Role of Matrix Metalloproteinase-2 in the Development of Atherosclerosis among Patients with Coronary Artery Disease

Coronary artery disease (CAD) is a caused by atherosclerotic plaque buildup in the coronary arteries that supply blood and oxygen to the heart. Matrix metalloproteinase (MMP) is a family of zinc-dependent endopeptidase that is involved in various stages of atherosclerosis as demonstrated in in vitro and in vivo studies. MMP-2 is associated with both stable and unstable atherosclerotic plaque formation. The current review aimed to identify the role of MMP-2 in atherosclerosis development among CAD patients. Literature search was conducted through four online databases and only studies that were published from 2018 until February 2023 were included. The risk of bias was assessed by using the Newcastle–Ottawa Scale. A total of 10,622 articles were initially identified, and only eight studies that fulfilled the selection criteria were included in this review. The results showed that MMP-2 levels and activity were higher in patients with unstable CAD than those with stable CAD and healthy subjects. There was a significant association between MMP-2 levels and cardiovascular disease with MMP-14 levels, which is a pro-MMP-2 activator. In addition, two single nucleotide polymorphisms of the MMP-2 gene (rs243865 and rs243866) were significantly associated with the development of atherosclerosis. In conclusion, MMP-2 plays a crucial role in the development of atherosclerosis among patients with CAD and could be a potential target for CAD therapy.


Introduction
Coronary artery disease (CAD) is the commonest type of heart disease involving the formation of plaque in the lumen of coronary arteries. A plaque is a buildup of fatty material that causes a heart attack by constricting the vessel lumen and obstructing blood flow to the heart [1,2]. CAD is one of the cardiovascular diseases (CVDs) with the highest estimated prevalence (53%) in 2019 compared with other CVDs [3]. Globally, it was estimated that 244.1 million people were living with CAD in 2020 [4]. Countries in Central and South Asia have the highest prevalence and mortality rate of CAD [4].
The biological root of CAD is atherosclerosis. Atherosclerosis begins with infiltration and deposition of cholesterol in the arterial wall, migration of vascular smooth muscle cells (VSMCs) to the intima, and development of fibrous matrix and plaque [5]. Over time, this plaque grows and impairs the blood flow to the heart muscle, subsequently causing angina [6]. There are two types of plaque, namely stable and unstable plaques. Stable plaque is associated with a stable CAD or chronic coronary syndrome (CCS) which usually presents with angina that is relieved by rest. CCS is further divided into two types; obstructive coronary and ischemia with nonobstructive coronary arteries (INOCA). An obstructive coronary present with blockage that is greater than or equal to 50% of the vessel diameter, while INOCA indicates ischemia with less than 50% blockage.
Meanwhile, an unstable plaque is prone to rupture, causing thrombosis to occur. Plaque thrombosis results in a nearcomplete or complete occlusion of the arterial lumen. This condition is known as unstable CAD or acute coronary syndrome (ACS) [7]. Myocardial infarction with ST-elevation (STEMI), non-ST-elevation (NSTEMI), unstable angina (UA), and myocardial infarction with the nonobstructive coronary arteries (MINOCA) are all examples of ACS [8]. Most ACS patients may experience symptoms such as substernal chest pain, shortness of breath, sweating, nausea, and lightheadedness [9,10]. About 60%-65% of myocardial infarction cases in young people are caused by plaque rupture [11].
A group of proteolytic enzymes known as matrix metalloproteinases (MMPs) is involved in various stages of atherosclerosis development as demonstrated in in vitro and in vivo studies [12][13][14][15][16][17][18]. MMPs are a family of zinc-dependent endoproteases which are secreted by endothelial cells, VSMCs, fibroblasts, osteoblasts, macrophages, neutrophils, and lymphocytes [19]. MMPs are made up of 28 members which are classified based on their structures and organization of their structural domains [20]. Each MMP contains at least three homologous protein domains; signal peptide, propeptide, and catalytic domains. The signal peptide domain directs MMPs to the secretory pathway, whereas the propeptide domain is removed when MMPs are activated. The zinc-binding region is located in the catalytic domain, which is crucial for MMPs to function as proteases. Additionally, the hemopexin domain interacts with tissue inhibitors of MMPs (TIMP) and is involved in substrate binding [21]. MMP-2 activity is inhibited by TIMP-2, whereas MMP-9 activity is inhibited by TIMP-1 [22].
One of the 28 members of the MMP family is MMP-2. MMP-2 belong to the gelatinase group, which has a gelatinbinding site in their catalytic domain ( Figure 1). MMP-2 is associated with both stable and unstable atherosclerotic plaques. During stable atherosclerotic plaque development, MMP-2 is involved in VSMC accumulation in the fibrous cap that shields the plaque. MMP-2 is the first proteinase shown to be responsible for the migration and proliferation of VSMCs [23]. According to a study by Sluijter et al. [24], MMP-2 activity increased in the VSMCs of fibrous-type atherosclerotic plaque, indicating that MMP-2 is strongly linked to the development of stable plaque. Consistent with this, MMP-2 was found to enhance plaque stability by accumulating VSMCs into the fibrous cap of atherosclerotic apolipoprotein-E knockout (ApoE-/-) mice [25].
However, the gelatin-binding site in the MMP-2 catalytic domain made MMP-2 capable of degrading gelatins in the extracellular matrix (ECM) of the fibrous cap enclosing the atherosclerotic plaque, hence promoting plaque instability [21,26]. Gelatinase is an important enzyme in acute myocardial infarction (AMI) since it can degrade the fibrillar collagen of ECM [27]. According to a study by Zeng et al. [28], atherosclerotic plaque instability in ACS patients may be predicted by high circulating levels of MMP-2. Recently, the activation of the transforming growth factor-beta/suppressor of mothers against decapentaplegic (TGF-/Smad) pathway revealed an increase in MMP-2 levels in rats with myocardial infarction [29]. Meanwhile, the histopathological study showed that MMP-2 was mostly found in fatty streaks and fibroatheromas with hemorrhage and calcification [30]. Since MMP-2 plays a vital part in the progression of atherosclerotic plaque, this review sought to systematically investigate the role of MMP-2 in atherosclerosis development in CAD patients.

