A total of 300 AMI patients and 100 age and sex matched control subjects were enrolled in this case control study. From these patients and controls, the study groups were selected based on their habit of smoking and the present study included both smokers and non smokers with AMI and their respective controls. The study included 222 AMI patients with the habit of smoking along with 62 normal controls with the habit of smoking but without the presentation of AMI. Non-smokers (n=78) with AMI were also studied along with 38 normal controls without the habit of smoking and without the presentation of AMI.

All patients admitted to the Intensive Coronary Care Unit (ICCU) with a diagnosis of AMI presenting within 24 hours of onset of chest pain were included in the study and myocardial infarction was diagnosed in these patients by at least 0.1-mv ST segment elevation in two or more contiguous limb leads or 0.2-mv ST segment elevation in two or more chest leads associated with typical chest pain. We have included only the patients diagnosed with AMI. Exclusion criteria were patients with cardiogenic shock, cerebrovascular accident, significant hepatic or renal diseases, clear evidence of infection anywhere in the body. The normal volunteers had no past history or evidence of cardiovascular disease, hypertension or diabetes mellitus. The present study does not include control subjects with a history of neoplastic, hepatic, infectious or autoimmune disease or any surgical procedure in the preceding 6 months. Written informed consent was obtained from each subjects prior to the study. The study was started only after obtaining the ethical clearance from the institutional ethics committee. The work was carried out in the Department of Cardiology at Amala Cancer Hospital and Research Centre, Thrissur, Kerala, India.

In patients included in the study, detailed history was taken and complete physical examination was carried out. A 12 lead ECG with V3R and V4R was recorded immediately on admission and repeated after 2hrs, 6hrs, 12hrs, 24hrs, 48hrs and pre-discharge. 10 ml blood was withdrawn for laboratory analysis from the peripheral vein on admission in heparinised tubes. After centrifugation the plasma samples were frozen and stored at -80^oC until the MMP assay was carried out. Serial creatine kinase assays were also done to confirm the myocardial infarction and it was done at 2hrs, 6hrs, 12hrs, 24hrs, and 48hrs after admission. Chest X- ray was done at the time of discharge from the ICCU. An echocardiographic examination was performed at the time of discharge or at the end of the first week or early second week after admission.

The patients were allowed to relax and on the second day after the admission they were subjected to an oral questionnaire as described in our Performa, in order to collect the history of these patients and also to find out whether they have the habit of smoking.

The blood collected from the patients at the time of admission was used for the assay of MMPs. MMP assay is based on the principle that the antibodies immobilized on a bead matrix, in combination with enzyme-labeled antibodies, directed against different antigenic sites on the same MMP molecule [11,12]. Upon addition of an MMP containing specimen, the result is an MMP molecule being sandwiched between the solid phase and enzyme labeled antibodies. After removing unbound enzyme-labeled antibody, the bead containing the sandwich is incubated with enzyme substrate and O-phenylenediamine, resulting in the development of color. The activity of the peroxidase enzyme is proportional to the amount of antigen, MMP, so that MMP concentration in specimens can be determined from a standard curve.

The assay mixture contained 50μl of standard or specimen with 300μl enzyme labeled antibody solution and one anti-MMP coated bead. The mixture was incubated at 17-27⁰C for 1 hour. The reaction was then stopped by the addition of 3ml of washing solution and it was aspirated and this was repeated at least 3 times. Each washed bead was then transferred in to a clean fresh tube and 300μl of coloring solution was added and incubated at 17-27⁰C for 1 hour. The reaction was then stopped by the addition of 1.5ml of stop solution. Using deionized water as blank, the absorbance for the standard curve solutions and specimens were taken at 492 nm (A492).

The statistical analysis was done by using Sigma stat version 2 and the values obtained were expressed as mean + SD . Statistical significance was done by using the z-test and by the analysis of variance (ANOVA). The z-value was determined and the results obtained by z-test were expressed in terms of probability (p). z-value > 2.88 (p<0.01) and >1.96 (p<0.05) were considered to be statistically significant. The inter group comparison have been done by using the one-way analysis of variance (ANOVA) using the same programme.

[Table/Fig-1] represents the values of MMP2 and MMP9 in both normal as well as in AMI patients irrespective of the smoking habits. When compared to the normal individuals (n=100) the values of both MMPs were found to be significantly (p<0.01) increased in all the AMI patients (n=300). [Table/Fig-2] represents the values of MMP2 and MMP9 in both normal as well as in AMI patients with and without the habit of smoking. Irrespective of the smoking habit, Both MMPs were found to be significantly elevated (p<0.01) in AMI patients when compared to their respective controls. [Table/Fig-3] represents the Values of MMP2 and MMP9 in AMI patients with and without the habit of smoking. MMP2 does not show any significant difference in patients with and without the habit of smoking. Whereas MMP9 was significantly (p<0.01) elevated in AMI patients with the habit of smoking when compared to AMI patients without the habit of smoking.

