Crystal growth and molecular modeling studies of inhibition of struvite by phosphocitrate.

The inhibition by phosphocitrate of struvite crystal formation and growth has been examined in the present study. Crystal growth in a gel matrix was controlled by phosphocitrate in a dose-dependent manner. The effects of inhibition were followed using scanning electron microscopy, optical microscopy, and single crystal X-ray analysis. The presence of phosphocitrate induced very strong, crystal face specific inhibition of struvite, leading to total cessation of crystal growth when sufficient concentration of the inhibitor was made available. Crystal growth studies and results from molecular modeling indicated strong affinity of phosphocitrate to (101) faces of struvite. This in turn led to an alteration in the expression of these faces and the development of a characteristic arrowhead struvite morphology. Similar changes were not observed in the presence of identical concentrations of citrate, acetohydroxamic acid, and N-sulfo-2 amino tricarballylate (an analog of phosphocitrate), emphasizing the unique interaction of phosphocitrate with the struvite crystal lattice.

Phosphocitrate inhibits a basic calcium phosphate and calcium pyrophosphate dihydrate crystal-induced mitogen-activated protein kinase cascade signal transduction pathway.

Calcium deposition diseases caused by calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate (BCP) crystals are a significant source of morbidity in the elderly. We have shown previously that both types of crystals can induce mitogenesis, as well as metalloproteinase synthesis and secretion by fibroblasts and chondrocytes. These responses may promote degradation of articular tissues. We have also shown previously that both CPPD and BCP crystals activate expression of the c-fos and c-jun proto-oncogenes. Phosphocitrate (PC) can specifically block mitogenesis and proto-oncogene expression induced by either BCP or CPPD crystals in 3T3 cells and human fibroblasts, suggesting that PC may be an effective therapy for calcium deposition diseases. To understand how PC inhibits BCP and CPPD-mediated cellular effects, we have investigated the mechanism by which BCP and CPPD transduce signals to the nucleus. Here we demonstrate that BCP and CPPD crystals activate a protein kinase signal transduction pathway involving p42 and p44 mitogen-activated protein (MAP) kinases (ERK 2 and ERK 1). BCP and CPPD also cause phosphorylation of a nuclear transcription factor, cyclic AMP response element-binding protein (CREB), on serine 133, a residue essential for CREB's ability to transactivate. Treatment of cells with PC at concentrations of 10(-3) to 10(-5) M blocked both the activation of p42/p44 MAP kinases, and CREB serine 133 phosphorylation, in a dose-dependent fashion. At 10(-3) M, a PC analogue, n-sulfo-2-aminotricarballylate and citrate also modulate this signal transduction pathway. Inhibition by PC is specific for BCP- and CPPD-mediated signaling, since all three compounds had no effect on serum-induced p42/P44 or interleukin-1beta induced p38 MAP kinase activities. Treatment of cells with an inhibitor of MEK1, an upstream activator of MAPKs, significantly inhibited crystal-induced cell proliferation, suggesting that the MAPK pathway is a significant mediator of crystal-induced signals.

Inhibition of calcium pyrophosphate dihydrate crystal formation in articular cartilage vesicles and cartilage by phosphocitrate.

Articular cartilage vesicles (ACV), isolated by differential centrifugation of adult hyaline articular cartilage collagenase digests, mineralized in the presence of calcium and ATP. Mineral analysis by microscopy, chemical analysis, energy-dispersive analysis, and infrared spectroscopy revealed crystals resembling calcium pyrophosphate dihydrate (CPPD). Adult articular cartilage also underwent ATP-dependent mineralization, supporting the contention that vesicles in situ fostered adult articular cartilage mineralization. Phosphocitrate (PC) is a recognized in vitro inhibitor of hydroxyapatite and calcium oxalate monohydrate crystal formation, but it is not known whether PC can similarly restrict CPPD crystal development. In the present study we examine the effect of PC, citrate, and n-sulfo-2-amino-tricarballylate (SAT, a PC analogue) on the ATP-induced CPPD crystal formation in both ACV and articular cartilage models. Only PC (10-1000 microM) blocked both the ATP-dependent and -independent mineralization in ACV in a dose-dependent fashion. At 1 mM, SAT and citrate blocked the ATP-independent mineralization. Similarly, only PC blocked both the ATP- and non-ATP-dependent mineralization in native articular cartilage slices. PC, SAT, and citrate had no effect on ACV nucleoside triphosphate pyrophosphohydrolase activity, suggesting that none of these agents blocked mineralization through the inhibition of nucleoside triphosphate pyrophosphohydrolase activity, which generates inorganic pyrophosphate from ATP.

