Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Angiotensin II Type 2 Receptor Modulates Synovial Macrophage Polarization by Inhibiting GRK2 Membrane Translocation in a Rat Model of Collagen-Induced Arthritis.

doi: 10.4049/jimmunol.2000561

Figure Lengend Snippet: FIGURE 2. The expression of AT1R/AT2R and the number of M1/M2 SMs are upregulated in RA patients’ synovial tissues. (A and B) IF staining is shown for CD68 (green); AT1R, AT2R, TGM2, and iNOS (red) and DAPI (blue). The single colors were merged to a combined picture. Original mag- nification 3400. (C) The AT1R and AT2R expression in PBMCs of RA patients was evaluated by ELISA after cell lysis. (D) Correlation analyses of AT1R and AT2R expression on PBMCs with inflammatory index were demonstrated by Pearson correlation test. Data are presented as mean 6 SEM, with 15 control and 32 RA patients. *p , 0.05, **p , 0.01 versus control group.

Article Snippet: Rabbit anti-rat AT2R was obtained from Novus Biologicals (catalog no. NBP1-77368; Toronto, ON, Canada).

Techniques: Expressing, Staining, Enzyme-linked Immunosorbent Assay, Lysis, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Angiotensin II Type 2 Receptor Modulates Synovial Macrophage Polarization by Inhibiting GRK2 Membrane Translocation in a Rat Model of Collagen-Induced Arthritis.

doi: 10.4049/jimmunol.2000561

Figure Lengend Snippet: FIGURE 3. AT2R activation attenuates the clinical symptoms of CIA rats. Sprague Dawley rats were immunized with CCII to establish the CIA model. The onset of clinical symptoms of arthritis was ∼day 14 after the first injection. CIA rats were randomly divided into different groups as follows: untreated CIA rats receiving an intra-articular injection of same volume of normal saline twice a week; CGP42112 group receiving treatment with intra-articular injection of CGP42112 (5, 10, and 20 mg/kg) twice a week; etanercept group receiving 3 mg/kg twice a week by i.p. injection was used as a positive control treatment. (A) The photographs of representative secondary paws from each group are presented at day 34. (B) Body weight was measured every 4 d. (C) Secondary paw swelling was checked measuring paw volume every 4 d. Data are shown as mean 6 SEM (n = 6). *p , 0.05, **p , 0.01 versus control group; #p , 0.05, ##p , 0.01 versus CIA group.

Article Snippet: Rabbit anti-rat AT2R was obtained from Novus Biologicals (catalog no. NBP1-77368; Toronto, ON, Canada).

Techniques: Activation Assay, Injection, Saline, Positive Control, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Angiotensin II Type 2 Receptor Modulates Synovial Macrophage Polarization by Inhibiting GRK2 Membrane Translocation in a Rat Model of Collagen-Induced Arthritis.

doi: 10.4049/jimmunol.2000561

Figure Lengend Snippet: FIGURE 4. AT2R activation improves arthritic joints histopathology and modulates spleen T cells subset and serum cytokines level of CIA rats. Un- treated CIA rats received an intra-articular injection of same volume of normal saline twice a week; CGP42112 group received treatment with intra-articular injection of CGP42112 (5, 10, and 20 mg/kg) twice a week. Etanercept, 3 mg/kg twice a week, was used as a positive control treatment. By doing this, we tried to choose an optimal concentration of CGP42112 for our further investigation. (A) Representative micrographs of H&E-stained histological sections of the joints are shown. The histological sections show the synoviocytes (S), the pannus (P), the inflammatory cells (I), the bone (B), the cartilage (C), and articular cavity (A). Original magnification 3200. (B) Representative x-ray images are shown. Arrow a represents soft tissue swelling, Arrow b represents monosaccate or multisaccate destruction of bone, Arrow c shows narrowing of the joint space. (C) Representative tables of the cytokines level in serum. (D) The level of production of the T cell cytokines IL-4, IL-17, and IFN-g. Data are mean 6 SEM (n = 5 or 6). **p , 0.01 versus control group; #p , 0.05, ##p , 0.01 versus CIA group.

