Journal: Purinergic Signalling

Article Title: Blockage of the adenosine A 2B receptor prevents cardiac fibroblasts overgrowth in rats with pulmonary arterial hypertension

doi: 10.1007/s11302-023-09952-z

Figure Lengend Snippet: Cardiac fibroblasts (CFs) isolated from MCT-treated rats overexpress the adenosine A 2B receptor subtype. Shown are representative immunofluorescence confocal microscopy images of RV myocardium sections ( A ) and isolated CFs from the RV and LV myocardium ( B ) of CTRL and MCT-treated rats stained against A 2B AR (green) and vimentin (red). Nuclei are stained in blue with DAPI. Micrographs are representative of at least five different individuals and were obtained with a laser-scanning confocal microscope using the same acquisition settings. Scale bar: 30 μm. In C , shown are representative immunoblots to document the relative amounts of the A 2B AR in CFs isolated from the RV myocardium of CTRL and MCT-treated rats allowed to grow in culture for 28 days; gels were loaded with 150 μg protein amounts. Two protein species were recognized by the A 2B AR antibody (#AAR-003, Alomone Inc., Jerusalem, Israel) corresponding to a peptide near the predicted molecular weight of the A 2B AR (~37 kDa) and a higher molecular mass (~45 kDa) isotype. Please note that the naturally occurring A 2B AR isotype is highly enriched in CFs isolated from the RV myocardium of MCT-treated rats compared to their CTRL littermates. Both bands fully disappeared after pre-adsorption of the A 2B AR primary antibody with a 10-fold molar excess of the A 2B AR blocking peptide (#BLP-AR003, Alomone Inc., Jerusalem, Israel) corresponding to amino acid residues 147–166 of the human A 2B AR second extracellular loop (negative control). The rat urinary bladder (RB) was used as a positive control for the A 2B AR. β-Tubulin (55 kDa) was used as a reference protein. Each bar represents pooled data from three different individuals; three replicas were performed in each experiment. The vertical bars represent SEM. * p < 0.05 (Student’s unpaired t -test) represents significant differences compared to the CTRL group

Article Snippet: Cultured cells were fixed in 4% PFA in PBS for 10 min, washed 3 times in PBS (10 min each), and, subsequently, incubated with blocking buffer I (10% FBS, 1% BSA, 0.1% Triton X, 0.05% NaN 3 ) for 1 h. Primary antibodies diluted in blocking buffer II (5% FBS, 1% BSA, 0.1% Triton X, 0.05% NaN 3 ) were applied [mouse anti-porcine vimentin 1:250 (DAKO); goat anti-human DDR2 1:25 (Santa Cruz); mouse anti-human α-smooth muscle actin (SMA)-FITC 1:250 (Sigma); rabbit anti-human A 2B AR (2nd extracellular loop, 36 kDa) 1:50 (#AAR-003, Alomone Labs)] and the slides incubated overnight at 4 °C.

Techniques: Isolation, Immunofluorescence, Confocal Microscopy, Staining, Microscopy, Western Blot, Molecular Weight, Adsorption, Blocking Assay, Negative Control, Positive Control

Journal: Purinergic Signalling

Article Title: Blockage of the adenosine A 2B receptor prevents cardiac fibroblasts overgrowth in rats with pulmonary arterial hypertension

doi: 10.1007/s11302-023-09952-z

Figure Lengend Snippet: Selective A 2B receptor blockage with PSB603 (100 nM) attenuates NECA-induced overgrowth of cardiac fibroblasts (CFs) from the RV of MCT-treated rats. NECA (10 μM) with or without PSB603 (100 nM) was incorporated in culture media throughout the whole assay. The ordinates represent NECA- and/or PSB603-induced changes in cell growth (MTT assay, A ) and type I collagen production (Sirius Red assay, B ) compared to the control situation using the same cell batch in the absence of test drugs at culture days 7, 14, 21, and 28. Zero represents the similarity between the two values (drug vs. control); positive and negative values represent facilitation or inhibition of either cell growth or type I collagen production relative to control data obtained at the same time points. Each bar represents pooled data from 5 (CTRL) and 6 (MCT) animals; 3 to 4 replicas were performed for each experiment. The vertical bars represent SEM. * p < 0.05 (ANOVA, one-way analysis of variance) represents significant differences from control values obtained in the absence of test drugs; # p < 0.05 (ANOVA, one-way analysis of variance) represents significant differences compared to the effect of NECA alone

