Expression of tardigrade extremotolerance-associated proteins improves recovery but not acute stress response of human microvascular endothelial cells

Vascular endothelial cells (VECs) play a critical role in tissue and organ homeostasis, wound healing, and immune function. VECs are commonly utilized for in vitro cell culture and in vivo tissue engineering applications which experience intrinsic and extrinsic stressors. Severe stress can overwhelm VECs adaptive stress response mechanisms inducing damage and loss of cell viability. Improving VEC stress tolerance is therefore of increasing interest. In this study we assess the transgenic expression of tardigrade extremotolerance-associated proteins (TEAPs), specifically CAHS3, MAHS, LEAM, and Dsup for improved VEC tolerance to heat shock and hyperosmotic stress. In vitro studies of all TEAP-expressing VECs demonstrate up to 45% decrease in population metabolic activity in response to 24 h incubation in hyperosmotic media solution without recovery. Interestingly, acute hyperosmotic stress treatments followed by a 24 h recovery period showed significant increase in population metabolic activity for CAHS3 (186%), Dsup (202%), and LEAM (223%), while MAHS (161%) showed non-significant increases. Similarly, heat shock treatments followed by a recovery period showed significant increases for CAHS3 (198%), Dsup (182%), MAHS (195%), and LEAM (156%). Our results demonstrate that TEAP expression in VECs confer context dependent protective effects with respect to heat shock and hyperosmotic stress, motivating further exploration of TEAPs stress tolerance function and mechanism of action in mammalian cells.