| Vascular disease is a major complication of diabetes mellitus.Inflammation,oxidative stress,and endothelial dysfunction play critical roles in the development of vascular complications induced by diabetes and hyperglycemia.Under hyperglycemia and diabetic conditions,the vascular endothelium is challenged by inflammation,reactive oxygen species with resultant endothelial dysfunction.Vascular smooth muscle cells(VSMC),as an important component of the vasculature,may be functionally impaired by high glucose and lipid and thus contribute to the increased incidence of vascular complications.Zinc finger transcription factor KLF5 is an important mediator of cardiovascular remodeling and is also implicated in the activation of inflammatory pathways following exposure to inflammatory stimuli.For example,vascular injury induces the expression of KLF5 in adult VSMC,which in turn activates many genes involved in cardiovascular remodeling,including inducible NO synthase(iNOS),PDGF-A/B,Egr-1,plasminogen activator inhibitor-1(PAI-1),and vascular endothelial growth factor(VEGF)receptors.Nuclear factor ?B(NF-?B)family of transcription factors,particularly p65 and p50,are well known to regulate inflammation and immune responses and plays a critical role in activating the pro-inflammatory genes.However,an actual relationship between KLF5 and NF-?B in the context of diabetic vascular complications is not fully understood.In the cardiovascular system,iNOS is mainly expressed in VSMC,and its expression is markedly induced by inflammation.iNOS is reportedly implicated in many human diseases associated with inflammation.It has been shown that impaired cardiovascular function in type 2 diabetes mellitus(T2DM)is partially attributed to abnormal overexpression of iNOS in cardiovascular tissues of diabetic rats.The increased release of NO derived from iNOS is responsible for the development of diabetic vascular complications.Certainly,sustained release of large amounts of nitric oxide(NO)through the iNOS has been linked to the production of harmful oxidative products such as peroxynitrite.Indeed,nitrotyrosine(tyrosine nitration,a permanent marker of protein nitrosylation)is significantly increased in the vascular wall of diabetic mice,indicating that in diabetes chronic hyperglycemia can increase both oxidative stress and peroxynitrite formation.However,although diabetic vascular complications are associated with increased levels of inflammatory markers and peroxynitrite formation,mutual regulation between KLF5,NF-?B and iNOS in diabetic vascular complications,as well as the underlying mechanisms,is not known.In this study,we investigated how KLF5,NF-?B and peroxynitrite derived from iNOS regulate inflammatory response of VSMC in the context of high glucose.Part? iNOS-derived peroxynitrite mediates high glucose-induced KLF5 expression and nitration in VSMCObjective: To investigate the role of KLF5 and iNOS of cultured VSMC in high glucose.Methods:1 qRT-PCR and Western blot were performed to examine the expression of KLF5 and iNOS in the vasculars tissues of human with or without diabetes.Confocal immunofluorescence was used to detect KLF5 and iNOS in VSMC of human vascular tissue that with or without diabetes.2 Nondiabetic VSMC were treated with different concentrations of glucose(5.5-25 mM)or treated with 25 mM glucose for various times(0-12 h),qRT-PCR and Western blot were performed to examine the expression of KLF5 and iNOS.VSMC were cultued with high glucose(25 mM)for 12 h,immunofluorescence assay were used to detect KLF5 and iNOS.3 Endogenous KLF5 was knocked down by transfecting VSMC with KLF5-specific siRNA(si-KLF5)or nonspecific siRNA(si-Ctrl),and then treated the cells with or without high glucose,western blot was performed to examine the expression of KLF5 and iNOS.4 ELISA was used to detect the peroxynitrite(ONOO–)formation during low glucose or high glucose.5 VSMC were treated with different concentrations of SIN-1(0-100 ?M)or treated with 100?M SIN-1 for various