Cyclophilin D Mediates TSH-induced Endothelial And Mitochondrial Oxidative Injuries

Background:Endothelial cells(ECs)are in a dynamic equilibrium with their environments and constitute a defensive barrier in the vasculature,controlling vascular permeability,smooth muscle tone,inflammatory and immune responses,angiogenesis,and thromboresistance.In many vascular diseases,the endothelium is thus both origin and victim.The interactions within the redox reactions are richly elaborated in an oxygen-dependent life and contribute to spatiotemporal organization for differentiation,development,and adaptation to the environment.Disruption of the balance between oxidants and anti-oxidants in favor of the oxidants leads to the occurrence of oxidative stress,in which the damaging effects of reactive oxygen species(ROS)exceed the ability of biological systems to neutralize the oxidizing agents and to repair cellular damage.Studies in human subjects and animals have demonstrated that oxidative stress and the associated endothelial dysfunction significantly correlate to the classic cardiovascular risk factors such as hypercholesterolemia,diabetes mellitus and chronic smoking,and also play important roles in the development of vasculopathies,including atherosclerosis,hypertension,restenosis after angioplasty,angiogenesis,cardiac injury associated with post-ischemic reperfusion and heart failure.Subclinical hypothyroidism(SCH)is characterized by elevated serum thyroid stimulating hormone(TSH)levels with normal serum thyroid hormone concentrations.It is a common thyroid disease with a prevalence ranging from 4%to 20%in adults,and the prevalence is progressively increasing.Although defined as an asymptomatic state,SCH is proposed to lead adverse consequences including systemic hypothyroid symptoms,neuromuscular dysfunction,progression to overt hypothyroidism,hypercholesterolemia and cardiovascular dysfunction.The higher serum TSH level has been recognized as a nonconventional risk foctor responsible for the increased risk of cardiovascular diseases(CVD)in SCH.Although with strong positive relationship to increased markers of oxidative stress and endothelial dysfunction in serum,the effects and underlying mechanisms of elevated TSH on ECs still remain to be established.Although with lower content,mitochondria are crucial in mainting redox homeostasis and cellular functions of ECs.Imbalance of redox state in mitochondria leads to excessive generation of mitochondrial ROS,which serve as kindling radicals to activate nicotinamide adenine dinucleotide phosphate(NADPH)oxidases,xanthine oxido-reductase and uncoupled endothelial nitric oxide synthase(eNOS),with endothelial dysfunction as a result.The mitochondrial permeability transition pore(mPTP)is a key regulator of mitochondrial homeostasis.Transient opening of the pore is important for release of ions and metabolites to maintain mitochondrial health.However,under extreme stress,excessive activation of mPTP can be triggered to aggravate mitochondrial degeneration,leading to matrix expansion and mitochondrial membrane rupture,which dissipates the mitochondrial membrane potential,deregulates Ca2+ homeostasis,generates and releases pathological mitochondrial ROS.Cyclophil:in D(CypD),a protein with peptidyl-prolyl cis-trans isomerase(PPIase)activity,is a critical regulator of the mPTP opening.It resides in the mitochondrial matrix in resting state.Once activated,CypD translocates to the mitochondrial inner membrane and binds to the mPTP constitutes,for instance ANT or F1F0 ATP synthase,to facilitate mPTP opening.Although Marcu,R et al.confirmed the effect of CypD on endothelial proliferation and angiogenesis,it remains to determine whether CypD-mediated mPTP opening and altered mitochondrial redox state are involved in the effects of TSH on ECs.Here we report for the first time that elevated TSH triggers mitochondrial oxidative stress in ECs and shed light onto the crucial role of CypD in modulating TSH-induced mitochondrial and endothelial perturbations.Objectives:1.To observe that whether elevated serum TSH could induce oxidative damage in endothelial cells and mitochondria.2.To observe whether CypD mediates TSH-induced oxidative damage in endothelial cells and mitochondria.3.To demonstrate the effect of TSH on CypD and confirm the related signaling pathways.Methods:1.Serum samples of SCH and euthyroidism subjects were obtained from the epidemiological research project from 2013-2014(Effects of L-thyroxine Replacement on Serum Lipid and Atherosclerosis in Hypothyroidism,NCT01848171).2.General information of the human subjects,including age,gender,past history and drug use,is recorded through questionnaires.Physical examination was used for collection of height,weight,waist circumference,hip circumference,blood pressure and so