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Phosphocitrate and its analogue N-sulpho-2-amino tricarballylate inhibit aortic calcification
Atherosclerosis, 52 (1984) 191-198 Elsevier Scientific Publishers Ireland. Ltd.
191
ATH 03495
Phosphocitrate and its Analogue N-Sulpho-2-Amino Tricarballylate Inhibit Aortic Calcification R. Shankar Biochemistry Department,
*, S. Crowden
and J.D. Sallis
The University of Tasmania, G. P.O. Box 252C, Hobart, Tasmania (Australia)
- 7001
(Received 7 November, 1983) (Revised, received 16 January, 1984) (Accepted 18 January, 1984)
Summary This study reports the ability of phosphocitrate and its enzyme-resistant analogue N-sulpho-2-amino tricarballylate to inhibit aortic calcification. Dystrophic calcification of aorta was induced by transplanting fresh aortic segments in Millipore chambers to the peritoneal walls of recipient rats. Daily intraperitoneal injection of the new inhibitors remarkably reduced calcium accumulation by the aortae and prevented the appearance of hydroxyapatite-like crystalline structures. Phosphocitrate was the most effective of the anti-calcifying agents tested, preventing aortic calcification at 1 pmole/day/rat. N-sulpho-2-amino tricarballylate was less effective, reducing aortic calcification by 60% at 10 pmoles/day/rat. The new inhibitors might prove therapeutically useful in man to arrest soft tissue calcification. Key words:
Anti-calcifying agent - Aortic calcification 2-amino tricarballylate
- Phosphocitrate
- N-Sulpho-
Introduction Calcification is an inevitable phase of advanced atherosclerotic lesions. The contribution of medial calcification to the pathogenesis of atherosclerosis still remains an open question but there is considerable evidence to suggest that medial This work was supported by the National Heart Foundation of Australia and The University of Tasmania. * To whom correspondence should be addressed. 0021-9150/84/$03.00
0 1984 Elsevier Scientific Publishers Ireland, Ltd.
192
calcification may be an important predisposing condition to the formation of atherosclerosis in humans and in experimental animal models [l-3]. Despite elegant studies on the chemical composition and crystalline architecture of calcium deposits in atherosclerotic lesions, very little is known about the true molecular events that lead to such deposition. In terms of clinical significance, these deposits are of paramount importance because they alter the rheology of the vessel wall and are often the site of ulceration and formation of microthrombi. Anti-calcifying agents, such as the diphosphonates and pyrophosphate, have been shown to exert some effects both in preventing calcium deposition and towards the regression of the atherosclerotic process [4-61. However, the diphosphonates are poor therapeutic agents in these situations because of the inevitable osteomalacia that accompanies any long-term therapy with these agents [7]. Nevertheless, such studies highlight a role for the calcium ion in the pathobiology of atherosclerosis in events other than those associated with the terminal phase. Phosphocitrate (PC) is a relatively new anti-calcifying agent which has been shown to be useful in preventing ectopic calcification such as nephrocalcinosis [8]. So far, evidence for a similar effect on bone mineralization has not been established despite the strong in vitro affinity of PC for hydroxyapatite [9-111. Our earlier studies with PC indicated that it may be susceptible to in vivo enzymatic degradation by phosphohydrolases, especially in the kidney [lo]. This finding prompted the search for a more stable analogue of PC which would be resistant to enzymic degradation. As a result, the synthesis of N-sulpho-Zamino tricarballylate (SAT) has recently been accomplished in our laboratory [12]. The present study then reports experiments designed to assess if the new inhibitors PC and SAT are capable of arresting aortic calcification. A possible therapeutic role of these agents in atherosclerosis and arteriosclerosis is considered. Methods The model for aortic calcification was essentially that used and devised by Rim [13]. Adult male hooded Wistar rats (200 g) raised on normal rat chow were used throughout the study. Thoracic aortae were aseptically removed from donor rats under halothane anaesthesia. The aortae were stripped of adventitia, cut into l-cm segments and stored in ice-cold Hank’s balanced salt solution until ready for grafting. Each piece of aorta was placed in a separate 0.22 pm Millipore membrane chamber and 2 such chambers were transplanted to the peritoneal wall of the recipient rat under anaesthesia. After recovery, the rats were placed in individual cages and maintained on standard rat chow and water ad lib for the remainder of the experimental period. The following experimental design was used to test PC, SAT and the diphosphonate, ethane-1-hydroxy-l,l-diphosphonate (EHDP). Rats were randomly allocated into groups of 6. The control group received daily intraperitoneal injections of isotonic saline (0.1 ml) and the test groups received daily intraperitoneal injections of various concentrations of the inhibitor compounds from day 2 to day 15. On the 16th day, the rats from all groups were killed and the aortae from the Millipore
193
capsules content.
