| 1 IntroductionPulmonary arterial hypertension (PAH) is a life-threatening disease, contributing to the morbidity and mortality of the patients with various lung and heart diseases. PAH has a multifactorial pathology and the pathogenesis remains poorly understood. It has been reported that a variety of cell types, including endothelial cells, smooth muscle cells, as well as inflammatory cells and platelets, may be implicated in the progress of PAH. Vascular smooth muscle cells (VSMCs) exist in the medial wall of blood vessels, which are normally quiescent and express a differentiated phenotype to maintain vascular tone. Under pathological conditions, VSMCs switch to a "synthetic" phenotype, secret inflammatory cytokines and contribute to the vascular pathogenesis. Now, VSMCs proliferation of pulmonary artery has been considered as one of the major causes for pulmonary arterial remodeling. While progressive pulmonary arterial remodeling is the characteristic of PAH, which plays a central role in the persistent deterioration and the difficult reverse of disease.The role of immune system in the progress of PAH has received considerable attention in recent years. However, how the immunomodulatory mechanisms might contribute to the pathogenesis of the disease have not been fully illuminated. CD4+CD25+ regulatory T cells (Tregs), as a specific subpopulation of T cells, participated in the regulation of immune response and controlled the progression of autoimmune diseases. While, deficiency or dysfunction of Tregs would disrupt immune homeostasis and lead to many pathological conditions.Few therapies may be effective in pulmonary arterial structure remodeling and following development of PAH. Tregs have been documented exert a benefit effect in the progress of many disease. However, the role of Tregs in the development of PAH remains unknown. In the present study, we aimed to investigate whether Tregs affected the development of PAH and investigate the underlying mechanism.2 Objectives(1) To assess the beneficial effects of Tregs on inflammation and remodeling of pulmonary artery in hypoxic mice.(2) To elucidate the inherent molecular mechanisms of Tregs-mediated beneficial effects on pulmonary hypertension induced by hypoxia.3 Methods3.1 AnimalsSixty male C57BL/6J mice (10 weeks) were randomly divided into three groups with twenty mice in each group: normoxia group, control group and Tregs group. The mice in normoxia group were maintained in a room air and exposure to normoxia environment. While the mice in control group and Tregs group were exposure to hypoxic conditions (10% O2) used a liter ventilated chamber (Flufrance apparatus, Cachan, France) for four weeks. All animals were kept in the same room and had access to standard mouse chow and water. All animal procedures were reviewed and approved by the Animal Care and Use Committee of Shandong University.3.2 Isolation and adoptive transfer studySpleens were isolated from the mice and a single cell suspension was prepared. Then, purified Tregs were isolated with CD4+CD25+ Regulatory T cell Isolation Kit (Miltenyi Biotec). The average purity of Tregs was>97%. Before the one day of exposure to hypoxic conditions, the mice in control group and Tregs group were injected with PBS or Tregs (106 cells) through the tail vein, respectively.3.3 Hemodynamic MeasurementsThe mice were anesthetized with intraperitoneal ketamine (6mg/100 g) and xylazine (1 mg/100 g). Then, the mice were placed in a supine position and the trachea was cannulated. A 26-gauge needle was passed percutaneously into the thorax via a subxyphoid approach and the right ventricular systolic pressure (RVSP) were measured and recorded using a miniature pressure transducer digitized by a data acquisition system. During the operation, the mice breathe room air spontaneously and the heart rate was maintained between 300 and 600 beats/min.Following measurement of RVSP, The right venteicle (RV) was isolated from the left venteicle (LV) and the septum (S) and weighed, respectively. Then, the Fulton’s index (RV/LV+S) was calculated.3.4 Lipid