[Table/Fig-1]:

Values of MMP2 and 9 in normal subjects and in AMI patients, values are expressed as Mean + SD, **p<0.01

[Table/Fig-2]:

Values of MMP2 and MMP9 in control and in AMI patients with and without the habit of smoking, Values are expressed as Mean + SD, **p<0.01

[Table/Fig-3]:

Values of MMP2 and MMP9 in AMI patients with and without the habit of smoking, values are expressed as mean + SD, **p<0.01

Cigarette smoking independently increases the coronary atherosclerotic disease, cerebrovascular disease and peripheral vascular diseases [13]. Inflammation and arterial wall oxidative stress are central in the pathogenesis of atherosclerosis [14]. The ability of cigarette smoke to induce vascular inflammation and oxidative stress appears fundamental to the broad effects of smoking on vascular pathophysiology [15]. The well described role of MMPs in the pathogenesis of smoking related chronic obstructive pulmonary disease serves as a paradigm for considering the potential role of MMPs in smoking related Cardio Vascular Diseases [16]. Activated macrophages, mast cells, T-lymphocytes, endothelial cells and vascular smooth muscle cells are the principle sources of MMPs in the vasculature. Cigarette smoke by increasing vascular inflammation and vascular ROS has the potential to increase the MMP expression by each of these cell types [17]. This is well evident from the present study as both MMP2 and MMP9 are found to be significantly elevated in all the AMI patients when compared to normal healthy individuals. This could indicate increased proteolysis in the plaques by an enhanced expression of these MMPs. The studies conducted by Kai H et al., also indicate increased expression of MMP2 and MMP9 in patients with acute coronary syndrome[18], supporting our findings. MMP system components are expressed in atherosclerotic tissues and in their active form they may contribute to vascular remodeling and plaque disruption [19].

However there are certain important findings obtained from this study. Both MMP2 and MMP9 were found to be elevated in all the AMI patients with and without the habit of smoking and out of these two MMPs, MMP9 was found to be predominantly elevated in AMI patients with the habit of smoking than MMP2 in patients with the habit of smoking. These findings are similar to the findings obtained by the study conducted by Garvin P et al., from Sweden [20]. They have demonstrated the elevated levels of circulating MMP-9 in middle aged normal population with cardiovascular risk factors including cigarette smoking. The significant elevation of MMP9 in AMI smokers when compared to that of MMP2 may be due to the increased expression of MMP9 in vasculature and also due to the availability of its substrate (type IV collagen) in the atherosclerotic plaque, which can contribute to the increased activity of MMP9. Further cigarette smoke could induce and inhibit the MMPs activity at multiple levels [21]. Presence of reactive oxygen species (ROS) and decreased nitric oxide activity due to cigarette smoke can induce the MMP transcription [22,23] and which may be another reason for the increased expression of this MMP. The monocyte interaction with collagen and platelets is required for the leukocytes to synthesize MMP9 and that the interactions are produced particularly in areas of the vessel where inflammatory phenomena develop in response to injury [24], especially by smoking. Thus MMP9 plays a significant role in the progress of extra cellular matrix degradation in the plaques [25] in AMI patients with the habit of smoking.

Epidemiological studies have shown that MMP9 levels are elevated in the circulation of patients with acute coronary syndromes [26]. Studies have also shown the increased expression of MMP9 along with other MMPs in endothelial cells exposed to cigarette smoke condensate [27]. Activity of MMP is regulated at the levels of gene transcription, proenzyme activation and endogenous inhibitors of MMPs. Cigarette smoke induced inflammation and oxidative stress have the potential to induce and inhibit MMP activity at the multiple levels. Cigarette smoke may increase MMP expression via activation of inflammatory transcription factors [28]. It is also associated with increased circulating levels of TNF-alpha and IL-1beta as well as increased monocyte expression of IL-1beta [29]. Nicotine and cotinine of the cigarette smoke directly stimulates vascular smooth muscle cells (VSMCS) collagenase, stromelysin and gelatinase expression, which will lead to plaque rupture [30]. This is well-evident from our studies as our results show increased activity of both MMP2 and MMP9 in all the AMI patients and also the increased activity of MMP9 in AMI patients with the habit of smoking.

Through the present study we could able to prove the increased activity of both MMP2 and MMP9 in AMI patients. Further the activity of MMP9 was found to be more in AMI patients with the habit of smoking and can be used as a risk predictor for future AMI as well as a potential indicator of AMI in subjects with the habit of smoking.