| Background:Despite the well-developed interventional therapy and drug treatment, coronary atherosclerotic heart disease remains the leading cause of morbidity and mortality in China, and its incidence is still rising. Atherosclerosis is the most important pathological change throughout coronary heart disease. Atherosclerosis could locate in all arteries including coronary artery, cerebral arteries and hindlimb artery, the acute arterial occlusion due to embolism or thrombosis would lead to a sudden decrease in the blood flow to the supplied tissue, resulting in necrosis and fatality. At present the main treatments are revascularization and thrombolytic therapy. However, they showed inefficient in multi-vessel or restenosis patients, plus their complications and contraindications should not be overlooked. It is necessary to explore new approaches.The collateral circulation produced in the ischemic region could improve the prognosis. Well-developed collateral circulations may potentially limit the size of infarction, improve ischemia tissue function, reduce future events resulting in better survival rates. Since the slow-process of collateral circulation formation could not totally satisfy the need of the ischemic tissue, the therapeutic angiogenesis is necessary by increasing the concentration of pro-angiogenic factors to promote the angiogenesis.TLRs (toll-like receptor) are transmembrane receptor family whose function is to recognize pathogen-associated molecular patterns (PAMPs) and play an important role in inflammatory, immunity response and tumorigenesis. TLR2, whose coding gene locates on4q32, is the important member in TLR family. TLR2expressed most abundantly in peripheral blood leukocytes could recognize bacteria and endogenous ligands to activate NF-kB and AP-1. After the ischemia injury, the apoptosis cells could induce host defense and wound healing response, releasing inflammatory cytokines and recruiting inflammatory cells which are important in tissue injury and angiogenesis. TLR2deficiency could induce the renal injury, leukocytes invading in renal ischemia/reperfusion. The inhibition of TLR2could block the transcription of NF-kB and AP-1in cardiomyocyte oxidative stress in vitro. Therefore, TLR2played an important role in I/R injure repairment.The angiogenesis promoted by activating TLR2signalling has been reported in many essays, however, the researches focused on tumorigenesis. For example, the Helicobacter pylori acts through TLR2to activate the MAPK cascade, leading to COX-2-denpedent prostaglandin E2release and thereby contributing to cancer cell invasion and angiogenesis. The relationship and mechanism between TLR2and angiogenesis in ischemia injure are still not clear.The evidences on human TLR2showed that TLR2highly-expressed on monocytes was an important risk factor for arteriosclerotic, and peripheral blood monocytes in unstable angina were characterized by elevated TLR2levels. There is no evidence about the relationship between coronary collateral circulation and serum TLR2expression in patients with coronary heart disease.Objective:In the present study, we use TLR2-/-mice (C57BL/6background) and wide type C57BL/6mice (WT) to investigate whether TLR2gene plays a role in post-ischemia tissue necrosis, as well as the subsequent neovascularization in a acute hindlimb ischemia model.Then we try to study the relationship between serum TLR2and the neovascularization in patients with coronary artery disease. Methods:HUVECs were used to clarify effect of TLR2agonist Pam3CSK4in vitro. A simple scratch was made to clarify effect of Pam3CSK4on migration of HUVECs; The HUVECs pre-treated with Pam3CSK4were seeded on upper chamber, then the separated lymphocytes were added to upper chamber, transwell was used to detect effect of Pam3CSK4on the permeability of HUVECs. In vivo, WT and TLR2-/-mice weighing18to25g (8-to10-week-old) were anesthetized with intraperitoneal injections of4%chloral hydrate. Under sterile conditions, the proximal portion of the left femoral artery including superficial and deep branch as well as the distal portion of the saphenous artery were exposed and ligated. A sham procedure (dissection of vessels without ligation) was performed on the contralateral right leg. After surgery, a semiquantitative assessment of the ischemic damage to the limb was performed with a modification of ischemic score (0=no change,1=mild discoloration,2=moderate/severe discoloration,3=necrosis,4-amputation;8mice per group) and ischemia limb function score (0=toe bending,1=planta bending,2=planta unbending without drawing limbs,3=drawing limbs). We measured blood flow in both limbs using a Laser Doppler blood perfusion