Discussion

In the present cross-sectional study, elevated homocysteine concentrations were positively associated with the occurrence of chronic total coronary occlusion in adults in southwest China. In addition, subgroup analysis showed that older age, hypertension, and diabetes may promote the onset of chronic total coronary occlusion. The association between quartiles of homocysteine and the prevalence of chronic total coronary occlusion was stronger in elderly individuals and those with hypertension and diabetes, whereas interaction analysis showed no interaction between the groups.

Chronic total coronary occlusion is the result of untimely recanalisation of acute coronary occlusion or progression of atherosclerosis. Arteriosclerosis is characterised by unremitting inflammatory damage to the arterial intima with increased permeability to plasma, deposition of plasma lipids, and fibrosis and calcification of so-called plaques. ^{Reference Schaffer, Verdoia, Cassetti, Marino, Suryapranata and De Luca13} Atherosclerosis is the most common pathological process that leads to cardiovascular disease, including myocardial infarction. The formation of atherosclerotic plaques is closely related to the proliferation of vascular endothelial cells, migration of vascular smooth muscle cells, lipid deposition in arterial walls, and remodelling of the extracellular matrix. Individuals with prior myocardial infarction have a higher mean homocysteine concentration than those without coronary artery disease. ^{Reference Agoşton-Coldea, Mocan, Seicean, Gatfossé and Rosenstingl14} Homocysteine is a known independent risk factor for atherosclerosis, ^{Reference Pang, Liu and Zhao15} and is considered a marker for the progression of atherosclerosis. ^{Reference Guthikonda and Haynes16 Reference Sreckovic, Sreckovic and Soldatovic–18} Homocysteine may promote atherosclerosis through several mechanisms, such as smooth muscle cell proliferation, endothelial dysfunction, oxidative damage, increased collagen synthesis, decreased high-density lipoprotein–cholesterol synthesis, and deterioration of the elastin of the arterial wall. ^{Reference Shenoy, Mehendale, Prabhu, Shetty and Rao11,Reference Zhang, Bai, Luo, Xiao, Wu and Ye19,Reference Zhao, Chen and Liu20} Its elevation can contribute to thin fibrous cap atherosclerosis, ^{Reference Liu, Wang and Wang21} ischaemia of the arterial wall, endothelial cell apoptosis, haemorrhage, inflammation, and intimal micro-abscesses, ultimately leading to the formation of fragile plaque. ^{Reference McCully22} Persistent elevation of homocysteine is a predictor of plaque progression. ^{Reference Zhang, Bai, Luo, Xiao, Wu and Ye19} This may lead to unstable plaque rupture and local thrombosis, eventually resulting in chronic total coronary occlusion. ^{Reference Shenoy, Mehendale, Prabhu, Shetty and Rao11} Previous studies did not find an association between homocysteine concentration and coronary collateral formation, but elevated homocysteine was noted in individuals with single-vessel chronic total occlusion. ^{Reference Sayar, Terzi and Bilsel23} These data are consistent with the present findings.

Homocysteine-induced atherosclerosis is associated with multiple cellular molecular mechanisms. The role of homocysteine in endothelial dysfunction is thought to be mediated by oxidative stress, nuclear factor-κB activation, and inhibition of endothelial nitric oxide synthase. ^{Reference Basu, Jenkins and Stoner24} Homocysteine binds to N-methyl-D-aspartate receptors, leading to oxidative stress, cytoplasmic calcium influx, apoptosis, and endothelial dysfunction. ^{Reference McCully17} Homocysteine can produce oxidative stress, contributing to the growth of atherosclerotic vascular lesions by interfering with low concentration endothelial nitric oxide synthase-derived nitric oxide production, mediating the loss of the endothelial antioxidant system, and increasing the concentrations of intracellular reactive oxygen species. ^{Reference Esse, Barroso, Tavares de Almeida and Castro25} Homocysteine stimulates proliferation of endothelial cells by generating reactive oxygen species through the autoxidation of its sulfhydryl group, as well as by inhibiting antioxidant enzymes, activating NADPH oxidase and matrix metalloproteinases, and inducing apoptosis through mitochondrial alterations. Homocysteine can also stimulate the proliferation of smooth muscle cells ^{Reference Ganguly and Alam26} and damage vascular collagen fibres. In addition, homocysteine can enhance low-density lipoprotein–cholesterol lipid oxidation and promote coagulation, enhance platelet aggregation, and alter the thrombotic properties of endothelial cells, thereby leading to atherosclerosis and thrombosis. ^{Reference Hankey and Eikelboom27} Homocysteine inhibits scavenger receptor group B member 1 via the DNMT3b/SP1 pathway ^{Reference Guo, Zhang and Yang28} and inhibits LXRα-mediated ABCA1/ABCG1-dependent cholesterol efflux from macrophages. ^{Reference Jin, Bian and Wang29} Additionally, homocysteine increases the production of inflammatory interleukin-1β in macrophages. ^{Reference Xie, Ding and Zhang30} Oxidative stress, proinflammatory factor activation, and genomic homocysteine DNA methylation play important roles in the progression of atherosclerosis. ^{Reference Hai and Zuo31} Hyperhomocysteinemia promotes atherogenesis by altering histone acetylation patterns and regulating miRNA expression. ^{Reference Hai and Zuo31} And finally, homocysteine induces SMAD7 promoter hypermethylation and activates nuclear factor-κB to promote vasculopathy. ^{Reference Wei, Chao and Gao32}