on.Venous blood samples were drawn between 8:00 A.M.and 10:00 A.M.after a minimum 10-hour fasting.Lipid profiles and fasting plasma glucose(FPG)were quantified using a BECKMAN Chemistry Analyzer AU5800 System(Beckman Coulter,Tokyo,Japan).Serum free triiodothyronine(FT3),free thyroxine(FT4),and TSH levels were measured by chemiluminescence methods(Cobas E601;Roche,Basel,Switzerland).3.Animal models.3.1 Generation of TT-KO mice.We utilized a Cre/LoxP strategy to yield thyroid-specific TSH receptor(TSHR)-knockout(TT-KO)mice.Tshrflox/flox mice were crossed with the heterozygous mice expressing TPO-driven Cre recombinase.Mice homozygous for the floxed gene and heterozygous for TPO-Cre(TT-KO)were used as experimental animals.Treatments to TT-KO mice.After discontinuing breast feeding at 4 weeks old,all male TT-KO mice were supplemented with exogenous T4(Sigma)to keep normal thyroid hormone levels.Mice aged 9-10 weeks were subcutaneously injected with freshly prepared TSH(TT-KO+TSH,7 mIU/g·d,Sigma)or solvent(TT-KO+solvent,same volume as TSH)for additional 2 weeks before sacrificed.To evaluate the consequences of pharmacological blocking of CypD in vivo,we injected cyclosporin A(CsA,15 mg/kg-d,Sandimmune,Novartis)or PBS(same volume as CsA)to TT-KO mice for 4 weeks prior to TSH or solvent co-injection for another 2 weeks(TT-KO+solvent,TT-KO+TSH,TT-KO+CsA,TT-KO+CsA+TSH).3.2 Tshr-/-mice.Tshr-/-mice and the littermate male Tshr+/+ mice were obtained by breeding heterozygous mice.After discontinuing breast feeding at 4 weeks old,male Tshr-/-mice were supplemented with exogenous T4 to keep normal thyroid hormone levels.3.3 CypD KO mice.CypD KO and wild type mice were got by breeding heterozygous mice.4.Cell culture Human umbilical vein endothelial cells(HUVECs,ATCC)and Human aortic smooth muscle cells(HA-VSMCs,ATCC)were cultured according to manufacturer's instructions.5.Genetic knockdown of CypD For the genetic knockdown of CypD,HUVECs were transfected with PPIF-shRNA,and cells transfected with ADV1-NC were used as controls.6.Serum endothelin-1(ET-I)levels of humans and serum TSH levels of mouse models were measured using Elisa Kits.7.Serum total T4 levels of mouse models were measured by total T4 RIA Kits.8.Whole body metabolic status of mice Mouse whole body metabolic status,indicated by oxygen consumption(VO2),carbon dioxide production(VCO2),heat production,physical activity and food intake were measures by the metabolic chambers(PhenoMaster,TSE Systems,Germany).9.Vasodilation measurement We adopted acetylcholine(Ach)or sodium nitroprusside(SNP)to the mouse mesenteric arteries for the measurement of endthelium dependent or independent vasodilation,respectively.10.Intracellular oxidative stress was measured by 2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA)staining.11.Mitochondrial superoxide in nouse aorta,HUVECs and HA-VSMCs were measured by MitoSox Red(Invitrogen)staining.12.Mitochondrial membrane potential were measured by tetramethylrhodamine methyl ester(TMRM,Invitrogen)staining.13.We adopted immunofluorescence staining for the measurement of ET-1 and 8-hydroxydeoxyguanosine(8-OHdG)in mouse aorta and HUVECs.14.Nitrate reduction was used as an indirect measurement for nitric oxide released from HUVECs.15.Western Blotting analysis was adopted for the measurement of eNOS,p-eNOS,Akt,p-Akt,catalase,CypD,AMPK,p-AMPK and SIRT3 proteins.16.Co-immunoprecipitation(CoIP)was used to measure the lysine acetylation of CypD.17.Measurement of mitochondrial swelling Freshly isolated mitochondria were suspended in swelling assay buffer.Mitochondrial swelling was immediately recorded with addition of calcium(1?mol/mg protein)on SpectraMax M2/M2e(Molecular Devices)for 11min at 540nm.18.Measurement of mitochondrial respiratory chain complexes activities For enzymatic assays of respiratory chain complexes ?-?,sufficient mitochondria of tissues or cells were isolated.The spectrophotometric kinetic assays for mitochondrial respiratory chain enzymatic activities were performed by using a microplate reader(Molecular Devices,USA).19.Mitochondrial oxygen consumption rate(OCR)was measured using the XF96 Analyzer(Seahorse Bioscience).20.Adenine ucleotide levels were determined by high-performance liquid chromatography(HPLC).Energy charge =[ATP + ADP/2]/[ATP + ADP +AMP].21.Statistical analysis All statistical analyses were performed using SPSS version 22.0 for Windows(Chicago,IL,USA).Values for quantitative data are expressed as mean ±standard deviation,or median(inter-quartile range).The relationship between TSH and ET-1 levels was performed by simple linear regression analysis.Differences between means were compared using One-Way ANOVA(Dunnett's t or LSD test)or Independent-Samples T Test.Differences were considered significant at p<0.05.Results:1.Serum TSH levels positively