Experimental Study On Anti-spasmodic Treatment And Prevention Of Coronary Artery Bypass Grafts

With a history of over 60 years, coronary artery bypass grafting (CABG) hasbecome one of the most common operations in cardiac surgery. Although the vein graftis still widely used, clinical practice has proved that the long-term effect is notsatisfactory, showing a 10-year maximum patency rate only 70%. According to thefollow-up data, great saphenous vein bypass showed higher incidence of atherosclerosisand vascular restenosis as compared with arterial grafts. In recent years, CABG hasbecome popular gradually in our country. With rapid development of surgicaltechniques and accumulation of clinical experience, there is growing recognition thatgraft choice, vessel harvesting and perioperative care are critical to the CABG outcome,which are the same important as skilled CABG surgical procedures. Since the greatsaphenous graft is not the ideal choice for CABG, people turn to radial artery that hasonce been discarded as arterial conduit. As an arterial bypass, radial artery has lowerincidence of atherosclerosis and higher short- and long-term patency rate, furthermore,radial artery conduit showed greater flexibility in surgical anastomosis due to itsoptimal length and internal diameter. Radial artery, an arterial graft with excellentelasticity, can better tolerate systemic pressure. Additionally, forearm complicationswere much fewer after radial graft harvesting. However, radial artery also has severaltroublesome shortcomings such as vasospasm tendency. Although the pathogenesis ofradial artery spasm is still unclear, it is well believed to be resulted from a combinationof factors such as mechanical injury during operation, vascular wall ischemia, local vasoactive substance relaese, neurohumoral and endocrine changes, difference in drugsensibility, vascular perfusion insuficiency due to intraoperative hemodynamicinstability, blood vessel dilation due to high blood pressure and so on. Meanwhile,intrinsic factors such as radial artery wall structure and physiology should also beconsidered. Therefore, doctors use vasoactive drugs in the prevention and treatment ofvascular spasm. This study aims to explore the best vasoactive drugs or compound,optimize drug dose and timing, as well as evaluate the final outcome, which will lay asolid foundation for future clinical trials and clinical experience accumulation. Inaddition, it is well evidenced that radial artery graft vasospasm is related to abnormalnitric oxide(NO) level. Based on rabbit CABG model and gene transfer technology,carotid artery graft was transfected with endothelial nitric oxide synthase (eNOS) geneand the transfection efficiency was assessed by various detective measures. Thesubsequent experiments were performed to compare graft spasm before and after genetransfection in order to explore the effect of eNOS gene transfection on graftvasospasm (feasibility and effectiveness of graft spasm prevention and treatment werestudied as well), which provided a theoretical basis for the clinical application of genetherapy. The whole thesis consists of four sections.Section 1 In vitro bypass graft model establishment forantispasmodic studiesUnder complex and ever-changing internal environment, vascular tissue is inresponse to various factors. In vivo study on etiology, pathogenesis, prevention andtreatment of vascular graft spasm can not guarantee that the vascular wall was underonly a single experimental factor, leading to a poor comparability between differentexperimental groups. Thus the results of in vivo study are not convincing. In this set ofexperiments, after being assessed and harvested, radial arteries were cut into 3mm-longvascular rings, which were mounted in Magnus bath and connected with otherequipments to collect experimental data such as resting tension. With tissue bath technique and previous experimental experience, we observed the effects of thevasoactive agents and other factors on resting tension of vascular ring. In this study, wealso explored to develop a more sophisticated experimental system, which could bettersimulate the in vivo environment for optimal vascular viability. Based onconcentration-resting tension curve for different vasoactive drug in human radial artery,the optimal drug concentration was determined for the subsequent experiments. Theendothelial integrity and its response to vasoactive drugs were assessed as well. Aimingto accumulate laboratory experience, this part made a preliminary experimental andequipment preparation for the subsequent experiments.Section 2 Effects of different factors on CABG graftsand analysis of graft spasmIn this section, we investigated the effects of different drugs or factors on theradial artery spasm. Vasospasm prevention and treatment with different drug or mixturewere also discussed. The subsequent experiments were based on the experimentalmodel mentioned in Section 1 and the vascular ring preparation technique mentionedpreviously(in brief, the human radial artery was harvested and cut into vascular rings,which were mounted in the organ bath chamber and then subject to a series of tests forvascular smooth muscle viability and endothelial integrity). First challenged withphenylephrine (10^-5M), vascular rings were incubated respectively with a certainconcentration of antispasmodic agent or mixture, namely nitroglycerin, verapamil,papaverine, phenoxybenzamine, milrinone, and the verapamil and nitroglycerin mixture(VG mixture). Vasorelaxant effect of these drugs (effect onset and efficacy) wasobserved at different time point and resting tension was recorded. Meanwhile, anotherset of experiments were performed. After 30-min pre-incubation with either verapamil,papaverine, phenoxybenzamine or VG mixture, potassium chloride (final concentrationof 60mmol/L) was added in the organ bath chamber and then vasoconstriction wasobserved subsequently. Finally, after 30-min pretreatment of different antispasmodic