Experimental Study On Coronary Artery Bypass Graft And Prevention Of Restenosis After The Operation

Background: CABG has been generally acknowledged as one of the most operative therapy for coronary heart diseases, but the invaded CABG procedure doing inevitable impairment to coronary artery and bridge vessels, at the same time, the old lesions unceasingly progressing or the bridges vessel occuring accelerated atherosclerosis after operation all lead to restenosis. How to improve the rate of short-term and long-term vessel patency after CABG is the probe of coronary artery surgery continues to vex surgeons at present.Restenosis after CABG was a complex biological process. Injury of VEC caused by CABG evoking several linkaged pathologic processes is an important etiological factor of restenosis. VEC is the main regulator managing structure and function of vessel in coronal microcirculation. The balance of relaxation factors and contraction factors expressed by VEC plays a major role in maintaining normal vessel tension. VEC can express several intercellular adhesion factors regulating the start and expanding of inflammation and maintaining the stable of hemorheology in coronal microcirculation. In addition, VEC can also express several growth regulating factors contribute to the plerosis and renewing of structure of coronary artery. Injury of CABG results in alteration of phenotype in impaired or regenerated VEC, and the disturbing of expressing biological active factors can develop succeeded interaction and several chain reactions with other pathologic factors. These changes further make coronary artery losting self-regulational dilatation and developing spasm, inflammation and secondary restenosis diseases (such as thrombosis, abnomal migration and proliferation of VSMC, excessive ECM or lipid deposition and atherosclerosis).Injury caused by CABG and lumen shear stress after bypass graft can up-regulate VEC expressing intercellular adhesion factors ( such as P-selectin, PECAM-1, β2-integrin, ICAM-1 and vWF), and evoke many immune identification molecules and inflammation factors (such as TGF-α, IL-6, LT-α and IL-10) gene developingpolymorphism changes. Those changes result in the destruction of regulating balance of inflammation and immune reaction, evoking inflammation of local intima of coronary artery. Inflammation is another important link of pathogenesis of coronary stricture after CABG, for that many factors derived from inflammation can activate the pathogenesis thrombosis and hyperplasia of endometrium, yield interaction with lipid deposition, facilitate atherosclerosis and evoke the reactive migration and proliferation of VSMC.VSMC migration from media to intima and proliferation are central events in vascular pathobiology and play another major role in deterioration of stenotic and restenotic lesions after CABG. The modulation of some phenotypes of VSMC lead to migrate and proliferate abnormally after injury or ischemia, and recent studies have suggestted that p53 and p53 targets such as the CDK inhibitor p21 are critical regulators of human VSMC proliferation and apoptosis in restenosis after CABG. Increasing expression of p53 after injury limits VSMC proliferation in vivo, correlates with apoptosis and a low level of cell proliferation. Stenotic lesions after CABG involves pathological changes in the normal structure and function of the arterial wall that depend critically on interactions between vessel wall cells and their extracellular environment, and the increasing expression of type VIII collagen after injury promot VSMC migration and proliferation in vivo. p53 and type VIII collagen are two important marker of phenotype modulation of VSMC.It is thus clear that, endothelial protection, suppression of inflammation, thromobosis and inhibiting abnomaly reactive migration and proliferation of VSMC are the key of a succeed preventional therapy.Objectives: To investigate the mechanism of restenosis and explore an operative preventional therapy, a typical and safe animal mimesis of local coronary artery stricture and myocardial infarction is essential. Impairment of magnus artery in vivo or vessel in vitro (such as common carotid artery) has been widespread accepted as the replacement at present, but because of discrepancy with coronary artery in structure and physiology, the replacement can not scientifically reflect the true mechanisim of diseases or therapy. Many scholars advocate to prevent restenosis after CABG via endothelial protection. Although the improving of technology of CABG such as 'off pump' can decrease injury to VEC in some extent, but the injury of vessel and cardiac muscle can hardly be avoided, and the effect is limited because of old coronary artery disease. To date, drug therapy(such as application of L-arginine, antagonist of calcium ions, angiotention-converting enzyme inhibitor agent