Medial vascular calcification (VC) is a degenerative

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1 Phosphonoformic Acid Prevents Vascular Smooth Muscle Cell Calcification by Inhibiting Calcium-Phosphate Deposition Ricardo Villa-Bellosta, Víctor Sorribas Objective The role of inorganic phosphate in the pathogenesis of vascular calcification (VC) has been studied extensively in recent years. Phosphonoformic acid (PFA), an inhibitor of type II Pi transporters, has been traditionally used to study the involvement of Pi transport in VC, because PFA also prevents calcium deposition in vitro. However, aortic vascular smooth muscle cells (VSMCs) only express PFA-resistant, type III transporters (Pit-1 and Pit-2). Therefore, in this article we have studied the mechanism of VC prevention by PFA. Methods and Results Radiotracer Pi uptake in rat VSMCs was not inhibited at the concentrations at which PFA prevents calcification. Alternative mechanisms whereby PFA could prevent calcification, such as cytotoxicity or phosphodiesterase inhibition, have also been excluded. The progression of calcification also took place in fixed cells. The kinetics of VC prevention by PFA, pyrophosphate, phosphonoacetate, and bisphosphonates was similar in live and fixed cells, showing mean effective concentrations in the micromolar range. Conclusions PFA mainly prevents VC through a physicochemical mechanism that is independent of any cellular metabolic activity, including Pi transport. Conversely, PFA seems to act similarly to its chemical analogues, inorganic pyrophosphate, and bisphosphonates, as suggested decades ago. (Arterioscler Thromb Vasc Biol. 2009;29: ) Key Words: phosphonoformic acid bisphosphonate vascular calcification phosphate transport Pit-1 Medial vascular calcification (VC) is a degenerative process of the large arteries that increases mortality risk in patients with chronic kidney disease. 1 This ectopic calcification takes place in the medial layer of the arterial wall, mainly as a response to renal-derived insults such as uremic toxins, hyperphosphatemia, type 2 diabetes, and ageing. 2,3 Primary cultures of vascular smooth muscle cells (VSMCs) are a common in vitro model that, despite several limitations, has led to the understanding that VC is a regulated process of both phenotype trans-differentiation and the expression of osteogenes, which ends in the production of a calcifying extracellular matrix. 4 6 More specifically, VSMCs have been used to study the calcification induced by a high concentration of inorganic phosphate (Pi), 4 which has led to the proposal that an increase in Pi influx through the plasma membrane could be responsible for the changes that end in the calcification process. 7 This argument was initially based on the fact that phosphonoformic acid (PFA), an inhibitor of renal Pi transport, also prevents VC in cultured VSMCs 4,8 12 and in osteoblast-like cells. 13 More recently, the involvement of the Pi transporter Pit-1 in the calcification of VSMCs was shown by interfering the expression of Pit-1, which consequently prevented Pi-induced calcification. 7 However, although the participation of Pit-1 in VC has been irrefutably shown, the specific role played by Pit-1 in the pathogenesis of calcification still needs to be determined. Several findings also suggest that this role is more complicated than initially expected. For example, we recently published a kinetic analysis of Pi transport in VSMCs, showing that Pi transport is saturated under physiological Pi concentration ( 1.1 mmol/l). 14 This transport system exhibits an Na-dependent component, which is mediated by Pit-1, Pit-2, and a saturable Na-independent uptake. Saturation implies that an increase in Pi influx can only be mediated through an enhanced expression of functional Pi transporters (ie, the capacity) in the plasma membrane. 14 However, such an increase has not been definitely observed in VSMCs, and we even found an inverse correlation: insulin prevents the calcification of VSMCs induced with 2 mmol/l Pi, but it increases Pi transport. 