Article Snippet: Cultured cells were fixed in 4% PFA in PBS for 10 min, washed 3 times in PBS (10 min each), and, subsequently, incubated with blocking buffer I (10% FBS, 1% BSA, 0.1% Triton X, 0.05% NaN 3 ) for 1 h. Primary antibodies diluted in blocking buffer II (5% FBS, 1% BSA, 0.1% Triton X, 0.05% NaN 3 ) were applied [mouse anti-porcine vimentin 1:250 (DAKO); goat anti-human DDR2 1:25 (Santa Cruz); mouse anti-human α-smooth muscle actin (SMA)-FITC 1:250 (Sigma); rabbit anti-human A 2B AR (2nd extracellular loop, 36 kDa) 1:50 (#AAR-003, Alomone Labs)] and the slides incubated overnight at 4 °C.

Techniques: MTT Assay, Control, Inhibition

Journal: Cells

Article Title: Adenosine A 2B Receptor Antagonism Interferes with TGF-β Cellular Signaling Through SMAD2/-3 and p65-Nf-κB in Podocytes and Protects from Phenotypical Transformation in Experimental Diabetic Glomerulopathy

doi: 10.3390/cells14120890

Figure Lengend Snippet: Evaluation of the effect of A 2B AR antagonism on renal alterations in experimental DN. ( A ) The experimental design consisted of inducing diabetes in rats through the administration of streptozotocin, with control rats receiving citrate buffer. Treatment with the A 2B AR antagonist MRS1754 (0.5 mg/kg every 48 h) was initiated at week 4 post-diabetes induction. After 8 weeks of treatment, the renal functional and histological alterations were evaluated. The graphs depict the protein-to-creatinine ratio in the urine ( B ), the serum angiotensin 1–8 (Ang II) levels ( C ), and glucosuria ( D ) in the experimental groups. Means ± SD of the samples from 6 animals in each experimental condition are shown. *, p < 0.05 vs. control; #, p < 0.05 vs. diabetes.

Article Snippet: The samples were incubated with an antibody against A 2B AR (dilution of 1:250, cat. orb234999, Biorbyt Ltd., Cambridge, UK), anti-α-SMA (dilution of 1:100, cat. sc-130617, Santa Cruz Biotech, Dallas, TX, USA), anti-ZO1 (dilution of 1:100, cat. 61-7300, Thermo Fisher Scientific, USA), anti-nephrin (dilution of 1:100, cat. PA5-106921, Thermo Fisher Scientific, USA), and anti-Snail (dilution of 1:100, cat. ab53519, Abcam, Cambridge, UK) overnight at 4 °C.

Techniques: Control, Functional Assay

Journal: Cells

Article Title: Adenosine A 2B Receptor Antagonism Interferes with TGF-β Cellular Signaling Through SMAD2/-3 and p65-Nf-κB in Podocytes and Protects from Phenotypical Transformation in Experimental Diabetic Glomerulopathy

doi: 10.3390/cells14120890

Figure Lengend Snippet: Immunohistochemical analysis of glomerular alterations in diabetic rats. ( A ) The panels show representative immunofluorescent images of the detection of A 2B AR, α-SMA, ZO-1, nephrin, and Snail in renal tissue from control rats, diabetic nephropathy (DN) rats, and diabetic rats treated with the A 2B AR antagonist MRS1754 (DN + MRS1754). The images emphasize changes in the levels of these proteins within the glomerulus. Nuclear staining with DAPI is in blue. The bar indicates 50 µm. ( B ) The graphs correspond to the means ± SD of relative fluorescent units (RFUs) of a quantitative analysis of A 2B AR, α-SMA, ZO-1, nephrin, and Snail in glomeruli from the control (white bar), diabetic (black bar), and MRS1754-treated diabetic (grey bar) rat groups. The means in the control group were normalized to 1. Images of 12 glomeruli from 2 control, 4 diabetic, and 4 treated diabetic rats were analyzed. *, p < 0.05 vs. control; #, p < 0.05 vs. diabetes.

Article Snippet: The samples were incubated with an antibody against A 2B AR (dilution of 1:250, cat. orb234999, Biorbyt Ltd., Cambridge, UK), anti-α-SMA (dilution of 1:100, cat. sc-130617, Santa Cruz Biotech, Dallas, TX, USA), anti-ZO1 (dilution of 1:100, cat. 61-7300, Thermo Fisher Scientific, USA), anti-nephrin (dilution of 1:100, cat. PA5-106921, Thermo Fisher Scientific, USA), and anti-Snail (dilution of 1:100, cat. ab53519, Abcam, Cambridge, UK) overnight at 4 °C.

