| Summary: | 博士 === 國立臺灣大學 === 臨床醫學研究所 === 90 === 英文簡述（Summary）
Dyslipidemia and ensuing atherosclerosis have been implicated in the pathophysiology of coronary heart disease (CHD). A number of prospective studies have established that the risk of cardiac morbidity and mortality is directly related to the concentration of plasma total or low-density lipoprotein (LDL) cholesterol. Furthermore, lipid-lowering therapy has been found to reduce the risk of cardiovascular events in both high-risk individuals and patients with manifest CHD. However, whether dyslipidemia influences the severity of myocardial infarction (MI) is still uncertain. It is noteworthy that, in the Scandinavian Simvastatin Survival Study, the mortality rate of definite MI in the placebo group was 28% higher than that in the simvastatin group. This finding suggests that dyslipidemia may have an adverse effect on the evolution of MI. Likewise, a number of animal and human studies have revealed that hypercholesterolemia can increase the susceptibility of myocardium to ischemic insults by various nonatherosclerotic mechanisms.
To examine the hypothesis that dyslipidemia can aggravate myocardial vulnerability in the clinical setting of acute MI and this deleterious effect is not entirely due to the well-known atherogenic properties of dyslipidemia, we performed a retrospective study on 165 patients with a first MI admitted to the coronary care unit from January 1992 to December 1993. All patients underwent measurements of serum lipid profiles 1 week and 3 months after MI, a radionuclide ventriculographic study, and a coronary angiographic study. The patients were divided into 3 groups according to their 3-month serum total cholesterol levels (group 1, <200 mg/dL; group 2, 200 to 240 mg/dL; group 3, >240 mg/dL). Groups 1, 2, and 3 consisted of 66, 59, and 40 patients, respectively. Group 3 had a higher Gensini score than groups 1 and 2, although this was not statistically significant (P=0.13). The postinfarct left ventricular ejection fraction (LVEF) was highest in group 1 (53 ± 13%), at mid level in group 2 (43 ± 14%), and lowest in group 3 (35 ± 11%)(P<0.0001). A significant negative correlation between 3-month LDL cholesterol and postinfarct LVEF after adjustment for all confounding variables was found. The product of peak creatine kinase (CKMAX) and time to CKMAX, and patency of the infarct-related artery, rather than variables of coronary atherosclerosis, were also independent predictors of the postinfarct LVEF. In this study, we have demonstrated, for the first time, that dyslipidemia had a detrimental effect on postinfarct LVEF. Moreover, the detrimental effect of dyslipidemia on postinfarct LVEF was independent of the extent of MI and the patency rate of infarct-related artery. These findings suggest that dyslipidemia may adversely influence the evolution of MI even after the establishment of a patent infarct-related artery, which indicates that reperfusion injury may be the pathogenic link mediating the deleterious effect of dyslipidemia on postinfarct left ventricular systolic function. The deleterious effect of dyslipidemia on postinfarct LVEF being independent of the size of MI estimated by cardiac enzyme elevation is another very intriguing finding. Because the cardiac enzymes are released only when cardiomyocytes break down and cardiomyocytes typically rupture during necrosis but not apoptosis, we therefore speculated that cardiomyocyte apoptosis may play a role in the deleterious effect of dyslipidemia on postinfarct LVEF. In fact, it has been reported that apoptosis is a significant contributor to myocardial cell death as a result of reperfusion injury. However, whether the extent of cardiomyocyte apoptosis following ischemia and reperfusion varies in different pathophysiological background is still uncertain. Accordingly, we designed an experiment to investigate whether prolonged hypercholesterolemia increases the extent of experimental myocardial ischemia-reperfusion injury by aggravating cardiomyocyte apoptosis in a rabbit model (Study #1). Although a large number of genes have been reported to be involved in the regulation of apoptotic cell death, the antiapoptotic Bcl-2 and proapoptotic Bax play major roles in regulating myocardial apoptosis following ischemia and reperfusion. We thus determined the effects of hypercholesterolemia on the expression of Bcl-2 and Bax in the ischemic and non-ischemic myocardium to identify the underlying molecular mechanisms induced by hypercholesterolemia.
