This Article Abstract Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jono, S. Articles by Morii, H. Search for Related Content PubMed PubMed Citation Articles by Jono, S. Articles by Morii, H.

(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1135-1142.)
© 1997 American Heart Association, Inc.

Articles

Parathyroid Hormone–Related Peptide as a Local Regulator of Vascular Calcification

Its Inhibitory Action on In Vitro Calcification by Bovine Vascular Smooth Muscle Cells

Shuichi Jono; Yoshiki Nishizawa; Atsushi Shioi; ; Hirotoshi Morii

From the Second Department of Internal Medicine, Osaka City University Medical School, Osaka, Japan.

Correspondence to Atsushi Shioi, MD, Second Department of Internal Medicine, Osaka City University Medical School, 1-5-7 Asahi-machi, Abeno-ku, Osaka 545, Japan.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract In the present study, we investigated the role of parathyroid hormone–related peptide (PTHrP) in vascular calcification by using an in vitro calcification model. We demonstrated that the expression of PTHrP decreased in the progression of bovine vascular smooth muscle cell (BVSMC) calcification and that inhibition of calcification by etidronate (EHDP) and levamisole restored PTHrP secretion, suggesting that the expression of PTHrP is associated with calcification. PTHrP (1-34) and PTH (1-34) dose-dependently inhibited BVSMC calcification. Protein kinase A (PKA) and protein kinase C (PKC) inhibitors completely blocked the inhibitory effect of PTHrP, suggesting that both PKA and PKC may be involved in its signaling pathway. Moreover, PTHrP inhibited alkaline phosphatase (ALP) activity, implying that the impact on ALP may contribute to its action on calcification. Furthermore, the PTHrP antagonist, PTHrP (7-34), dose-dependently increased calcium deposition by BVSMC. Interestingly, PTHrP production by BVSMC dramatically increased in the presence of EHDP, and PTHrP (7-34) partially antagonized the inhibitory effect of EHDP on BVSMC calcification. These results suggest that PTHrP may regulate vascular calcification as an autocrine/paracrine factor.

Key Words: atherosclerosis • bisphosphonates • protein kinase • ß-glycerophosphate • parathyroid hormone

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Calcification is commonly associated with atherosclerosis^{1 2} and it has important clinical implications, especially in coronary arteries.^{3 4 5 6} Calcification was generally considered to be a passive, degenerative, and end-stage process of atherosclerosis.^{1 7 8} However, there are increasing evidences for the presence in calcified atherosclerotic lesions of matrix vesicles,^{9 10} osteoblastic differentiation factors such as bone morphogenetic protein-2a (BMP-2a),¹¹ and calcium binding proteins typically associated with skeletal metabolism including osteopontin^{12 13 14 15} and matrix Gla protein,¹² suggesting that calcification associated with atherosclerosis is an organized, regulated process similar to mineralization in bone tissue.¹⁶ Moreover, disturbance of calcium and phosphate metabolism in atherosclerotic plaques may contribute to the development of vascular calcification. However, the mechanism of vascular calcification remains to be clarified.

To facilitate clarifying the mechanism of vascular calcification, we developed an in vitro calcification system in which diffuse calcification can be induced by culturing bovine vascular smooth muscle cells in the presence of ß-glycerophosphate.¹⁷ In this model, we demonstrated that expression of alkaline phosphatase is functionally important in BVSMC calcification and that osteopontin mRNA is expressed exclusively in calcified BVSMC.¹⁷

Parathyroid hormone–related peptide was originally identified as a pathogenic factor for malignancy-associated hypercalcemia.^{18 19 20} The homology of the amino-terminal region of PTHrP, with the corresponding domain of parathyroid hormone, and the resultant capacity of both molecules to interact with a common receptor²¹ account for the ability of PTHrP to mimic the various effects of PTH on calcium and phosphate homeostasis and on skeletal metabolism.^{22 23 24 25 26} Unlike PTH, however, whose expression is restricted to the parathyroid gland, PTHrP is produced in diverse normal adult^{27 28 29} and fetal^{30 31} tissues and may play a wide variety of physiological roles, mainly as an autocrine and/or paracrine factor.³² In vascular smooth muscle, PTHrP can act as a vasorelaxant and exert hypotensive effect in vivo.^{33 34 35 36} Its expression is reported in cultured vascular smooth muscle cells³⁷ as well as medial SMC in vivo³⁸ and upregulated by serum,³⁷ mechanical stretch,³⁹ and vasoconstrictive substances such as angiotensin II, thrombin, and endothelin.^{37 39 40} Additionally, PTHrP can modulate the growth-stimulating effect of angiotensin II on rat VSMC.⁴⁰ Therefore, it is possible to regard PTHrP as a local modulator in vascular smooth muscle.

In the present study, we investigated the potential role of PTHrP in vascular calcification by using an in vitro calcification model.¹⁷ We first demonstrated the inverse relationship between the expression of PTHrP and BVSMC calcification. Moreover, we found that exogenous PTHrP inhibits BVSMC calcification through activating both PKA and PKC pathways. The functional role of endogenous PTHrP was proved by demonstrating that PTHrP antagonist, PTHrP (7-34), dose-dependently increased this calcification. Interestingly, PTHrP production by BVSMC dramatically increased in the presence of etidronate (EHDP) and PTHrP (7-34) partially antagonized the inhibitory action of EHDP on BVSMC calcification. These results suggest that PTHrP may regulate vascular calcification as an autocrine and/or paracrine factor.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Reagents
Media, fetal bovine serum (FBS), and sodium pyruvate were purchased from Gibco. ß-Glycerophosphate and l-2,3,5,6-tetrahydro-6-phenylimidazo[2,1-b]thiazole (l-tetramisole, levamisole) were obtained from Sigma. Ethane-1-hydroxy-1,1-diphosphonic acid (etidronate, EHDP) was kindly provided by Sumitomo Pharmaceuticals Co, Ltd, Osaka, Japan. Human PTHrP (1-34), human PTHrP (7-34), and human PTH (1-34) were obtained from Peptide Institute. PKA inhibitor (H89) and PKC inhibitors (calphostin C and chelerythrine chloride) were purchased from Seikagaku Co and LC Laboratories, respectively. Human PTHrP cDNA probe containing a 451–base pair fragment [corresponding to -5 to 446 in its full-length cDNA²⁰ ] was a kind gift of Dr M. Hino (Department of Clinical Hematology, Osaka City University Medical School, Osaka, Japan). Unless otherwise mentioned, all other reagents were obtained from Wako Pure Chemical Industries, Ltd.

