UNRAVELING THE MECHANISMS INVOLVED IN ENDOTHELIAL RESPONSE TO MICROGRAVITY

Exposure to real or simulated microgravity is sensed as a stress by mammalian cells, which activate a complex adaptive response. Culture of human endothelial cells for 10 days in real microgravity onboard the ISS resulted in the modulation of more than a thousand genes, some of which involved in stress response. We cultured human endothelial cells for 4 and 10 days in the Rotating Wall Vessel, a NASA developed surrogate system for bench-top microgravity research on Earth. We highlight the crucial role of the early increase of HSP70, since its silencing markedly impairs cell survival. Once HSP70 upregulation fades away after 4 days of simulated microgravity, a complex and articulated increase of various stress proteins - SIRT2, PON2, SOD2, p21, HSP27, P-HSP27 all endowed with cytoprotective properties – occurs and counterbalances the upregulation of the pro-oxidant TXNIP. Interestingly, TXNIP was the most overexpressed transcript in endothelial cells after space flight. We conclude that HSP70 upregulation sustains the initial adaptive response of endothelial cells to mechanical unloading and drives them towards the acquisition of a novel phenotype that maintains cell viability and function through the involvement of different stress proteins. We also demonstrated that mitophagy contributes to endothelial adaptation to gravitational unloading. After 4 and 10 days of exposure to simulated microgravity in the Rotating Wall Vessel, the amount of BNIP3, a marker of mitophagy, was increased and, in parallel, mitochondrial content and oxygen consumption were reduced, suggesting that HUVEC acquire a thrifty phenotype to meet the novel metabolic challenges generated by gravitational unloading. Moreover, we suggested that microgravity induced-disorganization of the actin cytoskeleton triggers stress adaptation and mitophagy, thus creating a connection between cytoskeletal dynamics and mitochondrial content upon gravitational unloading. We also found that Mg homeostasis was modulated in microgravity, since a reduction of total intracellular magnesium and modulation of its transporters was found in EC exposed to simulated microgravity. We also investigated a new 3D cell culture method, a microfluidic system where EC are cultured in 3D and in presence of fluid laminar flow, in perspective of using these systems for experiments in microgravity.