15 Finally, we have found that the expression of Pit-1 in the plasma membrane of VSMCs is very weak, but it is very strong in the endoplasmic reticulum (manuscript in revision). Therefore, the role of Pit-1 in VC pathogenesis will be scarcely understood until we have deciphered the cell physiology of this transporter in detail. PFA is a competitive inhibitor of renal Pi transport, 16,17 which is mainly carried out by type II Na/Pi cotransporters. 18 The inhibiting capacity of PFA in VSMC Pi transport is very weak, 14 Received October 1, 2008; revision accepted February 4, From the Laboratory of Molecular Toxicology, University of Zaragoza, Spain. Correspondence to Víctor Sorribas, Laboratory of Molecular Toxicology, University of Zaragoza, Veterinary Faculty, Calle Miguel Servet 177, Zaragoza, Spain. sorribas@unizar.es 2009 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at DOI: /ATVBAHA

2 762 Arterioscler Thromb Vasc Biol May 2009 showing a K i of 2.6 mmol/l. Similar K i values were obtained for the type III Pi transporters present in VSMCs: Pit-1 (2.7 mmol/l) and Pit-2 (4.6 mmol/l). The weak inhibition of Pit-1/2 has been corroborated using a 2-electrode voltage clamp in Xenopus oocytes. 18,19 These results indicate that when PFA has been used in other studies to prevent calcification of VSMCs at the usual concentration of 0.5 mmol/l, the inhibition of Pi transport is negligible in the presence of physiological or pathological (2 mmol/l) concentrations of Pi in the culture medium. Nevertheless, calcification is prevented. Therefore, in this study we decided to analyze the mechanism of VC inhibition by PFA, with the expectation that our findings would be able to clarify some of the question marks about this degenerative process. Methods Cell Culture and Transport Assays The isolation and culture conditions of rat thoracic aortae VSMCs and radionuclide transport assays have been described previously. 14 Cell culture reagents were from Gibco, 32 P-orthophosphoric acid was from PerkinElmer, and phosphonoformic acid was obtained from Sigma-Aldrich. Calcification Determination The calcification of VSMCs was identified by Alizarin red staining, as described. 15 For the quantification of calcium, VSMCs were treated with 0.6 N HCl overnight at 4 C. The content of calcium was then colorimetrically analyzed using a QuantiChrom Calcium Assay Kit (BioAssay Systems). Cytotoxicity Assays Total cell death was estimated using a Cytotoxicity Detection Kit (Roche Molecular Biochemicals) and corroborated by double staining of VSMCs with acridine orange/ethidium bromide, as described. 20 Cells grown on culture slides (BD Biosciences) were visualized using a Carl Zeiss Axiovert 200 mol/l fluorescent microscope. Apoptosis was studied by deoxynucleotidyl transferase (TdT)-mediated dutp nick-end-labeling (TUNEL) and using an ApoAlert DNA Fragmentation Assay kit (Clontech Laboratories Inc). Cells were fixed with 4% formaldehyde in PBS, permeabilized with 0.2% Triton X-100 in PBS, and counterstained with 10 g/ml propidium iodide in PBS. As a positive control, cells were treated with DNAse I, as suggested by the manufacturer. For counting the cells, VSMCs were trypsinized and counted in a Neubauer chamber. Studies on Cyclases VSMCs were grown in 24-well multidishes (Nunc) and incubated with the following drugs in a culture medium containing either 1 or 2 mmol/l Pi and 1 mmol/l PFA: 10 mol/l MDL-12,330A (cis-n-[2- Phenylcyclopentyl]-azacyclotridec-1-en-2-amine hydrochloride or cis- N-[2-phenylcyclopentyl]azacyclotridec-1-en-2-amine) in DMSO, an inhibitor of adenylyl cyclase; 1 mol/l ODQ (1H- 1,2,4 oxadiazolo[4,3- ]quinoxalin-1-one) in DMSO, an inhibitor of gualylyl cyclase; 1 mmol/l 8-CPT-cAMP (8-[4-chlorophenylthio]-adenosine 3,5 -cyclic monophosphate sodium); and 1 mmol/l 8-CPT-cGMP (8-[4- chlorophenylthio]-guanosine 3,5 -cyclic monophosphate sodium). All products were from Sigma. The medium containing the drugs was replaced every day with new medium until the day of the experiment. Progression of Calcification in Fixed Cells After 24 hours of incubation with 2 mmol/l Pi-containing medium, the cells were fixed by several methods: 100% methanol at 20 C for 5 minutes; 3% paraformaldehyde at room temperature for 10 minutes; 1% formaldehyde at room temperature for 10 minutes; 100% acetone for 3 minutes at 20 C; or the medium was simply aspirated, washed in PBS, and left to dry overnight. Cell death was checked by trypan blue exclusion and acridine orange/ethidium