| Part I The Correlation Between Peripheral Blood HCMV-MIR-UL112-3p Expression And Carotid Artery IMT1 BackgroundAtherosclerosis is a progressive disease characterized by the accumulation of lipid and fibrous components in the arteries.Inflammation plays an important role in the development of atherosclerosis and is the most important cause of cardiovascular diseases,including stroke,myocardial infarction,heart failure and hemangioma.Epidemiological studies have shown that there are many risky factors for atherosclerosis.However,the pathogenesis and etiology of atherosclerosis are not completely clear.Human cytomegalovirus belongs to herpes virus beta subfamily and is a double-stranded DNA virus.The positive rate of adult serum antibodies is over 60-90%worldwide.Like all herpes viruses,once infection occurs,HCMV will establish a lifelong latent infection in the host(salivary glands,white blood cells,etc.).Latent infection is often activated intermittently and reactivated.HCMV infection in vascular system is correlated with cardiovascular diseases such as atherosclerosis,restenosis and graft atherosclerosis.HCMV can aggravate the inflammatory response of diseased vessels by altering the expression of cell adhesion molecules,inducing endothelial dysfunction and interfering with cytokine signals.Ganciclovir,an antiviral drug,has been shown to reduce atherosclerosis associated with heart transplantation.HCMV encodes at least 26 mature mi RNAs,but the relevance of these in clinical pathologies remains uncertain.Hcmv-mi R-UL112-3p(mi R-UL112-3p)is the most widely studied type among HCMV encoded mi RNAs and can target both cellular and viral transcripts.Previous study indicates that mi R-UL112-3p is the only circulating HCMV mi RNA highly expressed in hypertensive patients,and this HCMV mi RNA was associated with increased risk of hypertension.Previous studies also showed that plasma HCMV Ig G or anti-CMV antibody level was associated with atherosclerosis.However,other clinical studies have not found an association between HCMV infection and atherosclerosis.In this study,we aimed to detect the mi R-UL112-3p and HCMV Ig G levels in the plasma/serum of patients assessed the relationship between HCMV infection and atherosclerosis.2 ObjectivesThe correlation between carotid IMT and inflammatory cytokines in peripheral blood and levels of HCMV Ig G,Ig M and mi R-UL112-3p in patients with atherosclerosis was analyzed retrospectively,and the correlation between HCMV infection and atherosclerosis was evaluated.3 Methods3.1 Research object and detection methodA total of 458 participants were prospectively enrolled from September 2012 to June2016 at the first affiliated hospital of Anhui medical university.Clinical datas were collected,including age,sex,duration of diabetes mellitus,body mass index(BMI),smoking,and history of hypertension.Blood samples were taken to test fasting blood glucose(FBG),postprandial blood glucose(PBG),total cholesterol(TC),triglycerides(TG),high-density lipoprotein cholesterol(HDL-c),and low-density lipoprotein cholesterol(LDL-c).Blood samples were also used for determining HCMV infection,including HCMV mi R-UL112-3p,Ig G and Ig M.Patients with diagnosed malignant disease,auto-immune disease other than diabetes mellitus,or immunosuppression were excluded.The IMT was measured to determine the extent of subclinical carotid atherosclerosis,using carotid ultrasonography(US),which was performed by a single ultrasonographer.Anti-HCMV Ig M and Ig G levels were determined by use of commercial ELFA test kits.Taq Man mi RNA assays were performed using Taq Man primers,specific for mi R-UL112-3p.Serum levels of TNF-?,IL-6,and IL-1?were determined using a commercially available specific enzyme-linked immunosorbent assay(ELISA)kit(R&D Systems),according to the manufacturer's directions.3.2 Statistical AnalysisEstablishment of database by Excel 2007,Statistical analyses were performed using the SPSS 18.0 for Windows.All variables are expressed as the mean±standard deviation(SD),unless otherwise indicated.The primary outcome measure was a categorical variable‘high'IMT,defined as the upper value of the mean IMT.Categorical variables were compared by?2 analysis.Continuous variables were compared by Student's t-test and the Mann–Whitney U test.To determine independent risk factors and the odds ratio(OR)for high IMT with confidence intervals(CIs)set at 95%,a multiple logistic regression model was used,including the variables of the positive rate of circulating hcmv-UL112-3p,HCMV Ig G titer,Framingham score,and the value of Log(IL-1?),Log(TNF-?),and Log(IL-6),all of which differed with a P<0.05 in univariate analysis,ensuring the absence of significant multicollinearity.Single linear univariate correlations(Pearson's correlation coefficients)and stepwise multivariate regression analyses were performed to evaluate the relationships between the value of the positive rate of circulating hcmv-UL112-3p or anti-HCMV Ig G titer and other variables.P values?0.05 were considered statistically significant.4.ResultsA total of 458 participants were included in the study,247(53.9%)of the 458participants were in the group of high IMT.Multivariate logistic regression analysis revealed four independent risk factors of high IMT:the positive rate of circulating hcmv-UL112-3p(OR 1.05,95%CI 1.01-1.07;P=0.004),higher anti-HCMV antibody titer(OR 1.04,95%CI 1.01-1.06;P=0.003),higher Framingham score(OR1.14,95%CI 1.02-1.27;P=0.018),and higher IL-1?concentrations(OR 2.96,95%CI 1.26-6.97;P=0.013).Circulating hcmv-UL112-3p had a significantly positive correlation with max-IMT(r=0.328,P<0.001),free T4 levels(r=0.247,P=0.032),and Log(TNF-?)