Effects Of Connexin43 Knockdown Dendritic Cells By RNA Interference On Atherosclerosis In ApoE-/-mice

It has been showed that atherosclerosis is a chronic inflammatory and autoimmunity disease.Dendritic cells,which can induce immunologic response and immune tolerance,are the most powerful antigen present cell in the immune system. Dendritic cells have reportedly form cell-to-cell contact between DCs through gap junctions.Connexin43 is intercellular channel allowing direct exchange of signalingmolecules.Connexin43 can be upregulated and form gap junction when the immune cells become exposed to inflammatory factors.In this study,we established the mouse bone marrow-derived dendritic cells in vitro.Then we used small-interfering RNA(siRNA) targeted against connexin43 and screened the most effective siRNA to block connexin43 expression in three candidates.The expression of surface costimulatory molecules and the stimulatory capacity to stimulate T cells was assessed after DCs were transfected with connexin43 siRNA.Furthermore,we study the effects of DC modified by RNAi on atherosclerosis in ApoE knockout mice. Partâ… connexin43 gene silencing in BM-DCObjective:To construct small interference RNA(siRNA) targeting connexin43 molecular of mice dendritic cells(DCs) and screening the most effective siRNA on blocking connexin43 by electroporation protocol.Methods:Generation and identification of bone marrow-derived DC.DCs were generated from bone marrow progenitor cells in C57BL/6 mice.DCs were cultured in complete medium supplemented with recombinant GM-CSF and recombinant mouse IL-4 and being activated with LPS to form mature DC(mDC)).2.The protocol of connexin43 knockdown in DCs and its effects on DC.SiRNA sequences were selected in accordance with the method.Three siRNA of which sequence specified to connexin43 mRNA were synthesized in vitro respectively.These siRNA with suitable doses were transfected into DCs by electroporation protocol.The experiments were included six groups:group 1,DCs;group 2,DCs electroporated without SiRNA;group 3,DCs plus non-silencing control siRNA;group 4,DCs connexin43 targetl dsRNA;group 5,.DCs plus connexin43 target2 dsRNA;group 6,DCs plus connexin43 target3 dsRNA.The expression levels of connexin43 mRNA and protein were assayed by semi-quantitative-RT PCR and western blot after siRNA transfection.The most effective siRNA in knockdown the connexin43 in DCs were selected after siRNA transfection.3.The viability of the electroporated DCs was determined by Trypan blue staining after siRNA transfection.Results:1.under the stimulation of rmGM-CSF and rmIL-4,about 1-2X107 DCs were propagated from bone marrow each mouse in vitro.The level of CD11c in DCs was above 90%,which could satisfied the experiment.the CDllc positive rate of the acquired DCs was above 90%through handful isolatfon.2.Three sequences specific to the connexin43 gene can used in DC transfection. Realtime PCR analysis and western blot test indicated that the expression of connexin43 gene in DCs was downregulated effectively by R2/siRNA(vs other groups,P<0.05),the others siRNA have no inhibiting effect on connexin43 expression compared with the negative control groups.3.The influences of connexin43 siRNA and electroporation on the viability of DCs were excluded by comparing cells that were electroporated with cells that were not.Conclusion:1.A great quantity of DCs with typical bioimmunological characteristic were propagated from mouse bone marrow Precursors under rmGM-CSF and rmIL-4 stimulation.2.R2/siRNA is the most effective siRNA sequence against connexin43 gene owing to its significant inhibition of connexin43 mRNA and protein.3.DCs transfected by electroporation show high viability.Partâ…¡Affection of connexin43 gene silencing on immunological characteristic of BM-DCObjective:After successed in transfection in DCs,siRNA-connexin43DC will be studied in vitro on cell phenotype and cell immunofunction to evaluate its tolerogenic ability.Methods:Three groups were established.RNAi/connexin43-DC group:DCs were transfected with siRNA-connexin43;control DC group;electroporated without siRNA DC group. The expression of connexin43,surface costimulatory molecules which are MHC-â…¡,CD4D,CD80 and CD86 was assessed by western blot and flow cytometric analysis respectively.The capacity of DCs to stimulate T cell proliferation was tested in an allogeneic MLR.The bioimmunological function was compared among the three groups to evaluate the tolerogenic potential of RNAi connexin43.Results:1.Compared with control DC group and electroporated without siRNA DC group,the expression of connexin43 was significantly decreased in RNAi/connexin43-DC group.2.the expression of the MHC classâ…¡,CD40,CD80,and CD86 were decreased in RNAi/connexin43-DC group.3.RNAi/connexin43-DC inhibited allgeneic T cell proliferative response in MLR(vs control DC group and electroporated without siRNA DC group,P<0.05,when T/DC=20:l,50:1 and 100:1,respectively).And the inhibition phenomena could not be reversed by electroporated stimulation(P>0.05)Conclusion:1.Silencing connexin43 using siRNA results in arrest of BM-DC maturation with reduced expression of the MHC classâ…¡,CD40,CD80,and CD86.Functionally, connexin43-silenced BM-DC showed a dramatically reduced capability to induce T cell proliferation.2.The immunological characteristic of BM-DC was not influenced by electroporation. Partâ…¢Effects of Connexin43 Knockdown Dendritic Cells by RNA interference on Atherosclerosis in ApoE-/- miceObjective:To study the effects of the DC treated with connexin43 siRNA on the atherosclerosis in ApoE-/- mice.Methods:Thirty animals were divided into into three groups as the following:group 1- treated by DC without siRNA;group 2- treated with PBS;group 3- treated by DC with connexin43 siRNA.DCs modified by siRNA were transfused into ApoE-/- mice by subcutaneous injection every week.The mice were individually recorded and pathologically and immunohistochemistry evaluated after 12 weeks treatment. Connexin43 protein expression levels in the aorta of the ApoE-/- mice was determined by western blot in the same time.Results:The atherosclerosis of arota in siRNA modifying group 3 is significantly pathological than group 1 and group 2(P<0.05),but no differences comparing with groups 1 and 2. Lower connexin43 protein level in group 3 than other groups(P<0.05).Conclusion:In ApoE-/- mice,DCs have been knocked down in connexin43 through RNAi could exhibit protective effect on atherosclerosis.This mechanism might be due to reducing capability of DC to induce T cell proliferation.And the decreased expression of connexin43 maybe involved in anti-atherosclerosis.