Atherosclerotic Plaque Vulnerability

BackgroudAcute cardiovascular disease and cerebrovascular disease are two common and multi-onset diseases, which have serious effects on the health of human beings, and their pathological basis is atherosclerosis (AS). Traditionally, the development of AS was thought to be a line procedure, during which lipid infiltrated into the intima of the vessel and plaques formatted blocked the vessel. So regression of the plaques was the main therapy strategy. However, recent studies show that plaque disruption and arterial thrombosis are recognized as critical to the onset of acute coronary syndromes (ACS) and a vulnerable plaque that is prone to rupture is vital to induce ACS. Studies show that a vulnerable plaque has (1) a thin fibrous cap;(2) a large lipid core;(3) many inflammatory cells such as macrophages infiltrated;(4) endothelial dysfunction;(5)progressive blood coagulation function Whether a plaque ruptures or not lies in two factors: the intrinsic factor- tissue characteristic and the extrinsic factor-tissue stress, and the former is decisive. Therefore, it is very significant to detect vulnerable plaques earlier and take effective interventions to prevent ACS. It is a logical way to research on the factors of vulnerability of plaques and take effective methods to prompt vulnerable plaques becoming stable.However, there are still many problems unsolved about the vulnerability of atherosclerotic plaques: (1) Research on the causes and treatment of plaque rupture is hampered by the lack of a suitable animal model. Traditional animal models of stable atherosclerotic plaques are not suitable to the study of vulnerable plaques because of different nature of the twokinds of plaques. Therefore, an animal model of vulnerable plaques is greatly warranted not only for basic research but also for imaging studies. (2) The current evidence of plaque rupture and thrombosis in ACS is largely based on cross-sectional and retrospective studies.There is no robust prospective outcome study on the vulnerable plaque rupture.The mechanism of plaque vulnerability and rupture is still unknown. (3) The cell and molecular biological mechanism of plaque rupture remains poorly understood. Whether are soluble inflammatory markers able to predict plaque rupture? (4) A high sensitive imaging technique to detect vulnerable plaque is lack. Whether are intravascular ultrasound (IVUS), high-frequency ultrasound imaging technique and accuracy densitometry(AD) which can identify tissue characteristics used together to detect the vulnerable plaques? (5) Are the risk factors in predicting plaque rupture able to be screened out by Losistic regression?All mentioned above formed the objectives. Methodsl.The animal model induction Sixty-four New Zealand White rabbits were randomly divided into two groups: group A(n=54) and group B(n=10). Rabbits in group A underwent balloon-induced abdominal aortic wall injury and then were fed on a diet of 1% cholesterol while rabbits in group B were only fed on a diet of 1% cholesterol. At the end of the eighth week, rabbits in group A were divided into two subgroups randomly: group A1 (n=27) and group A2 (n=27). Recombinant adenovirus carrying p53 and β galactosidase (LacZ) genes were injected through a catheter into the aortic segments rich in plaques in group A1 and A2, respectively. Two weeks later, 10 rabbits each in group A1 and A2 were killed to observe the occurrence of spontaneous plaque rupture, and the remaining rabbits in group A1, A2 and B all underwent pharmacological triggering with injection of Chinese Russell's viper venom (CRVV) and histamine.2. Laboratory examinations At the beginning of the experiment and 10th week, blood samples were collected for measuring the lipid profile, MDA and fibrinogen. Using ELISA method to measure concentrations of oxLDL, sVCAM-1, sICAM-1 and hsC-RP.3. Examinations of imaging features of vulnerable plaques At the beginning of the experiment, pre-and post-pharmacological triggering at the 10 week, the abdominal aortas were