Methodology
This review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline [31]. PRISMA checklist was reported in Tables 1 and 2. 2.1. Search Strategy. The literature search was conducted through four online databases (Ovid, Scopus, Pubmed, and Google Scholar) and only studies that were published from 2018 until February 2023 were included. The keywords used for the search were: (CD) OR (CAD) OR (ischemic heart disease) OR (ACS) OR (coronary atherosclerosis) OR (myocardial infarction) AND (MMP).

Study
Criteria. The articles were studied individually by two researchers (NS and AA) centered on the inclusion and exclusion criteria. The inclusion criteria include (1) full-text original articles in English language, (2) studies that reported MMP-2 in atherosclerotic CAD, and (3) clinical studies involving adult human patients with CAD, both male and female, of any ethnicity. While the exclusion criteria were: (1) not original articles, (2) published in languages other than English, (3) studies that not reported MMP-2 in atherosclerotic CAD, (4) articles including reviews, conference abstracts, editorials, newsletters, book, and book chapters, and (5) in vitro and in vivo studies.

Article Selection and Data
Extraction. Three phases of article selection were carried out. Initially, articles were excluded primarily on the title. Then, by analyzing the abstracts, articles that were not relevant to MMPs and   10b List and define all other variables for which data were sought (e.g., participant and intervention characteristics, funding sources). Describe any assumptions made about any missing or unclear information Table 5 Study risk of bias assessment 11 Specify the methods used to assess risk of bias in the included studies, including details of the tool(s) used, how many reviewers assessed each study and whether they worked independently, and if applicable, details of automation tools used in the process    Results, paragraph 1 and see Figure 2 16b Cite studies that might appear to meet the inclusion criteria, but which were excluded, and explain why they were excluded Table 3 Study characteristics 17 Cite each included study and present its characteristics.
Results, paragraph 2-5 and see Table 4 Risk of bias in studies 18 Present assessments of risk of bias for each included study Results, paragraph 2, line 3, and see Table 4, Results of individual studies 19 For all outcomes, present, for each study: (a) summary statistics for each group (where appropriate) and (b) an effect estimate and its precision (e.g., confidence/credible interval), ideally using structured tables or plots Results, see Table 4, column 5 Results of syntheses 20a For each synthesis, in brief summarize the characteristics and risk of bias among contributing studies Results, see Table 4, column 2 and 6 20b Present results of all statistical syntheses conducted. If meta-analysis was done, present for each the summary estimate and its precision (e.g., confidence/credible interval) and measures of statistical heterogeneity. If comparing groups, describe the direction of the effect Results, see   Mediators of Inflammation atherosclerosis were excluded. In Last, after reading the complete articles, articles that did not meet the inclusion requirements were excluded. For data extraction, two researchers (NS and AA) independently recorded the name of the studies' first author, the study's design, subject characteristics, participants' ages and genders, methods of MMP-2 measurement, and MMP-2 levels in atherosclerotic CAD into a table.
2.4. Risk of Bias Assessment. The Newcastle-Ottawa Scale (NOS) which has three domains for both case-control and cohort studies was used by two reviewers (NS and AA) to assess the quality of risk of bias [32]. For case-control studies, NOS evaluated the choice of study groups (including cases and controls), the comparability of the groups, and the determination of exposure for both the cases and the control groups. In contrast, for cohort study, NOS evaluated the choice of study groups (exposed and nonexposed), comparability across groups, and evaluation of outcome. There were eight items in each of the three domains that may be given a star rating. A minimum of one star or a maximum of two stars were assigned to each item. Studies with seven to nine stars were deemed to be of high-quality studies, while those with four to six stars were deemed to be of fair quality studies and those with one to three stars to be of low-quality studies.