In view of the significant contribution of cardiomyocyte apoptosis as a result of reperfusion injury, it is speculated that anti-apoptotic interventions, such as caspase inhibitors, may reduce myocardial reperfusion injury by attenuating cardiomyocyte apoptosis within the ischemic area at risk. On the other hand, in addition to apoptosis, various other mechanisms responsible for reperfusion injury have been identified, including the massive generation of oxygen free radicals and inflammatory cytokines. Mechanistically, apoptotic cell death is mediated by a family of aspartate-specific cysteine proteases known as caspases that include at least 14 members. Caspase-1, the first identified member of the caspase family, is responsible for the activation of executioner caspases involved in apoptosis progression in a variety of experimental paradigms. Furthermore, it modulates the inflammatory reaction by processing the maturation of interleukin-1b (IL-1b). Caspase-1 hence has the peculiarity of being involved in the activation of both apoptosis and inflammation, through the intermediate of the pro-inflammatory cytokine IL-1b. Based on these background researches, we speculated that pharmacological inhibition of the caspase-1 cascade might have greater protective effects on myocardial ischemia-reperfusion injury in the context of hypercholesterolemia. In Study #2, we examined whether pharmacological inhibition of the caspase-1 cascade, using Ac-Tyr-Val-Ala-Asp-CH2Cl (Ac-YVAD.cmk), after myocardial ischemia have greater protective effects on myocardial ischemia-reperfusion injury in diet-induced hypercholesterolemic rabbits.
Although the observed adverse effect of dyslipidemia on postinfarct LVEF was mostly ascribed as a consequence of dyslipidemia-associated deleterious effects on the evolution of MI, we still can not exclude the possibility that it may in part reflect the baseline left ventricular systolic function. Large-scale clinical trials have shown that long-term treatment with lipid-lowering therapy results in a significant reduction in the occurrence of heart failure among patient with coronary artery disease without previous evidence of congestive heart failure, suggesting dyslipidemia may have an adverse effect on left ventricular performance. Hence, we performed another hospital-based study to examine whether dyslipidemia has a detrimental effect on left ventricular systolic function and whether this effect is dependent on the corresponding severity of coronary atherosclerosis (Study #3).
Despite we studied issues regarding dyslipidemia and various aspects of CHD, the definition of dyslipidemia per se is still under debate. Although elevated levels of LDL cholesterol and low levels of HDL cholesterol are independent risk factors for CHD, several important issues in clinical management of dyslipidemic individuals remain to be solved. For example, there is a continuing debate as to whether subjects with high levels of both HDL and LDL cholesterol have an increased risk for CHD. The same problem confronts clinicians in managing subjects with low levels of both HDL and LDL cholesterol. These unresolved issues reflect the inadequacy of current LDL cholesterol-based guidelines. Recent studies have shown that the total cholesterol/HDL cholesterol ratio is a powerful lipoprotein predictor for the development of CHD. Although this concept has not been integrated into current clinical guidelines, it is suggested that using total cholesterol/HDL cholesterol ratio as a stratifying variable may help clinicians to better clarify the risk status of individuals with high levels of both HDL and LDL cholesterol as well as those with low levels of both HDL and LDL cholesterol. In Study #4, by using the 8-year follow-up data of CHD-free participants in a well-characterized Chinese population-based prospective cohort study─the Chin-Shan Community Cardiovascular Cohort (CCCC) study, we first assessed the efficacy of various lipid and lipoprotein measurements at baseline in predicting the risk for future coronary events. Then, we determined the associated risk of CHD in subgroups stratified by different lipid and lipoprotein screening strategies to evaluate the adequacy of current total and LDL cholesterol-based approach in lipid management.
Several epidemiologic studies have demonstrated that elevated serum total cholesterol and LDL cholesterol levels lose their predictive power for CHD as people age. However, the underlying mechanisms remained uncertain. To elucidate this issue, in Study #5, we assessed the efficacy of various lipid and lipoprotein measurements at baseline in predicting CHD risk in both the younger (aged 35 to 55 years) and older cohorts (aged ³56 years) by analyzing the same 8-year follow-up data of CHD-free participants in the CCCC study.
