Phosphocitrate blocks calcification-induced articular joint degeneration in a guinea pig model.

OBJECTIVE: Calcium deposition occurs frequently in osteoarthritic (OA) joints. However, evidence for a causal role of calcification in cartilage degeneration is inferential. The present study was undertaken to examine the role of calcification in OA disease progression and to evaluate a formulation of phosphocitrate (PC) as a potential therapeutic agent. METHODS: We have identified a guinea pig OA model in which meniscal calcification appears to correlate with aging and disease progression. We synthesized a new formulation of PC, [CaNa(PC)2(H2O)](n) (CaNaPC), which is a potent antimineralization agent and a specific inhibitor of crystal-induced biologic effects. After weekly treatment of guinea pigs with experimental OA with CaNaPC for 3 months, we examined calcification in menisci and cartilage degeneration. As a control, we examined whether similar CaNaPC treatment had any therapeutic effect in a hemi-meniscectomy model in which there is no known crystal involvement. RESULTS: Meniscal calcification correlated with cartilage degeneration in this animal model. PC treatment led to significant reduction of calcium deposits and arrested OA disease progression. Similar treatment had no effect in the hemi-meniscectomy model. CONCLUSION: CaNaPC diminishes mineralization in a cutaneous calcergy model and a model of OA in which intraarticular mineralization is a prominent feature. In the OA guinea pig model, inhibition of calcification is accompanied by diminished cartilage degeneration. CaNaPC has no therapeutic effect in the hemi-meniscectomy model. We conclude that pathologic calcification may initiate or amplify processes leading to cartilage degeneration and that CaNaPC may interrupt such a pathway.

Inhibition of calcium phosphate-DNA coprecipitates induced cell death by phosphocitrates.

Phosphocitrate [PC] is a powerful inhibitor of biological crystallization and a potential disease modifying drug for crystal associated diseases such as crystals associated osteoarthritis [OA]. Recently, it has been reported that a new PC complex salt, calcium sodium PC [CaNaPC], is much more powerful than its precursor, sodium PC [NaPC], in reducing the size of chemically-induced calcified plaques in rat when examined using a calcergy assay (1). The molecular mechanisms underlying such a superior activity as a calcification inhibitor over its precursor NaPC are currently unknown. In order to evaluate the potential of CaNaPC as a disease modifying drug for crystals associated OA, we examined and compared CaNaPC and its precusor NaPC using several cell- based assays. CaNaPC was found to have an inhibitory potency similar to that of NaPC toward preventing the stimulating effects of basic calcium phosphate [BCP] crystals on the induction of MMP1, thymidine uptake and endocytosis. However, CaNaPC proved much more powerful than NaPC in the inhibition of amorphous calcium phosphate-DNA coprecipitates-induced cell death. These results suggest that the superior anti-calcification activity of NaCaPC over NaPC observed in rat is probably due to its superior activity in the inhibition of the effects associated with amorphous calcium phosphate clusters/aggregates/precipitates but not the effects associated with BCP crystals. Since amorphous calcium phosphate clusters/aggregates/precipitates are precursors of BCP crystals and coexist with calcium-containing crystals in calcified tissues (2-6), these amorphous clusters/aggregates/precipitates, similar to BCP crystals, may have played a significant role in pathological calcifications and in the development of crystals associated diseases such as crystals associated OA. The superior activity of CaNaPC over its precursor NaPC in the inhibition of amorphous calcium phosphate-DNA coprecipitates-induced cell death may, at least in part, explain its powerful anti-calcification activity in vivo. The findings suggest that CaNaPC through a dual action of inhibiting both the detrimental biological effects of formed BCP crystals and preforming amorphous calcium phosphate clusters/aggregates/precipitates, could present as a better disease-modifying drug for crystals associated OA than its parent NaPC.

Basic calcium phosphate crystal-induced prostaglandin E2 production in human fibroblasts: role of cyclooxygenase 1, cyclooxygenase 2, and interleukin-1beta.