Results
A total of 10,622 articles were obtained from four online databases: Ovid (2,894), Scopus (5,089), PubMed (2,637), and Google Scholar (2). The articles were published between 2018 and February 2023. Then, 1,601 articles were excluded due to replication. Following the assessment of the title and abstract of the articles, 8,951 articles were further excluded. The remaining 64 articles' complete texts were attained and thoroughly assessed. From these 64 articles, only eight were selected to be included in this study. The 56 excluded articles were listed with their respective exclusion reason in Table 3. Figure 2 displays the article selection process. Table 4 summarized the details of all the final eight studies. Four studies were case-control clinical trials, three studies were cross-sectional studies and one study was a cohort study by design. Quality assessment using NOS revealed that the score range of the studies reviewed was between 4 and 9 (fair to high quality). All the subjects were patients diagnosed with CAD. The subject's age ranged from 18 to 79 years old. Most of the studies measured MMP-2 levels in the serum [33][34][35], plasma [38,40], and genomic DNA isolated from the subject's blood [39]. Additionally, two studies also investigated MMP-2 expression in the coronary atherosclerotic plaque [36,37].
A potent natural anti-inflammatory agent, curcumin was found to significantly inhibit the MMP-2 activity in the serum of CAD patients in comparison to the placebo group (p<0:001) [33]. In addition, Sai et al. [34] demonstrated a higher MMP-2 levels in the serum of AMI patients compared with the healthy subjects. There was also a significantly lower serum MMP-2 levels in AMI patients posttreatment with benazepril and rosuvastatin, compared with those who only received rosuvastatin (p<0:05) [34].
Moreover, Li et al. [35] showed a significantly higher MMP-2 serum levels in patients with unstable CAD than in stable CAD and healthy groups. Murashov et al. [37] also demonstrated a higher expression of MMP-2 by 7.8 times in unstable atherosclerotic plaques compared with stable atherosclerotic plaques via immunohistochemistry (IHC) analysis (p<0:05). MMP was mostly expressed in the cytoplasm of foamy macrophages in atheromatous core and caps of unstable plaques [36]. However, MMP-2 expression did not significantly differ between the three types of unstable atherosclerotic plaque (degenerative-necrotic type, lipid type, and inflammatory-erosive type) [37]. A study by Owolabi et al. [40] found no significant difference in MMP-2 levels between the AMI and stable CAD groups at any time point.
Besides, Melin et al. [38] found that plasma MMP-2 levels were higher in Type 1 diabetic patients with high-MMP-14 levels. High-MMP-14 levels was associated with increased CVD in this group of patients [38]. Malkani et al. [39] found two MMP-2 gene single nucleotide polymorphisms (SNPs); namely rs243865 and rs243866 in patients with atherosclerosis. The CA, CG, and TA haplotypes of the MMP-2 gene were significantly connected with atherosclerosis [39].

