Abstract
Aim: To investigate the influence of type 2 diabetes (T2D) and hyperglycaemia on blood vessels and angiogenic markers in the dental pulp.
Methodology: Extracted non-carious permanent molar teeth were collected from patients with well-controlled T2D (n = 10) and non-T2D (controls) (n = 10). The pulp was examined qualitatively using haematoxylin and eosin and Van Gieson stains. Immunohistochemistry (IHC) identified the primary receptor for VEGF, VEGFR2, and the endothelial cell marker CD34. Primary human dental pulp cell (hDPC) lines (n = 3) were established from tissue explants and cells were grown in media containing 5.5 mM D-glucose (control), 12.5 mM (prediabetes) and 25 mM (T2D) D-glucose under normoxic conditions for 24, 48 and 72 h. Assays for metabolic activity (PrestoBlue) and cell viability (Crystal Violet staining) assessed the hDPC response to hyperglycaemia. The expression of angiogenic genes VEGFA, KDR, FLT-1, ANGPT1, ANGPT2, TIE1 and TEK were analysed using quantitative real-time polymerase chain reaction. ELISAs were used to quantify the level of expressed protein for VEGFA, ANG1, ANG2, TIE1, and TIE2 in the media. Data analyses were performed using GraphPad Prism and anova tests at p < .05.
Results: Blood vessels in T2D samples had thicker walls and stained strongly for elastin and collagen compared with non-T2D samples. VEGFR2 protein was not seen in any T2D samples but consistently detected in healthy specimens. Culturing healthy cells in high glucose (25 mM) significantly reduced cell viability at 24 h compared to the control (p = .005) and 12.5 mM glucose (p = .001) but the metabolic activity was not greatly affected by glucose and time. VEGFA mRNA and VEGFA protein expression were detected in the hDPCs in the presence of hyperglycaemia over time; however, the primary receptor, VEGFR2/KDR, was not detected. Genes for the ANG1 and ANG2 and their receptors were expressed at all glucose concentrations but hyperglycaemia upregulated ANG2 mRNA. Proteins for all growth factors were detected in the media however proteins for TIE1 and TIE2 receptors were not.
Conclusion: T2D and hyperglycaemia may impair the angiogenic response in the pulp similar to other body site. The scarcity of VEGFR2 and increased expression of ANG2 in response to hyperglycaemia suggests that VEGF and ANG-Tie1/Tie2 signalling may be compromised.