In Study #1, twenty-eight male New Zealand White Rabbits had been fed with standard chow (control, n = 14) or chow supplemented with 1% cholesterol (hypercholesterolemic, n = 14) for 8 weeks. Anesthetized rabbits were then subjected to 30 minutes of left circumflex artery occlusion followed by 4 hours of reperfusion. Apoptosis was identified as “DNA ladders” by gel electrophoresis and confirmed histologically using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. The infarct size (% of risk region) was significantly greater in hypercholesterolemic rabbits than control (39 ± 6% vs. 23 ± 2%, P=0.02). Very few TUNEL-positive cardiomyocytes could be identified in the non-ischemic regions in both groups, consistent with an absence of DNA laddering. In contrast, TUNEL-positive cardiomyocytes were significantly displayed in the ischemic, non-necrotic myocardium and DNA ladder occurred in all animals. The percentage of TUNEL-positive cardiomyocytes in the ischemic non-necrotic myocardium was significantly higher in hypercholesterolemic rabbits compared with control (40 ± 5% vs. 17 ± 1%, P<0.001). Western blot analysis showed that, in the non-ischemic myocardium, hypercholesterolemic rabbits exhibited an approximately 50% increase in the expression of Bcl-2 (P<0.05), but not Bax, than control rabbits. However, compared with control, hypercholesterolemic rabbits exhibited a more pronounced decrease in the expression of Bcl-2 (42 ± 4% vs. 26 ± 2%, P<0.01) and a similar extent of increase in the expression of Bax in the ischemic myocardium. Furthermore, hypercholesterolemic rabbits were associated with markedly increased activation of caspase-1 and caspase-3 within the ischemic myocardium than control rabbits. This study demonstrates that although hypercholesterolemia is associated with an increased myocardial Bcl-2/Bax ratio at baseline, it still significantly exacerbates cardiac reperfusion injury, not only by increasing the infarct size, but also by increasing the extent of cardiomyocyte apoptosis.
After demonstrating that hypercholesterolemia is associated with greater myocardial ischemia-reperfusion injury, in which apoptosis and inflammation-mediated necrosis both play a key role, we performed Study #2. In this study, sixty male New Zealand White Rabbits, fed with standard chow or chow supplemented with 1% cholesterol for 8 weeks, were subjected to 30 minutes of left circumflex artery occlusion followed by 4 hours of reperfusion. An intravenous bolus of Ac-YVAD.cmk (1.6 mg kg-1) or vehicle was given 20 minutes after coronary occlusion in each group. Apoptosis was also identified as “DNA ladders” by gel electrophoresis and confirmed histologically using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. The infarct size (% of risk region) assessed by triphenyltetrazolium chloride staining was significantly greater in cholesterol-fed rabbits than in normally fed ones (41±6% vs. 26±3%, P=0.003). Postischemic administration of Ac-YVAD.cmk markedly decreased infarct size from 26±3% to 12±2% in normally fed rabbits (P=0.005) and from 41±6% to 14±2% in cholesterol-fed rabbits (P<0.001). In the ischemic non-necrotic area, the percentage of TUNEL-positive cardiomyocytes was significantly greater in vehicle-treated cholesterol-fed rabbits compared with normally fed ones (39.0±2.3% vs 15.5±0.8%, P<0.001), whereas treatment with Ac-YVAD.cmk markedly reduced the percentage of TUNEL-positive cardiomyocytes from 15.5±0.8% to 2.2±0.1% in normally fed rabbits (P<0.001) and from 39.0±2.3% to 2.2±0.1% in cholesterol-fed rabbits (P<0.001). Although myocardial IL-1b levels and activity of both caspase-1 and caspase-3 in the ischemic area were significantly higher in vehicle-treated cholesterol-fed rabbits compared to normally fed ones, Ac-YVAD.cmk treatment resulted in a reduction not only of IL-1b and caspase-1, but also of caspase-3 in both normally fed and cholesterol-fed rabbits. Furthermore, no differences in IL-1b levels and activity of caspase-1 and caspase-3 were observed between Ac-YVAD.cmk-treated normally fed and cholesterol-fed rabbits. This study demonstrates that injection of a selective caspase-1 inhibitor after myocardial ischemia completely abolished the detrimental effect conferred by hypercholesterolemia on myocardial ischemia-reperfusion injury by attenuating both necrotic as well as apoptotic cell death pathways through inhibition of IL-1b production and activation of caspase-1 and caspase-3.