Cell Culture and In Vitro Calcification
Bovine vascular smooth muscle cells were obtained by an explant method as previously described.¹⁷ Briefly, medial tissue was separated from segments of bovine aorta. Small pieces of tissue (1 to 2 mm³) were placed in a 10-cm culture dish and cultured for several weeks in Dulbecco's modified Eagle's medium (DMEM) (high glucose, 4.5 g/L of glucose) containing 15% FBS and 10 mmol/L sodium pyruvate supplemented with 100 U/mL of penicillin and 100 µg/mL of streptomycin (growing medium) at 37°C in a humidified atmosphere containing 5% CO₂. Cells that had migrated from the explants were collected and maintained in the growing medium. The cells up to passage 8 were used for the experiments. BVSMC calcification was induced as previously described.¹⁷ Briefly, BVSMC were cultured in the growing medium. After confluence, the cells were inoculated in DMEM (high glucose, 4.5 g/L) containing 15% FBS and 10 mmol/L sodium pyruvate in the presence of 10 mmol/L ß-glycerophosphate supplemented with 100 U/mL of penicillin and 100 µg/mL of streptomycin (calcification medium) for 10 days. The medium was replaced with fresh medium every 2 days. In the time-course experiments, the beginning day of culture in calcification medium was defined as day 0.

Quantification of Calcium Deposition
The cells were decalcified with 0.6N HCl for 24 hours. The calcium content of HCl supernatant was determined colorimetrically by o-cresolphthalein complexone method (Calcium C-test Wako; Wako Pure Chemical Industries).⁴¹ After decalcification, the cells were washed three times with phosphate-buffered saline (PBS) and solubilized with 0.1N NaOH/0.1% sodium dodecyl sulfate (SDS). The protein content was measured with a BCA protein assay kit (Pierce). The calcium content of cell layer was normalized by protein content.

Measurement of PTHrP
The secretion of PTHrP by BVSMC was assessed by measuring PTHrP content of the culture supernatant with a immunoradiometric assay kit (PTHrP IRMA kit, Mitsubishi Kagaku). The supernatant was collected in the presence of 10 µg/mL of aprotinin and 1 mmol/L EDTA after the fresh medium containing 15% FBS was incubated for 48 hours with BVSMC in a 6-well plate. The content of PTHrP in the medium containing 15% FBS incubated for 48 hours without the cells was estimated as the background. The PTHrP level of the background was 1.38±0.04 [(fmol/well), mean±SEM (n=21)]. The net quantity of PTHrP secreted from BVSMC was estimated by subtracting the PTHrP content of the background from that in the cell culture supernatant. Finally, the data were normalized by the protein content of the cell layer.

Alkaline Phosphatase Assay
After the cells were washed twice with PBS, the cellular proteins were solubilized with 1% Triton X-100 in 0.9% NaCl and centrifuged, and the supernatants were assayed for ALP activity as described previously.⁴² One unit was defined as the activity producing 1 nmol of p-nitrophenol for 30 minutes. Protein concentrations were determined with a BCA protein assay kit (Pierce).

RNA Isolation and Northern Blot Analysis
Total RNA was isolated from BVSMC by extraction with acid guanidinium thiocyanate-phenol-chloroform. Twenty micrograms of total RNA were electrophoresed on 1% agarose gels containing formaldehyde and transferred to a nylon filter (Hybond N, Amersham). Blots were prehybridized at 37°C for 24 hours in a buffer containing 50% formamide, 3x SSC (1x SSC; 0.15 mol/L NaCl and 15 mmol/L sodium citrate, pH 7.4), 50 mmol/L Tris-HCl (pH 7.5), 0.1% SDS, 20 µg/mL denatured salmon sperm DNA, and 1x Denhardt's solution and then hybridized at 37°C for 48 hours with cDNA probe for human PTHrP which was labeled with [-³²P]dCTP (3000 Ci/mL; New England Nuclear) by use of a random priming method (Megaprime cDNA labeling system, Amersham). Blots were washed and autoradiographed with x-ray film at -70°C. The amounts of RNA were quantified by densitometric scanning and normalized by comparison with glyceraldehyde-3-phosphate dehydrogenase (GAPDH).⁴³

Statistics
In certain experiments, data were analyzed for statistical significance by ANOVA with post hoc analysis unless otherwise stated. These analyses were performed with the assistance of a computer program (StatView version 4.11, Abacus Concepts).

	Results

Top Abstract Introduction Methods Results Discussion References

 
PTHrP Expression in the Development of Calcification
We first examined PTHrP production by BVSMC in the development of calcification. In the presence of ß-glycerophosphate, calcium deposition dramatically increased in a time-dependent manner (on day 9, calcified BVSMC versus uncalcified control: 682.4±23.4 versus 7.9±2.8 µg/mg protein, mean±SEM, (n=3) (Fig 1A). Secretion of PTHrP decreased as early as 2 days in calcified BVSMC and reduced to 6.5% of the uncalcified control on day 10 (0.43±0.08 versus 6.59±0.66 fmol/mg protein, mean±SEM, n=3) (Fig 1B). These results suggest that PTHrP secretion decreases in the progression of BVSMC calcification. We next examined the expression of PTHrP mRNA in BVSMC during this calcification process by Northern blot analysis. A 1.4-kb PTHrP mRNA was detected in both calcified and uncalcified BVSMC. The expression of PTHrP mRNA clearly decreased late in the calcification process and resulted in 38% of the uncalcified control on day 8 (Fig 2). These results suggest that its gene expression also decreases in the progression of calcification. Since EHDP (a bisphosphonate) and levamisole (a specific inhibitor of ALP), as previously reported, inhibited BVSMC calcification in a dose-dependent manner,¹⁷ we utilized these reagents to examine whether inhibition of calcification may affect PTHrP production. EHDP dose-dependently increased PTHrP secretion compared with the calcified control and even more than the uncalcified control (Fig 3A). Levamisole partially increased PTHrP production as compared with that in calcified control (Fig 3B). These results suggest that calcification may affect PTHrP expression in BVSMC.

