Matrix metalloproteinases in COVID-19: underlying significance

Abstract The role of matrix metalloproteinases (MMPs) in pathogenesis and severity of coronavirus disease 2019 (COVID-19) is under extensive exploration. MMPs are a family of extracellular proteases involved in a variety of physiological and pathological processes and conditions. The role of MMPs in COVID-19 stems from the pathogenesis resulting in the release of chemokines and pro-inflammatory markers which cause pulmonary oedema. In addition, the approaches to treatment of COVID-19 often are associated with some complications like acute lung injury due to extracellular matrix remodelling. In this respect, the aim of this review is to summarize, interpret and evaluate the significance of matrix metalloproteinases in SARS-CoV-2 infection in terms of the severity of the condition and delve into potential treatment from this perspective as well as highlighting the physiological and protective role of some MMPs.


Introduction
Coronavirus disease 2019 (COVID-19) which is caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a member of the coronavirus family causing upper respiratory tract infections, has been estimated to be responsible for around 6.5 million deaths worldwide.The SARS-CoV-2 virion is composed of a single stranded RNA which is encapsulated by a lipid bilayer and surface proteins of three families: membrane (M), envelope (E) and spike (S) proteins [1].The routes of transmission include droplets, contact and fomites which are infected.
The role of matrix metalloproteinases (MMPs) in COVID-19 stems from the pathogenesis resulting in the release of chemokines and pro-inflammatory markers which cause pulmonary oedema [2].Based on the classification of these MMPs, each one has established a different role whether it be physiological or pathological.As a result, MMPs could be used to identify the severity of the COVID-19 stage to assess the true damage to the lungs [3].This is highlighted by MMP-3 showing evidence of increased levels as the WHO stages progress [3].Furthermore MMP-9 was positively correlated with the immune cells displayed during inflammation, which shows its involvement in COVID-19 but not as a direct biomarker of severity [3].There is also evidence that some MMPs can exert some protective effects: both MMP-7 and MMP-9 have protective effect through shedding of E-Cadherin and Syndecan-1 [4].In addition, approaches to therapy of COVID-19 often are associated with some complications like Acute lung injury (ALI) due to extracellular matrix remodelling, thus giving rational basis for anti-protease therapy [5].
Taking into account the extensive studies on MMPs and their inhibitors TIMPs in the pathogenesis of COVID-19, in this review we aimed to summarize, interpret and evaluate the significance of matrix metalloproteinases in SARS-CoV-2 infection in terms of the severity of the condition, to delve into potential treatment from this perspective as well as highlighting the physiological and protective role of some MMPs.

Methodology
International databases, such as PubMed, Google Scholar, EBSCO, Scopus, Web of Science, were searched using keywords: COVID-19, SARS-CoV-2, MMPs, metalloproteinase, TIPMs, therapy.We summarized the results and conclusions from the papers.The papers included in the review are mainly from the period of 2020-2022.

Pathogenesis of SARS-CoV-2 infection
SARS-CoV-2 attaches to the alveolar cells once entering the respiratory tract and attaches to the Angiotensin-converting enzyme 2 receptors (ACE2 receptors) and the transmembrane protease serine 2 (TMPRSS2), (both of which appear on the cell surface) through its spikes and enters the cell through endocytosis or through direct fusion through the cell membrane [6].After gaining access into the cell, the virus hijacks the cell production capabilities and, as the virus replicates, it damages the cells causing a cellular response which includes release of interleukins and cytokines.Interleukins act in a paracrine manner and cause other surrounding cells to prepare for antiviral defence.The damage to cells is detected by macrophages which switch on the immune response through the release of Tumour Necrosis Factor alpha (TNF-α), Interleukins (IL)-1, IL-6, IL-8, and chemokines which are pro-inflammatory markers [7].TNF-α and IL-1 increase vessel permeability and cell adhesion.The series of events leads to interstitial oedema which is followed by pulmonary oedema [8].
The key biological role of matrix metalloproteinase is to cleave extracellular matrix (ECM) protein and glycoproteins.When involved in biological processes such as tissue repair, wound healing, embryogenesis, endometrial regulation, cell proliferation, angiogenesis and primary tooth resorption, MMPs require careful regulation to ensure their function in accordance to the cell's needs [12].If there is deregulation of MMP activity, it can lead to the progression of a variety of pathological conditions such as cancer, asthma and COPD, lung and liver fibrosis and emphysema [13].In terms of cell processes, some MMPs (MMP-1 and MMP-13) cleave the N-terminal domain of Protease-activated receptors (PARs), triggering in this way the intracellular signalling pathway resulting in the inflammatory process [11,14].

