Inflammation Process After Artery Injury And Effect Of Oral L-arginine And Autologous Bone Marrow Mononuclear Cells Local Delivery On Inflammation

Background: It has been well established that atherosclerosis, as well as restenosis after percutanous coronary intervention (PCI) and transplant arteriosclerosis (TA) after coronary artery bypass grafting surgery (CABG) is inflammatory disease of the vessel wall. Recruitment of monocytes and lymphocytes from the peripheral blood to the intima of the vessel wall is a primordial event in early lesions, in which vascular cell adhesion molecule-1 (VCAM-1) plays an important role. Increased expression of VCAM-1 gene is known to be regulated by Nuclear Factor-κB (NF-κB). Outcome of vessel inflammation is a result of the balance of reendothelialization and neointimal proliferation. Drugs able to inhibit the activation of NF-kB and upregulation of VCAM-1 or block monocytes infiltration may accelerate reendothelialization and impede neointimal proliferation, thus improve repair of injured arteries. Nitrous oxide (NO) has been found confer vascular protection after arterial injury through mediation of endothelium-dependent relaxation. Whether NO impose its influence through inhibiting inflammation is not clear. Stem cells can differentiate into both endothelial cells (EC) and smooth muscle cells (SMC). Transplantation of culture-modified mononuclear cells or cytokine-induced mobilization of circulating endothelial progenitor cells (EPC) enhances repair of denuded arteries. Yet no specific surface epitope is definite that can be used to screen out all the stem cells and that can exclusively accelerate reendotheliliazation without differentiating into SMC. So we try to establish artery injury model to observe expression of VCAM-1 and its relationship with time course of NF-kB activation. AS rabbit treated with L-arginine (LA) will be used to determine LA/NO's effect on inflammation. Autologous bone marrow mononuclear cells (BMMNCs) without management after being isolated from bone marrow were delivery locally to freshly denuded endothelium to observe their influence on vessel repair and inflammation factors.Part1Purpose: To investigate VCAM-1 expression and its regulation in AS. Methods: Establish rabbit AS model with ballooning and high cholesterol diet. Sixteen rabbits were divided randomly into 2 groups: AS group and control group, 8 animals eachgroup, and crucified 30d after procedure. Activation of NF-kB was measured with Gel shift (EMSA). In situ hybridization (ISH) and Western Blot were adopted to determine the expression of VCAM-1 mRNA and protein. Nuclear translocation of P65 subunit was observed with immunohistochemistry (IH). Venous Blood was collected before and 3d> 1(^ 2(^ 30d after operation. Serum level of sVCAM-1 was detected with ELISA. Results: AS model was successfully established in AS group. Lipid plaque and remarkable noeintima proliferation could be seen in injured artery. Expression of VCAM-1 mRNA and protein were much higher in AS group than that in control group. NF-kB activation was confirmed by EMSA and P65 nuclear translocation in IH. Monocytes accumulated in injured artery of AS group. Serum level of sVCAM-1 increased gradually after procedure and was higher than that in control group (p<0.05). Conclusion: AS model has been successfully established. It can be seen that increased VCAM-1 expression and NF-kB activation as well as monocytes recruitment participate in AS. Their interaction participates in inflammation process of AS. Increased level of serum sVCAM-1 in AS can be a marker of local inflammation.Part 2Purpose: To investigate LA/NO's effects on inflammation after artery injury. Methods: Three groups of animals were use in this part: AS group (AS model same as the first part), LA group (treated with oral LA from the 2nd day after operation) and C group (control group), 8 rabbits each group. The methods for samples collection and detection are the same as the first part. Results: VCAM-1 mRNA and protein were decreased in LA group than in AS group. NF-kB activation and monocytes recruitment was also inhibited in LA group than in AS group. Serum level of sVCAM-1 was high in AS group (13.98±4.33 ng/ml) and much lower in LA group (5.11±1.49 ng/ml), p<0.01, while no difference between LA group and C group (3.02±0.07 ng/ml). Conclusion: Oral LA can inhibit NF-kB activation. Both local and circulating VCAM-1 expression was depressed by LA/NO. Monocytes adhesion to local artery can be blocked by LA/NO. LA/NO improves AS partly through its effects on inflammation. Reduction of serum level of sVCAM-1 is a sign of drug benefit.Part 3Purpose: To investigate time course of NF-kB activation after artery endothelium denudation and its relationship with local VCAM-1 expression. Methods: Deendothelialization injury of rat left common carotid artery was produced by ballooning (3 times) with PTCA catheter. Crucify 6 rats before and at every time points after procedure: 12h, 24h, Id, 2d, 3d, 7d, 14d. NF-kB activation was measured by EMS A. IH was used to detect local VCAM-1 expression 0 Results: Scanning electron microscopy showed that endothelium was completely denudated. Significant noeintima proliferation was seen at the 14th day after injury. NF-kB was activated at 12h and culminated at the 1st to the 3rd days after operation, and then decreased gradually. Its activation was higher at 14d after operation than control, but there was no statistically significance. No local VCAM-1 expression could be detected immediately after denudation. At the 7th day after operation, VCAM-1 was positive on the surface of endothelium and in the medium SMC. At the 14th day after operation, VCAM-1 was still positive on the surface of uncovered endothelium, while more abundant expression was seen in the bottom of noeintima near internal elastic lamina. Conclusion: NF-kB activation and VCAM-1 expression participate in the inflammation of rat denudated artery. Activation and inactivation of NF-kB occur before the expression and disappearance of VCAM-1. NF-kB is a regulator of VCAM-1.Part 4Purpose: To investigate the effect of autologous BMMNCs local delivery on artery repair after denudation and its mechanism. Methods and Results: Rats were divided into two groups: operation group (denude rat left carotid artery) and BMMNC group (local delivery autologous BMMNCs to freshly injured artery). Bone marrow was obtained by lumbar spinous process puncture from 450g Sprague-Dawley rats. BMMNCs were isolated by Ficoll gradient centrifugation and labeled with CM-Dil. Deendothelialization injury of left common carotid artery was produced by ballooning (3 times) with PTCA catheter. BMMNCs solution including (5.04±2.90)xl0 cells was administered into the freshly denuded intimal surface from the left outer carotid artery and incubated for 30 mins, while control group receive saline only. Rats were killed on day 14. Evan's blue staining revealed less remaining denuded area in treated group (19.07±5.25% vs. 58.28±11.40%, p<0.01). Intima to media (I/M) ratio was also decreased significantly in BMMNCs group...