View larger version (16K): [in this window] [in a new window]   Figure 1. Calcium deposition (A) and PTHrP secretion (B) during BVSMC calcification. The cells were cultured as described in "Methods." ß-GP (+) and (-) indicate the presence and absence of ß-glycerophosphate, respectively. Calcified group is indicated by closed circle and uncalcified control is indicated by open circle. A, The calcium contents were measured at the indicated times by o-cresolphthalein complexone method, normalized by cellular protein content, and are presented as mean±SEM. The differences compared with uncalcified control at each time point were statistically significant (*P<.05, Scheffè's test). B, The contents of PTHrP of the BVSMC culture supernatants were measured at the indicated times by immunoradiometric assay, corrected, and normalized as described in "Methods." The data are presented as mean±SEM. The differences compared with uncalcified control at each time point were statistically significant (*P<.05, Scheffè's test).

View larger version (34K): [in this window] [in a new window]   Figure 2. Northern blot analysis of PTHrP during BVSMC calcification. The cells were cultured as described in "Methods." Twenty micrograms of total RNA from BVSMC at the indicated times were electrophoresed, blotted, and probed with cDNA of human PTHrP. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ß-GP (+) and (-) indicate the presence and absence of ß-glycerophosphate, respectively. Calcified culture is indicated by closed circle and uncalcified control is indicated by open circle. Upper panel, Autoradiograph of Northern analysis of PTHrP. Lower panel, Densitometric analysis of the autoradiograms was performed and the results are presented as the ratio of PTHrP to GAPDH. The Northern blot illustrated is representative of two independent experiments.

View larger version (16K): [in this window] [in a new window]   Figure 3. The effects of etidronate (EHDP) (A) and levamisole (B) on PTHrP secretion by BVSMC. The cells were cultured in calcification medium (see "Methods") for 48 hours in the presence of the indicated dose of EHDP and levamisole and then PTHrP assay was initiated as described in "Methods." After 48-hour culture in the presence of the indicated dose of the reagent, PTHrP contents were measured by immunoradiometric assay, corrected, and normalized. The data are presented as mean±SEM. The differences compared with calcified control [ß-GP (+)] were statistically significant (*P<.05, Scheffè's test). ß-GP (+) and (-) indicate the presence and absence of ßglycerophosphate, respectively.

Effect of Exogenous PTHrP on BVSMC Calcification
Since PTHrP expression may be regulated by calcification, we next examined the effect of human PTHrP (1-34) on BVSMC calcification. Human PTHrP (1-34) inhibited this calcification in a dose-dependent manner and the calcium content decreased to 49% of the calcified BVSMC at 10^-7 mol/L (114.7±5.2 versus 233.7±16.5 µg/mg protein, mean±SEM, n=3) (Fig 4), suggesting that the amino-terminal region of PTHrP has the activity to inhibit calcification. Since PTHrP exerts its effect through a common receptor for PTH and PTHrP, we examined the effect of human PTH (1-34) on calcification. Human PTH (1-34) also inhibited BVSMC calcification in a dose-dependent manner and the maximal inhibitory effect (67% of the control culture) was observed at 10^-7 mol/L (157.3±6.1 versus 233.7±16.5 µg/mg protein, mean±SEM, n=3) (Fig 4). These results suggest that exogenous PTHrP inhibits calcification through a common receptor for PTH/PTHrP.

View larger version (22K): [in this window] [in a new window]   Figure 4. The effect of human PTHrP (1-34) and human PTH (1-34) on BVSMC calcification. The cells were cultured in calcification medium for 72 hours in the presence of the indicated dose of human PTHrP (1-34) or human PTH (1-34). The calcium contents were measured by o-cresolphthalein complexone method, normalized by cellular protein content, and are presented as mean±SEM. The differences compared with calcified control (CTL) were statistically significant (*P<.05, Scheffè's test).

Since protein kinase A and protein kinase C are involved in the PTH/PTHrP receptor signaling pathway, we examined whether PKA inhibitor (H89) and PKC inhibitors (calphostin C and chelerythrine chloride) can affect the inhibitory effect of PTHrP on BVSMC calcification. To confirm the specificity of PTHrP effect, we used a PTHrP antagonist, human PTHrP (7-34). Human PTHrP (7-34) (10^-7 mol/L) completely antagonized the effect of human PTHrP (1-34) (10^-7 mol/L) in a stoichiometric manner (Fig 5), suggesting that the inhibitory effect is specific for PTHrP. H89 (0.1 µmol/L), calphostin C (50 nmol/L), and chelerythrine chloride (1 µmol/L) also completely blocked the inhibitory effect of PTHrP (Fig 5). Higher doses of these reagents increased calcification even more than the calcified control (Fig 5). These data suggest that PTHrP exerts its inhibitory effect through activating both PKA and PKC pathways. Since ALP plays an important role in BVSMC calcification, as previously reported,¹⁷ we examined the effect of PTHrP on ALP activity in BVSMC. Human PTHrP (1-34) depressed ALP activity ranging from 10^-9 to 10^-7 mol/L and at 10^-7 mol/L ALP activity decreased to 74% of the control culture (1221±95 versus 1647±77 U/mg protein, mean±SEM, n=3) (Fig 6), suggesting that the inhibitory effect of PTHrP on calcification may be due to decreased ALP activity.

View larger version (20K): [in this window] [in a new window]   Figure 5. Antagonism of PKA and PKC inhibitors against PTHrP-induced inhibition of BVSMC calcification. The cells were cultured in calcification medium for 72 hours in the presence of the indicated reagents, and then calcification assay was initiated as described in "Methods." The calcium contents were measured by o-cresolphthalein complexone method, normalized by cellular protein content, and are presented as mean±SEM. The differences compared with calcified control were statistically significant (*P<.05, Scheffè's test). ß-GP, ß-glycerophosphate. The (+) and (-) indicate the presence and absence of the corresponding reagent, respectively.

View larger version (15K): [in this window] [in a new window]   Figure 6. The effect of human PTHrP (1-34) on ALP activity. The cells were cultured in calcification medium for 72 hours in the presence of the indicated dose of human PTHrP (1-34). ALP activities were measured, normalized by cellular protein content, and are presented as mean±SEM. The differences compared with control (CTL) were statistically significant (*P<.05, Scheffè's test).

