ABSTRACT

Objective:

Smoking causes cardiovascular risk, which may alter the stability between the production and degradation of the extracellular matrix. Matrix metalloproteinase-9 (MMP-9) is a zinc-containing endopeptidase that degrades the extracellular matrix and is involved in tissue remodelling and several physiological processes. As a result, smoking-induced elevated serum MMP-9 levels, particularly at a younger age, raise the risk of coronary heart disease (CHD). Thus, this study aimed to determine the possible relationship between smoking-induced circulating MMP-9 and the risk of cardiovascular disease in young smokers.

Methods:

In this cross-sectional study, the patients were divided into three groups. Each group contains 120 study participants. Group one consisted of 120 healthy individuals with no physical and mental illness, group two consisted of 120 active smokers with a heart disease, and group three consisted of 120 active smokers with a heart disease and diabetes, who attended Sri Ramaswamy Memorial Hospital for cardiology checkup at the age of 20-55 years. The serum MMP-9, high-sensitivity C-reactive protein (hs-CRP), and apolipoprotein-E (APO-E) levels were analyzed using the ELISA method, and the lipid levels were measured enzymatically using AU480 automatic analyzer (Beckman Coulter).

Results:

Compared with non-smokers, the study shows that the mean serum MMP-9, hs-CRP, and APO-E levels were significantly higher in smokers (p<0.001). A strong relationship was also found between MMP-9 and hs-CRP, APO-E, smoking load, and smoking intensity.

Conclusions:

A significant association was found between cigarette smoking with MMP-9, and relative exposure to circulating inflammation markers plays a potential role in the pathogenesis of CHD.

INTRODUCTION

Smoking is an environmental risk factor that is associated with different genetic factors and multifactorial disorders¹. The mechanism by which smoking induces cardiovascular risk is still unclear to some extent². Smoking can increase the risk of cardiovascular disease, which is adversely related to the smoking burden and smoking intensity³. Other than active smoking, passive smoking also increases the cardiovascular risk by altering the serum lipid levels in young smokers⁴. The chemical components of cigarette smoke, especially nicotine, can increase the fatty acid level by stimulating the release of adrenaline, leading to an increase in cardiovascular risk⁵. Free fatty acids can increase the secretion of triglycerides (TGL) and low-density lipoprotein (LDL) and the release of cholesterol from the hepatic circulation⁶. Smoking also produces free radicals that can alter the coagulation system⁷ and may accelerate the formation of plaque in the arteries⁸. Moreover, smoking promotes inflammation, which leads to the formation of atherosclerotic lesions and the progression of cardiovascular events. A recent study found that chronic inflammation results in atherosclerotic plaque development and eventual rupture⁸. However, regarding the predictors of cardiovascular disease, the markers for general inflammation are related to acute-phase reactants and proinflammatory cytokines including high-sensitivity C-reactive protein (hs-CRP)⁹. Specifically, the tissue necrosis factor-a and proinflammatory cytokines such as interleukin-1 (IL-1) regulate the synthesis of matrix metalloproteinase-9 (MMP-9) through the mesenchymal stem cells¹⁰. A recent study reported that smoking may modify the integrity of the extracellular matrix by shifting the balance between MMP synthesis and breakdown, potentially leading to the development of cardiovascular disease¹¹. MMP is the family of zinc-containing zymogene endopeptidases that degrade extracellular matrix proteins, play an important role in the extracellular matrix for tissue remodeling, and contribute to various physiological processes^12,13. MMP-9 is a collagenase enzyme with a molecular weight of 92 kDa. In the basement membrane, MMP-9 breaks proteoglycan proteins, type 4 collagen, and interstitial proteins¹⁴. It is now the most researched protein in connection to cardiovascular disease. Another study showed that in a high inflammatory setting, the activity of MMP-9 led to the increase in the formation of atherosclerotic plaque¹⁵. Thus, increased MMP-9 levels are linked to a higher risk of cardiovascular disease development, especially in smoking-induced inflammatory conditions. MMP-9 can be used as a therapeutic target and biomarker for future CHD¹⁶.

In Tamil Nadu, which is located the southern part of India, the smoking rate is likely to increase, which may enhance the prevalence of patients with CHD because of lifestyle changes and increased smoking rate at an early age. Thus, this study aimed to investigate the link between circulating MMP-9 levels and CHD risk in young smokers.