and so on) have little effect in preventing restenosis. Investigates of gene therapy are still in stage of background investigation, there are a lot of probes and risks to be put into practice.In our studies, we adopted a method of ligating canine LAD to make a research model of local coronary artery stenosis and injury of VEC and myocardial infarction, and explored whether or not such method is a safe and resonable suit to the study in coronary artery surgery area, by analyzing pathological and physiological changes after ligation. We applicated ligustrazine via vein injection at different times before ligation, and try to find out the preventional effect of ligustrazine to acute ischemical reperfusion injury of local VEC and cardiac muscle after ligation. After that, we performed CABG operation, the left pediculated ITA were anastomosed to the distal part LAD posterio to the ligating part, and observed the inhibiting effects of ligustrazine on the injury of local VEC and cardiac muscle caused by invaded procedure and ischemia reperfusion of CABG, and analyzed the inhibiting effects of ligustrazine on inflammation, thromobosis and reactive migration and proliferation of VSMC after injury, to explore whether or not ligustrazine has an operative pharmacological preventional effect on restenosis after CABG.Methods: Experiments were performed in two stages. In first phase: We fabricated a desease model of local coronary artery stricture and injury of VEC and myocardial infarction by ligating canine LAD. 45 aged healthy cross-breed dogs (14.79±6.74) kg were disparted into three groups: Ligustrazine (TMP) group , verapamil (VER) group , control (CON) group, each group had 15 animals. All animals were performed operations to ligate the near part of the LAD 0.5 cm part from the origin through left minimal thoracic incision, to interdict 75% of the flux respectively under the instruction of measuring coronary artery flow rate by an aseptic detecting head of doppler ultrasound on surface of heart. We used ligustrazine in TMP group via vein injection lOmg/kg in 20min before ligation, and lOmg/kg/d in two days before operation, and used verapamil in VER group 0.5mg/kg in 20min before ligation and 0.5mg/kg/d in two days before operation, and saline in CON group at same time as a contrast. Blood samples collected from right atrium appendage before ligation and in 5min, 15min, 30min, 45min, 60min after ligation, were storaged and measured under instruction of thespecifications of different test kits. Endothelium-derived factors ET-1, TXB2, 6-keto-PGFia were measured using immunoradiometric method, and NO was detected by nitrate reductase method. Before ligation and in 30min, 2min, 3h, 6h, 12h, 24h after ligation, blood samples were collected via vein to measure plasma CTnT by 'one-step' method and sP-selectin by ELISA. During the operation, ABP, LAP and HR were continuously mearured by an electrocardioscanner.In second stage: All stock dogs feeded with forage made with high cholesterol formula after ligating LAD for two months, were regrouped into three groups again: TMP group(15), VER group(15), CON group(lO). All dogs were performed CABG operation, and left pediculated ITA were grafted to the LAD posterio ligating part. Ligustrazine (lOmg/kg/d) were used in TMP group via vein injection in 3d before CABG and 7d after operation continuously, and 800mg/L ligustrazine solution were used to wash local anastomosis sites. Verapamil was used in VER group (0.5mg/kg/d) at the same times before and after operation, and 40mg/L solution were used to wash. Saline was used in CON group as a constrast. Blood sample was collected from right atrium before anastomosis and in 5min, 15min, 30min, 45min, 60min after anastomosis. ET-1 were measured using immunoradiometric method, and NO was detected by nitrate reductase method. We collected blood via vein before anastomosis and in 3h, 6h after anastomosis to measure plasma CTnT, and gathered blood sample before anastomosis and in 30min, 2h, 6h, 12h, 24h after Anastomosis to measure plasma sP-selectin by ELISA. In 2h, 12h after CABG, we cut parts of local cardiac muscle supply by LAD, and homogenized the myocardium tissues to analyze P-selectin expression by Western blotting. All animals were killed 3 weeks later, LAD and local cardiac muscle were harvested. Some segments of LAD tissues were homogenized to detected type VIII collagen and p53 expression by Western blot analysis, and parts of LAD were harvested and snap frozen in liquid nitrogen to be detected the expression of p53 and type VID collagen in vivo by immunohistochemistry. Other tissue segments of LAD and the patches of topical cardiac muscle stained by HE and Masson were observed the pathological changes by optical microscope, and segments of LAD and topical cardiac muscle of three groups were also observed the changes of ultrastructure and