Endogenous PTHrP May Modulate BVSMC Calcification
As we demonstrated that exogenous PTHrP inhibits BVSMC calcification, we next investigated the role of endogenous PTHrP in this calcification. Human PTHrP (7-34) dose-dependently increased BVSMC calcification and its stimulatory effect plateaued at 10^-8 mol/L (Fig 7). Moreover, the calcium content increased to 169% of the control at 10^-7 mol/L (189.1±29.0 versus 111.7±6.7 µg/mg protein, mean±SEM, n=3). These results suggest that endogenous PTHrP may prevent calcification. From a stoichiometric point of view, the level of biologically potent PTHrP secreted from calcified BVSMC was estimated to be 10^-8 mol/L. To examine the involvement of PKA and PKC pathways in the effect of endogenous PTHrP, we examined the effects of PKA and PKC inhibitors on calcification. H89 and chelerythrine chloride dose-dependently increased calcium deposition and the maximal increases were 194% (0.5 µmol/L) and 200% (5 µmol/L), respectively (Table). Calphostin C significantly increased calcification ranging from 50 to 150 nmol/L, and at 150 nmol/L the calcium content increased to 179% of the control (Table). In the absence of ß-glycerophosphate, however, these inhibitors themselves did not significantly increase calcium content compared with the control (control versus H89 0.5 µmol/L versus Calphostin C 150 µmol/L versus Chelerythrine chloride 5.0 µmol/L: 8.40±3.31 versus 23.18±14.433 versus 12.22±2.47 versus 14.78±3.31 µg/mg protein, mean±SEM, n=3, respectively). Although these actions of PKA and PKC inhibitors were not specific for inhibiting PTHrP signaling pathway, these data support the possible involvement of both PKA and PKC pathways in the action of endogenous PTHrP.

View larger version (17K): [in this window] [in a new window]   Figure 7. The effect of human PTHrP (7-34) on BVSMC calcification. The cells were cultured in calcification medium for 72 hours in the presence of the indicated dose of human PTHrP (7-34). The calcium contents of cell layer were measured by o-cresolphthalein complexone method, normalized by cellular protein content, and are presented as mean±SEM. The differences compared with calcified control (CTL) were statistically significant (*P<.05, Scheffè's test).

View this table: [in this window] [in a new window]   Table 1. Effects of PKA and PKC Inhibitors on BVSMC Calcification

Interestingly, PTHrP secretion by BVSMC dramatically increased in the presence of 100 µg/mL of EHDP, and on day 8 the level of PTHrP in the EHDP-treated group increased to 231% of the calcified control (12.03±0.40 versus 5.20±0.68 fmol/mg protein, mean±SEM, n=3) (Fig 8). Additionally, we confirmed that EHDP dose-dependently increased PTHrP production ranging from 10 to 100 µg/mL in the absence of ß-glycerophosphate (CTL versus EHDP 100 µg/mL: 5.34±0.57 versus 7.98±0.77 fmol/mg protein, mean±SEM, n=3, respectively). Therefore, we examined whether human PTHrP (7-34) can antagonize the inhibitory effect of EHDP on calcification. As the half-maximal effective dose (ED50) and the maximal effective dose were, from the results of Fig 3A, estimated to be 10 µg/mL and 50 µg/mL, respectively, we used these two doses for the experiments. Human PTHrP (7-34) dose-dependently blocked the effect of 10 µg/mL EHDP and at 10^-7 mol/L EHDP effect was completely inhibited (Fig 9). The effect of 50 µg/mL EHDP was not neutralized significantly by PTHrP (7-34), although it appeared to have some effect (Fig 9). These results further support that endogenous PTHrP act as a local regulator of calcification.

View larger version (18K): [in this window] [in a new window]   Figure 8. The effect of EHDP on PTHrP secretion during BVSMC calcification. The cells were cultured as described in "Methods." PTHrP contents were measured by immunoradiometric assay, normalized by cellular protein content, and are presented as mean±SEM. The differences compared with calcified control [ß-GP (+)] at each time point were statistically significant (*P<.05, Scheffè's test). ß-GP, ß-glycerophosphate; EHDP, etidronate. The (+) and (-) indicate the presence and absence of the reagent, respectively.

View larger version (27K): [in this window] [in a new window]   Figure 9. The effect of human PTHrP (7-34) on EHDP-inhibited BVSMC calcification. The cells were cultured in calcification medium for 72 hours in the presence of the indicated reagent(s). The calcium contents were measured by o-cresolphthalein complexone method, normalized by cellular protein content, and are presented as mean±SEM. The differences compared with the corresponding EHDP-treated control were statistically significant (*P<.05, Scheffè's test). ß-GP, ß-glycerophosphate. The (+) and (-) indicate the presence and absence of the indicated reagent, respectively.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In this study, we have demonstrated that PTHrP expression in BVSMC decreases in the progression of calcification (Figs 1 and 2) and that inhibition of calcification by either EHDP or levamisole increases PTHrP secretion (Fig 3A and 3B), suggesting that calcification may directly modulate PTHrP expression in vascular smooth muscle. The expression of PTHrP in VSMC is induced by vasoactive peptides, serum, and mechanical stretch, and the levels of its expression are correlated with growth capacity of VSMC.^{37 39 40} In coronary atherosclerotic lesions, PTHrP was overexpressed and its expression was closely related to the severity of coronary atherosclerosis.⁴⁴ However, in calcified atherosclerotic lesions PTHrP expression was significantly lower than that of uncalcified lesions.⁴⁴ In this study, therefore, we have demonstrated in vitro this relationship between PTHrP expression and calcification that had been observed in vivo. However, the mechanism of regulation of PTHrP expression during BVSMC calcification remains to be elucidated.

There is a time lag of PTHrP down-regulation in the calcification process between its secretion and gene expression. In the development of BVSMC calcification, PTHrP secretion decreased as early as 2 days, while its gene expression reduced late in the process (on day 6) (Fig 1 and Fig 2, respectively). These observations suggest that the regulatory mechanisms between PTHrP secretion and its gene expression may be different. In fact, PTHrP secretion by rat Leydig tumor cells is regulated by extracellular calcium concentration at a post-transcriptional level.⁴⁵ Therefore, it is likely that concentrations of extracellular calcium and phosphate may change in the presence of ß-glycerophosphate (under the calcifying condition), which contributes to the regulation of PTHrP secretion by BVSMC.

