Endothelial Aldehyde Dehydrogenase 2 as a Target to Maintain Vascular Wellness and Function in Ageing

Endothelial cells are the main determinants of vascular function, since their dysfunction in response to a series of cardiovascular risk factors is responsible for disease progression and further consequences. Endothelial dysfunction, if not resolved, further aggravates the oxidative status and vessel wall inflammation, thus igniting a vicious cycle. We have furthermore to consider the physiological manifestation of vascular dysfunction and chronic low-grade inflammation during ageing, also known as inflammageing. Based on these considerations, knowledge of the molecular mechanism(s) responsible for endothelial loss-of-function can be pivotal to identify novel targets of intervention with the aim of maintaining endothelial wellness and vessel trophism and function. In this review we have examined the role of the detoxifying enzyme aldehyde dehydrogenase 2 (ALDH2) in the maintenance of endothelial function. Its impairment indeed is associated with oxidative stress and ageing, and in the development of atherosclerosis and neurodegenerative diseases. Strategies to improve its expression and activity may be beneficial in these largely diffused disorders

Involvement of bradykinin B2 receptor in pathological vascularization in oxygen-induced retinopathy in mice and rabbit cornea

The identification of components of the kallikrein-kinin system in the vitreous from patients with microvascular retinal diseases suggests that bradykinin (BK) signaling may contribute to pathogenesis of retinal vascular complications. BK receptor 2 (B2R) signaling has been implicated in both pro-inflammatory and pro-angiogenic effects promoted by BK. Here, we investigated the role of BK/B2R signaling in the retinal neovascularization in the oxygen-induced retinopathy (OIR) model. Blockade of B2R signaling by the antagonist fasitibant delayed retinal vascularization in mouse pups, indicating that the retinal endothelium is a target of the BK/B2R system. In the rabbit cornea assay, a model of pathological neoangiogenesis, the B2 agonist kallidin induced vessel sprouting and promoted cornea opacity, a sign of edema and tissue inflammation. In agreement with these results, in the OIR model, a blockade of B2R signaling significantly reduced retinal neovascularization, as determined by the area of retinal tufts, and, in the retinal vessel, it also reduced vascular endothelial growth factor and fibroblast growth factor-2 expression. All together, these findings show that B2R blockade reduces retinal neovascularization and inhibits the expression of proangiogenic and pro-inflammatory cytokines, suggesting that targeting B2R signaling may be an effective strategy for treating ischemic retinopathy

Inhibition of cell cycle progression by the hydroxytyrosol-cetuximab combination yields enhanced chemotherapeutic efficacy in colon cancer cells

Hydroxytyrosol (HT), a polyphenol of olive oil, downregulates epidermal growth factor (EGFR) expression and inhibits cell proliferation in colon cancer (CC) cells, with mechanisms similar to that activated by the EGFR inhibitor, cetuximab. Here, we studied whether HT treatment would enhance the cetuximab inhibitory effects on cell growth in CC cells. HT-cetuximab combination showed greater efficacy in reducing cell growth in HT- 29 and WiDr cells at concentrations 10 times lower than when used as single agents. This reduction was clearly linked to cell cycle blockade, occurring at G2/M phase. The cell cycle arrest in response to combination treatment is related to cyclins B, D1, and E, and cyclin-dependent kinase (CDK) 2, CDK4, and CDK6 down-regulation, and to the concomitant over-expression of CDK inhibitors p21 and p27. HT and cetuximab stimulated a caspase-independent cell death cascade, promotedtranslocation of apoptosis-inducing factor (AIF) from mitochondria to nucleus and activated the autophagy process. Notably, normal colon cells and keratinocytes were less susceptible to comboinduced cell death and EGFR downregulation. These results suggest a potential role of diet, containing olive oil, during cetuximab chemotherapy of colon tumor. HT may be a competent therapeutic agent in CC enhancing the effects of EGFR inhibitors

Bradykinin B2 receptor contributes to inflammatory responses in human endothelial cells by the transactivation of the fibroblast growth factor receptor FGFR-1

Elevated levels of bradykinin (BK) and fibroblast growth factor-2 (FGF-2) have been implicated in the pathogenesis of inflammatory and angiogenic disorders. In angiogenesis, both stimuli induce a pro-inflammatory signature in endothelial cells, activating an autocrine/paracrine amplification loop that sustains the neovascularization process. Here we investigated the contribution of the FGF-2 pathway in the BK-mediated human endothelial cell permeability and migration, and the role of the B2 receptor (B2R) of BK in this cross-talk. BK (1 µM) upregulated the FGF-2 expression and promoted the FGF-2 signaling, both in human umbilical vein endothelial cells (HUVEC) and in retinal capillary endothelial cells (HREC) by the activation of Fibroblast growth factor receptor-1 (FGFR-1) and its downstream signaling (fibroblast growth factor receptor substrate: FRSα, extracellular signal–regulated kinases1/2: ERK1/2, and signal transducer and activator of transcription 3: STAT3 phosphorylation). FGFR-1 phosphorylation triggered by BK was c-Src mediated and independent from FGF-2 upregulation. Either HUVEC and HREC exposed to BK showed increased permeability, disassembly of adherens and tight-junction, and increased cell migration. B2R blockade by the selective antagonist, fasitibant, significantly inhibited FGF-2/FGFR-1 signaling, and in turn, BK-mediated endothelial cell permeability and migration. Similarly, the FGFR-1 inhibitor, SU5402, and the knock-down of the receptor prevented the BK/B2R inflammatory response in endothelial cells. In conclusion, this work demonstrates the existence of a BK/B2R/FGFR-1/FGF-2 axis in endothelial cells that might be implicated in propagation of angiogenic/inflammatory responses. A B2R blockade, by abolishing the initial BK stimulus, strongly attenuated FGFR-1-driven cell permeability and migration

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

Targeting endothelial-to-mesenchymal transition: the protective role of hydroxytyrosol sulfate metabolite

Purpose: Endothelial-to-mesenchymal transition (EndMT) plays an important role in pathogenesis of a number of inflammatory diseases. Hydroxytyrosol (HT) and, particularly, its major plasma metabolite HT-3O sulfate (HT-3Os) are known olive oil antioxidant and anti-inflammatory polyphenols which exert benefits against vascular diseases by improving endothelial function. However, to date the HT-3Os role in EndMT is not well known. Methods: To investigate the HT-3Os effects on EndMT in the inflamed endothelium, we used an in vitro model of endothelial dysfunction, challenging endothelial cells (EC), human umbilical EC (HUVEC) and human retinal EC (HREC) with Interleukin-1β (IL-1β), an inflammatory agent. HREC were used as a specific model to investigate HT-3Os effects on vascular retinal diseases. Results: We found that IL-1β treatment-induced EndMT phenotype in both cell models, also changing cell morphology. HT-3Os protected EC against IL-1β effects, recovering cell morphology and phenotype. Mechanistically, HT-3Os targeting fibroblast growth factor receptor 1 FGFR1 expression and let-7 miRNA, controlled transforming growth factor beta (TGF-β) signalling in EC, downregulating transcription factors expression (SNAI1 and ZEB2) and gene expression of late EndMT markers (FN1, VIM, NOTCH3, CNN1, MMP2 and MMP9). Conclusion: These results demonstrate that HT-3Os blunts pathological EndMT in inflamed EC, maintaining high let-7 miRNA expression and preventing activation of TGF-β signalling