Role Of NHE1 And MiR-29b In Diabetic Endothelial Dysfunction And Prevention

BackgroundsThe risk of cardiovascular morbidity and mortality is significantly higher in patients with diabetes,compared with non-diabetic individuals.In particular,diabetes is associated with a poor outcome after vascular occlusion,such as ischemic heart disease.Angiogenesis is critical for re-establishing the blood supply to the surviving myocardium after myocardial infarction(MI)and,consequently,to the recovery of cardiac function.Angiogenesis dependents on cell proliferation,migration,and capillary tubulogenesis in endothelial cells.However,the molecular mechanism responsible for impaired angiogenesis in diabetes remains largely unknown.All eukaryotes contain an intracellular fluid whose pH value is known as the intracellular pH(pHi)value.A slight change of pHi value,even in a physiological range,possibly alters cellular functions.The pHi value is critically controlled by Na+/H+ exchanger 1(NHE1),as a plasma membrane protein in mammalian cells.It exchanges intracellular H+ for extracellular Na4 to increase pHi value.Under pathological stimuli.NHE1 is activated,leading to intracellular alkalinization and cell dysfunctions in cardiac remodeling.By contrast,we and others reported that intracellular acidosis(1A)by NHE1 inhibition prevented endothelial dysfunction,and inhibited vascular hypertrophy and cardiomyopathy in diabetes,demonstrating the vital role of NHE1 activation in diabetic vascular complications.However,whether NHE1 activation plays an important role in diabetes-impaired angiogenesis has been incompletely understood.Akt is a serine/threonine kinase regulating essential cellular functions including survival,proliferation and metabolism in vascular homeostasis and angiogenesis.Many of the angiogenic functions attributed to vascular endothelial growth factor are mediated by intracellular activation of Akt signaling.In endothelial cells,Akt inactivation is also involved in endothelial dysfunction in diabetes,which is similar to NHE1 activation.Thus,we hypothesized that Akt activity is potentially related to pHi value and hyperglycemia may activate NHE1 to induce intracellular alkalinization,causing Akt inactivation.The present study was designed to investigate the role and mechanism of NHE1 inhibition or Akt activation in HG impaired endothelial function,and then find out the effects of NHE1,Akt and NHE1 selective inhibitor cariporide in angiogenesis and cardiac functions recovery after MI in diabetic mice.Objectives1.To incdue intracellular acidosis(IA)in HUVECs and investigate whether Akt is activated by IA in endothelial cells2.To detect the effects of HG on NHE1 activity and pHi value in HUVECs.3.To study if NHE1 inhibition or Akt overexpression reversed HG impaired tube formation and cell migrations in HUVECs4.The role of NHE1 and Akt in postischemic angiogenesis and heart function recovery in diabetic mice following MI5.To study the effects of cariporide on myocardial dysfunction and angiogenesis in diabetic mice following MIMethods1.Cell culture and Induction of IAECM was used to culture human umbilicus vessel endothelial cells(HUVECs).IA was induced by using the NH4C1 pulse method.First,HUVECs balanced with Tyrode solution for 90 minutes,then 30 mM NH4C1 solution was used to culture HUVECsfor 5 minutes.IA was induced by washout of NH4C1 with Tyrode solution containing cariporide or Na+-free Tyrode solution.2.Transfection of siRNA into HUVECsHUVECs got transfection with siRNA of NHE1 to examine its effect on tube formation and cell migration.3.Lentivirus infection to HUVECsA lentiviral vector expressing constitutively active Akt(lenti-Akt)was constructed from Akt cDNA.HUVECs were infected with lentivirus in antibiotic-free medium with 2%FCS overnight.The aim of lenti-Akt transfection is to find out its effect on tube formation and cell migration.4.Measurement of pHiThe pHi value was measured in HUVECs by using the pH-sensitive fluorescent dye BCECF.5.Measurement of NHE1 activity in HUVECsAfter treatment of HUVECs,NHE1 activity was determined by measuring the initial rate of pHi recovery following IA using NH4C1 pulse method.6.In vitro tube formation assayCultured HUVEC were seeded on cell culture dishes coated with growth factor reduced Matrigel(BD Biosciences)and cultured medium containing 0.5%FCS with or without HG.After 24 hours,photographs were taken through a microscope.7.