| Objective Surgical bypass using autogenous vein provided an effective treatment for many patients with coronary or peripheral therosclerosis. However, the vein graft restenosis occured in a significant percentage. The pathophysiology of vein graft failure involved a complex mechanism. Conventional pharmacotherapy had limited impact on graft failure. Rapamycin was an immunosuppressive agent which also exhibited antiproliferative properties. Rapamycin coated stents had been demonstrated to suppress restenosis in experimental and clinical studies of percutaneous coronary catheter intervention. PLGA (hydroxy acid and lactic acid copolymer) as a good carrier of controlled release drug could increase the drug retention time in the body, release drug at a constant velocity. However, the study of local infusion intravascular with rapamycin nanoparticles inhibiting neointimal hyperplasia in vein graft had not been reported. Therefore, the aim of this study was to observe the effect of vein graft after local infusion intravascular with rapamycin PLGA nanoparticles on animal experiment and to investigate the effect of plasma adrenomedullin and endothelin-1 in patients with on-pump and off-pump coronary artery bypass grafting on clinical trial. The specific purpose on animal experiment of the present study was described as follows: the established vein bypass graft models in rabbits were verified if vein graft restenosis could be caused. The correlation of pathological examination and color Doppler echocardiography was analyzed to evaluate the reliability of color Doppler echocardiography in detecting the degree of vein graft stenosis in the rabbit model. A good bioadhesive rapamycin nanoparticle was prepared, and its physicochemical and biological characterizations were evaluated. The perfect biological compatibility and drug-release capability of rapamycin nanoparticle were verified. After rapamycin nanoparticle was applied locally through intravascular infusion in vein graft, its concentrations in vein tissue and plasma were tested, the side effects of liver and renal function monitored, the vein graft intimal and plasma adrenomedullin (ADM) and endothelin-1 (ET-1) levels detected. The relationship between vein graft restenosis and plasma adrenomedullin, between vein graft restenosis and plasma endothelin-1 were analyzed.Methods The study was divided into two parts. One part was animal experiment. The other was clinical trial.Firstly, the vein bypass graft model, which external jugular vein was end-side anastomosed at the proximal and distal side of the ligated carotid artery was established in a rabbit. Eighteen rabbits were conducted. Six rabbits were euthanized at the seventh, fourteenth, and twenty-eighth postoperative day respectively and pathological examinations of the vein graft were made. Before enthanizing, Color Doppler echocardiography was done to detect the thickness of endothelium, internal diameter, the maximum flow velocity, the minimum flow velocity, pulsatility index (PI) and resistance index (RI) of the graft vein. The contralateral external jugular vein was regarded as control. The correlation of color Doppler echocardiography and pathological examination was analyzed.Secondly, rapamycin nanoparticle was prepared and its physicochemical characterization was evaluated. Solvent diffusion was applied to prepare for bioadhesive rapamycin nanoparticle, using different concentrations carbopol 940 or PVA as stabilizers. The physicochemical characterizations were evaluated in terms of morphology, average particle size and particle size distribution, Zeta potential, encapsulation efficiency, the drug loading and in vitro release. The biological characterization of the nanoparticle was evaluated in a rabbit model. The experiment model was divided into two groups. Each group had five rabbits. One group was treated with free nanoparticles saline infused into graft vein through a self-made drug infusion device. The other group was treated similarily with rapamycin nanoparticle At the seventh day after the treatment, the morphology of vein graft was analyzed under microscope to observe perivascular inflammatory reaction.Thirdly, the pharmacological behavior of different concentrations rapamycin nanoparticles treatment locally on vein graft was investigated. The rabbit vein graft model was divided into the low concentration group and the high concentration group according to treated rapamycin nanoparticles concentrations (100μg/ml and 500μg/ml). Each group had twenty rabbits. At the first, seventh, fourteenth and twenty-eighth postoperative day, five rabbits were euthanized respectively for detection of rapamycin concentrations in plasma and vein tissue by high performance liquid chromatography (HPLC). Their liver and kidney function were tested at the same time. At the twenty-eighth postoperative day, pathological examinations were made for the vein graft intimal thickness.Lastly, the variation of plasma endothelin-1 (ET-1), adrenomedullin (ADM) levels and vein graft intimal after local application of rapamycin nanoparticles were