We have clearly demonstrated that exogenous PTHrP (1-34) and PTH (1-34) inhibit BVSMC calcification in a dose-dependent manner (Fig 4). Although there is some controversy regarding the actions of PTH and PTHrP on osteoblastic cells, they appear to inhibit various osteoblastic functions such as collagen synthesis,^{46 47} ALP activity,^{48 49} and bone nodule formation.⁵⁰ Moreover, in vivo studies using mice lacking both copies of the PTHrP gene suggest that PTHrP through activating PTH/PTHrP receptor is involved in retarding calcification of cartilage.^{51 52}

As we demonstrated in Fig 7, addition of PTHrP antagonist increased BVSMC calcification in a dose-dependent manner. Furthermore, we also revealed that PTHrP antagonist partially blocked the inhibitory effect of EHDP action on BVSMC calcification (Fig 9). These results suggest that PTHrP plays an inhibitory role in this calcification in an autocrine/paracrine manner. However, in order to directly prove the role of endogenous PTHrP, it is necessary to examine the effect of antisense oligonucleotides or overexpression of antisense RNA to inhibit endogenous PTHrP production on BVSMC calcification.⁵³

PTH/PTHrP receptor is a G protein–linked receptor that has seven transmembrane-spanning domains.²¹ The activated PTH/PTHrP receptor activates PKA and PKC signaling pathways.⁵⁴ Both PKA and PKC pathways are involved in the various biological effects of PTH/PTHrP. To clarify which pathway is involved in the inhibitory effect of PTHrP on BVSMC calcification, we examined the effects of PKA and PKC inhibitors on the action of exogenous PTHrP. As we demonstrated, both PKA and PKC inhibitors completely blocked the action of exogenous PTHrP, suggesting that both PKA and PKC pathways are activated in the signaling of the inhibitory action on calcification (Fig 5). Furthermore, as shown in the Table, both PKA and PKC inhibitors themselves increased calcium content of the cell layer. Although these actions are not specific for PTHrP signaling in this case, it is likely that some endogenous inhibitory factors including PTHrP may exert their effects through activating both PKA and PKC pathways. Another possibility is that activation of PKA and PKC may inhibit BVSMC calcification through inducing the expression of endogenous PTHrP.^{39 55} Therefore, the roles of PKA and PKC in vascular calcification should be clarified.

Finally, it is important to consider whether all BVSMC from the aortic media have the capacity to calcify their extracellular matrix, since Watson et al⁵⁶ reported that only a subpopulation of BVSMC undergoes in vitro calcification. We have recently examined the levels of ALP activity of BVSMC clones with cytochemical staining. Among 398 BVSMC clones, 204 clones (51%) were positive for ALP, suggesting that not all BVSMC can undergo in vitro calcification in the presence of ß-glycerophosphate (unpublished data). However, whether these BVSMC clones have the phenotypic features of pericytes, so-called calcifying vascular cells⁵⁶ remains to be clarified.

	Selected Abbreviations and Acronyms

 

ALP = alkaline phosphatase BVSMC = bovine vascular smooth muscle cell EHDP = etidronate PKA = protein kinase A PKC = protein kinase C PTH = parathyroid hormone PTHrP = PTH-related peptide

	Acknowledgments

 
The authors thank Dr M. Hino and Sumitomo Pharmaceuticals Co, Ltd, for kindly providing human PTHrP cDNA probe and etidronate (EHDP), respectively.

Received August 15, 1996; accepted October 11, 1996.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Blumenthal HT, Lansing AI, Wheeler PA. Calcification of the media of the human aorta and its relationship to intimal arteriosclerosis, aging and disease. Am J Pathol. 1944;20:665-687.

2. Frink RJ, Achor RWP, Brown JAL, Kincaid OW, Brandenburg RO. Significance of calcification of the coronary arteries. Am J Cardiol. 1970;26:241-247.[Medline] [Order article via Infotrieve]

3. McCarthy J, Palmer F. Incidence and significance of coronary artery calcification. Br Heart J. 1974;36:499-506.[Free Full Text]

4. Locker TH, Schwartz RS, Cotta CW, Hickman JR. Fluoroscopic coronary artery calcification and associated coronary disease in asymptomatic young men. J Am Coll Cardiol. 1992; 19:1167-1172.

5. Fitzgerald PJ, Ports TA, Yock PG. Contribution of localized calcium deposits to dissection after angioplasty: an observational study using intravascular ultrasound. Circulation. 1992;86:64-70.[Abstract/Free Full Text]

6. Rumberger JA, Schwartz RS, Simons DB, Sheedy PF, Edwards WD, Fitzpatrick LA. Relation of coronary calcium determined by electron beam computed tomography and lumen narrowing determined by autopsy. Am J Cardiol. 1994;93:1169-1173.

7. Yu SY. Calcification processes in atherosclerosis. Adv Exp Med Biol. 1974;43:403-425.[Medline] [Order article via Infotrieve]

8. Moon JY. Factors affecting arterial calcification associated with atherosclerosis. Atherosclerosis. 1972;16:119-126.[Medline] [Order article via Infotrieve]

9. Kim KM. Calcification of matrix vesicles in human aortic valve and aortic media. Fed Proc. 1976;35:156-162.[Medline] [Order article via Infotrieve]

10. Tanimura A, McGregor DH, Anderson HC. Calcification in atherosclerosis, I: human studies. J Exp Pathol. 1986;2:261-272.[Medline] [Order article via Infotrieve]

11. Boström K, Watson KE, Horn S, Wortham C, Herman IM, Demer LL. Bone morphogenetic protein expression in human atherosclerotic lesions. J Clin Invest. 1993;91:1800-1809.

12. Shanahan CM, Cary NRB, Metcalfe JC, Weissberg PL. High expression of genes for calcification-regulating proteins in human atherosclerotic plaques. J Clin Invest. 1994;93:2393-2402.

13. Giachelli CM, Bae N, Almeida M, Denhardt DT, Alpers CE, Schwartz SM. Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques. J Clin Invest. 1993;92:1686-1696.

14. Hirota S, Imakita M, Kohri K, Ito A, Morii E, Adachi S, Kim H-M, Kitamura Y, Yutani C, Nomura S. Expression of osteopontin messenger RNA by macrophages in atherosclerotic plaques: a possible association with calcification. Am J Pathol. 1993;143:1003-1008.[Abstract]

15. Fitzpatrick LA, Severson A, Edwards WD, Ingram RT. Diffuse calcification in human coronary arteries: association of osteopontin with atherosclerosis. J Clin Invest. 1994;94:1597-1604.

16. Doherty TM, Detrano RC. Coronary arterial calcification as an active process: a new perspective on an old problem. Calcif Tissue Int. 1994;54:224-230.[Medline] [Order article via Infotrieve]

17. Shioi A, Nishizawa Y, Jono S, Koyama H, Hosoi M, Morii H. ß-Glycerophosphate accelerates calcification in cultured bovine vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 1995;15: 2003-2009.

