COVID-19 pneumonia is characterized by diffuse alveolar damage and infiltration of monocytes, macrophages, and lymphocytes in pulmonary interstitium, which blocks alveolar gas exchange and induces ARDS. Patients with severe COVID-19 have high inflammatory response to SARS-CoV-2 with overproduction of inflammatory cytokines, known as macrophage activation syndrome (MAS) or cytokine storm. Activated inflammatory macrophages play a crucial role in matrix destruction by producing matrix metalloproteinases (MMPs), such as MMP-2 and MMP-9.
Several studies have found elevated serum MMP-2 and MMP-9 levels in many chronic inflammatory conditions including chronic obstructive pulmonary disease. Nevertheless, these MMPs are strictly regulated by their specific inhibitor of metalloproteinases (TIMPs), which controls its proteolytic activity.
SARS-CoV-2 invades host cells via two receptors: angiotensin-converting enzyme 2 (ACE2) and CD147. The overexpression of CD147 enhances the release and the activation of MMPs and the invasive potential during the differentiation of monocyte into macrophages. The aim of this study is to evaluate in Covid-19 the imbalance between MMP-2 and MMP-9 and their inhibitors (TIMP-2 and TIMP-1, respectively) that may cause excessive degradation of tissue, a condition that is often related to chronic inflammatory diseases.
Plasma MMP-2, MMP-9, TIMP-1 and TIMP-2 levels of will be evaluated by ELISA while MMP-2 and MMP-9 activity will be assessed by zymography. Gelatinases and their inhibitors mRNA expression will be evaluated through quantitative real time PCR (qPCR) in PBMCs. Moreover, by flow cytometry analysis the expression of CD147 on monocytes will be evaluated and the results will be compared to MMPs and TIMPs results
The evaluation of balance between gelatinases (MMP-2 and MMP-9) and their natural inhibitors (TIMP-1 and TIMP-2) in patients with COVID-19 could be a novel disease marker as well as a therapeutic target.
Current management of COVID-19 is supportive, and respiratory failure from acute respiratory distress syndrome (ARDS) is the leading cause of mortality.
Proteases, and especially matrix metalloproteinases (MMPs), are known to be involved in degradation of extracellular matrix (ECM). MMPs are secreted in a latent form, which needs activation to exercise its catalytic activity. The activity of MMPs is inhibited by specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). MMP-2 is essentially inhibited by TIMP-2, and MMP-9 is inhibited by TIMP-1 (Legrand et al., 1999).
The imbalance between MMPs and TIMPs has been proposed to play a role in the pathogenesis and evolution of ARDS. Indeed, the level of MMP-9 in bronchoalveolar lavage (BAL) fluid was elevated in the early phase of the disease, whereas the MMP-9/TIMP-1 ratio remained elevated in late-phase ARDS compared with healthy subjects. Regarding the level of MMP-9 and MMP-2, similar results were obtained in BAL fluid of ARDS patients compared with healthy subjects (Lanchou et al., 2003).. Hence, Delclaux et al. reported a correlation between epithelial lining fluid albumin and the sum of activated MMP-9 and MMP-2, suggesting an involvement of these MMPs in the increase of alveolar-capillary permeability and therefore in the evolution of the disease (Delclaux et al., 1996).
The aim of this study is to longitudinally evaluate the imbalance between MMPs and TIMPs that may cause excessive degradation of tissue in hospitalized patients with COVID-19 pneumonia. This approach can contribute to better understand COVID-19 pathogenesis investigating the expression and function of MMP-2 and MMP-9 and their inhibitors and providing potential new therapeutic options in the future.
Patients with COVID-19 who admitted to the Policlinico Umberto I Hospital, Sapienza University of Rome will be enrolled and will be evaluated at the admission (T0) and after seven (T1) and 14 days (T2).
For each patient with COVID-19 pneumonia enrolled, the following information was extracted from electronic medical records: age, sex, medical history, symptoms, laboratory findings, chest computed tomography (CT) or radiology findings.
As control group, healthy donors (HDs) with similar age and sex, absence of symptoms, negative swab for SARS-CoV-2 detection and negative serostatus for SARS-CoV-2 will be enrolled.
The MMP-2 and MMP-9 will be investigated by: (1) mRNA expression in PBMC (qPCR), (2) plasma activity (zymography) and by (3) plasma levels (ELISA). (1) Total cellular RNA will be isolated from PBMC using the QiagenRNeasy mini kit according to the manufacturer's instructions, and its quantity and quality determined spectrophotometrically, then will be stored at -80°C until use. Complementary DNA (cDNA) will be prepared by reverse transcription using random primers and will be amplified using specific primers for the human MMP-2 and MMP-9 sequence in a real time PCR system. Negative control (PCR mix without sample cDNA) and positive controls will be included. Amplification of human GAPDH, a relatively invariant internal reference RNA, will be performed in parallel, and MMP-2 and MMP-9 mRNA relative quantification will be determined using the ¿delta delta Ct¿ method. (2) Heparin plasma will be used to detect MMP-2 and MMP-9 activity by zymography as white bands of digestion on the blue background of the gel and will be identified by co-localization on the zymogram with human MMP-2 and MMP-9 standard (ALEXISBiochemicals). Quantitation of MMP-9 activity will be performed using computerized image analysis (ImageMaster1D, PharmaciaBiotech) through one-dimensional scanning densitometry (UltroscanXL, PharmaciaBiotech). MMP-2 and MMP-9 activity was expressed as optical density (OD)xmm2, representing the scanning area under the curves, which takes into account both brightness and width of the substrate lysis zone.
The TIMP-1 and TIMP-2 will be investigated by: (1) mRNA expression in PBMC (qPCR) as previously described using specific primers for the human TIMP-1 and TIMP-2 sequences (2) plasma levels (ELISA) using commercial kits which detects both the free and complexed form of the proteins.
Finally, the comparison between the expression of CD147 on monocytes of peripheral whole blood and MMPs and TIMPs results could be useful to understand completely the regulation process for MMP-2 and MMP-9 activity.