Role of adhesion molecules and NO in vein graft disease

Autologous vein grafts remain the only surgical alternative for many types of vascular reconstruction, but obliterative stenosis (arteriosclerosis) often follows. The Pathogenesis of this disease is poorly understood, and no successful clinical interventions have been identified. Recently, many investigators have developed mouse genetic models in which genes are either overexpressed, deleted, or mutated. Such models have considerable advantages over other animal systems in that they overcome the need to administer factors or their inhibitors, which can be problematic and often difficult to quantify. We have established a mouse model of vein graft arteriosclerosis, which could be very useful for studying the mechanism of the pathogenesis as well as for therapeutic intervention of vein graft disease. In the present proposal, we will use such a mouse model to focus on the role of endothelium in the formation of vein graft arteriosclerosis. First, animal models for vein-grafted arteriosclerosis will be established by transplantation of jugular veins or vena cava to carotid arteries of wild-type C57BL/6J mice. At various time points, the grafted veins will be examined using en face immunofluorescence and immunohistochemistry to localize adhesion molecules (ICAM-1, ELAM-1 and VCAM-1), and to analyse the cellular composition of adhered cells. Second, to analyse effects of endothelial adhesion molecules (ICAM and ELAM) on grafted arteriosclerosis, ICAM-/-and ELAM-/- mice will be used as vein graft recipients and donors. The severity of arteriosclerosis in grafted veins will be compared to those from wild-type mice. Third, to determine the role of nitric oxide (NO) in the development of vein grafted arteriosclerosis, eNOS-/- or iNOS-/- mice will be used to establish the vein graft model and the lesions will be compared to these of wild-type C57BL/6J as will proliferation and apoptosis of vascular smooth muscle cells derived from eNOS and iNOS knockout mice. Finally, we will initiate work on gene therapy by transferring eNOS and iNOS ex vivo to the vein fragments and then transplanting them to the mouse. All techniques described in this project have been successfully used by the author, and all key reagents and mice are available. This study is timely and may yield important information concerning the mechanisms of graft vein disease development, especially the effects of adhesion molecules and NO in the formation of atherosclerosis. Such findings could provide a basis for therapeutic intervention in vascular diseases.

Autologous vein grafts remain the only surgical alternative for many types of vascular reconstruction, but the patency rate is limited due to obliterative stenosis of the grafted vessels. The pathogenesis of this disease is poorly understood, and no successful clinical intervention is available. Recently, we established the first mouse model of vein bypass graft arteriosclerosis, which has been proven to be a powerful tool. Using this model in gene-deleted animals the present project has designed to study the pathogenesis of vein-graft disease focusing on the role of adhesion molecules and nitric oxide. Intercellular adhesion molecule-1 (ICAM-1), a surface glycoprotein of the immunoglobulin superfamily. The major known functions of ICAM-1 relate to its role in cell adhesion and migration. Several lines of evidence have suggested that ICAM-1/MAC-1-dependent cellular interaction is involved in a number of inflammatory processes and in arteriosclerosis via mononuclear cell adhesion and interactions. However, it remains unknown whether ICAM-1 plays a causal role in the development of vein bypass graft arteriosclerosis. Using our mouse model, we studied the role of intercellular adhesion molecule-1 (ICAM-1) in the development of vein graft arteriosclerosis in ICAM-1-deficient mice. Neointimal hyperplasia of vein grafts in ICAM-1 -/- mice were reduced 30% to 50% compared to wildtype controls. Immmunofluorescent analysis revealed that increased ICAM-1 expression was observed on the endothelium and smooth muscle cells (SMCs) of the grafted veins in wildtype, but not ICAM-1 -/- , mice. Mac-1 (CD11b/18) positive cells adhering to the surface of vein grafts in ICAM-1 -/- mice were significantly less as identified by en face immunofluorescence, and these positive cells were more abundant in intimal lesions of vein grafts in wildtype mice. When TNF-alpha-stimulated SMCs were incubated with mouse spleen leukocytes, numbers of adhered cells to ICAM-1 -/- SMCs were significantly lower than those to ICAM-1 +/+ SMCs, which was markedly blocked by pretreatment of leukocytes with the anti-MAC-1 antibody. Matrix metalloproteinases (MMPs) can degrade most of the vascular extracellular matrix components, including elastin and collagen. Their expression could be regulated by the interplay of inflammatory cells and cytokines present in the vein grafts. Tissue inhibitors of MMPs (TIMP) are native inhibitors of MMPs. Using our mouse model, we studied the effects of local gene transfer of TIMP-2 on vein graft remodeling. Mouse isogeneic vessels of the vena cava veins were grafted end-to-end into carotid arteries and then enveloped with the replication- defective recombinant adenoviruses overexpressing human TIMP-2 (RAdTIMP-2) or beta-galactosidase (RAdLacZ). In the untreated group, vessel wall thickening was observed as early as 1 week after surgery and progressed to 4-, 10-fold original thickness in grafted veins at 4 and 8 weeks, respectively. RAdLacZ vector treatment significantly enhanced neointima lesions at 8 weeks, which was completely blocked by RAdTIMP-2 gene overexpression. Interestingly, RAdTIMP-2 gene transfer resulted in a reduction in vessel diameter of grafted veins. Thus, this mouse model has been proven to be useful in gene transfer studies. Our findings demonstrate that local TIMP-2 gene transfer significantly reduces vein graft diameter, i.e. remodeling to an artery-like vessel via inhibition of matrix metalloproteinase activity. In summary, we provide the first evidence that ICAM-1 is critical in the development of venous bypass graft arteriosclerosis, which provides essential information for therapeutic intervention to vein graft disease in patients undergoing bypass operations. We have also provide the first evidence that this model can be used for studying gene therapy for vein graft atherosclerosis.