(ell migrationScratch test was applied to detect the migration of cells.After cells were inoculated into 24 well plates,a cross was scratched in the middle of the bottom of’plates with steriled 200ul tip.Took a picture of the intersection of the scratch zero hour,one day.two days,three days,four days respectively after the scratch to observe the healing power,to count the number of cell migration,and to calculate the migration rate.8.Animal modelMale wild-type C57BL/6J mice.8-12 weeks of age,20-25 g,were housed in temperature-controlled cages and given free access to water and normal chows.A low-dose(50 mg/kg/day for 5 consecutive days)STZ induction regimen was used to induce pancreatic islet cell destruction and persistent hyperglycemia.Hyperglycemia was defined as a random blood glucose level of>300 mg/dl for>2 weeks after injection.Mice were subjected to myocardial infarction by ligation of left anterior descending coronary artery(LAD).First,anesthetize the mice with 2%isoflurane inhalation.Then,open the mice chest at the 4th intercostal space and extrude the heart smoothly and gently out of the hole.MI was created by permanent LAD ligation using a 6-0-suture line.9.Cardiac function evaluation by EchocardiographyTwo weeks after MI surgery,echocardiography with standard parasternal was conducted in the left lateral recumbent position.Systolic or diastolic left ventricular internal diameter(LVIDs or LVIDd),Left ventricular fractional shortening(%FS)and ejection fraction(%EF)were calculated by the echocardiography system.10.Histology and immunohistochemistryThe heart tissue was paraffin-embedded and sectioned(5μm)for staining.HE staining was used to examining the infarct size.Immunohistochemistry was used to examining αSMA、NHE1、Akt、pAkt and endomucin.11.Western blot analysisProtein was extracted from cells and heart tissue of different groups.The expression of pAkt.Akt.NHE1 PTE,pGSK were evaluated by Western blot.Results1.IA increases Akt phosphorylation in HUVECsAkt phosphorylation and activity were increased in IA model induced by NH4C1 pulse plus inhibition of NHE1 by Tyrode solution or Tyrode solution containing cariporide.2.Akt phosphorylation is determined by pHi value in HUVECsHUVECs were exposed to a series of K+-rich pH buffers containing nigericin,in which pHi value is equal to extracellular pH(pHe)value.Low pHi value(6.4-7.0)increased Akt phosphorylation,compared with pHi value at 72-7.4.3.PI3K is involved in IA-induced Akt activation in endothelial cellsPI3K Inhibitor wortmannin significantly abolished the increase of Akt phosphorylation in cells at pHi value of 6.4,but not altered the level of Akt phosphorylation in cells at pHi value of 7.4,revealing that PI3K-PDK1 signaling may mediate IA-induced Akt phosphorylation.4.High glucose(HG)activates NHE1 and reduces Akt phosphorylation in HUVECsHG significantly increased both NHE1 activity from 60 minutes and pHi value from 2 hours.But Akt phosphorylation and activity gradually decreased beginning from 2 hours after incubation with HG.5.NHE1 activation is required for HG-decreased Akt phosphorylation in HUVECsNHE1 inhibitor-cariporide bypassed the reductions of Akt phosphorylation and activity in HG-treated cells,suggesting that HG-induced Akt inactivation is through NHE1 activation.NHE1 siRNA,but not control siRNA.blocked the effects of HG on Akt phosphorylation in HUVECs,further supporting that NHE1 activation is required for HG-reduced Akt phosphorylation in HUVECs.6.HG impairs tubulogenesis in endothelial cells via NHE1 activation and Akt inhibitionHG dramatically decreased the tube formation in HUVECs transfected with control siRNA but not in HUVECs transfected with NHE1 siRNA.Akt upregulation promoted tube formation in HG treated HUVECs.7.HG via NHE1 activation or Akt inhibition inhibits cell migrations in HUVECsHG inhibited the migration rates in HUVECs transfected with control siRNA or infected with lentivirus expressing vector.but not in cells transfected with NHE1 siRNA or infected with lentivirus expressing Akt cDNA.8.Hyperglycemia via NHE1 activation or Akt inhibition inhibits ischemia-induced angiogenesis in mice heartsResults from immunohistochemistiy of a-SMA and endomucin showed that capillary density exhibited a robust increase in ischemic hearts from diabetic mice with NHE1 deficiency or Akt overexpression.9.NHE1 deficiency or Akt overexpression attenuates the delayed recovery of heart function in diabetic mice following MIEchocardiographic analysis showed that deficiency of NHE1 or overexpression of Akt improved the heart function in diabetic mice after MI.10.Cariporide promotes angiogenesis in the ischemic myocardium of diabetic mice and improves heart functions in diabetic mice after MIThe results of α-SMA and endomucin immunohistochemistry showed that administration of cariporide increased the capillary and arteriolar densities in ischemic hearts of diabetic mice.And cariporidc steadily decreased the LVIDs and LVIDd.and increased FS and EF in diabetic mice following MI.Conclusion1.IA increases Akt phosphorylation in HUVECs.2.HG impairs tubulogenesis and migrations in endothelial cells via NHE1 activation and Akt inhibition.3.NHE1 deficiency or Akt overexpression promotes