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LI Hong Zheng, WANG Qin, ZHANG Yi Yuan, WANG Jin Dong, WU Hong Juan, ZHANG Mo Gen, LI Ji Chen, LIU Zhi Jun. Onset of Coronary Heart Disease is Associated with HCMV Infection and Increased CD14+CD16+ Monocytes in a Population of Weifang, China[J]. Biomedical and Environmental Sciences, 2020, 33(8): 573-582. doi: 10.3967/bes2020.076
Citation: LI Hong Zheng, WANG Qin, ZHANG Yi Yuan, WANG Jin Dong, WU Hong Juan, ZHANG Mo Gen, LI Ji Chen, LIU Zhi Jun. Onset of Coronary Heart Disease is Associated with HCMV Infection and Increased CD14+CD16+ Monocytes in a Population of Weifang, China[J]. Biomedical and Environmental Sciences, 2020, 33(8): 573-582. doi: 10.3967/bes2020.076

Onset of Coronary Heart Disease is Associated with HCMV Infection and Increased CD14+CD16+ Monocytes in a Population of Weifang, China

doi: 10.3967/bes2020.076
Funds:  This work was funded by the National Natural Science Foundation of China [81471048]; the Natural Science Foundation of Shandong Province [ZR2019MC059]; and Shandong Province Government-Sponsored Overseas Study Project
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  • Author Bio:

    LI Hong Zheng, male, born in 1996, Master of science, majoring in mechanism and intervention of virus infection

    WANG Qin, female, born in 1972, Doctor of philosophy, majoring in pathogenic biology and immunology

