Role of CMV & Interferon-γ in Myocardial infarction , Angina and Hypertension

Aims: There is limited epidemiologic evidence relating CMV specifically to primary coronary atherosclerosis. Its association with atherosclerosis and restenosis appears to have merit and needs to be studied further. Cardiovascular disease being an inflammatory process leads to detectable rise in inflammatory markers like Interferon-γ. The aim of this study was to evaluate the role of CMV and Interferon-γ in cardiovascular disease. Methods: Study was conducted on 63 randomly selected cardiovascular disease patients and 29 healthy controls. ELISA for detection of IgG antibodies against CMV were detected (Calbiotech Diagnostics, USA). Interferon-γ levels were determined by ELISA (Diaclone, USA). Relevant investigations, clinical history & examination were recorded. Results: Of 63 cases 41 (65.08%) were positive for IgG antibodies against CMV and 8 (27.58%) of 29 controls were IgG positive (p<0.001). Among CMV seropositives 19 (46.34%) were MI patients, 13 (31.70%) were hypertensives and 9 (21.95%) were angina patients. The mean value of Interferon-γ for cases was 32.13pg/ml, the mean of controls was 11.32pg/ml (p<0.0001). Among CMV IgG seropositives the mean value of Interferon-γ in hypertensives was 12.76pg/ml, in angina patients was 32.48pg/ml and in MI patients was 67.10pg/ml. Conclusion: In our study CMV seropositivity was significantly associated with CVD cases. CMV seropositivity increased with severity of disease. Mean value of Interferon-γ was higher among cases reflecting role of inflammatory aetiology in CVD. Mean value of Interferon-γ increased with severity of disease infection clearly indicating the role of inflammatory markers along with CMV in cardiovascular disease.


Introduction
The known risk factors of Cardiovascular Disease (CVD) are not sufficient to explain all the epidemiological variables and fluctuations of the disease.These observations fuel renewed interest in a link between CVD and infectious agents.Infectious etiology with regard to CVD has been in the limelight for more than 20 years 1 .The hypothesis of infectious aetiology of atherosclerosis is one of the most interesting areas of vascular research, it states that more than one infectious agent could play a role in atherogenesis and atherothrombosis.This entire process may involve pro-inflammatory mechanisms like interleukins and cytokines.
Infectious agents are considered as significant factors in the pathogenesis of atherosclerosis because atherogenic processes resemble chronic inflammation 2 -a process that may be promoted by microorganisms like cytomegalovirus (CMV) [3][4][5] .CMV is a relatively newer pathogen with regard to atherosclerosis.This association was proposed in the 1980's.The hypothesis of infectious aetiology is very attractive because it would provide us with a powerful platform for prevention and treatment of a disease which represents an escalating public health problem worldwide and especially in India.Even after extensive research the role of infectious agents in CVD still remains an enigma.
Cytomegalovirus is a member of Betaherpesviridae in the subfamily of Herpesviridae.Most people are infected with CMV at some point in life although the age of infection varies worldwide.As with other Herpes viruses CMV establishes a latent infection in the host and may reactivate later during a period of immunosuppression.CMV commonly causes intrauterine infection and prenatal damage to foetus, leading to congenital abnormality.In adults and older children it may cause a syndrome resembling EBV mononucleosis.In adults it is usually asymptomatic.Almost two decades ago several investigators suggested a role for herpes viruses in CVD.The Atherosclerosis Risk in Communities Study in 2000 correlated pre-existing high CMV titres and traditional risk factors with carotid atherosclerosis, incident MI or CHD death 6 .Seroepidemiologic, histopathologic, in vitro and animal studies have investigated possible links between human atherosclerosis and human herpes viruses, primarily CMV 7 .Studies have linked Cytomegalovirus to three arterial diseases: Primary atherosclerosis, Post angioplasty restenosis and post transplantation atherosclerosis.An association has been observed between the presence of CMV and restenosis following coronary angioplasty. 8There is limited epidemiologic evidence relating CMV specifically to primary coronary atherosclerosis.Its association with atherosclerosis and restenosis appears to have merit and needs to be studied further.Cardiovascular disease being an inflammatory process leads to detectable rise in inflammatory markers e.g.Interferon-γ.Over the last decade substantial advances in basic and experimental science have established the role of inflammation in CVD 9 , from initiation to progression.Thus inflammation may be considered as a surrogate marker of increased risk of adverse cardiovascular events.The development of interest in inflammatory markers is also due to the fact that reduction in levels of systemic inflammation by medical intervention could lead to significant reduction in adverse cardiovascular events.The aim of this study was to study the association of Cytomegalovirus with Cardiovascular Disease & Evaluation of inflammatory markers as potential predictors of risk for cardiovascular events.

