An Experimental Study Of Gene And Drug Therapies To Stabilizethe Vulnerable Plaques

BackgroundRecent studies show that atherosclerosis is a chronic inflammatory disease that is caused by multiple processes, including infiltration of inflammatory cells, proliferation of smooth muscle cells, increase in extra cellular matrix and thrombus formation. Morphological features of vulnerable plaques include a thin fibrous cap and a large core of lipid with many inflammatory cells such as monocytes/macrophages. The risk of plaque rupture depends on plaque type rather than plaque size. Therefore, major determinants of plaque vulnerability and rupture are progressive lipid accumulation (core formation) and cap weakening due to ongoing inflammation with collagen degradation (macrophage-related) and impaired healing and repair (vsmc-related). In early atherosclerotic lesions, accumulation of monocytes/macrophages is noted in the intima, while the accumulation of these inflammation cells is one of the reasons that lead to the rupture of plaques. Recruited macrophages to the vessel wall promote progression of atherosclerosis by producing and releasing various cytokines, chemokines and growth factors. Among these mediators monocyte chemoattractant protein-1 (MCP-1) is a potent and specific chemokine for the recruitment and activation of monocytes/macrophages. It coulddirect the migration of monocytes from the blood stream to the vessel wall and plays an important role in developing atherosclerosis.MCP-1 belongs to a CC chemokine subfamily of chemokines, it was the first one cloned in this family. Yoshimura isolated the cDNA encoding human monocyte chemoattractant protein-1 (MCP-1) from glioma cell line culture fluid in 1989. Screening of a cDNA library from total poly (A) RNA of glioma cell line U-105MG yielded a clone that coded for the entire MCP-1. Nucleotide sequence analysis and comparison with the amino acid sequence of purified MCP-1 showed that the cDNA clone comprises a 53-nucleotide 5'-non-coding region, an open reading frame coding for a 99-residue protein of which the last 76 residues correspond exactly to pure MCP-1, and a 389-nucleotide 3'-untranslated region. The hydrophobicity of the first 23 residues is typical of a signal peptide.Jarnagin and others deleted the N-terminal amino acids 2 to 8, called mutant of the MCP-1 gene or 7ND, they found the amino terminus of MCP-1 was essential as truncation of residues 2-8 ([l+9-76]huMCP-l) results in a protein that cannot stimulate chemotaxis. It should be possible to trap CCR2 in conformational states that are unable to stimulate all of the pathways required for chemotaxis.Egashira and his research group transfected the N-terminal deletion mutant of the MCP-1 gene into the ApoE-KO mice' skeletal muscles, found that it not only attenuated the development of early atherosclerotic lesions but also limited progression of preexisting atherosclerotic lesions.In this regard, the promise of 7ND gene therapy in atherosclerosis is high, but it has not been used in the vulnerable plaque model. This strategy is a challenge that highlights the need for therapeutic options in stabilizing vulnerable plaques.Therefore, the primary aim of our study was to test the hypothesis that targeting MCP-1 might be a useful and practical form of gene therapy in the stabilization of the vulnerable plaques .The mutant MCP-1 lacking N-terminal amino acids 2 through 8, might block the MCP-1 signal as dominant-negative inhibitor of MCP-1.It is known that statin and other drugs were effective in stabilizing the vulnerableplaque, but matrix metalloproteinase inhibitor, immunosuppressant, and Chinese traditional medicine haven't been administered to the vulnerable plaques model. Both doxycycline and rapamycin are macrolides antibiotic and now they have been recognized as as matrix metalloproteinase inhibitor and immunosuppressant, respectively. Rapamycin has also proven effective in inhibiting restenosis in humans when eluted from implanted stents. Chinese traditional medicine-tongxinluo has proved valuable in modulating the liplid profile and improving the function of injuried endothelium. The aim of the study was to investigate the possibility and molecular biological mechanisms of these drugs in stabilizing vulnerable plaques in animal models. Objectives1 .To establish a vulnerable plaque model with a simple, efficient and time-saving technique which is mimic to plaques in patients and is available for assessment of gene and drug treatment. And furthermore to evaluate the merits of pathology, sonography and serum tests in detecting vulnerable plaques.2.To construct an eukarytic expressing vector of GFP-fused huMCP-1 mutant -7ND, which lacked the N-terminal amino acids 2 through 8 of huMCP-1 by recombinant PCR, and to evaluate the efficiency and mechanisms of this vector in stabilizing vulnerable plaques.3. To explore the possibility and mechanisms of drug treatment with higher-dose simvastatin, doxycycline, rapamycin and Chinese traditional medicine-tongxinluo in stabilizing vulnerable plaques. Methods1. Establishment of the rabbit vulnerable plaque modelOne hundred and twenty male New Zealand White Rabbit were randomly grouped into three groups: group A (n=8), group B (n=16), group C (n=96). Rabbits in group A were fed standard chow , rabbits in group B were fed cholesterol-rich diet (a normal chow supplemented with 1% cholesterol), while rabbits in group C were underwent balloon-induced abdominal aortic wall injury and then fed the cholesterol-rich diet. At 8th week, rabbits in group C were randomly divided into eightsubgroups: group Cl was baseline (n=16), C2 was gene treatment group (n=20), C3 was simvastatin treatment group(n=10, 5mg/kg/d), C4 was doxycycline intervention group(n=10, lOmg/kg/d), group C5 was administered rapamycin(n=10, 0.5mg/kg/d), group C6 was given tongxinluo(n=10, lg/kg/d), group C7(n=10) changed to standard chow as regression control group, group C8(n=10) was control group. The treatment groups and control group were still fed the cholesterol-rich diet. At the end of eight weeks, the rabbits in group A, B and Cl all underwent pharmacological triggering with injection of Chinese Russell's viper venom (CRVV) and histamine, while gene treatment group was triggered at 28th after transfection and the other groups at the end of 12 weeks after intervention.2. The experiment of gene therapy(1) To establish expressing vector of GFP-fused huMCP-1 mutant (7ND): By using recombinant PCR to delete the N-terminal amino acids 2 through 8 of human monocyte chemoattractant protein-1 and gene cloning technique to construct pIRES-EGFP-7ND eukarytic expressing vector.(2) In vitro: The pIRES-EGFP-7ND was transfected into HK293 with lipofectamine mediated. After transfection of 48h, the 7ND expression in transfected HK293 were observed and detected under fluorescence microscope and by ELISA respectively. The 7ND mRNA was detected by RT-PCR. The transwell chamber was used to assay 7ND's chemotaxis.(3) In vivo: Randomly divided group C2 into group C2a (pIRES-EGFP-7ND transfected group, n=10) and group C2b (pIRES-EGFP transfected group, n=10), subsequently transfected them with lipofectamine into the rabbits' skeletal muscles. After transfection of 3 days, randomly sacrificed one rabbit in each group and observed the expression of 7ND under fluresence microscope. The expression of 7ND was detected by ELISA before and after transfection of Id, 3d, 7d, 14d, 28d.3. Laboratory examinations: Blood samples were collected in different period to measure lipid profile and fibrinogen. Using sandwich ELISA to quantify the amount of different inflammation mediators such as hs-CRP, IL-8, MMP-1, P-selectin and MCP-1.4. Blood drug concentration monitoring: The blood doxycycine concentration in group C4 was monitored by using HPLC after treatment of one day, one week, four weeks, eight weeks and twelve weeks respectively.5. Examinations the features of vulnerable plaques: The rabbits' atheroslcerosis plaques in abdominal aortas were detected with high frequency ultrasound, AD technique and intravascular ultrasound (IVUS) respectively. Intima-media thickness (IMT), blood flow velocity (Vp, Vm, VTI), tissue characteristic, plaque area, plaque distribution and plaque rupture were examinated. The rabbits as models were underwent the examinations above at the beginning of the experiment, pre-and post-pharmacological triggering at 8th week, rabbits in gene therapy group at 28 days after transfection, while the other groups at the end of 12 weeks after intervention.6. Histologic evaluation: The abdominal aorta were excised