Techniques: Immunohistochemical staining, Control, Staining

Journal: Cells

Article Title: Adenosine A 2B Receptor Antagonism Interferes with TGF-β Cellular Signaling Through SMAD2/-3 and p65-Nf-κB in Podocytes and Protects from Phenotypical Transformation in Experimental Diabetic Glomerulopathy

doi: 10.3390/cells14120890

Figure Lengend Snippet: A 2B AR antagonism prevents the loss of podocyte epithelial markers in diabetic glomerulopathy. ( A ) Representative Western blot analysis showing the levels of epithelial ZO-1 and nephrin, as well as mesenchymal α-SMA and Snail, in purified glomeruli from the different rat groups. ( B ) The graphs represent the means and SD of the levels of epithelial and mesenchymal markers relative to β-actin. The means in the control group were normalized to 1. ( C ) Representative Western blot analysis of the Snail distribution in cell fractions purified from the kidneys of experimental rat groups. ( D ) The graph represents the ratio of the Snail subcellular distribution. The mean in the control was normalized to 1. The number of animals analyzed in each experimental group was 5 in ( B ) and 2 in ( D ). *, p < 0.05 vs. control; #, p < 0.05 vs. DN; &, p < 0.05 vs. control.

Article Snippet: The samples were incubated with an antibody against A 2B AR (dilution of 1:250, cat. orb234999, Biorbyt Ltd., Cambridge, UK), anti-α-SMA (dilution of 1:100, cat. sc-130617, Santa Cruz Biotech, Dallas, TX, USA), anti-ZO1 (dilution of 1:100, cat. 61-7300, Thermo Fisher Scientific, USA), anti-nephrin (dilution of 1:100, cat. PA5-106921, Thermo Fisher Scientific, USA), and anti-Snail (dilution of 1:100, cat. ab53519, Abcam, Cambridge, UK) overnight at 4 °C.

Techniques: Western Blot, Purification, Control

Journal: Cells

Article Title: Adenosine A 2B Receptor Antagonism Interferes with TGF-β Cellular Signaling Through SMAD2/-3 and p65-Nf-κB in Podocytes and Protects from Phenotypical Transformation in Experimental Diabetic Glomerulopathy

doi: 10.3390/cells14120890

Figure Lengend Snippet: A 2B AR antagonism attenuates inflammatory mediators in diabetic rats. ( A ) The graphs depict the levels of the chemokines MCP-1 and CCL3 released from glomeruli and the urinary TGF-β levels in the rat experimental groups. n = 6 samples per duplicate. *, p < 0.05 vs. control; #, p < 0.05 vs. DN; &, p < 0.05 vs. control. ( B ) The heat map highlights changes in the transcript levels of selected inflammation-related genes in the experimental rat groups. The table shows values related to a decrease in the transcripts in the DN + MRS1754 group vs. the DN group.

Article Snippet: The samples were incubated with an antibody against A 2B AR (dilution of 1:250, cat. orb234999, Biorbyt Ltd., Cambridge, UK), anti-α-SMA (dilution of 1:100, cat. sc-130617, Santa Cruz Biotech, Dallas, TX, USA), anti-ZO1 (dilution of 1:100, cat. 61-7300, Thermo Fisher Scientific, USA), anti-nephrin (dilution of 1:100, cat. PA5-106921, Thermo Fisher Scientific, USA), and anti-Snail (dilution of 1:100, cat. ab53519, Abcam, Cambridge, UK) overnight at 4 °C.

Techniques: Control

Journal: Cells

Article Title: Adenosine A 2B Receptor Antagonism Interferes with TGF-β Cellular Signaling Through SMAD2/-3 and p65-Nf-κB in Podocytes and Protects from Phenotypical Transformation in Experimental Diabetic Glomerulopathy

doi: 10.3390/cells14120890

Figure Lengend Snippet: The antagonism of A 2B AR blocks TGF-β/SMAD3 signaling in podocytes. Differentiated podocytes were incubated in medium containing 25 mM D-glucose (HG) and treated with 10 ng/mL TGF-β and 50 nM MRS1754. ( A ) Upper panel shows the detection of activated phospho-SMAD2/-3 following 0.5 h of treatments in total protein extracts from three independent experiments through Western blot. Lower panel is detection of β-actin as loading control; n = 3. ( B ) Podocytes were treated as above for 48 h, refreshing TGF-β and MRS1754 at 24 h, and harvested for a ChIP-qPCR analysis. The graphs depict normalized means and the SEM occupancy of SMAD3 to the collagen type IA2 and fibronectin promoters; n = 5. *, p < 0.05 vs. HG; #, p < 0.05 vs. TGF-β. ( C ) The graphs show normalized means and the SEM enrichment of H3K9ac at SMAD3 target promoters; n = 5. *, p < 0.05 vs. HG; #, p < 0.05 vs. TGF-β.