profileSerum levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) were determined with an enzymatic assay.3..5 Cell cultureHuman peripheral blood mononuclear cells (PBMCs) were prepared from healthy volunteers and Tregs were isolated from PBMCs by use of a CD4+CD25+ Regulatory T cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s protocol for the further experiment.Human pulmonary artery smooth muscle cells (HPASMCs) were cultured in DMEM containing 10% fetal bovine serum (FBS) at 37℃ in a 5% CO2 and 95% air atmosphere. The cells at up to passage 4 were used for the subsequent experiments. HPASMCs were placed in hypoxic conditions (1% O2,5% CO2) in a cell culture pretreated without T cells (control group) or with Tregs (5×105/well, Tregs group) in the presence of anti-CD3 antibody (50ng/ml) at 37℃ for 48 hours referred prior study. At last, floating T cells were discarded, HASMCs were collected.3.6 Measurement of HPASMC proliferationThe proliferation of HPASMCs was determined by MTT assay. Briefly, HPASMCs were seeded in 96-well plates at a density of 5,000 cells each well. Following exposure to hypoxic conditions without or with Tregs treatment, HPASMCs were incubated with 10 uL MTT (5mg/mL)/well reagent for 4 h at 37℃. The supernatant was carefully removed, and 75 μL/well dimethylsulfoxide (DMSO) was added to dissolve the formazan crystals. Then samples were analyzed at 570 nm by use of a Varioskan Flash multifunction plate reader (Thermo Scientific, MO, USA).For cell counting, HPASMCs were seeded in a 6-well plate at the same density, and then treated with the above conditions. At the end of experiment, cells were washed with PBS, harvested with trypsin, and counted with a hematocytometer.3.7Quantitative real-time PCRTotal RNA was extracted from the lungs of the mice,or HPASMCs. In the in vivo experiment, the mRNA expression of Foxp3,MCP-1, IL-1β, IL-6 and IL-10 was analyzed.In the in vitro experiment, the mRNA expression of cyclin D1, CDK4 and p27 was analyzed too.3.8 Western blot analysisTotal proteins were extracted from the lung of the mice or HPASMCs. The protein expression of Foxp3,MCP-1, IL-1β, IL-6 and IL-10 in the lung of the mice and cyclin Dl, CDK4 and p27 in HPASMCswere analyzed by western blot.4 Results4.1 Body weight and lipid profile studiesThere were no significant differences in body weight and serum levels of TC, TG, LDL-C and HDL-C among the three groups (data not shown).4.2 Adoptive transfer of Tregs enhanced Foxp3 expressionFoxp3 is exclusively expressed in Tregs and is a requisite factor for the maturation and function of Tregs. In this study, Foxp3 mRNA expression in the lung of the mice was measured to assess whether the intravenously injected Tregs entered the tissues. We found that Foxp3 mRNA was significantly enhanced in the mice treated with Tregs than those in control group (data not shown). These results confirmed that the exogenous Tregs entered the tissues.4.3 Adoptive transfer of Tregs improve the hypoxia-induced pulmonary hypertension and vascular remodelingThe hemodynamic parameters of mice were measured before euthanasia. Compared with normoxia group, the mice development a increases in RVSP expose to hypoxia. However, after Tregs treatment, the RVSP was significantly reduced. (P<0.05, figure 1A). Chronic severe PAH is a cardiopulmonary disease and RV hypertrophy should also be assessed. Expose to chronic hypoxia promoted right ventricular hypertrophy4. In this study, we measured Fulton’s index for assessment of the right ventricular hypertrophy. The result showed that exposure to hypoxia was associated with an increase of Fulton’s index, while the increased index was partly reversed in the Tregs group (P<0.05, figure 1B). The results suggested that Tregs could improve the hypoxia-induced pulmonary hypertension and vascular remodeling.4.4 Tregs downregulated proinflammatory cytokine expression and upregulated anti-inflammatory cytokine expression in vivoIn the mice, the mRNA expression of proinflammatory cytokine (MCP-1, IL-1β and IL-6) in the lung assessed by RT-PCR, which was significantly