STUDY GROUP:
The study was conducted overa period of 11

STATISTICAL ANALYSIS:
Each parameter as it was measured was compared between cases and controls to see if their mean level was similar.For this a two group Students's t test or Wilcoxon Ranksum test was applied as appropriate.

SAMPLE COLLECTION AND TRANSPORTATION:
5 ml blood was obtained by venepuncture taking all aseptic precautions.Serum was separated from sample by centrifugation and stored at-20ºC.Enzyme immunoassay was performed for detection of IgG antibodies against CMV (Calbiotech Diagnostics, USA).Levels of Interferon-γ were estimated by Enzyme immunoassay (Diaclone, USA) following manufacturer's instructions.Standard curve was prepared against which the sample readings were plotted.

Demography of the study & control group:
Of the 63 cardiovascular disease cases, majority of patients 45 (71.43%) were in the age group 41-60 years with maximum clustering 22 (34.92%) in the age group 56-60 years followed by 12 (19.05%) in the age group 46-50 years.The mean age distribution was 52.7±8.9 years in cases and 54.01±8.1 years in controls.In the cases 38 (60.31%) were male and 25 (39.68%) were female.In the control group 18 (62.5%)were male and 11 (37.5%) were female.Out of 63 cases, highest number of patients were of myocardial infarction 28 (44.4%)followed closely by primary hypertension 21 (33.33%) and 14 (  3. The conventional risk factors with or without CMV infection were not strongly associated with CVD.After adjusting for traditional risk factors using multiple logistic regression CMV independently emerged as a single significant risk factor in causation of CVD Cases in each study group were also analysed with respect clinical details such as haemogram, neutrophilia, ESR, renal function test, liver function test, ECG, cardiac enzymes Echo, ultrasound and angiography was performed.However no clear association was observed except for the presence of ECG findings and cardiac enzymes in patients of angina and MI respectively.These clinical variables along with the traditional risk factors were also compared among the CMV seropositive and seronegative populations and it was observed that there was no significant difference in the prevalence of traditional risk factors in either group.On analysing the role of CMV seropositivity in the study groups majority 19 (46.34%) were MI patients, 13 (31.70%)were hypertensives and 9 (21.95%) were angina patients.Value of Interferon-γ ranged from 5.46-17.05pg/ml among controls and 8.09-638.84pg/ml among cases.The mean value of Interferon-γ for cases was much higher at 32.13 than the mean of controls which 11.32pg/ml (p=0.026).Among the cases there were two groups; 41 (65.07%) were CMV seropositive and 22 (34.92%) were CMV seronegative.Distribution of CMV seropositives and seronegatives according to study group is given in Table 4. On comparing the value of Interferon-γ in the study groups between CMV seropositives and seronegatives the following results were obtained.Among hypertensives CMV seropositives had marginally higher value at 12.76 pg/ml whereas seronegatives had 10.06pg/ml.Among angina patients CMV seropositives had 32.48 pg/ml IFN-γ whereas seronegatives had 9.73 pg/ml IFN-γ.Finally among MI patients CMV seropositives had a much higher level of IFN-γ at 67. 10  As a result, the Indian subcontinent suffers from a tremendous loss of productive working years due to CVD deaths: an estimated 9.2 million productive years of life were lost in India in 2000, with an expected increase to 17.9 million years in 2030 (almost ten times the projected loss of productive life in the United States) 13 .
Strangely the traditional risk factors were not significant contributors to CVD in the present study while CMV emerged as a significant risk factor for CVD.This startling observation strengthens the role of CMV alone and more so when it activates proinflammatory cytokines like Interferon-γ in pathogenesis of cardiovascular disease over and above the traditional risk factors.
MANOVA test was used to study CMV seropositivity, Interferon-gamma and traditional risk factors as independent variables and CVD outcome as dependent variable.CMV seropositivity and Interferon-gamma In our study CMV was not only significantly associated with CVD cases (p<0.001) but CMV seropositivity increased with severity of disease (from hypertension to MI) underlining its role in progression of the disease.The results of a study [14] demonstrated a similar prevalence of CMV seropositivity in patients with coronary artery disease and in an age and sex matched control group.Earlier studies have mostly reported association of CMV infection with coronary atherosclerosis in transplanted hearts, following coronary angioplasty, or in carotid arteries [17,18] .In a previous investigation from the Framingham Heart Study, no association between clinically apparent herpes virus infection and coronary heart disease was documented