MATERIALS and METHODS

Study Subjects and Pattern

In this cross-sectional study, the patients were divided into three groups. Each group contained 120 study participants. Group one consisted of 120 healthy individuals with no physical and mental illness, group two consisted of 120 active smokers with a heart disease, and group three consisted of 120 active smokers with a disease and diabetes, who attended Sri Ramaswamy Memorial Hospital for cardiology checkup at the age of 20-55 years. During regular cardiovascular health assessments, a standard questionnaire is used to acquire information about the patient’s history and lifestyle. To determine whether patients fit the inclusion/exclusion criteria and are qualified to take part in the trial, a simple questionnaire was initially administered to each participant.

Inclusion criteria: Men aged 20-55 years who were either non-smokers or smoked at least five cigarettes per day for more than 1 year were included. CHD was defined as coronary angiography revealing >50% stenosis.

Exclusion criteria: The study excluded participants with hepatic disorders, renal disease, cardiovascular accidents, and systemic illness. Previous smokers, alcoholics, individuals with a family history of cardiovascular disease, and individuals taking medications such as steroids and lipid-lowering drugs were also excluded.

Quantifying smoking exposure: Smoking behaviors were determined by self-report. Smoking status, smoking burden (smoking duration) and smoking intensity (number of cigarettes smoked per day) were calculated. Smoking was defined as smoking at least five cigarettes per day for more than 1 year¹⁷.

Ascertainment of Covariates

Age, sex, educational background, and other covariates, as well as prior medical and health histories, were based on self-reports. Standard techniques were used to acquire anthropometric measures such as height and body weight. Blood pressure at rest was measured after 2 min of relaxation. Systolic blood pressure of >140 mmHg and diastolic blood pressure of >90 mmHg are considered signs of hypertension. In this study, fasting levels of plasma glucose >126 mg/dL or a glycated hemoglobin level >6.5% were used to diagnose diabetes mellitus.

Data Collection

Individuals were advised to report to the hospital between 8:00 and 9:00 a.m. before laboratory measurements. After a 12-h fast, 5 mL of venous blood was aseptically extracted in a plane tube using a vacutainer. The serum samples were obtained by centrifuging blood samples at 2000 × g for 10 min at 4 °C, which were then stored at -20 °C before further analysis.

Laboratory Assays

The AU480 automated analyzer (Beckman coulter) was used to quantify fasting blood glucose (FBG), total cholesterol (TC), TGL, high-density lipoprotein-cholesterol (HDL-C), LDL-C, and very-LDL-cholesterol levels enzymatically. A sandwich enzyme-linked immunosorbent assay was used to assess the serum MMP-9, hs-CRP, and apolipoprotein-E (APO-E) concentration in accordance with the manufacturing protocol (Abbkine, Inc. China).

Ethical Consideration

The study was approved by the Ethical Committee of SRM Hospital, SRMIST, and India (IEC no. 1763, date: 22.08.2019). All study participants provided written consent.

Statistical Analysis

Statistical analysis was conducted using IBM SPSS Statistics, version 22 (IBM Corp., Armonk, NY, USA). Numeric values are expressed as the mean and standard deviation (SD). A One-Way analysis of variance (ANOVA) was used to evaluate the differences between the three groups. For p<0.001, p<0.05, and p>0.05, differences were judged as extremely significant, significant, or non-significant, respectively. The correlations between variables were determined using Pearson’s correlation coefficient (r) or Spearman correlations, and linear regression analysis was performed to show the relationship between MMP-9 and hs-CRP, APO-E, smoking load, and smoking intensity.

RESULTS

Demographic and Quantitative Parameters of the Study Population

Tables 1 and 2 show the demographic and baseline characteristic data of all the three groups. A significant difference was found in participants’ weight, body mass index, waist-hip ratio, systolic blood pressure, number of cigarettes smoked per day (smoking intensity), and smoking duration (smoking load). Compared with group 1, groups 2 and 3 had significantly higher FBG and lipid profile. The study group did not receive any kind of lipid-lowering treatment.

The serum MMP-9, hs-CRP, and APO-E levels were presented as mean ± SD. As shown in Table 2, group 2 was found to have higher blood MMP-9, hs-CRP, and APO-E levels, followed by group 3, which were gradually rising when compared with those of group 1 (p<0.001), indicating that smoking induces inflammation.