were
retrieved
for microscopic
analysis
and
determination
of calcium
Histology All aortae were fixed in 10% buffered formol-saline and embedded in paraffin wax. Serial sections were stained with hematoxylin and eosin for histological evaluation and von Kossa for the histochemical detection of insoluble calcium phosphate salts. Electron microscopy Aortae were fixed in ice-cold 2.5% glutaraldehyde for 3 h, post-fixed in 1% osmium tetroxide and embedded in epon. Thin sections were cut and the ultrastructural details were examined in a transmission electron microscope (Hitachi H300 series). For scanning electron-microscopic studies deparaffinised carbon-coated serial sections of aortae on perspex mounts were used. The sections were X-ray microanalysed for semi-quantitation of calcium and phosphate. Calcium analysis The calcium content of the aortae was analysed by atomic tometry after digesting the tissue at 80°C in a 1 : 2 mixture and 34 % hydrogen peroxide (v/v).
absorption spectrophoof 70% perchloric acid
Materials Phosphocitrate (PC) was synthesized from triethyl citrate and cyanoethyl phosphate by the method of Williams and Sallis [14]. SAT was prepared by reacting 2-amino tricarballylate vith pyridine-sulphur trioxide as described by Brown and Sallis [12]. EHDP was a gift from the Proctor and Gamble Co. (Cincinnati, U.S.A.). Results The effect of PC and SAT on aortic calcification is shown in Fig. 1. Daily intraperitoneal administration of 1 pmole of PC per rat totally abolished aortic calcification. In the control group of animals, the mean calcium content of aortae was 1.2 k 0.36 pmoles/mg dry weight aorta. By contrast, in the PC-treated group (1 pmole/day), calcium uptake by the aorta was reduced to 0.09 + 0.03 pmole/mg dry weight aorta. Intraperitoneal administration of equimolar amounts of sodium phosphate and sodium citrate had no detectable effect on the development of aortic calcification (not shown here). EHDP, on the other hand, required 5 times the effective concentration of PC to prevent aortic calcification. SAT was a weaker inhibitor, reducing aortic calcium accumulation to 0.48 + 0.11 pmole/mg dry weight aorta at 10 pmoles/day/rat. Of the 4 inhibitors tested, PPi proved to be weakest in this system, the order of inhibitory potential of these compounds being PC > EHDP > SAT > PPi. Light-microscopic examination confirmed the findings. Sections of aorta from control groups of animals indicated heavy calcium phosphate deposits in the medial
194
0
1 INHl~lfOR
5 CONCENTRATION
10 pmole / day
Fig. 1. Effect of inhibitors on calcium accumulation by aorta. Phosphocitrate (PC), N-sulpho-2-amino tricarballylate (SAT), ethane-1-hydroxy-l.l-diphosphonate (EHDP) and pyrophosphate (PPi) were the inhibitors tested. The data plotted represent mean+ SD of 8 aortic segments at each dose level.
Fig. 2. Light-microscopic study on the effect of phosphocitrate on aortic calcification. Sections of aortae were stained by the van Kossa technique and counterstained with van Gieson. The aortae from the control group (A) showed extensive deposition of insoluble calcium phosphate salts in the medial elastic layers. Similar sections from the phosphocitrate-treated group (1 pmoie/day/200 g rat) (8) were remarkably free of mineral deposits.
Fig. 3. Transmission electron-microscopic study on the effect of phosphocitrate on aortic calcification. Thin plastic sections of aortae were stained with uranyl acetate and lead citrate ( X 58400). A: The sections from the control aortae showed extensive cellular degeneration with heavy calcification (CF). There was also increased lipid accumulation associated with the calcification front seen as electron-dense droplets in the centre (LD). B: Similar sections from the phosphocitrate-treated group (1 pmole/day/200 g rat) were not only free from mineral deposits and lipid droplets but also showed considerably less cellular degeneration. VS
lOOOK
VS 25K
15 20 E DAX Normal rat aorta
15
VS. 1000K
EDAX
VS.25K C-
15
20 Control
20
Phosphockrate
-
DI
EDAX
15
(lpmol/day)
SAT
20
EDAX
(lO!Jmol/day)
Fig. 4. Energy dispersive X-ray microanalyses (EDAX) of uncalcified aorta from a normal rat (A), untreated control (B), phosphocitrate-(PC), 1 pmole/day (C) and N-sulpho-2-amino tricarballylate(SAT), 10 pmole/day (D)-treated aortae. The vertical scales of A and C are 40 X that of E and D. The calcium and phosphorus peaks are represented by their atomic numbers along the horizontal axis. The control aortae showed strong peaks corresponding to calcium and phosphorus. Similar peaks were absent in the PC-treated group and the peak heights were much shorter in the SAT-treated group.
