| The prevalence of cardiovascular disease increased rapidly in the last decade.According to a survey published by American Heart Association in 2013,cardiovascular disease was the leading diagnostic group of hospital discharges in 2010,representing around 5.8 million patients.Many of these patients require invasive interventions including balloon angioplasty,stent implantation,or arterial bypass surgery.Neointimal hyperplasia is a physiologic response to vascular injury that also occurs after stent or graft implantation.Too much neointimal hyperplasia may cause vascular lumen stenosis or even occlusion of the vessel,leading to failure of the graft.Graft complications increase medical cost and negatively affect patient experience.Since neotinimal hyperplasia is the leading cause of graft failure,surgeons and scientists are trying to find solutions to inhibit neointimal hyperplasia.Various new methods including drug-eluting stents or grafts have been tested in clinical and animal models,with both positive and negative results.While arterial disease has a higher chance of requiring surgical intervention,venous disease is much less likely to require stent placement or bypass grafting except in particular situations.In the case of critical inferior vena cava injury or when tumors invade into the IVC,venous bypass surgery may be a lifesaving treatment.The venous system is a very low velocity system with a higher incidence of thrombus formation.Understanding the mechanism of vessel response to invasive intervention is essential to prevent or at least control venous neointima hyperplasia.Strategies to inhibit venous neointimal hyperplasia may lower the rate of graft stenosis.Bovine pericardial patches are widely used in vascular surgery,but details about its effect on vessel healing and mechanisms to prevent neointimal hyperplasia remain unclear.To study this process,we sought to develop a method for conjugating nano-particles containing therapeutic agents to the bovine pericardial patch.We used a rat inferior vena cava venotomy model to place a pericardial patch containing nanoparticles into the IVC and examine its effects on vessel healing and on the development of neointimal hyperplasia.We hypothesized a pericardial patch could be conjugated with nanoparticles containing rapamycin which could be used to reduce the degree of neointimal hyperplasia in our animal model.Objective:1,To understand the healing process of bovine pericardial patch angioplasty of the IVC after venotomy.2.To explore whether nano-particles containing rapamycin can be conjugated to the bovine pericardial patch.3,To examine whether nano-particles containing rapamycin conjugated to a bovine pericardial patch can inhibit venous neointima hyperplasia.Method:Young(6–8 week old)male Wistar rats were used for patch implantation(n = 91).Patches were inserted into either the IVC(n=32)or subcutaneously d(n=12).To examine the healing process and mechanism of healing,the animals were sacrificed on postoperative days 1,3,7,or 30.To determine whether nanoparticles containing rapamycin can inhibit neointimal hyperplasia,16 rats were treated with the nanoparticle rapamycin patch and 16 rats were treated with a nanoparticle control patch,15 rats were treated with nanoparticle rapamycin.Animals were sacrificed on postoperative days 7 or 30.Changes to the neotinima and protein expression were measured using HE staining,immunohistochemistry,immunofluorescence,and Western blot.Result:1.The neointima and adventitia appeared at day 7 and became thicker by day 30(p<0.0001).2.Compared to day 7,the endothelial confluence at day 30 was significantly larger(p=0.0048).There was no significant change in smooth muscle cell mean density(p=0.9224)but there were fewer mesenchymal cells(p=0.001),macrophages(p=0.0001),and leukocytes(p<0.0001).Endothelial progenitor cells(EPCs),smooth muscle progenitor cells(SMPCs),and venous progenitor cells(VPCs)were detected in the formation the neointima at day 7 and day 30.3.The smooth muscle cells and macrophages expressed VCAM,ICAM and CD29.4.Compared to day 7,the proliferation index at day 30 was significantly lower(p<0.0001)without a change in the apoptosis index(p=0.7383).5.Both at day 7 and day 30,the endothelial cells and smooth muscle cells expressed Eph-B4.Compared to day 7,the collagen-1 mean density was significantly higher(p=0.0001),but there was no elastin expression.At day 7 and day 30,there were some Ephrin-B2 positive cells in the neointima,but the endothelial cells were Ephrin-B2 negative.6.Compared to day 7,the mean density of MMP-2,MMP-3 and MMP-14 was significantly lower(p=0.0011,p=0.0074,p=0.043).7.Scanning electronic microscope showed the nano-particle rapamycin conjugated to the collagen fibers of the bovine pericardial patch and rapamycin release could be detected for at least 3 weeks.8.Compared to the nano-particle control group,at day 7,the neointima and the adventitia were significantly thinner(p=0.0010,p=0.0010).There was no significant difference between the nano-particle control group and the control group(p=0.0787).At day 30,the neointima and the adventitia remained significantly thinner(p=0.0046,p=0.0026)than the nano-particle control group.There remained no significant difference between the nano-particle control group and the control group(p=0.5921).There was no significant difference of the endothelial confluence between nanoparticle rapamycin group and nano-particle control group both at day 7 and day 30(p=0.5914,p=0.8025).9.Compared to the NP-control group,there were fewer macrophages both at day 7 and day 30 in the NP-rapamycin group(p=0.0028,p=0.0248).Compared to the NP-control group,there was reduced mean density of smooth muscle cells in the NPrapamycin group(p=0.0103,p=0.0093).At day 7,there was a significantly lower percentage of proliferating cells in the nano-particle rapamycin group compared to the nano-particle control group(p=0.0039)and there was also a lower smooth muscle cell proliferation index in the nano-particle rapamycin group(p=0.0018).At day 30,there was no significant difference of the proliferation index between nano-particle rapamycin group and nano-particle control group(p=0.1883).Both at day 7 and day 30,there was no significant difference in the apoptosis index between nano-particle rapamycin group and nano-particle control group(p=0.7338,p=0.7118).10,Nano-particle rhodamine is mainly metabolized in rat liver and kidney with only trace amounts of rhodamine detected in other tissues.The nano-particle rhodamine could be detected on the patch at 24 h after implantation.Conclusion:1.A variety of mature and progenitor cells participated in the formation of neointima after IVC venotomy repaired using a bovine pericardial patch in rats.The percentage of different cell types changed at different time points.The proliferation index also changed at different time points as did the protein expression patterns.2.In the process of neointimal formation,the neointima developed structure and identity similar to that of normal vein.3.Different molecules regulated cell recognition and adhesion.4.Nano-particle rapamycin can be successfully conjugated to bovine pericardial patches.5.Nano-particle rapamycin conjugated bovine pericardial patches can inhibit venous neointimal hyperplasia. |
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