angiogenesisand improves heart functions in diabetic mice following MI.4.Cariporide promotes angiogenesis in the ischemic myocardium of diabetic mice and improves heart functions in diabetic mice after MI.BackgroundsLovastatin is used for the treatment of hypercholesterolemia because it inhibits HMG-CoA reductase,and thus prevents HMG-CoA’s conversion into mevalonic acid.Independent of cholesterol-lowering activity,there are increasing evidences that statins have a potential benefit against cardiovascular diseases(CVD).Oxidative stress is believed to cause endothelial dysfunction,an early event and a hallmark in CVD.including atherosclerosis and hypertension.At this standpoint,it is widely accepted that statins prevent endothelial dysfunction by decreasing oxidative stress in vascular cells.However,the targets for stain-reduced oxidative stress in CVD have not been completely elucidated.Our and other’s previous studies have proven that activation of proteasome links to oxidative stress and consequent endothelial dysfunction in CVD.Proteasomes are responsible for the degradation of a large number of protein targets throughout eukaryotic cells.The function of proteasomes is regulated by activators that bind to the ends of the 20S core catalytic particle in a single or double fashion and open the ends of the core proteasome to allow entry of protein targets.As one of proteasome activators(PAs).PA200 is broadly expressed in mammalian tissues and it has been suggested to play a crucial role in response to DNA damage.MicroRNA(miRs)are approximately 20-nucleotide,single-stranded RNA molecules that target mRNA through partial complementarity and regulate gene expression through inhibition of translation or transcript degradation.Recently,Anna S et al reported that microRNAs play an important role in the regulation of proteasome activity.Furthermore,miR-29b replacement inhibits proteasomes in myeloma cells by targeting PSME4 that encodesPA200.Available data suggest that statin,such as lovastatin,exerts its effects by inhibiting the proteasome activity in osteoblast,and vascular endothelial and smooth muscle cells.Thus,it is interesting to establish if statin via regulation of miR-29b inhibits proteasome activity in CVD.In our studies,we characterized how lovastatin regulates PA200 and determined the role of miR-29b in proteasome activition.And we established rat models of diabetes and dyslipidemia.investigated the the mechanism of lovastatin and miR-29b in oxidative stress-mediated endothelial dysfunction.Objectives1.To study the effect of lovastatin on the expression of miR-29b;2.To investigate the effect of lovastatin on proteasome activity and its mechanism;3.To establish rat models of diabetes and dyslipidemia,investigate the the mechanism of lovastatin and miR-29b in oxidative stress-mediated endothelial dysfunction.Methods1.Cell cultureECM was used to culture human umbilicus vessel endothelial cells(HUVECs).2.Generation of lentivirus vector and infection to cellsThe lentivirus containing scr-miR,anti-miR29b,pre-miR29b,or full length PA200 cDNA.was generated by GenePharma Company(Shanghai,China).HUVECs were infected with lentivirus overnight in antibiotics-free medium supplemented with 2%FBS.The cells were then washed and incubated in fresh medium for an additional 12 h before experimentation.3.Transfection of siRNA into HUVECsHUVECs got transfection with siRNA of PA200 to examine its effect on inhibition of proteasome activity by lovastatin.4.Proteasome activity assayProteasome activity was monitored continuously by detection of free 7-amido-4-methylcoumarin with a fluorescence plate reader at 380/460 nm at 37℃.5.Detection of ROS productions in cells and tissuesROS productions in cultured cells were assayed by measuring the DHE fluorescence,combined with High Performance Liquid Chromatography(HPLC).Cells were incubated with DHE(100μM)for 30 min.washed,harvested and homogenized,and subjected to methanol extraction.O2-production was determined by conversion of DHE into oxyethidine.HPLC was performed by using a C-18 column to separate and quantify oxyethidium and ethidium.O2-production was determined by conversion of DHE into oxyethidine.To measure ROS production in the artery in situ,fresh frozen sections of carotid arteries were isolated from rats,and were stained with 10μM DHE for 30 min,rinsed,and observed by fluorescent microscopy.6.RNA quantificationTotal RNA was isolated using a Trizol-based RNA isolation protocol.RNA was quantified by Nanodrop.First strand cDNA synthesis was performed for each RNA sample using the Superscript H RT Kit,and real time quantitative polymerase chain reaction was performed using Taqman PCR reagents.7.Preparation of isolated rat aortic ringsRats were sacrificed under anesthesia by intravenous injection with pentobarbital sodium(30mg/kg).The