bromide staining. Calcification was then performed as it was for live cells. Figure 1. Inhibition of Pi transport and calcification in VSMCs. Cells were analyzed for calcification (squares) and Pi uptake (triangles). For the calcification effect, cells were incubated in a medium containing 2 mmol/l Pi, and for the transport effect, cells were incubated with 0.1 mmol/l 32 P-H 3 PO 4 for 12 minutes. Dose-Response Assays VSMCs were induced to calcify using 2 mmol/l Pi for 24 hours and then incubated for 6 additional days in the presence of increasing concentrations of PFA, phosphonoacetic acid (PAA), pyrophosphate, dichloromethylenediphosphonic acid (DMDP), or sodium ibandronate (all from Sigma). In some experiments, VSMCs were fixed with 1% formaldehyde after the 24-hour induction of calcification. In all cases, new medium containing 2 mmol/l Pi and the corresponding drug was added every day. The mean effective constants on the prevention of calcification were calculated by fitting the data to a sigmoidal dose-response equation using nonlinear regression, as explained. 17 Data Analysis Every experiment was repeated at least 3 times with similar results, and when quantifiable, the data are shown as the mean SE. The significances of differences were evaluated by a 1-way analysis of variance and a Tukey multicomparison post test. Results PFA Prevention of Calcification Is Independent of Pi Transport In a previous study we showed that PFA was a very poor inhibitor of Pi transport in aortic VSMCs (K i of 2.6 mmol/ L). 14 In this study, we proposed to verify whether or not the inhibition of Pi-induced VC by PFA matched the inhibition of Pi transport in VSMCs. Figure 1 shows that although 0.16 mmol/l PFA prevents VC by 90%, total uptake of Pi is not affected up to 1 mmol/l PFA. Calcification was always preceded by the induction of Msx2 and Cbfa1 after 24 hours of incubation with 2 mmol/l Pi, and Bmp2 was not significantly increased (unpublished data, 2008). Our kinetic results unequivocally show that PFA is preventing VC through a mechanism other than the inhibition of Pi transport. PFA Prevention of Calcification Is Independent of Cytotoxicity We subsequently checked to see whether PFA was cytotoxic to VSMCs at the concentrations at which it prevented VC, given that chemical damage could prevent or alter the gene expression and phenotypic changes that seem to underlie VC pathogenesis. Total cell death was determined by the activity

3 Villa-Bellosta and Sorribas Phosphonoformic Acid and Calcification 763 Figure 2. Cytotoxicity of PFA. A, LDH activity in VSMCs incubated with PFA. B, Staining of VSMCs with acridine orange/ ethidium bromide treated as in A. C, The effect of PFA on cell density. D, Apoptosis analysis using a TUNEL assay. Positive control, cells treated with DNAse I. of cytosolic lactate dehydrogenase (LDH) in culture medium. Figure 2A shows that cell death was not observed even after 9 days of incubation with 10 mmol/l PFA. Double staining of the cells with acridine orange/ethidium bromide (AO/EB) and phase contrast illumination showed that the cell density was lower at 10 mmol/l PFA (Figure 2B) as a consequence of cell detachment. Attached cells were not dead, as shown by the failure of EB to enter the cells (the nuclei do not fluoresce red), and only green fluorescence is evidenced from the DNA-intercalated acridine orange, which is taken up by live cells. 20 The decrease in cell number was corroborated by direct cell counting of trypsinized cells, thereby showing that the number was significantly lower with 2.5 mmol/l PFA after 8 days of treatment (Figure 2C). Although the morphology of the cells in Figure 2B did not evidence any sign of apoptosis, we also determined the presence of this phenomenon in PFA-treated cells by a TUNEL assay. As shown in Figure 2D, no sign of apoptosis-induced DNA fragmentation was observed in PFA-treated cells at any assayed concentration. In any event, no sign of toxicity was observed at the PFA concentrations usually used to prevent VC, wherefore the possibility of toxicity was also discarded. PFA Prevention of Calcification Is Independent of Nucleotide Cyclase Activity PFA is used as an antiviral drug (Foscarnet) because of its ability to inhibit viral polymerases. Similarly, it has also been