Specific inhibition of basic calcium phosphate and calcium pyrophosphate crystal-induction of metalloproteinase synthesis by phosphocitrate.

Calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate (BCP) crystal deposition diseases are a group of heterogeneous arthritides which are a significant source of morbidity in the elderly. Both crystals induced mitogenesis and metalloproteinase (MP) synthesis and secretion by fibroblasts and chondrocytes which may promote degradation of intra-articular tissue. We have previously shown that phosphocitrate (PC), an inhibitor of hydroxyapatite crystallization, specifically blocks BCP crystal-induced mitogenesis in 3T3 cells. This led us to examine the effect of PC on BCP and CPPD crystal induction of MP synthesis in human fibroblasts. PC (10(-3) to 10(-4) M) specifically inhibited the crystal-induced collagenase and stromelysin mRNA accumulation while having no effect on epidermal growth factor-induced or basal levels of mRNA for both enzymes. Western blots (collagenase) of conditioned media confirmed that PC blocked crystal-induced proteinase secretion as well. Moreover, PC (10(-3) M) also blocked the crystal induction of c-fos and c-jun. Since FOS and JUN proteins form a transacting activator (AP-1) for expression of collagenase and stromelysin genes, PC may block the synthesis of both enzymes by inhibiting the transcription of c-fos and c-jun.

Liposome and multiple emulsion formulations augment the anticalcifying efficacy of phosphocitrate in a cutaneous calcergy model.

The anticalcifying agent phosphocitrate was incorporated into phosphatidylcholine/cholesterol liposomes by reverse-phase evaporation. The compound was entrapped to the extent of 11.6% (mol mol-1 of lipid) and the liposomes exhibited prolonged retention of the compound when incubated with rat plasma. Phosphocitrate's ionic contribution in solution adversely influenced the encapsulation efficiency but improvements were made through ion-pairing with the quaternary ammonium detergent centrimide, or with the inclusion of stearylamine in the lipid phase. The liposomal dose that could be practically administered in-vivo was restricted to 2.5 mg phosphocitrate kg-1 day-1. The formulation of a multiple emulsion preparation of phosphocitrate, however, offered an alternative delivery mode permitting infrequent dosing to be successfully investigated. In a rat calcergy model, both vehicles effectively reduced the formation of induced subcutaneous calcified plaques at doses for which the phosphocitrate salt alone was inactive. The current formulations demonstrate that the therapeutic efficacy of phosphocitrate can be markedly improved through an appropriately designed drug delivery system, signalling a new approach for the future therapeutic application of this compound.

Scanning electron microscopy and molecular modeling of inhibition of calcium oxalate monohydrate crystal growth by citrate and phosphocitrate.

Binding of citrate and phosphocitrate to calcium oxalate monohydrate crystals has been studied using scanning electron microscopy (SEM) and molecular modeling. Phosphocitrate structure has been resolved using low temperature X-ray analysis and ab initio computational methods. The (-1 0 1) crystal surface of calcium oxalate monohydrate is involved in binding of citrate and phosphocitrate, as shown by SEM and molecular modeling. Citrate and phosphocitrate conformations and binding energies to (-1 0 1) faces have been obtained and compared to binding to another set of calcium-rich planes (0 1 0). Difference in inhibitory properties of these compounds has been attributed to better coordination of functional groups of phosphocitrate with calcium ions in (-1 0 1). Relevance of this study to design of new calcium oxalate monohydrate inhibitors is discussed.

Controlling influence of phosphocitrate in vitro and in vivo on calcium oxalate crystal formation and growth.

Calcium oxalate (CaOx) crystallization in the presence of phosphocitrate (PC) was studied by both in vitro and in vivo techniques. Crystals of the monohydrate (COM) and the dihydrate (COD) forms were generated under controlled conditions in a silica gel matrix. Our data indicated only COD crystals formed when PC was present, inferring that the COD to COM transformation was being impeded. COD crystals were smaller in size than controls and there was evidence of interpenetral twinning. An in vivo study using a rat bladder implant model noted similar findings. Scanning electron microscopy (SEM) revealed that implants recovered from PC treated rats had primarily COD crystals deposited, whereas both the surface and inner layers of encrusted implants from normal rats contained predominantly COM crystals. Infrared (IR) analysis confirmed the visual findings indicating quantitatively that there was a higher proportion of COD present on the implants recovered from the treated rats than in the controls. It is concluded that although total CaOx crystallization cannot be eliminated by PC, its action could assist in reducing the harmful nature of such crystallites in the urine.

Phosphocitrate prevents disease progression in murine progressive ankylosis.

