Metformin inhibits vascular calcification in female rat aortic smooth muscle cells via the AMPK-eNOS-NO pathway.

Metformin exhibits diverse protective effects against diabetic complications, such as bone loss. Here, we investigated the effect of metformin on vascular calcification, another type 2 diabetes complication. In female rat aortic smooth muscle cells (RASMCs), we observed that metformin significantly alleviated ß-glycerophosphate-induced Ca deposition and alkaline phosphatase activity, corresponding with reduced expression of some specific genes in osteoblast-like cells, including Runx2 and bone morphogenetic protein-2, and positive effects on α-actin expression, a specific marker of smooth muscle cells. Mechanistic analysis showed that phosphorylation levels of both AMP-activated protein kinase (AMPK) and endothelial nitric oxide synthase (eNOS) were increased with NO overproduction. After inhibition of either AMPK or eNOS with the pharmacologic inhibitors, compound C or Nω-Nitro-L-arginine methyl ester, NO production was lowered and metformin-meditated vascular protection against ß-glycerophosphate-induced Ca deposition was removed. Our results support that metformin prevents vascular calcification via AMPK-eNOS-NO pathway.

Bone morphogenetic protein-7 inhibits vascular calcification induced by high vitamin D in mice.

Vascular calcification refers to the deposition of calcium phosphate in cardiovascular tissues, including arteries and myocardium. Vascular calcification is frequently associated with cardiovascular disease. Recently, bone morphgenetic protein-7 (BMP-7) has been proposed to play an inhibitory role in vascular calcification, but its inhibitory effect has not been fully elucidated. We therefore tested the hypothesis that BMP-7 inhibits vascular calcification by using two conditions, high levels of vitamin D and phosphate, each of which could enhance vascular calcification. C57BL/6 mice were treated for 3 days with high vitamin D (500,000 IU/kg/day) in the presence or absence of recombinant human BMP-7 (rhBMP-7). Expression levels of osteopontin and osteocalcin, markers of the osteoblastic phenotype, were assessed by immunohistochemical staining or Western blotting analysis. Vitamin D increased calcium staining in thoracic aortas and hearts and the expression levels of osteopontin and osteocalcin in mice. Importantly, pretreatment for 7 days and subsequent treatment for 3 days with rhBMP-7 (10 microg/kg/day) abolished the vitamin D-mediated increases in the above parameters. In addition, human aortic smooth muscle cells (HASMCs) were cultured with high beta-glycerophosphate, a phosphate donor, for 2 weeks in the presence or absence of rhBMP-7. High beta-glycerophosphate increased expression levels of osteopontin and osteocalcin as well as calcium staining in HASMCs, but these changes were attenuated by treatment with BMP-7. Thus, BMP-7 inhibits vascular calcification associated with high levels of vitamin D or phosphate. We propose that BMP-7 treatment may be helpful in reducing the risks of cardiovascular disease related to vascular calcification.

Taurine inhibits osteoblastic differentiation of vascular smooth muscle cells via the ERK pathway.

Vascular calcification develops within atherosclerotic lesions and results from a process similar to osteogenesis. Taurine is a free beta-amino acid and plays an important physiological role in mammals. We have recently demonstrated that vascular smooth muscle cells (VSMCs) express a functional taurine transporter. To evaluate the possible role of taurine in vascular calcification, we assessed its effects on osteoblastic differentiation of VSMCs in vitro. The results showed that taurine inhibited the beta-glycerophosphate-induced osteoblastic differentiation of VSMCs as evidenced by both the decreasing alkaline phosphate (ALP) activity and expression of the core binding factor alpha1 (Cbfalpha1). Taurine also activated the extracellular signal-regulated protein kinase (ERK) pathway. Inhibition of ERK pathway reversed the effect of taurine on ALP activity and Cbfalpha1 expression. These results suggested that taurine inhibited osteoblastic differentiation of vascular cells via the ERK pathway.

The effect of phospholipase A2 on mitochondrial glycerol-3-phosphate oxidation.

Glycerol-3-phosphate oxidation in brown adipose tissue mitochondria of cold-adapted hamster is strongly inhibited by phospholipase A2 (PLA2). Our data show that the glycerol-3-phosphate branch of the respiratory chain is sensitive to PLA2 action more than the succinate branch and that the transfer of reducing equivalents from the glycerol-3-phosphate dehydrogenase to artificial electron acceptor is especially sensitive to the PLA2 action.

Levamisole and inorganic pyrophosphate inhibit beta-glycerophosphate induced mineralization of bone formed in vitro.

The addition of the organic phosphate, beta-glycerophosphate, to the culture media of osteoid forming cells or tissues will induce formation of mineralized bone. The mechanisms behind this phenomenon are not clearly understood. In order to gain new understanding of organic phosphate induced mineralization of bone it was decided to attempt to inhibit this process in two fundamentally different ways. Firstly, the reversible inhibitor of alkaline phosphatase, Levamisole, was used to help define the role of alkaline phosphatase in mineralization in vitro. Secondly, inorganic pyrophosphate, a known inhibitor of hydroxyapatite (HA) formation was also used. It was hypothesized that inorganic pyrophosphate, in addition to its ability to block HA formation, might also interfere with organic phosphate access to alkaline phosphatase and thereby prevent mineralization. The data show that mineralization is blocked when alkaline phosphatase activity is inhibited by Levamisole prior to but not after osteoid maturation. Inorganic pyrophosphate blocks organic phosphate induced mineralization whether added before or after osteoid formation. Organic phosphate effects on alkaline phosphatase activity are reversed by the addition of inorganic pyrophosphate either before or after osteoid formation. These findings suggest a role for alkaline phosphatase in organic phosphate induced mineralization. The data show further that inorganic pyrophosphate may effect mineralization of bone not only by blocking apatite formation but possibly by modulating organic phosphate metabolism.

Effects of phosphonic acid analogues of glycerol-3-phosphate on the growth of Escherichia coli.

The four-carbon phosphonate, 3,4-dihydroxybutyl-1-phosphonate, is similar to glycerol-3-phosphate in its ability to inhibit cell growth of Escherichia coli strain 8 cultured in low-phosphate synthetic medium supplemented with either succinate or casein hydrolysate as the sole carbon source. The three-carbon phosphonate, 2,3-dihydroxypropyl-1-phosphonate, does not appear to exhibit a similar effect. The inhibition caused by the four-carbon phosphonate differs from that caused by glycerol-3-phosphate in at least three ways. (i) Its inhibitory effect is not offset by the presence of glucose in the culture medium. (ii) It is capable of exerting its inhibitory effect on cells containing an active aerobic glycerol-3-phosphate dehydrogenase. (iii) Its inhibitory effect is maintained in synthetic medium containing high concentrations of inorganic phosphate. The four-carbon phosphonate appears to be bacteriostatic and inhibits the uptake of labeled glycerol-3-phosphate by E. coli strain 8.