18. Burtis WJ, Wu T, Bunch C, Wysolmerski JJ, Isogna KL, Weir EC, Broadus AE, Stewart AF. Identification of a novel 17 000-dalton parathyroid hormone-like adenylate cyclase-stimulating protein from a tumor associated with humoral hypercalcemia of malignancy. J Biol Chem. 1987;161:7151-7156.

19. Strewler GJ, Stern PH, Jacob JW, Eveloff J, Klein RF, Leung SC, Rosenblatt M, Nissenson RA. Parathyroid hormonelike protein from human renal carcinoma cells: structural and functional homology with parathyroid hormone. J Clin Invest. 1987;80:1803-1807.

20. Suva LJ, Winslow GA, Wettenhall REH, Hammonds RG, Moseley JM, Diefenbach-Jaggar H, Rodda CP, Kemp BE, Rodriguez H, Chen EY, Hudson PJ, Martin TJ, Wood WI. A parathyroid hormone-related protein implicated in malignant hypercalcemia: cloning and expression. Science. 1987;237:893-896.[Abstract/Free Full Text]

21. Jüppner H, Abou-Samra A-B, Freeman M, Kong XF, Schipani E, Richards J, Kolakowski LF Jr, Hock J, Potts JT Jr, Kronenberg HM, Segre GV. A G protein-linked receptor for parathyroid hormone and parathyroid hormone-related peptide. Science. 1991; 254:1024-1026.

22. Kemp BE, Moseley JM, Rodda CP, Ebeling PR, Wettenhall REH, Stapleton D, Diefenbach-Jaggar H, Ure F, Michelangeli VP, Simmons HA, Raisz LG, Martin TJ. Parathyroid hormone-related protein of malignancy: active synthetic fragments. Science. 1987;238: 1568-1570.

23. Rabbani SA, Mitchell J, Roy DR, Hendy GN, Goltzman D. Influence of the amino-terminus on in vitro and in vivo biological activity of synthetic parathyroid hormone-like peptides of malignancy. Endo-crinology. 1988;123:2709-2716.[Abstract/Free Full Text]

24. Rodan SB, Isogna KL, Vignery AMG, Stewart AF, Broadus AE, D'souza SM, Bertolini DR. Factors associated with humoral hypercalcemia of malignancy stimulate adenylate cyclase in osteoclastic cells. J Clin Invest. 1983;72:1511-1515.

25. Botella A, Rekik M, Delvaux M, Davicco MJ, Barlet JP, Frexinos J, Bueno L. Parathyroid hormone (PTH) and PTH-related peptide induce relaxation of smooth muscle cells from guinea pig ileum: interaction with vasoactive intestinal peptide receptors. Endocrinology. 1994;135:2160-2167.[Abstract]

26. Caulfield MP, McKee RL, Goldman ME, Duong LT, Fisher JE, Gay CT, DeHaven PA, Levy JJ, Roubini E, Nutt RF. The bovine renal parathyroid hormone (PTH) receptor has equal affinity for two different amino acid sequences: the receptor binding domains of PTH and PTH-related protein are located within the 14-34 region. Endo-crinology. 1990;127:83-87.[Abstract/Free Full Text]

27. Merendino JJ Jr, Isogna KL, Milstone LM, Broadus AE, Stewart AF. A parathyroid hormone-like protein from cultured human keratinocytes. Science. 1986;231:388-390.[Abstract/Free Full Text]

28. Thiede MA, Rodan GA. Expression of a calcium-mobilizing parathyroid hormone-like peptide in lactating mammary tissue. Science. 1988;242:278-280.[Abstract/Free Full Text]

29. Kramer S, Reynolds FH Jr, Castillo M, Valenzuela DM, Thorikay M, Sorvillo JM. Immunological identification and distribution of parathyroid hormone-related protein polypeptides in normal and malignant tissues. Endocrinology. 1991;128:1927-1937.[Abstract/Free Full Text]

30. Senior PV, Heath DA, Beck F. Expression of parathyroid hormone-related protein mRNA in the rat before birth: demonstration by hybridization histochemistry. J Mol Endocrinol. 1991;6: 281-290.

31. Moseley JM, Hayman JA, Danks JA, Alcorn D, Grill V, Southby J, Horton MA. Immunohistochemical detection of parathyroid hormone-related protein in human fetal epithelia. J Clin Endocrinol Metab. 1991;73:478-484.[Abstract/Free Full Text]

32. Broadus AE, Stewart AF. Parathyroid hormone-related protein: structure, processing, and physiological actions. In: Bilezikian JP, Levine MA, Marcus R, eds. The Parathyroids. New York, NY: Raven Press; 1994:259-294.

33. Roca-Cusachs A, DiPette DJ, Nickols GA. Regional and systemic hemodynamic effects of parathyroid hormone-related protein: preservation of cardiac function and coronary and renal flow with reduced blood pressure. J Pharmacol Exp Ther. 1991;256:110-118.[Abstract/Free Full Text]

34. DiPette DJ, Christenson W, Nickols MA, Nickols GA. Cardiovascular responsiveness to parathyroid hormone (PTH) and PTH-related protein in genetic hypertension. Endocrinology. 1992;130: 2045-2051.

35. Nickols GA, Nana AD, Nickols MA, DiPette DJ, Asimakis GK. Hypotension and cardiac stimulation due to the parathyroid hormone-related protein, humoral hypercalcemia of malignancy factor. Endocrinology. 1989;125:834-841.[Abstract/Free Full Text]

36. Winquist RJ, Baskin EP, Vlasuk GP. Synthetic tumor-derived human hypercalcemic factor exhibits parathyroid hormone-like vasorelaxation in renal arteries. Biochem Biophys Res Commun. 1987;149: 227-232.

37. Hongo T, Kupfer J, Enomoto H, Sharifi B, Giannella-Neto D, Forrester JS, Singer FR, Goltzman D, Hendy GN, Pirola C. Abundant expression of parathyroid hormone-related protein in primary rat aortic smooth muscle cells accompanies serum-induced proliferation. J Clin Invest. 1991;88:1841-1847.

38. Burton DW, Brandt DW, Deftos LJ. Parathyroid hormone-related protein in the cardiovascular system. Endocrinology. 1994;135: 253-261.