OBJECTIVE: Mice with progressive ankylosis, a spontaneous arthropathy, were treated with phosphocitrate (PC) in vivo to determine the effect of PC on disease progression. METHODS: Two groups of mice with progressive ankylosis (matched for age, weight, and sex) were treated parenterally for 6 weeks with either PC or saline vehicle. RESULTS: Clinically, histologically, and microradiographically, there were significant differences in disease progression and severity in the PC-treated and the saline-treated mice. CONCLUSION: PC appears to inhibit disease progression in murine progressive ankylosis.

Adsorption and mineralization effects of citrate and phosphocitrate on hydroxyapatite.

The adsorption of citrate and phosphocitrate ions by hydroxyapatite (HAP) surfaces and their influence on the constant composition growth kinetics of HAP have been investigated. Phosphocitrate was strongly adsorbed to HAP and inhibited crystal growth. When HAP surfaces containing preadsorbed citrate were exposed to phosphocitrate, the uptake of the latter markedly increased. The two additives behaved synergistically in their HAP crystal growth inhibition.

Inhibition of basic calcium phosphate crystal-induced mitogenesis by phosphocitrate.

Basic calcium phosphate crystals control the traverse of cells from the Go/G1 to S-phase of the cell cycle and initiate proliferation by rendering fibroblasts competent to respond to insulin-like growth factors in plasma. Simultaneous addition of phosphocitrate [a powerful inhibitor of hydroxyapatite crystallization] to cells exposed to basic calcium phosphate crystals caused a dose-dependent inhibition of crystal-induced DNA synthesis and c-fos transcription. This inhibition was specific for crystal-induced mitogenesis, since similar concentrations of phosphocitrate had no effects on either PDGF or 10% calf serum-induced thymidine incorporation and c-fos transcription.

Evidence in vitro for an enzymatic synthesis of phosphocitrate.

A biological synthesis of phosphocitrate is described from precursors, citrate and adenosinetriphosphate reacting in the presence of rat liver homogenate. Identity of the newly formed product was examined by enzymatic digestion of reactions mixtures, HPLC chromatography and 1H-NMR spectra. Authenticity of product was established by comparison to chemically synthesized phosphocitrate. Recognition of the existence of a biologically synthetic pathway adds credence to the known presence of phosphocitrate in mitochondria and a postulated role to control calcium phosphate deposition in that organelle.

Influence of probucol on early experimental atherogenesis in hypercholesterolemic rats.

A possible cellular action for probucol in early atherogenesis was investigated. In diet-induced hypercholesterolemic rats, probucol reduced aortic accumulation of cholesterol without ameliorating monocyte attachment to the arterial endothelium. Under the imposed conditions, circulating cholesterol levels were not significantly altered by probucol. 125I-labeled acetyl LDL uptake and degradation studies with mouse peritoneal macrophages revealed that probucol had an inhibitory effect on the scavenger receptor pathway. The data suggested that the observed beneficial effects of probucol were not related to an early cholesterol-induced injury phase which might involve calcium. Instead, probucol probably through its free radical scavenging property, intervened at a subsequent cellular level to restrict lipid accumulation.

Protection of crystal-induced polymorphonuclear leukocyte membranolysis by phosphocitrate.

The protection afforded by phosphocitrate, a phosphorylated polycarboxylic acid, against crystal-induced membrane damage to polymorphonuclear leukocytes was studied in vitro. Membranolysis was assessed by nitro blue tetrazolium salt reduction, lactate dehydrogenase release, and scanning electron microscopy. Phosphocitrate protected strongly against hydroxyapatite crystal-induced damage, an action attributable to crystal surface binding of phosphocitrate rather than to the membrane. The ability of phosphocitrate to prevent hydroxyapatite crystallization, together with its membrane protective effect against preformed crystals, would suggest that the compound might have a useful future role against crystal-induced arthropathies.

Reduction of infection stones in rats by combined antibiotic and phosphocitrate therapy.

The potential of phosphocitrate to inhibit infection stones in rats when combined with an antibiotic was studied. A significant reduction occurred in both the number and weight of recovered stones from rats receiving combined treatment with amoxycillin (50 mg./kg. body wt./day and phosphocitrate (112 mumol./kg. body wt./day) for four weeks. The inhibitory responses were attributed to the intact phosphocitrate molecule as administration of citrate in equimolar concentrations did not mimic the observed effects of phosphocitrate. In comparison with non-infected controls, antibiotic treatment alone failed to eliminate total stone growth. However, composition of the stone reverted from predominantly struvite to a mixture of struvite and newberyite as urinary parameters normalized. The studies highlight the usefulness of phosphocitrate to restrict magnesium salt deposition in vivo.

Retardation of calcification of bovine pericardium used in bioprosthetic heart valves by phosphocitrate and a synthetic analogue.