reported that PFA (IC 50 in the micromolar range) strongly inhibits the activity of adenylyl and guanylyl cyclases and the accumulation of camp and cgmp in some cells through interaction with the pyrophosphate (PPi)-binding site. 21 To check whether this inhibiting activity was involved in the prevention of VC by PFA, VSMCs were induced to calcify using 2 mmol/l Pi for 4 days, in the presence or absence of 1 mmol/l PFA and the indicated drugs (supplemental Figure I, available online at The effect of PFA on Pi-induced calcification was not mimicked by the specific inhibitor of adenylyl cyclase, MDL-12330A (10 mol/l daily). Similarly, the effect of PFA on Pi-induced calcification was not blunted by the concomitant addition of 8-CPT-cAMP (1 mmol/l daily), a potent and membranepermeable analogue of camp. VC also was not induced by 8-CPT-cAMP (1 mmol/l daily) in a 1 mmol/l Pi-containing medium. Identical results were obtained when studying the role of guanylyl cyclase using the specific inhibitor ODQ (1 mol/l daily) or 8-CPT-cGMP (1 mmol/l daily). Therefore, PFA prevents calcification through a mechanism that is also different from the inhibition of adenylyl or guanylyl cyclases. VSMC Calcification Progresses in the Absence of Cellular Activity In addition to the action of cyclases and polymerases, PFA can alter many other metabolic processes by acting as a substitute for PPi. However, we directly tested the possibility that PFA could simply inhibit the formation or deposition of calcium phosphate products in a newly synthesized calcifying matrix (ie, the progression of calcification). We first stimulated calcification by incubating VSMCs with 2 mmol/l Pi for 24 hours to induce the expression of procalcifying genes (Figure 3A and unpublished data, 2008). 4,9,22 Every day, some of the cells (a vertical row of wells, labeled with the respective day number) were subsequently lysed using different methods (100% methanol, 3% paraformaldehyde, or 1% formaldehyde), and all the wells were further incubated

4 764 Arterioscler Thromb Vasc Biol May 2009 Figure 3. VC progression in dead cells. A, Effect of time of fixation on calcification progression. B, Calcium content of fixed VSMCs incubated with 2 mmol/l. C, The effect of the lysis method on the extent of VSMC calcification with control (white bars) or 2 mmol/l Pi (black bars) medium. with a new medium containing 2 mmol/l Pi for 5 additional days. This procedure was repeated every day, meaning that new cells from a new row of wells were lysed according to the respective method and that all the wells were kept incubated with additional Pi-calcifying medium. After 5 days of incubation with 2 mmol/l Pi, all cells were stained with alizarin red. As shown in Figure 3A, cells calcified at the same intensity whether they were alive or dead when incubated with 2 mmol/l Pi for a total of 5 days, after the initial 24 hours of induction with the same medium. Similarly, when cells were induced to calcify using 2 mmol/l Pi for 24 hours and then lysed, further incubation using a 2 mmol/l Pi medium showed progressive calcification over time, as shown in Figure 3B. Calcification showed the same intensity and progression rate, regardless of the method used to lyse the cells, including paraformaldehyde, formaldehyde, methanol, acetone, or even overnight drying (Figure 3C). PFA Prevents the Progression of Calcification in Fixed Cells The previous method of calcification using lysed cells was then used to study the effect of PFA on VC in the absence of cellular activity or metabolism. In a first experiment (Figure 4A), VSMCs grown in a 24-well plate were induced to calcify using 2 mmol/l Pi for 24 hours. Then the cells were lysed with 1% formaldehyde and kept in 2 mmol/l Pi for 6 days in the presence of increasing concentrations of PFA. The quantification of calcium deposition revealed that PFA prevented calcification in a dose-dependent way. The inset of Figure 4A shows Alizarin red staining of cells incubated for 24 hours with 2 mmol/l Pi, which were then fixed and incubated for 5 additional days with 2 mmol/l Pi in the absence or presence of 1 mmol/l PFA. In a second experiment (Figure 4B), VSMCs were incubated with 2 mmol/l Pi from 1 to 5 days and were respectively divided into 5 groups. Each group of cells