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Endocrinology Vol. 139, No. 3 1411-1419
Copyright © 1998 by The Endocrine Society

ARTICLES

The Integrin Ligand Echistatin Prevents Bone Loss in Ovariectomized Mice and Rats

Michiko Yamamoto1,2, John E. Fisher2, Michael Gentile, J. Gregory Seedor, Chih-Tai Leu, Sevgi B. Rodan and Gideon A. Rodan

Department of Bone Biology and Osteoporosis Research, Merck Research Laboratories, West Point, Pennsylvania 19486

Address all correspondence and requests for reprints to: Gideon A. Rodan, M.D., Ph.D., Department of Bone Biology and Osteoporo-sis Research, WP26A-1000, Merck Research Laboratories, West Point, Pennsylvania 19486. E-mail: rodan{at}merck.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Integrins that bind RGD (arginine-glycine-aspartic acid) containing peptides, especially the vitronectin receptor {alpha}vß3, have been implicated in the regulation of osteoclast function. Echistatin, an RGD-containing snake venom peptide with high affinity for ß3 integrins, as well as nonpeptide RGD mimetics, were shown to inhibit osteoclastic bone resorption in vitro and in vivo. To evaluate the role of RGD-binding integrins in bone metabolism, we examined by several methods the effects of echistatin on ovariectomy (OVX)-induced bone loss in mice and rats. First, we confirmed that echistatin binds in vitro with high affinity (Kd, 0.5 nM) to {alpha}vß3 integrin purified from human placenta and established a competitive binding assay to measure echistatin concentrations in serum. We find that echistatin infused for 2 or 4 weeks at 0.36 µg/h·g body weight (~50 nmol/day·mouse) completely prevents OVX-induced cancellous bone loss in the distal femora of ovariectomized mice. Echistatin has no effect on uterine weight, body weight, and femoral length changes induced by OVX, nor does it cause any apparent changes in major organs other than bone. In OVX rats, echistatin infusion at 0.26 µg/h·g for 4 weeks effectively prevents bone loss, evaluated by dual energy x-ray absorptiometry of the femur, by femoral ash weight, and by bone histomorphometry of the proximal tibia. At effective serum concentrations of 20–30 nM, measured at the end of the infusion period, echistatin maintains histomorphometric indices of bone turnover at control levels but does not decrease osteoclast surface. In conclusion, these results provide in vivo evidence, at the level of bone histology, that RGD-binding integrins, probably {alpha}vß3, play a rate-limiting role in osteoclastic bone resorption and suggest a therapeutic potential for integrin ligands in the suppression of bone loss.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BONE loss caused by estrogen deficiency is due, both in humans and experimental animals, primarily to increased osteoclastic bone resorption (1, 2, 3). Osteoclasts are multinucleated cells of hemopoietic origin that resorb bone by attaching to the bone surface via a highly specialized and polarized structure (4, 5). The molecular mechanisms by which osteoclasts attach to bone and initiate resorption are not fully understood. Interaction between osteoclasts and the extracellular matrix via integrins was proposed to play a role in this process (6, 7). Integrins are heterodimeric glycoprotein receptors composed of {alpha}- and ß-subunits that recognize a wide range of cell- and extracellular matrix-associated ligands (8). Many integrins interact with extracellular matrix proteins at sites containing the tripeptide sequence, arginine-glycine-aspartic acid (RGD) (9). This interaction is inhibited competitively by small synthetic peptides containing RGD (10) as well as by a class of RGD-containing peptides from snake venoms called disintegrins (11). Echistatin is a 49-amino acid disintegrin isolated from the venom of the viper Echis carinatus (12).