descending aorta isolated by removing the adhering perivascular tissue caref’ully was cut into rings(3-4 mm in length).8.Establishments of diabetes and dyslipidemiaFor diabetic model of rats,a low-dose STZ(50 mg/kg/day.5 consecutive days.I.P.)in induction regimen was used to induce pancreatic islet cell destruction and persistent hyperglycemia.Hyperglycemia was defined as a random blood glucose level of>300 mg/dl for>2 weeks after injection.The dyslipidemia model in rats has been established by injection of native LDL(4 mg/kg)via the sublingual vein under ether anesthesia.9.Measurements of MDA,SOD and GSH-PxThe determinations of MDA content,SOD activity and GSH-Px activity in aortic tissues or serum were assayed by using commercial kits as recommend by the protocol.10.Western blot analysisProtein was extracted from cells and aortic tissue of different groups.The expression of PA200 were evaluated by Western blot.RESULTS1.Lovastatin increases miR-29b expression in endothelial cellsLovastatin increases miR-29b expression in endothelial cells,which is in time-or dose-dependent manner.2.Lovastatin decreases proteasome activity in endothelial cellsThe impacts of lovastatin on the proteasome activity in vitro were determined by exposing endothelial cells to lovastatin as indicated concentrations or times.3.MiR-29b mediates lovastatin-induced suppression of proteasome activity in endothelial cellsLovastatin still dramatically reduced proteasome activity in HVEC’s infected with scr-miR lentivirus,but not in endothelial cells infected with anti-miR-29b lentivirus.The suppression of lovastatin on proteasome activity was further enhanced if cells were overexpressed with miR-29b.4.PA200 is a target of-miR-29b in endothelial cells treated with lovastatinLovastatin significantly decreased PA200 protein level in endothelial cells.However.the downregulation of PA200 induced by lovastatin was abolished by anti-miR-29b and further amplified by overexpression of pre-miR-29b in HUVECs.5.Inhibition of proteasome activity by lovastatin is PA200-dependentAlthough]ovastatin reduced proteasome activity in vector-infected cells.it failed to inhibit proteasome activity in endothelial cells with overexpression of PA200.Knockdown of PA200 mirrored the effects of lovastatin on suppression of proteasome activity.6.Blockage of miR-29b abolishes the effects of lovastatin on suppressing ROS productions induced by multiple cardiovascular risk factorsMultiple cardiovascular risk factors,including ox-LDL.H2O2,TNF-α and HG.dramatically increased ROS productions by measuring DHE fluorescent density in endothelial cells,but treatment with lovastatin reversed these abnormalities.Lovastatin reduced ROS productions induced by ox-LDL or HG in scr-miR-infected cells,and these suppressions were abolished in cells infected with anti-miR-29b lentivirus.7.Lovastatin prevents endothelial dysfunction induced by multiple oxidants in isolated rat descending aortic arteries ex vivoBy incubations of rat aortic rings with LPC of the major metabolite of ox-LDL.DPPH of an exogenous free radical,and H2O2 of an endogenous ROS.all oxidants impaired acetylcholine-induced endothelium-dependent relaxation.Pre-incubation of aortic rings with lovastatin bypassed these detrimental effects induced by oxidants including LPC,DPPH and H2O2-8.Lovastatin improves endothelial dysf’unction induced by high glucose both ex vivo and in vivoHG but not high osmatic decreased acetylcholine-induced vessel relaxation in isolated rat aortic rings.This was attenuated by lovastatin in dose-dependent manner.Type 1 diabetes model in rat was induced by injection of STZ.Lovastatin dose-dependently rescued the impairment of endothelial function induced by hyperglycemia.Furthermore,increased MDA level and decreased SOD and GSH-Px activities in blood from diabetic rats were reversed by lovastatin treatments.9.Lovastatin improves endothelial dysfunction in dyslipidemia ratsAdministration of lovastatin at 2 or 4mg/kg/day.prior to LDL injection,rescued LDL-induced impairment of endothelial function in rats.Besides,LDL-induced oxidative stress,as decreased SOD activity and increased MDA level in serum,were also reversed by lovastatin treatment.10.Lovastatin reduces ROS productions in models of diabetes,dyslipidemia.and hyperhomocysteinemia in ratsAll risk factors of CVD including diabetes and dyslipidemia,dramatically increased ROS productions in carotid arteries from rats,and administration of-lovastatin abolished these alterations in those rats.Conclusion1.Lovastatin increases miR-29b expression anddecreases proteasome activity in endothelial cells2.PA200 is a target of miR-29b in endothelial cells treated with lovastatin3.Lovastatin via suppression of oxidative stress prevents endothelial dysfunction in models of diabetes and dyslipidemia in rats...