Recently, the Department of Cardiovascular Medicine team at the Second Xiangya Hospital of Central South University published an article titled "High-sensitivity C-reactive protein may be a biomarker of high-density lipoprotein dysfunction and structural remodeling in patients with coronary atherosclerotic heart disease" in the Chinese Journal of Cardiovascular Diseases (online). Based on nuclear magnetic resonance (NMR) spectroscopy, the article explored indicators that can reflect high-density lipoprotein (HDL)-mediated cholesterol efflux capacity (CEC) in patients with coronary heart disease (CHD). The article gave special thanks to ProteinT for its support in NMR detection and analysis. Based on the advantages of NMR spectroscopy—non-destructive detection of trace samples, high-throughput full-process automation, unparalleled reproducibility, and an exclusive database platform—ProteinT continues to explore and research the early warning, medication guidance, and long-term monitoring of cardiovascular and cerebrovascular diseases, promoting more precise screening and medication guidance for cardiovascular and cerebrovascular diseases and common cancers.
Research Background
The Global Burden of Disease study and the China Cardiovascular Health and Disease Report show that atherosclerotic cardiovascular disease, represented by coronary atherosclerotic heart disease (CHD), remains the leading cause of death worldwide, and the mortality rate of CHD in China is still rising year by year, making the prevention and treatment of CHD urgent. Although current therapies that lower low-density lipoprotein cholesterol (LDL-C) levels have achieved significant benefits in the prevention and treatment of CHD, even when LDL-C is lowered to a low value of 0.78 mmol/L, about 10% of patients still experience cardiovascular events—that is, residual risk persists—which urges us to actively seek new intervention targets. In the 1980s, multiple epidemiological studies consistently found that high-density lipoprotein (HDL) cholesterol (HDL-C) levels were negatively correlated with the incidence of CHD events, so HDL-C was once considered an important cardiovascular protective factor. However, subsequent clinical trials of drugs that raise HDL-C and genetic studies did not support this view. HDL is a group of highly heterogeneous lipoprotein particles in plasma, 8-13 nm in size, with functions including mediating cholesterol efflux from foam cells, antioxidation, and anti-endothelial cell inflammation. At present, multiple large-scale clinical trials have confirmed that the HDL cholesterol efflux capacity (CEC) measured in vitro can independently predict cardiovascular risk in healthy populations and independently predict the incidence of adverse events and mortality in CHD patients, having important clinical value. However, the measurement of CEC requires in vitro cell models and isotopes, which limits its use in routine clinical work. Therefore, in this study we hoped to find an indicator that reflects CEC in CHD patients.
Research Methods
Consecutively enrolled were 36 patients with coronary heart disease diagnosed by coronary angiography (CHD group) and 61 non-coronary heart disease patients (non-CHD group) hospitalized in the Department of Cardiovascular Medicine of the Second Xiangya Hospital of Central South University from June to July 2018. Fasting blood was collected the morning after admission, and plasma samples were obtained after centrifuging whole blood. The plasma samples were aliquoted and frozen at -80 °C for the detection of CEC and HDL structure. The detection of CEC mainly involved the following five steps: obtaining HDL by heparin-manganese precipitation, loading macrophages with lipids, upregulating the expression of cholesterol efflux channel proteins, cholesterol efflux, and measuring isotope content. Nuclear magnetic resonance spectroscopy (NMRS) was used to detect the structure of HDL. Baseline characteristics and routine biochemical indicators of the study subjects were collected, and CEC and HDL structural parameters were measured. Pearson correlation analysis was used to find variables related to CEC, and multiple linear regression analysis was used to further evaluate the relationship between the variables and CEC.
Research Results
1. Clinical Characteristics
All patients in the CHD group were diagnosed with acute coronary syndrome, including 4 cases of ST-segment elevation myocardial infarction, 14 cases of non-ST-segment elevation myocardial infarction, and 18 cases of unstable angina. Among them, 29 patients (80.6%) had a blood collection time at least 7 d after the onset of the disease. Compared with the non-CHD group, patients in the CHD group were older, had more males, more diabetes, hypertension, and current smokers, and lower plasma total cholesterol, HDL-C, and LDL-C, but higher hs-CRP [1.76 (0.88-4.05) vs. 0.91 (0.32-1.87), Z=2.89, P=0.004].
2. Relationship Between High-Density Lipoprotein Cholesterol and Cholesterol Efflux Capacity
The CEC in the CHD group was lower than that in the non-CHD group [(11.9%±2.3%) vs. (13.0%±2.2%), t=-2.32, P=0.022, Figure 1], and the difference was statistically significant. In the non-CAD group, HDL-C was positively correlated with CEC (r=0.358, P=0.006, Figure 2A), but this correlation was not statistically significant in the CAD group (r=0.216, P=0.206, Figure 2B). NMRS verified this result, i.e., HDL-C by NMRS was positively correlated with CEC in the non-CHD group (r=0.416, P=0.001, Figure 2C), but had no correlation with CEC in the CAD group (r=0.065, P=0.708, Figure 2D). NMRS also simultaneously measured the content of apolipoprotein A-I (apoA-I), apoA-II, phospholipids, triglycerides, and free cholesterol in total HDL and each HDL subgroup. Univariate correlation analysis of CEC with the above indicators revealed that in the non-CHD group, CEC was positively correlated with total HDL phospholipids, total HDL free cholesterol, total HDL apoA-I, and total HDL apoA-II (all P<0.05), but in the CHD group, all the above correlations disappeared.