times(0-12 h),q RT-PCR and Western blot were performed to examine the KLF5 expression.6 VSMC were stimulated with high glucose(25 mM)in the presence or absence of the different concentrations of FeTPPS,co-immunoprecipitation experiments were carried out to detect KLF5 nitration.7 VSMC were cultured in medium containing 5.5 mM or 25 mM glucose and treated with or without 100 ?M SIN-1,followed by treatment with or without FeTPPS(100 ?M)for 12 h.The cell lysates were immunoprecipitated with anti-KLF5 or anti-3-NT antibody,and the resulting precipitates were analyzed by Western blotting using anti-3-NT or anti-KLF5 antibody.Immunofluorescence staining of 3-nitrotyrosine(3-NT),KLF5,and the nucleus on VSMC.8 Immunofluorescence staining of 3-nitrotyrosine(3-NT),KLF5,and the nucleus on splenic artery sections of non-diabetic(non-DM)and diabetic(DM)patients.Results:1 KLF5 and iNOS expressions are upregulated in VSMC of diabetic patientsThe expression of KLF5 and iNOS was about 3-fold up-regulated in diabetic vascular tissue at the mRNA levels.Moreover,the levels of KLF5 and iNOS proteins were also markedly increased in diabetic vascular tissue compared with that in nondiabetic vascular tissue.Confocal immunofluorescence of SM ?-actin,KLF5,and iNOS showed that KLF5 and iNOS expression was up-regulated,as evidenced by the increased fluorescence intensity in diabetic vascular tissues,and KLF5,iNOS,and SM ?-actin are co-localized in VSMC.2 High glucose induces the expression of KLF5 and iNOS in cultured mouse VSMCThe addition of glucose to the cells led to a dose-and time-dependent increases in KLF5 and iNOS mRNA levels,with mRNA levels of KLF5 or iNOS being more than 2-fold up-regulated in 25 Mm glucose-treated VSMC.Simultaneously,glucose also increased the level of KLF5 and iNOS proteins in a dose-and time-dependent manner,reaching its maximal effects at 25 mM of glucose.Immunofluorescence assay showed that treatment of cultured VSMC with high glucose also markedly induced KLF5 and iNOS expression.When knocked down endogenous KLF5,the results of western blot demonstrated the marked attenuation of high glucose-induced iNOS expression in si-KLF5-transfected cells compared with cells transfected with nonspecific siRNA,suggesting that high glucose-induced iNOS expression is mediated by KLF5.The results of ELISA demonstrated that treatment of VSMC with high glucose increased peroxynitrite(ONOO–)formation 2-fold over that observed with normal medium,implying that iNOS-mediated peroxynitrite generation may be involved in vascular complications induced by high glucose.3 High glucose induces KLF5 nitration by iNOS-mediated peroxynitrite in VSMCUnder normoglycemic conditions(glucose 5.5 mM),SIN-1 dose-and time-dependently increased the expression of KLF5 at mRNA and protein levels,with a maximum increase at 100 ?M SIN-1 or at 12 h after SIN-1 treatment.Co-immunoprecipitation experiments showed that FeTPPS dose-dependently decreased the levels of nitrated tyrosine present in anti-KLF5 immunoprecipitates.Likewise,a similar result was obtained by reciprocal immunoprecipitation with anti-3-nitrotyrosine(NT)antibodies,showing that tyrosine nitration in KLF5 was obviously less in cells treated with high glucose and 100 ?M FeTPPS than high glucose alone.In further experiments,we also demonstrated that high glucose or SIN-1 markedly increased tyrosine nitration in KLF5,and the increased KLF5 nitration was abrogated by FeTPPS.Furthermore,confocal immunofluorescence staining of nitrated tyrosine and KLF5 showed that KLF5 nitration was induced by high glucose or SIN-1,and FeTPPS abolished high glucose-induced tyrosine nitration in KLF5.As expected,the levels of nitrated tyrosine in KLF5 were significantly higher in diabetic human vascular tissue than nondiabetic vascular tissue.Summary:KLF5 and iNOS expressions are upregulated in VSMC of diabetic patients.High glucose induces the expression of KLF5 and iNOS in cultured mouse VSMC.iNOS-derived peroxynitrite mediates high