The effects of reactive oxygen species (ROS) on different cellular types are variable. In some conditions they can be harmful metabolites, but they can also act as intracellular messengers that are able to activate different transcription factors. Based on previous reports in which ROS were shown to stimulate the proliferation of mesenchymal cells, this study was carried out to assess this effect on bovine aortic endothelial cells (BAECs). When cells were incubated with glucose oxidase (GO), an enzyme that generates H2O2 continuously, a significant increase in BAEC proliferation was detected. BAEC proliferation was measured by the incorporation of [3H]-thymidine in the DNA of BAECs, and also by an increase in the number of cells. The effect observed with GO was maximal at 8-24 h. Catalase abolishes proliferation. We also tested the ability of GO to phosphorylate tyrosine residues in endothelial cell proteins. A significant increase in tyrosine phosphorylation was found, which might constitute the molecular basis for proliferative effect of GO. In conclusion, these results demonstrate the ability of H2O2 to stimulate BAEC proliferation at least under certain experimental conditions. We suggest a general activation of the cascade of tyrosine phosphorylation as one of the possible cellular mechanisms responsible for GO-induced BAEC proliferation.

Despite the presence of vitamin D receptor (VDR) in endothelial cells, the effect of vitamin D on endothelial function is unknown. An unbalanced production of vasoactive endothelial factors such as nitric oxide (NO) or endothelin-1 (ET-1) results in endothelial dysfunction, which can alter the normal cardiovascular function. Present experiments were devoted to assess the effect of active vitamin D (calcitriol) on the synthesis of endothelial vasoactive factors. The results were that, in cells, calcitriol increased ET-1 and NO productions, which were measured by ELISA and fluorimetric assay, respectively. Calcitriol also increased endothelin-converting enzyme-1 (ECE-1) and endothelial-nitric oxide synthase (eNOS) activities, their mRNA (qPCR), their protein expressions (Western-blot), and their promoter activities (transfection assays). Calcitriol did not change prepro-ET-1 mRNA. The effect was specific to VDR activation because when VDR was silenced by siRNA, the observed effects disappeared. Mechanisms involved in each upregulation differed. ECE-1 upregulation depended on AP-1 activation, whereas eNOS upregulation depended directly on VDR activation. To evaluate the in vivo consequences of acute calcitriol treatment, normal Wistar rats were treated with a single ip injection of 400 ng/kg calcitriol and euthanized 24 h later. Results confirmed those observed in cells, that production and expression of both factors were increased by calcitriol. Besides, calcitriol-treated rats showed a slight rise in mean blood pressure, which decreased when pretreated with FR-901533, an ECE-1 antagonist. We conclude that calcitriol increases the synthesis of both ET-1 and NO in endothelial cells. However, the ET-1 upregulation seems to be biologically more relevant, as animals acutely treated with calcitriol show slight increases in blood pressure.

Summary: Hyperphosphatemia is related to some pathologies, affecting vascular cell behavior. This work analyzes whether high concentration of extracellular phosphate induces endothelial senescence through up‐regulation of endothelin‐1 (ET‐1), exploring the mechanisms involved. The phosphate donor β‐glycerophosphate (BGP) in human endothelial cells increased ET‐1 production, endothelin‐converting enzyme‐1 (ECE‐1) protein, and mRNA expression, which depend on the AP‐1 activation through ROS production. In parallel, BGP also induced endothelial senescence by increasing p16 expression and the senescence‐associated β‐galactosidase (SA‐ß‐GAL) activity. ET‐1 itself was able to induce endothelial senescence, increasing p16 expression and SA‐ß‐GAL activity. In addition, senescence induced by BGP was blocked when different ET‐1 system antagonists were used. BGP increased ROS production at short times, and the presence of antioxidants prevented the effect of BGP on AP1 activation, ECE‐1 expression, and endothelial senescence. These findings were confirmed in vivo with two animal models in which phosphate serum levels were increased: seven/eight nephrectomized rats as chronic kidney disease models fed on a high phosphate diet and aged mice. Both models showed hyperphosphatemia, higher levels of ET‐1, and up‐regulation in aortic ECE‐1, suggesting a direct relationship between hyperphosphatemia and ET‐1. Present results point to a new and relevant role of hyperphosphatemia on the regulation of ET‐1 system and senescence induction at endothelial level, both in endothelial cells and aorta from two animal models. The mechanism involved showed a higher ROS production, which probably activates AP‐1 transcription factor and, as a result, ECE‐1 expression, increasing ET‐1 synthesis, which in consequence induces endothelial senescence.

In mammalians, advancing age is associated with sarcopenia, the progressive and involuntary loss of muscle mass and strength. Hyperphosphatemia is an aging-related condition involved in several pathologies. The aim of this work was to assess whether hyperphosphatemia plays a role in the age-related loss of mass muscle and strength by inducing cellular senescence in murine myoblasts and to explore the intracellular mechanism involved in this effect. Cultured mouse C2C12 cells were treated with 10 mM beta-glycerophosphate (BGP] at different periods of time to induce hyperphosphatemia. BGP promoted cellular senescence after 24 h of treatment, assessed by the increased expression of p53, acetylated-p53 and p21 and senescence associated β-galactosidase activity. In parallel, BGP increased ILK expression and activity, followed by mTOR activation and autophagy reduction. Knocking-down ILK expression increased autophagy and protected cells from senescence induced by hyperphosphatemia. BGP also reduced the proliferative capacity of cultured myoblasts. Old mice (24-months-old] presented higher serum phosphate concentration, lower forelimb strength, higher expression of p53 and ILK and less autophagy in vastus muscle than young mice (5-months-old]. In conclusion, we propose that hyperphosphatemia induces senescence in cultured myoblasts through ILK overexpression, reducing their proliferative capacity, which could be a mechanism involved in the development of sarcopenia, since old mice showed loss of muscular strength correlated with high serum phosphate concentration and increased levels of ILK and p53.