detected with high frequency ultrasound , intravascular ultrasound (IVUS), aortography and AD technique,respectively. Intima-media thickness(IMT),plaque area,plaque distribution ,tissue characteristic and vascular remodeling pattern were examinated.4.Hemodynamics examination Before and after the rabbits were pharmacologically triggered at the 10th week, blood flow velocity (Vp, Vm, VTI), systolic blood pressure (SBP),diastolic blood pressure (DBP), dp/dtmax and -dp/dtmax of abdomial aorta were measured.5. Histopathology The abdominal aorta were processed and examined by HE stain, Masson's trichrome stain and immunohistochemical stain of a -smooth muscle cell actin,CD68, p53, VCAM-K ICAM-K NF-kB p65> IKB^ CD40 , MMP-land MMP-9 respectively.The intimal surface was examined with scanning electron microscope to detect plaque rupture and thrombus.Using transmission microscope to observe the ultrastructure of the rupture segments.6. RT-PCR To determine the mRNA expressions of p53,NF-KB, 1KB, IL-2,IL-6,,MCP-1,VCAM-1,ICAM-1 JIMP-l.MMP-1,and MMP-9 in the position of ruptured and nonruptured plaques.7. Western blot To examine the protein expressions of p53, NF-kB, 1KB, VCAM-1 and ICAM-1 in the position of ruptured and nonruptured plaques..8. Cellular apoptosis Vascular smooth muscle cell apoptosis rate was assayed by agarose-gel electrophoresis, terminal deoxynucleotidyl transferase end-labeling (TUNEL) and cell cytometry, respectively.9. Logistic regression Using Logistic regression method to analyse the risk factors of vulnerable atherosclerotic plaques.Results1. Establishing an animal model: After given balloon-induced abdominal aortic wall injury and a high cholesterol diet ,an animal model of atherosclerotic plaques was dulplicated .An animal model of vulnerable plaques was built after tranfectinghuman wild-type p53 genes in the location of plaques.In group A, plaques with a large lipid core were formed. p53 gene over-expression resulted in a marked increase in cellular apoptosis and accumulation of inflammatory cells within the plaques, and a significant decrease of vascular smooth muscle cells (VSMCs) and the thickness of the fibrous caps Although spontaneous plaque rupture was rare, plaque rupture and thrombosis occurred in 12 rabbits (85.7%)and 20 lesions after pharmacological triggering.2.Lab examination(l)Lipid profile: The total serum cholesterol at baseline was( 2.02 ± 0.62)mmol/L, with a LDL level of (0.71 ±0.42)mmol/L and a TG level of( 1.10 + 0.46)mmol/L .After 10 weeks on the 1 %cholesterol diet, the TC level, LDL level and TG level had increased to (31.05 + 9.89 )mmol/L (P<0.001) ,(27.76 + 8.67)mmol/L (PO.001) and (2.04 + 2.11 )mmol/L (PO.05) .respectively. There was no significant difference among the three groups.(2)Fibrinogen level: There was no significant difference among the three groups before pharmacological triggering. After pharmacological triggering,the level was increased significantly,but there was no significant difference among the three groups.The level of plaque rupture group was significantly higher than nonruptured group.(3)MDA and oxLDL:Only the level of oxLDL in plaque ruptured group was significantly higher before pharmacological triggering After pharmacological triggering, the levels of MDA and oxLDL were significantly higher compared with pre-triggering.(4)Only concentrations of hsC-RP were significantly higher in plaque-ruptured rabbits than in nonruptured ones before pharmacological triggering (P<0.05). After triggering, concentrations of hsC-RP, sVCAM-1 and sICAM-1 were significantly higher in plaque ruptured group than in nonruptured group.3. Ultrasound measurement: IMT of AS model group was thicker markedly than the nomal level,Ds,Dd,Vp and Vm also increased greatly.IMT of Group Ai and A2 was significantly more than that of Group B(p<0.01).Ds and Dd in Group Ai weresignificantly bigger than Group B IMT and Vp were significantly higher in plaque ruptured group than nonruptured group(p<0.05).IMT correlated well with TC,TG and LDL,respectively (r=0.366, PO.Ol; r=0.324, P<0.05; r=0.284, P<0.05). Sensitivity, distinctiveness,positive forecasting value and negtive forecasting value of ultrasound in diagnosing plaque rupture and thrombosis was 55.5%,80%,78.9% and 57.1%,respectively.4. AD analysis: Values of AIIc% in Group Ai and A2 were significantly lower than Group B.AIIc% in plaque ruptured group was lower significantly than nonruptured group.IMT correlated negatively with AIIc%( r=-0.376, PO.05).5.1ntravascular ultrasound: Minimum of vessel diameter (Vdmin), external elastic membrance area (EEMA),plaque area (PA),lumen area stenosing (LAS%),eccentric index (El) and remodeling index(RI)in Group Ai and A2 were higher significantly than group B.EEMA,PA,LAS%,EI and RI of ruptured plaques were significantly higher than nonruptured ones. The positions of ruptured plaques mainly showed positive remodeling,while nonruptured plaques showed no remodeling or negative remodeling. Sensitivity,distinctiveness,positive forecasting value and negtive forecasting value of IVUS in diagnosing plaque rupture and thrombosis was 77.8%,90%,91.3% and 75%,respectively.PA correlated well with IMT, TC(r=0.356, P <0.05 and r=O.5O5, p<0.01, respectively)and correlated negatively with AIIc%(r=-0.537, P <0.01).In measuring the length of thrombus,IVUS showed positively well with pathology(r=0.729, P <0.01),but they had no correlation regarding cross-area of thrombus.Both hsC-RP and sVCAM-1 showed positive correlations with RI(r=0.496 and 0.634, P <0.05 and <0.01 ,respectively).sVCAM-1 correlated well with EEMA(r=0.645, PO.Ol).6. Abdominal aortography: Abdomial aorta showed extensive constriction, especially at the inferior extremity and common iliac artery, and the right femoral artery which was injuried by balloon was almost occluded. After pharmacological triggering, the position of plaque rupture showed complex changes:niche shadow, eclipsed shap and line structure.Ellipse ,ribbon or irregulated shap was showed at the location of thrombus.7. Hemodynamics detection of abdomial aorta : There was no significant difference of Vp, systolic blood pressure (SBP), diastolic blood pressure(DBP),heart rate (HR), dp/dtmax and -dp/dtmax of abdomial aorta among the three groups.At the moment of using histamine, markers shown above increased greatly, and Vp, SBP,HR and dp/dtmax showed significant difference compared with pre-triggering.Compared with nonruptured group,Vp, SBP and dp/dtmax were significantly higher in plaque rupture group after triggering.8. Histopathology analysis(l)Morphological analysis: In Group Ai, only one p53-treated plaque in the ten rabbits developed spontaneous rupture before pharmacological triggering. After triggering, it was found that plaque disruption and thrombosis occurred in 12 rabbits and 20 lesions. There were 12 p53-treated plaques having developed disruption and thrombosis. In Group A2, LacZ-treated plaques did not demonstrate spontaneous rupture and thrombosis. After triggering, only 5 rabbits and 6 lesions had disruption and thrombi. There were 2 LacZ-treated plaques having developed disruption and thrombosis. The rate of plaque rupture in the position of gene injection was significantly different between Group Ai and A2 (P<0.01) and the lengths and cross-section areas of the thrombi formed in the position of gene injection were significantly different (P<0.05,respectively) (Table 1).While rabbits in Group B did not occur plaque disruption and thrombosis. All white thrombi were firmly attached to the arterial wall. These thrombi were cylindrical and had round edges. Red clots were loosely attached to the end of the white thrombi.(2)Measurement of vessel area: Vessel areas of plaque ruptured segments were significantly larger than reference segments and showed positive arterial remodeling. Corresponding nonruptured segments showed no remodeling or negative remodeling. Vessel areas of plaque ruptured segments were significantly larger than nonruptured vessels,but there was no significant difference between their reference segments.