| This study was divided into three sections.Section A:AngtiotensinⅡPromotes the Expression of Connexin 43 in Dendritic Cell and the Gap Junctional Intercellular Communication between Dendritic cellsBackground:As a key member of the rennin-angiotensin system(RAS),angiotensinⅡ(AngⅡ) plays a crucial role in the formation,progression,plaque rupture of atherosclerosis through its proinflammatory activity,but the mechanism is far from clearly understood.Objects:This study was aimed to explore the role of Ang II in the expression of connexin(Cx)43,costimulatory molecule CD40,CD80,CD86 and antigen presenting molecule major histocompatibility complex-Ⅱ(MHC-Ⅱ) in dendritic cells(DC),in the formation of the gap junctional intercellular communications(GJIC) between DCs,and in the stimulatory capability of DC on T lymphocyte.Methods:Mouse bone marrow-derived dendritic cells were treated with 10-6 mol/L AngⅡfor 5,15,30,60 minutes(min) or incubated with different concentrations(10-8 mol/L,10-7 mol/L,10-6 mol/L) of AngⅡfor 30 min,in some experiments,DCs were incubated with 10-6 mol/L AngⅡor 100ng/ml LPS alone or with AngⅡplus LPS in the presence or absence of 10-5 mol/L valsartan(Val) or 5×10-5 mol/L heptanol(Hep) for 24 hours,the expression of Cx43 was detected by Western blotting analysis,the scarification labeling immunofluorescence tracing technology was adopted to detect the GJIC between DCs,CD40,CD80,CD86 and MHC-Ⅱwere examined by flow cytometry,and the T cell stimulatory ability of DCs was determined by Allo-MLR assay.Results:AngⅡsignificantly stimulated the expression of Cx43 both in concentration and time depending manner.The peak stimulatory function happened in the concentration of 10-6 mol/L(relative expression:0.51±0.03 versus Control 0.23±0.03,P < 0.05) and at 30 min(relative expression:0.59±0.08versus 0 min 0.19±0.05,P < 0.01), after 1 hour,the function still remained.LPS also significantly induced the expression of Cx43(relative expression:0.78±0.03 versus Control 0.40±0.06,P<0.01),this effect was enhanced by 10-6 mol/L AngⅡ(relative expression:1.01±0.08 versus LPS 0.78±0.03,P < 0.05),and the enhancement was inhibited by AngⅡtype 1 receptor antagonist valsartan(relative expression:0.69±0.09 versus AngⅡ+LPS 1.01±0.08, P< 0.01).AngⅡsignificantly potentialized the GJIC between DCs,this effect was inhibited by Val,and eliminated by Hep.AngⅡalone could not promote T lymphocyte proliferation(stimulation index(SI):SI:1.45±0.15 versus Control 1.05±0.10,P> 0.05),but could amplify the T lymphocyte proliferation inducing effect of LPS(SI: 3.91±0.19 versus LPS 3.15±0.21,P<0.01),this amplification could be inhibited by Val(SI:2.84±0.17 versus AngⅡ+LPS 3.91±0.19,P<0.01),and be attenuated by Hep(SI:1.81±0.13 versus AngⅡ+LPS 3.91±0.19,P<0.01).Conclusions:This is the first study about the effect of AngⅡon the expression of Cx43 in DCs and the gap junctional intercellular communications between DCs.Our findings include:1.AngⅡcan induce the production of Cx43 in DCs and stimulate the GJIC between DCs.2.AngⅡfail to induce the expression of co-stimulatory molecules of DCs such as CD40,CD80,CD86 and the antigen presenting molecule MHC-Ⅱ,but it can effectively intensify the T lymphocyte proliferation inducing effect of LPS. These findings indicate that AngⅡmay contribute to the maturation and effective activation of DCs in a cooperative way.The molecular mechanisms and the effect of AngⅡon DCs in vivo remain to be further elucidated.Section B:Angiotensin-(1-7) Enhances AngiotensinⅡInduced Phosphorylation of ERK1/2 in Mouse Bone Marrow-Derived Dendritic CellsBackground and purposes:It is well known that angiotensin-(1-7)(Ang-(1-7)) counterbalances vasoconstrictive and proliferative functions of AngⅡ,some of those actions are via inhibition of AngⅡinduced activation of mitogen-activated protein kinases(MAPK).This study