Low extracellular Ca2+: a mediator of endothelial inflammation

Background. Recent studies have suggested that vitamin D and an imbalance in calcium homeostasis may have an impact on the cardiovascular system. The aim of this study was to assess the impact of different concentrations of extracellular Ca 2+ on human umbilical vein cord endothelial cells (HUVEC) by measuring its effect on parameters involved in the pathogenesis of vascular inflammatory responses. Methods. HUVEC were grown in the 3.5, 4.5 or 5.8 mg/dL concentration of extracellular Ca 2+ for 2―3 weeks. Expression of adhesion molecules was analysed by flow cytometry. Endothelial nitric oxide synthase (eNOS), receptor of advanced glycation end-product (RAGE) and interleukin-6 (IL-6) mRNA expressions were determined by real-time PCR. eNOS, inhibitor kappa Bα (IκBα) and phosphorylated IκBα protein levels by Western blot, eNOS activity by conversion of [ 14 C]-arginine to [ 14 C]-citrulline, IL-6 and osteoprotegerin (OPG) secretion by ELISA and DNA-binding activity of nuclear factor kappa B (NFκB)-p65 were assayed colorimetrically in nuclear extracts. Results. In the presence of low Ca 2+ (3.5 mg/dL), protein expressions and activity of eNOS were diminished, while the protein expressions of intercellular adhesion molecule-1 (ICAM-1), as well as the mRNA expressions of RAGE and IL-6, were elevated. The protein secretions of IL-6 and OPG were also stimulated in low Ca 2+ concentration. At this concentration, the DNA-binding activity of NFκB was enhanced, probably due to the decreased level of IκBα. Conclusions. These results suggest that lower extracellular ionized Ca 2+ may play a relevant role in modifying endothelial cells functions.