and examined by red oil O stain, picrosirius red stain and immunohistochemical stain of RAM-11 (anti-rabbit macraphage), a -actin (anti-smooth muscle cell), MCP-1, NF- k B p65, MMP-1, MMP-3, MMP-12, TIMP-1 and P-selectin in local lesions respectively, and the vulnerability index was calculated by dividing the area of (macraophage +lipid) by that of (SMC+collagen). The intimal surface was examined with scanning electron microscope, using transmission microscope to observe the ultrastructure of the smooth muscle cell.7. Reverse-transcriptase polymerase chain reaction: To determine the mRNA expression of various inflammation medicators such as MCP-1, MMP-1, MM-3, MMP-12 and others in the abdominal arterial atherosclerosis lesions.8. Western-blot: To examine the protein expressions of MCP-1, MMP-1, MM-3, MMP-12 and others in the lesions.9. Statistic analysis: Continuous variables are reported as mean±deviazione standard (SD) and have been compared using t test, categorical variables by x2 test. The relationship between variables was estimated by correlation. For all analysis, a p value less than 0.05 was considered significant, using a statistical programme (spss version 11.0).Results1 .The rabbit vulenerable plaque model(1) After given balloon-induced abdominal aortic wall injury and cholesterol-rich diet for eight weeks, the rabbit vulnerable plaque model was successfully produced. Plaque rupture and thrombosis occurred in six rabbits (54.5%) from the remaining eleven rabbits in group Cl after pharmacological triggering, while there were no plaque rupture in group A and B. Three rabbits had acute ischemic stroke in brain in group Cl, and one in group C4, C7 and C8 respectively. Group Cl was divided into plaque ruptured group and nonruptured group according to the events occurred or not, which includes the plaques ruptured or acute ischemic stroke in brain attacked.(2) Lipid profile: TC was increased from( 2.12 ±0.50)mmol/l to( 28.0 ± 8.09)mmol/l ( PO.05) by 27 folds, TG from (0.98±0.56)mmol/l to (3.08± 2.47)mmol/l by 2 folds, LDL from( 0.76 + 0.52) mmol/1 to (24.9±7.50)mmol/l by 31 folds in group Cl after 8 weeks' cholesterol-rich diet, marked increase than group A( P<0.05 ) , there was no significant difference of lipid profile between group B and group Cl.(3) Fibrinogen level: There was a significant difference in group Cl before and after pharmacological triggering (PO.05) . The level was significantly increased in group B than group Cl after pharmacological triggering (.PO.05) .(4) The level of hs-CRP, IL-8, IL-18, MMP-1, P-selectin, MCP-1 had significant difference between plaque ruptured group and nonruptured group (PO.05), whereas TIMP-1, IL-10, MMP-3, MMP-12 had no significant difference (P>0.05).(5) Ultrasound measurement: Values of IMT in plaque ruptured group were markedly thicker than nonruptured group (1.22 ± 0.35) mm vs (0.79 ±0.17) mm, Vp also increased greatly than nonruptured group(112.8 ± 26.75)cm/s vs (78.5 ± 23.54)cm/sfP<0.05). AD analysis: Values of AIIc% in plaque ruptured group were significantly lower than nonruptured group (62.4+12.36) %vs (78.0 ±10.56) %(P<0.05).(6) Intravascular ultrasound: External elastic membrance area (EEMA), plaque area (PA) and lumen area stenosis(LAS%) in plaque ruptured group weresignificantly higher than nonruptured group (P<0.05).(7) Histologic evaluation: Immunohistochemistry showed the expressions of RAM-11, MCP-1, MMP-1, NF- k B and P-selectin in plaque ruptured group were significantly higher than nonruptured group ,whereas the expression of a-actin was lower than nonruptured group (P<0.05). The expressions of MMP-3, MMP-12, TIMP-1 had no significant difference between the two groups (P>0.05). Vulnerability index in plaque ruptured group was higher than nonruptured group (3.13 + 1.8) vs(0.86 + 0.27) (PO.05). Vulnerability index correlated well with hs-CRP, MMP-1, EEMA, PA, LAS%andIMT (r=0.74, 0.6, 0.54, 0.57, 0.87, 0.88, respectively, PO.05), but had no correlation with MCP-1, IL-8, L-18, P-selectin, Vp R Allc%. The thickness of cap in plaque ruptured group were significantly thinner, compared with nonruptured group (102 + 52) u m vs (182 + 40 ) u m (PO.05). The ratio of thickness of the plaque cap and intima-media was significantly different between the two groups (0.10 + 0.12) vs (0.31+0.11) (PO.05).