glucose-induced KLF5 expression and nitration in VSMC.Part? Nitration of KLF5 and NF-?B p50 interactions induced by high glucose cooperatively induce pro-inflammatory gene expressionObjective: To elucidate the molecular mechanism that nitration of KLF5 and NF-?B p50 interactions induced by high glucose cooperatively induce pro-inflammatory gene expression.Method:1 VSMC were treated with different concentrations of glucose for 12 h or with 25 mM glucose for the indicated times.NF-?B p50 expression was determined by Western blotting.2 VSMC were incubated with low glucose(5.5 mM),high glucose(25 mM),SIN-1(100 ?M),or high glucose plus FeTPPS(100 ?M)for 12 h.The expression of NF-?B p50 was analyzed by Western blotting.3 VSMC were treated with low glucose(5.5 mM),high glucose(25 mM),SIN-1(100 ?M),or high glucose plus FeTPPS(100 ?M)for 12 h.The cell extracts were subjected to immunoprecipitation with anti-NF-?B p50 or anti-3-NT antibody,and the resulting precipitates were analyzed by Western blotting with anti-3-NT or anti-NF-?B p50 antibody.In addition,the cell extracts were subjected to immunoprecipitation with anti-NF-?B p50 or anti-KLF5 antibody,and the resulting precipitates were analyzed by Western blotting with anti-3-NT,anti-KLF5 or anti-NF-?B p50 antibody.4 VSMC were incubated with low glucose(5.5 mM),high glucose(25 mM),SIN-1(100 ?M)or high glucose plus FeTPPS(100 ?M)for 12 h.The expression of IL-1? and TNF-? mRNA and protein was analyzed by Western blotting and q RT-PCR.VSMC were treated as above,and the expressions of IL-1?,TNF-? and KLF5 were analyzed by confocal fluorescence immunohistochemistry.5 VSMC were transfected with either si-KLF5 or si-Ctrl for 24 h,and then treated with low or high glucose for 12 h.VSMC were transfected with either Ad-GFP or Ad-KLF5 for 24 h,and then treated with low or high glucose for 12 h.The expression of KLF5,IL-1? and TNF-? mRNA and protein was analyzed by Western blotting and qRT-PCR.6 The expression of KLF5,iNOS,NF-?B p50,IL-1? and TNF-? was analyzed by qRT-PCR and Western blotting in the thoracic and abdominal aorta of control(NC)and KLF5?/? mice treated or not with STZ for 12 weeks.7 Immunofluorescence staining of KLF5,smooth muscle ?-actin(SMA),and the nucleus on the aortic sections of control(NC)and KLF5?/? mice treated or not with STZ for 12 weeks.8 Immunofluorescence staining of KLF5,3-nitrotyrosine(3-NT),NF-?B p50,and the nucleus on the aortic sections of control(NC)and KLF5?/? mice treated or not with STZ for 12 weeks.Results:1 Nitration of KLF5 and NF-?B p50 induced by high glucose facilitate their interaction with each otherHigh glucose increased the level of NF-?B p50 protein in a dose-and time-dependent manner,with maximum increase at 25 mM glucose or at 12 h after glucose exposure.Exposure of VSMC to SIN-1(100 ?M)also increased NF-?B p50 expression,and FeTPPS treatment abolished high glucose-induced NF-?B p50 expression.CoIP showed that the nitration of NF-?B p50 was markedly increased(4-5 fold)following treatment of VSMC with high glucose and SIN-1.Similarly,high glucose-induced nitrationof NF-?B p50 was abrogated in FeTPPS-treated VSMC.The results of co-immunoprecipitation experiments showed that exposure of high glucose or SIN-1 increased the levels of NF-?B p50 present in anti-KLF5 immunoprecipitates.Likewise,reciprocal immunoprecipitation with anti-NF-?B p50 showed temporal co-precipitation of KLF5 in response to high glucose or SIN-1.Notably,the nitration level of the immunoprecipitated NF-?B p50 or KLF5 also increased markedly in high glucose-or SIN-1-treated VSMC,and FeTPPS treatment reduced the high glucose-induced tyrosine nitration and the interaction of KLF5 with NF-?B p50.Laser confocal microscopy showed that the co-localization of NF-?B p50 and KLF5 could not be observed in normoglycemia-cultured VSMC,as well as in the high glucose plus FeTPPS-treated cells.However,high glucose and SIN-1 not only up-regulated the expression of NF-?B p50 and KLF5,but also promoted their interaction,as