Can angiotensin II affect the inflammatory response of human dental pulp cells in vitro?

Figures

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  Figure 1. Target genes used in RT-qPCR reactions for HPF and HPFD, with respective TaqMan® Assay IDs.

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  Figure 2. Phenotypic characterization of pulp cells by fibroblast surface protein (FSP) staining. HPF (A, B, and C) and HDPF (D, E, and F) showed positive staining for FSP-1 protein (green). Cell nuclei were stained with DAPI- blue (4’,6- diamidino-2-phenylindole dihydrochloride). Negative controls are also shown. Images were acquired using confocal microscopy (TCS model, SPE, Leica®, Mannheim, Germany) with a 40× objective lens. Scale bars represent 20 μm.

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  Figure 3. qPCR analysis of RAS components by HDPF stimulated without (Control) or with lipopolysaccharide from Escherichia coli (EcLPS) (1μg/mL) or ANG II (1 μM) for six and 24 hours. Relative expression levels of the target mRNA relative to RPL13 mRNA from four donors in triplicates (n = 4) are shown in graphs. The mean values for each patient are represented as a symbol to compare the stimulated values with their respective control group in the same experimental period by the non-paired t test. * indicates significant difference with the respective control in the same experimental period (p<0.05).

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  Figure 4A. qPCR analysis of cytokines/chemokines by human deciduous dental pulp fibroblasts (HDPF) stimulated with or without (Control) lipopolysaccharide from Escherichia coli (EcLPS) or angiotensin II (ANG II, 1 μM) for six (4A) and 24 hours (4B). Relative expression levels of the target mRNA normalized to RPL13 mRNA from four donors in triplicates (n = 4) are shown in graphs. The following targets were analyzed: CCL2, CXCL12, TNF-α, vascular endothelial growth factor (VEGF), interleukin-1α (IL-1α), and interleukin-1β (IL-1β). Mean values for each donor are represented by individual symbols. Expression levels in stimulated groups were compared to their respective control group at the same time point using an unpaired t-test. Asterisks indicate statistically significant differences to the respective control group at the same time point (p<0.05).*

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  Figure 4B. qPCR analysis of cytokines/chemokines by human deciduous dental pulp fibroblasts (HDPF) stimulated with or without (Control) lipopolysaccharide from Escherichia coli (EcLPS) or angiotensin II (ANG II, 1 μM) for six (4A) and 24 hours (4B). Relative expression levels of the target mRNA normalized to RPL13 mRNA from four donors in triplicates (n = 4) are shown in graphs. The following targets were analyzed: CCL2, CXCL12, TNF-α, vascular endothelial growth factor (VEGF), interleukin-1α (IL-1α), and interleukin-1β (IL-1β). Mean values for each donor are represented by individual symbols. Expression levels in stimulated groups were compared to their respective control group at the same time point using an unpaired t-test. Asterisks indicate statistically significant differences to the respective control group at the same time point (p<0.05).*

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  Figure 5A. qPCR analysis of cytokines/chemokines by human dental pulp fibroblasts (HPF) stimulated with or without (Control) angiotensin II (ANG II, 1 μM) or interleukin-1β (IL-1β, 0.1 ng/mL) in isolation or combined for three, six, and 24 hours. Relative expression levels of the target mRNA normalized to RPL13 mRNA from three donors in triplicates (n=3) are shown in graphs. The following targets were analyzed: IL-6, IL-8, TNF-α (5A), CCL2, COX-2, and AT1R (5B). Mean values for each donor are represented by individual symbols. Expression levels in stimulated groups were compared to their respective control group at the same time point using an unpaired t-test. Asterisks indicate statistically significant differences to the respective control group at the same time point (p<0.05).

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  Figure 5B. qPCR analysis of cytokines/chemokines by human dental pulp fibroblasts (HPF) stimulated with or without (Control) angiotensin II (ANG II, 1 μM) or interleukin-1β (IL-1β, 0.1 ng/mL) in isolation or combined for three, six, and 24 hours. Relative expression levels of the target mRNA normalized to RPL13 mRNA from three donors in triplicates (n=3) are shown in graphs. The following targets were analyzed: IL-6, IL-8, TNF-α (5A), CCL2, COX-2, and AT1R (5B). Mean values for each donor are represented by individual symbols. Expression levels in stimulated groups were compared to their respective control group at the same time point using an unpaired t-test. Asterisks indicate statistically significant differences to the respective control group at the same time point (p<0.05).