  • Corresponding author: LIU Zhi Jun, Associate Professor, E-mail: zhijun.liu@wfmc.edu.cn
  • ΔThese authors contributed equally to this work.
  • Received Date: 2019-11-02
  • Accepted Date: 2020-05-25
  •   Objective  To investigate the relationship between human cytomegalovirus (HCMV) infection and peripheral blood CD14+CD16+ monocytes in the pathogenesis of coronary heart disease (CHD), and to elucidate the mechanism of pathogenesis in CHD by analyzing the correlation between infection, inflammation, and CHD, to provide a basis for the prevention, evaluation, and treatment of the disease.  Methods  In total, 192 patients with CHD were divided into three groups: latent CHD, angina pectoris, and myocardial infarction. HCMV-IgM and -IgG antibodies were assessed using ELISA; CD14+CD16+ monocytes were counted using a five-type automated hematology analyzer; mononuclear cells were assessed using fluorescence-activated cell sorting; and an automatic biochemical analyzer was used to measure the levels of triglyceride, cholesterol, high- and low-density lipoprotein cholesterols, lipoprotein, hs-CRp and Hcy.  Results  The positive rates of HCMV-IgM and -IgG were significantly higher in the CHD groups than in the control group. HCMV infection affects lipid metabolism to promote immune and inflammatory responses.  Conclusion  HCMV infection has a specific correlation with the occurrence and development of CHD. The expression of CD14+CD16+ mononuclear cells in the CHD group was increased accordingly and correlated with acute HCMV infection. Thus, HCMV antibody as well as peripheral blood CD14+CD16+ mononuclear cells can be used to monitor the occurrence and development of CHD.
  • ΔThese authors contributed equally to this work.
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  • [1] Arbab-Zadeh A, Fuster V. The risk continuum of atherosclerosis and its implications for defining CHD by coronary angiography. J Am Coll Cardiol, 2016; 68, 2467−78. doi:  10.1016/j.jacc.2016.08.069
    [2] Wong ND. Epidemiological studies of CHD and the evolution of preventive cardiology. Nat Rev Cardiol, 2014; 11, 276−89. doi:  10.1038/nrcardio.2014.26
    [3] Herrmann J, Kaski JC, Lerman A. Coronary microvascular dysfunction in the clinical setting: from mystery to reality. Eur Heart J, 2012; 33, 2771−82b. doi:  10.1093/eurheartj/ehs246
    [4] Wang JW, Zhou ZQ, Hu DY. Prevalence of arterial stiffness in North China, and associations with risk factors of cardiovascular disease: a community-based study. BMC Cardiovasc Disord, 2012; 12, 119. doi:  10.1186/1471-2261-12-119
    [5] Li X, Zhang Y, Wang M, et al. The prevalence and awareness of cardiometabolic risk factors in Southern Chinese population with coronary artery disease. Sci World J, 2013; 2013, 416192.
    [6] Opadijo OG, Akande AA, Jimoh AK. Prevalence of coronary heart disease risk factors in Nigerians with systemic hypertension. Afr J Med Med Sci, 2004; 33, 121−5.
    [7] Wang L, Yao D, Wu T. Prevalence of overweight and smoking patients with coronary heart disease in rural China. Aust J Rural Health, 2004; 12, 17−21. doi:  10.1111/j.1440-1584.2004.t01-1-00543.x
    [8] Amaral N, Okonko DO. Metabolic abnormalities of the heart in type II diabetes. Diab Vasc Dis Res, 2015; 12, 239−48. doi:  10.1177/1479164115580936
    [9] Kucharska-Newton A, Griswold M, Yao ZH, et al. Cardiovascular disease and patterns of change in functional status over 15 years: findings from the atherosclerosis risk in communities (ARIC) study. J Am Heart Assoc, 2017; 6.
    [10] Nuutinen S, Ailanen L, Savontaus E, et al. Melanocortin overexpression limits diet-induced inflammation and atherosclerosis in LDLR(-/-) mice. J Endocrinol, 2018; 236, 111−23. doi:  10.1530/JOE-17-0636
    [11] Yamada S, Senokuchi T, Matsumura T, et al. Inhibition of local macrophage growth ameliorates focal inflammation and suppresses atherosclerosis. Arterioscler Thromb Vasc Biol, 2018; 38, 994−1006. doi:  10.1161/ATVBAHA.117.310320
    [12] Zhao R, Ghazzawi N, Wu J, et al. Germinated brown rice attenuates atherosclerosis and vascular inflammation in low-density lipoprotein receptor-knockout mice. J Agric Food Chem, 2018; 66, 4512−20. doi:  10.1021/acs.jafc.8b00005
    [13] Spence JD, Norris J. Infection, inflammation, and atherosclerosis. Stroke, 2003; 34, 333−4. doi:  10.1161/01.STR.0000054049.65350.EA
    [14] Levi M, van der Poll T, Schultz M. Infection and inflammation as risk factors for thrombosis and atherosclerosis. Semin Thromb Hemost, 2012; 38, 506−14. doi:  10.1055/s-0032-1305782
    [15] Kozarov E, Huber K, Wojta J. Infection-associated biomarkers of inflammation in atherosclerosis. Curr Pharm Des, 2015; 21, 1776−82. doi:  10.2174/1381612821666141129173343
    [16] Tang-Feldman YJ, Lochhead SR, Lochhead GR, et al. Murine cytomegalovirus (MCMV) infection upregulates P38 MAP kinase in aortas of Apo E KO mice: a molecular mechanism for MCMV-induced acceleration of atherosclerosis. J Cardiovasc Transl Res, 2013; 6, 54−64. doi:  10.1007/s12265-012-9428-x
    [17] Heybar H, Alavi SM, Farashahi Nejad M, et al. Cytomegalovirus infection and atherosclerosis in candidate of coronary artery bypass graft. Jundishapur J Microbiol, 2015; 8, e15476.
    [18] Kawasaki M, Arai Y, Takayama M, et al. Carotid atherosclerosis, cytomegalovirus infection, and cognitive decline in the very old: a community-based prospective cohort study. Age (Dordr), 2016; 38, 29. doi:  10.1007/s11357-016-9890-5
    [19] Jia YJ, Liu J, Han FF, et al. Cytomegalovirus infection and atherosclerosis risk: a meta-analysis. J Med Virol, 2017; 89, 2196−206. doi:  10.1002/jmv.24858
    [20] Datta SK, Tumilowicz JJ, Trentin JJ. Lysis of human arterial smooth muscle cells infected with herpesviridae by peripheral blood mononuclear cells: implications for atherosclerosis. Viral Immunol, 1993; 6, 153−60. doi:  10.1089/vim.1993.6.153
    [21] R Horváth, J Cerný, J Benedík Jr, et al. The possible role of human cytomegalovirus (HCMV) in the origin of atherosclerosis. J Clin Virol, 2000; 16, 17−24. doi:  10.1016/S1386-6532(99)00064-5
    [22] Lee YL, Liu CE, Cho WL, et al. Presence of cytomegalovirus DNA in leucocytes is associated with increased oxidative stress and subclinical atherosclerosis in healthy adults. Biomarkers, 2014; 19, 109−13. doi:  10.3109/1354750X.2013.877967
    [23] Tsirpanlis G, Chatzipanagiotou S, Ioannidis A, et al. Serum and peripheral blood mononuclear cells infectious burden: correlation to inflammation and atherosclerosis in haemodialysis patients. Nephrology (Carlton), 2005; 10, 256−63. doi:  10.1111/j.1440-1797.2005.00414.x
    [24] Sacre K, Hunt PW, Hsue PY, et al. A role for cytomegalovirus-specific CD4+CX3CR1+ T cells and cytomegalovirus-induced T-cell immunopathology in HIV-associated atherosclerosis. AIDS, 2012; 26, 805−14. doi:  10.1097/QAD.0b013e328351f780
    [25] Pant S, Deshmukh A, Gurumurthy GS, et al. Inflammation and atherosclerosis--revisited. J Cardiovasc Pharmacol Ther, 2014; 19, 170−8. doi:  10.1177/1074248413504994
    [26] Popovic M, Smiljanic K, Dobutovic B, et al. Human cytomegalovirus infection and atherothrombosis. J Thromb Thrombolysis, 2012; 33, 160−72. doi:  10.1007/s11239-011-0662-x