(r=0.509,P<0.001)inmultivariate correlation analysis.5.ConclusionsIn a word,this study has clinical significance since it is the first report to evaluate the relationship between carotid atherosclerosis and the prevalence of mi R-UL112-3p in Asian population.These findings provide insights into the important role of HCMV in the pathogenesis of atherosclerosis and chronic inflammation.Part II Human Cytomegalovirus Promotes The Activation Of TGF-?1 In Human Umbilical Vein Endothelial Cells By MMP-2After Endothelial Mesenchymal Transition1.BackgroundHuman cytomegalovirus(HCMV)belongs to the?-herpesvirus family and is a double stranded DNA virus.HCMV infection is very common all over the world,and the virus will remain in the body for the rest of one's life once infected.It has been found that HCMV is associated with a variety of cardiovascular diseases such as atherosclerosis(AS),coronary heart disease and hypertension.HCMV infection is also related to the dysfunction of vascular endothelial cells.Vascular endothelial cells(VEC)are an important regulatory factor that can participate in a variety of physiological and pathological processes of cardiovascular disease.Recent studies have shown that endothelial dysfunction and its associated cardiovascular diseases can be induced through a process known as endothelial mesenchymal transition(End MT).In the process of End MT,endothelial cells(ECs)lose specific endothelial markers such as vascular endothelial cadherin(VE-cadherin)and leukocyte differentiation antigen 31(CD31),obtain the mesenchymal cell markers alpha smooth muscle actin(?-SMA),fibroblast specific protein 1(keratin)and fibronectin,and acquire migratory,invasive and proliferative phenotypes.The occurrence of End MT is regulated by many cytokines,TGF-?1 is the key cytokine for the development of End MT and an important cytokine in vascular pathophysiology.Increased TGF-?1 can promote the occurrence of cardiovascular diseases,such as pulmonary hypertension(PAH)and atherosclerosis,and blocking the expression of TGF-?1 can inhibit the PAH process and the formation of unstable AS plaques.After 5hours of CMV infection in human fibroblasts,the promoter of TGF-?1 could be activated by HCMV IE gene,which increased the expression of TGF-?1 by 4times.The activation factors of latent TGF-?1 mainly contain protease(plasmin),metalloproteinase(MMPs),thrombospondin(TSP-1),?v?6 and?v?8.Thus,HCMV infected vascular endothelial cells may be involved in the pathogenesis of AS by inducing the expression or activation of TGF-?1.2.ObjectivesTo observe the effect of HCMV infection on End MT of vascular endothelial cells and discuss the possible molecular mechanism of HCMV infection in the occurrence and progression of atherosclerosis.3.Methods3.1 Cell and Virus CultureHUVECs(Human Umbilical vein endothelial cells)were cultured with Human Endothelial serum-free medium containing b FGF(20 ng/ml),EFG(10 ng/ml)and human plasma fibronectin(10?g/ml)(Life Technologies,Carlsbad CA,USA)at37?.HELF and mink lung epithelium cells were cultured with DMEM containing 10%fetal bovine serum(Life Technologies,Carlsbad CA,USA)at 37?.The HCMV TR virus strain was cultured in HELF cells.The supernatant of the viral culture was centrifuged at 4?,16000×g for 2 h,and the virus was re-suspended with Human Endothelial-SFM and stored at-80?.The virus was placed in a crosslinked chamber(Bio-Rad,Hercules CA,USA)and irradiated with 150 m J UV rays to inactivate the virus.3.2 Virus Titer DetectionIn 96-well cell culture plate,2.5 x 10~4HELFs were added to each hole for overnight incubation.The next day,100?l diluted virus suspension was added to each pore and incubated overnight at 37 C and 5%CO2 incubator.The next day,the culture medium was discarded and washed with PBS three times.Absolute ethanol was added to each pore for 50?l and fixed at room temperature for 20 minutes.Absolute ethanol was discarded and immediately hydrated with PBS for 15 minutes.Wash 3 times with PBS,add 20?l diluted HCMV IE monoclonal antibody to each pore,incubate at room temperature for 2 hours.Wash 3 times with PBS,add 20?l diluted goat anti-mouse Ig G-FITC into each hole,incubate at room temperature for 1 hour.Wash 3 times with PBS,add 70%glycerol 20?l