In Vitro Characterization of a Tissue Renin-Angiotensin System in Human Nucleus Pulposus Cells

Figures

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  Figure 1. Immunofluorescence analysis of the angiotensin II type 1 (AGTR1) and 2 (AGTR2) and MAS1 receptor, as well as angiotensin II (AngII) and angiotensin-converting enzyme 2 (ACE2). Representative images are shown. n = 4 biological replicates with 2 technical replicates each were evaluated via immunofluorescence. DAPI was used to visualize cell nuclei. Scale bar, 20 μm.

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  Figure 2. Cell metabolic activity analysis in human NP cells treated with angiotensin II (0.1 µM–10 µM) after 24 h, 48 h, and 72 h of exposure. Cell metabolic activity normalized to the baseline (day 0/beginning of drug exposure) is shown. n = 2 biological replicates and n = 3 technical replicates per donor were assessed. * p < 0.05.

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  Figure 3. Illustration of tRAS axis ratios (AGTR1/AGTR2, AGTR1/MAS1) [11] and inflammatory to anti-inflammatory gene expression markers (Il-6/IL-10, IL-8/IL-10, and TNF-α/IL-10) after 24 h (A) and 48 h (B) of drug or control exposure. Ratios of pro-inflammatory to anti-inflammatory markers are indicators for the inflammatory state of the intervertebral disc and low back pain [38,39]. AGTR1/AGTR2 and AGTR1/MAS1 gene expression ratios were positively correlated with the pro-inflammatory/anti-inflammatory marker ratios Il-6/IL-10, IL-8/IL-10, and TNF-α/IL-10. Spearman correlation coefficients are shown in the matrix. Fold change values obtained from the 2^(−DeltaDeltaCt) method and normalized to the baseline (day 0) are shown. n = 4 biological replicates and n = 3 technical replicates per donor were assessed. Inflammatory stimulus indicates the groups exposed to TNF-α. Correlation of ratios was assessed by taking the mean from the technical replicates for each group of the respective donors and calculating the correlation coefficient between the ratios (n = 24 per ratio; 4 donors x 6 groups (with the mean used for the three technical replicates per group). Lines are connected for better…

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  Figure 4. Illustration of indirect nitric oxide (NO2) concentrations in the medium for the study groups after 24 h (A) and 48 h (B) of drug/control exposure. Nitric oxide was measured indirectly from nitrite release from the metabolism of nitric oxide. n = 4 biological replicates and n = 3 technical replicates per donor were assessed. Mean and standard error of the mean (SEM) are shown. #,* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

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  Figure 5. Cell metabolic activity analysis in human NP cells for candesartan (“Cand”) (1 µM, 10 µM, 100 µM), the MAS-receptor agonist AVE 0991 (“AVE”) (1 µM, 10 µM, 100 µM), and the AGTR2-receptor agonist CGP-42112A (“CGP”) (0.1 µM, 1 µM, 10 µM) with 4 h, 24 h, and 48 h of exposure. DMSO (0.1 µL/mL, 1 µL/mL, 10 µL/mL; shown in µL volume on the x-axis) was used as the solvent control. Cell viability normalized to the baseline (day 0/timepoint of drug exposure) is shown. n = 2 biological replicates and n = 3 technical replicates per donor were assessed. Mean and standard deviation (SD) are shown.

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  Figure 6. Gene expression analysis of inflammation-related markers in human NP cells for candesartan (1 µM, 10 µM, 100 µM), the MAS-receptor agonist AVE 0991 (1 µM, 10 µM, 100 µM), and the AGTR2-receptor agonist CGP-42112A (0.1 µM, 1 µM, 10 µM) with 4 h and 24 h of exposure in inflammatory settings (TNF-α addition). DMSO (0.1 µL/mL, 1 µL/mL, 10 µL/mL; shown in µL volume on the x-axis) was used as the solvent control. Fold change values normalized to the baseline (day 0) are shown. n = 4 biological replicates and n = 3 technical replicates per donor were assessed. Mean and standard error of the mean (SEM) are shown. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

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  Figure 7. Gene expression analysis of tissue-degenerative and phenotype-modulating markers in human NP cells for candesartan (1 µM, 10 µM, 100 µM), the MAS-receptor agonist AVE 0991 (1 µM, 10 µM, 100 µM), and the AGTR2-receptor agonist CGP-42112A (0.1 µM, 1 µM, 10 µM) with 4 h and 24 h of exposure in inflammatory settings (TNF-α addition). DMSO (0.1 µL/mL, 1 µL/mL, 10 µL/mL; shown in µL volume on the x-axis) was used as the solvent control. Fold change values normalized to the baseline (day 0) are shown. n = 4 biological replicates and n = 3 technical replicates per donor were assessed. Mean and standard error of the mean (SEM) are shown. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

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  Figure 8. Gene expression analysis of tRAS markers in human NP cells for candesartan (1 µM, 10 µM, 100 µM), the MAS-receptor agonist AVE 0991 (1 µM, 10 µM, 100 µM), and the AGTR2-receptor agonist CGP-42112A (0.1 µM, 1 µM, 10 µM) with 4 h and 24 h of exposure in inflammatory settings (TNF-α addition). DMSO (0.1 µL/mL, 1 µL/mL, 10 µL/mL; shown in µL volume on the x-axis) was used as the solvent control. Fold change values normalized to the baseline (day 0) are shown. n = 4 biological replicates and n = 3 technical replicates per donor were assessed. Mean and standard error of the mean (SEM) are shown. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

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  Figure 9. Protein alterations of IL-6, IL8, IL10, IL-8/IL-10 ratio, IL6-IL-10 ratio, as well as intracellular and secreted angiotensin II and iNOS in human nucleus pulposus cells after 24 h of exposure to candesartan (10 µM), the MAS-receptor agonist AVE 0991 (10 µM), and the AGTR2-receptor agonist CGP-42112A (1 µM) in inflammatory settings (TNF-α addition). DMSO (1 µL/mL) shown in µL volume on the x-axis) was used as the solvent control. Fold change values are shown. For the iNOS analysis, fold change in relative band intensity of GAPDPH and control are shown. n = 3 biological replicates and n = 2 technical replicates per donor were assessed in the ELISA analysis. n = 2 biological replicates were assessed for the western blot analysis. ImageJ was used for Western blot quantification [37]. Mean and standard deviations (SD) are shown.* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

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  Figure 10. Illustration of tissue renin-angiotensin system (tRAS) mechanisms in humans [11]. Local renin-angiotensin systems are found in human nucleus pulposus cells. The angiotensin II receptor type 2 (AGTR2) and the Ang1–7/Mas receptor (MasR) axis counteract the pro-inflammatory effects (increasing intracellular reactive oxygen species (ROS) of accumulating angiotensin II concentrations (AngII) on angiotensin II receptor type 1 (AGTR1). The inhibition of the pathological axis (AGTR1) and the stimulation of the protective axis could show beneficial effects on the inflammatory and degenerative state of cells. ARBs: angiotensin II type 1 receptor blockers.