196 TABLE
1
PERCENTAGE MASS FRACTIONS” OF CALCIUM TREATED AND UNTREATED RAT AORTAE Inhibitor (dose/day/200 None PC (1 pmol) SAT (10 pmol) EHDP (5 pmol) PPi (10 pmol)
g rat)
AND
PHOSPHORUS
IN
No. of observations
Calcium
Phosphorus
6 6 6 6 6
21.15 0.15 9.8 0.28 10.28
18.02+6.5 0.13*0.04 8.4 k4.4 0.15+0.04 9.1 k3.7
* 7.3 50.03 +4.8 f 0.015 * 4.6
INHIBITOR-
a Mean f 1 SD.
elastic layers whereas the aortae from animals receiving the effective concentration PC were remarkably free from such deposits (Fig. 2). Only a few deposits were seen in aortae from rats receiving lower concentrations of PC, providing a clear indication of the potency of this compound in preventing aortic calcium accumulation. Transmission electron micrographs revealed calcification changes around the medial elastic layers with extensive cellular degeneration and lipid accumulation in the control aortae. These observations were in close agreement with the findings of Kim [13]. Similar examination of aortae from PC-treated rats showed considerably less cellular degeneration and no calcium deposits (Fig. 3). Scanning electron microscopy coupled with X-ray microanalysis of aortic sections permitted the quantitation of calcium and phosphorus present in the aortae in terms of percentage mass fractions. The results are shown in Table 1 and Fig. 4. X-ray microanalysis not only showed that PC effectively abolished calcium phosphate deposition but also confirmed that the crystal salt deposited in the control aortae was in fact hydroxyapatite. Discussion Pathological calcification of arteries appears to originate from initial injury to the endothelial cell lining [15]. Such an injury together with plasma Ca2+ concentration which is almost lo4 times higher than the cytoplasmic concentration, may allow the net entry of calcium and phosphate into medial layers and initiate the process of calcification. Medial calcification is an age-related phenomenon and this factor appears to be an important pre-requisite for the formation of the intimal plaque [3]. Ca2+ itself has recently been shown to be a potent stimulator of smooth muscle cell proliferation, a phase central to the pathology of atherosclerosis [16]. Therefore, any agent which can abolish or reduce medial and atherosclerotic calcification may prove to be an important agent against the development of atherogenesis. In the present study an aortic transplant model was used to demonstrate the anti-calcifying potential of two new inhibitors, namely, PC and SAT. Although the model unfortunately does not reproduce many of the factors that accompany
197
pathological calcification in humans (e.g. dynamic blood flow, some humoral factors, etc.) it does display the ability to induce calcification slowly and consistently without the direct intervention of exogenous agents. The data obtained showed that both new inhibitors were effective in arresting aortic calcification. Histochemistry, electron microscopy and chemical analyses confirmed the effectiveness of the two inhibitors with PC being superior to SAT. In addition, electron microscopy also established that hydroxyapatite was the calcium salt deposited in the control aortae. One uncertainty in the therapeutic value of PC is its stability under in vivo conditions. Our earlier work, in vitro, indicated that PC was hydrolysable, albeit slowly, in the presence of phosphohydrolases. Additional studies with [ 32P]PC given to rats exogenously, indicated that PC could be selectively degraded in the kidney [lo]. More recent studies [8] and the data presented here, however, suggest that PC is perhaps more stable than originally thought. PC is a natural inhibitor present in biological tissues [lo] and, therefore, would not be expected to produce any adverse effects when given exogenously. In addition, the cleavage products of administered PC, namely, inorganic phosphate and citrate are also natural metabolites and even on complete hydrolysis would only marginally increase their tissue and plasma levels. This is in contrast to many other synthetic anti-calcifying agents which can lead to secondary side effects. Only limited studies with regard to the actions of PC on tissues have so far been documented, but Reddi et al. have shown that PC has little effect on the in vivo matrix-induced model of bone formation [ll]. Thus, the formidable anti-calcifying ability of PC together with a lack of any deleterious action on tissues would make it an attractive agent to arrest arterial calcification. While SAT, the enzyme-resistant analogue of PC, is much less effective in arresting aortic calcification, the compound does appear to have other advantages. Preliminary pharmacokinetic studies with [3sS]SAT have shown that given orally, it is absorbed well and excreted unchanged by the kidneys. Whilst further studies on both short- and long-term toxicity are still in progress, administration.of SAT at 10 pmole/day for 2-6 weeks resulted in no change in the rate of growth of the animals and no deaths. Examination of kidney, liver, bone and gut revealed no gross or histological abnormalities. Urinalysis was also normal with no proteinuria and normal leucocyte excretion (unpublished data). Therefore, despite its limited potency, SAT may still prove to be useful in arresting soft tissue calcification if administered orally in large doses. Because aortic calcification and calcium fluxes are significant contributory factors in the development and acceleration of atherosclerosis [5,15], the present study has potential clinical implications. The pathobiology of atherogenesis involves many Ca2+-dependent intermediate steps such as platelet aggregation, smooth muscle cell proliferation and lipoprotein-binding to smooth muscle cell receptors [5,17]. It is possible that anti-calcifying agents can exert their effects by interfering with any of these intermediate steps. Although our present studies do not reveal the mechanism(s) of PC and SAT inhibition, the indications are that if PC or its analogue is capable of altering Ca2 + fluxes in tissues, they may prove useful agents in preventing or ameliorating the incidence of atherosclerosis.
