Angiotensin-II Stimulated Cytokines Expression by Human Deciduous Pulp Fibroblasts

Objectives: This study aimed to evaluate the toxicity of different concentrations of angiotensin II (ANG II) and their capacity to stimulate gene expression of inflammatory cytokines by human deciduous tooth pulp fibroblasts.
Methods: ANG II (10^-7 M and 10^-6 M) were tested for their cytotoxicity and ability to stimulate the expression of inflammatory cytokines by HPDF. Primary fibroblasts were seeded in 96-well plates (1 x 10⁴ cells/well) and 24-well plates (2.5 x 10⁵ cells/well) for the evaluation of toxicity and gene expression, respectively. After 24 hours culture medium containing 10^-7 M or 10^-6 M ANG II was added to wells in triplicate in a final volume of 200μL/well (96-well plate) and 500μL/well (24-well plate). DMEM was added to the wells, as control. After the periods of 1, 3, 6, 12 and 24 hours, culture medium was removed and 50μL/well of the LIVE/DEAD fluorescent solution (2μM calcein and 4μM ethidium homodimer) were added. Plates were read at wavelengths of 485/530nm and 530/645nm. In the 96-well plates 230μL/well of the lysis solution from the RNA extraction kit was added. Quantitative expression of mRNA for SRA receptors AT1 and MAS as well as for cytokines (IL-1α, IL-1β, IL-2, IL-3, IL-4, IL- 8, IL-9, IL-15, IL-17A, IL-18, TGF-β, MCP-1 (CCL2), INF- α, INF-γ , TNF- α, were analyzed by RT-qPCR. RPL13 was used as the reference gene.
Results: Both Ang II concentrations showed no toxic effect on cells at any experimental times. HPDF express AT1 receptor gene and this expression was higher at 10⁶ M concentration after 24h. On the other hand, the concentration of 10⁷ did not promote any change in this expression. For MAS a low mRNA expression was detected. Expression of IL-6, IL-1b, IL-15, INF-a1 and TGF-b did not change with both concentrations at any time. For IL-8, an increase in gene expression with 10⁶ was observed in the 6-hour period, while the concentration of 10⁷ did not promote significant changes. For CCL2, mRNA expression was increased when 10⁷ ANG II was used in 12-hour period. IL-4, IL-18 and IL-1a cytokines showed low expression while no expression was detected for IL-2, IL-3, IL-9, IL-17A and INF-γ cytokines.
Conclusions: Ang II is capable of increasing mRNA expression of AT1 receptor and some cytokines involved in the inflammatory response of human deciduous pulp fibroblasts.