UNVEILING CARDIAC IL-33/ST2L PATHWAY DEREGULATION IN ANIMAL MODEL OF OBESITY AND CARDIOVASCULAR DISEASE

Obesity has become an increasing public health issue worldwide; it is estimated than over 65% of American adult population is overweight and 35% are actually obese. Criteria for obesity definition is traditionally linked to Body Mass Index (BMI). Although an important correlation exists between BMI and body fat deposit, the BMI criteria cannot actually predict body composition in any given individual due to variability determined by age, sex, race and ethnicity. Besides the weight of an individual, both fat localization and type represent more important estimators of cardiovascular disease. Importantly, the accumulation of visceral fat – more than subcutaneous fat – is linked to cardiovascular disease severity and mortality. Among ectopic fat depots, pericardial and epicardial fat accumulation are intriguingly important. Epicardial adipose tissue (EAT) represents a biological active tissue that secretes adipokines known to promote low-grade inflammation and diabetic vascular complications. The cause is due to the shared blood supply between EAT and myocardium, as indeed EAT provides the 50–70% of the energy used for contraction trough the release of free fatty acids (FFAs). It is intuitive that molecular/metabolic alteration of EAT will be reflected on its secretome and on cardiac metabolism. EAT thickness seems to be associated to ventricular myocardial mass and importantly to myocardial steatosis. A plethora of works highlighted the role of IL-33 and its receptor ST2 within cardiac dysfunction and inflammation associated with obesity. Once secreted in activated or damaged cells, IL-33 modulates the functions of various immune cells through ST2 binding, affecting the proliferation of T cells, macrophages, and innate lymphoid cells. Accordingly, IL-33/ST2 pathway is involved in lipid metabolic diseases and - following its role as immune sensor to infection and stress – it is linked to pro-fibrotic remodeling of myocardium and more in general in cardiovascular diseases. Our group demonstrated in coronary artery disease (CAD) a direct correlation between EAT thickness and IL-33/ST2 signalling imbalance further highlighting a role for EPAC protein involved in cAMP signal transduction In this PhD Project we aimed to further investigated the IL-33/ST2 effects on cardiac remodeling in obesity, focusing on the molecular pathway that links adipose/cardiac-derived IL-33 to development of fibrosis or hypertrophy. We choose Zucker Fatty rats (ZF) and Zucker Diabetics Fatty rats (ZDF) to overcome the problems linked to tissue availability and to the homogeneity of human EAT samples that need to be collected during cardiac surgery. Since genetic animal models do not fully recapitulate human pathology, we developed in vitro models to mimic adipose and myocardial relationship in vitro. Indeed, we evaluated the effects of visceral adipose tissue (VAT)-derived cellular medium in the modulation of adipose secretome and how they affect myocardial gene expression. Finally, we compared obtained results with those derived from Diet induced obesity (DIO) mice, a naturally occurring model of obesity that reliably resembles human disease. Following both molecular and proteomic analysis, we demonstrated a dysregulation of IL-33/ST2 signalling in both adipose and cardiac tissue, where they affected myocardial gene expression and determined a pro-fibrotic signature. In Zucker rats, pro-fibrotic effects were counteracted by ghrelin-induced IL-33 secretion, whose release influenced transcription factor expression (such as MEF2a), but also increased sST2 and not cardioprotective ST2L form. In this context we observed a reduction of EPAC signalling, that is promoted by VAT secretome and linked to ST2 isoforms balancing regulation. Similar results were obtained in ZDF rats, as both models did not develop evident alteration of cardiac architecture, although we described the increase of the pro-fibrotic signature. To avoid the genetic bias linked to leptin mutation and ghrelin up-regulation we repeated our analysis in DIO mice that fully recapitulates human obesity and presents gradual appearance of hyperglycaemia and progression of metabolic syndrome. Thanks to the analysis of cardiac proteome, we observed an enrichment in proteins and networks involved in extra-cellular matrix remodeling and ventricular function. Interestingly, IL-33 was considered among the possible up-stream regulators of this process. In conclusion, this PhD project demonstrated a dysregulation of IL-33/ST2 signalling in obesity, that directly correlate with Epac expression in ZF rats. The alteration of these pathways in adipose tissue could influence IL-33/ST2 expression and hypertoric/fibrotic response in cardiac tissue. Importantly the effect of IL-33 signalling could be modulated by hormones (such as Ghrelin) and other stimuli. Importantly, the final effect of IL-33 signalling activation is dependent on several factor, as cell types’ origin and balancing of ST2 isoforms. Noteworthy, extreme importance has to be ascribed to the animal models used in in vivo experiments, the chronic or acute condition as such as the time of IL-33 secretion. This way, it is reasonable that to define a unique protective role of IL-33 is over-simplistic and further studies are needed to confirm and unveil mechanisms of IL-33 as a gene expression regulator in cardiac obesity.