198
Acknowledgements We thank Mr. J.E. Jordan and Miss L.C. Ward for expert technical assistance, Mr. W. Jablonski for scanning electron microscopy and Mr. R. J. Tennent for transmission electron microscopy. References 1 Bluementhal, H.T., Lansing, AI. and Wheeler, P.A., Calcification of media of the human aorta and its relation to intimal atherosclerosis, aging and disease, Amer. .I. Path., 20 (1944) 665. 2 Lansing, A.I., Alex, M. and Rosenthal, T.B., Calcium and elastin in human atherosclerosis, J. Gerontol., 5 (1950) 112. 3 Moon, J.Y., Factors affecting arterial calcification associated with atherosclerosis, Atherosclerosis, 16 (1972) 119. 4 Rosenblum, I.Y., Flora, L. and Eisenstein, R., The effect of disodium ethane-1-hydroxy-1,1-diphos phonate (EHDP) on a rabbit model of atherosclerosis, Atherosclerosis, 22 (1975) 411. 5 Kramsch, D.M., Aspen, A.J. and Rozler, L.J., Atherosclerosis-prevention by agents not affecting abnormal levels of lipids, Science, 213 (1981) 1511. 6 Fleisch, H., Schibler, D., Mearki. J. and Frossard, I., Inhibition of aortic calcification by means of pyrophosphate and polyphosphates, Nature (Lond.), 207 (1965) 1300. 7 Schenk, R., Merz, W.A., Muhlbauer, R., Russell, R.G.G. and Fleisch. H., Effect of ethane-l-hydroxyl,l-diphosphonate (EHDP) and dichloromethylene disphosphonate (Cl,MDP) on calcification and resorption of cartilage and bone in the tibia1 epiphysis and metaphysis of rats. Calcif. Tiss. Res.. 11 (1973) 196. 8 Tew, W.P., Mallis, C.D., Howard, J.E. and Lehninger. A.L., Phosphocitrate inhibits mitochondrial and cytosolic accumulation of calcium in kidney cells in viva, Proc. Nat. Acad. Sci. (USA), 78 (1981) 5528. 9 Williams, G. and SaIlis, J.D., Structure-activity relationship of inhibitors of hydroxyapatite formation. B&hem. J., 184 (1979) 181. 10 Williams, G. and Sallis, J.D., The source of phosphocitrate and its role as an inhibitor of calcium phosphate and calcium oxalate crystallization. In: L.H. Smith, W.G. Robertson and B. Finlayson (I%.), Urolithiasis - Clinical and Basic Research, Plenum Press, New York, NY, 1981. p. 559. 11 Reddi, A.H., Meyer. J.L., Tew, W.P., Howard, J.E. and Lehninger, A.L., Influence of phosphocitrate. a potent inhibitor of hydroxyapatite crystal growth, on mineralization of cartilage and bone. Biochem. Biophys. Res. Commun., 97 (1980) 154. of N-sulfo-2-amino tricarballylate 12 Brown, M.R. and Sallis, J.D., The synthesis and characterization An analogue of phosphocitrate and inhibitor of calcification, Anal. Biochem., 132 (1983) 115. of rat aorta in Millipore chambers, Metab. Bone Dis. Rel. 13 Kim, K.M., Matrix vesicle calcification Res., 1 (1978) 213. and analytical 14 Williams, G. and Sallis, J.D., The synthesis of unlabeled and 32P-labeled phosphocitrate systems for its identification, Anal. B&hem.. 102 (1980) 365. N. Engl. J. Med., 295 (1976) 369,420. 15 Ross, R. and Glomset. J.A., The pathogenesis of atherosclerosis, of aortic 16 Nakao, J., Ito, H., Ooyama, T., Chang, W.-C. and Murota, S.-I., Calcium dependency smooth muscle cell migration induced by 12-L-hydroxy-,5.8.10,14-eicosatetraenoic acid, Atherosclerosis, 46 (1983) 309. 17 Charnley-Campbell, J.H., Nestel, P. and Campbell, G.R.. Smooth muscle metabolic activity in atherogenesis - LDL metabolism and response to serum mitogens differ according to phenotype. In: G.R.V. Born, A.L. Catapano and R. Paoletti (Eds.), Factors in Formation and Regression of the Atherosclerotic Plaque, Plenum Press, New York. NY, 1982, p. 115.