Discussion
The aim of this review was to determine the role of MMP-2 in atherosclerosis development in CAD patients. MMP-2 levels were discovered to be higher in CAD patients than in healthy individuals. Meanwhile, amongst patients with CAD, MMP-2 levels were higher in unstable than in stable atherosclerotic plaque, indicating that MMP-2 plays a role in the vulnerability and severity of the atherosclerotic plaque. However, one study did not support such findings, in which no significant difference in MMP-2 levels was found in acute MI and stable CAD [40]. This might be because of the nature of the study that examined MMP-2 levels following cardiac catheterization, which is a procedure to clear the blockage that might disturb the MMP-2 levels. MMP-2 was also found to be related to high levels of MMP-14. MMP-14 is a pro-MMP-2 activator that is linked to the incidence of CVD. Two SNPs of the MMP-2 gene (rs243865 and rs243866) were associated with atherosclerosis. Moreover, treatment of CAD patients with ACE inhibitor, lipid-lowering drug, and anti-inflammatory compound decreased MMP-2 activity and levels, indicating that the inhibition of MMP-2 is useful in treating CAD.
Atherosclerosis is a chronic inflammatory disease involving the deposition of cholesterol, fats, blood cells, and other substances that form a plaque inside the vessel wall [41]. It is the main underlying cause of CAD, in which the atherosclerotic plaque buildup causes narrowing of the arterial lumen and disrupts the blood supply to the heart [42]. In the worstcase scenario, the atherosclerotic plaque could rupture, leading to thrombus formation and complete occlusion of the coronary arteries that presents as ACS [13].
MMPs participate in various stages of atherosclerosis development, from lesion initiation to plaque rupture. MMP-2 is widely distributed in endothelial cells, VSMCs,      Mediators of Inflammation leukocytes, platelets, adventitia, and dermal fibroblasts [14], and is essential for the development of atherosclerosis [15-18, 43, 44]. Atherosclerosis starts with endothelial dysfunction that promotes infiltration of low-density lipoprotein (LDL) into the intima, which becomes oxidized to form oxidized LDL (oxLDL). MMP-2 promotes endothelial dysfunction by causing proteolytic degradation of endothelial nitric oxide synthase (eNOS) and its cofactor, heat shock protein 90 (HSP90). This leads to reduced endothelial nitric oxide (NO) production [15]. Incubating bovine coronary artery endothelial cells with recombinant MMP-2 significantly decreased NO release. Furthermore, MMP-2 cleaves HSP90 into several fragments, thus disrupting eNOS activity [15].
Subsequently, the macrophages take up oxLDL in the intima to form foam cells. Circulating and activated platelets facilitate this stage of atherosclerosis formation. MMP-2 expressed by the circulating platelets is higher in patients with CAD compared with healthy people [16]. A study by Cheung et al. [45] found the increased release of MMP-2 from platelets activated by oxidative stress in acutely ill neonates. MMP-2 expressed by the activated platelets has an important role in facilitating monocyte infiltration into the intima to become macrophages [16]. This platelet MMP-2 interacts with endothelial cell's protease-activated receptor 1 (PAR-1) and increases vascular cell adhesion molecule 1 (VCAM-1) expression. VCAM-1 encourages monocyte adherence and infiltration via endothelial cells into the subintimal layer, which contributes to the development of atherosclerosis [16,17].
OxLDL is also responsible for the migration of VSMCs into the intima. The VSMCs migrate from media into the intima, forming fibrous cap of the atherosclerosis plaque [46]. The sphingomyelin/ceramide/sphingosine-1-phosphate (Spm/Cer/S1P) pathway is one of the signaling pathways involved in VSMC proliferation induced by oxLDL [18]. By activating this pathway, MMP-2 stimulates oxLDL-induced VSMCs proliferation in atherosclerosis [18]. It is also supported by the silencing of MMP-2 gene inhibits the proliferation of oxLDL-induced VSMCs [18].
