Synergism of MSC-secreted HGF and VEGF in stabilising endothelial barrier function upon lipopolysaccharide stimulation via the Rac1 pathway.
Synergism of MSC-secreted HGF and VEGF in stabilising endothelial barrier function upon lipopolysaccharide stimulation via the Rac1 pathway.
复制标题
MSC 分泌的 HGF 和 VEGF 通过 Rac1 途径在脂多糖刺激下协同稳定内皮屏障功能
DOI：
10.1186/s13287-015-0257-0
复制
发表时间：
2015-12-16
影响因子：
7.5
通讯作者：
Qiu HB
中科院分区：
文献类型：
--
作者：
Yang Y;Chen QH;Liu AR;Xu XP;Han JB;Qiu HB
背景间充质干细胞（MSCs）通过旁分泌肝细胞生长因子（HGF）稳定急性肺损伤中的内皮屏障功能。由间充质干细胞分泌的血管内皮生长因子（VEGF）是内皮通透性的另一个关键调节因子；然而，其在调节渗透率中的作用仍存在争议。此外，MSCs分泌的HGF和VEGF之间是否发生相互作用尚不完全清楚。方法建立人肺微血管内皮细胞（HPMECs）与间充质干细胞条件培养基（CM）共培养模型，经缺氧培养24 h。VEGF和HGF在MSC-CM中的存在分别被抗VEGF和抗HGF抗体中和。为了确定mscs分泌HGF和VEGF的作用和机制，我们采用重组人源化HGF和重组人源化VEGF与hpmes共培养。此外，我们使用RhoA抑制剂C3转移酶和Rac1抑制剂NSC23766来抑制与MSC-CM相同剂量的MSC-CM或VEGF/HGF处理的hpmes中RhoA和Rac1的活性。然后检测内皮细胞旁和细胞间通透性。western blot检测VE-cadherin、occludin和caveolin-1蛋白在hpmec中的表达。免疫荧光法检测粘附体连接蛋白，包括F-actin和VE-cadherin。结果smsc - cm处理可显著降低脂多糖诱导的内皮细胞旁和细胞间通透性，而HGF抗体预处理或VEGF和HGF抗体预处理均可显著抑制这种通透性。此外，MSC-CM处理增加了内皮细胞间粘附连接蛋白VE-cadherin和occludin的表达，降低了caveolin-1蛋白的表达。MSC-CM处理可减少内皮细胞凋亡，诱导内皮细胞增殖；然而，HGF抗体预处理或HGF和VEGF抗体预处理均可抑制MSC-CM的治疗效果。此外，当用Rac1抑制剂NSC23766预处理hpmes时，MSC-CM和VEGF/HGF降低内皮细胞旁和跨细胞通透性的作用减弱。结论MSCs分泌hgf可保护内皮屏障功能；然而，MSCs分泌的VEGF可能与HGF协同稳定内皮细胞屏障功能。Rac1是msc分泌的VEGF和HGF调节内皮细胞通透性的途径。
BackgroundMesenchymal stem cells (MSCs) stabilise endothelial barrier function in acute lung injury via paracrine hepatocyte growth factor (HGF). Vascular endothelial growth factor (VEGF), which is secreted by MSCs, is another key regulator of endothelial permeability; however, its role in adjusting permeability remains controversial. In addition, whether an interaction occurs between HGF and VEGF, which are secreted by MSCs, is not completely understood.MethodsWe introduced a co-cultured model of human pulmonary microvascular endothelial cells (HPMECs) and MSC conditioned medium (CM) collected from MSCs after 24 h of hypoxic culture. The presence of VEGF and HGF in the MSC-CM was neutralised by anti-VEGF and anti-HGF antibodies, respectively. To determine the roles and mechanisms of MSC-secreted HGF and VEGF, we employed recombinant humanised HGF and recombinant humanised VEGF to co-culture with HPMECs. Additionally, we employed the RhoA inhibitor C3 transferase and the Rac1 inhibitor NSC23766 to inhibit the activities of RhoA and Rac1 in HPMECs treated with MSC-CM or VEGF/HGF with the same dosage as in the MSC-CM. Then, endothelial paracellular and transcellular permeability was detected. VE-cadherin, occludin and caveolin-1 protein expression in HPMECs was measured by western blot. Adherens junction proteins, including F-actin and VE-cadherin, were detected by immunofluorescence.ResultsMSC-CM treatment significantly decreased lipopolysaccharide-induced endothelial paracellular and transcellular permeability, which was significantly inhibited by pretreatment with HGF antibody or with both VEGF and HGF antibodies. Furthermore, MSC-CM treatment increased the expression of the endothelial intercellular adherence junction proteins VE-cadherin and occludin and decreased the expression of caveolin-1 protein. MSC-CM treatment also decreased endothelial apoptosis and induced endothelial cell proliferation; however, the effects of MSC-CM treatment were inhibited by pretreatment with HGF antibody or with both HGF and VEGF antibodies. Additionally, the effects of MSC-CM and VEGF/HGF on reducing endothelial paracellular and transcellular permeability were weakened when HPMECs were pretreated with the Rac1 inhibitor NSC23766.ConclusionHGF secreted by MSCs protects the endothelial barrier function; however, VEGF secreted by MSCs may synergize with HGF to stabilise endothelial cell barrier function. Rac1 is the pathway by which MSC-secreted VEGF and HGF regulate endothelial permeability.