lower in the Tregs groups than those in the control groups (P<0.05, Figure 2A-C). The protein of MCP-1 IL-1β and IL-6 showed the similar results (P<0.05, Figure 2D-G). In addition, the levels of anti-inflammatory cytokine was also assessed, and we found the mRNA and protein expression of IL-10 was significantly upregulated in the Tregs groups than that in the control groups (P<0.05, Figure 3). These results showed that Tregs lowered proinflammatory cytokine expression and upregulated anti-inflammatory cytokine expression.4.5 Tregs reduced hypoxia induced HPASMCs proliferation in vitroWe evaluated the effects of Tregs on HPASMCs proliferation under hypoxic conditions. MTT assay showed that Tregs inhibited HPASMCs proliferation compared with the hypoxia group. The cell counting assay showed the similar results (P<0.05, figure 4). We believed that the regulation of Tregs on HPASMC proliferation might be one important mechanism of anti-PAH.4.6Tregs arrested HPASMCs in G1/GO-phase under hypoxic conditionThe cell proliferation depends on cell cycle transition from G1/G0 to G2/S-phase. In this study, we determined whether Tregs affected the cell cycle of HPASMCs. As shown in figure 5A, compared with control group, Tregs treatment arrested more percentage of HPASMCs in the G1/G0 phase (P<0.05).4.7 Tregs reduced cyclin D1 and CDK4 expression, but increased p27 expressionPrevious studies showed that cyclin D1, CDK4 and p27 played a key role in cell proliferation and cell cycle. In vitro experiment, the levels of cyclin D1, CDK4 and p27 in HPASMCs were assayed. As we expected, compared with the control group, the mRNA and protein expression of cyclin D1 and CDK4 were markedly reduced in Tregs treated-HPASMCs, while Tregs treatment enhanced hypoxia-induced p27 gene expression (P<0.05, figure 5B-H). These results showed that the regulation of Tregs on cell cycle might be another mechanism on its anti-PAH effects.4.8 Tregs decreased Akt and ERK1/2 phosphorylationIt has reported that Akt and ERK pathway are involved in HPASMCs proliferation and progression of PAH3. Thus, we assessed Akt and ERK protein expression by western blot in vitro. We found that compared with the control group, Tregs downregulated the phosphorylation of Akt and ERK, which might be another mechanism of the protection of Tregs (P<0.05, figure 6).5 Conclusion(1) Tregs protected against hypoxia induced PAH in mice.(2) The major mechanisms might involve that Tregs significantly suppressed inflammatory response, enhanced anti-inflammatory cytokine levels, inhibited HPASMCs proliferation, and regulated the cell cycle.(3) Tregs may be a new target for the treatment of PAH.1 Introduction;Pulmo打ary arterial hyperte打sio打(PAH) is 打ot just 汪 disease but a range of diseases,collectively,it is a serious threat 化 human heal化,its pathogenesis remains unclear. It is a commo打 feature of complex vascular endothelial proliferation and sustained progress in pulmonary vascular remodeling, vascular smooth muscle cell proliferatio打 plays an i打dispensable role in the vascular remodeling process. Hypoxic Pulmonary Hypertension is an important clinical subtypes,our common among patients with chronic obstructive pulmonary disease and pulmonary fibrosis often associated with hypoxic pulmo打ary hypertension, i打 which patients with ac山e exacerbation of chronic obstructive pulmonary disease reported 91% associated with pulmonary hypertension, which can further lead to right ventricular hypertrophy or right ve打tricular atte打uation,which directly determi打es the prognosis of patients.Currently there are still not sure of pulmonary hypertension treatment.Argi打ase(Argnase Arg) is acting on 化e enzymes of 化e urea cycle,ca打 be CO打verted i打to ure过,Argi打ine and ornithine, i\rgin过se 2 mai打ly in the kid打ey,vascular smootii muscle cells of 化e mi1:ochondrial matrix, it is a打oxic induce过 human pulmonary artery smoo化 muscle cell proliferation specific Arginase isoforms.Recently, more and more studies have found in hypoxic pulmonary hypertension, the Argi打ine was significantly increased and led 化 the proliferatio打 of pulmonary