MMPs are also participate in modulating the progression of stable atherosclerotic plaque to unstable plaque by degrading the ECM [47,48]. Degradation of ECM causes thinning of the plaque fibrous cap, and facilitates the plaque to become unstable and prone to rupture [49,50]. An unstable atherosclerotic plaque is responsible for the life-threatening clinical conditions such as AMI [51]. Previous study showed that 4-Hydroxynonenal (HNE), a highly reactive product of lipid peroxidation enhanced MMP-2 production in the fibrous cap's VSMCs by activating the activating tyrosine kinase/ Nuclear factor kappa beta (Akt/NF-ĸβ) signaling pathways [43]. There was an increase in MMP-2 mRNA and protein expression in VSMCs following exposure to HNE, indicating     Initially, MMP-2 cleaves eNOS or its cofactor, HSP90 to cause endothelial dysfunction and facilitate infiltration of LDL into the intima. Then, MMP-2 expressed by activated platelets promote monocyte transmigration into the intima through endothelial PAR-1 activation. MMP-2 also facilitates oxLDL-induced VSMCs migration to the intima, forming fibrous cap in the atherosclerotic plaque. Last, MMP-2 stimulates fibrous cap degradation and rupture of atherosclerotic plaque via several pathways including Akt-NF-ĸβ pathway which is induced by HNE, and NADPH oxidase pathway which is induced by ANG II and ROS. Akt = activating tyrosine kinase, ANG II, angiotensin II; eNOS, endothelial nitric oxide synthase; HSP90, heat shock protein 90; HNE, hydroxynoneal; LDL, low-density lipoprotein; MMP-2, matrix metalloproteinase-2; MT1-MMP, membrane Type1-matrix metalloproteinase; NAD(P)H, nicotinamide adenine dinucleotide phosphate; NO, nitric oxide; NF-ĸβ, nuclear factor kappa beta; oxLDL, oxidized low density lipoprotein; PAR-1, protease-activated receptor 1; ROS, reactive oxygen species; Sm/Cer/S1P, sphingomyelin/ceramide/ sphingosine-1-phosphate; VCAM-1, vascular cell adhesion molecule 1. Included studies Sources of funding Mogharrabi et al. [33] Mashhad University of Medical Sciences Research Council Sai et al. [34] Not stated Li et al. [35] Not stated Murashov et al. [36] Not stated Murashov et al. [37] Not stated Melin et al. [38] Research and Development Fund of Region Kronoberg, Växjö, Sweden, and by the Research Council of South Eastern Sweden (FORSS), Linköping, Sweden, and the Southern Healthcare Region, Lund, Sweden Malkani et al. [39] Not stated Owolabi et al. [40] American Heart Association (11CRP7300003), and National Institutes of Health (1P20 GM103492) that HNE regulates MMP-2 production at transcriptional level [43]. Given that HNE is consistently present in a significant amount in the unstable atherosclerotic plaque, it is postulated that HNE accelerates plaque rupture through enhanced production of MMP-2 [52]. This is supported by previous studies that demonstrate a higher MMP-2 expression and activity in vulnerable regions of the atherosclerotic plaque [53,54]. Furthermore, a vasoactive peptide, angiotensin II (ANG II) has been found to enhance MMP-2 mRNA expression in the fibrous cap's VSMCs by activating NADPH oxidase in a p47phox-dependent manner [44]. VSMCs isolated from wildtype (WT) mice showed a significant increase in MMP-2 mRNA expression following exposure to ANG II. In contrast, VSMCs isolated from p47phox−/− mice secreted less MMP-2 compared with the VSMCs of WT mice. This indicates that ANG II-stimulated MMP-2 expression is dependent on the p47phox subunit of NADPH-oxidase. Besides, MMP-2, p47phox and ANG II were colocalized in VSMCs-rich areas of atherosclerotic plaques, suggesting their functional interactions [55]. Furthermore, ANG II stimulates reactive oxygen species (ROS) production that alters the plaque proteolytic balance by enhancing VSMC's NADPH oxidase-dependent release of MMP-2. This eventually leads to plaque instability [44]. The relevant signaling pathways related to MMP-2 and the various stages of atherosclerosis development are summarized in Figure 3.

Study Limitation
A few limitations have been identified from this review. First, there is only one study that focuses on association of MMP-2 gene polymorphisms with the occurrence of atherosclerosis and CAD. Hence, more studies on MMP-2 gene polymorphisms in association with CAD are needed. Second, there is a limited data on MMP-2 signaling pathways that is based on human study. Most of the data on the signaling pathways were derived from in vitro and in vivo preclinical studies. Therefore, further studies related to MMP-2 signaling pathways using human samples are needed in the future.

Conclusion
To conclude, this review has identified some studies that demonstrate the role of MMP-2 in atherosclerosis progression among patients with CAD. MMP-2 expression and activity are found to be higher in patients with CAD. Besides, MMP-2 participates in various stages of atherosclerotic plaque development, which is the main pathophysiology underlying CAD. This review could increase our understanding on the role of MMPs, specifically MMP-2 in atherosclerosis development among CAD patients, which could help in early disease diagnosis and development of targeted therapy via the MMP-2 signaling pathway.

Disclosure
The review protocol has been registered with the International Platform of Registered Systematic Review Protocols (registration number INPLASY202340058) [56].