Article Snippet: Rabbit anti-rat AT2R was obtained from Novus Biologicals (catalog no. NBP1-77368; Toronto, ON, Canada).

Techniques: Activation Assay, Histopathology, Injection, Saline, Positive Control, Concentration Assay, Staining, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Angiotensin II Type 2 Receptor Modulates Synovial Macrophage Polarization by Inhibiting GRK2 Membrane Translocation in a Rat Model of Collagen-Induced Arthritis.

doi: 10.4049/jimmunol.2000561

Figure Lengend Snippet: FIGURE 5. AT2R activation regulates AT1R expression and SM polarization. CIA rats were treated with intra-articular injection of CGP42112 (20 mg/kg) twice a week and etanercept (3 mg/kg) twice a week by i.p. injection. Untreated CIA rats receiving an intra-articular injection of same volume of normal saline twice a week were used as control. (A) The number of SM infiltration. (B and C) The population of CD11b+AT1R+SM and CD11b+AT2R+SM. (D and E) The population of CD11b+CD86+ M1-type SM and CD11b+CD163+ M2-type SM were directly determined by flow cytometry in freshly digested synovial tissue single-cell suspension. Data are mean 6 SEM (n = 6). **p , 0.01 versus control group; #p , 0.05, ##p , 0.01 versus CIA group.

Article Snippet: Rabbit anti-rat AT2R was obtained from Novus Biologicals (catalog no. NBP1-77368; Toronto, ON, Canada).

Techniques: Activation Assay, Expressing, Injection, Saline, Control, Cytometry, Suspension

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Angiotensin II Type 2 Receptor Modulates Synovial Macrophage Polarization by Inhibiting GRK2 Membrane Translocation in a Rat Model of Collagen-Induced Arthritis.

doi: 10.4049/jimmunol.2000561

Figure Lengend Snippet: FIGURE 6. AT2R activation inhibits NO expression. The NO expression was used to evaluate the optimal dose concentration of AT2R agonist CGP42112 and AT2R antagonist PD123319 for further experiments. (A) BMDMs were pretreated with different concentrations of AT2R agonist CGP42112 for 2 h before incubation with LPS (1 mg/ml, 24 h). Data are mean 6 SEM (n = 6). **p , 0.01 versus control group. #p , 0.05, ##p , 0.01 versus LPS (1 mg/ml) group. (B) Half an hour before AT2R agonist CGP42112 treatment, we give different concentration of AT2R antagonist PD123319 to block the effect of CGP42112. IL-4 (10 ng/ml, 24 h) was used as positive control. Data are mean 6 SEM (n = 6). **p , 0.01 versus control group; ##p , 0.01 versus CGP42112 (1026 M) group; $$p , 0.01 versus PD123319 (1028 M) group.

Article Snippet: Rabbit anti-rat AT2R was obtained from Novus Biologicals (catalog no. NBP1-77368; Toronto, ON, Canada).

Techniques: Activation Assay, Expressing, Concentration Assay, Incubation, Control, Blocking Assay, Positive Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Angiotensin II Type 2 Receptor Modulates Synovial Macrophage Polarization by Inhibiting GRK2 Membrane Translocation in a Rat Model of Collagen-Induced Arthritis.

doi: 10.4049/jimmunol.2000561

Figure Lengend Snippet: FIGURE 7. AT2R activation regulates AT1R expression and BMDM polarization. (A) BMDMs were pretreated with CGP42112 or PD123319 and then incubated with LPS (1 mg/ml, 4 h), and Western blot was used to detect the AT1R/AT2R expression after AT2R activation. (B) BMDMs were pretreated with CGP42112 or PD123319 and then incubated with LPS (1 mg/ml, 24 h), and BMDMs polarization marker expression were detected by Western blot after AT2R activation. Data are shown as mean 6 SEM (n = 4). *p , 0.05, **p , 0.01 versus control group; ##p , 0.01 versus LPS group; $p , 0.05, $$p , 0.01 versus LPS plus CGP group; $p , 0.05, $$p , 0.01 versus LPS plus CGP group.