artery snioo化 muscle cells. Exp巧iments show 化at inh化iting 化e activity of Argmine ca打reduce pulmo打ary artery smoo化 muscle cell proliferation,reduce pulmonary artery remodeli打g,but the exact mechanism is 打ot clear. Increased expression of Arginase 2while hypoxia-induced mecha打ism is still unclear and i*equires further elucidation.Inflammatory r*espo打se i打 the pathogenesis of pulmonary hypertension has been more and more attention. There is sufficient evidence 化at hypoxia-induced pulmonary hypertension in the case, the content of IL-6 also increased significantly,so far,noreport whether IL-6 and otiier inflammatory mediators can affect 化e expi^essio打 of八rginase,which feed we propose to suppress the expressio打 of t:he enzyme Arginine can ease 化e development of hypoxic pulmonary hypertension,1:he specific mechanisms, induding inh化ition of IL-6 and o化er inflammatory mediators,can inhibit Arginase 2 expression by reducing the expression of D1 and CDK4, and partially Kciuced Akt and E民K phosphorylatio打 and other processes 化 reduce pulmonary artery smooth muscle cell proliferation, reduce pulmonary artery remodeling. Based on 化ese ideas, we designed and implemented a series of i打 vivo and in vitro experiments.2Objectives(1)In vivo test: observed Arginase inhibhoi-s on pulmonary artery remodeling.(2)I打 vitro tests: To investigate the mechanism of elevated Argi打ase a打d Arginase inWbUoirs reduce the molecular mechanisms of cell proliferation.3Methods3.1a凸imal protocal Sixty male C57BL/6J mice(10 weeks) were randomly divided into three groups wil:h twenty mice in each group: normoxia group, control group and BEC group. The mice in normoxia group were maintained in 过 room air and exposure to normoxic environment. While the mice in control group and Tregs group were exposure to hypoxic CO打ditio打s(10〇/G 〇2) used a liter ventilated chamber(Flufrance apparatus,Cachan,France) for four weeks. All animals wei*e kept i打 the same room and had access to standard mouse chow and water. All animal procedures were reviewed and approved by the Animal Care and Use Committee of Shandong University.3.2Body weight and Lipid profile Serum levels of total cholesterol(TC), triglyceride(TG), low density lipoprotein cholesterol(LDL-C) and high density lipopro1;ei打 cholesterol(HDL-C) were determined with 3H cnzym泣tic 过ss3y i打 cxpcrimc打10 weeks 3打d 4w6 cks.3.3F山l:on,s index Fulto打’s i打dex(RV / LV + S) is the propoitio打 of the left ventricle and right ventricle and interventricular septum weight, it can i:eflect the extent of the right ventricular hypertrophy,and is an important indicator to determine the right ventricular hypertrophy and attenuation. After the mice were sacrificed, the mice with ventricular septal left ventricle and right ventricle as a whole, peeled and quality were measured to calculate Fulton’s index.3.4Hist:opal:hology Preparation of hypoxic P山monary tissue sections of mice, using HE staining detect the angiogenesis and using immu打ohistochemical method wereto de1:ect the proliferation of pulmonary artery smooth muscle cells and expression of IL-6.3.5Cell C山化re The human pulmonary artery smooth muscle cells(HPASMCs) spread to the fourth generation i打 DMEM medium containing fe1:al calf serum,and o打 cultured under hypoxic conditions(1% 02, 5% C02), divided into a control group, IL-6 group, IL-6antagonist group,A民G interference group and 良EC groups. IL-6 was added into IL-6intervention group,IL-6 antagonist group 如id BEC groups were added 化 IL-6 receptor antagoni巧 and BEC co-culture i*espectively,the control group did not join any interve打tio打 agents,they also cultured for 48 hours; AJRG interference group A民G2added i打 tArgeting siRNA,cultured for 120 hours.3.6Measurement of HPASMC prol瓜ration The proliferation of HPASMCs was determined by MTT assay.Briefly, HPASMCs were seeded in 96-well plates at a density of 5,000 cells each well. Following exposure1:0 hypoxic con出tions without or with intervention agents. Then samples were analyzed at 570 nm by use of 过 Varioskan Flash multifunction plate reader.For cell coimti打g,MPASMGs were seeded i打 a 6-wdI plate 过t th目 