Table_1_Cirrhotic Endothelial Progenitor Cells Enhance Liver Angiogenesis and Fibrosis and Aggravate Portal Hypertension in Bile Duct-Ligated Cirrhotic Rats.doc

BackgroundCirculating cirrhotic endothelial progenitor cells (EPC) interact with both liver sinusoidal endothelial cells (LSEC) and hepatic stellate cells (HSC) and promote angiogenesis in vitro. This study evaluated the effect of cirrhotic and control EPCs on hepatic angiogenesis, microcirculation, and fibrosis in vivo in rat models of cirrhosis.MethodologyAnimal models of cirrhosis were prepared by bile duct ligation (BDL). Circulating EPCs isolated from healthy human and cirrhotic blood were characterized by flow cytometry, cultured and administered through the tail vein in BDL rats after 2 weeks of ligation. The cells were given thrice a week for 2 weeks. The untreated group of BDL rats received only saline. Fibrosis was evaluated by Masson’s trichrome staining. Dedifferentiated LSECs were identified by the expression of CD31, and activated HSCs were marked as alpha-SMA-positive cells and were studied by immunohistochemistry and western blotting in saline-, healthy EPC-, and cirrhotic EPC-treated rats. In vivo, hepatic and systemic hemodynamic parameters were evaluated. Liver functions were evaluated.ResultsIn comparison to controls, BDL rats revealed an increase of fibrosis and angiogenesis. Among the treated rats, cirrhotic EPC-treated rats had increased fibrosis grade as compared to healthy EPC-treated and saline-treated rats. There was an increase of both fibrosis and angiogenesis markers, alpha-SMA and CD31 in cirrhotic EPC-treated rats as compared to healthy EPC-treated and saline-treated rats in immunohistochemistry and western blot studies. Cirrhotic EPC-treated BDL rats had high portal pressure and portal blood flow with significantly elevated hepatic vascular resistance in comparison with healthy EPC- and saline-treated BDL animals, without significant differences in mean arterial pressure. Cirrhotic EPC-treated BDL rats also showed a substantial increase in the hepatic expression of angiogenic receptors, VEGFR2 and CXCR4 in comparison with saline-treated rats.ConclusionThe study suggests that transplantation of cirrhotic EPCs enhances LSEC differentiation and angiogenesis, activates HSCs and worsens fibrosis, thus resulting in hepatic hemodynamic derangements in BDL-induced cirrhosis.

背景：循环性肝细胞癌内皮祖细胞（EPC）与肝窦内皮细胞（LSEC）和肝星状细胞（HSC）相互作用，并在体外促进血管生成。本研究评估了肝细胞癌和对照EPC对肝硬化大鼠模型中肝脏血管生成、微循环和纤维化的影响。方法：通过胆管结扎（BDL）制备肝硬化动物模型。从健康人和肝硬化血液中分离的循环EPC通过流式细胞术进行表征，并在结扎2周后通过尾静脉在BDL大鼠中培养和给药。细胞每周给予三次，连续两周。未处理的BDL大鼠仅接受盐水。通过Masson三色染色评估纤维化。通过CD31表达鉴定去分化LSEC，通过α-SMA阳性细胞标记活化HSC，并在盐水、健康EPC和肝硬化EPC处理的大鼠中进行免疫组化和蛋白质印迹分析。在体内，评估肝脏和全身血流动力学参数。评估肝功能。结果：与对照组相比，BDL大鼠表现出纤维化和血管生成的增加。在处理的大鼠中，与健康EPC处理和盐水处理的大鼠相比，肝硬化EPC处理的大鼠纤维化程度增加。在免疫组化和蛋白质印迹研究中，与健康EPC处理和盐水处理的大鼠相比，肝硬化EPC处理的大鼠纤维化和血管生成标记物α-SMA和CD31增加。与健康EPC和盐水处理的大鼠相比，肝硬化EPC处理的大鼠门脉压力和门脉血流量高，肝血管阻力显著升高，平均动脉压无显著差异。与盐水处理的大鼠相比，肝硬化EPC处理的大鼠肝中血管生成受体VEGFR2和CXCR4的表达显著增加。结论：本研究表明，肝硬化EPC的移植增强了LSEC的分化、血管生成，激活了HSC，并加剧了纤维化，从而导致胆管结扎诱导的肝硬化中的肝脏血流动力学紊乱。