agent in the same way as described previously, the vascular rings were mounted inMagnus bath chamber and challenged with phenylephrine(10^-5M). Vascular spasticityand vosospasm duration were observed at different time point which might provideguidance for optimal timing of clinical application. The results showed that theseantispasmodic drugs relieved vascuar ring spasm with different duration, and that thesevasorelaxant drugs differed in effect onset and efficacy. The VG mixture relievedvascular ring spasm most rapidly (P＜0.05), followed in descending order bynitroglycerin, verapamil, milrinone and papaverine (P＜0.05). Phenoxybenzamineshowed the slowest onset, ranking at the bottom of the list (P＜0.01). In the second partof pretreatment experiment, 60mmol/L KCl only induced slight vasoconstriction ineither phenoxybenzamine, verapamil, or VG mixture group. Stronger contraction wasshown obviuosly in either nitroglycerin or papaverine group. The last part of thisexperiment was designed for time-effect study on these antispasmodic agents. Based ona four-hour-long observation of resting tension after 30 min antispsmodic pretreatment,we found that the best for graft spasm prevention was phenoxybenzamine (novasoconstriction was observed within the whole process of experiment), then VGmixture (vasorelaxant duration was two hour). Milrinone group showed a graduallyincreasing vasoconstriction, which peaked at a value only about sixty percent of themaximum resting tension; however, papaverine played a minor role in the prevention ofvasospasm. Our study showed that phenoxybenzamine and VG mixture were ideal forthe prevention of radial artery spasm. Although phenoxybenzamine showed a slowerantispasmodic onset, its duration was the longest, lasting over 4 hours. Inferior tophenoxybenzamine in the efficacy of vasospasm prevention, VG mixture still couldrelieve vascular ring spasm rapidly and effectively. Further study is needed to decidewhether mixture of the three (phenoxybenzamine, verapamil and nitroglycerin) canextend duration of vascular relaxant effect. Section 3 Experimental study on eNOS gene transfection ofcarotid artery in rabbit CABG modelPrevious experiments have proved that radial artery spasm is related to a relativedecrease in eNOS quantity and activity, resulting in the reduced local release of NO. Inthis study, transgene technology was applied to induce an excessive eNOS expression,leading to vasorelaxation by inhibiting voltage-dependent and receptor-dependent ionchannels. We first tried to establish a rabbit CABG model with autologous femoral-carotid bypass. A replication defective adenovirus vector encoding eNOS (AdCMVeNOS)gene was transfected into rabbit vascular graft(autologous femoral artery). Rabbits weresacrificed 30 days after eNOS gene transfection and the targeted vascular segmentswere harvested. Exogenous gene expression was detected by RT-PCR methods; eNOSprotein, proliferating ceil nuclear antigen (PCNA), smooth muscle cells (myofibers)and actin (alpha-actin) expression in the vascular wall were determined by SABCimmunohistochemical method; PCNA and eNOS protein content were detected bysemi-quantitative western blot analysis. We compared the eNOS-tranfected grafts andnon-transfected femoral arteries, analyzed the eNOS expression pattern, eNOS activity,NO level in the vascular wall and so on, as well as discussed the origin of difference,feasibility and effectiveness of eNOS gene transfection. Our work provided atheoretical basis for the clinical application of gene therapy in this aspect. Our studyproved that normal rabbit femoral artery wall showed only a weak eNOS geneexpression, but eNOS-transfected graft had a significant increase in eNOS proteinexpression(P＜0.01). Total NOS activity and NO content in the vascular wall increasedsignificantly than those before gene transfection(P＜0.01). PCNA andα-actingexpression were decreased significantly in the vascular wall after gene transfection(P＜0.01). Thus, this experiment proved that gene transfer technology can upregulateeNOS gene expression and enzyme production in the vascular wall further increase theNO concentration, highlighting the bright future of gene transfection technology in solving clinical problems associated with eNOS lack or insufficiency on the gene orprotein level.Section 4 Experimental study on the changes of graft spasmodiccharacteristics after eNOS gene transfectionConventional therapy can not prevent arterial graft spasm effectively. In order tofind an effective therapy to control the graft vasospasm and confirm a hypothesis thateNOS gene upregulation can relieve vasospasm by increasing NO production to inhibitthe voltage-dependent and receptor-dependent pathway, we first established, inaccordance with the method mentioned previously, a rabbit CABG model withautologous femoral-carotid bypass. Vascualr grafts (arteries) were transfected witheNOS gene and the targeted vessles were harvested 30 days later. Comparison beforeand after eNOS gene transfection was performed as follows: 1. Challenged withphenylephrine at different concentration to induce vasospasm of varying degrees, thesevascular grafts were used to compare the changes of graft spasmodic characteristicsafter eNOS gene transfection (a genetic manipulation leading to various NO content inarterial vascular wall). 2. Vascular contraction of antispasmodic-pretreated vascularring was studied after incubation with 60 mM KCl solution. 3. After phenylephrinechallenge (10^-5M)to induce vasospasm, the vascular rings were incubated with VGmixture(see Section 2)to study its vasorelaxant property at different time point. Ourstudy showed that eNOS-transfected grafts were superior to non-transfected ones inboth vascular contractility and drug sensitivity to antispasmodics (P＜0.01). Thissuggests eNOS gene transfection can relieve vascular spasm and reduce the incidenceof arterial graft spasm. In conclusion, eNOS gene transfection is somewhat valuable forclinical application.