Study of vascular smooth muscle cell calcification induced by hyperphosphate and intervented by phosphonoformic acid

Objective： To evaluate the effects of different concentrations of phosphate on calcium deposition and osteocalcin level in cultured bovine aortic smooth muscle cell, investigate the mechanism of hyperphosphatemia to evoke calcification of vascular smooth muscle cell and observe the effects of phosphonoformic acid（PFA） in different concentrations on vascular calcification. Methods： The bovine aortic smooth muscle cells （BASMC） were cultured.Calcium deposition and the expression of osteocalcin of BASMC in different concentrations of phosphate （1.5 mmol/L and 2.0 mmol/L） and PFA were determined by o-cresolphthalein complexone and radioimmunity methods, respectively. Osteocalcin mRNA expressions were determined by RT-PCR. Results： After six or nine days of BASMC cultured, the calcium deposition in Pi 2.0 mmol/L group was more than that in Pi 1.5 mmol/L group[（77.187 ± 11.692） lag/（mg · protein） vs（25.768 ± 1.750）lag/（mg · protein）, P 〈 0.01 and（125.399 ± 16.677）lag/（mg · protein） vs（29.046 ± 2.635）lag/（mg · protein）, P 〈 0.01 respectively]. The calcium deposition was dependent on time and dosage of phosphate treatment. After 72 h culture the osteocalcin in Pi 2.0 mmol/L group was more than that in Pi 1.5 mmol/L grouplin supematant,（1.503 ± 10^-2 ± 2.601 × 10^-3）ng/（ lag o protein） vs（2.981 × 10-3 ± 8.382 × 10-34）ng/（ lag · protein）, P 〈 0.001], the same was found in osteocalcin mRNA expression[OC/GAPDH, （1.906 ± 0.132） vs（0.748 ± 0.037）, P〈 0.001]. Compared to Pi 1.5mmol/L group,bovine smooth muscle cells（BSMC） cultured in media containing Pi 2.0 mmol/L phosphate levels increased calcium deposition[On day 6,（77.187 ± 11.692） la g/（mg · protein） vs （25.768 ±1.750） la g/（mg · protein）, P 〈 0.001]. Elevated phosphate treatment of BSMCs also enhanced the expression of the osteoblastic differentiation marker osteocalcin[On day 3, Pi 2.0 mmol/L group vs Pi 1.5mmol/L group,（1.503 × 10^-2 ± 2.601 × 10^-3 ）ng/（ lag · protein） vs（2.981× 10^-3 ± 8.382 × 10^-4）ng/（ μg · protein）, P 〈 0.001]. PFA decreased ciacium deposition and osteocalcin expression statistically[Pi 2.0 mmol/L±PFA1.0 mmol/L group vs Pi 2.0mmol/L group, ciacium deposition, （37.729 ± 5.899） lμg/（mg · protein） vs （77.187 ± 11.692）μg/（mg ·protein）, P 〈 0.001]; Osteocalcin in supernatant, （4.529 ± 10^-3 ± 1.250 × 10^-3）ng/（ μ g · protein） vs（1.503 × 10^-2 ± 2.601 × 10^-3） ng/（ μg · protein）, P〈 0.001; osteocalcin mRNA expression, OC/GAPDH, （0.642 ± 0.092） v s （1.89 ± 0.165）, P 〈 0.01]. Conclusion： Hyperphosphate may directly promote calcium deposition and the osteocalcin expression of B ASMCs. It may be a new explanation for the phenomenon of vascular calcification in hyperphosphatemic conditions. Hyperphosphatemia is an independent factor to stimulate vascular calcification. PFA can inhibit calcium deposition and osteocalcin expression induced by elevated phosphate.PFA may be a new medicine to treat vascular calcification induced by elevated phosphate.

Synthesis of N-(methoxycarbonyl or isopropylcarbamoyl- methoxyphosphonyl)-α-amino acid ester and their stereomers

<正> N-(methoxycarbonyl-methoxyphosphonyl)-α-amino add esters (I) were synthesized via the reaction of the corresponding phosphonyl chloride with amino acid ester hydrochlorides in the presence of a base. Compound I was aminated to yield N-(isopropylcarbainoyl-methoxyphosphonyl)-α-amino acid esters (II). With l-amino acids as starting materials, the isomers of products I and II were separated and their configurations were confirmed by the single crystal X-ray diffraction of II.