shown by their co-localization in the nucleus.2 KLF5 and NF-?B p50 interactions induced by high glucose cooperatively induce pro-inflammatory gene expressionqRT-PCR and Western blot showed that high glucose or SIN-1 increased both protein and mRNA levels of IL-1? and TNF-?,with their mRNA levels being more than 2.5-fold up-regulated in high glucose-or SIN-1-treated VSMC.The high glucose-elicited increases in TNF-? and IL-1? expression were further confirmed by immunofluorescent staining for TNF-? and IL-1? in high glucose-or SIN-1-treated VSMC,with FeTPPStreatment leading to abrogation of high glucose-mediated effects in VSMC.Knockdown of KLF5 dramatically reduced TNF-? and IL-1? expression at transcription and translation levels regardless of high glucose stimulation,whereas transfecting with a control siRNA did not affect high glucose-mediated inflammatory effects.Conversely,the overexpression of KLF5 significantly increased TNF-? and IL-1? expression at both mRNA and protein levels regardless of the presence of high glucose.Importantly,the expression of IL-1? and TNF-? was no longer up-regulated by high glucose alone or together with SIN-1 in KLF5-knocked down VSMC.3 VSMC-specific knockout of KLF5 reduces inflammatory cytokine expression in the vascular tissues of type 1 diabetic miceThe mouse body weight was significantly decreased at 12 weeks after STZ injection compared with controlmice,and blood glucose level in STZ-injected mice significantly increased at 3 weeks after STZ injection,with a 3.7-fold increase at 12 weeks.However,weight loss and increased blood glucose level induced by STZ were greatly reduced in KLF5?/? mice compared with C57BL/6 mice,suggesting that KLF5 deletion in VSMC has protective effects on STZ-induced type1 diabetes.Diabetic mice induced by STZ injection for 12 weeks showed a significant increase in mRNA and protein levels of KLF5,iNOS,and NF-?B p50.Immunofluorescent staining for KLF5 and SM ?-actin(SMA)also confirmed that KLF5 expression was obviouslyup-regulated in VSMC of the STZ-diabetic mice,as evidenced bythe increased fluorescence intensity of KLF5 and its co-localization with SMA in VSMC,whereas in KLF5?/?mice,KLF5 expression in VSMC could not be detected.Notably,knockout of KLF5 in VSMC greatly decreased mRNA and protein expression of iNOS and NF-?B p50 regardless of STZ treatment.qRT-PCR and Western blot showed that mRNA and protein levels of IL-1? and TNF-? were markedly up-regulated at 12 weeks after STZ injection,with a 2.5-4-fold increase at their mRNA levels.Likewise,knockout of KLF5 in VSMC completely abrogated the STZ-induced up-regulation of inflammatory cytokines IL-1? and TNF-?.Consistent with the results in cultured VSMC,tyrosine nitration in KLF5 and NF-?B p50 was obviously increased in vascular tissues of STZ-diabetic mice.Immunofluorescent staining for KLF5,NF-?B p50,and nitrated tyrosine also showed a marked increase in KLF5 and NF-?B p50 nitration in STZ-diabetic vascular tissues,as shown by a substantial co-localization of these two molecules with nitrated tyrosine,and deletion of KLF5 in VSMC abolished the STZ-induced KLF5 and NF-?B p50 nitration.Summary:Nitration of KLF5 and NF-?B p50 interactions induced by high glucose cooperatively induce pro-inflammatory gene expression.VSMC-specific knockout of KLF5 reduces inflammatory cytokine expression in the vascular tissues of type 1 diabetic mice.Part ? 17?-estradiol-induced interaction of ER? with NF-?B p50 decreases high glucose-induced interaction of KLF5 with NF-?B p50,contributing to the suppression of IL-1? and TNF-? gene expressionObjective: To explore the molecular mechanism of 17?-estradiol suppress the expression of inflammion gene in VSMC.Method:1 VSMC were treated with different doses of 17?