39. Noda M, Katoh T, Takuwa N, Kumada M, Kurokawa K, Takuwa Y. Synergistic stimulation of parathyroid hormone-related peptide gene expression by mechanical stretch and angiotensin II in rat aortic smooth muscle cells. J Biol Chem. 1994;269:17911-17917.[Abstract/Free Full Text]

40. Pirola CJ, Wang HM, Kamyar A, Wu S, Enomoto H, Sharifi B, Forrester JS, Clemens TL, Fagin JA. Angiotensin II regulates parathyroid hormone-related protein expression in cultured rat smooth muscle cells through transcriptional and post-transcriptional mechanisms. J Biol Chem. 1993;268:1987-1994.[Abstract/Free Full Text]

41. Gitelman HJ. A modified uronic acid carbazole reaction. Anal Biochem. 1967;4:521-531.

42. Bessey OA, Lowry OH, Brock MJ. Method for rapid determination of alkaline phosphatase with 5 cubic millimeters of serum. J Biol Chem. 1946;164:321-329.[Free Full Text]

43. Fort P, Piechaczyk M, Sabrouty SE, Dani C, Jeanteur P, Blanchard JM. Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate-dehydrogenase multigenic family. Nucleic Acids Res. 1985;13:1431-1442.[Abstract/Free Full Text]

44. Nakayama T, Ohtsuru A, Enomoto H, Namba H, Ozeki S, Shibata Y, Yokota T, Nobuyoshi M, Ito M, Sekine I, Yamashita S. Coronary atherosclerotic smooth muscle cells overexpress human parathyroid hormone-related peptides. Biochem Biophys Res Commun. 1994;200: 1028-1035.

45. Rizzoli R, Bonjour J-P. High extracellular calcium increases the production of a parathyroid hormone-like activity by cultured Leydig tumor cells associated with humoral hypercalcemia. J Bone Miner Res. 1989;4:839-844.[Medline] [Order article via Infotrieve]

46. Kano J, Sugimoto T, Fukase M, Chihara K. The direct involvement of cAMP-dependent protein kinase in the regulation of collagen synthesis by parathyroid hormone (PTH) and PTH-related peptide in osteoblast-like osteosarcoma cells. Biochem Biophys Res Commun. 1992;184:525-529.[Medline] [Order article via Infotrieve]

47. Kream BE, Rowe DW, Givorek SC, Raisz LR. Parathyroid hormone alters collagen synthesis and procollagen mRNA levels in fetal rat calvaria. Proc Natl Acad Sci USA. 1980;77:5654-5658.[Abstract/Free Full Text]

48. Majeska RJ, Rodan GA. Alkaline phosphatase inhibition by parathyroid hormone and isoproterenol in a clonal rat osteosarcoma cell line. Possible mediation by cAMP. Calcif Tissue Int. 1982; 34:59-66.

49. Jongen JWJM, Bos MP, van der Meer JM, Herrmann-Erlee MPM. Parathyroid hormone-induced changes in alkaline phosphatase expression in fetal calvarial osteoblasts: differences between rat and mouse. J Cell Physiol. 1993;155:36-43.[Medline] [Order article via Infotrieve]

50. Bellows CG, Ishida H, Aubin JE, Heersche JNM. Parathyroid hormone reversibly suppresses the differentiation of osteoprogenitor cells into functional osteoblasts. Endocrinology. 1990;127: 3111-3116.

51. Amizuka N, Warshawsky H, Henderson JE, Goltzman D, Karaplis AC. Parathyroid hormone-related peptide-depleted mice show abnormal epiphyseal cartilage development and altered endochondral bone formation. J Cell Biol. 1994;126:1611-1623.[Abstract/Free Full Text]

52. Karaplis AC, Luz A, Glowacki J, Bronson RT, Tybulewicz VLJ, Kronenberg HM, Mulligan RC. Lethal skeletal dysplasia from targeted disruption of the parathyroid hormone-related peptide gene. Genes Dev. 1994;8:277-289.[Abstract/Free Full Text]

53. Kaiser SM, Laneuville P, Bernier SM, Rhim JS, Kremer R, Goltzman D. Enhanced growth of a human keratinocyte cell line induced by antisense RNA for parathyroid hormone-related peptide. J Biol Chem. 1992;267:13623-13628.[Abstract/Free Full Text]

54. Bringhurst FR, Jüppner H, Guo J, Urena JP, Potts JJ, Kronenberg HM, Abou SA, Segre GV. Cloned, stably expressed parathyroid hormone (PTH)/PTH-related peptide receptors activate multiple messenger signals and biological responses in LLC-PK1 kidney cells. Endocrinology. 1993;132:2090-2098.[Abstract/Free Full Text]

55. Ikeda K, Okazaki R, Inoue D, Ogata E, Matsumoto T. Transcription of the gene for parathyroid hormone-related peptide from the human is activated through a cAMP-dependent pathway by prostaglandin E1 in HTLV-1-infected T cells. J Biol Chem. 1993;268:1174-1179.[Abstract/Free Full Text]

56. Watson KE, Boström K, Ravindranath R, Lam T, Norton B, Demer LL. TGF-b1 and 25-hydroxycholesterol stimulate osteoblast-like vascular cells to calcify. J Clin Invest. 1994;93: 2106-2113.

This article has been cited by other articles:

				F. G. Graciolli, K. R. Neves, L. M. dos Reis, R. G. Graciolli, I. L. Noronha, R. M. A. Moyses, and V. Jorgetti Phosphorus overload and PTH induce aortic expression of Runx2 in experimental uraemia Nephrol. Dial. Transplant., May 1, 2009; 24(5): 1416 - 1421. [Abstract] [Full Text] [PDF]

				S. C. Palmer, J. C. Craig, and G. F. M. Strippoli Taking aim at targets Nephrol. Dial. Transplant., May 1, 2009; 24(5): 1358 - 1361. [Full Text] [PDF]

				R. Shroff, M. Egerton, M. Bridel, V. Shah, A. E. Donald, T. J. Cole, M. P. Hiorns, J. E. Deanfield, and L. Rees A Bimodal Association of Vitamin D Levels and Vascular Disease in Children on Dialysis J. Am. Soc. Nephrol., June 1, 2008; 19(6): 1239 - 1246. [Abstract] [Full Text] [PDF]

				G. Elder and K. S. Kumar Calciphylaxis associated with chronic kidney disease and low bone turnover: management with recombinant human PTH-(1-34) NDT Plus, April 1, 2008; 1(2): 97 - 99. [Full Text] [PDF]

				Y. Orita, H. Yamamoto, N. Kohno, M. Sugihara, H. Honda, S. Kawamata, S. Mito, N. N. Soe, and M. Yoshizumi Role of Osteoprotegerin in Arterial Calcification: Development of New Animal Model Arterioscler Thromb Vasc Biol, September 1, 2007; 27(9): 2058 - 2064. [Abstract] [Full Text] [PDF]