Article Snippet: The samples were incubated with an antibody against A 2B AR (dilution of 1:250, cat. orb234999, Biorbyt Ltd., Cambridge, UK), anti-α-SMA (dilution of 1:100, cat. sc-130617, Santa Cruz Biotech, Dallas, TX, USA), anti-ZO1 (dilution of 1:100, cat. 61-7300, Thermo Fisher Scientific, USA), anti-nephrin (dilution of 1:100, cat. PA5-106921, Thermo Fisher Scientific, USA), and anti-Snail (dilution of 1:100, cat. ab53519, Abcam, Cambridge, UK) overnight at 4 °C.

Techniques: Incubation, Western Blot, Control, ChIP-qPCR

Journal: Purinergic Signalling

Article Title: Blockage of the adenosine A 2B receptor prevents cardiac fibroblasts overgrowth in rats with pulmonary arterial hypertension

doi: 10.1007/s11302-023-09952-z

Figure Lengend Snippet: Cardiac fibroblasts (CFs) isolated from MCT-treated rats overexpress the adenosine A 2B receptor subtype. Shown are representative immunofluorescence confocal microscopy images of RV myocardium sections ( A ) and isolated CFs from the RV and LV myocardium ( B ) of CTRL and MCT-treated rats stained against A 2B AR (green) and vimentin (red). Nuclei are stained in blue with DAPI. Micrographs are representative of at least five different individuals and were obtained with a laser-scanning confocal microscope using the same acquisition settings. Scale bar: 30 μm. In C , shown are representative immunoblots to document the relative amounts of the A 2B AR in CFs isolated from the RV myocardium of CTRL and MCT-treated rats allowed to grow in culture for 28 days; gels were loaded with 150 μg protein amounts. Two protein species were recognized by the A 2B AR antibody (#AAR-003, Alomone Inc., Jerusalem, Israel) corresponding to a peptide near the predicted molecular weight of the A 2B AR (~37 kDa) and a higher molecular mass (~45 kDa) isotype. Please note that the naturally occurring A 2B AR isotype is highly enriched in CFs isolated from the RV myocardium of MCT-treated rats compared to their CTRL littermates. Both bands fully disappeared after pre-adsorption of the A 2B AR primary antibody with a 10-fold molar excess of the A 2B AR blocking peptide (#BLP-AR003, Alomone Inc., Jerusalem, Israel) corresponding to amino acid residues 147–166 of the human A 2B AR second extracellular loop (negative control). The rat urinary bladder (RB) was used as a positive control for the A 2B AR. β-Tubulin (55 kDa) was used as a reference protein. Each bar represents pooled data from three different individuals; three replicas were performed in each experiment. The vertical bars represent SEM. * p < 0.05 (Student’s unpaired t -test) represents significant differences compared to the CTRL group

Article Snippet: Both bands completely disappear after pre-adsorption of the A 2B AR primary antibody with a 10-fold molar excess of the A 2B AR blocking peptide (#BLP-AR003, Alomone Inc., Jerusalem, Israel), which corresponds to amino acid residues 147–166 of the human A 2B AR second extracellular loop (negative control).

Techniques: Isolation, Immunofluorescence, Confocal Microscopy, Staining, Microscopy, Western Blot, Molecular Weight, Adsorption, Blocking Assay, Negative Control, Positive Control

Journal: Purinergic Signalling

Article Title: Blockage of the adenosine A 2B receptor prevents cardiac fibroblasts overgrowth in rats with pulmonary arterial hypertension