apoptosis by electronic microscope.Statistical analysis Results of all measurements data are expressed as Mean±Std.Statistical analysis was performed by repeated measure analysis of variance followed by a paired two-tailed student's t test using SAS 8.2 version, to analyze the difference among groups and within group. Statistical significance was set at P< 0.05.Results: In first phase: Measurements of plasma NO, ET-1, TXB2, 6-keto-PGF)a, sP-selectin and CTnT all had significant difference among three groups in diferent time after ligating LAD. Model, Time and Time*Group all has significance ( PO.01) . Contrast with before ligation, measurements of plasma NO, 6-keto-PGFia in different times after ligation had notable descent in VER and CON group (PO.05), and ET-1, TXB2, CTnT and sP-selectin had siganificant step-up (P>0.05), but measurements of all plasma samples had not siganificant changes in TMP group compared with the measurements before ligation (PO.01). Curves of changes of ABP, LAP and HR in three groups had siganificant differences after ligating LAD.In CABG experiments: Measurements of plasma NO, ET-1, sP-selectin and CTnT also had significant difference among three groups in diferent time after anastomosis. Model, Time and Time*Group all has significance ( PO.01) .Within TMP group, conmpared with measurements before anastomosis, the measurements of plasma NO had not notable descent, and ET-1, CTnT, sP-sel had not striking step-up (P>0.05), but all measurement of different times after anastomosis had siganificant changes in VER and CON groups compared with measurements before anastomosis. Protein extracted from homogenate cardiac muscle of representative TMP, VER and CON groups after CABG were analyzed for P-selectin expression by Western blotting . The expression of P-selectin was very low in nomal cardiac muscle, but was up-regulated after CABG, a far more intense expression pattern was found in VER group and CON group. Type VIII collagen expression in injuried left anterior descending of ligustrazine groups were significantly lower than those of other two groups. p53 expression of injuried arteries were obviously up-regulated in ligustrazine group. Type VIII collagen expression in uninjured proximal arteries anterior anastomotic site was minimal. In contrast, specific staining for type VIII collagen in vivo of sections from distal injured arteries posterio anastomotic site of three groups all showed obvious, and they were robust in VER, CONTROL groups than those in TMP group. Special stain of immunochemistry demonstrated that p53 were not detectable in normal vessels and VER and CON group.In contrast, positive p53 were detectable in the specimens of TMP group. Under optical and electronic microscope, there were only some mild injury and structure damage in endothecium of coronary artery, the resilient layer had not become distict thinner in TMP group. In later two groups, there were serious damages and absence of endouthelium in endothecium of coronary artery, the resilient layer had notablely become thinner, and the vessel wall had notable hyperplasia and transfer of VSMC with more vacuole inside, and there were serious infarction and fibrosis after infarction in cardiac muscle.Conclusions: 1. The injury of local VEC and cardiac muscle caused by ischemia after blocking 75% influx by ligating canine LAD is significant, and this blocking level does not result in the death of model animal. Such method to make a research model of local coronary artery stenosis, VEC injury and myocardial infarction by ligating canine LAD, is a safe and reasonable, suit to the study of coronary artery surgery.2. Analyzing the results of the changes of measurements in different time before and after ligating LAD, including: endothelium derived factors, CTnT and sP-selectin, suggestting that ligustrazine has a significant effect of suppressing acute ischemical reperfusion injury of local VEC and cardiac muscle after ligating LAD, and may have the role inhibiting the pathogenesis inflammation , thrombosis caused by VEC injury.3. The invading procedure of CABG doing notable impairment to coronary artery and bridge vessel, and causing a serial linkage pathological processes, are the major reason of restenosis after the operation. On the basis of analyzing the results of the changes of measurements in different time before and after CABG, including: endothelium derived factors, CTnT and sP-selectin, and observing local pathological changes, suggestting that ligustrazine can relieve such injury, and supress hyperplasia of endometrium, lipid deposition, atherosclerosis, abnomal reactive migration and proliferation of VSMC evoked by VEC injury, thus develop profitable affect to the thickening of neonate intima and replastics of vessels after CABG. Ligustrazine may have an operative pharmacological preventional effect on restenosis after CABG.