Elevated homocysteine is a characteristic feature of ageing. Homocysteine levels are higher in elderly individuals with coronary artery disease than in those without it. ^{Reference Dimitrova, DeGroot, Myers and Kim33} Many factors contribute to age-related increase in homocysteine levels, including vegetarian diet, ^{Reference Obersby, Chappell, Dunnett and Tsiami34} deficiency of vitamins B6 and B12, folic acid and hypertension, ^{Reference Ostrakhovitch and Tabibzadeh35} and lack of exercise. ^{Reference Nygård, Vollset and Refsum36} Age-dependent reduction in cystathionine beta-synthase activity increases serum homocysteine levels. ^{Reference Ostrakhovitch and Tabibzadeh35,Reference Nordström and Kjellström37,Reference Wang, Sun and Zhou38} N-nitration of Trp208, Trp43, and Tyr223, and loss of thiolate coordination inhibit cystathionine beta-synthase activity and lead to age-related hyperhomocysteinemia. ^{Reference Ostrakhovitch and Tabibzadeh35} Increased homocysteine levels are associated with arteriosclerotic outcomes in elderly individuals and are considered an independent risk for cardiovascular disease. ^{Reference Mangge, Becker, Fuchs and Gostner39}

Hypertension is a recognised contributor to atherosclerosis, causing vascular smooth muscle proliferation, atheromatous plaque, atheroma formation, and luminal narrowing. ^{Reference Pessina, Pauletto, Rossi and Valle40} Renin, catecholamines, and angiotensin that are associated with hypertension can also alter the metabolism of the arterial wall, thereby leading to endothelial damage. ^{Reference Pessina, Pauletto, Rossi and Valle40} Increased arterial stiffness is a warning sign of coronary events in hypertensive patients. ^{Reference Boutouyrie, Tropeano and Asmar41} Besides an independent effect, a multiplicative effect of hypertension and hyperhomocysteinemia substantially increases the risk of coronary artery disease. Strong interactive effects were observed between homocysteine levels and increasing age or hypertension. ^{Reference Gupta, Sharma, Garg, Kaur, Bedi and Vij42} These findings suggest a causal link between homocysteine and blood pressure, and elevated blood pressure may mediate some of the cardiotoxic effects of homocysteine. ^{Reference Lim and Cassano43} The individuals with hypertension and high levels of homocysteine had more severe coronary atherosclerosis and more diffuse atherosclerosis than those without this association, and the presence of both hypertension and hyperhomocysteinemia was associated with more severe coronary artery disease. ^{Reference Montalescot, Ankri and Chadefaux-Vekemans44} Hypertension is a major link between hyperhomocysteinemia and coronary artery disease occurrence. ^{Reference Ghassibe-Sabbagh, Platt and Youhanna45} Previous studies have noted increased concentrations of haemostatic or proinflammatory cytokines, growth factors, and heat-shock proteins in the plasma of hypertensive individuals, all of which may promote atherosclerosis. ^{Reference Parissis, Korovesis, Giazitzoglou, Kalivas and Katritsis46,Reference Pockley, De Faire, Kiessling, Lemne, Thulin and Frostegård47} Diabetes impairs vascular endothelial function. ^{Reference Hu, Xu and Wang48} Insulin resistance leads to less phosphatidylinositol-3-kinase/protein kinase B signalling, impaired endothelial nitric oxide synthase expression, and decreased nitric oxide bioavailability, resulting in endothelial dysfunction in diabetes individuals with hyperhomocysteinemia. ^{Reference Janus, Szahidewicz-Krupska, Mazur and Doroszko49} In the combined metabolic state of hyperhomocysteinemia and hyperglycaemia, endothelin 1 synthesis is increased in endothelial cells and mitochondrial reactive oxygen species produced. ^{Reference Sethi, Lees, Douthwaite, Dawnay and Corder50} In addition, homocysteine increases the activity of hydroxymethyleneglycol coenzyme A, which increases cholesterol synthesis, and low-density lipoprotein–cholesterol damages endothelial nitric oxide synthase activity by increasing oxidative stress, which in turn leads to endothelial cell dysfunction. ^{Reference Hermida and Balligand51} Additionally, in individuals with diabetes and hyperhomocysteinemia, abnormal vascular smooth muscle cell activity thickens the wall of arteries to add to the pathologic changes of atherosclerosis. ^{Reference Begum, Hockman and Manganiello52}

The study has several limitations. First, this was a single-centre study performed on a Chinese population, which may have limited the generalisability of the results. The issue needs to be further explored at a larger number of institutions and in a larger number of other ethnic groups. Second, this cross-sectional study only showed an association between elevated homocysteine and the incidence of chronic total coronary occlusion, and a large multicenter prospective study is needed to validate the predictive power of homocysteine on the risk of chronic total coronary occlusion and evaluate whether homocysteine-lowering therapy can reduce the incidence of adverse events in patients with chronic total coronary occlusion who have elevated homocysteine. Third, this study only included subjects who completed coronary angiography, so some populations may have been missed and there may have been selection bias. Fourth, this was a retrospective observational study and responses to medication history may have been inaccurate and folic acid and vitamin B12 levels were not measured, and although we made statistical adjustments for confounders in our multivariate logistic regression model, we may not have completely eliminated their effects. Finally, we did not consider whether any given patient’s diagnosis at admission was acute coronary syndrome or chronic coronary ischaemic syndrome, and we did not collect details on the patient’s chronic total coronary occlusion lesions, such as site, number, length, whether they were combined with calcification, or collateral circulation, so we were unable to perform an analysis of these relevant conditions.