Article Snippet: Rabbit anti-rat AT2R was obtained from Novus Biologicals (catalog no. NBP1-77368; Toronto, ON, Canada).

Techniques: Activation Assay, Expressing, Incubation, Western Blot, Marker, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Angiotensin II Type 2 Receptor Modulates Synovial Macrophage Polarization by Inhibiting GRK2 Membrane Translocation in a Rat Model of Collagen-Induced Arthritis.

doi: 10.4049/jimmunol.2000561

Figure Lengend Snippet: FIGURE 8. AT2R activation suppresses the phosphorylation of ERK1/2 and inhibits NF-kB signaling. BMDMs were pretreated with CGP42112 or PD123319 and then incubated with LPS (1 mg/ml, 4 h). (A) Western blot analysis depicting the phosphorylated and total protein forms of p38 MAPK, ERK1/2, and JNK in MAPK signaling. (B) The phosphorylation of IkB and NF-kB p50/p65 subunit nuclear expression was also detected by Western blot. Data are presented as mean 6 SEM (n = 4). *p , 0.05, **p , 0.01 versus control group; #p , 0.05, ##p , 0.01 versus LPS group; $$p , 0.01 versus LPS plus CGP group.

Article Snippet: Rabbit anti-rat AT2R was obtained from Novus Biologicals (catalog no. NBP1-77368; Toronto, ON, Canada).

Techniques: Activation Assay, Phospho-proteomics, Incubation, Western Blot, Expressing, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Angiotensin II Type 2 Receptor Modulates Synovial Macrophage Polarization by Inhibiting GRK2 Membrane Translocation in a Rat Model of Collagen-Induced Arthritis.

doi: 10.4049/jimmunol.2000561

Figure Lengend Snippet: FIGURE 9. AT2R activation inhibits GRK2 membrane translocation and promotes the coexpression with p-ERK1/2. BMDMs were pretreated with CGP42112 or PD123319 before the addition of LPS (1 mg/ml, 4 h). (A) The membrane protein levels of GRK2 were determined by flow cytometry (n = 5). (B) The total, membrane, and cytoplasm protein levels of GRK2 were determined by Western blot analyses (n = 4). (C) Confocal imaging of GRK2 translocation in BMDMs, Original magnification 3400. (D) LPS-stimulated BMDMs with CGP42112 or PD123319 were immunoprecipitated using an anti-GRK2 Ab and subjected to Western blotting to visualize ERK1/2 to evaluate its association with the GRK2 in cytoplasm. Coimmunoprecipitation (IP): anti-GRK2; immunoblot (IB): p-ERK1/2 and GRK2. (n = 3). **p , 0.01 versus control group; #p , 0.05, ##p , 0.01 versus LPS group; $p , 0.05, $$p , 0.01 versus LPS plus CGP group.

Article Snippet: Rabbit anti-rat AT2R was obtained from Novus Biologicals (catalog no. NBP1-77368; Toronto, ON, Canada).

Techniques: Activation Assay, Membrane, Translocation Assay, Cytometry, Western Blot, Imaging, Immunoprecipitation, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Angiotensin II Type 2 Receptor Modulates Synovial Macrophage Polarization by Inhibiting GRK2 Membrane Translocation in a Rat Model of Collagen-Induced Arthritis.

doi: 10.4049/jimmunol.2000561

Figure Lengend Snippet: FIGURE 10. The possible underlying mechanism of AT2R anti- inflammatory role on SM. The coexpression between p-ERK1/2 and GRK2 in cytoplasm is increased after AT2R activation, and the de- creased dissocation of p-ERK1/2 fails to promote IkB phosphorylation. Thus, the activation of NF-kB is downregulated because of low phosphorylation of IkB, reduced nuclear translocation, and a low level of inflammatory cytokines.