				I. Z. Jaffe, Y. Tintut, B. G. Newfell, L. L. Demer, and M. E. Mendelsohn Mineralocorticoid Receptor Activation Promotes Vascular Cell Calcification Arterioscler Thromb Vasc Biol, April 1, 2007; 27(4): 799 - 805. [Abstract] [Full Text] [PDF]

				D. A. Towler Calciotropic Hormones and Arterial Physiology: "D"-lightful Insights J. Am. Soc. Nephrol., February 1, 2007; 18(2): 369 - 373. [Full Text] [PDF]

				M. Ketteler, G. Schlieper, and J. Floege Calcification and Cardiovascular Health: New Insights Into an Old Phenomenon Hypertension, June 1, 2006; 47(6): 1027 - 1034. [Full Text] [PDF]

				Y. Sato, R. Nakamura, M. Satoh, K. Fujishita, S. Mori, S. Ishida, T. Yamaguchi, K. Inoue, T. Nagao, and Y. Ohno Thyroid Hormone Targets Matrix Gla Protein Gene Associated With Vascular Smooth Muscle Calcification Circ. Res., September 16, 2005; 97(6): 550 - 557. [Abstract] [Full Text] [PDF]

				K. A. Hruska, S. Mathew, and G. Saab Bone Morphogenetic Proteins in Vascular Calcification Circ. Res., July 22, 2005; 97(2): 105 - 114. [Abstract] [Full Text] [PDF]

				C. Henley, M. Colloton, R. C. Cattley, E. Shatzen, D. A. Towler, D. Lacey, and D. Martin 1,25-Dihydroxyvitamin D3 but not cinacalcet HCl (Sensipar(R)/Mimpara(R)) treatment mediates aortic calcification in a rat model of secondary hyperparathyroidism Nephrol. Dial. Transplant., July 1, 2005; 20(7): 1370 - 1377. [Abstract] [Full Text] [PDF]

				D. Somjen, Y. Weisman, F. Kohen, B. Gayer, R. Limor, O. Sharon, N. Jaccard, E. Knoll, and N. Stern 25-Hydroxyvitamin D3-1{alpha}-Hydroxylase Is Expressed in Human Vascular Smooth Muscle Cells and Is Upregulated by Parathyroid Hormone and Estrogenic Compounds Circulation, April 5, 2005; 111(13): 1666 - 1671. [Abstract] [Full Text] [PDF]

				D. A. Towler Inorganic Pyrophosphate: A Paracrine Regulator of Vascular Calcification and Smooth Muscle Phenotype Arterioscler Thromb Vasc Biol, April 1, 2005; 25(4): 651 - 654. [Full Text] [PDF]

				M. Abedin, Y. Tintut, and L. L. Demer Mesenchymal Stem Cells and the Artery Wall Circ. Res., October 1, 2004; 95(7): 671 - 676. [Abstract] [Full Text] [PDF]

				S. M. Moe and N. X. Chen Pathophysiology of Vascular Calcification in Chronic Kidney Disease Circ. Res., September 17, 2004; 95(6): 560 - 567. [Abstract] [Full Text] [PDF]

				G. M. London, C. Marty, S. J. Marchais, A. P. Guerin, F. Metivier, and M.-C. de Vernejoul Arterial Calcifications and Bone Histomorphometry in End-Stage Renal Disease J. Am. Soc. Nephrol., July 1, 2004; 15(7): 1943 - 1951. [Abstract] [Full Text] [PDF]

				T. M. DOHERTY, H. UZUI, L. A. FITZPATRICK, P. V. TRIPATHI, C. R. DUNSTAN, K. ASOTRA, and T. B. RAJAVASHISTH Rationale for the role of osteoclast-like cells in arterial calcification FASEB J, April 1, 2002; 16(6): 577 - 582. [Abstract] [Full Text] [PDF]

				M. COZZOLINO, A. S. DUSSO, and E. SLATOPOLSKY Role of Calcium-Phosphate Product and Bone-Associated Proteins on Vascular Calcification in Renal Failure J. Am. Soc. Nephrol., November 1, 2001; 12(11): 2511 - 2516. [Full Text] [PDF]

				S. Jono, M. D. McKee, C. E. Murry, A. Shioi, Y. Nishizawa, K. Mori, H. Morii, and C. M. Giachelli Phosphate Regulation of Vascular Smooth Muscle Cell Calcification Circ. Res., September 29, 2000; 87 (7): e10 - e17. [Abstract] [Full Text] [PDF]

				H. Koshiyama, Y. Nakamura, S. Tanaka, and J. Minamikawa Decrease in Carotid Intima-Media Thickness after 1-Year Therapy with Etidronate for Osteopenia Associated with Type 2 Diabetes J. Clin. Endocrinol. Metab., August 1, 2000; 85(8): 2793 - 2796. [Abstract] [Full Text]

				K. Mori, A. Shioi, S. Jono, Y. Nishizawa, and H. Morii Dexamethasone Enhances In Vitro Vascular Calcification by Promoting Osteoblastic Differentiation of Vascular Smooth Muscle Cells Arterioscler Thromb Vasc Biol, September 1, 1999; 19(9): 2112 - 2118. [Abstract] [Full Text] [PDF]

				T. Wada, M. D. McKee, S. Steitz, and C. M. Giachelli Calcification of Vascular Smooth Muscle Cell Cultures : Inhibition by Osteopontin Circ. Res., February 5, 1999; 84(2): 166 - 178. [Abstract] [Full Text] [PDF]

				S. Jono, Y. Nishizawa, A. Shioi, and H. Morii 1,25-Dihydroxyvitamin D3 Increases In Vitro Vascular Calcification by Modulating Secretion of Endogenous Parathyroid Hormone–Related Peptide Circulation, September 29, 1998; 98(13): 1302 - 1306. [Abstract] [Full Text] [PDF]

				S. Jono, C. Peinado, and C. M. Giachelli Phosphorylation of Osteopontin Is Required for Inhibition of Vascular Smooth Muscle Cell Calcification J. Biol. Chem., June 23, 2000; 275(26): 20197 - 20203. [Abstract] [Full Text] [PDF]

This Article Abstract Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jono, S. Articles by Morii, H. Search for Related Content PubMed PubMed Citation Articles by Jono, S. Articles by Morii, H.