Hypoxic Stress Induces Complement-Mediated Lysis of Mesenchymal Stem Cells by Downregulating Factor H and CD59

Abstract

Background: Factor H and membrane inhibitor of reactive lysis (CD59) are key regulators of complement activation. Mesenchymal stem cells (MSCs) secrete Factor H and express CD59 to protect themselves from complement-mediated damage. Severe hypoxia found to decrease the survival chances of MSCs after transplantation; however, little is known about the impact of severe hypoxia on modulating the complement system activity and its effect on MSCs survival. Our study seeks to explore the effect of severe hypoxia on modulating the complement cascade in MSCs.

Methods: Human adipose tissue-derived MSCs (hAD-MSCs) were cultured under severe hypoxia using 400 μM Cobalt Chloride (CoCl2) for 48 h. The protein expressions of survival marker; Phosphoinositide 3-kinases (PI3K), and pro-apoptotic marker; Caspase-3 were assessed using western blotting. The level of complement system related factors; Factor H, CD59, C3b, iC3b, C5b, C9, and the complement membrane attack complex (MAC) were analyzed using Elisa assays, western blotting, and immunocytochemistry.

Results: Our results showed for the first time that severe hypoxia can significantly impair Factor H secretion and CD59 expression in MSCs. This has been associated with upregulation of MAC complex and increased level of cell lysis and apoptosis marked by downregulation of PI3K and upregulation of Annexin v and Caspase-3.

Conclusion: The loss of Factor H and CD59 in hypoxic MSCs can initiate their lysis and apoptosis mediated by activating MAC complex. Preserving the level of Factor H and CD59 in MSCs has significant clinical implication to increase their retention rate in hypoxic conditions and prolong their survival.