(8) Electronic microscopy: At the 20th day after balloon-injuried, scanning electron microscopy showed there were small thrombi on the endothelium, the endothelia cells were disorder, transmission microscopy found smooth muscle cells were mainly excreting phenotypes and were disarranged, while in the nucleus, some heterochromosomes spread around the nucleus membrance. After triggering at the end of eight weeks, scanning electron microscopy showed plaque ruptured and larger thrombi formation.(9) Reverse-transcriptase polymerase chain reaction: The mRNA expressions of MCP-1, MMP-1, MMP-3, MMP-12 in plaque ruptured group were significantly higher than noneruptured group in abdominal arterial atherosclerotic lesions (PO.05).(10) Western blot: The protein expressions of MCP-1, MMP-1, MMP-3, MMP-12, NF- k B in plaque ruptured group were higher than noneruptured group(P0.05).2. Gene therapy(1) The N-terminal amino acids 2 through 8 of human monocyte chemoattractantprotein-1 were deleted by recombinant PCR and tested by DNA sequencing, then the recombinant eukarytic expression vector pIRES-EGFP-7ND was successfully constructed.(2) The pIRES-EGFP-7ND transfected HK293 cells with lipofectamine mediated displaying green fluorescence were observed under fluorescence microscopes after transfection of 48h , mRNA and protein of 7ND were detected, the latter assayed by ELISA was about (50+12) pg/ml.(3) The transwell chamber was used to assay its chemotacxis activity. It showed cell counts of monocyte cell were 12 + 9 /high power lens in transfected group vs 50 + 15/high power lens in control group with moving distance of (65.9+10.6) u m vs (104.3 +12.5) um(P<0.05).(4) In vivo: Green fluorescence was expressed in the rabbits at the day 3 after transfected pIRES-EGFP-7ND, which was mediated by lipofectamine into the rabbits' skeletal muscles. It was still detected till 28 days after transfection.(5) The results of ELISA indicates that there were significant difference of the expression level of human MCP-1 between 7ND group and control group, the expression was the highest in the former group on the third day after transfection (326±18pg/ml). It was still expressed at the 28th day, while there was no expression in control group (group C2b).There were no significant difference expression level of the rabbits' MCP-1 between the two groups, but there was markedly decreased of hs-CRP, IL-8, IL-18, MMP-1, P-selectin in 7ND group than control group (PO.05).(6) Ultrasound measurement: Values of IMT in 7ND group were markedly lower than control group (0.79±0.12) mm vs (1.32±0.26 ) mm(P<0.05). AD analysis: Values of AIIc% in 7ND group were significantly higher than control group (77.0±10.22) %vs (53.6±12.52) % (PO.05).(7) Intravascular ultrasound: External elastic membrance area (EEMA), plaque area (PA), lumen area stenosing (LAS%) in 7ND group, were significantly lower than control group (P<0.05). At the end of 28 days, the pharmacological triggering lead to one rabbit dead and two rabbits' plaque rupture in control group, while there was no case of plaque rupture happened in 7ND group.(8) Histologic evaluation: Immunohistochemistry showed that expressions of MCP-1, MMP-1, MMP-3, MMP-12 and P-selectin in 7ND group were much lower than control group(P0.05). Vulnerability index in 7ND group was much lower than control group (0.63±0.12) vs (2.29±0.51)(PO.05). Compared with control group, the thickness of cap in 7ND group were significantly higher (221 ±20) u m vs(160±40) U m (PO.05), while the thickness of the intima-media was significantly thinner in 7ND group (PO.05).(9) Electronic microscopy: Scanning electron microscopy showed there were less monocytes cells in 7ND group than control group, while transmission microscopy found smooth muscle cells were slightly injuried in 7ND group than control group, heterochromosomes spreaded around the nucleus membrance, with integrity nucleus membrane.(10) Reverse-transcriptase polymerase chain reaction: The mRNA expressions of MCP-1, MMP-1, MMP-3, MMP12, P-selectin in 7ND group were significantly lower than control group in the abdominal arterial atherosclerosis lesions (PO.05).