Correlation Analysis

Table 3 and Figure 1 show Pearson’s correlation between serum MMP-9, hs-CRP, and APO-E levels in the CHD group. A positive correlation was found between MMP-9 and hs-CRP (r=0.3776, p<0.0001), APO-E (r=0.4039, p<0.0001), TC (r=0.3204, p=0.0003), TGL (r=0.3881, p<0.0001), LDL-C (r=0.5003, p<0.0001), number of cigarettes smoked per day (r=0.3411, p=0.00013) and smoking duration (r=0.3175, p=0.0004).

Table 4 and Figure 2 also show a correlation between the serum MMP-9, hs-CRP, and APO-E levels in group 3. A positive correlation was found between MMP-9 and hs-CRP (r=0.3776, p<0.0001), APO-E (r=0.4614, p<0.0001), TC (r=0.4858, p<0.0001), TGL (r=0.3917, p<0.0001), LDL (r=0.4689, p<0.0001), number of cigarettes smoked per day (r=0.4287, p<0.0001), and smoking duration (r=0.3638, p<0.0001), and a negative correlation was found with HDL-C levels (r=-0.3705, p<0.0001).

DISCUSSION

To our knowledge, this is the first-ever report on MMP-9 in young smokers. Smoking habits continuously increase the risk of cardiovascular disease and peripheral vascular disease¹⁸. Modifiable risk factors such as high blood pressure or high cholesterol levels do not explain clearly the relationship between cigarette smoking and CHD risk¹⁹. However, certain studies revealed that smoking increases the levels of circulating MMP-9, which raises the likelihood of cardiovascular disease²⁰.

The results of this study reveal a significant positive link between cardiac risk factors, notably cigarette smoking, hs-CRP, and MMP-9. Furthermore, some studies have shown a correlation between higher circulating MMP-9 levels, smoking status, and inflammatory markers including hs-CRP and IL-6²¹. The study also showed that MMP-9 significantly correlated with APO-E levels. This may be due to the shedding of the lipoprotein receptor by MMP-9. MMP-9 is an endopeptidase that can bind and proteolyze (i.e., shedding) lipoprotein receptors²².

In this study, smokers with CHD (both with and without diabetes) had significantly higher blood MMP-9 levels than normal controls (non-smokers) (p<0.001). Compared with groups 2 and 1, group 3 had significantly higher MMP-9 levels, with a significance value of 0.001. This may reflect abnormal extracellular matrix metabolism group 3²³. MMP-9 is also a novel regulator of cholesterol metabolism. Furthermore, the findings show that the dysregulation of MMP-9 activity alters hepatic transcriptional responses to dietary cholesterol, potentially leading to metabolic disorders such as atherosclerosis and CHD²⁴.

Smoking accelerates inflammation and the oxidative modification of lipids and prospectively slows down the activity of MMPs at various levels. By activating inflammatory transcription factors, smoking increases the MMP expression²⁵. Moreover, smoking increases the monocyte expression of IL-beta cells²⁶. Cigarette smoke contains nicotine, and the predominant metabolite cotinine increases the production of vascular smooth muscle cell collagenase and gelatinase, which may lead to plaque rupture²⁷.

Different mechanisms have been anticipated to explain how smoking stimulates MMP both in vitro and in vivo²⁸. In vitro, tobacco smoke induces MMP-9 expression through the endothelial cells²⁹. Similarly, exposure to smoking induces MMP-1 expression through the human fibroblasts. Moreover, tobacco smoke induces proteolysis when the cadmium present in the smoke is inhaled, and increased exposure may lead to cardiovascular disease as it increases in the aorta of smokers¹².

The main limitation of this study is the small sample size, despite the solid conclusion on the link between smoking and CHD risk. Some factors such as age, sex, individual condition, environment, and experimental procedure may affect the results, which may influence the interpretation of the results. Analyzing the activities of endogenous tissue inhibitors of metalloproteinase-1 (TIMP-1) is necessary because it creates a balance between MMP-9 and TIMP-1³⁰. However, this study did not analyze TIMP-1 concentration. Our finding regarding MMP-9 in young smokers gives evidence that cigarette smoking increases the circulating MMP-9 levels at a young age.

CONCLUSION

This study reveals a substantial link between serum MMP-9 levels and CHD risk in young smokers. The results of this study indicate that an increase in MMP-9 levels, particularly in smoking-induced inflammatory conditions, is related to CHD risk in young smokers.