In Study #3, 114 consecutive patients with stable angina and a positive exercise thallium-201 myocardial perfusion single-photon emission computed tomography were studied. All patients underwent measurement of serum lipid profiles, right-sided heart catheterization, left ventriculography, and selective coronary arteriography. Mean serum levels of total cholesterol and triglycerides were 4.5 mmol/L and 1.4 mmol/L, respectively. In univariate analysis, a significant positive correlation between serum high-density lipoprotein (HDL) cholesterol and LVEF (r = 0.49, P<0.0001) was found. Patients in the lower tertile of serum HDL cholesterol had a significantly lower mean LVEF than those in the upper tertile (55.9 ± 15.2 vs. 72.8 ± 6.8 %, P<0.0001). Stepwise multiple linear regression analysis revealed that LVEF significantly correlated with HDL cholesterol (P<0.0001), the Gensini score (P=0.008), and diabetes mellitus (P = 0.08)(r = 0.55, P<0.0001). In subgroup analysis of patients with angiographically normal coronary arteries, serum HDL cholesterol was still significantly associated with LVEF. The present study demonstrated an independent association between low HDL cholesterol and subclinical left ventricular systolic dysfunction in Chinese patients with stable angina whose serum levels of total cholesterol and triglycerides were relatively low. Moreover, the correlation between HDL cholesterol and baseline LVEF remained significant even in patients with normal coronary angiograms, suggesting HDL cholesterol might influence left ventricular systolic performance through extra-atherosclerotic mechanisms.
In Study #4, we assessed the efficacy of various lipid and lipoprotein measurements at baseline in predicting the risk for CHD and determined the associated risk of CHD in subgroups stratified by different lipid and lipoprotein screening strategies to evaluate the adequacy of current total and LDL cholesterol-based approach in lipid management. We analyzed data from the Chin-Shan Community Cardiovascular Cohort study, a Chinese population-based prospective cohort study since 1990. During an 8-year follow-up, 213 (6.7%) of 3,159 CHD-free participants (aged ³35 years) developed CHD. The total cholesterol/HDL cholesterol ratio was the most powerful lipoprotein discriminator of future CHD (hazard ratio, 1.21 for 1.0 increment in ratio; P<0.001). Subjects with “high-risk” LDL cholesterol levels (>160 mg/dL) and low total cholesterol/HDL cholesterol ratios (£5) had an incidence of CHD similar to those with low levels of both LDL cholesterol (£130 mg/dL) and total cholesterol/HDL cholesterol ratios (4.9% vs. 4.6%). On the other hand, subjects with “low-risk” LDL cholesterol levels (£130 mg/dL) and high total cholesterol/HDL cholesterol ratios (>5) had a 2.5-fold higher incidence of CHD than those with similar LDL cholesterol levels but low total cholesterol/HDL cholesterol ratios (P<0.001). Compared to using LDL cholesterol level of 130 mg/dL as the cut-off point, using total cholesterol/HDL cholesterol ratio of 5 was associated with superior specificity (73% vs. 59%, P<0.001) and accuracy (72% vs. 58%, P<0.001) and similar sensitivity (50% vs. 53%).
In Study #5, we first assessed the efficacy of various lipid and lipoprotein measurements at baseline in predicting CHD risk in the younger and older cohorts to understand whether the predictability differed between both age groups. In the younger cohort (35 to 55 years), total cholesterol and LDL cholesterol levels were independently predictive of CHD risk, whereas in the older cohort (³56 years), HDL cholesterol level was a better predictor of CHD risk than total cholesterol or LDL cholesterol levels. Among the lipoprotein measurements examined, the ratio of total to HDL cholesterol was the most powerful lipoprotein predictor of CHD risk in both cohorts. We then divided both cohorts into subgroups of different lipoprotein phenotypes on the basis of serum LDL cholesterol levels and the ratios of total to HDL cholesterol at baseline. Compared with those with an LDL cholesterol level of £130 mg/dL and a total cholesterol/HDL cholesterol ratio of £5, individuals with an LDL cholesterol level of >130 mg/dL and a total cholesterol/HDL cholesterol ratio of >5 (the “high LDL-low HDL cholesterol” phenotype) had an increased CHD risk in both the younger and older age groups (hazard ratios, 2.10 and 2.17, respectively). By contrast, only older individuals with an LDL cholesterol level of £130 mg/dL and a total cholesterol/HDL cholesterol ratio of >5 (the “isolated low HDL cholesterol” phenotype) had an increased CHD risk (hazard ratio, 3.04), whereas the younger counterparts did not. It is noteworthy that individuals with an LDL cholesterol level of >130 mg/dL and a total cholesterol/HDL cholesterol ratio of £5 did not have an increased CHD risk regardless of their age. The present study demonstrated that CHD risk associated with the high LDL-low HDL cholesterol phenotype developed early in adult life, whereas CHD risk associated with the isolated low HDL cholesterol phenotype developed in later life.