same de打sity,and the打treated with the above conditions. At the end of experiment, cells were washed with PBS,harvested with trypsin,a打d counted with a hematocytometer.3.7Arginase kinetics.To de化ct the activity of Arginase, we lysed the cells produce a mixture of smooth muscle samples. Described in the librattire(Am J Physiol Limg Cell Mol Physiol 2009Dec; 297(6):.. LI 151-9) The me1;hod fbr 也e sample wil:h a colorimetric determination of urea. Each sample(300 jil) was added to 1.5-ml chromogenic reagent [5 mg of thiosemicarbazide,250 mg of diacetyl monoxime,and 25 mg of FeCl];n 150 ml of16.7%(vol/vol) H2SO4 and 13.3%(vol/vol) H3PO4]. The mixtures were vortexed,boiled at 100〇C for 5 mi打,and 化en cooled to room temperature. Each sample wi化化e added chromogenic reagent(200 |xl) was pipetted into a microplate;n duplicate, and the diffei’e打ce i打 absorbance at 530 nra was compared with a urea sta打dard curve. The Arginase activi巧 was calculated as amount of urea(limo^/min, and velocity was determmed as |imol/(mg protein/min). The effects of Arginase inh化ition were assessed by adding ei化er vehicle or 化e 八rginase inhibiU)r,BEC(1 mM),to L-Arginine(ImM),and the assay was performed as described above. L-Arginine at 1mM were used because hypoxia-induced Arginase activity was augmented at these CO 打 cen 化 atio 打 s.3.8 siRNA For expliciting the effects of Arginase 2 gene silencing on the proliferation of hPASMC,we use DharmaFECT I reagent instructions, fbr Arginase2 RNA interference(si民NA),.In control group,Saline ca打 be added in place of siRNA.hPASMC put in 6-well plates using PBS. siRNA-DharmaFECT mixture(1 ml), and fresh medium(1 ml) were placed in 6-weH plates,and cultured u打der hypoxic oxygen24 hours. Removing the culture medium, the cells were washed, added SmGM,incubated 24 hours in hypoxia of oxyge打,then 也e cell count or Wesl;em blot analysis of proteins extracted row.义9 RT- PCR Total RNA was extract;ed from the lungs of the mice,or HPASMCs. In the m vzVo experiment, the mRNA expression of ARG2,cyclin Dl,CDK4,p27 was analyzed.In the in vitro experiment, the mRNA expression of IL-6, ARG2, cyclin Dl,CDK4, p27 was analyzed 1:oo.3.10 Western blot Total prot;eins were extracted from the lung of the mice or HPASMCs. The pro1:ei打e邵ression ofARG2, c:yclin Dl, CDK4, p27 in 化e lung of 化e mice and比-6, ARG2,cyclin D1, CDK4, p27, Akt,ERK in HPASMCswere analyzed by western blot.4 民 esults4.1 Body weight and lipid pro挂e shidies There were no sig打ificant differences i打 body weight and serum levels of 了C, TQ LDL-C and HDL-C among the three groups(data not shown).4.2. BEC inhibits pulmonary artery smooth muscle cell proliferation in mice HE staining of histopathologic analysis showed; nonnoxic pulmonary artery wall thickening did not change sig打ificantly in 也e control group and 化e pulmonary artery walls are 也ickened when BEC occurs. The extent of wall thickening was significantly higher than the control group BEC group(P <0.05) and normoxic group(P <0.05), theexl:ent of wall thickening i打 BEC group than i打 the normal group(P <0.05).Pa1;hological a smool:h muscle tissue sections were stained analysis: the degree of smoo也 muscle 化ickening i打化e control group was significantly higher 化an BEC group(P <0.05) and normoxic group(P <0.05),the extent of wall thicke打 in control group is higher 化an BEC group(P <0.05). Intima and adventitia hypoxic pulmonary vascular,IL-6 expre巧io打 in group was increased,relative 化 the control group,化e increasing was significa打tly(P <0.05),whereas there wos no significant diffei*ence relative 1;o 化e BEC group(P = 0.09).4.3BEC inhibited 化e expression of Arginase I打 vivo experiments, we fbund by 民T-PC艮 me化od,m the lung tissue of mice of BEC group,the expressio打 ARG2 mRNA significantly was lower 化an 化at of control group,while the control group ARG2 mRNA of expression was higher than that of noraioxic group, but no significant difference betwee打 the BEC group and 打ormoxic group; extracted by the expression level of the pro1:ein i打 lung tissue A民G2 also similar results. This indicates 化at,under hypoxic conditions,化e level of expression of mouse AHG2 