MATRIX METALLOPROTEINASES IN HUMAN HEALTH AND DISEASE: A MULTISYSTEM APPROACH TO UNDERSTANDING THEIR MOLECULAR FUNCTION AND CLINICAL RELEVANCE IN REPRODUCTION, MULTIPLE SCLEROSIS, AND FIBROSIS

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases involved in the remodelling of the extracellular matrix (ECM). Among MMPs, gelatinase A (MMP-2) and gelatinase B (MMP-9) are important contributors to physiological and pathological processes, such as wound healing and reproduction. On the other hand, MMP dysregulation may be involved in numerous diseases, including cancer, neuroinflammation and fibrosis. Throughout this PhD thesis, the pleiotropic and multifaceted role of MMPs was investigated using a multisystem approach in three diverse pathophysiological conditions: human reproduction, multiple sclerosis (MS), and Dupuytren's disease (DD).In the reproductive context, MMPs are implicated in ECM remodelling during follicle growth, ovulation, corpus luteum formation, angiogenesis and embryo implantation. The composition and function of follicular fluid (FF) were analysed in n=126 women undergoing assisted reproductive techniques (ART). The study characterised the gelatinolytic activity of MMP-2 and MMP-9, both in the FF and FF-derived extracellular vesicles (FF-EVs) isolated by ultracentrifugation. Zymographic analysis showed a higher activity of proMMP-2 and less variability among patients, while proMMP-9 showed a lower activity and was more variable, suggesting distinct regulatory mechanisms. In FF-EVs, the active form of MMP-2 was also detected in 70% of the analysed samples, indicating a specific compartmentalisation of active enzyme. A significant inverse correlation emerged between patient age and MMP activity levels. Furthermore, the positive correlation between MMP-9 and C-reactive protein (CRP) suggested a link between gelatinase and the inflammation status of the patients. In vitro functional studies showed that FF from young women stimulates cell migration and reduces E-cadherin expression, indicating a possible effect on endometrial receptivity mediated by mechanisms similar to the epithelial-mesenchymal transition (EMT). These results propose MMPs as potential biomarkers predictive of oocyte quality and ART success.In the neurological context, a multisystemic analysis was conducted on biological fluids from multiple sclerosis patients. MS is a chronic neuroinflammatory disease with blood-brain barrier (BBB) impairment, demyelination and neurodegeneration. Cerebrospinal fluid (CSF), serum, and plasma samples were obtained from n=62 patients with MS and compared with n=31 patients with other neurological diseases (OND), further classified as inflammatory and non-inflammatory diseases. High-resolution proteomic CSF profiling (LC-MS/MS) identified n=892 proteins, with upregulated and downregulated proteins involved in different aspects of MS aetiology and progression. A special focus was deserved for the concomitant IGFBP-2 upregulation and IGF-2 downregulation in MS patients, also because IGFBP-2 was the leading discriminating marker by machine learning classification analysis. In parallel, zymographic analysis was conducted on MS biological fluids. In CSFs, MMP-2 and MMP-9 were mainly detected as proenzymatic forms, with higher expression of proMMP-2. Gelatinase activity was surprisingly lower overall in MS patients than in OND controls. Interestingly, there was a prominent decrease in active MMP-2 during relapse vs. remission. Correlation analysis also revealed a positive association between proMMP-2 and CRP cerebrospinal fluid levels, thereby connecting matrix remodelling to subclinical inflammatory events. To analyse the peripheral compartments, gelatinolytic activity was also examined in the plasma and serum of MS patients compared to controls. Serum zymography detected higher expression of proMMP-9, as well as its dimer and proMMP-9/TIMP-1 complex. Generally, also in serum the gelatinase activity was also considerably lower in MS patients. Serum CRP levels were also considered, and correlation analysis demonstrated a negative correlation with proMMP-2, suggesting a differential association between gelatinase activity and systemic inflammation across central and peripheral compartments. These findings highlight the dual role of MMPs in MS and remark on their potential as biomarkers of disease stage and therapeutic response. The elevated levels of proMMP-9 in serum compared with plasma are most likely due to leukocyte degranulation during clotting and highlight the need for additional work to clarify the clinical relevance of matrix-specific MMP activity in peripheral compartments.Finally, in Dupuytren’s disease, a fibrotic disorder of the palmar fascia, MMP-14 (MT1-MMP) is highly involved in extracellular matrix remodelling and fibroblast contractility. Primary fibroblasts from genotyped patients were analysed to assess the impact of a functional MMP14 SNP. Conversely to the expectations, homozygous AA cells showed higher proMMP-2 activity, with minimal active MMP-2 in other genotypes. Anti-MMP-14 antibody treatment unexpectedly increased proMMP-2 activity across all genotypes, without affecting total MMP-2 levels. However, MMP-14 protein expression was reduced after the treatment, confirming antibody efficacy. These results suggest a genotype-dependent modulation of gelatinase activity and a functional dissociation between expression and activation. Future proteomic analysis of fibroblast media will lead to the identification of downstream targets involved in fibrosis, in order to evaluate the therapeutic potential of MMP-14 inhibition.In conclusion, this PhD thesis illustrates MMPs as central molecular actors of tissue remodelling in numerous biological settings. Their expression, activation, and extracellular vesicle-mediated delivery are strictly controlled in response to physiological demands and pathological insults. Through the synergy of biochemical, molecular, proteomic, and functional techniques, this thesis provides an integrative view of MMPs as candidate biomarkers and therapeutic targets in human reproduction, neuroinflammation, and fibrotic disorders.