    [27] Du Y, Zhang G, Liu Z. Human cytomegalovirus infection and coronary heart disease: a systematic review. Virol J, 2018; 15, 31. doi:  10.1186/s12985-018-0937-3
    [28] Britt WJ. Human cytomegalovirus: propagation, quantification, and storage. Curr Protoc Microbiol, 2010; Chapter 14, Unit 14E 3.
    [29] Hamprecht K, Kagan KO, Goelz R. Hyperimmune globulin to prevent congenital CMV infection. N Engl J Med, 2014; 370, 2543. doi:  10.1056/NEJMc1405377
    [30] Westphal M, Lautenschlager I, Backhaus C, et al. Cytomegalovirus and proliferative signals in the vascular wall of CABG patients. Thorac Cardiovasc Surg, 2006; 54, 219−26. doi:  10.1055/s-2006-923891
    [31] Fiorentini S, Luganini A, Dell'Oste V, et al. Human cytomegalovirus productively infects lymphatic endothelial cells and induces a secretome that promotes angiogenesis and lymphangiogenesis through interleukin-6 and granulocyte-macrophage colony-stimulating factor. J Gen Virol, 2011; 92, 650−60. doi:  10.1099/vir.0.025395-0
    [32] Jakovljevic A, Knezevic A, Nikolic N, et al. Herpesviruses viral loads and levels of proinflammatory cytokines in apical periodontitis. Oral Dis, 2018; 24, 840−6. doi:  10.1111/odi.12829
    [33] Sinclair J, Sissons P. Latent and persistent infections of monocytes and macrophages. Intervirology, 1996; 39, 293−301. doi:  10.1159/000150501
    [34] Geng S, Chen K, Yuan R, et al. The persistence of low-grade inflammatory monocytes contributes to aggravated atherosclerosis. Nat Commun, 2016; 7, 13436. doi:  10.1038/ncomms13436
    [35] Woollard KJ, Geissmann F. Monocytes in atherosclerosis: subsets and functions. Nat Rev Cardiol, 2010; 7, 77−86. doi:  10.1038/nrcardio.2009.228
    [36] Yang FJ, Shu KH, Chen HY, et al. Anti-cytomegalovirus IgG antibody titer is positively associated with advanced T cell differentiation and coronary artery disease in end-stage renal disease. Immun Ageing, 2018; 15, 15. doi:  10.1186/s12979-018-0120-0
    [37] Belge KU, Dayyani F, Horelt A, et al. The proinflammatory CD14+CD16+DR++ monocytes are a major source of TNF. J Immunol, 2002; 168, 3536−42. doi:  10.4049/jimmunol.168.7.3536
    [38] Zhang Q, Qian G, Ding Z. Xuemaitong granules attenuate carotid atherosclerosis by decreasing the expression of CD14+CD16+ monocytes, IL-6, TNF-alpha, and hsCRP. Genet Mol Res, 2014; 13, 7519−27. doi:  10.4238/2014.September.12.19
    [39] Adam E, Melnick JL, Probtsfield JL, et al. High levels of cytomegalovirus antibody in patients requiring vascular surgery for atherosclerosis. Lancet, 1987; 2, 291−3.
    [40] Juhl D, Vockel A, Luhm J, et al. Comparison of the two fully automated anti-HCMV IgG assays: abbott architect CMV IgG assay and biotest anti-HCMV recombinant IgG ELISA. Transfus Med, 2013; 23, 187−94. doi:  10.1111/tme.12036
    [41] Fabricant CG, Fabricant J, Litrenta MM, et al. Virus-induced atherosclerosis. J Exp Med, 1978; 148, 335−40. doi:  10.1084/jem.148.1.335
    [42] Biolatti M, Gugliesi F, Dell'Oste V, et al. Modulation of the innate immune response by human cytomegalovirus. Infect Genet Evol, 2018; 64, 105−14. doi:  10.1016/j.meegid.2018.06.025
    [43] Guo N, Zhang N, Yan L, et al. Down-regulation of single-stranded DNA-binding protein 1 expression induced by HCMV infection promotes lipid accumulation in cells. Braz J Med Biol Res, 2017; 50, e6389. doi:  10.1590/1414-431x20176389
    [44] Ziegler-Heitbrock L. The CD14+ CD16+ blood monocytes: their role in infection and inflammation. J Leukoc Biol, 2007; 81, 584−92. doi:  10.1189/jlb.0806510
    [45] Reeves MB, Sinclair JH. Circulating dendritic cells isolated from healthy seropositive donors are sites of human cytomegalovirus reactivation in vivo. J Virol, 2013; 87, 10660−7. doi:  10.1128/JVI.01539-13
    [46] Ismahil MA, Hamid T, Bansal SS, et al. Remodeling of the mononuclear phagocyte network underlies chronic inflammation and disease progression in heart failure: critical importance of the cardiosplenic axis. Circ Res, 2014; 114, 266−82. doi:  10.1161/CIRCRESAHA.113.301720
    [47] Jiang S, Li D, Li J, et al. Correlation between high-density lipoprotein and monocyte subsets in patients with stable coronary heart disease. Med Sci Monit, 2015; 21, 3129−35. doi:  10.12659/MSM.894485
    [48] Ozdogru I, Inanc MT, Eryol NK, et al. CD14+ monocyte levels in subgroups of acute coronary syndromes. Coron Artery Dis, 2007; 18, 519−22. doi:  10.1097/MCA.0b013e3282ef4e7c
    [49] Park YM. CD36, a scavenger receptor implicated in atherosclerosis. Exp Mol Med, 2014; 46, e99. doi:  10.1038/emm.2014.38
    [50] Ibrahim S, Siddiqui AA, Siddiqui AR, et al. Sociodemographic factors associated with IgG and IgM seroprevalence for human cytomegalovirus infection in adult populations of Pakistan: a seroprevalence survey. BMC Public Health, 2016; 16, 1112. doi:  10.1186/s12889-016-3772-8
    [51] Zhang X, Xie Y, Zhou H, et al. Involvement of TLR4 in oxidized LDL/beta2GPI/anti-beta2GPI-induced transformation of macrophages to foam cells. J Atheroscler Thromb, 2014; 21, 1140−51. doi:  10.5551/jat.24372
    [52] Izadi M, Fazel M, Saadat SH, et al. Cytomegalovirus localization in atherosclerotic plaques is associated with acute coronary syndromes: report of 105 patients. Methodist Debakey Cardiovasc J, 2012; 8, 42−6.
    [53] Miyajima S, Naruse K, Kobayashi Y, et al. Periodontitis-activated monocytes/macrophages cause aortic inflammation. Sci Rep, 2014; 4, 5171.
    [54] Gautier EL, Jakubzick C, Randolph GJ. Regulation of the migration and survival of monocyte subsets by chemokine receptors and its relevance to atherosclerosis. Arterioscler Thromb Vasc Biol, 2009; 29, 1412−8. doi:  10.1161/ATVBAHA.108.180505
    [55] Huang Y, Wang JS, Yin HJ, et al. The expression of CD14(+)CD16(+) monocyte subpopulation in coronary heart disease patients with blood stasis syndrome. Evid Based Complement Alternat Med, 2013; 2013, 416932.
    [56] Schlitt A, Heine GH, Blankenberg S, et al. CD14+CD16+ monocytes in coronary artery disease and their relationship to serum TNF-alpha levels. Thromb Haemost, 2004; 92, 419−24. doi:  10.1160/TH04-02-0095
    [57] Chavez-Sanchez L, Garza-Reyes MG, Espinosa-Luna JE, et al. The role of TLR2, TLR4 and CD36 in macrophage activation and foam cell formation in response to oxLDL in humans. Hum Immunol, 2014; 75, 322−9. doi:  10.1016/j.humimm.2014.01.012
    [58] Stellos K, Bigalke B, Borst O, et al. Circulating platelet-progenitor cell coaggregate formation is increased in patients with acute coronary syndromes and augments recruitment of CD34+ cells in the ischaemic microcirculation. Eur Heart J, 2013; 34, 2548−56. doi:  10.1093/eurheartj/eht131
    [59] Tarkin JM, Joshi FR, Rudd JH. PET imaging of inflammation in atherosclerosis. Nat Rev Cardiol, 2014; 11, 443−57. doi:  10.1038/nrcardio.2014.80
    [60] Sbrana F, Cocci F, Papa A, et al. Routine laboratory tests to risk-stratify patients with chronic coronary artery disease. J Cardiol, 2013; 61, 132−7. doi:  10.1016/j.jjcc.2012.09.005
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Onset of Coronary Heart Disease is Associated with HCMV Infection and Increased CD14+CD16+ Monocytes in a Population of Weifang, China