Scanner (at pre-operation, post-1day, post-3day, post-7day, post-14day, TLR2-/-mice versus WT mice, respectively). Equal amount of adductor muscles of both legs were harvested over dry ice for western blot to evaluate the VEGF expression. The expression of CD31was detected in gastrocnemius ischemic muscles by immunohistochemistry. Serum IL-6, TNF-a level was measured by ELISA in1,3,7,14day after surgery. In addition, lymphocytes isolated from TLR2-/-mice and WT mice were observed in trans-endothelial migration, respectively.In the clinical study,87consecutive subjects underwent coronary angiography were recruited and given the written informed content. All the subjects were divided into three groups according to the angiograms and collateral formation:the control group,26subjects whose angiograms showed no visible diseased vessel without collateral circulation; poor collateral group,39subjects with more than75%stenosis in at least one coronary artery without visible collateral circulation; good collateral group,22patients with more than75%stenosis in at least one coronary artery with well-developed collateral circulation. Collateral score were recorded according to the Rentrop classification. Then the blood samples were collected before the angiography, and the serum were examined by ELISA for the level of TLR2.Results:Pam3CSK4enhanced the migration of HUVEC in vitro, and the trans-endothelial migration of lymphocyte was enhanced after pretreatment of HUVECs with Pam3CSK4(102.29±10.60/20X HP versus68.69±10.57/20X HP, P<0.001). In vivo, hindlimb ischemia was induced in TLR2-/-and WT mice. The function of ischemia limb was recovered from the3rd day post-surgery in WT mice compared with7th day in TLR2-mice. Ischemia limb function score was significantly higher in TLR2"'" mice at postoperative day1,3and7(1.88±0.92versus1.10±0.85, P=0.011;1.54±0.88versus0.81±0.83, P=0.031;1.56±0.53versus0.67±0.77, P=0.008). Laser Doppler scanning showed that limb perfusion was significantly lower in TLR2-/-mice at postoperative day7and14(39.15±3.71versus52.40±2.93, P=0.001;33.47±1.69versus47.43±4.27, P=0.013), but at postoperative day1and3, the difference was not statistically significant(24.05±6.69versus26.50±9.16, P>0.05;38.14±8.82versus39.61±9.45, P>0.05). Moreover, TLR2-/-mice displayed reduced CD31expression when tested at postoperative day7and14(7.63±1.41/40X high power field(HP) versus14.17±4.07/40X HP, P=0.001;7.44±1.42/40X HP versus13.67±2.92/40X HP, P<0.001) at the site of collateral vessel development. Western blot showed reduced and delayed-increasing VEGF expression in TLR2" mice, suggesting a reduced neovascularization process in the absence of TLR2gene. In ELISA, TLR2-/-mice showed reduced IL-6level at postoperavtive day1,3,7and14(5.85±0.94ng/ml versus11.79±5.80ng/ml, P=0.043;5.80±0.80ng/ml versus18.91±7.03ng/ml, P=0.021;5.60±2.39ng/ml versus19.95±10.81ng/ml, P=0.003;4.68±0.57ng/ml versus13.68±7.80ng/ml, P=0.002); and lower levels of TNF-a was observed in TLR2-/-mice at postoperative day1,7and14(15.18±5.09ng/ml versus50.80±25.97ng/ml, P=0.021;27.35±9.44ng/ml versus44.04±9.68ng/ml, P=0.005;35.69±15.16ng/ml versus 55.74±20.08ng/ml, P=0.046). Besides, lymphocytes isolated from TLR2-/-mice and WT mice trans-endothelial migration showed the ability of lymphocytes from TLR2' mice was weakened(60.92±12.27/20X HP versus145.07±14.49/20X HP, P<0.001).In the clinical study, the serum level of TLR2showed no significantly difference among three groups (1380.1±891.36pg/ml,1033.9±860.57pg/ml,1433.8±805.75pg/ml, p>0.05), but the patients with level of Rentrop score3showed a significantly higher TLR2than other patients. The multiple ordinal logistic regression showed the level of TLR2was a strong independent association factor with the collateral Rentrop score (p=0.025).Conclusions:1,TLR2agonist enhanced migration of HUVEC in vitro, and increased the lymphocyte-permeability of HUVECs.2,In the mice acute hindlimb ischemia model by ligation of femoral artery, TLR2-/-mice showed delayed function recovery of ischemia limb, compared with C57BL/6wild-type mice. It is related with less collateral vessels developed in ischemia region in TLR2-/-mice than in C57BL/6wild-type mice.3. The less collateral vessles developed in ischemia region in TLR2-/-mice after ischemia injure could be related to reduced serum inflammatory cytokines TNF-a and IL-6levels, decreased VEGF expression, and weakened trans-endothelial migration of lymphocytes in TLR2knockout mice.4,The serum TLR2level was strongly correlated with collateral circulation in coronary heart disease patients, and the correlation was independent of age, diabetes mellitus, BMI, heart function and hyperlipemia. |