(3) p53 gene over-expression in the location of the plaque resulted in a thinner cap and a marked decrease of the vascular smooth muscle cells(VSMCs). There were many inflammatory cells infiltrating into the rupture positions and the thrombi. Thecaps at the rupture positions were broken. The thrombi were firmly attached to the arterial wall, which had many platelets ,fibrins, red blood cells. In group Ai, there were four plaques showing the existence of intraplaque haemorrage.(4) Compared with Group A2 and B, the thickness of cap in group Ai was significantly thinner (P<0.01,0.05,respectively). No difference was found in relation to the thickness of the intima-media between Ai and A2, but the two groups were significantly different compared with group B (P<0.05). The ratio of thickness of the plaque cap and intima-media was significantly different between Group.Ai and B.9. Electron microscopy(1) Scanning electron microscopy showed the ruptured fissures of plaques and the thrombi which were made up of platelets, fibrins and red blood cells. In the rupture location, myofibrils were disorder and on bundle distribution,with some macrophages loosely spreading in them.(2) Transmission microscopy found smooth muscle cells were mainly excreting phenotypes and were disarranged ,with some lipid droplets, lysosomes, glycogens and apoptotic bodies diffusing in the plasm of them. While in the nucleus, some heterochromosomes spread around the nucleus membrance. After p53 gene tranfecting, the number of VSMC became less and their bodies became smaller, with large number of lysosomes, glycogens and apoptotic bodies diffusing in them.10. Immunohistochemistry: Immunohistochemistry showed that expressions of CD68, a-actin , p53, VCAM-1, ICAM-1,NF- k B, 1KB, CD40, MMP-1, MMP-9 were found in the atherosclertic plaques. By using PAbl801, the human p53 postive cells were significantly more in the position of p53 transfection. Staining for a -smooth muscle cell actin, CD68-postive cells (macrophages), VCAM-1JCAM-1, NF- k B, MMP-1,MMP-9 in the ruptured positions and thrombi were more significantly than those in non-ruptured plaques. IKB,CD40 positive cells were more in the position of ruptured plaque,but there was no significant difference between the two groups.ll.RT-PCR(l)Only the expressions of MMP-1 and p53 mRNA in Group Ai were significantly higher than Group B, there was no significant difference of mRNA expressions of VCAM-l.ICAM-1, NF- k B,IKB,MMP-l,MMP-9,IL-2,IL-6 and MCP-1 among the three groups.(2) The mRNA expressions of p53 gene,VCAM-l,ICAM-l, NF- k B p65,IL-2,MMP-l and MMP-9 were significantly higher in plaque ruptured group than nonruptured group.But there was no significant difference of mRNA expressions of IL-6, TIMP1, MCP-1 and 1KB between ruptured and nonruptured groups.12.Western blot: The protein expressions of p53, VCAM-1, ICAM-l,NF-kappa B and 1KB were found in the atherosclerotic plaques.Protein of p53 was higher in the location of p53 gene injection.Compared with nonruptured plaques,expressions of VCAM-l,ICAM-l,NF-kappa B were higher ,IKB also increased but slightly.13. Celluar apoptosis(l)TUNEL:Positive cells showed circular and shrinkage shaps,amounted to (2.5 ±0.8)% in Group Ah (1.0 + 0.3)% in Group A2 and (0.9±0.4)% in Group B, comparing with group A2 and B, positive cells in group Ai were significantly different(P<0.01 respectively).(2)Results of flow cytometer showed distribution of cell cytle in p53 gene injection :GO-G1 74.2%±8.2%,G2-M 0.25%±0.03%,S 25.55%±3.2%.Ratio of celluar apoptosis in Group Ai ,A2 and B were 8.04%±1.2%,6.89%±1.20%,and 6.61 + 1.11% ,respectively.Compared with GroupA2 and B,there were significant difference in Group Ai(p<0.05).(3) DNA ladder:Some bands appeared in the position of p53 gene injection using DNA agarose-gel electroporesis method which proved the existence of the celluar apoptosis.14. Risk factors of plaque vulnerability.In Logistic regression analysis,risk factors were plaque area(PA) and eccentric index(EI)measured by IVUS,sVCAM-l obtained by EL1SA and AIIc% measured by AD technique. Conclusions...