Study On The Mechanism Of High Glucose-induced Endothelial Cell Apoptosis

Diabetes mellitus,a chronic endocrine disease due to absolute or relative insulin deficiency,is characterized by hyperglycemia and other metabolic disorders of fat,protein and electrolytes.Epidemiological data indicated that diabetes-induced cardiovascular complications have been proved to be the leading cause of diabetes-related death and disability.Vascular endothelial cell apoptosis has been considered as an initial event and pathological basis of diabetic cardiovascular complications.Mitochondria are commonly referred to as cellular power plants critical to produce energy.Besides,mitochondria also play a central role in cell apoptosis.When damages occur to mitochondria,the mitochondrial membrane permeability could increase and Cytc was released from mitochondria which would activate caspase3 and induce cell apoptosis.Increasing evidence indicated that voltage-dependent anion channel(VDAC),an abundant protein located in the outer mitochondrial membrane,plays a critical role in the regulation of mitochondrial membrane permeability via interacting with HK2、Bcl-2 and Bax.HK2 and Bcl-2reduce but Bax increases mitochondrial permeability.In the pathological state,whether the expression levels of these protein affect mitochondrial membrane permeability and Cytc release remains unknown.AIM: In order to investigate the molecular mechanism of hyperglycemia-induced apoptosis of vascular endothelial cells.METHODS: HUVEC cells were used to mimic the pathological status of endothelial cells in the condition of hyperglycemia.The viability and apoptosis of HUVEC cells were detected by MTT and Hoechst staining respectively.The mitochondrial membrane potential was detected by JC-1 staining.The transcription level of Hexokinase2(HK2)m RNA was detected by reverse transcription-polymerase RT-PCR and the luciferase assay.Western blot and immune coprecipitation were used to detect the expression of VDAC1,HK2,Bcl-2,and Bax on the mitochondrial outer membrane and their interaction.HUVECs were infected by adenovirus expressing vector of HK2 for the purpose of overexpressing HK2.The viability and apoptosis of HUVEC cells,the mitochondrial membrane potential,the transcription of HK2,the expression of VDAC1,HK2,Bcl-2,and Bax on the mitochondrial outer membrane and their interaction,the transcription of Bcl-2 and the expression level of CREB and NF κB(P65),which are transcription factors of Bcl-2 were detected under the condition of high glucose.RESULTS: The apoptosis of HUVEC cells incubated in medium containing 16.5 or33 mM glucose was increased remarkably:(1)The viability of cells was reduced;(2)The concentration of nucleus chromatin was increased;(3)Mitochondrial membrane potential was reduced;(4)Bcl-2 protein expression levels were reduced,the activation of caspase3 was increased,mitochondrial Cytc was reduced and cellular Cytc remained unchanged,mitochondrial Bax was reduced and cellular Bax remained unchanged,mitochondrial and cellular HK2 were both decrease by western blot;(5)The interactions between VDAC1 and HK2 as well as Bcl-2 were reduced,while the Bax and VDAC1 interactions were compensatorily enhanced;(6)HUVECs were infected by adenovirus expressing vector of HK2,the regulator of VDAC1.Overexpression of HK2 is able to protect against the apoptosis caused by high glucose(33 mM),the viability of HUVEC cells has been reversed;(7)The concentration of nucleus chromatin has been reversed;(8)Mitochondrial membrane potential has been reversed;(9)Overexpression of HK2 effectively reversed the expression levels of Bcl-2、activation of caspase 3 and mitochondrial Cytc、Bax and HK2;(10)HK2 overexpression recovered the high glucose-reduced interactions between VDAC1 and HK2 as well as Bcl-2,and attenuated the enhanced interactions between VDAC1 and Bax;(11)High glucose significantly reduced HK2 m RNA evels and protein levels;(12)When MG132 was used to block HK2 protein degradation,the reduction in HK2 protein levels still remained;(13)PPARγ is confirmed to be the transcription factor of HK2 in HUVECs coexpressing PPARγwith luciferase reporter vector containing HK2 promotor by the luciferase assay;(14)High glucose reduced m RNA levels of endogenous PPARγ;(15)High glucose reduced protein levels of endogenous PPARγ but did not influence exogenous flag-tagged PPARγ level;(16)Overexpression of HK2 reversed high glucose-induced suppression of Bcl-2 m RNA level;(17)CREB and NF-κB(p65)were confirmed to be the transcription factor of Bcl-2 by the luciferase assay in HUVECs co-expressing CREB or NF-κB(p65)with luciferase reporter vector containing Bcl-2 promotor;(18)Overexpression of HK2 reversed high glucose-induced suppression of phosphorylation of CREB and NF-κB(p65).CONCLUSION: High glucose reduced HK2 expression via inhibiting the transcription activity of PPARγ,which impaired the interaction between HK2 and VDAC1,thus compensatorily enhanced that between Bax and VDAC1.Reduced interaction between Bax and VDAC1 increased mitochondrial permeability eventually inducing apoptosis of HUVEC cells.