Khaswaneh Ramada R, Abu-El-Rub Ejlal, Alzu'bi Ayman, Almahasneh Fatimah A, Almazari Rawan A, Ai-Jariri Heba F, Al-Zoubi Raed M
Department of Basic Medical Sciences, Faculty of Medicine, Yarmouk University, Irbid, 211-63, Jordan.
Surgical Research Section, Department of Surgery, Hamad Medical Corporation, Doha, Qatar.
Tissue Eng Regen Med. 2025 Jan;22(1):105-112. doi: 10.1007/s13770-024-00678-6. Epub 2024 Nov 1.
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.
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.
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.
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.
补体因子H和反应性溶解膜抑制剂（CD59）是补体激活的关键调节因子。间充质干细胞（MSC）分泌补体因子H并表达CD59以保护自身免受补体介导的损伤。研究发现，严重缺氧会降低MSC移植后的存活几率；然而，关于严重缺氧对调节补体系统活性的影响及其对MSC存活的作用知之甚少。我们的研究旨在探讨严重缺氧对调节MSC中补体级联反应的影响。
使用400μM氯化钴（CoCl2）在严重缺氧条件下培养人脂肪组织来源的MSC（hAD-MSC）48小时。使用蛋白质印迹法评估存活标志物磷酸肌醇3激酶（PI3K）和促凋亡标志物半胱天冬酶-3的蛋白表达。使用酶联免疫吸附测定（ELISA）、蛋白质印迹法和免疫细胞化学分析补体系统相关因子补体因子H、CD59、C3b、iC3b、C5b、C9和补体膜攻击复合物（MAC）的水平。
我们的结果首次表明，严重缺氧可显著损害MSC中补体因子H的分泌和CD59的表达。这与MAC复合物上调以及细胞裂解和凋亡水平增加有关，表现为PI3K下调以及膜联蛋白v和半胱天冬酶-3上调。
缺氧的MSC中补体因子H和CD59的缺失可通过激活MAC复合物引发其裂解和凋亡。维持MSC中补体因子H和CD59的水平对于提高其在缺氧条件下的保留率和延长其存活时间具有重要的临床意义。
