kokina, I., Uotila, S., Ahtialansaari, T., Laitinen, T., Hakumaki, J., Laakso, M., Herzig, K‐H., Ylä‐Herttuala, S. VEGF‐A, VEGF‐D, VEGF receptor‐1, VEGF receptor‐2, NF‐B, and RAGE in atherosclerotic lesions of diabetic Watanabe heritable hyperlipidemic rabbits. FASEB J. 20, E1550 –E1559 (2006)

Gene therapy to prevent occlusion of venous bypass grafts.

Revascularization with vein grafts is standard surgical therapy for occlusive arterial diseases. Autologous saphenous vein grafts are important conduits for repairing blocked coronary arteries and are used in the majority of vein graft procedures. Up to 50% of saphenous vein grafts will be occluded during the first decade after surgery. Vein graft occlusion occurs as a result of neointimal hyperplasia, which takes place in response to hemodynamic changes and vessel wall injury, and is characterized by the migration and proliferation of vascular smooth muscle cells. Intimal hyperplasia is further complicated by the concomitant development of atherosclerosis and thrombosis. In the absence of effective pharmacological interventions for the treatment and prevention of occlusive vein graft disease, gene therapy has emerged as a potential therapeutic alternative. Gene therapy could improve vein graft patency by reducing early thrombosis, neointimal hyperplasia and atherosclerosis. In this review we will summarize the emerging applications of gene therapy as a therapeutic tool in occlusive vein graft disease.

Adventitial gene transfer of VEGFR-2 specific VEGF-E chimera induces MCP-1 expression in vascular smooth muscle cells and enhances neointimal formation.

BACKGROUND The role of vascular endothelial growth factors (VEGFs) in neointimal formation has been controversial. VEGF receptor (R)-2 signaling pathway is crucial in bringing about the effects of VEGFs including vasodilatation, endothelial cell migration and proliferation. In this study we have used an established adventitial gene transfer technique, in vitro studies and a novel VEGF-E/PlGF chimera that binds specifically to VEGFR-2, to investigate the role of VEGFR-2 in neointimal formation. METHODS Intimal hyperplasia was induced in the carotid arteries of cholesterol fed male New Zealand White rabbits using a silastic collar. Adenoviral vectors encoding VEGF-E chimera (1×10(9) pfu/ml) were transferred to the adventitia of the carotid arteries either alone or together with adenoviruses encoding soluble VEGFR-2 (sVEGFR-2). Adenoviruses encoding LacZ were used as controls. All animals were sacrificed 7 days after the gene transfer. RESULTS Significant increases in neointimal formation, proliferating cells, inflammatory responses and adventitial angiogenesis were observed in the VEGF-E chimera transduced arteries. The number of medial smooth muscle cells expressing VEGFR-2 was significantly (p<0.001) higher. MCP-1 mRNA levels were significantly (p<0.01) increased in the VEGF-E chimera transduced arteries and transduced rabbit aortic smooth muscle cells (p<0.05). Soluble VEGFR-2 (sVEGFR-2) significantly inhibited VEGF-E chimera induced neointimal formation (p<0.01), cellular proliferation (p<0.01), inflammatory responses (p<0.01) and adventitial angiogenesis (p<0.01). CONCLUSIONS The results indicate that VEGFR-2 mediated signaling could aggravate neointimal formation and suggest a potential therapeutic role of sVEGFR-2 in inhibiting neointimal formation and adventitial angiogenesis.

Periadventitial angiopoietin 1 gene transfer induces angiogenesis in rabbit carotid arteries

This study was performed to evaluate angiogenic responses of angiopoietin-1 (Ang1) in vivo after adenovirus-mediated gene transfer in the periadventitial space of the rabbit carotid arteries using a collar technique. Adenoviruses encoding LacZ and vascular endothelial growth factor (VEGF) receptor-1-Ig fusion protein (VEGF-R1-Ig) adenoviruses were used as controls. Increased neovessel formation was seen in adventitia of the Ang1 transduced arteries 7 days after the gene transfer. Neovessels in the Ang1 transduced arteries were large in size and well perfused. Ang1 binds to Tie2 (tyrosine kinase with immunoglobulin and epidermal growth factor homology domain) receptors, which were expressed in the endothelium of the neovessels. When VEGF-R1-Ig was used with Ang1, it resulted in a decrease in the number of neovessels, which implies that VEGF-A or some other VEGF-R1 ligand(s) play a crucial role in angiogenesis occurring in response to Ang1. There were no significant differences in the total number of capillaries in the adventitia of the VEGF-R1-Ig transduced arteries as compared to LacZ controls. Neointima formation was not increased in the Ang1 transduced arteries as compared to the controls. We conclude that in the periadventitial space Ang1 shows angiogenic activity and is a potentially useful factor for the induction of therapeutic vascular growth in vivo.