19
. Recent prospective studies have also failed to demonstrate an association of CMV infection with risk of cardiovascular disease [20][21][22][23] .Our study contrasts with these observations from populationbased studies, and it indicates a strong association between CMV infection and risk of cardiovascular disease.This could be due to cryptic factors in the Indian population possibly genetic or racial or even related to increasingly stressful lifestyles.In another study among control patients matched to surgically treated cases of atherosclerosis, high CMV antibody titres were more common in those who subsequently developed coronary events than in the remainder(45% v 26%), but this comparison was not adjusted for potential confounding variables [24] .
Detection of CMV IgG in hypertensives in our study had a good Sn of 61.90%, a high Specificity of 72.41%, PPV 61.90%, NPV 72.41% and OR of 4.27.In Angina patients Sensitivity was 64.68%, Sp was 72.41%, PPV 52.94%, NPV 80.77%, OR was 4.73, whereas in MI patients Sensitivity was 67.86%, Sp was 72.41%, PPV 70% and OR was 5.54.The OR is extremely high in all the three subgroups, CMV infection itself has atherogenic potential 17 and being intracellular pathogen also had the ability to induce release of Interferon-γ 25 CMV being a chronic infection fluctutating between the poles of periods of latency and reactivation may lead to release of proinflammatory cytokines including IFN-γ which may accelerate atherogenesis ultimately causing plaque rupture.
In conclusion the inflammatory response to CMV should be studied in detail.It can be hypothesized that the adverse clinical outcome is not solely due to infection per se but also due to the inflammatory response to infection.CMV, an omnipresent virus which infects practically every individual in childhood may be getting reactivated in a subset of individuals who are under stress due to modern work culture and stressful lifestyles.Herpes simplex 1 viruses are known to get reactivated under stress, CMV may also be getting reactivated under similar conditions.CMV on reactivation may be associated with hyperplasia and atherogenesis of the coronary arteries, the concomitant raised Interferon-γ leading to rupture of the plaque leading to MI.In our study angina was associated with raised CMV titres and moderately elevated levels of Interferon-γ while MI was associated with raised CMV titres and highly elevated titres of Interferon-γ while least Interferon-γ titres were observed in hypertension which explains the preeminent role played by Interferon-γ in concert with CMV in the progression of cardiovascular disease.However this is just a baseline study and larger prospective studies need to be undertaken before any concrete conclusions can be made.

Stable angina was defined as chest pain occurring
on physical activity, begins slowly and gets worse and is relieved on medication or rest.Ethical clearance was obtained from Institutional Ethical Committee Jawaharlal Nehru Medical College, AMU, Aligarh.
3. Patients with any other septic foci.4. Patient with history of antibiotic intake in the last 1 month.CONTROL GROUP: Control group consisted of 29 age, sex and other variable matched healthy volunteers.A detailed Out of 63 cases 41 (65.08%) were positive for IgG antibodies against CMV and 8 (27.58%) of 29 controls were IgG positive( p<0.001).Of 41 CMV IgG seropositive, 26 (63.41%) were males and 15 (36.58%) were females.Majority 9 (21.95%)cases were in the age group 46-50 years with scattering of cases 23 (56.09%) in the age group 46-60 years as shown in Table 2. Unexpectedly CVD manifested in a relatively younger age group with the youngest patient being 35 years old.There were 3 & 5 patients in the 36-40 and 41-45 yr age group.The relatively young population of 30-50 years (43.9%) was associated with CMV followed by 51-55 years and 56-60 years.This shift in age could be attributed to a change in lifestyle compounded by CMV reactivation.Detection of CMV was significantly more (p<0.001)with CVD.Sensitivity of this test was 65.08%, specificity was72.41%,positive predictive value 83.67%, negative predictive value 48.84% and Odds ratio was 4.89.On evaluating the role of conventional risk factors in CMV infected individuals 21 (51.2%) had past history of cardiac event, 14 (34.1%) were smokers, 11 (26.8%) were obese, 10 (24.4%) had deranged lipid profile, 7 (17.1%)had family history and only 5(12.2%) had diabetes as shown in Table

Table 2 .
pg/ml whereas seronegatives Cytomegalovirus IgG Seropositivity according to age

Table 3 .
Distribution of Risk factors in seropositive and seronegatives population.

Table 4 .
Number of CMV seropositive & seronegatives patients in each study group whose IFN-γ was estimated

Table 5 .
Comparision of value of Interferon-γ in the study groups between CMV seropositives ans seronegatives.The highest mean Interferon-γ levels were seen in MI patients with CMV infection clearly indicating an active role of CMV along with inflammatory markers in the pathogenesis of cardiovascular disease.