191
ATH 03495
Phosphocitrate and its Analogue N-Sulpho-2-Amino Tricarballylate Inhibit Aortic Calcification R. Shankar Biochemistry Department,
*, S. Crowden
and J.D. Sallis
The University of Tasmania, G. P.O. Box 252C, Hobart, Tasmania (Australia)
- 7001
(Received 7 November, 1983) (Revised, received 16 January, 1984) (Accepted 18 January, 1984)
Summary This study reports the ability of phosphocitrate and its enzyme-resistant analogue N-sulpho-2-amino tricarballylate to inhibit aortic calcification. Dystrophic calcification of aorta was induced by transplanting fresh aortic segments in Millipore chambers to the peritoneal walls of recipient rats. Daily intraperitoneal injection of the new inhibitors remarkably reduced calcium accumulation by the aortae and prevented the appearance of hydroxyapatite-like crystalline structures. Phosphocitrate was the most effective of the anti-calcifying agents tested, preventing aortic calcification at 1 pmole/day/rat. N-sulpho-2-amino tricarballylate was less effective, reducing aortic calcification by 60% at 10 pmoles/day/rat. The new inhibitors might prove therapeutically useful in man to arrest soft tissue calcification. Key words:
Anti-calcifying agent - Aortic calcification 2-amino tricarballylate
- Phosphocitrate
- N-Sulpho-
Introduction Calcification is an inevitable phase of advanced atherosclerotic lesions. The contribution of medial calcification to the pathogenesis of atherosclerosis still remains an open question but there is considerable evidence to suggest that medial This work was supported by the National Heart Foundation of Australia and The University of Tasmania. * To whom correspondence should be addressed. 0021-9150/84/$03.00
0 1984 Elsevier Scientific Publishers Ireland, Ltd.
192
calcification may be an important predisposing condition to the formation of atherosclerosis in humans and in experimental animal models [l-3]. Despite elegant studies on the chemical composition and crystalline architecture of calcium deposits in atherosclerotic lesions, very little is known about the true molecular events that lead to such deposition. In terms of clinical significance, these deposits are of paramount importance because they alter the rheology of the vessel wall and are often the site of ulceration and formation of microthrombi. Anti-calcifying agents, such as the diphosphonates and pyrophosphate, have been shown to exert some effects both in preventing calcium deposition and towards the regression of the atherosclerotic process [4-61. However, the diphosphonates are poor therapeutic agents in these situations because of the inevitable osteomalacia that accompanies any long-term therapy with these agents [7]. Nevertheless, such studies highlight a role for the calcium ion in the pathobiology of atherosclerosis in events other than those associated with the terminal phase. Phosphocitrate (PC) is a relatively new anti-calcifying agent which has been shown to be useful in preventing ectopic calcification such as nephrocalcinosis [8]. So far, evidence for a similar effect on bone mineralization has not been established despite the strong in vitro affinity of PC for hydroxyapatite [9-111. Our earlier studies with PC indicated that it may be susceptible to in vivo enzymatic degradation by phosphohydrolases, especially in the kidney [lo]. This finding prompted the search for a more stable analogue of PC which would be resistant to enzymic degradation. As a result, the synthesis of N-sulpho-Zamino tricarballylate (SAT) has recently been accomplished in our laboratory [12]. The present study then reports experiments designed to assess if the new inhibitors PC and SAT are capable of arresting aortic calcification. A possible therapeutic role of these agents in atherosclerosis and arteriosclerosis is considered. Methods The model for aortic calcification was essentially that used and devised by Rim [13]. Adult male hooded Wistar rats (200 g) raised on normal rat chow were used throughout the study. Thoracic aortae were aseptically removed from donor rats under halothane anaesthesia. The aortae were stripped of adventitia, cut into l-cm segments and stored in ice-cold Hank’s balanced salt solution until ready for grafting. Each piece of aorta was placed in a separate 0.22 pm Millipore membrane chamber and 2 such chambers were transplanted to the peritoneal wall of the recipient rat under anaesthesia. After recovery, the rats were placed in individual cages and maintained on standard rat chow and water ad lib for the remainder of the experimental period. The following experimental design was used to test PC, SAT and the diphosphonate, ethane-1-hydroxy-l,l-diphosphonate (EHDP). Rats were randomly allocated into groups of 6. The control group received daily intraperitoneal injections of isotonic saline (0.1 ml) and the test groups received daily intraperitoneal injections of various concentrations of the inhibitor compounds from day 2 to day 15. On the 16th day, the rats from all groups were killed and the aortae from the Millipore
193
capsules content.