correlated to ET-1 levels in humans with SCHIn SCH patients with TSH>= 10 mIU/L,serum TSH levels positively correlated to ET-1 levels at baseline.Moreover,when TSH declined to normal levels in these subjects after LT4 therapy,serum ET-1 levels were significantly reduced.2.TSH impaired endothelial functions with enhanced oxidative stressWe confirmed increased ET-1 expression and oxidative stress(measured by DCFH-DA staining)in aorta of TT-KO+TSH mice.Mesenteric arteries of TT-KO+TSH mice were less responsive to Ach,indicating that TSH inhibited endothelium-dependent vasodilation.Interestingly,Ach elicited similar relaxation response in TT-KO groups when arteries were pretreated with N-nitro-L-arginine methyl ester(L-NAME,the inhibitor of eNOS).While compared to Tshr+/+ mice,Tshr-/-mice significantly resisted to TSH-induced decline of endothelium-dependent vasodilation.Moreover,we confirmed that elevated TSH per se significantly impaired endothelial functions in vitro.TSH dose or time dependently increased ET-1 expression,decreased eNOS and Akt activities and inhibited NO release.Besides,our data demonstrated that in cultured HUVECs,elevated TSH levels also reduced catalase expression,increased intracellular oxidative stress and DNA oxidative injury in a dose-dependent manner.3.Elevated TSH induced excessive mPTP opening and mitochondrial oxidative damages in aorta and ECsAortic mitochondria from TT-KO+TSH mice showed a greater mitochondrial swelling than the ones from TT-KO+solvent mice,indicating excessive mPTP opening in TT-KO+TSH mouse aorta.Besides,elevated serum TSH levels resulted in mitochondrial depolarization(TMRM staining)and significantly increased aortic mitochondrial ROS generation(MitoSox Red staining)in TT-KO+TSH mouse aorta.In cultured HUVECs,elevated TSH significantly increased sensitivities of mPTP to Ca2+ overload.Mitochondrial respiratory complex I-III activities were significantly reduced,paralled with notable increment in mitochondrial ROS production.Besides,mitochondrial oxygen consume rates(OCR)relating to basal respiration,ATP production and maximal respiration were markedly blunted with TSH exposure.4.Blocking cyclophilin D attenuated TSH-induced mitochondrial and endothelial perturbationsTo confirm the specific effects of CypD in the process of TSH-induced mitochondrial and endothelial perturbations,we reduced CypD expression in HUVECs with PPIF shRNA transfection and cells transfected by ADV1-NC were used as controls.CypD deficient ECs displayed significant resistance to TSH-induced mitochondrial ROS over-production.Besides,the impaired mitochondrial OCR were markedly ameliorated.Moreover,CypD defeciency attenuated TSH-triggered endothelial perturbations,exhibiting as lowered ET-1 expression and increased eNOS phosphorylation at Ser1177.Compared to those from their WT littermates,mesenteric arteries from CypD KO mice significantly resisted to TSH-induced decline of endothelium-dependent vasodilation.To evaluate the consequences of pharmacological blocking of CypD,we adopted 1 ?mol/L CsA to the cultured HUVECs 1 hour before TSH co-incubation for 24 hours.Results showed that CsA pretreatment not only attenuated TSH-triggered excessive mPTP opening and mitochondrial ROS generation,but also ameliorated mitochondrial OCR.ATP production and energy charge were reduced by TSH,both of which were reversed by CsA pretreatment.Functionally,CsA reversed TSH-induced catalase reduction and ET-1 increment.In vivo,we found that pretreatment with CsA(15 mg/kg-d)attenuated TSH-triggered mitochondrial swelling in mouse aorta.Besides,TSH-induced decreased aortic mitochondrial complex I and III activities were reversed by CsA and the excessive ROS production in aortic mitochondria was accordingly reduced.Furthermore,pretreatment with CsA inhibited TSH-induced excessive ET-1 expression in aorta and endothelium.Pharmacological inhibition of CypD did reverse TSH-induced decline of endothelium-dependent vasorelaxation.5.TSH enhanced CypD acetylation via inhibiting AMPK/SIRT3 signaling pathwayIn comparison with the control group,TSH exposure did't significantly increase CypD expression,but did markedly elevate CypD acetylation.TSH inhibited AMPK phorphorylation at Thr-172 and decreased SIRT3 expression in dose-dependent manner.With AICAR(the activator for AMPK activation)co-incubation,HUVECs exposed to TSH showed enhanced AMPK phosphorylation(Thr-172)and SIRT3 expression,leading to a reversal in the increment of CypD acetylation caused by TSH exposure.Conclusions:1.Elevated TSH levels lead to decreased endothelium-dependent vasodilation and the mitochondrial oxidative stress is the underlying mechanism.2.CypD mediates TSH-induced mitochodnrial and endothelial perturbations.3.TSH enhanced CypD acetylation via inhibiting AMPK/SIRT3 signaling pathway in ECs.