The Mechanistic Study That ATR001 Monoclonal Antibody Ameliorates Atherosclerosis

Part I The mechanistic study that McAb-ATR did not affect Gq or Gi2/i3 pathwayObjective: AT1R is a typical 7-transmembrane GPCRs and plays an important role in the development of cardiovascular diseases such as hypertension,coronary heart disease,and heart failure.When biased ligands bind to GPCRs,they could be functionally selective or biased towards either G-protein dependent pathways(Gs,Gq,G12/13,Gi2/i3)or G-protein independent signaling pathways(beta-arrestin1/2).The main purpose of this section is to study the key sites and characteristics of McAb-ATR binding to AT1 R,the effects and mechanisms of McAb-ATR on intracellular signaling pathways Gq,Gi2/i3,Ca2+ and the difference from ARBs after McAb-ATR binding to AT1 R.Moreover,we also investigated the effect of McAb-ATR on RAS and plasminogen activator inhibitor-1(PAI-1)in Apolipoprotein E knock-out(Apo E-/-)mice with western-type diet(WTD)-induced atherosclerosis.Method: Male BALB/c mice(8 weeks old)were immunized with ATRQ?-001 vaccine.The hybridoma cell clone that stably secreted McAb-ATR was cultured by monoclonal antibody technology.Firstly,various tool cells stably transfected with different plasmids were constructed to explore the downstream effects of McAb-ATR.The mechanism of McAb-ATR was explored by various assays.And McAb-ATR was compared with ARBs Losartan.The specificity of McAb-ATR binding to AT1 R was detected by immunofluorescence,enzyme-linked immunosorbent assay(ELISA)and radioligand assay of AT1 R binding.Bioluminescence resonance energy transfer measurement(BRET)were used to analyze proteins Gq,Gi2/i3 interacting with AT1 R.Intracellular Ca2+ concentration measurements were used to examine the effect of McAb-ATR on Ang ?-induced intracellular Ca2+ release.Moreover,Apo E-/-mice(male,7 weeks old)were randomly allocated into 4 groups: Control group(n=13),20 ?g McAb-ATR group(n=13),100 ?g McAb-ATR group(n=13)and Valsartan group(n=13).The Apo E-/-mice were fed WTD for 18 weeks.The Apo E-/-mice received the following treatments for 18 weeks: 5 mg/kg Valsartan perorally every day,or 20 ?g McAb-ATR,100 ?g McAb-ATR or phosphate-buffered saline(PBS)injected into the tail vein every week.After 18 weeks,Apo E-/-mice had been sacrificed using cervical dislocation,PRA and plasma angiotensin II concentration of Apo E-/-mice were measured.PAI-1 concentration in serum of Apo E-/-mice was detected by ELISA.All images were analyzed with Image-Pro Plus software.Result: 1.The immunofluorescence,ELISA,and radioligand assay of AT1 R binding results demonstrated that McAb-ATR could specifically bind to Phe182-His183-Tyr184 site of AT1 R ECL2,and the binding of McAb-ATR to AT1 R did not affect the interaction between Ang ? and AT1 R.2.Our BRET assay results indicated that in contrast to Losartan,McAb-ATR did not influence Ang ?-induced uncoupling of heterotrimeric G proteins(Gq or Gi2/i3).3.The intracellular Ca2+ concentration measurement revealed that McAb-ATR had no impact on Ang ?-induced Gq-dependent intracellular Ca2+ release.Moreover,the mutation of Phe182 and Tyr184 site of AT1 R ECL2 did not affect Ca2+ release.In contrast,intracellular Ca2+ concentration was significantly suppressed by Losartan.4.In the RAS assay,Valsartan significantly caused feedback activation of PRA and Ang ? concentration.No RAS feedback activation was observed in McAb-ATR group.5.The results of ELISA in serum of Apo E-/-mice showed that in contrast to Valsartan,McAb-ATR could significantly decrease the level of PAI-1.Conclusion: McAb-ATR could specifically bind to AT1 R,at which Phe182-His183-Tyr184 of AT1 R ECL2 was the key binding site.In contrast to ARBs,McAb-ATR non-competitively bound to AT1 R without affecting Ang ?