The Role Of Cdc42 On Endothelial Regeneration And Vascular Repair In Acute Lung Injury

Acute lung injury(ALI)and its severe state,acute respiratory distress syndrome(ARDS)are popularly known clinic critical diseases with a high mortality that threaten the human healthy,daily life and production.Nowadays,there are no available specific pharmacologic therapies for ALI/ARDS.Therefore,to elucidate the pathogenesis and to seek for novel ways to treat ALI/ARDS have the practical significance.The main pathogenesis of ALI/ARDS is lung inflammation and pulmonary capillary disrupted that lead to pulmonary edema.In the process,the pulmonary microvascular endothelium is the first barrier system to defend injury which is vulnerable to noxious stimuli directly or indirectly,resulting in development of ARDS.Therefore,it is of great significance to promote vascular endothelial cells regeneration and vascular repair that make an effort to fix vascular integrity and to reduce neutrophils infiltrating and lung edema.But the mechanism of vascular recovery is so complex that researches are still exploring the regulatory factors related to the ability of pulmonary microvascular endothelial regeneration.Cell division cycle 42(cell division cycle),referred to as Cdc42,is an important member of the Rho family protein,25kD.It has many important biological activities,such as regulating cell cycle,promoting cell proliferation,migration,maintaining cell polarity,and so on.In recent years,researchers use Cre/Loxp conditional gene knockout technique to construct animal,organs or tissues specific-Cdc42 knockout model,to explore the regulatory role and mechanism of Cdc42.In this study,we first constructed a systemic vascular endothelial specific knockout Cdc42 gene in mice,and established a model of acute lung injury in order to further investigate the effects of Cdc42 on vascular endothelial injury and repair.It is possible to provide a potential therapeutic target for the promotion of endothelial cell regeneration and vascular repair after lung injury.Methods:1 In vivo experiment1.1 The expression of Cdc42-GTP in ALI lungs.1.2 The construction of endothelial cell specific knockout Cdc42 mice and ALI mode.1.3 The left lungs harvested were fixed,paraffin embedded and sliced for HE or IHC staining.The right lungs were grinding for western blot analysis.2 In vitro experiment2.1 The pulmonary microvascular endothelial cells were cultured and knocked out Cdc42 gene by cre/loxp technique.2.2 Human pulmonary microvascular endothelial cell lines were cultured and knocked down Cdc42 transfected with siRNA.Use BrdU staining for cell proliferation,wound healing assay for cell migration.The TEER and FITC-dextran test were used to estimate cell barrier function.The Matrigel-formation assay was used for estimating the alibility of vascular network.2.3 The cells were harvested for western blot.The downstream protein related to Cdc42 were detected.2.4 Statistical analysis was computed using SPSS.Results:1.The Cdc42-GTP expression in ALI lungs was correlated with severity of injury.The expression of Cdc42GTP in lung tissue was significantly changed after LPS treatment.Compared with the control group,the expression of Cdc42GTP in LPS group decreased at day 1 and increased at day 7 after LPS treatment(P<0.05).2.Stably construction of endothelial cell specific-knockout Cdc42 gene mice and primary lung microvascular cellsThe expression of Cdc42 protein in Cdc42 KO group was significantly lower than that of control group,and the difference was statistically significant(P<0.05).The results in cell mode are the same as the animal ones.3.Stably construction of Cdc42 protein knockdown HPMVE cell lines.The expression of Cdc42 protein in siCdc42 group was significantly lower than that of siControl group,and the difference was statistically significant(P<0.05).4.Loss of Cdc42 aggravated inflammatory cells infiltration and pulmonary vascular leakage and impaired proliferation after ALI.The levels of pulmonary perivascular infiltration of neutrophils,pulmonary microvascular leakage rate in KO lungs were significantly higher than the control lungs,the difference was statistically significant(P<0.05).And the proliferation of pulmonary vascular endothelial cells in KO mice was significantly lower that the control mice.5.Cdc42 deficiency disrupted endothelial cells proliferation,migration and vascular formation.Compared with the control group,the vascular endothelial proliferation,migration and angiogenesis ability of siCdc42 group decreased significantly,the difference was statistically significant.(P<0.05).6.Cdc42 knockdown downregulated the expression of PAK2/Akt signaling pathway.Cdc42 knockdown downregulated the expression of PAK1/AKT,VE-cadherin.And there is no significantly change of p-P38/P38,p-JNK/JNK.Conclusion:1.Cdc42 defect aggravated inflammatory cell infiltration,pulmonary microvascular leakage and damaged pulmonary microvascular barrier function in ALI.Thus,Cdc42 may be a therapeutic target for improving ALI/ARDS.2.In vivo,knockout of Cdc42 reduced pulmonary microvascular proliferation;in vitro,knockdown of Cdc42 decreased proliferation,migration and tube formation in pulmonary microvascular endothelial cells.Therefore,Cdc42 is an important regulator of endothelial regeneration and vascular repair after lung injury.3.Downregulation of Cdc42 expression decreased the expression of PAKI/AKT signaling pathway.Therefore,Cdc42 may regulate the endothelial regeneration and vascular repair through PAK1/Akt signaling pathway.