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.

VEGF-DdeltaNdeltaC mediated angiogenesis in skeletal muscles of diabetic WHHL rabbits.

BACKGROUND: Arterial occlusive disease is often associated with diabetes mellitus and hypercholesterolaemia which may reduce angiogenic potential of several growth factors. Accordingly, the usefulness of therapeutic angiogenesis in the presence of diabetes and hypercholesterolaemia has remained unclear. We evaluated angiogenic effects of the mature form of vascular endothelial growth factor-D (VEGF-D(deltaNdeltaC)) in skeletal muscles in the presence of severe diabetes and hypercholesterolaemia. METHODS: Intra muscular injections of adenoviruses encoding human VEGF-D(deltaNdeltaC) (AdVEGF-D(deltaNdeltaC)) were given in the hind limbs of a group of diabetic hypercholesterolaemic rabbits and adenoviruses encoding LacZ (AdLacZ) were used as a control. All animals were killed 6 days after the gene transfer. RESULTS: Capillary count, capillary area, capillary permeability and perfusion were significantly higher in the AdVEGF-D(deltaNdeltaC) transduced muscles compared with the AdLacZ controls. Expressions of endothelial nitric oxide synthase (eNOS) and VEGF receptor(R)-2 were also significantly increased in the VEGF-D(deltaNdeltaC) transduced muscles, along with an increased expression of angiopoietins (Angs) and neuropilin-2 (NP-2). Furthermore, VEGF-D(deltaNdeltaC) gene transfer to the skeletal muscles increased localized recruitment of cells with endothelial progenitor-like characteristics. CONCLUSIONS: VEGF-D(deltaNdeltaC) gene transfer can induce efficient angiogenesis in the presence of severe diabetes and hypercholesterolaemia by upregulating eNOS and VEGFR-2 expression. VEGF-D(deltaNdeltaC) appears to be a promising agent for inducing therapeutic angiogenesis even in cases with severe diabetes and hypercholesterolaemia.

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 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.

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.

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.

Adenovirus-mediated gene transfer of placental growth factor to perivascular tissue induces angiogenesis via upregulation of the expression of endogenous vascular endothelial growth factor-A.

Placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family that binds specifically to VEGF receptor (VEGFR)-1. However, the mechanism of PlGF- and VEGFR-1-mediated angiogenesis has remained unclear and some in vitro studies suggest that VEGF-A/VEGFR-2 signaling may also play a role in PlGF-mediated angiogenesis. To clarify these issues we evaluated angiogenic responses in a well-characterized periadventitial angiogenesis model using adenovirus-mediated PlGF-2 (AdvPlGF-2) gene transfer. We also investigated the roles of VEGFR-1 and VEGFR-2 in PlGF-2-mediated angiogenesis. Using a periadventitial collar technique, AdvPlGF-2 (1 x 10(9) plaque-forming units/ml) was transferred to the adventitia of New Zealand White rabbits alone or together with adenoviruses encoding soluble VEGFR-1 (sVEGFR-1) or soluble VEGFR-2 (sVEGFR-2). Adenoviruses encoding LacZ were used as controls. All animals were killed 7 days after gene transfer. Increased neo-vessel formation, upregulation of endogenous VEGF-A expression, and a significant inflammatory response were seen in AdvPlGF-2-transduced arteries. The neo-vessels were large and well perfused. sVEGFR-1 and sVEGFR-2 suppressed the angiogenic response of PlGF-2 by 80 and 71.7%, respectively. We conclude that adenovirus-mediated PlGF-2 gene transfer to vascular tissue increases endogenous VEGF-A expression and produces significant angiogenesis. Both sVEGFR-1 and sVEGFR-2 can inhibit PlGF-2-mediated angiogenesis. PlGF-2 is a potentially useful candidate for the induction of therapeutic angiogenesis in vivo.

Angiogenesis-dependent and independent phases of intimal hyperplasia.

BACKGROUND: Neointimal vascular smooth muscle cell (VSMC) proliferation is a primary cause of occlusive vascular disease, including atherosclerosis, restenosis after percutaneous interventions, and bypass graft stenosis. Angiogenesis is implicated in the progression of early atheromatous lesions in animal models, but its role in neointimal VSMC proliferation is undefined. Because percutaneous coronary interventions result in induction of periadventitial angiogenesis, we analyzed the role of this process in neointima formation. METHODS AND RESULTS: Local injury to the arterial wall in 2 different animal models induced periadventitial angiogenesis and neointima formation. Application of angiogenesis stimulators vascular endothelial growth factor (VEGF-A165) or a proline/arginine-rich peptide (PR39) to the adventitia of the injured artery induced a marked increase in neointimal thickening beyond that seen with injury alone in both in vivo models. Inhibition of either VEGF (with soluble VEGF receptor 1 [sFlt1]) or fibroblast growth factor (FGF) (with a dominant=negative form of FGF receptor 1 [FGF-R1DN]), respectively, signaling reduced adventitial thickening induced by VEGF and PR39 to the level seen with mechanical arterial injury alone. However, neither inhibitor was effective in preventing neointimal thickening after mechanical injury when administered in the absence of angiogenic growth factor. CONCLUSIONS: Our findings indicate that adventitial angiogenesis stimulates intimal thickening but does not initiate it.

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.

Gene therapy for cardiovascular diseases.

Gene therapy is a rapidly evolving field of medicine, which potentially offers new treatments for cardiovascular diseases. With the use of gene transfer methods it is possible to modify somatic cells in blood vessels and myocardium to overexpress or inhibit pathologically important proteins and achieve therapeutic effects. Prevention of restenosis after vascular interventions such as percutanous coronary angioplasty (PTCA), percutanous peripheral angioplasty (PTA) or stent implantation, prevention of venous graft failures and therapeutic angiogenesis are the major aims of experimental studies and clinical gene therapy. The promise of gene therapy in the treatment of cardiovascular diseases remains high. Experimental studies have established the proof of principle that gene transfer to cardiovascular system can achieve therapeutic effects. First human clinical trials provided initial evidence of the feasibility and safety of the novel therapy. There are also first successful reports on the prevention of neointimal hyperplasia and promotion of therapeutic angiogenesis in clinical trials. However, there are still important questions regarding utility, efficiency and safety of gene therapy in the treatment of cardiovascular diseases. In this review we discuss the rapid progress in cardiovascular gene therapy, the development of delivery systems and vectors, most promising therapeutic genes and results of the recent human clinical trials.

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.