Mechanism Of Lipoxin A₄ Regulating Endothelial Permeability In Human Umbilical Vein Endothelial Cells

Background: Vascular endothelium ,the largest endocrine organ to regulate the balance in cardiocerebral vascular system, are getting more and more attention.The nomal biological function of endothelial cells will be disordered if they are damaged. The most obvious phenomenon is a mass of increase of vascular endothelial permeability ,which could result in many diseases,like atherogenesis,hypertension,edema caused by preeclampsia. Thus, how to protect endothelial cells from impairment and reduce permeability to a normal level undoubtfully becomes the hot topic in some fields invovled in preventing cardiocerebral vascular diseases.There is no doubt that inflammatory oxidative stress plays a critical role in disfunction of endothelial cells and is able to cause microvascular dysfunction and cardiovascular diseases.Meanwhile, It is well-known that lipoxins are the first endogenous anti-inflammation and pro-resolution chemical substances identified in vivo. Some studies have showed that the level of lipoxins was very low in patients'blood with preeclampsia and hypertension compared with health population.So these evidences strongly suggest that lipoxins probabley exert an important effect of anti-oxidation so as to regulate endothelial permeability.Objective: To explore how lipoxins could inhibite endothelial hyperpermeability induced by LPS and possible mechanism.Methods: Human unbilical vein endothelial cell lines (CRL-1730) were cultured and treated with LXA₄ for 30 minutes before stimulation with lipopolysaccharide (LPS). Morphological change of HUVECs was observed by hematoxylin and eosin (H&E); Endothelial permeability was measured by albumin clearance across the monolayer; The production of ROS was detected by ROS kit; the expression of FPRL2 was identified by Real-time PCR, the mRNA expression of VE-cadherin,TRPC1 was tested by RT-PCR; Western blotting method was used to measured the expression of VE-cadherin,β-catenin; Immunofluorescent assay was used to detect the expression of VE-cadherin which was also observed the change of gap size; intracellular Ca²⁺ concentration was determined by Calcium-imaging technology.Results:â‘´The expression of FPRL2 in CRL-1730 cell lines is similar with that in primary HUVECs and LXA₄ could upregulate the expression of FPRL2.⑵HUCECs became much small,out of shape and cell junctions were destroyed and even emerged very large gap by LPS treatment alone,compared with vehicle group but the morphological change was not obvious in LPS + LXA₄ group.⑶Compared with control group,endothelial permeability was significantly increased in LPS group (**P<0.01) and the level of permeability was downregulated to the normal by LPS+LXA₄ treatment (**P<0.01), LXA₄ was no dose-dependent manner.â‘·Compared with control group,the mRNA or protein expression of VE-cadherin,β-Catenin were inhibited and the mRNA of TRPC1 was increased by LPS treatment(*P<0.05), meanwhile, all but TRPC1 expressions of them were upregulated by LPS combined with LXA₄ treatment compared with LPS alone(*P<0.05)⑸The production of ROS was higher by LPS treatment than that without any treatment(*P<0.05), LPS and LXA₄ co-treatment reduced the production of ROS induced by LPS. (*P<0.05)⑹LXA₄ suppressed the increase of intracellular Ca²⁺ concentration by LPS treatment(**P<0.01). Conclusions: Lipoxins inhibited endothelial hyperpermeability induced by LPS through decreasing the production of ROS and downregulating Ca²⁺ signaling to suppress disassembly of VE-cadherin/β-Catenin.