were
retrieved
for microscopic
analysis
and
determination
of calcium
Histology All aortae were fixed in 10% buffered formol-saline and embedded in paraffin wax. Serial sections were stained with hematoxylin and eosin for histological evaluation and von Kossa for the histochemical detection of insoluble calcium phosphate salts. Electron microscopy Aortae were fixed in ice-cold 2.5% glutaraldehyde for 3 h, post-fixed in 1% osmium tetroxide and embedded in epon. Thin sections were cut and the ultrastructural details were examined in a transmission electron microscope (Hitachi H300 series). For scanning electron-microscopic studies deparaffinised carbon-coated serial sections of aortae on perspex mounts were used. The sections were X-ray microanalysed for semi-quantitation of calcium and phosphate. Calcium analysis The calcium content of the aortae was analysed by atomic tometry after digesting the tissue at 80°C in a 1 : 2 mixture and 34 % hydrogen peroxide (v/v).
absorption spectrophoof 70% perchloric acid
Materials Phosphocitrate (PC) was synthesized from triethyl citrate and cyanoethyl phosphate by the method of Williams and Sallis [14]. SAT was prepared by reacting 2-amino tricarballylate vith pyridine-sulphur trioxide as described by Brown and Sallis [12]. EHDP was a gift from the Proctor and Gamble Co. (Cincinnati, U.S.A.). Results The effect of PC and SAT on aortic calcification is shown in Fig. 1. Daily intraperitoneal administration of 1 pmole of PC per rat totally abolished aortic calcification. In the control group of animals, the mean calcium content of aortae was 1.2 k 0.36 pmoles/mg dry weight aorta. By contrast, in the PC-treated group (1 pmole/day), calcium uptake by the aorta was reduced to 0.09 + 0.03 pmole/mg dry weight aorta. Intraperitoneal administration of equimolar amounts of sodium phosphate and sodium citrate had no detectable effect on the development of aortic calcification (not shown here). EHDP, on the other hand, required 5 times the effective concentration of PC to prevent aortic calcification. SAT was a weaker inhibitor, reducing aortic calcium accumulation to 0.48 + 0.11 pmole/mg dry weight aorta at 10 pmoles/day/rat. Of the 4 inhibitors tested, PPi proved to be weakest in this system, the order of inhibitory potential of these compounds being PC > EHDP > SAT > PPi. Light-microscopic examination confirmed the findings. Sections of aorta from control groups of animals indicated heavy calcium phosphate deposits in the medial
194
0
1 INHl~lfOR
5 CONCENTRATION
10 pmole / day
Fig. 1. Effect of inhibitors on calcium accumulation by aorta. Phosphocitrate (PC), N-sulpho-2-amino tricarballylate (SAT), ethane-1-hydroxy-l.l-diphosphonate (EHDP) and pyrophosphate (PPi) were the inhibitors tested. The data plotted represent mean+ SD of 8 aortic segments at each dose level.
Fig. 2. Light-microscopic study on the effect of phosphocitrate on aortic calcification. Sections of aortae were stained by the van Kossa technique and counterstained with van Gieson. The aortae from the control group (A) showed extensive deposition of insoluble calcium phosphate salts in the medial elastic layers. Similar sections from the phosphocitrate-treated group (1 pmoie/day/200 g rat) (8) were remarkably free of mineral deposits.
Fig. 3. Transmission electron-microscopic study on the effect of phosphocitrate on aortic calcification. Thin plastic sections of aortae were stained with uranyl acetate and lead citrate ( X 58400). A: The sections from the control aortae showed extensive cellular degeneration with heavy calcification (CF). There was also increased lipid accumulation associated with the calcification front seen as electron-dense droplets in the centre (LD). B: Similar sections from the phosphocitrate-treated group (1 pmole/day/200 g rat) were not only free from mineral deposits and lipid droplets but also showed considerably less cellular degeneration. VS
lOOOK
VS 25K
15 20 E DAX Normal rat aorta
15
VS. 1000K
EDAX
VS.25K C-
15
20 Control
20
Phosphockrate
-
DI
EDAX
15
(lpmol/day)
SAT
20
EDAX
(lO!Jmol/day)
Fig. 4. Energy dispersive X-ray microanalyses (EDAX) of uncalcified aorta from a normal rat (A), untreated control (B), phosphocitrate-(PC), 1 pmole/day (C) and N-sulpho-2-amino tricarballylate(SAT), 10 pmole/day (D)-treated aortae. The vertical scales of A and C are 40 X that of E and D. The calcium and phosphorus peaks are represented by their atomic numbers along the horizontal axis. The control aortae showed strong peaks corresponding to calcium and phosphorus. Similar peaks were absent in the PC-treated group and the peak heights were much shorter in the SAT-treated group.