doi: 10.3967/bes2020.076
Funds:  This work was funded by the National Natural Science Foundation of China [81471048]; the Natural Science Foundation of Shandong Province [ZR2019MC059]; and Shandong Province Government-Sponsored Overseas Study Project
  • Author Bio:

  • Corresponding author: LIU Zhi Jun, Associate Professor, E-mail: zhijun.liu@wfmc.edu.cn
  • ΔThese authors contributed equally to this work.

Abstract:   Objective  To investigate the relationship between human cytomegalovirus (HCMV) infection and peripheral blood CD14+CD16+ monocytes in the pathogenesis of coronary heart disease (CHD), and to elucidate the mechanism of pathogenesis in CHD by analyzing the correlation between infection, inflammation, and CHD, to provide a basis for the prevention, evaluation, and treatment of the disease.  Methods  In total, 192 patients with CHD were divided into three groups: latent CHD, angina pectoris, and myocardial infarction. HCMV-IgM and -IgG antibodies were assessed using ELISA; CD14+CD16+ monocytes were counted using a five-type automated hematology analyzer; mononuclear cells were assessed using fluorescence-activated cell sorting; and an automatic biochemical analyzer was used to measure the levels of triglyceride, cholesterol, high- and low-density lipoprotein cholesterols, lipoprotein, hs-CRp and Hcy.  Results  The positive rates of HCMV-IgM and -IgG were significantly higher in the CHD groups than in the control group. HCMV infection affects lipid metabolism to promote immune and inflammatory responses.  Conclusion  HCMV infection has a specific correlation with the occurrence and development of CHD. The expression of CD14+CD16+ mononuclear cells in the CHD group was increased accordingly and correlated with acute HCMV infection. Thus, HCMV antibody as well as peripheral blood CD14+CD16+ mononuclear cells can be used to monitor the occurrence and development of CHD.

ΔThese authors contributed equally to this work.
LI Hong Zheng, WANG Qin, ZHANG Yi Yuan, WANG Jin Dong, WU Hong Juan, ZHANG Mo Gen, LI Ji Chen, LIU Zhi Jun. Onset of Coronary Heart Disease is Associated with HCMV Infection and Increased CD14+CD16+ Monocytes in a Population of Weifang, China[J]. Biomedical and Environmental Sciences, 2020, 33(8): 573-582. doi: 10.3967/bes2020.076
Citation: LI Hong Zheng, WANG Qin, ZHANG Yi Yuan, WANG Jin Dong, WU Hong Juan, ZHANG Mo Gen, LI Ji Chen, LIU Zhi Jun. Onset of Coronary Heart Disease is Associated with HCMV Infection and Increased CD14+CD16+ Monocytes in a Population of Weifang, China[J]. Biomedical and Environmental Sciences, 2020, 33(8): 573-582. doi: 10.3967/bes2020.076
    • Coronary heart disease (CHD) affects cardiac function (hypoxia, ischemia, and necrosis) due to stenosis and the obstruction of arterial blood vessels[1-3]. The pathological changes are mainly caused by atherosclerosis (AS), which has a high mortality rate in European and American countries. In China, it currently affects about 11 million patients and this number is increasing every year[4, 5]. Epidemiological studies have shown that classical risk factors, such as hypertension, hyperlipidemia, smoking, obesity, and diabetes cause AS, but the etiology of the disease still remains unclear[6-8]. In recent years, the prevalence of these risk factors has successfully reduced; however, there is still high incidence of cardiovascular diseases with AS[9]. Further research studies have revealed that chronic, repeated, and persistent inflammation might be an important cause of AS[10-12]. Moreover, infective factors and their inflammatory reactions are closely associated with the disease[13-15]. Some reports suggest that human cytomegalovirus (HCMV) may be involved in the initiation of AS[16-19]. HCMV infection and the number of peripheral blood mononuclear cells have been shown to be independent risk factors for AS; in addition, different subsets of mononuclear cells seem to play different roles in the AS inflammatory response[20-22]. HCMV infection can initiate the process of AS formation, as elucidated by the finding that regulatory T lymphocytes are reduced in HCMV-infected patients[23, 24], which disturbs the balance of immune suppression, thus inducing a highly inflammatory state, and further leading to the occurrence and development of coronary artery disease in patients with CHD. Inflammatory response is one of the important factors that cause AS[25]. Studies have shown that HCMV antigen and antibody form immune complexes in the AS lesion[21, 26]. These are deposited on the vascular wall and induce vascular endothelial cells, macrophages, smooth muscle cells, foam cells, and infiltrated T lymphocytes to express the mononuclear cell chemical AS-1 (MCp-1). This subsequently stimulates macrophages to release many inflammatory cytokines such as interleukin (IL)-1, -6, -8, and -10, and tumor necrosis factor-alpha (TNF-α). These factors activate monocytes in the blood and induce migration into the arterial intima[27].