Article Snippet: Rabbit anti-rat AT2R was obtained from Novus Biologicals (catalog no. NBP1-77368; Toronto, ON, Canada).

Techniques: Activation Assay, Phospho-proteomics, Translocation Assay

Journal: Biology of Reproduction

Article Title: Activation of angiotensin type 2 receptor attenuates testosterone-induced hypertension and uterine vascular resistance in pregnant rats ^†

doi: 10.1093/biolre/ioab051

Figure Lengend Snippet: Effect of AT2R agonist C21 on Ang II receptor and eNOS expression in placental vessels isolated from control and PE women. Placental vessels were incubated with and without AT2R agonist C21 (1 μM) for 24 h in phenol red-free DMEM with 1% charcoal-stripped fetal bovine serum and then processed for protein quantification. Representative Western blots for AT1R, AT2R, eNOS, and β-actin are shown at top; blot density obtained by densitometric analysis normalized to β-actin is shown at bottom. Values are means ± SEM of six patients per group. *P ≤ 0.05 versus Vehicle control. #P ≤ 0.05 versus PE without C21.

Article Snippet: Assays for rat (AT 2 R-Rn00560677_s1, AT 1 R-Rn02758772_s1, eNOS-Rn02132634_s1, β-actin-Rn00667869_ m1) and human (AT 2 R-Hs02621316_s1, AT 1 R-Hs00258938_m1, eNOS-Hs01574659_m1, β-actin-Hs01060665_ g1) were obtained by Assay-on-Demand (Applied Biosystems; Thermo Scientific).

Techniques: Expressing, Isolation, Control, Incubation, Western Blot

Journal: Biology of Reproduction

Article Title: Activation of angiotensin type 2 receptor attenuates testosterone-induced hypertension and uterine vascular resistance in pregnant rats ^†

doi: 10.1093/biolre/ioab051

Figure Lengend Snippet: Effect of AT2R agonist C21 on Ang II receptor and eNOS expression in UA from control and T dams. On GD 20, UA was isolated from control and T dams with and without C21 administration and processed for (A) quantitative RT-PCR analysis for mRNA and (B) Western blotting for protein expression of AT1R, AT2R, and eNOS. Representative blots for AT1R, AT2R, eNOS, and β-actin are shown on the top and normalized densitometric analysis are shown at the bottom. Both mRNA and protein expression were normalized to β-actin. Values are means ± SEM of six rats per group. *P ≤ 0.05 versus Vehicle control. #P ≤ 0.05 versus T without C21.

Article Snippet: Assays for rat (AT 2 R-Rn00560677_s1, AT 1 R-Rn02758772_s1, eNOS-Rn02132634_s1, β-actin-Rn00667869_ m1) and human (AT 2 R-Hs02621316_s1, AT 1 R-Hs00258938_m1, eNOS-Hs01574659_m1, β-actin-Hs01060665_ g1) were obtained by Assay-on-Demand (Applied Biosystems; Thermo Scientific).

Techniques: Expressing, Control, Isolation, Quantitative RT-PCR, Western Blot

Journal: Kidney international

Article Title: The angiotensin II type 2 receptors protect renal tubule mitochondria in early stages of diabetes mellitus.

doi: 10.1016/j.kint.2018.06.006

Figure Lengend Snippet: Figure 1 | Transgenic angiotensin II type 2 receptor (AT2R) expression in renal tubules. Nonradioactive in situ hybridization for transgenic AT2R mRNA in kidneys of 8-week-old male transgenic rats

Article Snippet: Immunohistochemistry was performed as previously described.12 Primary antibodies were used in 1:50 (rat AT2R [sc-7420, Santa Cruz Biotechnology, Dallas, TX], HA tag [sc7392, Santa Cruz], and rat Ki-67 [Dako]), 1:100 (human AT2R [ab19134, Abcam]), or 1:500 (VDAC1 [ab14734, Abcam]) dilution for 1 hour or overnight, and then either Alexa Fluor 488, Alexa Fluor 647, Alexa Fluor 532, or Alexa Fluor 647 (1:500 dilution) conjugated secondary antibodies or a biotinylated secondary antibody (for Ki-67) (anti-mouse IgG, Sigma, 1:200 dilution) was used.