was lysed at the end of the incubation day. Then the cells were treated as follows: (1) no additional medium, to determine the level of calcification obtained every day (first horizontal row of wells, top); (2) a medium containing 2 mmol/l Pi, to maintain the calcification of fixed cells (second horizontal row of wells); (3) a medium with 2 mmol/l Pi plus 1 mmol/l PFA, to prevent further calcification of fixed cells (third horizontal row of wells); and (4) control with 1 mmol/l Pi, to establish the PFA prevention level (fourth horizontal row of wells, bottom). Once again, PFA completely prevented the calcification of cells in proportion to the calcification time before lysis, as indicated in the bottom row of wells. Kinetics of Calcification Prevention The fact that PFA prevents calcification in dead cells suggests that it directly prevents the deposition of calcium phosphate, similarly to PPi or bisphosphonates. To support this possibility, we compared the kinetics of calcification prevention by different hydrolysable (PPi) and nonhydrolysable agents, whose molecular structures are shown in Figure 5A. Confluent VSMCs were made quiescent overnight and incubated 6 days with 2 mmol/l Pi, in the presence of different drugs (Figure 5B). The PFA effect on calcium deposition resulted in a mean Figure 4. PFA prevents calcification in fixed cells. A, Dose-response effect of PFA on calcification of fixed VSMCs. Inset, Effect of PFA on calcification of fixed cells for 5 days. B, Time-course of PFA effect on calcification of VSMCs fixed at different days.

5 Villa-Bellosta and Sorribas Phosphonoformic Acid and Calcification 765 Figure 5. Kinetics of calcification inhibition. A, Chemical structure of phosphonoformate-related compounds. B, Prevention of calcification in live cells incubated for 6 days with increasing concentrations of the indicated drugs. C, Prevention of calcification as in B using fixed cells. Data refer to EC 50 values. effective concentration (EC 50 ) of 61 mol/l, whereas the chemically analogous phosphonoacetic acid (PAA) was the weakest inhibitor of calcification. The geminal bisphosphonate ibandronate was the strongest inhibitor (EC mol/l), followed by PPi and by dichloromethylenediphosphonic acid. An identical experiment was performed on cells that had been induced to calcify with 2 mmol/l Pi, and they were then fixed with 1% formaldehyde and incubated 6 more days with 2 mmol/l Pi (Figure 5C). In all cases, the mean effective concentrations were very similar to the values in live cells (see values in fit curves), but they were slightly higher for ibandronate and PPi, which suggests that all compounds prevent calcification mainly by direct inhibition of mineralization, although minor effects on cellular activity cannot be discarded. Discussion Phosphonoformic acid is a phosphonocarboxylic acid analogous to inorganic pyrophosphate (PPi; Figure 5A), and it has been used as an antiviral agent 23 and as an experimental inhibitor of renal Pi transport. 16 The antiviral activity of PFA (Foscarnet) is based on inhibiting the formation of phosphodiester bonds generated by viral polymerases, but PFA also inhibits the biochemical reactions that are similarly catalyzed by adenylyl and guanylyl cyclases. 21 In both cases (polymerases and cyclases), the effects by PFA are related to the structural similarity with PPi and to competition for the same binding site. Therefore, it is most likely that PFA alters many other biochemical reactions in the cell related to PPi. The inhibition of renal Pi reabsorption is an exception, because PFA competitively inhibits type II mediated Pi transport via direct interaction with the renal transporters. 16,18 PFA has been successfully used to prevent calcification of VSMCs in vitro, but this effect has not been related to PPi. Instead, the PFA effect has been empirically interpreted as an inhibition of the type III, SLC20 Pi transporters (Pit-1 and Pit-2), 4,8 12 which are expressed in VSMCs (type I and II are not), 14 and as an extrapolation of the effect of PFA on type II Pi transporters. 7 This interpretation has been maintained despite reports indicating that PFA is not an inhibitor of Pit-1 and Pit-2. 