-induced uncoupling of heterotrimeric G proteins(Gq or Gi2/i3),Gq-dependent intracellular Ca2+ release.McAb-ATR did not cause RAS feedback activation,and could reduce PAI-1 level.Part II The mechanistic study that McAb-ATR biasly regulated beta-arrestin pathwayObjective: The first section has explored the effects of McAb-ATR on Gq and Gi2/i3 signaling pathways.The main purpose of this section is to study the effects and mechanisms of McAb-ATR on intracellular beta-arrestin signaling pathways.And McAb-ATR was compared with ARBs.Additionally,the effect of McAb-ATR on the beta-arrestin-mediated peroxisome proliferator activated receptor gamma(PPAR?)and I?B/NF?B p65 pathway was investigated in vivo and in vitro.Method: Firstly,BRET were used to analyze proteins beta-arrestin interacting with AT1 R.Western blotting(WB)detects the inhibitory effect of McAb-ATR on Ang ? or S?-Ang-induced ERK1/2 phosphorylation.The effect of McAb-ATR on Ang ?-induced AT1 R internalization was detected by immunofluorescence.In addition,the effects of McAb-ATR on PPAR? and I?B/NF?B p65 signaling pathway were detected by WB and immunofluorescence of Apo E-/-mouse aorta protein and RAW264.7 macrophages,and immunohistochemistry of the serial cryosections of Apo E-/-mouse aortic root plaque lesions.Result: 1.Our BRET assay results indicated that in contrast to Losartan,McAb-ATR did not influence Ang ?-induced beta-arrestin recruitment.2.The WB assay revealed that McAb-ATR and Losartan effectively suppressed Ang ? or S?-Ang-induced beta-arrestin2-dependent ERK1/2 phosphorylation at 10 minutes in AT1R-CHO-K1 cells.Moreover,the mutation of Phe182 and Tyr184 site of AT1 R ECL2 also inhibited beta-arrestin2-dependent ERK1/2 phosphorylation at 10 minutes.Subsequent WB assays confirmed that McAb-ATR effectively suppressed Ang ?-induced beta-arrestin2-dependent ERK1/2 phosphorylation in 10-60 minutes in CHO-K1 cells transiently expressing the AT1 R DRY-AAY mutant.3.The confocal microscopy results indicated that beta-arrestin-mediated AT1 R internalization induced by Ang ? was retained in McAb-ATR group.However,the phenomenon was diminished by Losartan.4.In vivo and in vitro,the results showed that McAb-ATR not only increased the expression of PPAR?,but also promoted its translocation from cytoplasm to nucleus.6.McAb-ATR increased the expression of I?B and inhibited the expression of NF?B p65.Meanwhile,McAb-ATR inhibited NF?B p65 nucleus translocation.Conclusion: McAb-ATR and ARBs inhibited Ang ?-or S?-Ang-induced beta-arrestin2-dependent slower and prolonged ERK1/2 phosphorylation.However,in contrast to ARBs,McAb-ATR did not affect Ang ?-induced beta-arrestin recruitment,or beta-arrestin-mediated AT1 R internalization.Furthermore,McAb-ATR regulated beta-arrestin-mediated PPAR? and I?B/NF?B p65 pathway in vivo and in vitro.Part III The mechanistic study that McAb-ATR inhibited atherosclerosis through beta-arrestin2 pathwayObjective: The previous sections have demonstrated that McAb-ATR could biasedly regulate beta-arrestin pathway without affecting Gq or Gi2/i3 pathway,but the role of beta-arrestin1 or 2 in the function of the antibody remains unknown.The main purpose of this section is to study the connection between the inhibitory effect of McAb-ATR on atherosclerosis and beta-arrestin1 or beta-arrestin2 in vivo and in vitro.Method: Low-density lipoprotein receptor knockout(LDLr-/-),Arrb1+/+,Arrb2+/+,Arrb1-/-and Arrb2-/-mice and wire injury of the mouse carotid artery model and bone marrow transplantation + WTD-induced LDLr-/-mice atherosclerotic model was used to explore the mechanism of McAb-ATR in atherosclerotic model.Arrb1+/+,Arrb2+/+,Arrb1-/-and Arrb2-/-mice(male,8 weeks old)were randomly allocated into 16 groups: sham surgery group: Arrb1-/-group(n=6),McAb+ Arrb1-/-group(n=6),Arrb1+/+ group(n=6),McAb+ Arrb1+/+ group(n=6),Arrb2-/-group(n=5),McAb+ Arrb2-/-group(n=5),Arrb2+/+group(n=5)and McAb+ Arrb2+/+ group(n=4);surgery group(Injury): Arrb1-/-group(n=17),McAb+ Arrb1-/-group(n=16),Arrb1+/+ group(n=14),McAb+ Arrb1+/+ group(n=12),Arrb2-/-group(n=15),McAb+ Arrb2-/-group(n=13),Arrb2+/+ group(n=15)and McAb+ Arrb2+/+ group(n=13).The surgery group received wire injury of the mouse carotid artery surgery.The mice received the following treatments for 4 weeks: PBS or 