The interface between cardiovascular disease and inflammation: hydrogen sulfide and annexin A1 pathways as novel pharmacological targets

Cardiovascular diseases are a heterogeneous group of disorders of the heart and the vasculature, in which different factors are responsible for their onset. In particular, among the various factors contributing the development of vascular disorders there are hyperlipidaemia, hyperglycaemia, cigarette smoke, obesity and hyperhomocysteinemia. Many of the pathologies that are part of vascular disorders, as hypertension, diabetes, atherosclerosis and heart failure, are characterized by two main conditions: oxidative stress and excessive prolonged inflammatory response. The interception of these dual aspects delineates the concept of inflammation-based vascular diseases (IBVD). In this context, it is of note to underline the decline of the nitric oxide (NO) and hydrogen sulfide (H2S) levels, molecules that share vasorelaxant and anti-inflammatory properties. Deficiency of these two gaseous mediators, normally produced within the body, targets vascular endothelium resulting into one of the classical IBVD example. In this project, we aimed to dissect the molecular mechanisms underlying the vascular impairment observed in hyperglycaemia and the cross talk between NO and H2S in IBVD, with respect to vascular inflammatory response. Indeed, we used in vitro, ex vivo and in vivo models to perform multiple approaches tackling different sides of disease conditions. We used bovine aortic endothelial cells (BAEC) and aorta rings harvested from mice to study the impact of hyperglycaemia and the effect of a novel mitochondrial-specific H2S donor (AP123). In addition, we also performed specific experiments on human macrophages aiming to evaluate the effect of H2S pathway in the physiological response by specialized pro-resolving molecules (SPMs) in inflammatory conditions. First, we found that the decline NO levels associated to hyperglycaemia is paralleled by similar outcomes in H2S levels. This impairment can be corrected in endothelial cells when AP123 was used as H2S donor and the underlying mechanism is based on a transcriptional effect on eNOS, involving the activation of cAMP response binding protein (CREB) by PI3K. Furthermore, we found that this mechanism is also operative in the whole vascular tissue as demonstrated by ex vivo experiments. In the context of experimental inflammation, we found that CSE pathway is downregulated in endothelial cells following administration of TNF-α. Interestingly, the beneficial role of H2S has been confirmed by administration of AP123. Therefore, the modulation of inflammatory response by H2S has been also shown to be part of the mechanism of action of “unrelated” anti-inflammatory drug such as clodronate. To evaluate whether H2S activity could influence changes in the lipid mediators’ activity, we evaluated the effect of slow-releasing H2S donor, AP123, and a specific CSE inhibitor, PAG, on the concentrations of SPMs in M1 macrophages, stimulated with LPS, at two different time points. We found that the majority of LM are differentially regulated between two treatments, involving pro- and anti-inflammatory mediators. However, the administration of 1nM AP123, at 45 min, mainly triggered the involvement of anti-inflammatory lipid mediators, while H2S donor (100nM and 1μM, 45min) seems to positively modulate the levels of a particular pro-resolving mediator, referred to as RvD2n3-DPA. Overall, our studies suggest that H2S is a crucial vasculoprotective mediator in IBVD at vascular level and H2S-releasing molecules may open new perspectives in the therapy of H2S deficiency conditions with a pleiotropic mechanism not only related to a simple “replacement” effect.