MMPs as biomarkers of COVID-19 severity
Objectively viewing MMPs as severity biomarkers allows us to understand the implication of MMPs.A study with 48 controls and 108 COVID-19 patients, classified on the basis of the World Health Organization (WHO) stage, has presented the serum levels of MMP-3 and 9 at hospital admission and at 1 week from hospitalization.The results demonstrate an increase in serum MMP-3 at admission as the stages progress, while after a week from hospitalization, MMP3 decreases.These results have suggested a role of MMP-3 in the early steps of lung inflammation [3].
Concerning MMP-9, the results of its assessment at hospital admission have shown higher serum levels in COVID-19 patients than in controls, but without correlation with the stage.The serum MMP-9 continued to rise further especially in patients with WHO stages 3 and 5-7.Based on these findings, it has been suggested that MMP-9 may have a role in the late phases of the inflammatory process, specifically during the repair phase [3].MMP-3 is produced by endothelial cells and is released due to inflammatory cytokines [15].Its role in the pathogenesis of lung damage is still not well clarified, but the endothelial damage MMP-3 causes in COVID-19 and other conditions has been well documented [16,17].The data also show that the levels of MMP-3 increase early in infection, being in a positive correlation with the COVID-19 WHO stage, which supports the suggestion of using MMP-3 as an early marker of severity [3].
A recent study indicates that MMP-1 enzyme activity and its plasma levels are highly deregulated in COVID19 patients [18].It reported that both MMP-1 and VEGF were elevated in the hospitalized patients in comparison to the mild/moderate cases.Based on the observed positive correlation between those two inflammatory factors, as well as considering the ability of MMP-1 to act as a potent ligand for protease-activated receptor-1 (PAR1), it was suggested that MMР-1/РAR1/ VЕGFR2/VEGF-А signalling might be overactivated in endothelial cells of patients with COVID-19, especially with more severe disease [18].Thus the elevated levels and activity of MMP-1 through stimulation of the expression of vascular permeability factor (VEGF-A), might contribute to aggravation of the patients making them more susceptible to interstitial and pulmonary oedema [19].
Analysis of proteomic data from lung biopsies of COVID-19 patients has suggested that MMP-1, MMP-2, MMP-7, MMP-8 and MMP-14 show a significant increase as compared to non-COVID-19 patients [2].In addition, the levels of MMP-2 and MMP-8 in tracheal aspirate fluid, as well the levels of the active form of MMP-2 were reported significantly higher in non-survived COVID-19 patients than in survived.All these data have allowed the authors to hypothesize that MMPs, especially MMP-2/MMP-8 axis were strongly associated with lung COVID-19 severity [2].

MMP-7 and MMP-9 as biomarkers for severity in obese patients
The serum levels of MMP-7 and MMP-9 in obese diabetic patients were significantly higher than those in the non-obese non-diabetic group [20].It was subsequently found that patients who developed acute respiratory distress syndrome (ARDS) who were obese and diabetic had a higher level of MMP-7, indicating that it can be viable as a marker of severity.The level of LPS in obese diabetic patients with COVID-19 was lower, which indicates that the macrophages with M2 phenotypes would be more active due to LPS being a primary activator of pro-inflammatory phenotypes (M1).The serum levels of macrophage activation markers in obese diabetic patients one week after admission in hospital showed that patients with ARDS had significantly higher levels of MMP-7 and MMP-9 in the serum than non-ARDS patients.This was due to macrophages being polarized towards the anti-inflammatory and pro-healing phenotypes (M2) in obese diabetic COVID-19 patients with significant upregulation of the pro-fibrotic markers MMP-7, MMP-9, PDGF and TGF-β.Thus, high levels of MMP-7 and MMP-9 are associated with ARDS in severe COVID-19 disease among obese-diabetic patients [20].Both markers also were significantly elevated in obese diabetic patients who developed ARDS compared to those who did not develop ARDS during the follow-up period [20].MMP-7 and MMP-9 are upregulated in response to many factors such as mechanical ventilation, NF-kB, chemokines like CCL6, cytokines like TNF-α and IL-1 and IL-6, which rise in COVID-19 infections [21,22].