14,18,19 The PFA analogue, PPi, is also a strong inhibitor of VC and a substrate for alkaline phosphatase. PPi prevents VC by a physicochemical reaction that inhibits the formation and aggregation of calcium phosphate crystals, prevents the transition of amorphous calcium phosphate into hydroxyapatite, and delays the aggregation of apatite crystals The same mechanism of calcification inhibition has been attributed to PPianalogous drugs, the (geminal) bisphosphonates (Figure 5A), 27,28 and even PFA. 29 This seminal work on PFA 29 shows that the chemical structure for preventing hydroxyapatite formation requires the presence of at least one phosphate group and an acidic group in another position, which can be either a phosphate or a carboxylic acid group. Bisphosphonates bind to the calcium of hydroxyapatite by chemisorption with oxygen from each phosphonate group. 27 The more potent tridentate bisphosphonates bind at a third location, such as the oxygen of a hydroxyl group of the central carbon. Therefore, it is very likely that PFA acts similarly with the corresponding carboxylic group. Our results are compatible with the possibility that PFA acts mainly like PPi or a bisphosphonate when inhibiting VC. First, we show that PFA is a very poor inhibitor of Pi transport in VSMCs (Figure 1), yet conversely it is a strong inhibitor of VC and provides an EC 50 of 61 mol/l (Figure 5B). If Pi transport inhibition were the real mechanism of VC prevention by PFA, then according to the K i for PFA (2.6 mmol/l), 49 mmol/l PFA should be necessary to reduce Pi transport in VSMCs to half the rate in the presence of 2 mmol/l Pi to prevent calcification, such as in hyperphosphatemic patients. 14 This concentration of PFA is 803 times higher than the experimental value that we obtained

6 766 Arterioscler Thromb Vasc Biol May 2009 for calcification prevention (61 mol/l, Figure 5B); moreover, Pi transport in VSMCs is saturated at the physiological Pi concentration (1.1 mmol/l), and it is not significantly increased at 2 mmol/l Pi. 14 Second, PFA is not cytotoxic at the low concentrations that are necessary to inhibit calcification (Figure 2). Third, the effect of PFA is not dependent on the inhibition of adenylyl or guanylyl cyclases or the level of camp or cgmp in the cell (supplemental Figure I). And fourth, and more importantly, PFA prevents the progression of calcification in the VSMCs that have been lysed using any method. In other words, after the induction of calcification, both the calcification progression (Figure 3) and the prevention mechanism by PFA (Figure 4) are processes that seem to be essentially independent of any biochemical/metabolic activity, Pi transport included. The conclusion that PFA mainly prevents vascular calcification by direct physicochemical inhibition of mineralization, as proposed decades ago for PPi, bisphosphonates, 27,28 or PFA, 29 is clearly observed in Figure 5. A comparison of the kinetics of calcification inhibition by PFA, PAA, bisphosphonates, or PPi shows that all of them exhibit EC 50 in the micromolar range, which is in the range of the affinities of bisphosphonates for hydroxyapatite chemisorption. 30 Most importantly, inhibition is almost identical in cells that were either alive or fixed after induction of calcification with 2 mmol/l Pi, especially for the case of PAA, PFA, and DMDP. PPi and ibandronate show slightly higher EC 50 values in fixed cells, which suggests the existence of additional mechanisms in vivo in the vascular cell, similar to the direct actions of bisphosphonates on osteoblasts and osteoclasts. 31 In conclusion, PFA prevents vascular calcification by a mechanism that is independent of Pi transport inhibition. Our findings suggest that PFA mainly and directly inhibits mineralization and hydroxyapatite formation, acting similarly to PPi and to bisphosphonate drugs. This study also supports the leading role of pyrophosphate and other calcifying inhibitors in the balance of ectopic calcification. Sources of Funding This work was supported by a grant from the Spanish Ministry of Education and Science (BFU /BFI to V.S.) and by a predoctoral fellowship from the Government of Aragón, Spain (B086/2007 to R.V.B.). None. Disclosures References 1. Qunibi WY. Cardiovascular calcification in nondialyzed patients with chronic kidney disease. 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