(11) Western-blot: The protein expressions of MCP-1, MMP-1, MMP-3, MMP-12 and P-selectin were lower in 7ND group than control group in the abdominal arterial atherosclerosis lesions (PO.05).3. Medication treatment(1) The weight of rabbit in group C5 (rapamycin group) after treatment was modest decrease than before (2.52±0.37) kg vs (3.81±0.51)kg (PO.05). There was no significant difference in other groups.(2) Lipid profile: Compared with group C8 (control group), TC, TG and LDL were significantly decreased in group C3 (simvastatin group) and C6(tongxinluo group). There was also significantly difference of lipid profile between the two groups (PO.05).(3) Fibrinogen level: It was significantly decreased in group C6 (tongxinluo group) compared with control group C8 (1.45±0.32) g/1 vs(2.76±1.23) g/1 (PO.05).(4) Compared with group C8 (control group), the level of hs-CRP, IL-8, IL-18, MMP-1, P-selectin, MCP-1 in other groups were markedly decreased (P<0.01-0.05).(5) During the treatment the blood concentration of doxycycline in group C4(doxycycline group) was monitored by HPLC, it was maintained at the level of 2.32 ± 0.36 ug/ml.(6) Ultrasound measurement: Compared with group C8, the values of IMT in mediation groups were markedly lower, C5 (rapamycin group) was the thinnest among these group , (0.56±0.12)u m vs (1.33±0.26)u m (PO.05) . Compared with group C8, the values of AHc% were significantly higher in group C5 (rapamycin group) (81.3±9.8) % vs(63.6±12.5)% (PO.05).(7) Intravascular ultrasound: Compared with group C8, the external elastic membrance area (EEMA), plaque area (PA) and lumen area stenosis (LAS%) were much lower in other groups but group C7 (regression control group). At the end of 12 weeks, the pharmacological triggering led to one rabbit dead in group C5 (rapamycin group) and group C8 respectively and two rabbits' plaques rupture in group C8, while there was no case of plaque rupture happened in other groups.(8) Histologic evaluation: Compared with group C8, the expressions of MCP-1, MMP-1 showed by immunohistochemistry in the medication group were much lower (P<0.05). Compared with group C8, the vulnerability index in other groups were much lower expecially in group C3 (simvastatin group) and group C5 (rapamycin group), while the thickness of plaque cap in other groups was signinficantly thicker and the intima-media thickness was significantly decrease(P<0.05, respectively).(9) Reverse-transcriptase polymerase chain reaction: Compared with group C8, the mRNA expressions of MCP-1, MMP-1, MMP-3, MMP12, P-selectin in treatment group were significantly lower in abdominal arterial atherosclerosis lesions.(10) Western-blot: Compared with group C8, the protein expressions of MCP-1, MMP-1, MMP-3, MMP-12, P-selectin found in treatment group were much lower in abdominal arterial atherosclerosis lesions (P<0.05).Conclusions1. The method of balloon-induced abdominal aortic wall injury together with a cholesterol-rich diet and drug triggering is a simple, efficient and time-saveing way to establish rabbit vulnerable plaque model which is mimic to human disease and usefulfor gene and drug treatment experiments. Ultrasound measurements and inflammatory markers correlated closely with vulnerability index measured pathologically.2. The N terminal amino acids 2 through 8 of human monocyte chemoattractant protein-1 can be deleted by recombinant PCR, and successfully constructed into pIRES-EGFP-7ND eukarytic expressing vector. The effects of pIRES-EGFP-7ND eukarytic expressing vector transfection in vitro and in vivo show that the vector can suppress monocyte recruitment, adhesion, chemotaxis and lead to a change of the contents in the vunlnerable plaques with more smooth cells, more collagen and less macrophages. Therefore,t his novel therapy is effective in stabilizing vulnerable plaques.3. Different mechanisms of simvastatin, rapamycin, doxycycline and Chinese traditional medicine-tongxinluo contribute to the plaque stabiliy produced by these drugs. Lipid and anti-inflammation mechanisms contribute to the beneficial effects of simvastatin in plaque stability, rapamycin has an efficient effect of inhibiting proliferation of intima and expression of inflammation factors, doxycycline inhibits the expression of MMPs resulting in stabilizing vulnerable plaques and Chinese traditional medicine-tongxinluo has effects of lowering lipid and plasma fibrinogen levels and of anti-inflammation leading to stabilization of vulnerable plaques.