OBJECTIVE: To elucidate the mechanism of basic calcium phosphate (BCP) crystal-induced prostaglandin E(2) (PGE(2)) production in human foreskin fibroblasts (HFFs), to identify the signaling pathway involved in the induction of cyclooxygenase 2 (COX-2) messenger RNA (mRNA) by BCP crystals, to examine the effect of BCP crystals on interleukin-1beta (IL-1beta) mRNA expression, and to investigate the potential of phosphocitrate to abrogate the BCP crystal-induced effects. METHODS: PGE(2) levels were quantified using a commercial enzyme immunoassay kit. COX-2 and COX-1 transcript levels were quantified using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Induction of IL-1beta and COX-2 mRNA was examined by end-point RT-PCR. COX-2 protein expression was assessed by Western blotting. RESULTS: PGE(2) production measured 4 and 30 hours after BCP crystal treatment was higher in BCP crystal-treated (mean +/- SEM 1,891 +/- 273 pg/microg and 1,792 +/- 233 pg/microg, respectively) than in untreated (88 +/- 5 pg/microg and 205 +/- 93 pg/microg, respectively) HFFs. The PGE(2) produced after 4 hours was sensitive to inhibition with NS398, a selective COX-2 inhibitor, implying that it was COX-2 mediated, whereas the PGE(2) produced at 30 hours could not be completely inhibited by NS398. Real-time RT-PCR demonstrated a 23-fold increase in COX-2 mRNA that was maximal at 4 hours, whereas analysis of mRNA for COX-1 showed up-regulation of transcript peaking at 24 hours poststimulation (1.75-fold increase). The protein kinase C and phosphatidylinositol 3-kinase signal-transduction inhibitors bisindolylmaleimide I and LY294002, respectively, blocked BCP crystal-induced COX-2 mRNA in HFFs. In addition, BCP crystals were found to up-regulate the proinflammatory cytokine IL-1beta (maximal at 8 hours). The induction of both COX-2 and IL-1beta by BCP crystals was attenuated when the cells were treated with phosphocitrate. CONCLUSION: These findings indicate that BCP crystals may be an important amplifier of PGE(2) production through induction of the COX enzymes and the proinflammatory cytokine IL-1beta.

Phosphocitrate inhibits calcium hydroxyapatite induced mitogenesis and upregulation of matrix metalloproteinase-1, interleukin-1beta and cyclooxygenase-2 mRNA in human breast cancer cell lines.

Microcalcifications containing calcium hydroxyapatite (HA) are often associated with malignant human breast lesions. Frequently, they are the only mammographic features that indicate the presence of a tumoural lesion. We previously reported the induction of both mitogenesis and prostaglandin E2 (PGE2) production and the increased activities of matrix metalloproteinases (MMPs) MMP-2 and MMP-9 in normal human mammary epithelial cells and breast cancer cell lines, treated with HA. In the present study we attempted to elucidate the mechanism of these biological effects. Firstly, we found that direct cell-crystal contact was required for induction of mitogenesis as the effect was not merely a result of isotopic exchange of calcium into the culture medium. Treatment with bafilomycin A1, a proton pump inhibitor, abrogated HA-induced mitogenesis to control cell levels. These results suggest that phagocytosis and intracellular crystal dissolution is required for HA-induced mitogenesis. We also demonstrated that the increase in prostaglandin E2, previously reported, is due, at least in part, to HA-induced upregulation of cyclooxygenase-2 (COX-2) in Hs578T cells. An accumulation of MMP-1 mRNA was also shown in response to HA stimulation in Hs578T cells. Furthermore, a HA-induced increase in interleukin-1beta (IL-1beta), a potent inducer of MMP-1 gene expression, was demonstrated in Hs578T cells at 2 and 4 h. Treatment with phosphocitrate (PC) (a naturally occurring inhibitor of calcium phosphate crystallisation, which is known to block a number of HA-induced biological effects in other cell types) blocked HA-mediated mitogenesis, as well as, COX-2, MMP-1 and IL-1beta induction, at the transcriptional level. These results show that calcium HA crystals are capable of exerting significant biological effects on surrounding cells which can be abrogated by PC and emphasise the role of calcium HA in amplifying the pathological process involved in breast cancer.

Inhibitors of articular calcium crystal formation.

The coexistence of calcium phosphate and calcium pyrophosphate crystals in osteoarthritis is a well-described phenomenon. In addition to deposition in articular cartilage, the crystals induce numerous changes to biochemical parameters in the surrounding fluid. This survey discusses crystal-inhibitor interactions whereby defining the molecular structure of crystals assists in an optimal inhibitor design. Crystal growth studies in the presence and absence of a test compound can generate data on crystal face changes. Together with knowledge of the inhibitors' molecular structure, computer modeling can portray crystal-inhibitor interactions. Few inhibitors have been tested in both in vitro and in vivo phases. One exception is phosphocitrate. This compound inhibits both the development of specific calcium crystals and crystal-induced intracellular changes. Animal studies confirm a strong action on pathologic calcification. The recently described phosphocitrate in a mixed calcium and sodium salt, yet to be tested in a guinea pig osteoarthritis model, appears to be an even more powerful inhibitor.