100 ?g McAb-ATR injected into the tail vein every week.Four weeks later,the injured carotid artery was extracted,fixed in phosphate-buffered formalin,embedded in paraffin,and then sectioned and stained with hematoxylin-eosin(HE)staining.A biological microscope was used to capture all images of injured carotid artery sections.Image-Pro Plus software was used to analyze neointima formation on the injured carotid artery.LDLr-/-mice(male,8 weeks old)were randomly allocated into 8 groups: Arrb1-/-group(n=7),McAb+ Arrb1-/-group(n=7),Arrb1+/+ group(n=7),McAb+ Arrb1+/+ group(n=7),Arrb2-/-group(n=7),McAb+ Arrb2-/-group(n=7),Arrb2+/+group(n=7)and McAb+ Arrb2+/+ group(n=7).LDLr-/-mice were lethally irradiated with 9.5 Gy of radiation to eliminate most of BM-derived and endogenous BM cells.Approximately 2×106 BM cells from donor mice(Arrb1+/+,Arrb2+/+,Arrb1-/-,or Arrb2-/-mice)were injected into each irradiated mouse.After BM transplantation,the irradiated mice were fed normal chow diet for 6 weeks,followed by WTD for 16 weeks.The mice received the following treatments for 16 weeks: PBS or 100 ?g McAb-ATR injected into the tail vein every week.After 16 weeks,LDLr-/-mice had been sacrificed using cervical dislocation,the whole aortas were dissected and opened and then subjected to Oil Red O staining for neutral lipids.The serial cryosections(6 ?m)of LDLr-/-mouse aortic root plaque lesions were then stained with Oil Red O and hematoxylin.Images of the whole aortas were captured with a digital camera,whereas those of the tissue sections were captured under a biological microscope.All images were analyzed with Image-Pro Plus software.In addition,WB of Apo E-/-mouse aorta protein detects the effects of McAb-ATR on cholesterol metabolism including ATP-binding cassette subfamily G member 1(ABCG1),phenotype transformation of macrophage including inducible nitric oxide synthase(i NOS),mannose receptor C-type 1(CD206),chitinase-like 3(YM-1)and arginase 1(Arg-1).And Oil Red O staining of foam cell detects the effects of McAb-ATR on the cholesterol metabolism in the mouse RAW264.7 macrophages.WB detected the effects of McAb-ATR on cholesterol metabolism and phenotype transformation of bone marrow-derived macrophages(BMDMs)from Arrb1+/+,Arrb2+/+,Arrb1-/-,or Arrb2-/-mice.Result: 1.The wire injury of the mouse carotid artery model result showed that with McAb-ATR administrated to the mice in Arrb1+/+,Arrb2+/+,and Arrb1-/-+ wire injury group,the vascular injuries were obviously ameliorated.However,no significant difference in neointimal area was observed between Arrb2-/-mice injected with McAb-ATR and those injected with PBS after wire injury.2.The bone marrow transplantation + WTD-induced LDLr-/-mice atherosclerotic model result showed that McAb-ATR could diminish the atherosclerotic lesion area in the mice with Arrb1+/+,Arrb2+/+,or Arrb1-/-mouse BM transplant.However,no significant difference of plaque lesions in the whole aortas or aortic roots were observed between the mice transplanted with BM from Arrb2-/-mice injected with McAb-ATR and those transplanted with BM from Arrb2-/-mice injected with PBS.3.WB of Apo E-/-mouse aorta protein showed 100 ?g McAb-ATR could increase the expression of ABCG1.4.In Oil Red O staining of foam cell experiment the lipid deposition were observed more intuitively in Raw264.7 macrophages.Ang ? and oxidized low-density lipoprotein(ox LDL)promoted cholesterol deposition and this role was inhibited by McAb-ATR.5.WB of Apo E-/-mouse aorta protein showed McAb-ATR up-regulated Arg-1,YM-1 and CD206,and down-regulated i NOS expression.6.The WB assay revealed that McAb-ATR upregulated ABCG1 and Arg-1 and downregulated i NOS in BMDMs from Arrb1+/+,Arrb2+/+,and Arrb1-/-mice,whereas it had no effect on the BMDMs from the Arrb2-/-mice.Conclusion: McAb-ATR inhibited atherosclerosis through beta-arrestin2,rather than beta-arrestin1 pathway.McAb-ATR regulated cholesterol metabolism and accelerated M2 phenotype transformation of macrophages through beta-arrestin2,not beta-arrestin1 pathway.