G Rashid1, J Bernheim, J Green, S Benchetrit. 1. Renal Physiology Laboratory, Department of Nephrology and Hypertension, Meir Medical Center, Kfar-Saba, Israel.
Abstract
BACKGROUND: We showed previously that parathyroid hormone (PTH) may stimulate the endothelial expression of pro-atherosclerotic and pro-inflammatory markers. Considering the impact of PTH on vasculature, we decided to evaluate its effect on mRNA and intra-cellular protein expressions of endothelial vascular endothelial growth factor (VEGF) taking into account that VEGF may play a role in the pathogenesis of endothelial dysfunctions. MATERIALS AND METHODS: Human umbilical vein cords endothelial cells (HUVEC) were stimulated for 24 h with 10(-12)-10(-10) mol L(-1) PTH. The VEGF-165 mRNA expression (critical in stimulating endothelial cell proliferation) was evaluated by RT/PCR and the intra-cellular VEGF protein expression by flow cytometry. The pathways by which PTH may have an effect on VEGF expression were also evaluated. RESULTS: PTH (10(-10) mol L(-1)) significantly increased VEGF-165 mRNA expression (P < 0.05). The addition of 50 nmol L(-1) protein kinase C (PKC) and 10 micromol L(-1) protein kinase A (PKA) inhibitors significantly reduced the VEGF-165 mRNA expression (P = 0.01). We also examined whether nitric oxide (NO) may be involved in the PTH-induced stimulation of VEGF-165 expression. Pre-treatment of the cells with 200 micromol L-nitro arginine methyl ester (L-NAME, NO synthase inhibitor) was found to inhibit VEGF-165 mRNA expression (P = 0.006). VEGF protein could not be detected in the medium of HUVEC but it was present in the cell cytoplasm. PTH had no significant effect on cytoplasmatic VEGF protein expression. CONCLUSION: The stimulatory effect of PTH on endothelial VEGF-165 mRNA expression is partly through PKC and PKA pathways and is also NO dependent.
BACKGROUND: We showed previously that parathyroid hormone (PTH) may stimulate the endothelial expression of pro-atherosclerotic and pro-inflammatory markers. Considering the impact of PTH on vasculature, we decided to evaluate its effect on mRNA and intra-cellular protein expressions of endothelial vascular endothelial growth factor (VEGF) taking into account that VEGF may play a role in the pathogenesis of endothelial dysfunctions. MATERIALS AND METHODS:Human umbilical vein cords endothelial cells (HUVEC) were stimulated for 24 h with 10(-12)-10(-10) mol L(-1) PTH. The VEGF-165 mRNA expression (critical in stimulating endothelial cell proliferation) was evaluated by RT/PCR and the intra-cellular VEGF protein expression by flow cytometry. The pathways by which PTH may have an effect on VEGF expression were also evaluated. RESULTS:PTH (10(-10) mol L(-1)) significantly increased VEGF-165 mRNA expression (P < 0.05). The addition of 50 nmol L(-1) protein kinase C (PKC) and 10 micromol L(-1) protein kinase A (PKA) inhibitors significantly reduced the VEGF-165 mRNA expression (P = 0.01). We also examined whether nitric oxide (NO) may be involved in the PTH-induced stimulation of VEGF-165 expression. Pre-treatment of the cells with 200 micromol L-nitro arginine methyl ester (L-NAME, NO synthase inhibitor) was found to inhibit VEGF-165 mRNA expression (P = 0.006). VEGF protein could not be detected in the medium of HUVEC but it was present in the cell cytoplasm. PTH had no significant effect on cytoplasmatic VEGF protein expression. CONCLUSION: The stimulatory effect of PTH on endothelial VEGF-165 mRNA expression is partly through PKC and PKA pathways and is also NO dependent.
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