Tatsuya Sawamura, MD, PhD
Professor, Department of Physiology, Shinshu University
LDL is a “bad” cholesterol in the blood that is involved in the progression of arteriosclerosis. Particular focus has been placed on modified LDL, such as LDL that has been oxidized, and its action. Lectin-like oxidized LDL receptor-1 (LOX-1) is an oxidized LDL receptor that we identified in vascular endothelial cells1,2). LOX-1 binds to oxidized LDL, causing endothelial dysfunction in forms such as apoptosis, release of inflammatory cytokines, production of reactive oxygen species, and a decrease in NO. LOX-1 is expressed in smooth muscle, macrophages, and platelets, and LOX-1 causes the progression of atherosclerotic plaque, i.e. the proliferation of smooth muscle cells, the formation of foam cells, and the aggregation of activated platelets. In addition, LOX-1 acts as an adhesion molecule for leukocytes and activated platelets. Moreover, LOX-1 acts to promote inflammation by binding to CRP (Fig. 1). In various models of cardiovascular disease, inhibition of LOX-1 results in improvements such as decreased lipid deposition in arteries in hyperlipidemia3), improved vascular permeability dependent upon CRP, decreased infiltration of neutrophils due to inflammation, decreased intimal thickening due to arterial injury, and decreased myocardial damage due to ischemia-reperfusion. In other words, LOX-1 acts as an exacerbating factor in each stage of atherosclerotic disease (Fig. 2).
The expression of LOX-1 increases starting in the initial stages of atherosclerotic disease, and LOX-1 binds to a range of ligands. What follows is a description of development of disease markers based on measurement of LOX-1 and its ligands.
I. The LOX index as an indicator of cardiovascular disease
1. LAB (modified LDL)
In clinical practice, the amount of LDL is measured as an index of bad cholesterol, but truly bad LDL is LDL that has been modified, like the oxidized form of LDL. Quantification of modified LDL is essential to accurate diagnosis and assessment of the risk of atherosclerotic disease. Conventional measurement of oxidized LDL used monoclonal antibodies that recognized only some of the forms of oxidized LDL. This method of measurement is unable to detect every form of oxidized LDL because of its varied modifications. In addition, oxidized LDL is merely one form of modified LDL. Other modifications of LDL vary and include malondialdehyde modification, acetylation, carbamylation, glycosylation, and generation of negatively charged lipoproteins. Oxidized LDL is commonly known to be present in atherosclerotic plaque. Over the past few years, however, research has revealed increased amounts of other forms of modified LDL in the blood due to smoking and conditions such as diabetes mellitus, chronic kidney disease, acute coronary syndrome, and cerebral infarction.
LOX-1 binds to modified LDL (including oxidized LDL) and causes endothelial dysfunction. Thus, we utilized the function of the LOX-1 receptor to measure different forms of modified LDL with bioactivity in order to possibly develop a biomarker of atherosclerotic disease. In specific terms, we used sandwich ELISA with anti-LOX-1 (extracellular domain) and anti-apoB antibodies to detect lipoprotein containing apoB (LOX-1 ligands containing apoB, or LAB) binding to LOX-1 (Fig. 3)4). Results of animal experiments revealed that the level of LAB in the blood increased in hyperlipidemic apoE-deficient mice4) and hypercholesterolemic WHHL rabbits. Results also revealed that reducing the level of LAB in the blood resulted in a reduction in the size of atherosclerotic plaque. These findings suggest that LAB promotes arteriosclerosis and that the amount of LAB in the blood may reflect the progression of disease.
Thus, we measured both LAB and sLOX-1, and we defined the LOX index as the product of the two to indicate interaction between the receptor and its ligands (Fig. 3). Using data from the Suita study, we examined the usefulness of the LOX index in assessment of the risk of developing atherosclerotic disease5). The Suita Study is a cohort study that the National Cerebral and Cardiovascular Center has conducted since 1989. The Study has examined the development of and prognosis for cardiovascular disease in urban residents of the City of Suita in Osaka Prefecture (6,485 men and women, ages 30-79 years). We conducted a follow-up of individuals who underwent a routine health checkup in 1994, and we studied 2,295 such individuals (1,094 men, 1,201 women) who did not have a history of coronary artery disease or stroke. Our study endpoints were ① initial onset of coronary artery disease or stroke, ② death, ③ moving away from the City of Suita, ④ and December 31, 2007. Over an average follow-up period of 11 years, 68 individuals developed coronary artery disease and 91 suffered a stroke (with 60 of those 91 suffering cerebral infarction).
LAB and sLOX-1 levels were measured in subject sera. The mean LAB level was 516.1±17.1 ng/mL for men and 782.3±23.7 ng/mL for women. The mean sLOX-1 level was 1060.1±8.6 pg/mL for men and 797.8±0.2 pg/mL for women. Subjects were divided into a 1st to a 4th quartile based on their levels of LAB and sLOX-1, with lower levels constituting the 1st quartile. The relationship between measured levels and results of the health checkup was then examined. For both men and women, higher sLOX-1 levels signaled a higher rate of smoking, but a correlation between LAB and sLOX-1 and the prevalence of hypertension and diabetes mellitus was not noted.