observed in rabbit vein graft models. Thirty rabbits were conducted and divided into the experimental group which high concentrations of rapamycin nanoparticles (500μg/ml) was used to deal with vein graft in the same way, the control group (peripheral venous injection with the same rapamycin nanoparticles) and the blank group (without treatment). At preoperation, postoperation immediately, the 1st and 7th postoperative day, the blood samples were extracted to measure the concentrations of plasma ET-1 and ADM by radioimmunoassay in three groups. At the twenty -eighth postoperative day, the graft external jugular vein in rabbits were removed for Hematoxylin and Eosin(HE) and Van Gieson(VG) eosin to analyze the intimal thickness, internal diameter, intimal/media thickness ratio and collagen volume index. The correlation of plasma ET-1 and ADM concentrations and the intimal thickness, internal diameter, intimal/media thickness ratio and collagen volume index of vein graft were analyzed by pearson correlation.On clinical trial, patients with on-pump and off-pump coronary artery bypass grafting and general thoracotomy were conducted. At preoperation, postoperation immediately, the 1st, 7th and 14th postoperative day, the concentrations of plasma ET-1 and ADM were measured by radioimmunoassay. Results 1. Compared with the contralateral external jugular vein, the intimal of vein graft was thickened, the internal diameter narrowed, the maximun and minimun flow velocity slowed, PI and RI increased on color Doppler echocardiography after operation. There were significant statistical difference (P<0.05). And with the extension of time, the above mentioned criteria had changed more obviously. Which implied that there was significant statistical difference (the seventh postoperative day vs. the fourteenth postoperative day, the seventh postoperative day vs.the twenty-eighth postoperative day, and the fourteenth postoperative day vs. the twenty-eighth postoperative day). On pathological examination, the intimal of vein graft after operation was thickened, the internal diameter narrowed, too. Compared with the contralateral normal external jugular vein, the difference had statistical significance (P<0.05). And there were significant statistical difference between the different time point (the seventh postoperative day vs.the fourteenth postoperative day, the seventh postoperative day vs.the twenty-eighth postoperative day, and the fourteenth postoperative day vs. the twenty-eighth postoperative day). The results of color Doppler echocardiography and pathological examination showed positive correlation in vein graft intimal thickness and internal diameter (r = 0.905, r = 0.971, r = 0.928; r = 0.924, r = 0.829, r = 0.940).2. The rapamycin nanoparticle was successfully prepared with solvent diffusion. Its average diameter was about 200nm and encapsulation efficiency was about more than 70 percent. The surface of nanoparticle had negative charge. The drug release in vitro was fit to Higuchi release model and was performed with sustained-release phase. No neutrophil infiltration was found in perivascular tissue at the seventh postoperative day both in rapamycin nanoparticles treatment group and saline control group. The biological compatibility of rapamycin nanoparticle was proved perfect.3. At the first day after treatment with low and high concentration rapamycin nanoparticles, a certain concentration of rapamycin in the vein tissue could be detected. The high concentration rapamycin nanoparticle was about (8.88±0.26)μg/10 mg and the low concentration was (6.24±0.24)μg/10 mg. At the seventh day after operation, the drug concentration arrived at its peak. At the fourteenth day after operation, the drug concentration stood at the peak level steadily. At the twenty-eighth day after operation, the drug concentration began to decline. The rapamycin in plasma could be detected at the first postoperative day, but its concentration was very low. The high concentration rapamycin nanoparticle was about (1.00±0.03)μg/10mg, the low concentration was (0.51±0.02)μg/10mg. Compared with the vein tissue, there was significant difference (P<0.05). At the seventh postoperative day, the drug concentration in plasma reached the peak point, however it was still at a low level. It declined significantly at the fourteenth postoperative day, and declined to zero at the twenty-eighth postoperative day. The drug concentrations of plasma and vein tissue in the high concentration group were always higher than those in the low concentration group at the same postoperative day (P<0.05). The drug concentrations of vein tissue were always higher than those of plasma, too (P<0.05). The liver and kidney function were normal at each postoperative day both in low and high concentration rapamycin nanoparticles group. At the twenty-eighth postoperative day, the graft vein intimal thickness got less significant in high concentration rapamycin nanoparticles than that in low concentration rapamycin nanoparticles (P<0.05).4. At the twenty-eighth postoperative day, compared with the normal