      HCMV is an opportunistic pathogen that belongs to the beta herpes virus subfamily[28]. It is highly species-specific and spreads only from person to person. HCMV infection is characterized by susceptibility, persistence, and repetition, and it can infect people of any age. Close contact with the body fluids of the infected persons causes it to spread in the human population; once infected, the host produces specific HCMV-IgM, HCMV-IgG, and other antibodies. The IgM and IgG antibodies produced are generally not affected by other factors, can be used as direct evidence of infection, and have predictive value for the condition[29]. The periodic activation of HCMV causes abnormal lipid metabolism and local chronic immune or inflammatory response. HCMV infection upregulates the endothelial adhesion molecules E-selectin and intercellular adhesion molecule-1 that favor the adhesion of monocytes[30]. HCMV infection also induces monocyte chemoattractant protein-1 (MCp-1), MCp-2, and macrophage colony-stimulating factor[31], and stimulates macrophages to produce and release interleukins such as IL-1, IL-6, IL-8, and IL-10, as well as inflammatory factors such as TNF-α. These cytokines mainly act to activate monocytes and promote their entry into the endothelium[32].

      Mononuclear macrophages play an important role in the pathogenesis of AS; one-third of the proliferating cells in the artery endothelial injury are monocyte macrophages. Sinclair et al.[33] found that HCMV is cytosolic in mononuclear macrophages to avoid degradation and elimination. HCMV persists in monocyte precursors in the bone marrow, so monocytes become carriers and transporters of HCMV. Under the combined action of adhesion molecules, chemokines, and cytokines, monocytes transfer HCMV to the site of vascular damage, enter the endothelium to transform into macrophages, and after taking in a large amount of cholesterol, turn into foam cells. Therefore, HCMV may initiate or promote the development of AS via the movement of infected mononuclear macrophages into the coronary arteries[34].

      From the early lipid aggregation to plaque formation and instability until rupture, a series of ischemic symptoms and malignant events are triggered during this process, in which the migration, aggregation, and inflammation of monocytes play a key role[35]. Yang et al.[36] detected HCMV and virus antibody IgM in peripheral blood mononuclear cells using PCR and ELISA. The results showed that the positive rates of HCMV and IgM antibody are significantly higher in patients with coronary heart disease (CHD) than in the controls. Furthermore, the study found that the ratio of the peripheral blood classical monocyte subgroup (CD14++CD16) in patients with acute coronary syndrome does not differ significantly from those with stable angina pectoris and controls, while the ratio of the CD14+CD16+ mononuclear cell subgroup is significantly higher. The group showed that CD14+CD16+ monocytes are more strongly associated with inflammation. Compared to CD14++CD16 mononuclear cells, CD14+CD16+ monocytes have a stronger ability to induce TNF and IL-6 in the process of inflammation, and upregulate the expression of inflammatory chemokine receptors more evidently[37, 38]. Based on the expression of CD14 and CD16, we divided monocytes into four subgroups: CD14++CD16, CD14++CD16+, CD14+CD16+, and CD14+CD16. The role of HCMV infection and CD14+CD16+ mononuclear cells in the development and pathogenesis of CHD and its interrelated mechanism were investigated based on HCMV infection and CD14+CD16+ mononuclear cells in 192 patients with CHD, to provide a reference for its prevention, evaluation, and treatment.

    • Between September 2017 and January 2018, we recruited 192 patients with cardiovascular disease from Weifang People’s Hospital and Yidu Central Hospital. All participants provided written informed consent for the study protocol, which was approved by the Ethics Committee of Weifang Medical University (reference number, wfmu2017012). Patients with heart disease caused by thyroid disease, rheumatic heart disease, non-rheumatic valvular disease, dilated or hypertrophic cardiomyopathy, hypertensive, congenital, pulmonary, and other heart diseases, were excluded. Patients were divided into three groups: latent CHD (n = 77), angina pectoris (n = 64), and acute myocardial infarction (n = 51). Seventy-nine healthy individuals were recruited as controls.

    • Smoking: Continuous smoking for > 12 months (not less than once per day) and still smoking, or long-term smoking for more than six months.

      Hypertension: A history of hypertension or systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 90 mmHg, or on antihypertensive drugs.

      Diabetes: Fasting blood glucose ≥ 7 mmol/L or blood glucose ≥ 11.1 mmol/L 2 h after a meal.

      Family history: First-degree relatives of women < 55 years of age (or males < 65 years of age) who have associated cardiovascular disease.

    • Peripheral venous blood was collected from patients and controls after fasting for at least 8 h and assessed as follows: The serum was obtained by collection in a standard coagulant tube, followed by separation from whole blood using centrifugation. HCMV-IgG and HCMV-IgM antibodies were detected by enzyme immunoassay (HCMV-IgM and HCMV-IgG detection kits were obtained from Beijing Baer Bioengineering Co. Ltd.). Four wells in the reaction plates were reserved for negative and positive controls (3 wells for negative control and 1 for positive control, according to the manufacturer’s protocol), and the reaction was stopped within 10 min. The detection parameters of the microplate reader were set according to the number of samples to be tested and the wells of the reaction plate; the double wavelength was set at 450/630 nm, and the absorbance (A value) of each reaction well was detected and recorded. The A values were classified as positive or negative, based on thresholds. PCR assays were used for measuring HCMV DNA in peripheral blood leukocytes to reduce false positives and improve sensitivity.