is i打creased, BEC ca打 inhibit the expressio打 in lung tissue of A民G2.4.4IL-6 antagonists inh化ited the expression of Argi打ase 2I打 vivo, IL-6 Immunohi巧ochemistry shows that in and around 化e pulmonary artery,BEC group a打d the CO打trol group expressed L-6 sig打ificantly,a signi巧cant difference compared with the normal group, but no significant difference between the control group and 化e BEC group;In vitro experiments,IL-6 was added to the culture medium of pulmo打ary artery smoo化 muscle cells, extracted cells A民G2mRNA and ARG2 pro化in,compared with 化e control group,expi*ession levels were significantly increased in IL-6 group; using the IL-6 recep1:or antagonist,we fbund that 也e expression levels of protein expression and ARGlmRNA of pulmonary artery smooth mu化le cells was decreased 化an that in 化e control group. This sugg巧ts 化at IL-6 can promote 化e expressio打 of ARG2.4.5Arginase II-siRNA, reduce the expression of Arginase2 Using siRNA interference pulmonary artery smooth muscle cells,can preve打t the expression of hypoxia-induced A民G2 prcrtein. reagent useing DharmaFECT tArgeting A民G2 siRNA was tra打sfected into hPASMC incubated for 120 hours at 1% 02(hypoxia). ARG2 analysis of prcrtein expression levels were analy之ed using the Western blot method. We found that compared with the control group, ARG2於cpressio打 level w化 significantly decreased in siRNAgroup.4.6BEC can reduce hypoxia-induced right ve打tricular hypeiirophy Chronic hypoxia can lead to right ventricular hypertrophy, we measured the Fulton’s index in this study,sho^ved that under hypoxic conditions may be associated with increased Fulton’s index? In BEC group its Fuhonis index can be reduced in some degree. This result showed that hypoxia-induced BEC may improve pulmonary artery pressure and right ventricular hypertrophy.4.7BEC and siRNA A?RG2 can reduce HPASMCs proliferation caused by hypoxia,We fbund 化at comparing with hypoxia group by MTT method,BEC can inh化it the proliferation of HPASMCs, tArgeting 化 A民G2 siRNA also inh化ited HPASMCs proliferatio打 signi巧cantly, cell count analysis has also bee打 a similar conclusion. We believe BEC o打 HPASMC inMbition is an important mechanism to slow progression of pulmonary hypertension.4.8BEC arrested HPASMCs in Gl/GO-phase under hypoxic condition The cell proliferation depends on cell cycle transitio打任om Gl/GO to G2/S-phase. I打this study, we determined whether BEC affected the cell cycle of HPASMCs.Compared with control group, Tregs treatment arrested more percentage of HPASMCs in 化e Gi/G〇 phase(户<0.05).4.9BEC reduced cyclin D1 and CDK4 expression, but increased p27 expression Previous studies showed that cyclin Dl, CDK4 and p27 played a key role in cell proliferation and cell cycle. In vitro experiment, the levels of cycli打 01,CDK4 and p27 i打 HPASMCs were assayed. As we expect:ed,compared with the control group,the mRNA and protein expre巧ion of cyclin D1 and CDK4 were markedly reduced in Tregs treated-HPASMCs, while BEC treatment enhanced hypoxia-induced p27 gene expressio打(户<0.05, figure 5B-H). These ^sults showed 也at the regulation of BEC0打 cell cycle might be another mechanism on its anti-PAH effects.4.10Tregs decreased Akt and ERKl/2 phosphorylation。has reported that Akt and ERK pathway are involved in HPASMCs proKferatio。and progression of PAtf. Thus, we assessed Akt and ERK protein expression by western blot in vitro. We found that compared with the control group, BEC downregulated the phosphorylation of Akt and ERK, which might be another mechanism of the prcrtection of BECCP<0.05, figure 巧.5 Conclusions(1)I打 a mouse model of hypoxia-induced pulmonary hyperte打sio打,BEC can reduce pulmo打ary vascular remodeling, reduce pulmonary artery pressure.(2)in culture, BEC ca打 inWbit Arginase 2 expression,by reducing the D1 and CDK4 expression, in parUo reduce 化e phosphorylatio打 of Akt and ERK and 0化er processes,reduce pulmonary artery smoo化 muscle cell proliferatio打 and pulmonary artery remodeling.(3) U打der hypoxic conditions,IL-6 can promote the expressio打 of mRNA and protein.of ARG2 in HPASMCs... |
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