doi: 10.1007/s11302-023-09952-z

Figure Lengend Snippet: Cardiac fibroblasts (CFs) isolated from MCT-treated rats overexpress the adenosine A 2B receptor subtype. Shown are representative immunofluorescence confocal microscopy images of RV myocardium sections ( A ) and isolated CFs from the RV and LV myocardium ( B ) of CTRL and MCT-treated rats stained against A 2B AR (green) and vimentin (red). Nuclei are stained in blue with DAPI. Micrographs are representative of at least five different individuals and were obtained with a laser-scanning confocal microscope using the same acquisition settings. Scale bar: 30 μm. In C , shown are representative immunoblots to document the relative amounts of the A 2B AR in CFs isolated from the RV myocardium of CTRL and MCT-treated rats allowed to grow in culture for 28 days; gels were loaded with 150 μg protein amounts. Two protein species were recognized by the A 2B AR antibody (#AAR-003, Alomone Inc., Jerusalem, Israel) corresponding to a peptide near the predicted molecular weight of the A 2B AR (~37 kDa) and a higher molecular mass (~45 kDa) isotype. Please note that the naturally occurring A 2B AR isotype is highly enriched in CFs isolated from the RV myocardium of MCT-treated rats compared to their CTRL littermates. Both bands fully disappeared after pre-adsorption of the A 2B AR primary antibody with a 10-fold molar excess of the A 2B AR blocking peptide (#BLP-AR003, Alomone Inc., Jerusalem, Israel) corresponding to amino acid residues 147–166 of the human A 2B AR second extracellular loop (negative control). The rat urinary bladder (RB) was used as a positive control for the A 2B AR. β-Tubulin (55 kDa) was used as a reference protein. Each bar represents pooled data from three different individuals; three replicas were performed in each experiment. The vertical bars represent SEM. * p < 0.05 (Student’s unpaired t -test) represents significant differences compared to the CTRL group

Article Snippet: Both bands fully disappeared after pre-adsorption of the A 2B AR primary antibody with a 10-fold molar excess of the A 2B AR blocking peptide (#BLP-AR003, Alomone Inc., Jerusalem, Israel) corresponding to amino acid residues 147–166 of the human A 2B AR second extracellular loop (negative control).

Techniques: Isolation, Immunofluorescence, Confocal Microscopy, Staining, Microscopy, Western Blot, Molecular Weight, Adsorption, Blocking Assay, Negative Control, Positive Control

Journal: Purinergic Signalling

Article Title: Blockage of the adenosine A 2B receptor prevents cardiac fibroblasts overgrowth in rats with pulmonary arterial hypertension

doi: 10.1007/s11302-023-09952-z

Figure Lengend Snippet: Cardiac fibroblasts (CFs) isolated from MCT-treated rats overexpress the adenosine A 2B receptor subtype. Shown are representative immunofluorescence confocal microscopy images of RV myocardium sections ( A ) and isolated CFs from the RV and LV myocardium ( B ) of CTRL and MCT-treated rats stained against A 2B AR (green) and vimentin (red). Nuclei are stained in blue with DAPI. Micrographs are representative of at least five different individuals and were obtained with a laser-scanning confocal microscope using the same acquisition settings. Scale bar: 30 μm. In C , shown are representative immunoblots to document the relative amounts of the A 2B AR in CFs isolated from the RV myocardium of CTRL and MCT-treated rats allowed to grow in culture for 28 days; gels were loaded with 150 μg protein amounts. Two protein species were recognized by the A 2B AR antibody (#AAR-003, Alomone Inc., Jerusalem, Israel) corresponding to a peptide near the predicted molecular weight of the A 2B AR (~37 kDa) and a higher molecular mass (~45 kDa) isotype. Please note that the naturally occurring A 2B AR isotype is highly enriched in CFs isolated from the RV myocardium of MCT-treated rats compared to their CTRL littermates. Both bands fully disappeared after pre-adsorption of the A 2B AR primary antibody with a 10-fold molar excess of the A 2B AR blocking peptide (#BLP-AR003, Alomone Inc., Jerusalem, Israel) corresponding to amino acid residues 147–166 of the human A 2B AR second extracellular loop (negative control). The rat urinary bladder (RB) was used as a positive control for the A 2B AR. β-Tubulin (55 kDa) was used as a reference protein. Each bar represents pooled data from three different individuals; three replicas were performed in each experiment. The vertical bars represent SEM. * p < 0.05 (Student’s unpaired t -test) represents significant differences compared to the CTRL group

Article Snippet: Two protein species were recognized by the A 2B AR antibody (#AAR-003, Alomone Inc., Jerusalem, Israel) corresponding to a peptide near the predicted molecular weight of the A 2B AR (~37 kDa) and a higher molecular mass (~45 kDa) isotype.

Techniques: Isolation, Immunofluorescence, Confocal Microscopy, Staining, Microscopy, Western Blot, Molecular Weight, Adsorption, Blocking Assay, Negative Control, Positive Control