Conclusions
In Studies #1 and #2, we showed that although hypercholesterolemia is associated with an increased myocardial Bcl-2/Bax ratio at baseline, diet-induced hypercholesterolemia significantly exacerbates cardiac reperfusion injury, not only by increasing the infarct size, but also by increasing the extent of cardiomyocyte apoptosis. The increased extent of cardiomyocyte apoptosis may adversely influence the left ventricular remodeling process and subsequently lead to poor prognosis. Because the magnitude of reperfusion-related cardiomyocyte apoptosis is significantly greater in hypercholesterolemic rabbits, we speculated that the administration of anti-apoptotic agents following the establishment of successful revascularization might provide additional benefits in salvaging the ischemic myocardium. Based on these findings, we subsequently demonstrated that pharmacological inhibition of caspase-1 as an adjunct to reperfusion results in a significant cardioprotection from ischemia-reperfusion insult. Furthermore, we presented the first evidence that pharmacological inhibition of caspase-1 completely abolished the detrimental effect conferred by hypercholesterolemia on myocardial ischemia-reperfusion injury. This cardioprotective effect is achieved not only by blocking the apoptotic pathway but also by inhibiting IL-1b-mediated inflammation. These observations indicate that inhibition of caspase-1 could be a promising therapeutic approach to attenuate myocardial damage caused by ischemia and reperfusion, particularly in those with concomitant hypercholesterolemia.
Our previous study revealed that dyslipidemia was independently associated with decreased postinfarct LVEF in patients with a first MI. Despite that dyslipidemia-associated adverse effect on the evolution of MI was assumed to be the most important mechanism, as shown in Studies #1 and #2, the present finding of the association between low serum HDL cholesterol and subclinical left ventricular systolic dysfunction in Study #3 suggests that dyslipidemia-associated deleterious effect on baseline left ventricular systolic function may partly contribute to the decreased postinfarct LVEF in dyslipidemic patients observed in our previous study. Moreover, it would be important to investigate whether left ventricular systolic dysfunction could be reversed by vigorous control of abnormal serum lipid profiles.
In Studies #4 and #5, we clearly demonstrated that current guidelines for lipid management may misclassify individuals with high levels of both HDL and LDL cholesterol as well as those with low levels of both HDL and LDL cholesterol. Using the ratio of total to HDL cholesterol as the initial screening tool can obviate this discrepancy. Therefore, the definition of dyslipidemia should be revised. We also confirmed that the differential predictability of various lipoprotein measurements for CHD in younger and older cohorts observed in most Western epidemiologic studies was also present in this Chinese population-based prospective study. Furthermore, by using LDL cholesterol level and the ratio of total to HDL cholesterol as the stratifying variables, we explicitly distinguished two atherogenic lipoprotein phenotypes and, for the first time, demonstrated that CHD risk associated with the high LDL-low HDL cholesterol phenotype is consistently increased across a wide age range, whereas CHD risk associated with the isolated low HDL cholesterol phenotype is evident only in older individuals. More importantly, both younger and older individuals with high levels of both HDL and LDL cholesterol did not have an excess risk for CHD. These findings not only delineate the atherogenic risk of different lipoprotein phenotypes, but also elucidate the confusing information regarding the relations between total cholesterol and LDL cholesterol levels and CHD risk in older individuals.
It is noteworthy that all published primary-prevention or secondary-prevention lipid-intervention trials included patients with an average total cholesterol/HDL cholesterol ratio far greater than 5. However, the average level of total cholesterol/HDL cholesterol ratio in Western populations is only 4.5. Why there was such a discrepancy is still uncertain. However, the absence of specific clinical trials that document the magnitude of benefit from drug therapy and the low absolute risk in CHD-free individuals with high levels of both HDL and LDL cholesterol allude to the need for more caution when considering aggressive lipid-lowering therapy in this clinical setting.
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