3. Factors Affecting CEC Cholesterol Efflux Capacity in the Coronary Heart Disease Group
To explore the factors affecting CEC in the CHD group, we performed correlation analysis of various clinical indicators with CEC. CEC had no correlation with patient age, body mass index, severity of coronary artery lesions (Gensini score), high-sensitivity troponin T, total cholesterol, triglycerides, or low-density lipoprotein cholesterol (Table 3). However, CEC was negatively correlated with hs-CRP (r=-0.351, P=0.036, Figure 3A). The 36 CAD patients were divided into a high hs-CRP group and a low hs-CRP group according to the median hs-CRP (1.75 mg/L). Except for high-sensitivity troponin T, there was no significant difference in baseline characteristics or the lipid and apolipoprotein content of total HDL between the two groups (Table 4), but the CEC in the high hs-CRP group was lower than that in the low hs-CRP group [(11.3%±2.2%) vs. (12.6%±2.1%), t=-1.83, P=0.038, Figure 3B], and the difference was statistically significant.
4. Components of High-Density Lipoprotein in the Coronary Heart Disease Group
NMRS was used to detect the structure of HDL in CHD patients with different levels of hs-CRP. The results showed that, compared with the low hs-CRP group, the largest HDL subgroup (HDL1) in the high hs-CRP group had higher lipid and apolipoprotein content, while the smallest HDL subgroup (HDL4) had lower lipid and apolipoprotein content (Figure 4). Correlation analysis showed that hs-CRP was positively correlated with the lipid and apolipoprotein content of HDL1, and negatively correlated with the lipid and apolipoprotein content of HDL4.
5. Multiple Linear Regression Analysis
In the CHD group, after adjusting for age, male sex, low-density lipoprotein cholesterol, diabetes, current smoking, body mass index, triglycerides, high-sensitivity troponin, and statin use, and further controlling for HDL-C/HDL-C by NMRS/HDL1 cholesterol/HDL4 cholesterol respectively, hs-CRP remained negatively correlated with CEC.
Discussion
For a long time, HDL-C has been widely recognized as a protective factor against atherosclerotic cardiovascular disease, but most clinical trials of drugs targeting HDL-C elevation, such as niacin and cholesteryl ester transfer protein inhibitors, ended in failure. Recently, an epidemiological study including 43,407 healthy people with a mean follow-up of 12.1 years found that extremely high HDL-C levels (≥2.32 mmol/L) were associated with an increased risk of cardiovascular death. This shows that predicting cardiovascular risk based on HDL-C levels alone is unreliable. HDL mainly exerts anti-atherosclerotic effects by mediating the reverse transport of cholesterol from beneath the vascular endothelium to the liver. CEC is an indicator measured in vitro and can be used to evaluate the initial step of reverse cholesterol transport, i.e., the step in which HDL mediates the removal of cholesterol from cells. Multiple studies have clearly established that CEC is an independent protective factor against cardiovascular events. However, the measurement of CEC has high experimental environment requirements and is time-consuming and labor-intensive. How to simply evaluate CEC in patients with coronary heart disease is a question worth exploring.
This study explored the function and structure of HDL in a small sample of non-CHD and CHD patients, and found that: (1) CEC in the CHD group was lower than that in the non-CHD group [(11.9%±2.3%) vs (13.0%±2.2%)], and the difference was statistically significant. In the non-CHD group, CEC was positively correlated with HDL-C (enzymatic method: r=0.358, P=0.006; NMRS: r=0.416, P=0.001), but in the CHD group, CEC had no correlation with HDL-C (enzymatic method: r=0.216, P=0.206; NMRS: r=0.065, P=0.708); (2) in the CHD group, CEC was negatively correlated with hs-CRP (r=-0.351, P=0.036), and this correlation persisted after adjusting for coronary heart disease risk factors and HDL-C, indicating that hs-CRP is a risk factor for CEC; (3) in the CHD group, the higher the hs-CRP, the more large-particle HDL and the less small-particle HDL, indicating that HDL is remodeled toward large particles.
1. Reduced HDL-C efflux capacity in patients with coronary heart disease
High-quality studies in the past have found that CEC in patients with coronary heart disease is significantly lower than that in healthy people, consistent with the results of this study. However, the literature is controversial regarding the relationship between HDL-C and CEC in patients with coronary heart disease. The correlation coefficient between HDL-C and CEC was 0.51 (P<0.0001) in the study by Khera et al., whereas it was -0.09 in the study by Zhang et al. The results of this study are consistent with those of Zhang et al., possibly because both this study and that of Zhang et al. enrolled patients with acute coronary syndrome, whereas Khera et al. enrolled patients with stable coronary heart disease and some healthy individuals. It can thus be seen that disease state affects CEC. In addition to coronary heart disease, patients with acute inflammation, end-stage renal disease, type 1/2 diabetes, and autoimmune diseases all have impaired CEC.