MMP targeted therapy for major complications of COVID-19
Given the function and involvement of MMPs in COVID-19, therapies can also be experimented and hypothesised to control and decrease the severity of the effects of the virus.One major complication of COVID-19 is acute lung injury (ALI), which results from epithelial cell damage that can lead to diffuse parenchymal injury [23].There is a sharp rise in pro-inflammatory mediators due to focal inflammation of the lung.It, in turn, induces a hyper-release of MMPs which further participate in airway remodelling [24].In addition, the elevated secretion and activation of MMPs cause liberation of bioactive chemokines that contribute to airway inflammation [25].In this respect, it has been supposed that inhibition of MMPs would most possibly ameliorate some adverse effects of acute lung injury (ALI) and reduce mortality from this disease, rather than the virus effects [4,26].

Natural anti-proteases
One of the mechanisms in the pathogenesis of acute lung injury (ALI) includes imbalance of the neutrophil derived proteases with serine and gelatinase protease activity and their natural endogenous inhibitors Tissue inhibitor of matrix metalloproteinase 2 (TIMP-2) and Secretory leukocyte protease inhibitor (SLPI) [4,26].An experimental study with animal models of ALI successfully demonstrated that the administration of polyclonal anti-TIMP-2 and anti-SLPI antibodies resulted in aggravation of lung injury, revealed by intensification of extravascular albumin leak, and neutrophil accumulation in bronchoalveolar lavage (BAL) fluids [27].The results of this early study prove the contribution of endogenous inhibitors in controlling the activity of proteases and support the rationality of manipulation of MMP activity as an appropriate therapeutic approach in conditions associated with acute lung injury.

Metabolic alteration of the AMPK pathways
In healthy lung, the integrity of the alveolar-capillary barrier is required, and the appropriate metabolism enables the normal function of endothelial cells.Important control mechanisms include the adenosine monophosphate-activated protein kinase (AMPK) pathways, which are vital for the energy metabolism of cells [28,29].
There is greater evidence to suggest that the SARS-CoV-2 infection causes deactivation of AMPK [28].This is probably performed by binding of SARS-CoV-2 nucleocapsid (N) to some host mRNA binding proteins as LARP1 (a mTOR-regulated translational repressor) and Casein kinase 2 (CK2, a serine/threonine protein kinase) [30].
Alternatively, a mice model of obesity and diabetes has shown that in skeletal muscle and adipose tissues there is inactivation of AMPK by TNF-α, which involves de novo synthesis of the ubiquitous serine/ threonine protein phosphatase called protein phosphatase 2 C (PP2C).Such deactivation of the central signalling AMPK pathways would result in an irregularity in cell metabolism and would eventually cause failures of organ metabolism leading to organ damage [29,31,32].
MMPs promote the activation of TNF-α, and TNF-α promotes the deactivation of AMPK.Thus, it can be inferred that the increased level of MMPs, increased level of TNF-α and decreased level of AMPK are synergic in causing irregularities in cell metabolism, which is regulated by AMPK in COVID-19 patients.Therefore, by potentially using drugs which inhibit MMPs, decrease TNF-α activity and subsequently increase AMPK activators, it would be possible to restore the functionality of cell metabolism [29].An example of a drug approved by the FDA as an MMP inhibitor is doxycycline, but others that have been tested have shown toxic effects in patients with left ventricular remodelling after myocardial infarction [33].
Perhaps lower doses of cocktails consisting of MMP inhibitors would be more suitable toward treatment; however, there are no approved methods in the field of cardiovascular or pulmonary disorders.Some benefits that could be obtained from such therapeutics include dampening of inflammation, which is the primary initiator in the pathogenesis in SARS-CoV-2.Other expected results are the blocking of MMP-dependent excessive cleavage of extracellular matrix components and normalization of AMPK activity by preventing the deactivation of AMPK by TNF-α.The final intended positive effects, like diminishing adverse extracellular matrix remodelling and restoring target organ metabolism, would limit organ damage by SARS-CoV-2 [29].