    • Whole blood was collected into EDTA-K2 anticoagulant tubes. First, routine analysis was performed using the BC-6800 automatic blood analyzer (Mindray Shenzhen, China) to record the number of white blood cells, mononuclear cells, and their proportions in whole blood. Following that, mononuclear cell CD14 and CD16 were detected using FACS (FACSCalibur, BD Biosciences, CA, USA). The cells were divided into four subgroups: CD14++CD16, CD14++CD16+, CD14+CD16, and CD14+CD16+ upon counting the percentages of 20,000 mononuclear cells expressing CD14 and CD16 (Figure 1).

      Figure 1.  FACS analysis of CD14+CD16+ monocytes in CHD patients and controls. (A) Area of R1 represents gated monocyte population. (B) Areas of R3-R6 represent CD14++CD16, CD14++CD16+, CD14+CD16, and CD14+CD16+ monocyte subsets, respectively. (C–D) CD14+CD16+ monocytes rates in CHD patients.

      The blood lipid indexes, such as triglycerides (TG), cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), lipoprotein A [Lp (a)], Hypersensitive C-reactive protein (hs-CRP) and Homocysteine (Hcy) were assessed after standard coagulant collection and serum separation by centrifugation at 3,500 rpm for 5 min using the BS-800 automatic biochemical analyzer (Mindray).

    • All data were analyzed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). Data are shown as the mean ± standard deviation; counts are expressed as positive and positive rates. All data were analyzed using the Chi-square and ANOVA tests. Multivariate logistic regression was used for correlation analysis. Differences between groups were considered to be statistically significant at a P value < 0.05.

    • To assess the relationship between the levels of HCMV antibodies and the incidence of various types of CHD, we first measured the IgM and IgG levels in the latent CHD, angina pectoris, myocardial infarction, and control groups. The positive rates of HCMV-IgM in the control, latent CHD, angina pectoris, and myocardial infarction groups (Table 1) were 19.0%, 33.8%, 40.6%, and 52.9%, respectively. The positive rates in the latent CHD, angina pectoris, and myocardial infarction groups were higher than in the control group (χ2 = 4.396, P = 0.036; χ2 = 8.094, P = 0.004; χ2 = 16.338, P < 0.001) The positive rates of HCMV-IgG in the control, latent CHD, angina pectoris, and myocardial infarction groups (Table 1) were 44.3%, 62.3%, 67.2%, and 80.3%, respectively. The positive rates in the latent CHD, angina pectoris, and myocardial infarction groups were higher than in the control group (χ2 = 5.094, P = 0.024; χ2 = 7.468, P = 0.006; χ2 = 16.621, P < 0.001) with a gradually increasing trend.

      Group (count)IgM (+)IgG (+)
      Count%P ValueCount%P Value
      Control (79)15 19.0/35 44.3/
      LCHD (77)26*33.80.03648*62.30.024
      AP (64)26*40.60.00443*67.20.006
      MI (51)27*52.9< 0.00141*80.3< 0.001
        Note. The tests were carried out in 4 groups: control, LCHD (latent coronary heart disease), AP (angina pectoris), and MI (myocardial infarction). Data are presented as the mean. Statistical significance of differences between groups was evaluated using the ANOVA test. *P < 0.05 versus control.

      Table 1.  Differences in IgM (+) and IgG (+) rates between the disease and control groups

    • To compare the CD14+CD16+ monocytes in CHD patients versus controls, we assayed the cells using FACS and found that there was an increase in the ratio of mononuclear cells and the expression of CD14+CD16+ mononuclear cells in the latent CHD, angina pectoris, and myocardial infarction groups (Figure 2A). Compared to the control group, the expression of CD14+CD16+ mononuclear cells in the latent CHD, angina pectoris, and myocardial infarction groups were significantly different (t = 4.1776, P < 0.001; t = 8.9747, P < 0.001; t = 16.2291, P < 0.05) (Figure 2A). In addition, there was a significant difference in the IgM (+) and IgM (–) CD14+CD16+ mononuclear cells among the groups (P < 0.05) (Figure 2B). The TG, TC, HDL-C, LDL-C, Lp (a), hs-CRp, and Hcy levels in the myocardial infarction, angina pectoris, and control groups were also significantly different (P < 0.05) (Table 2).

      Figure 2.  Monocytes and subgroups in latent CHD. LCHD (latent coronary heart disease), AP (angina pectoris), MI (myocardial infarction), and control groups. *P < 0.05 between groups.

      GroupTG (mmol/L)TC (mmol/L)HDL-C (mmol/L)LDL-C (mmol/L)Lp (a) (nmol/L)hs-CRp (mg/L)Hcy (µmol/L)
      Control 1.31 ± 0.473.70 ± 0.771.25 ± 0.331.86 ± 0.7852.6 ± 9.343.78 ± 0.9912.20 ± 2.60
      LCHD1.42 ± 0.523.88 ± 0.771.26 ± 0.453.11 ± 0.2370.95 ± 8.455.29 ± 1.3313.40 ± 2.60
      AP2.21 ± 0.21*5.44 ± 0.54*1.03 ± 0.25*3.48 ± 0.35*81.8 ± 8.44*8.27 ± 1.39*16.10 ± 3.60*
      MI2.62 ± 0.32*6.01 ± 0.80*0.93 ± 0.24*3.90 ± 0.49*95.45 ± 8.12*11.87 ± 2.95*19.50 ± 4.40*
        Note. The tests were carried out in 4 groups: control, LCHD (latent coronary heart disease), AP (angina pectoris), and MI (myocardial infarction). Data are expressed as mean ± standard deviation. *P < 0.05 versus control. Triglyceride (TG), Cholesterol (TC), High- and low-density lipoprotein cholesterols (HDL-C, LDL-C), Lipoprotein A [Lp (a)], Hypersensitive C-reactive protein (hs-CRP) and Homocysteine (Hcy).