196 TABLE
1
PERCENTAGE MASS FRACTIONS” OF CALCIUM TREATED AND UNTREATED RAT AORTAE Inhibitor (dose/day/200 None PC (1 pmol) SAT (10 pmol) EHDP (5 pmol) PPi (10 pmol)
g rat)
AND
PHOSPHORUS
IN
No. of observations
Calcium
Phosphorus
6 6 6 6 6
21.15 0.15 9.8 0.28 10.28
18.02+6.5 0.13*0.04 8.4 k4.4 0.15+0.04 9.1 k3.7
* 7.3 50.03 +4.8 f 0.015 * 4.6
INHIBITOR-
a Mean f 1 SD.
elastic layers whereas the aortae from animals receiving the effective concentration PC were remarkably free from such deposits (Fig. 2). Only a few deposits were seen in aortae from rats receiving lower concentrations of PC, providing a clear indication of the potency of this compound in preventing aortic calcium accumulation. Transmission electron micrographs revealed calcification changes around the medial elastic layers with extensive cellular degeneration and lipid accumulation in the control aortae. These observations were in close agreement with the findings of Kim [13]. Similar examination of aortae from PC-treated rats showed considerably less cellular degeneration and no calcium deposits (Fig. 3). Scanning electron microscopy coupled with X-ray microanalysis of aortic sections permitted the quantitation of calcium and phosphorus present in the aortae in terms of percentage mass fractions. The results are shown in Table 1 and Fig. 4. X-ray microanalysis not only showed that PC effectively abolished calcium phosphate deposition but also confirmed that the crystal salt deposited in the control aortae was in fact hydroxyapatite. Discussion Pathological calcification of arteries appears to originate from initial injury to the endothelial cell lining [15]. Such an injury together with plasma Ca2+ concentration which is almost lo4 times higher than the cytoplasmic concentration, may allow the net entry of calcium and phosphate into medial layers and initiate the process of calcification. Medial calcification is an age-related phenomenon and this factor appears to be an important pre-requisite for the formation of the intimal plaque [3]. Ca2+ itself has recently been shown to be a potent stimulator of smooth muscle cell proliferation, a phase central to the pathology of atherosclerosis [16]. Therefore, any agent which can abolish or reduce medial and atherosclerotic calcification may prove to be an important agent against the development of atherogenesis. In the present study an aortic transplant model was used to demonstrate the anti-calcifying potential of two new inhibitors, namely, PC and SAT. Although the model unfortunately does not reproduce many of the factors that accompany
197
pathological calcification in humans (e.g. dynamic blood flow, some humoral factors, etc.) it does display the ability to induce calcification slowly and consistently without the direct intervention of exogenous agents. The data obtained showed that both new inhibitors were effective in arresting aortic calcification. Histochemistry, electron microscopy and chemical analyses confirmed the effectiveness of the two inhibitors with PC being superior to SAT. In addition, electron microscopy also established that hydroxyapatite was the calcium salt deposited in the control aortae. One uncertainty in the therapeutic value of PC is its stability under in vivo conditions. Our earlier work, in vitro, indicated that PC was hydrolysable, albeit slowly, in the presence of phosphohydrolases. Additional studies with [ 32P]PC given to rats exogenously, indicated that PC could be selectively degraded in the kidney [lo]. More recent studies [8] and the data presented here, however, suggest that PC is perhaps more stable than originally thought. PC is a natural inhibitor present in biological tissues [lo] and, therefore, would not be expected to produce any adverse effects when given exogenously. In addition, the cleavage products of administered PC, namely, inorganic phosphate and citrate are also natural metabolites and even on complete hydrolysis would only marginally increase their tissue and plasma levels. This is in contrast to many other synthetic anti-calcifying agents which can lead to secondary side effects. Only limited studies with regard to the actions of PC on tissues have so far been documented, but Reddi et al. have shown that PC has little effect on the in vivo matrix-induced model of bone formation [ll]. Thus, the formidable anti-calcifying ability of PC together with a lack of any deleterious action on tissues would make it an attractive agent to arrest arterial calcification. While SAT, the enzyme-resistant analogue of PC, is much less effective in arresting aortic calcification, the compound does appear to have other advantages. Preliminary pharmacokinetic studies with [3sS]SAT have shown that given orally, it is absorbed well and excreted unchanged by the kidneys. Whilst further studies on both short- and long-term toxicity are still in progress, administration.of SAT at 10 pmole/day for 2-6 weeks resulted in no change in the rate of growth of the animals and no deaths. Examination of kidney, liver, bone and gut revealed no gross or histological abnormalities. Urinalysis was also normal with no proteinuria and normal leucocyte excretion (unpublished data). Therefore, despite its limited potency, SAT may still prove to be useful in arresting soft tissue calcification if administered orally in large doses. Because aortic calcification and calcium fluxes are significant contributory factors in the development and acceleration of atherosclerosis [5,15], the present study has potential clinical implications. The pathobiology of atherogenesis involves many Ca2+-dependent intermediate steps such as platelet aggregation, smooth muscle cell proliferation and lipoprotein-binding to smooth muscle cell receptors [5,17]. It is possible that anti-calcifying agents can exert their effects by interfering with any of these intermediate steps. Although our present studies do not reveal the mechanism(s) of PC and SAT inhibition, the indications are that if PC or its analogue is capable of altering Ca2 + fluxes in tissues, they may prove useful agents in preventing or ameliorating the incidence of atherosclerosis.