Tetracyclines
Tetracycline and chemically modified tetracyclines like CMT-3 (6-demethyl-6-deoxy-4-dedimethylamino-tetracycline) act as non-specific inhibitors and suppress MMP activity as well as transcription for MMPs.In a rat model of ventilator-induced lung injury, where elevated MMP-9 immunohistochemical expression and gelatinase activity were seen in injured lung, upon preadministration of CMT-3 before mechanical ventilation, the lung pathological changes were reduced [34].MMP-9 has an inhibitory effect on the soluble Receptor for advanced glycation end-products (sRAGE), which in turn contributes to increased inflammation in the lungs during complication of SARS-CoV-2 such as ALI [35].Once CMT-3 was administered, a proteomic analysis of lung homogenates showed that there were elevations in the sRAGE, which may play a role in decreasing inflammation caused by the RAGE ligands [36].

Batimastat
The pro-inflammatory cytokine TNF-α is seen as an active participant in the stimulation of the expression and activation of MMPs, so by inhibiting the inflammatory cytokine, it will be possible to prevent the cascade of events leading to MMP activation.The introduction of the synthetic MMP inhibitor, batimastat (BB-94) has been shown to reduce lung injury in rat models following acute pancreatitis [37].There was a clear reduction in proteinaceous exudate and neutrophil accumulation following pre-administration of batimastat 48 h before injury, and the histological grading of the lung injury was also improved due to batimastat [37].
Another study has provided evidence that MMP-9 increases neutrophil migration and alveolar capillary leakage.However, through the administration of batimastat there was a reduction in neutrophils due to the inhibition of TNF-α [38].

Role of MMP-7 and MMP-9 in alveolar epithelial repair
Despite the pathological implications that have been discussed so far, MMPs have a profound physiological role in the human body.MMP-7 is involved in epithelial repair through regulation of cell-cell and cell-matrix adhesion via shedding of E-cadherin and Syndecan-1 from the cell surface [4].Syndecan-1 is shed early after lung injury to allow cell migration, and E-cadherin shedding is delayed.An experimental in vitro and in vivo study has indicated that shedding of ectodomain of E-cadherin is carried on by MMP-7, and this process is required for epithelium repair possibly by facilitating migration of surviving cell trough the reorganized cell-cell contacts [39].
MMP-9 has a function in alveolar epithelial tissue repair, as an experiment using salbutamol in the beat agonist lung injury trial (BALTI) showed a great increase in MMP-9 in the day 4 salbutamol group, which was associated with a decrease in extravascular water.Furthermore, upon inhibition of MMP-9 with wounded alveolar epithelial cells, there was a reduction in epithelial repair proportional to the dose of inhibitor administered.This can suggest that MMP-9 has a function in alveolar epithelial repair, but the use of MMP inhibitor drugs such as doxycycline could in fact inhibit epithelial repair in SARS-CoV-2 patients suffering from ALI/ARDS [40].

Conclusions
Accumulated evidence in the literature indicates that MMPs have an underlying role in the severity of SARS-CoV-2 infection through the complications that result, such as ALI/ARDS (Acute respiratory distress syndrome).Furthermore, it is also clear that MMPs serve multiple, distinct roles based on their substrate specificity and expression and activation levels and localization, which opens the idea of specific and non-specific inhibition of MMPs as therapeutic approaches.The impact of non-specific inhibitors was positive as their inhibiting effect lowered MMPs such as MMP-9.Thus the inhibition of MMPs would work to reduce the impact of SARS-CoV-2 through improvement of the severe complications.However, further research showed that some MMPs are in fact involved in the alveolar repair and these inhibitory approaches may impair recovery.Thus, to estimate the benefits of such treatment, it is necessary to further evaluate the impact of the inhibition of inflammation vs. the impact of impaired alveolar recovery.However, the approach of specific MMP inhibition may be more effective as it would allow the natural MMP functionality for those that act in favour of physiological improvement.Despite this, more research is to be carried out to elucidate the pleiotropic functions of MMPs in the pathogenesis and repair of lung injury in order treatment to be more effective.

Figure 1 .
Figure 1.Subclasses of mmps according to their domain structural organization.modified from Ref. [11].