      Table 2.  Blood indexes of atherosclerosis (${{ \bar {\text{x}}}}$ ± SD)

    • To clarify whether HCMV infection is an independent factor leading to CHD, we tested possible predisposing factors in all patients and controls and found that the factors, smoking (P < 0.05), hypertension (P < 0.05), diabetes (P < 0.001), family history (P < 0.001), HCMV-IgM (P < 0.001), and HCMV-IgG (P < 0.001) showed significant association in the single-factor analysis (Figure 3), while age (P = 0.154) and body mass index (P = 0.140) did not (Table 3). Multivariate logistic regression analysis showed that hypertension, diabetes, family history, HCMV-IgM, and HCMV-IgG are independent factors affecting CHD.

      Figure 3.  Logistic regression analysis of risk factors (smoking, hypertension, diabetes, family history, HCMV-IgM, and HCMV-IgG) in CHD and control groups.

      Risk factorsControl group (n = 79)CHD group (n = 192)tP
      Age56.6 ± 4.257.5 ± 4.91.430.154
      Body mass index (kg/m2)23.3 ± 4.924.2 ± 4.41.480.140
        Note. Data are expressed as mean ± standard deviation. T-tests showed no significance.

      Table 3.  Single-factor analysis of general clinical data (risk factors) in CHD and control groups

    • CHD is a progressive disease that is common in the elderly; it has a high mortality rate in the western countries. The incidence rate of CHD in China's population aged > 60 years is ≥ 80%, and this rate is increasing every year. Our study showed that smoking, hypertension, diabetes, family history, and other risk factors are independent factors contributing to CHD. More importantly, this study also identified that the development of AS is closely associated with HCMV infection.

      The serological epidemiology, related molecular biology of HCMV, as well as animal studies together indicate that the occurrence and development of AS is closely associated with HCMV infection. Adam et al.[39] were the first to demonstrate that HCMV infection is associated with AS. Adam et al. found that the HCMV infection rate was 90% higher in the AS population than in the non-AS group (90% and 74%, respectively). In this study our findings showed that, the positive rate of a high antibody titer was greater in the AS population than in controls (P < 0.001, 75% to 26%); the titer was not affected by TG, TC, and other risk factors. These results suggest that the occurrence and development of atherosclerotic CHD is associated with HCMV infection.

      After HCMV infection, the IgM and IgG antibodies are generally not disturbed by other factors, and so can be used as direct evidence of infection and to predict the development of AS post-infection[40]. Our study showed that the positive rates of HCMV-IgM and HCMV-IgG were higher in the latent CHD (33.8% and 62.3%, respectively), angina pectoris (40.6% and 67.2%, respectively), and myocardial infarction (52.9% and 80.3%, respectively) groups (Table 1) than in the controls (19.0% and 44.3%, respectively). Molecular biology-based research studies have also provided direct evidence for the hypothesis that HCMV infection can lead to AS[16, 22]. Some animal experiments also support the hypothesis. Fabricant et al.[41] induced AS lesions in chickens with Marek disease virus and showed that their coronary, gastric, and celiac arteries displayed pathological changes.

      Second, HCMV infection affects lipid metabolism and promotes immune and inflammatory responses[42]. Chronic, persistent, cyclically repeated inflammation may be an important factor affecting the development and pathogenesis of CHD. It has been reported that TG and TC levels rise markedly after HCMV infection, and are not affected by diet[43]. Comparison of the blood AS indexes [TG, TC, HDL-C, LDL-C, and Lp (a)] (Table 2) revealed significant differences in these levels between the myocardial infarction, angina pectoris, and control groups (P < 0.05). Significant differences were also noted between the IgM (+) and IgM (–), IgG (+), and IgG (–) rates in the latent CHD, angina pectoris, myocardial infarction, and control groups (P < 0.05). However, the change in trend of HCMV infection and blood AS index could not be captured because of the low number of subjects in this study.

      Third, the expression of CD14+CD16+ mononuclear cells in the latent CHD, angina pectoris, and myocardial infarction groups showed increasing trends; correlations with acute HCMV infection were also found. Thus, assessment of HCMV antibodies and peripheral blood CD14+CD16+ monocytes may provide clues for the occurrence and development of AS. Among the cells proliferating in AS endothelial injury, one third are mononuclear macrophages, of which CD14+CD16+ monocytes have a strong pro-inflammatory effect[44]. Many cells, especially monocytes, macrophages, endothelial cells, and smooth muscle cells are susceptible to HCMV infection[26]; they play an important role in the development of atherosclerotic vascular diseases. It has been found that HCMV persists in the precursors of bone marrow mononuclear cells, which act as HCMV carriers[45]. Therefore, the virus can infect mononuclear macrophages in patients with CHD and promote its development. Monocytes and macrophages play important roles in the pathogenesis of CHD[46]. Several studies have found that different subgroups of monocytes play different roles[47]. There is evidence for a change in the proportion of monocyte subsets in the initial stage[48]. A potential new target for treatment is the chemotaxis of endothelial monocytes and their adherence to HCMV-infected endothelial cells. Increased CMV infection and adhesion molecule expression, induced by endothelial cell injury, may play a positive feedback role in the regulation of CD14+CD16+ monocyte expression. HCMV increases CD36 mRNA transcription resulting in high expression of CD36 receptor in CD14+CD16+ monocytes, which is a main mechanism to promote AS[49]. The results of our study show that an increase in IgM positivity in the CD14+CD16+ monocyte subgroup is associated with the virus. As the HCMV antibody IgM represents a recent infection, an acute HCMV infection might affect the expression of CD14+CD16+ monocytes[50]. Our findings revealed that the CD14+CD16+ mononuclear cells displayed features that are typical of mature and inflammatory functions, and thus the high expression of CD14+CD16+ monocytes should not be overlooked.

