Cbfa1 expression in vascular smooth muscle cells may be elevated by increased nitric oxide/iNOS

ABSTRACT Introduction: Vascular calcification is a common complication of chronic kidney disease. Osteoblast differentiation factor (Cbfa1) is present in histologic sections of arteries from patients with end-stage renal disease. Vascular smooth muscle cells (VSMC) can dedifferentiate to osteoblast-like cells, possibly by up-regulation of Cbfa1. There is evidence that the production of nitric oxide (NO) may have an important role in the regulation of osteoblast metabolism. The aim of this study is to evaluate whether increased NO/iNOS expression causes an increase in cbfa1 expression in VSMC. Methods: VSMC were obtained from renal artery of Wistar male rats, treated for 72 hours with lipopolysaccharide (LPS), ß-glycerophosphate (BGF), a donor of phosphate and aminoguanidine (AG), an inhibitor of iNOS, in the following groups: CTL (control), LPS, BGF, LPS + BGF, and LPS + AG. NO synthesis was determined by chemiluminescence. Cbfa1 and iNOS mRNA expressions were analyzed by RT-PCR, Cbfa1 protein expression by immunohistochemistry and cellular viability by acridine orange. Results: Cbfa1 and iNOS mRNA expressions were higher in LPS and LPS+ BGF vs CTL (p < 0.05), and they were lower in LPS+AG vs LPS (p < 0.05). The Cbfa1 in the groups LPS and LPS+BGF also resulted in a higher value compared to CTL (p < 0.05), and in LPS+AG it was lower compared to LPS (p < 0.05). NO was higher in LPS and LPS+BGF compared to CTL group (p < 0.05) and lower in LPS + AG compared to LPS group (p < 0.05). Cellular viability showed no statistical difference among groups. Conclusion: This study showed that increased NO/iNOS expression causes an increase in cbfa1 expression in VSMC.

Introduction: Vascular calcification is a common complication of chronic kidney disease. Osteoblast differentiation factor (Cbfa1) is present in histologic sections of arteries from patients with end-stage renal disease. Vascular smooth muscle cells (VSMC) can dedifferentiate to osteoblast-like cells, possibly by up-regulation of Cbfa1. There is evidence that the production of nitric oxide (NO) may have an important role in the regulation of osteoblast metabolism. The aim of this study is to evaluate whether increased NO/iNOS expression causes an increase in cbfa1 expression in VSMC. Methods: VSMC were obtained from renal artery of Wistar male rats, treated for 72 hours with lipopolysaccharide (LPS), ß-glycerophosphate (BGF), a donor of phosphate and aminoguanidine (AG), an inhibitor of iNOS, in the following groups: CTL (control), LPS, BGF, LPS + BGF, and LPS + AG. NO synthesis was determined by chemiluminescence. Cbfa1 and iNOS mRNA expressions were analyzed by RT-PCR, Cbfa1 protein expression by immunohistochemistry and cellular viability by acridine orange. Results: Cbfa1 and iNOS mRNA expressions were higher in LPS and LPS+ BGF vs CTL (p < 0.05), and they were lower in LPS+AG vs LPS (p < 0.05). The Cbfa1 in the groups LPS and LPS+BGF also resulted in a higher value compared to CTL (p < 0.05), and in LPS+AG it was lower compared to LPS (p < 0.05). NO was higher in LPS and LPS+BGF compared to CTL group (p < 0.05) and lower in LPS + AG compared to LPS group (p < 0.05). Cellular viability showed no statistical difference among groups. Conclusion: This study showed that increased NO/iNOS expression causes an increase in cbfa1 expression in VSMC.

IntRoductIon
The pathogenesis of vascular calcification is not completely understood in clinical practice. Therefore, understanding the regulatory mechanisms that control this calcification is essential for exploring therapeutic targets for potential clinical applications. Abnormalities in mineral metabolism, in particular hyperphosphatemia, frequently observed in chronic renal disease, have emerged as a key regulator of vascular calcification and been considered risk factors for cardiovascular mortality in this population 6,7 .
NO is a cell-signaling molecule with multiple roles, including the regulation of vascular tone and neurotransmission 8,9 . NO is produced by most cells of the body and its substrate is the L-arginine, which under the effect of nitric oxide synthase (NOS) generates L-citrulline and NO 10 . The NO synthesized by endothelial cells is crucial in maintaining vascular health and preventing the development of vascular diseases 11 . NOS presents two isoforms: the constitutive (cNOS) and the inducible (iNOS); the latter can be induced by agents as lipopolysaccharide (LPS) from several bacteria and inhibited by aminoguanidine (AG) 12 .

NOS expression has been detected in vivo and in vitro
in osteoblastic cell lineage 13 and there is evidence that the production of NO may have an important role in the regulation of osteoblast metabolism 14 .
Studies about the effect of NO on osteoblastic function are unclear. Some researchers have shown that NO donors increase cGMP production and alkaline phosphatase activity and induce bone nodule formation in vitro 15,16 . Therefore, the aim of the present study was to evaluate whether increased NO production by iNOS has an effect on Cbfa1 expression in VSMC from rat renal artery.

methods
This study was approved by the Institutional Animal Ethics Committee.

VSMc culture
Wistar male rats weighing 250 to 300 g were anesthetized with a mixture of ketamine (80 mg/kg) and xylazine (10 mg/kg) by intraperitoneal injection. After 72 hours, cell culture media were collected and stored at -20 °C for nitric oxide determination.
Cells were lysed with 2% sodium dodecyl sulfate (SDS) for protein determination.

StatiStical analySiS
Results were reported as mean ± standard error of mean    Dose-response curve of no synthesis (nmol/mg protein) in vsmc exposeD to lps (50, 100, 200, or 300 µ g/ml) for 72 hours Mean ± SEM, one-way analysis of variance (ANOVA); a p < 0.05 vs LPS 50 µg/mL        Although oxidative stress was not evaluated in this study, it should be remembered that reactive nitrogen species act in conjunction with reactive oxygen species, damaging cells and causing nitrosative stress 8 . These two species are therefore often referred to collectively and would have to be better evaluated in future studies.
Although there was an increase in Cbfa1 expression following increased NO/iNOS expression, it cannot be said it was due to either a direct or indirect path. To support this hypothesis, further investigations about mechanisms will be needed for a better understanding of the complex relationship between NO system and the Cbfa1 gene expression.

conclusIon
In conclusion, this study showed that increased NO/ iNOS expression causes an increase in cbfa1 expression in cultured VSMC, indicating that increased NO production may participate on Cbfa1 expression.