Angiotensin II type 2 receptor agonist Compound 21 attenuates pulmonary inflammation in a model of acute lung injury

Figure 1. (A) Arterial PO2 (mmHg), (B) arterial PCO2 (mmHg), (C) heart rate (min−1), and (D) mean arterial pressure (mmHg) at baseline (0 min), after pulmonary lavage (5 min), and over the entire observation period (240 min) in rats with mechanical ventilation only (control; n=9), surfactant washout by repeated pulmonary lavage and mechanical ventilation (LAV; n=9), or surfactant washout by repeated pulmonary lavage, mechanical ventilation, and direct stimulation of the AT2 receptor with C21 (0.03 mg/kg body weight; LAV+C21; n=9). Notes: Data are presented as mean±SD; multivariate nonparametric analysis for longitudinal data over the course of time (two-way MANOVA), p>0.05 for comparison between the LAV and LAV+C21 groups. Abbreviations: AT2, angiotensin II type 2; C21, Compound 21; MANOVA, multivariate analysis of variance; MAP, mean arterial pressure.

Figure 2. Wet/dry-weight ratio of lungs of rats with mechanical ventilation only (control; n=9), repeated pulmonary lavage and mechanical ventilation (LAV; n=9), and repeated pulmonary lavage, mechanical ventilation, and direct stimulation of the AT2 receptor with C21 (0.03 mg/kg body weight; LAV+C21; n=9). Notes: Data are presented as mean±SD, Mann–Whitney U-test (n=9). n.s., not significantly different compared with lavaged group. *p<0.05. Abbreviations: AT2, angiotensin II type 2; C21, Compound 21.

Figure 3. (A) Protein content of recovered BALF of the first consecutive lavage samples; ***p<0.001 when compared with control (lavage fluid, NaCl 0.9%); ##p<0.01 when compared with the first lavage. Data are presented as mean±SD, Mann–Whitney U-test (n=9). (B) Dynamic measurement of surface tension expressed as Gamma (mN/m) of recovered BALF of the first lavage samples; ****p<0.0001 compared with control (lavage fluid, NaCl 0.9%); ####p<0.0001 compared with minimal surface tension of the first lavage; data are presented as mean±SD, Mann–Whitney U-test (n=9). Abbreviations: BALF, bronchoalveolar lavage fluid; n.d., not detected.

Figure 4. Relative mRNA expression of (A) TNF-α, (B) IL-6, (C) IL-1β, (D) IL-10, (E) IL-4, and (F) TGF-β in lungs of rats with mechanical ventilation only (control; n=9), repeated pulmonary lavage and mechanical ventilation (LAV; n=9) and repeated pulmonary lavage, mechanical ventilation, and direct stimulation of the AT2 receptor with C21 (0.03 mg/kg body weight; LAV+C21; n=9). Notes: Lungs were harvested after 240 min of mechanical ventilation and real-time polymerase chain reaction was performed, GAPDH served as housekeeping gene, expression of control was set to 100%. Data are presented as mean±SEM, Mann–Whitney U-test, *p<0.05 compared with LAV group (n=9). Abbreviations: AT2, angiotensin II type 2; C21, Compound 21; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; n.d., not detected; SEM, standard error of the mean; TGF-β, transforming growth factor-beta; TNF-α, tumor necrosis factor-alpha.

Figure 5. Protein expression of (A) TNF-α and (B) IL-1β in lungs of rats with mechanical ventilation only (control; n=9), repeated pulmonary lavage and mechanical ventilation (LAV; n=9), and repeated pulmonary lavage, mechanical ventilation, and direct stimulation of the AT2 receptor with C21 (0.03 mg/kg body weight; LAV+C21; n=9). Notes: Lungs were harvested after 240 min of mechanical ventilation, and protein levels were analyzed using CBA and FACS analysis. Results are given as pg (of the respective cytokine) per mg protein of lung tissue. Data are presented as mean±SEM, Mann–Whitney U-test, *p<0.05 compared with LAV group (n=9). Abbreviations: AT2, angiotensin II type 2; C21, Compound 21; CBA, cytometric bead array; FACS, fluorescence-activated cell sorting; SEM, standard error of the mean; TNF-α, tumor necrosis factor-alpha.

Figure 6. Relative mRNA and protein expression of the AT2 receptor in the lung of rats with mechanical ventilation (control; n=9), repeated pulmonary lavage and mechanical ventilation (LAV; n=9), and repeated pulmonary lavage, mechanical ventilation, and direct stimulation of the AT2 receptor with C21 (0.03 mg/kg body weight; LAV+C21; n=9). Notes: Lungs were extracted after 240 min of mechanical ventilation and real-time polymerase chain reaction (left) or immunoblotting (right) was performed. GAPDH served as housekeeping gene and α-actinin served as loading control. Expression of control was set to 100%. Data are presented as mean±SEM, Mann–Whitney U-test, *p<0.05 compared with LAV group (n=9). Abbreviations: AT2, angiotensin II type 2; C21, Compound 21; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SEM, standard error of the mean.