A proportional hazard model was adjusted for sex, age, hypertension, diabetes mellitus, use of lipid-lowering drugs, BMI, smoking, alcohol consumption, and non-HDL. This multivariate-adjusted proportional hazard model was then used to determine the relative risk of developing disease for the LAB level, the sLOX-1 level, and the LOX index. Results for patients with an LAB level in the 4th quartile indicated that the incidence of stroke was 2.09 times higher, the incidence of cerebral infarction was 3.11 times higher, and the incidence of cardiovascular disease (coronary artery disease＋stroke) was 1.91 times higher. Thus, the incidence of disease was significantly higher, suggesting that a high LAB level signals a risk of developing these conditions. For patients with an sLOX-1 level in the 3rd quartile, the incidence of coronary artery disease was 2.13 times higher. However, significant differences between these patients and those with an sLOX-1 level in the 4th quartile were not noted.
For patients with an LOX index in the 4th quartile, the incidence of coronary artery disease was 2.09 times higher and the incidence of cardiovascular disease was 1.83 times higher. Thus, the incidence of disease was significantly higher. A finding worth noting is the fact that patients with an LOX index in the 2nd to the 4th quartile all had an incidence of cerebral infarction that was 3 or more times higher (Fig. 4)5). Another study involving the Suita cohort compared patients with hypertension to those with a normal blood pressure, and the study reported that the incidence of stroke was about 3 times higher in men with hypertension and about 2 times higher in women with hypertension6). Hypertension is the greatest risk factor for cerebral infarction, and a high LOX index signals a risk of disease on par with hypertension. As was mentioned earlier, the levels of LAB and sLOX-1 in the blood are not related to the prevalence of hypertension, but a high LOX index and hypertension may be independent risk factors. In the future, use of the two in combination will probably allow more precise assessment of the risk of cerebral infarction.
At the current point in time, a predictor of cerebral infarction has yet to be established. Numerous cohort studies (including the Suita Study) have reported that total cholesterol (TC), LDL, and non-HDL are not related to cerebral infarction7). Although the LAB level and the LOX index are not viable indices of cholesterol, the fact that they are closely associated with cerebral infarction is extremely interesting.
2. Prospects for the future
Of the previous prospective cohort studies that have analyzed the relationship between oxidized LDL and the risk of disease, only 1 study from the US has reported that a high level of oxidized LDL results in a 2-fold or greater increase in the incidence of metabolic syndrome 8). However, a recent study has reported the level of LAB in the blood of normal Japanese males is correlated with smoking, waist circumference, and development of hypertriglyceridemia and metabolic syndrome9). In addition, attempts to use the LAB level as a therapeutic index have begun. An intervention study of patients with hypercholesterolemia reported that pitavastatin resulted in a decrease in TC, LDL, and triglycerides as well as a decrease in LAB10). The study found no correlation between pitavastatin use and the extent of the decrease in LDL and LAB, so the decrease in LAB is presumably due to a mechanism other than pitavastatin’s lowering of LDL. Statins have multi-faceted action (including antioxidant action), and LAB levels may represent a composite result of using statins. Numerous studies will presumably measure LAB in the future, potentially revealing a relationship between LAB and disease and drug efficacy.
More precise indices of cholesterol are needed to facilitate early diagnosis of atherosclerotic disease. The LAB level and the LOX index are unique indices that reflect the modified state of LDL as well as levels of LDL. In the future, we hope to demonstrate the increased usefulness of the LOX index as a biomarker as we assemble additional evidence.
Reference
1) Sawamura T, Kume N, Aoyama T, et al: An endothelial receptor for oxidized low-density lipoprotein. Nature 386: 73-77, 1997.
2) Yoshimoto R, Fujita Y, Kakino A, et al. The discovery of LOX-1, its ligands and clinical significance. Cardiovasc Drugs Ther 25: 379-391, 2011.
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4) Sato Y, Nishimichi N, Nakano A, et al. Determination of LOX-1-ligand activity in mouse plasma with a chicken monoclonal antibody for ApoB. Atherosclerosis 200: 303-309, 2008.
5) Inoue N, Okamura T, Kokubo Y, et al. LOX index, a novel predictive biochemical marker for coronary heart disease and stroke. Clin Chem 56: 550-558, 2010.
6) Kokubo Y, Kamide K, Okamura T, et al. Impact of high-normal blood pressure on the risk of cardiovascular disease in a Japanese urban cohort: the Suita study. Hypertension 52: 652-659, 2008.
7) Okamura T, Kokubo Y, Watanabe M, et al. Low-density lipoprotein cholesterol and non-high-density lipoprotein cholesterol and the incidence of cardiovascular disease in an urban Japanese cohort study: the Suita study.
8) Hovoet P, Lee DH, Steffes M, et al. Association between circulating oxidized low-density lipoprotein and incidence of the metabolic syndrome. JAMA 299: 2287-2293, 2008.
9) Uchiba K, Suehiro A, Nakanishi M, et al. Association of atherosclerotic risk factors with oxidized low-density lipoprotein evaluated by LOX-1 ligand activity in healthy men. Clin Chim Acta 412: 1643-1647, 2011.
10) Matsumoto T, Fujita M, Sawamura T, et al. Pitavastatin reduces lectin-like oxidized low-density lipoprotein receptor-1 ligands in hypercholesterolemic humans. Lipids 45: 329-335, 2010.