OSTEOPROTEGERIN AS A NEW POSSIBLE PLAYER IN MITRAL VALVE PROLAPSE WITH SEVERE REGURGITATION: FROM IN VITRO TO HUMAN STUDIES

Background: Mitral valve prolapse (MVP) is a common valve pathology and affects more than 176 million people worldwide. Echocardiography is the diagnostic gold standard and surgical intervention represents the only successful treatment when the prolapse causes severe regurgitation and symptoms occur. To date, there are no reliable biomarkers available for the identification of this pathology. MVP is a disorder characterized by extracellular matrix (ECM) remodelling but molecular and cellular mechanisms are not fully understood. The disruption of ECM tight regulation and the constitutive activation of valve interstitial cells (VIC) have been linked to MVP, as well as valve endothelial cell (VEC) mesenchymal transition (EndMT). Osteoprotegerin (OPG) is involved in a myriad of physiological and pathological processes. In the vascular environment, both endothelial and smooth muscle cells constitutively secrete OPG. In order to gain further insights into the molecular mechanisms involved in MVP progression, we investigated the role of OPG during EndMT and we generated a predictive model able to identify MVP patients with high accuracy. Methods: Human VECs and VICs were isolated from posterior mitral valve leaflets of patients who underwent mitral valve repair. β-glycerolphosphate and ascorbic acid (βGAA) treatments were used to promote EndMT. Immunofluorescence, Western blot, quantitative and digital PCR were performed to evaluate VEC and VIC phenotypes. We assessed the oxidative stress status measuring the oxidized (GSSG) and the reduced (GSH) form of glutathione by liquid chromatography-tandem mass spectrometry method. OPG plasma levels were measured by enzyme-linked immunosorbent assay. These biochemical variables combined with clinical and demographic parameters were considered to generate a logistic regression model able to identify MVP patients with high sensitivity and specificity. Results: OPG was significantly elevated (309±49.8%) in prolapsed tissues when compared to healthy tissues (p<0.05). We carried out an in vitro system able to force EndMT in isolated VECs. During EndMT, VECs showed a significant up-regulation of OPG RNA levels (4.1±1.3 fold vs. untreated cells; p<0.05). In addition, VECs secreted more OPG in comparison to untreated cells (2369±564.7 pg/µg and 923.5±261.1 pg/µg of total protein, respectively; p<0.05). Moreover, OPG itself triggered autocrine effects (RNA levels): collagen I (Col1A1, +4.0±0.6; p<0.001); collagen III (Col3A1, +3.0±0.6; p<0.01); bone morphogenetic protein 4 (BMP4, +2.4±0.7; p<0.05) and versican (VCAN, +3.8±0.2; p<0.001 ). Furthermore, OPG treatments accelerated VEC migration (area closed: 43.3±3.9% treated cells vs. 21.8±2.6% untreated cells; p<0.001). OPG also promoted VICs proliferation (22.4% increment; p<0.05) and significantly up-regulated VIC RNA levels: Col1A1 (+2.24±0.29; p<0.001); Col3A1 (+1.58±0.29; p<0.05); BMP4 (+1.6±0.1; p<0.05); biglycan (BGN, +1.9±0.3; p <0.001); VCAN (+1.5±0.2; p <0.05); metalloproteinase 2 (MMP2, +1.6±0.1; p <0.01); smooth muscle actin (SMA, +1.6±0.2); and OPG (+1.5±0.2; p<0.05).Regarding plasma analyses, both oxidative stress (GSSG/GSH ratio) and OPG levels were significantly higher in patients compared to control subjects (0.116±0.007 vs. 0.053±0.013 and 1748±100.2 vs. 1109±45.3 pg/mL, respectively; p<0.001). Finally, the combination of these two variables with body-mass index allowed us to generate a regression model able to correctly identify 95% of patients and 90% of control subjects. Conclusions: Our results support EndMT as a possible mechanism involved in the pathogenesis of mitral valve degeneration. In addition, to the best of our knowledge, this is the first study to show a strong association between OPG, oxidative stress status and body-mass index in patients affected by MVP with severe regurgitation. Finally, since it is quite hard to believe that one single protein could discriminate two populations with high specificity and sensitivity, we believe that this approach could improve the identification of several signatures not only in mitral valve disease. As the mechanisms explaining these correlations are still unclear, further molecular studies along with clinical validations will be necessary to confirm our findings.