Pharmacological modulation of endothelial function during tissue remodeling in physiopathological conditions

Vascular endothelium plays a pivotal role in the maintenance of many biological functions, including angiogenesis, defined as the formation of new capillaries from pre-existing vasculature. The process is aimed at supplying nutrients and oxygenation to growing or healing tissues following injury and, in the context of wound healing, endothelial cells lining the inner surface of blood vessels, are continuously engaged in a crosstalk with other cell types, immunity cells or fibroblasts, to assure a correct progression in the process of regeneration. Disruption of endothelial functions, which can occur for several reasons, including systemic administration of certain dugs or hyperglycemia, leads to a variety of pathological cardiovascular consequences which are commonly featured by inflammation and a dysregulation in pro-angiogenic factors release. Despite many efforts have been made to address this issue, endothelial dysfunction continues to be one of the main causes of morbidity and mortality all over the world, for which new therapeutical approaches are needed to prevent the damage or revert cardiovascular disorders. The first aim of this thesis was to investigate the pro-angiogenic effect of erucin, a natural isothiocyanate with “smart” H2S-releasing properties, particularly abundant in the edible cruciferous plant Eruca sativa. In this study we characterized the pro-angiogenic effect of erucin on endothelial cells by using different functional in vitro assays aimed at evaluating cell migration and ability to organize in a capillary network. A special focus was paid to the molecular mechanisms involved in endothelial cell response to the compound by investigating the early activation of enzymes involved in angiogenesis, such as eNOS, ERK1/2, Akt. Secondly, we demonstrated erucin ability, alone or in combination with vascular endothelial growth factor (VEGF), to protect endothelial cells from high glucose-induced damage and recover impaired functional responses to physiological levels. In the second part of this topic we started to analyze the activity of the isothiocyanate in the context of wound healing by assessing in vitro its pro-migratory, pro- survival effect on dermal fibroblasts (NHDF) and keratinocytes (HaCaT). Lastly, a preliminary study using indirect co-cultures on NHDF and HUVEC, was carried out in order to investigate erucin ability to promote and sustain endothelial-stromal crosstalk, a fundamental step in wound healing. The second part of my thesis addressed the role of activated fibroblasts in the context of impaired wound healing characterized by eccessive inflammation, which underlies several pathologic conditions ranging from healing delay to fibrosis. The aim was to characterize the cellular and molecular events associated with the anti-inflammatory activity of photobiomodulation therapy on human dermal fibroblasts exposed to a mix of inflammatory cytokines followed by laser treatment. Results demonstrated laser ability to revert fibroblast inflammatory phenotype by reducing to basal levels pro-angiogenic factors, as VEGF, and inducible inflammatory key enzymatic pathways, as iNOS and COX-2/mPGES-1/PGE2, by retaining NF-kB transcription factor in a cytoplasmic localization. These molecular changes are accompanied by a shift in cell morphology attributed to a re-distribution of fundamental cytoskeletal proteins (Tubulin, F- actin, and α-SMA) to basal localization following laser treatments. In the third and final topic of this dissertation we discussed the importance of assuring endothelial safety during drug development. The cardiovascular system has proven to be particularly sensitive to a large variety of drugs, especially chemotherapeutic agents, which can promote or accelerate the onset of relevant cardiovascular diseases by impairing vascular integrity and tone. Recently, carbonic anhydrase IX (CA-IX), emerged as a promising new anticancer target for the treatment of solid hypoxic tumors and many efforts have been made to develop selective inhibitors for biomedical applications. In the last project presented, the safety profile of two CA-IX inhibitors, SLC-0111 and AA-06-05 on human endothelial cells was assessed.