external jugular vein, the vein graft intimal was thickened obviously, the internal diameter was significantly narrowed, the intima/media thickness ratio increased and the collagen volume index increased significantly in the blank group and the control group in vein graft HE and Van Gieson eison stain. The above mentioned indicators in the experimental group had no apparent change compared with the normal external jugular vein (P>0.05). There were significant difference between experimental group, the control group and the blank group (experimental group vs. control group, and experimental group vs. blank group). The plasma ET-1 and ADM concentrations had no significant statistical difference in three groups before operation (P>0.05). Immediately after operation, the plasma ET-1 and ADM concentrations were increased in three groups, but the experimental group increased slightly. Compared with the preoperative level, the difference had statistical significance (P<0.05). But between the control group and the blank group, there was no significant difference (P>0.05) in plasma ET-1 and ADM. There were significant difference between the experimental group and the control group, and between the experimental group and the blank group. At the first postoperative day, the plasma ET-1 and ADM concentrations started to decline. At the seventh postoperative day, the plasma ET-1 and ADM concentrations returned to the preoperative level in the experimental group, but in the blank group and the control group, they stood at a high level. There were strong correlations between plasma ET-1, ADM concentrations and the intimal thickness, internal diameter, intimal / media thickness ratio and collagen volume index of vein graft.5. In comparison of preoperative plasma ADM and ET-1 concentration, there was no significant difference in three groups. But they increased immediately after operation in the off-pump and on-pump groups, and increased more significantly in the on-pump group. Compared between the off-pump group and the on-pump group, there were significant difference (P<0.05).The plasma ADM and ET-1 levels started to decline at the first postoperative day, and continued to decline at the fourteenth postoperative day. But their concentrations in the off-pump group and the on-pump group still stood at a higher level than normal. The concentrations of plasma ADM and ET-1 in the on-pump group were higher than those in the off-pump group at each postoperative day. However, in the thoracotomy group, the concentrations of plasma ADM and ET-1 had almost not increased after operation. Compared with preoperative level, there was no significant difference (P>0.05). Their concentrations declined quickly and closed to normal level at the first postoperative day.Conclusion 1. The rabbit autologous graft vein model in which external jugular vein was end-side anastomosed at the proximal and the distal side of the same carotid artery could lead to vein graft restenosis. Color Doppler echocardiography was a new experimental technique which might be used to observe the vein graft stenosis continuously and noninvasively. Color Doppler echocardiography and pathological examination had good correlation in evaluation of vein graft restenosis. 2. The rapamycin nanoparticle which was prepared with solvent diffusion in the experiment had a certain biological adhesion, high encapsulation efficiency and slow-release effectiveness. It showed good biocompatibility with rabbit vessels without perivascular inflammatory reaction.3. Rapamycin nanoparticles could maintain high concentrations in the vein tissue after local application, but a few rapamycin nanoparticles in plasma could be detected. The different concentration of rapamycin nanoparticle could release different concentrations in the local tissues and plasma. The effective time of rapamycin nanoparticles could maintain for several months, and had no damage and no significant toxicity to other organization. The different concentration of rapamycin nanoparticle could inhibit graft vein intimal hyperplasia differently.4. Rapamycin nanoparticles were applied locally for the vein graft which could reduce the release of vasoactive peptide after the injury of endothelial cell, decrease the plasma concentration of ET-1 and ADM, and protect the vascular endothelia function. It could inhibit vein graft intimal hyperplasia, and prevent the restenosis of vein graft. The function of rapamycin nanoparticles applied through peripheral venous had no less. There were strong correlations between plasma ET-1, ADM concentration, and the intimal thickness, internal diameter, intimal/media thickness ratio and collagen volume index of vein graft.5. Both on-pump and off-pump coronary artery bypass grafting could cause endothelial dysfunction, increase plasma levels of ET-1 and ADM. But compared with the on-pump coronary artery bypass grafting patient, there was less affect in the off-pump coronary artery bypass graft patient. The effect of ET-1 and ADM in general thoracotomy had no less.
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