Concentrated Ambient PM2.5 And Endothelial Cdk5 Knockdown Are Involved In Cardio-cerebrovascular Damage

Background:Cardio-cerebrovascular diseases are a kind of diseases related to cardiovascular and cerebrovascular dysfunction with high incidence,disability and mortality.At present,they have become the primary threat to human health.Diverse factors especiallly PM2.5 are contributed to cardio-cerebrovascular diseases.PM2.5components are complex and diverse,which is closely related to the external climate,time,temperature,geographical environment,etc.The establishment of stable PM2.5research model in clinical studies is faced with great challenges,and the molecular mechanism of cardio-cerebrovascular toxicity caused by PM2.5 has not been clarified.Researchers have been working hard to explore toxicological mechanism of PM2.5 on cardio-cerebrovascular disorders and try to find reliable clinical treatment strategies for cardio-cerebrovascular protection.Endothelial cells are located in the inner wall of the vascular lumen as important components in vascular structure,forming a barrier for the communication between the inside and outside of the vessel.CDK5,a proline-guided threonine/serine protein kinase,plays a key role in cellular physiology processes such as migration,proliferation,apoptosis and senescence.In the central nervous system,Cdk5 is mainly involved in important biological processes such as neuronal migration,axon orientation,synaptic generation and transmission,and neuronal apoptosis.However,few studies have focused on the important role of Cdk5 in the cardiovascular and cerebrovascular system.The evidence on the connection as well as the molecular signal mechanism between Cdk5 and cardio-cerebrovascular diseases still needs to be further explored.Purpose:In our study,through concentrated ambient PM2.5 exposure（termed as CAP）for the mice model in which vascular endothelial Cdk5 knocked down specifically,we try to explore the important role of genetic factors and environmental factors in cardio-cerebrovascular toxicity and reveal the mechanism of vascular injury in order to provide a new theoretical basis and new ideas for clinical prevention and treatment of cardio-cerebrovascular diseases.Methods and Results:First of all,the importance of vascular endothelial cell Cdk5was proved in maintaining the biological function homeostasis of the central nervous system by bioinformatics analysis.Cell Counting Kit（CCK8）results revealed that PM2.5-DMSO extracts induced more significant damage to endothelial cells than lung epithelial cells.Therefore,Cdh5-Cre;Cdk5^f/n mouse line was constructed for subsequent experiments.Moreover,it was found by behavior test that mice in which Cdk5 knockdown on vascular endothelial cells had no abnormal behavior phenotype.Then we treated Cdh5-Cre;Cdk5^f/n and WT（Wild Type）mice with PM2.5 exposure for2 months and confirmed the integrity of BBB and no changes of cerebral inflammatory factor levels through western blotting,quantitative real time PCR,etc.Behavioral analysis further demonstrated that PM2.5 exposure and endothelial Cdk5 knockdown did not affect neuronal activity in central nervous system.To examine cerebrovascular physiological function,we use myograph,myogenic response and laser doppler flowmetry to verify no changes on cerebrovascular tension,myogenic activity and cerebral blood flow between four groups:WT+FA,WT+CAP,Cdh5-Cre;Cdk5^f/n+FA,Cdh5-Cre;Cdk5^f/n+CAP.In conclusion,CAP exposure and endothelial Cdk5knockdown have no effect on cerebral microvessels.In subsequent studies,we turned our attention to the role of environmental and genetic factors in the peripheral system.H&E staining of lung tissue showed that CAP treatment led to abnormal lung morphology in Cdh5-Cre;Cdk5^f/n and WT mice,while no changes of lung morphology in Cdh5-Cre;Cdk5^f/n mice when compared to WT.These findings suggest that PM2.5,not Cdk5 knockdown,could result in significant lung damage.Then,detecting vascular tension of the thoracic aorta,it showed increased vasoconstriction of thoracic aorta in Cdh5-Cre;Cdk5^f/n+CAP group with AngiotensinⅡor Norepinephrine treatment when compared to FA（Filter Air,FA）group,while WT mice showed no change in vascular tension between FA and CAP groups.In order to further explain the underlying molecular mechanism of vascular toxicity caused by PM2.5 and Cdk5,we utilize western blotting to certify that endothelial Cdk5 knockdown may elevate the expression of angiotensinⅡtype 1 receptor（AT1R）.And Cdh5-Cre;Cdk5^f/nmice showed a trend of increased AT1R expression after CAP exposure when compared with Cdh5-Cre;Cdk5^f/n+FA mice,while the expression of AT1R associated downstream signaling molecules Calmodulin and PKC did not change,suggesting that the increased vasoconstriction was not mediated by the classic PLC-DAG/IP3 signaling pathway.Conclusion:The synergistic effect of endothelial knockdown Cdk5 and PM2.5 can mediate increased peripheral artery contraction but have no effect in central nervous system.With PM2.5 treatment,there is a trend to increased AT1R expression in peripheral blood vessels of Cdh5-Cre;Cdk5^f/n mice,which leading to increased vasoconstriction.However,the PLC-DAG/IP3 signal pathway was not involved.