-Estradiol(E2)for 12 h.The expression of KLF5,iNOS,NF-?B p50,IL-1? and TNF-? mRNA and protein was analyzed by qRT-PCR and Western blotting.2 VSMC were treated with high glucose(25 mM),high glucose plus E2(100 nM),or high glucose plus E2 and ER antagonist-ICI182780(1 ?M).The expression of KLF5,iNOS,NF-?B p50,IL-1? and TNF-? was analyzed by qRT-PCR,Western blotting and immunofluorescence staining.3 VSMC were treated with high glucose(25 mM),high glucose plus E2(100 nM),or high glucose plus E2 and ER antagonist-ICI182780(1 ?M).The cell extracts were subjected to immunoprecipitation with anti-NF-?B p50 or anti-ER? antibody,and the resulting precipitates were analyzed by Western blotting with anti-ER? and anti-KLF5 or anti-NF-?B p50 antibody.KLF5,NF-?B p50 and ER? expression and co-localization were examined by immunofluorescence staining.Results:1 17?-estradiol decreases high glucose-induced the expression of KLF5,iNOS,NF-?B p50,IL-1? and TNF-?The addition of E2 to the VSMC led to a dose-dependent decreases in mRNA and protein levels of KLF5,iNOS,NF-?B p50,IL-1? and TNF-?,with their mRNA levels being reduced to less than 50% of the control upon exposure to 100 nM E2.2 17?-estradiol decreases high glucose-induced the expression of KLF5,iNOS,NF-?B p50,IL-1? and TNF-? through ER?The addition of 100 nM E2 to the VSMC decreases high glucose induced the expression of KLF5,iNOS,NF-?B p50,IL-1? and TNF-? in mRNA and protein levels,with their mRNA levels being reduced to less than 50% of the control.Pharmacological inhibition of ER? receptor activity by the ER? receptor antagonist ICI182780 completely abrogated inhibitory effects of E2 treatment on the up-regulation of KLF5,iNOS,NF-?B p50,IL-1? and TNF-? expression induced by high glucose,indicating that the inhibitory effect of E2 on these gene expressions is mediated through activation of estrogen receptors.3 17?-estradiol-induced interaction of ER? with NF-?B p50 decreases high glucose-induced interaction of KLF5 with NF-?B p50,contributing to the suppression of IL-1? and TNF-? gene expressionCo-immunoprecipitation experiments were conducted to test whether E2 affects KLF5 interaction with NF-?B p50 or ER? in VSMC.High glucose-induced increase in the interaction of KLF5 with NF-?B p50 was greatly reduced in high glucose plus E2-treated VSMC.Instead,ER? interaction with NF-?B p50 dramatically increased,showing that the level of ER? present in anti-p50 immunoprecipitates markedly increased compared with those treated with glucose alone.Likewise,reciprocal immunoprecipitation with anti-ER? also showed an obvious increase in the interaction of ER? with NF-?B p50 in high glucose plus E2-treated VSMC,which was completely abolished by ER? receptor antagonist.These findings suggested that E2-activated ER? can compete with KLF5 for binding to NF-?B p50,which in turn inhibits inflammation gene expression.To provide additional confirmation that E2-activated ER? competes with KLF5 for binding to NF-?B p50,the co-localization of NF-?B p50 with KLF5 or ER? was observed by laser confocal microscopy.As anticipated,treatment of VSMC with high glucose alone induced KLF5 and NF-?B p50 expression and promoted their co-localization predominately in the nucleus,whereas in high glucose plus E2-treated VSMC,NF-?B p50 and ER? were predominately co-localized.Summary:E2-induced increase in the interaction of ER? with NF-?B p50,accompanied by the decrease in NF-?B p50 interaction with KLF5,leads to the suppression of inflammation gene expression in VSMC.Conclusion:1 KLF5 and iNOS expressions are upregulated in VSMC of diabetic patients.2 High glucose induces the expression of KLF5 and iNOS in cultured mouse VSMC.3 High glucose induces KLF5 nitration by iNOS-mediated peroxynitrite in VSMC.4 Nitration of KLF5 and NF-?B p50 interactions induced by high glucose cooperatively induce pro-inflammatory gene expression.5 VSMC-specific knockout of KLF5 reduces inflammatory cytokine expression in the vascular tissues of type 1 diabetic mice.6 17?-estradiol-induced interaction of ER? with NF-?B p50 decreases high glucose-induced interaction of KLF5 with NF-?B p50,contributing to the suppression of IL-1? and TNF-? gene expression. |
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