Focusing on metabolism, oxidative stress and inflammation in endothelial cells: relevance for vascular diseases.

The endothelium consists of a monolayer of polygonal cells, endothelial cells (ECs), lining the internal surface of the vessels, and regulating vascular homeostasis. Thus, endothelial functions may serve as "cardiovascular risk barometer". On the other hand, endothelial dysfunction may occur upon a variety of insults. Endothelial dysfunction, regarded as an impairment of physiological functions, results in the acquisition of a pro-thrombotic, pro-inflammatory and pro-angiogenic phenotype which, in turn, predisposes to the onset of cardiovascular diseases as atherosclerosis, stroke and retinopathies. Many factors contribute to the onset of cardiovascular diseases including aging and inflammation. The latter is linked both to a process known as endothelial-to-mesenchymal transition (EndMT) and pathological neovascularization. In particular, EndMT is a process similar to the well-known epithelial-to-mesenchymal transition (EMT) by which ECs convert into mesenchymal ones. EndMT orchestrates some physiological processes, such as differentiation, but it can also contribute to some inflammatory-related diseases such as atherosclerosis. Neoangiogenesis, the formation of new vessels from pre-existing ones, is an intricate process finely orchestrated by pro- and anti-angiogenic factors that occurs in many physiopathological processes. Physiologically, this process is strictly and transiently controlled; however, there are pathological conditions in which this process is uncontrolled and results in an irregular growth of the aberrant vessels. In this case, angiogenesis is associated with inflammatory-related diseases, such as retinopathies. During my Ph.D. fellowship, these features of the endothelium have been studied by investigating the role of multiple different players. In fact, this thesis is divided into three topics that mainly focus on endothelium: the objective of the first one is represented by aldehyde dehydrogenase 2 (ALDH2), an enzyme responsible for the metabolism of endogenous and exogenous aldehydes, and its role in mitochondrial bioenergetic functions associated with the acquisition of premature senescent phenotype in ECs. To this end, the umbilical vein ECs (HUVEC) challenged with the ALDH2-targeting siRNAs or ALDH2 specific inhibitor were chosen as a study model. In this paper we have highlighted that inhibition of ALDH2 activity reduces mitochondrial respiration and reserve capacity and increases oxidative stress in endothelial cells, promoting the acquisition of a senescent phenotype. Another field of study was to document the effects of a metabolite of a polyphenol (hydroxytyrosol, HT) found in extra virgin olive oil called hydroxytyrosol-3-O sulphate (HT-3Os) in EndMT process in inflamed ECs. To this end, we used HUVEC and human retinal EC (HREC) challenged with Interleukin-1β (IL-1β), an inflammatory stimulus, mimicking an in vitro model of endothelial dysfunction. The results demonstrate that HT-3Os blunts pathological EndMT in inflamed ECs maintaining high let-7 miRNA expression and preventing activation of TGF-β signalling. Furthermore, in another study we investigated the role of HT in promoting the effects of cetuximab against colon cancer cells while preserving healthy intestinal cells. In the latter topic we investigated the role of Bradykinin (BK) and its Bradykinin B2 receptor (B2R)-mediated signalling in the process of retinal angiogenesis, as a process implicated in the most common retinal diseases. Moreover, we also went into detail with the study of the possible crosstalk between BK/B2R signaling and fibroblast growth factor-2/fibroblast growth factor receptor 1 (FGF-2/FGFR-1) pathway. In conclusion, the results of the first topic suggest that preserving ALDH2 may be a strategy that slows down the onset of senescent cells. In the second topic, the results provide biological evidences both on the protective role of HT-3Os against inflammatory insults in endothelium and that of its precursor in promoting the effects of cetuximab against colon cancer cells. Lastly, the results of the third topic suggest that targeting B2R signaling may be an effective strategy for treating retinopathies. Collectively, these results strengthen the importance of endothelial functions in the maintenance of vascular wellness and functioning, being endothelial dysfunction the therapeutic target to finely control redox state and inflammatory conditions by nutraceuticals or synthetic compounds.