2. In patients with coronary heart disease, cholesterol efflux capacity is negatively correlated with high-sensitivity C-reactive protein
This study also observed that, in patients with coronary heart disease, along with the decline in CEC, hs-CRP was negatively correlated with CEC. This phenomenon is very similar to that found by Vaisar et al. in an acute inflammation model. In that study, low-dose endotoxin was used to induce an acute inflammatory response in healthy subjects, and CEC decreased. Proteomics of HDL at that time revealed that serum amyloid A1 (SAA1) and SAA2 in HDL were significantly elevated, and SAA was negatively correlated with CEC. Although the reason has not been fully elucidated, in vitro experiments and SAA1/2 gene knockout mouse models have confirmed that SAA incorporated into HDL can itself cause a decrease in CEC. Because coronary heart disease is also an inflammatory disease, and CRP, similar to SAA, is an acute-phase protein secreted by the liver during systemic inflammatory responses, we speculate that the phenomenon observed in this study—a decline in CEC negatively correlated with hs-CRP—may be caused by increased SAA in HDL. On the other hand, inflammatory apoA-I modification can also impair CEC. In this study, total HDL apoA-I was correlated with CEC in healthy individuals but this correlation disappeared in patients with coronary heart disease, suggesting that inflammatory apoA-I modification may also be one of the reasons for the aforementioned phenomenon. Although the mechanism of inflammation in the occurrence and development of coronary heart disease has not been fully elucidated, hs-CRP is already a recognized risk marker for coronary heart disease. Patients with stable coronary heart disease and high baseline hs-CRP (≥2 mg/L) have a significantly higher risk of major adverse cardiovascular events (MACE) than those with low baseline hs-CRP. In the CANTOS study, patients with prior myocardial infarction and hs-CRP ≥2 mg/L were treated with canakinumab, a monoclonal antibody against interleukin-1β. Those whose hs-CRP levels were reduced to below 2 mg/L during treatment had a 25% reduction in MACE events, while those whose hs-CRP did not reach the target had no significant benefit. In contrast, another anti-inflammatory drug, methotrexate, provided no benefit in patients with prior myocardial infarction, accompanied by no significant reduction in hs-CRP. Based on the above results and the findings of this study, the reason (or at least one of the reasons) why hs-CRP can serve as a risk marker for coronary heart disease is that it can reflect HDL dysfunction.
3. High-density lipoprotein is remodeled toward large particles in coronary heart disease patients with high high-sensitivity C-reactive protein levels
Our study also found that in coronary heart disease patients with high hs-CRP levels, HDL in plasma is remodeled toward large particles. This is consistent with previous results obtained by electrophoretic methods to distinguish HDL subclasses. Mature HDL is a spherical particle formed by a monolayer of phospholipids encapsulating a hydrophobic lipid core (mainly containing cholesteryl esters and triglycerides), with apolipoproteins on the surface. According to particle size, HDL can be categorized into large and small particles. Epidemiological studies have found that in healthy populations, small-particle HDL is negatively correlated with the incidence of CHD, whereas large particles show no significant correlation. This study did not conduct an in-depth exploration of the mechanism of HDL remodeling, but it is worth mentioning that the aforementioned incorporation of SAA can also cause a dose-dependent increase in HDL volume.
4. Limitations of this study
This study has certain limitations. First, because the samples and data of this study came from another cross-sectional study whose original purpose was not to find markers of CEC, the sample size of CHD patients was relatively small. Second, in the multiple linear regression analysis, although hs-CRP remained negatively correlated with CEC after adjusting for cardiovascular risk factors (P<0.05), if the P value were corrected using the Bonferroni test, the correlation would lose statistical significance; therefore, we can only reach a conclusion of "possibly correlated," and further research is needed to reach a definitive conclusion. Third, half of the coronary heart disease patients enrolled in this study were diagnosed with myocardial infarction. The source of inflammation in patients with myocardial infarction (mainly necrotic myocardium) differs from that in patients with stable coronary heart disease (mainly the vessel wall or adipose tissue), and the mechanisms by which they affect HDL function may differ. In the future, large-scale clinical studies are still needed to separately explore the HDL structure and function of patients with stable coronary heart disease or acute-phase myocardial infarction, so as to further verify this conclusion. Finally, although this study speculates that systemic inflammation plays an important role in the structural and functional remodeling of HDL, we did not explore its mechanism in depth.
Research Results
In summary, in patients with coronary heart disease, HDL-C cannot reflect the CEC of HDL, but measuring hs-CRP can. The higher the hs-CRP level, the lower the CEC of HDL, accompanied by remodeling of HDL toward large particles. hs-CRP may be a marker of HDL dysfunction and structural remodeling in patients with coronary heart disease. Systemic inflammation may be the common mechanism behind these phenomena.