investigated the effects of Ang-(1-7) on AngⅡ-mediated cell signaling pathways in mouse bone marrow-derived dendritic cells(DC).Methods:The expression of receptor Mas and angiotensin-converting enzyme-related carboxypeptidase(ACE2) mRNA was examined by reverse transcription-polymerase chain reaction(RT-PCR),activation of MAPK was detected by immunoblotting after incubation of dendritic cells with AngⅡin the presence or absence of Ang-(1-7), valsartan,PD123319,and D-Ala7-Ang-(1-7).Results:AngⅡrapidly(5 min,10-7 mol/L) stimulated phosphorylation of extracellular signal-related kinase(ERK1/2)(relative expression:0.46±0.09versus 0 min 0.24±0.04, P < 0.05);this effect was partially inhibited by AngⅡtype 1(ATI) receptor antagonist valsartan(relative expression:0.72±0.05 versus AngⅡ0.91±0.13,P=NS) and significantly attenuated by AngⅡtype 2(AT2) receptor antagonist PD123319(relative expression:0.52±0.08 versus AngⅡ0.91±0.13,P<0.01).Ang-(1-7) alone also induced phosphorylation of ERK1/2(relative expression:0.57++++++++++0.05 versus control 0.35±0.04,P < 0.01),co-treatment of Ang-(1-7) and AngⅡmarkedly enhanced ERK1/2 phosphorylation(relative expression:1.20±0.20versus AngⅡ0.91±0.13,P< 0.05),the enhancement was eliminated by the Ang-(1-7) receptor antagonist D-Ala7-Ang-(1-7).Both Ang-(1-7) and AngⅡhad no effect on p38 and c-Jun N-terminal kinase(JNK) phosphorylation.Conclusions:AngⅡstimulates ERK1/2 phosphorylation via AT2 receptor in mouse DC,Ang-(1-7) enhances this effect.Generation of Ang-(1-7) by DC could thereby counteract on the proinfiammatory function of locally generated AngⅡSection C:Endogenous AngiotensinⅡPromotes the Expression of Connexin 43 and Maturation of Dendritic Cells in AtherosclerosisBackground and purposes:Our in vitro study revealed that Ang II could induce the production of Cx43 in DC and the gap junctional intercellular communication between DCs,but could not induce the expression of co-stimulatory molecules,yet there has evidence that AngⅡcan control the differentiation and maturation of DC.Therefore, we presented here an in vivo study to investigate whether the endogenous AngⅡcould promote the expression of Cx43 and maturation of DC in ApoE-/- mice.Methods:One kidney per mouse was clipped to generate endogenous high AngⅡApoE-/- mouse model.The models were divided into three groups,group 1,sham, group 2,two kidney one clip(2K1C),group 3,2K1C with valsartan(Val) 4mg/kg.d-1 intragastric administration.After 10 weeks of clipping,blood pressure(BP) and heart rate(HR) were measured,plasma concentrations of AngⅡand rennin activity were examined by radioimmunology assay,staining of oil-red O and anti-smooth muscle cell(SMC)α-actin were adopted to explore the morbidity and morphology of plaques,the expression of Cx43 and CD40 of DCs in atherosclerosis was detected by immunohistochemistry double staining.Results:The model was created successfully,2K1C ApoE-/- mice exhibited higher BP compared with sham(MBP:122±6 mmHg versus sham 88±3 mmHg,P<0.05),Val 4mg/kg.d-1 had no effect on BP(MBP:123±5 mmHg versus sham 88±3 mmHg,P < 0.01;versus 2K1C 122±6 mmHg,P = NS).2K1C ApoE-/- mice developed more plaques than sham mice did,and the plaques of 2K1C ApoE-/- mice were more instable,Val both reduced the plaque volume and stabilized the plaque.The expression of Cx43 and CD40 of DCs in 2K1C ApoE-/- mice atherosclerotic plaques was higher than that in sham mice plaques,Val could attenuate the expression.Conclusions:Endogenous AngⅡcan induce maturation of DCs in ApoE-/- mice atherosclerotic plaques by promoting the expression of Cx43 through AT1 receptor, thereby,accelerate atherosclerosis formation and progression.These observations shed light on the mechanism of atherosclerosis formation and progression,and may have prophylactic and therapeutic significance.
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