Blockage of the adenosine A2B receptor prevents cardiac fibroblasts overgrowth in rats with pulmonary arterial hypertension

Fig. 1. Histomorphometric analysis of the RV of rats with pulmonary arterial hypertension (PAH) induced by monocrotaline (MCT). Shown in A is the body weight and in B is Fulton’s index, which is the ratio between the mass of the RV over that of the LV plus the septum (RV/(LV+S)), of rats injected subcutaneously with saline (CTRL) or monocrotaline (MCT, 60 mg/kg) 21 days before the assessment. Tissue sections of the RV myocardium of both animal groups were stained with hematoxylin and eosin (H&E) (C) or Picro-Sirius Red (D). Scale bar: 50 μm. In E, shown is the average cross-section area (μm2) of at least 200 cardiomyocytes per animal group. In F, is represented the percentage of the Picro-Sirius Red positive staining (collagen deposition) to the total area of the micrograph, measured at least in 60 different tissue slices. Data are mean ± SEM, n = 5–9 animals/group. *p < 0.05 (Student’s unpaired t-test) represents significant differences compared to the CTRL group

Fig. 2. Immunophenotypic characterization of cardiac fibroblasts (CFs) isolated from the RV of CTRL and MCT-treated rats on culture day 7. Confocal microscopy images show that CFs exhibit positive immunoreactivity against vimentin (a reliable fibroblast marker; green, A), α-smooth muscle actin (α-SMA, a myofilament protein typically expressed by activated myofibroblasts; green, D), and discoidin domain receptor 2 (DDR2, a collagen-specific receptor tyrosine kinase specifically expressed in CFs and myofibroblasts; red, B and E). Panels C and F show a merge of vimentin and DDR2, and α-SMA and DDR2, respectively. Nuclei are stained in blue with DAPI; yellow denotes co-localization. Scale bar: 30 μm

Fig. 3. Cardiac fibroblasts (CFs) isolated from the RV of MCT-treated rats exhibit increased cell viability/proliferation (MTT assay) and produce higher amounts of type I collagen (Sirius Red assay) than cells from healthy controls. Panel A illustrates changes in viability/proliferation measured by the MTT assay (i) and type I collagen production assessed by the Sirius Red staining (ii) in CFs isolated from CTRL (n = 16) and MCT-treated (n = 18) rats. The ordinates represent absorbance determinations at 600 nm and 550 nm per well at certain time points (1, 7, 14, 21, and 28 days), for the MTT assay (Ai) and the Sirius Red staining (Aii), respectively. Data are mean ± SEM from an n number of individuals; the experiments were performed in triplicate. *p < 0.05 (ANOVA, one-way analysis of variance) compared to the CTRL group. In panel B, the red labelling in the upper panels represents vimentin-positive cells infiltrating RV interstitial spaces in CTRL and MCT-treated animals (Bi); scale bar: 100 μm. The binary (black and white) representation of the images (bottom panels) was used for automatic quantification by the ImageJ software® (version 1.8.0, U.S. National Institutes of Health,…

Fig. 4. Cardiac fibroblasts (CFs) isolated from MCT-treated rats overexpress the adenosine A2B 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 A2BAR (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 A2BAR 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 A2BAR antibody (#AAR-003, Alomone Inc., Jerusalem, Israel) corresponding to a peptide near the predicted molecular weight of the A2BAR (~37 kDa) and a higher molecular mass (~45 kDa) isotype. Please note that the naturally occurring A2BAR isotype is highly enriched in CFs isolated from the RV myocardium of MCT-treated rats compared to their CTRL…

Fig. 5. The enzymatically stable adenosine analogue, NECA (1–30 μM), concentration-dependently increases cell viability/growth and type I collagen production by cardiac fibroblasts (CFs) from the RV of CTRL and MCT-treated rats. The ordinates represent NECA (1–30 μM)-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 the adenosine analogue at culture days 7 (i), 14 (ii), 21 (iii), and 28 (iv). Zero represents the similarity between the two values (NECA 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 three to six animals performed in triplicate. 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 CTRL group

Fig. 6. Effects of the selective A2A receptor agonist, CGS216980 (3–10 nM), on cell viability/growth and type I collagen production by cardiac fibroblasts (CFs) from the RV of CTRL and MCT-treated rats. The ordinates represent CGS216980 (3–10 nM)-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 the A2AAR agonist at culture days 7, 14, 21, and 28. Zero represents the similarity between the two values (CGS216980 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 7 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

Fig. 7. Selective A2B 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

Fig. 8. Selective A2A receptor blockage with SCH442416 (100 nM) did not modify NECA-induced overgrowth of cardiac fibroblasts (CFs) from the RV of MCT-treated rats. NECA (10 μM) with or without SCH442416 (100 nM) was incorporated in culture media throughout the whole assay. The ordinates represent NECA- and/or SCH442416-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 8 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