The purpose of this study was to determine if phosphocitrate (PC), a naturally occurring inhibitor of calcification, and its synthetic analogue, N-sulpho-2-amino tricarballylate (SAT), administered either by daily injection or local delivery via Alzet osmotic minipump, could inhibit calcification of glutaraldehyde-preserved bovine pericardium used in bioprosthetic heart valves, subcutaneously implanted in rats. Local drug delivery, but not systemic administration, was effective. PC, administered by Alzet minipump (12 mg.kg-1.day-1), inhibited calcification significantly (tissue calcium = 5 +/- 2 micrograms/mg dry tissue, mean +/- SEM), compared with untreated or saline-treated controls (89 +/- 9 and 49 +/- 9 micrograms/mg, respectively). SAT, administered by the same route at both the same and a higher molar dosage, was less potent (tissue calcium = 26 +/- 9 micrograms/mg and 17 +/- 5 micrograms/mg, respectively). PC and SAT therapy were not associated with adverse effects. We conclude that locally administered PC and SAT can inhibit intrinsic calcification of bovine pericardium, with PC being more potent.

A possible antiatherogenic role for phosphocitrate through modulation of low density lipoprotein uptake and degradation in aortic smooth muscle cells.

The present study reports the influence of a phosphorylated polycarboxylic acid, phosphocitrate, on low density lipoprotein (LDL) metabolism in cultured rabbit aortic smooth muscle cells. Phosphocitrate profoundly influenced both LDL binding and degradation. At the maximal effective concentration (2 mM), phosphocitrate released approximately 90% of the receptor-bound [125I]LDL whilst the total amount of [125I]LDL degraded was reduced by 60%. Measurement of total cholesterol accumulation revealed that even in the presence of high concentrations of added LDL, phosphocitrate (2 mM) diminished cholesterol levels close to the basal levels seen in incubations in lipoprotein-deficient serum. Further, this inhibitory effect of phosphocitrate was demonstrable after 24 h at 37 degrees C. Phosphocitrate, a recognized anticalcifying agent, possesses a strong negative charge to size ratio at physiological pH. It is postulated that the observed effects probably arise from charge interference and/or its ability to modulate cellular calcium concentration.

Glycosaminoglycans as inhibitors of stone formation.

The presence of glycosaminoglycans in the kidney, bladder and urinary tract exerts influence on stone formation through prevention of growth and aggregation. The source of the urinary glycosaminoglycans is contentious with respect to the relative contribution of the kidney and bladder secretions to the overall inhibitory power. Mechanisms have been advanced for the action of glycosaminoglycans, and the active species involved appears dependent upon the degree of sulfation of the molecule. Thus, the ultimate role of glycosaminoglycans as urinary inhibitors of stone formation may well be dictated by the specific nature of the glycosaminoglycan present and its inherent acidic properties. The overall contribution of urinary glycosaminoglycans to protect against urolithiasis however still needs to be evaluated.

Atherogenesis. Mitigation of monocyte adhesion to arterial endothelium in hyperlipidemic rats by phosphocitrate, a phosphorylated polycarboxylic acid.

Phosphocitrate, a phosphorylated polycarboxylic acid ameliorates two early events in atherogenesis. When administered to rats on an atherosclerotic diet (112 mumol/kg body wt./day), it reduced monocyte adhesion to aortic endothelium from 34 +/- 7 cells/HPF for untreated rats to 1 +/- 1 cell/HPF, a value seen in normal, non-atherosclerotic rats. Transmission electron microscopy of aortic sections showed no evidence of subendothelial lipid accumulation in phosphocitrate-treated rats despite the high circulating plasma lipid levels. The mechanisms of action of phosphocitrate are unknown but the indications are that its influence may be mediated through its polyanionic chemical nature and/or its ability to modulate cellular calcium accumulation. In addition to its possible therapeutic value as an anti-calcifying and anti-atherogenic compound, phosphocitrate may prove useful as an experimental probe for studying the cellular basis of atherogenesis.

Phosphocitrate inhibition of 45Ca2+ uptake in rat aortic smooth muscle cells in primary culture.

Phosphocitrate inhibits 45Ca2+ uptake by rat aortic smooth muscle cells in primary culture. Unlike the Ca2+ channel blocking effect of diltiazem, the phosphocitrate effect is neither time dependent nor reliant on a depolarized membrane. Phosphocitrate and diltiazem together in maximum inhibitory concentration show an additive effect. The data suggest that different Ca2+ uptake mechanisms are involved. The potential exists for phosphocitrate to have a therapeutic role in atherogenesis through modulating Ca2+ movements in arterial smooth muscle.