prepared with PBS into each hole,and observe under inverted fluorescence microscope.3.3 Immunofluorescence stainingHCMV TR virus was inoculated in the HELF and HUVECs in 24-well plates on coverslips according to MOI=1 with or without recombinant active TGF-?1(ra TGF-?1,15 ng/ml)treatment for 5 d.The cells were fixed with 4%paraformaldehyde and permeabilized with 0.1%Triton X-100.They were incubated at 4°C overnight after addition of appropriately diluted primary antibodies.Then,they were washed and incubated at room temperature for one hour with Alexa Fluor 488 or Alexa Fluor 594labeled secondary antibodies.Then,they were incubated at room temperature for 15min with Alexa Fluor 488 labeled phalloidin and DAPI,and the slides were mounted after washing and observed under a fluorescence microscope.3.4 Western blotting analysisThe cells were harvested and lysed with radioimmunoprecipitation assay(RIPA)lysis buffer on ice for 30 min with shaking at 12 000 rpm/min.Total cellular protein was collected,and the concentration was determined by a BCA Kit.Equal amounts of protein(25?g/well)were separated by 10%SDS-PAGE electrophoresis.Then,it was electrotransferred to the PVDF membrane.After the transfer,the PVDF membrane was rinsed with TBS for 10 to 15 min,placed in TBS/T blocking buffer containing 5%(w/v)skimmed milk powder and shaken at room temperature for 2 hours.It was incubated at4°C overnight after adding primary antibodies at the appropriate dilution.Then,the membrane was washed with TBST three times(for 5 minutes each time).The membrane was incubated at 37°C for one hour with HRP labeled secondary antibody(1:10000),diluted with TBST containing 0.05%(w/v)skimmed milk powder.The gel was developed with ECL for 5 minutes.The protein bands were quantified using an Imagequant LAS4000.3.5 RNA extraction and Reverse transcription polymerase chain reactionTotal RNA was extracted from different groups using the RNeasy kit according to the manufacturer's manual.Their concentration and purity were detected with an Agilent2100 Bioanalyzer.1?g RNA was subjected to reverse transcription using the RT2 First Strand Kit.Real-time PCR was performed using the Fast Start Universal SYBR Green Master.RT-PCR primers were purchased from ABI.Reaction parameters were 95°C for45 sec,95°C for 5 sec and 60°C for 30 sec,with 40 cycles.The relative expression of genes was calculated using 18S RNA as control.The expression levels of TIMP-2,MT1-MMP and MT3-MMP m RNA in different treated HUVEC cells were detected by RT-PCR methods,they were performed using Platinum?Taq Green Hot Start DNA Polymerase according to the instructions.3.6 TGF-?1 content detectionThe contents of total TGF-beta 1 and activated TGF-beta 1 in the supernatant of mink pulmonary epithelial cells were determined by bioassay and ELISA kit,respectively.3.7 ImmunoprecipitationThe cells were harvested by centrifugation and lysed with pre-cooled RIPA lysis buffer containing protease inhibitor.MMP-2 antibody was added to the cells,and they were incubated at 4?overnight.Then,the protein A-agarose was added and incubated with them,and they were lysed with pre-cooled RIPA lysis buffer.The cells were re-suspended,SDS-PAGE loading buffer was added to them,and the resulting solution was boiled.Electrophoresis was carried out with 8%SDS-PAGE gel.TIMP-2,MT1-MMP and MT3-MMP were detected in the cell lysate before and after immunoprecipitation by the Western blot method.3.8 sh RNA transfectionHUVEC cells were transfected with MMP-2 sh RNA and its control plasmid via Amaxa cell line Nucleofector Kit V.The transfection was successful when the cells showed obvious green fluorescence under fluorescence microscope 24 hours later.The transfection steps were referred to the kit instructions.3.9 Statistical analysisThe data are expressed as the mean values±standard error mean(SEM).All analyses were conducted using Prism 5.0 software.Differences among groups were assessed using one-way analysis of variance(ANOVA)and Student's T test.P values<0.05 were considered an indicator of a significant difference.4.Results1)HCMV can proliferate in HUVECs but is not affected by ra TGF-?1;2)HCMV can infect HUVEC cells with End MT induced by TGF-?1;3)HCMV could induce the activation of TGF-?1 in HUVECs with End MT;4)The amount of newly synthesized activated TGF-?1 was positively correlated with the infective dose of TGF-?1 and HCMV;5)MMP-2 is involved in the up-regulation and activation of TGF-?1 in vascular endothelial cells with End MT induced by HCMV infection.5.ConclusionsIn summary,this study showed that HCMV could infect HUVECs with EndMT induced by TGF-?1.HCMV infection could enhance the autocrine release of TGF-?1 in vascular endothelial cells by an MMP cascade reaction.These findings suggested that HCMV infected arterial endothelial cells may contribute to fibrosis by activating TGF-?1,which is involved in cardiovascular disease. |
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