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Acknowledgements We thank Mr. J.E. Jordan and Miss L.C. Ward for expert technical assistance, Mr. W. Jablonski for scanning electron microscopy and Mr. R. J. Tennent for transmission electron microscopy. References 1 Bluementhal, H.T., Lansing, AI. and Wheeler, P.A., Calcification of media of the human aorta and its relation to intimal atherosclerosis, aging and disease, Amer. .I. Path., 20 (1944) 665. 2 Lansing, A.I., Alex, M. and Rosenthal, T.B., Calcium and elastin in human atherosclerosis, J. Gerontol., 5 (1950) 112. 3 Moon, J.Y., Factors affecting arterial calcification associated with atherosclerosis, Atherosclerosis, 16 (1972) 119. 4 Rosenblum, I.Y., Flora, L. and Eisenstein, R., The effect of disodium ethane-1-hydroxy-1,1-diphos phonate (EHDP) on a rabbit model of atherosclerosis, Atherosclerosis, 22 (1975) 411. 5 Kramsch, D.M., Aspen, A.J. and Rozler, L.J., Atherosclerosis-prevention by agents not affecting abnormal levels of lipids, Science, 213 (1981) 1511. 6 Fleisch, H., Schibler, D., Mearki. J. and Frossard, I., Inhibition of aortic calcification by means of pyrophosphate and polyphosphates, Nature (Lond.), 207 (1965) 1300. 7 Schenk, R., Merz, W.A., Muhlbauer, R., Russell, R.G.G. and Fleisch. H., Effect of ethane-l-hydroxyl,l-diphosphonate (EHDP) and dichloromethylene disphosphonate (Cl,MDP) on calcification and resorption of cartilage and bone in the tibia1 epiphysis and metaphysis of rats. Calcif. Tiss. Res.. 11 (1973) 196. 8 Tew, W.P., Mallis, C.D., Howard, J.E. and Lehninger. A.L., Phosphocitrate inhibits mitochondrial and cytosolic accumulation of calcium in kidney cells in viva, Proc. Nat. Acad. Sci. (USA), 78 (1981) 5528. 9 Williams, G. and SaIlis, J.D., Structure-activity relationship of inhibitors of hydroxyapatite formation. B&hem. J., 184 (1979) 181. 10 Williams, G. and Sallis, J.D., The source of phosphocitrate and its role as an inhibitor of calcium phosphate and calcium oxalate crystallization. In: L.H. Smith, W.G. Robertson and B. Finlayson (I%.), Urolithiasis - Clinical and Basic Research, Plenum Press, New York, NY, 1981. p. 559. 11 Reddi, A.H., Meyer. J.L., Tew, W.P., Howard, J.E. and Lehninger, A.L., Influence of phosphocitrate. a potent inhibitor of hydroxyapatite crystal growth, on mineralization of cartilage and bone. Biochem. Biophys. Res. Commun., 97 (1980) 154. of N-sulfo-2-amino tricarballylate 12 Brown, M.R. and Sallis, J.D., The synthesis and characterization An analogue of phosphocitrate and inhibitor of calcification, Anal. Biochem., 132 (1983) 115. of rat aorta in Millipore chambers, Metab. Bone Dis. Rel. 13 Kim, K.M., Matrix vesicle calcification Res., 1 (1978) 213. and analytical 14 Williams, G. and Sallis, J.D., The synthesis of unlabeled and 32P-labeled phosphocitrate systems for its identification, Anal. B&hem.. 102 (1980) 365. N. Engl. J. Med., 295 (1976) 369,420. 15 Ross, R. and Glomset. J.A., The pathogenesis of atherosclerosis, of aortic 16 Nakao, J., Ito, H., Ooyama, T., Chang, W.-C. and Murota, S.-I., Calcium dependency smooth muscle cell migration induced by 12-L-hydroxy-,5.8.10,14-eicosatetraenoic acid, Atherosclerosis, 46 (1983) 309. 17 Charnley-Campbell, J.H., Nestel, P. and Campbell, G.R.. Smooth muscle metabolic activity in atherogenesis - LDL metabolism and response to serum mitogens differ according to phenotype. In: G.R.V. Born, A.L. Catapano and R. Paoletti (Eds.), Factors in Formation and Regression of the Atherosclerotic Plaque, Plenum Press, New York. NY, 1982, p. 115.