The Effect Of NADPH Oxidase In Adventitia In The Transplant Vasculopathy

Background:Transplant vasculopathy (TV) is a proliferative vascular disease characterized by neointima formation. Ninety percent of the patients would develop into TV within 10 years after organ transplantation. In recent years, with the development of the immunosuppressive agent, the incidence of acute rejection after organ transplantation has been controlled. However, the chronic rejection after organ transplantation is still the main factor that constrains the long-term survival of grafts. How to decrease the vascular injury effectively and improve the long-term survival of grafts after transplantation is currently an important issue in the field of organ transplantation. The intimal hyperplasia is the core issue. Most research on it’s mechanism focus on the vascular smooth muscle cells(VSMCs) of the media, which can proliferate and migrate into the intima. Inflammatory cells infiltration in the vessel wall, endothelial cell injury and secreting a variety of cytokines are also considered to promote intima hyperplasia and vascular restenosis. Nevertheless, the adventitia has been regarded as a a layer of connective tissue, which provides effective protection and nutrition to the vessels for a long time. Its effect has been ignored largely. In recent years, some researchers pay more attention to the role of the adventitia, and find that the adventitia plays an important role in the vascular balloon injury model and the atherosclerosis (AS). And the research has shown that when vessels are subjected with stimulus, such as cytokines, tension and mechanical damage, the fibroblasts of the adventitia can be activated, which is characterized phenotypic changes and increased ability of proliferation and migration. These changes promote the reconstruction of transplanted vessels. That indicates the adventitia plays an important role in the diseases involved in vascular remodeling.In addition, the important role of NADPH oxidase in many cardiovascular diseases has been reported, for example, the initiation and development of atherosclerosis are closely related to NADPH oxidase activity. And some researchers have proved that NADPH oxidase participates in the oxidative stress of fibroblasts in the adventitia, which makes fibroblasts be activated, and then induces vascular remodeling.However, few people research on the expression of NADPH oxidase and how it works in the adventitia to result in TV. This study mainly explores the effect of NADPH oxidase, adventitial inflammation and angiogenesis in the adventitia on vascular remodeling. Objective:The thoracic-abdominal aorta transplantation model is established in rats. The adventitial activation in TV is proved through the experimental process in vivo and in vitro. The high expression of the protein and mRNA level of NADPH oxidase in fibroblasts of adventitia increases proliferative and migratory ability of fibroblasts. In our study, we mainly explore the effect of adventitia and fibroblasts activation, and the accelerating effect of adventitial inflammation and neovascularization on the neointimal formation and development in TV. Methods:In vivo:Remove the thoracic aorta of a male Fisher 344 rat as donor artery, and transplant it to abdominal aorta of a Lewis rat to establish experimental animal model. Transplanted vessels are regarded as experimental group; non transplanted thoracic aortas of F344 male rats are as normal control group. Remove transplanted vessels 0.5,3,7,14 days after transplantation respectively to perform the HE staining, measure the thickness of the intima, media and adventitia and the ratia of intima/media. Immunohistochemical staining detects expression of p47phox, gp91phox, CD68 and CD3. Separate the adventitia and detect the expression of gp91phox and p47phox of time course with QRT-PCR at the mRNA level.In vitro:Separate the adventitia of thoracic aorta of F344 and transplanted aorta(F344-Lewis) after transplantated 14 days respectively, and culture the fibroblasts. The primary cells of passage 3-5 are used for experimentation. Compare the ability of proliferation and migration of two kinds of primary cells; QPCR detects the expression of p47phox and gp91phox at mRNA level of two primary fibroblasts. p47phox-siRNA specifically silences p47phox gene and then we observe the changes of cell function:primary fibroblasts of 14 days after transplantation are divided into three groups, blank control group (group without transfection), negative control group and p47phox-siRNA transfection group. Brdu staining evaluates the ability of cell proliferation, and transwell chamber detects the ability of cell migration. Diapocynin non selectively suppresses NADPH oxidase and the cell function are tested:primary fibroblasts of 14 days after transplantation are divided into three groups, blank group, DMSO group (control group) and Diapocynin group. CCK-8 test evaluates the ability of cell proliferation and transwell chamber detects the ability of cells migration. Results:1. Adventitial hyperplasia precedes neointima formation. HE staining showed that the increase of the thickness in the adventitial layer was observed as early as 3 days after surgery, while no neointima was present. The adventitial thickness increased gradually from day 7 to day 14. Neointima formation could be found at day 7, which further increased at day 14. The thickness of the media did not change significantly with time course.2. NADPH oxidase expression was upregulated in the adventitia of transplanted vessel. The protein levels of both gp91phox and p47phox subunits were significantly upregulated as compared to control tissues, and there was a progressive increase in the expression of these genes in adventitia from day 3 to day 14. Seven days after thoracic aorta transplantation, gp91phox and p47phox positive cells began to be observed in the intima, and 14 days continue to increase. The mRNA levels of gp91phox and p47phox significantly increased at day 3 compared with the normal control group after transplantation, and continue to increase at day 7, further increase at day 14.3. Infiltration of Inflammatory cells in transplanted aorta. CD68+ monocytes appeared in the adventitia at day 3, increased at day 7, further increased at day 14; CD68+ monocytes appeared in the intima at day 7, increased at day 14. Only a few CD3+ T cells were present in the adventitia, which did not increase significantly with time course.4. The ability of fibroblasts proliferation and migration decrease after inhibition of expression of NADPH oxidase. After the transfection of p47phox-siRNA and the intervention of Diapocynin for allograft fibroblasts, the proliferative and migratory ability of primary cells were significantly be suppressed.5. Neovascularization in the adventitia correlates with neointima formation. The vwF-positive neovascular were not readily detectable in normal adventitia. However, the number of neovascular was significantly augmented at day 7 in the adventitia of grafts. A large number of neovasculars were present throughout the adventitial layer at day 14. Linear-regression analysis demonstrated that there was a significant correlation between the number of adventitial neovasculars and the average thickness of the neointima.Conclusion:1. Adventitial activation occurred earliest in TV. The high expression of NADPH oxidase is regarded as one of aspects of adventitial activation which plays an important role on neointima formation.2. The high expression of NADPH oxidase increased the ability of proliferation and migration of fibroblasts in adventitia, and may promote the neointimal formation.3. The macrophage infiltration and the increase in the number of neovascular in the adventitia may further promote vascular remodeling.