      Monocytes in the blood are induced by chemokines to migrate to the intima of blood vessels and change into macrophages, when their phagocytic lipid results in characteristic foam cells. This process is the initiation of AS[51]. Damage to the local endothelium and infiltration of lipids induces the self-proliferation of mononuclear macrophages and increases their number in the plaque[52], resulting in the formation of more foam cells after lipid phagocytosis[53]. So mononuclear macrophages play an important role in the pathogenesis of CHD. In addition, we also found that different subgroups of mononuclear cells play different roles. Evidence has shown that regulation of the ratios of monocyte subsets and changes in the functions of these subsets are potential new targets for AS therapy[54].

      The results of the present study showed that the number of monocytes, proportion of mononuclear cells, and expression of CD14+CD16+ mononuclear cells are higher in the latent CHD, angina pectoris, and myocardial infarction groups as compared to the control group, indicating the CD14+CD16+ monocyte subgroup is an independent risk factor for CHD. Huang et al.[55] examined the expression of the CD14+CD16+ monocyte subset in peripheral blood from different populations and found that the levels were significantly higher in acute coronary syndrome patients than in stable angina patients and controls. Further studies have shown that the number of CD14+CD16+ monocytes is negatively correlated with the level of high-density lipoprotein, and positively correlated with AS lipid changes. This suggests that there may be some correlation between the abnormal changes in CD14+CD16+ monocytes and blood lipids. Schlitt et al.[56] found that the proportion of CD14+CD16+ cells is higher in the blood of patients with CHD than in normal controls, and that the concentration of CD14+CD16+ cells is positively correlated with the concentration of TNF-α. People with increased CD14+CD16+ cells display a 5 times higher risk of CHD than the normal population. The local immune response in AS plaques is also a key factor in the occurrence of malignant events such as myocardial infarction, and most of the inflammatory factors are produced by mononuclear macrophages. Based on the expression of CD14 (LpS receptor) and CD16 (Fc gamma-III receptor) on the cell surface, monocytes are divided into four subgroups (CD14++CD16-, CD14++CD16+, CD14+CD16+, and CD14+CD16-); of these, different monocyte subsets have different effects on AS and play different roles in myocardial infarction. We observed in our study that the CD14+CD16+ mononuclear cells are more mature and inflammatory than the classical mononuclear cells (CD14++CD16-). Induced by lipopolysaccharide, CD14+CD16+ mononuclear cells increase the synthesis and secretion of TNF-α and matrix metalloproteinase, enhance the expression of inflammatory chemokine receptors on the membrane surface, promote the synthesis of cytokines IFN-γ and IL-6, and inhibit collagen fibrinolysis in smooth muscle cells. Therefore, the role of highly expressed CD14+CD16+ monocyte subsets in the pathogenesis of CHD cannot be ignored. During the development of AS, monocyte receptors and ligands also play important roles. For example, CD36 mediates the phagocytosis of OX-LDL by mononuclear cells to form foam cells[57], while CD40 and the cell surface p-Selectin glycoprotein ligand-1 form platelet-monocyte aggregates (PMAs). PMAs use intracellular signaling pathways that cause monocytes to secrete and express IL-6, chemokine-1, and extracellular matrix enzymes; the activated platelets also enhance the phagocytosis of monocytes, thus increasing the possibility of cardiovascular risk events. PMAs are significantly higher in case of acute myocardial infarction, as well as in patients with potential AS thrombus formation and at risk for hypertension and diabetes[58]. Because AS is a dynamic process of CHD, static imaging has certain limitations[59]. Hematological assessment is a simple and quick way to predict dynamic changes in AS plaques by labeling related markers[60]. Continuous assessment can make up for the limitations of imaging. Therefore, the detection of monocyte subsets has special clinical significance for the dynamic assessment and prognosis of CHD. Targeted cell therapy for AS is a promising and valuable clinical strategy based on the specific immunoregulation of the expression of different subsets of mononuclear cells. In conclusion, it is likely that HCMV infection and monocyte subsets play important roles in the development of AS.

      In summary, HCMV infection stimulates/inhibits lipid metabolism and increases activity of the immune and inflammatory systems. HCMV infection is correlated with the occurrence and development of CHD. Since the expression of peripheral CD14+CD16+ mononuclear cells in the CHD group was increased and correlated with acute HCMV infection, both HCMV antibody and CD14+CD16+ mononuclear cells can be used to monitor the occurrence and development of CHD. Thus, HCMV antibody and peripheral blood CD14+CD16+ mononuclear cells can be used to monitor the occurrence and development of CHD. However, it has to be noted that certain questions still remain and need to be explored. For example, if HCMV promotes coronary AS development, what is the breakthrough? What specific roles do the monocyte subsets play in the development of AS? Are there differences in the distribution of HCMV and monocytes in different parts of AS plaques? Are there different distributions at different stages of AS? Can the development of AS plaques be reduced by preventing HCMV infection or altering the function of specific types of monocytes? What is the relationship between HCMV infection and the expression of monocyte subsets? With the development of research and the emergence of new technologies, we will have a clearer understanding of HCMV infection and the types of monocytes, thus facilitating the development of a better strategy for the prevention and treatment of atherosclerotic diseases.

    • HL and QW performed the whole experiments, YZ responsible for the figures. JW, HW, MZ, and JL organized the content of the entire manuscript and wrote the whole sections. HL, QW and ZL contributed to the design of the work. All authors read and approved the final manuscript.

    • The authors report that they have no competing interests.

    • We thank Dr. IC Bruce for reading the manuscript.

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