Techniques: Transgenic Assay, Expressing, In Situ Hybridization

Journal: Kidney international

Article Title: The angiotensin II type 2 receptors protect renal tubule mitochondria in early stages of diabetes mellitus.

doi: 10.1016/j.kint.2018.06.006

Figure Lengend Snippet: Figure 3 | Colocalization of angiotensin II type 2 receptors (AT2Rs) and mitochondrial marker. Immunohistochemistry on kidney sections show colocalization of AT2Rs with the mitochondrial marker voltage-dependent anion-selective channel 1 (VDAC1) in rats: (a) healthy wild-type (WT) rats, (b) STZ-induced diabetes (28 days) in WT rats, and (c) transgenic rats (TGRs). (d) Immunohistochemistry on human influenza hemagglutinin (HA)–tagged AT2R-transfected rat proximal tubule cells demonstrates colocalization of AT2R with MitoTracker Deep Red. (e) Specificity of the anti-AT2R antibody was demonstrated by intense signals on kidney sections from TGRs and the lack of signals on kidney sections from AT2R knockout mice. (f) Maximum binding capacity for angiotensin II receptors as determined by receptor binding studies of mitoplasts after digitonin digestion (mitoplast fraction a and b with different purity). Full expression of an oxidative phosphorylation marker on mitoplasts indicates an intact electron transfer chain. (Continued)

Article Snippet: Immunohistochemistry was performed as previously described.12 Primary antibodies were used in 1:50 (rat AT2R [sc-7420, Santa Cruz Biotechnology, Dallas, TX], HA tag [sc7392, Santa Cruz], and rat Ki-67 [Dako]), 1:100 (human AT2R [ab19134, Abcam]), or 1:500 (VDAC1 [ab14734, Abcam]) dilution for 1 hour or overnight, and then either Alexa Fluor 488, Alexa Fluor 647, Alexa Fluor 532, or Alexa Fluor 647 (1:500 dilution) conjugated secondary antibodies or a biotinylated secondary antibody (for Ki-67) (anti-mouse IgG, Sigma, 1:200 dilution) was used.

Techniques: Marker, Immunohistochemistry, Transgenic Assay, Transfection, Knock-Out, Binding Assay, Expressing, Phospho-proteomics

Journal: Kidney international

Article Title: The angiotensin II type 2 receptors protect renal tubule mitochondria in early stages of diabetes mellitus.

doi: 10.1016/j.kint.2018.06.006

Figure Lengend Snippet: Figure 3 | (Continued) (g,h) Immunohistochemistry on human kidney sections show colocalization of AT2Rs with the mitochondrial marker VDAC1 in normal tissue from patients with a kidney tumor (g). In renal tissue from patients with minimal change disease, AT2R is downregulated and colocalizes rarely with mitochondria (h). To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

Article Snippet: Immunohistochemistry was performed as previously described.12 Primary antibodies were used in 1:50 (rat AT2R [sc-7420, Santa Cruz Biotechnology, Dallas, TX], HA tag [sc7392, Santa Cruz], and rat Ki-67 [Dako]), 1:100 (human AT2R [ab19134, Abcam]), or 1:500 (VDAC1 [ab14734, Abcam]) dilution for 1 hour or overnight, and then either Alexa Fluor 488, Alexa Fluor 647, Alexa Fluor 532, or Alexa Fluor 647 (1:500 dilution) conjugated secondary antibodies or a biotinylated secondary antibody (for Ki-67) (anti-mouse IgG, Sigma, 1:200 dilution) was used.

Techniques: Immunohistochemistry, Marker