It is now well established that the {alpha}vß3 integrin is highly expressed in osteoclasts from all species examined (13, 14, 15, 16). Echistatin colocalizes with the integrin {alpha}v-subunit on rat osteoclasts (17) and is more potent than RGD-containing small peptides in inhibiting the attachment and bone resorbing activity of isolated osteoclasts (17, 18). Fisher et al. (19) showed that systemic administration of synthetic echistatin inhibits the acute calcemic response to exogenous PTH in thyroparathyroidectomized (TPTX) rats. Recently, Crippes et al., (20) showed that anti {alpha}vß3 antibodies have similar effects and Engleman et al. (21) showed that RGD nonpeptide mimetics inhibit OVX-induced bone loss. These findings suggest that integrins, especially the vitronectin receptor {alpha}vß3, play a rate-limiting role in osteoclast function (22, 23, 24, 25).

To obtain further evidence at the level of the bone tissue for the role of integrins in bone resorption, we examined the effects of echistatin in OVX mice and rats. We found that echistatin effectively inhibits bone resorption by reducing bone turnover in OVX rodents.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Echistatin was either chemically synthesized by solid-phase methods (26) or produced by recombinant DNA technology (27) as described previously. The purity of the compound from each source was determined by HPLC and amino acid analysis. The biological activity of the synthetic peptide was shown to be identical to that of the native peptide (26). The recombinant peptide was equipotent to the synthetic echistatin in in vitro assays (17) and in an in vivo model (19), in which both recombinant and synthetic echistatin inhibited the calcemic response to PTH in TPTX rats with a similar dose response (our unpublished results). 125I-echistatin was prepared by iodination of echistatin (100 µg) with 10 mCi of Na125I (Amersham Corp., Arlington Heights, IL) using 10 µg Iodogen (Pierce Chemical Co., Rockford, IL). The radioligand was then purified by HPLC using C-4 columns and stored at -70 C. Antihuman vitronectin receptor polyclonal antiserum was purchased from Telios Pharmaceutical Inc. (San Diego, CA). This antiserum recognizes mainly {alpha}vß3 but was shown to cross-react weakly with {alpha}vß5. GRGDSPK peptide was synthesized using an automated peptide synthesizer (Applied Biosystems 430A Peptide Synthesizer, Foster City, CA).

In vitro studies
Purification of integrin {alpha}vß3 from human placenta. Full-term human placentae were obtained at parturition from local hospitals with informed consent. All placentae were stored at -20 C immediately after delivery. Placental extract was prepared as described by Pytela et al. (28). The extract was applied to a GRGDSPK-Sepharose column. The column was washed with 5 column volumes of HBS buffer (50 mM HEPES, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, pH 7.4)/50 mM octylglucoside/1 mM phenylmethylsulfonyl fluoride (PMSF). {alpha}vß3 was eluted with HBS buffer containing 100 mM octylglucoside, 20 mM EDTA, and 1 mM PMSF into tubes containing 25 mM CaCl2/MgCl2 to restore the functional receptor. Fractions were identified by immunoprecipitation with antibodies which recognize {alpha}vß3 and by 125I-echistatin binding.

Cross-linking and immunoprecipitation studies. Affinity purified pooled integrin fractions were cross-linked with 125I-echistatin (1700 Ci/mmol) with 0.2 mM bis(sulfosuccinimidyl) suberate (BS3) in the presence or absence of 1 µM unlabeled echistatin and immunoprecipitated with antihuman {alpha}vß3 polyclonal antibodies. The immunoprecipitates were electrophoresed in 7.5% SDS-PAGE gel under reducing conditions followed by autoradiography using Kodak X-OMAT AR film.

Echistatin binding to purified {alpha}vß3. The binding of 125I-echistatin to purifed {alpha}vß3 was determined by incubating 10 µg of purified integrin with 30 pM 125I-echistatin in binding buffer (100 mM Tris-HCl, pH 7.8, 100 mM NaCl, 1 mM CaCl2/MgCl2, 1% BSA) in the presence of increasing concentrations of unlabeled echistatin. Integrin-bound echistatin was separated from unbound by filtration (Skatron Cell Harvesting System 11021, Sterling, VA) on filters presoaked with 1.5% polyethyleneimine. The filters were washed with 6 ml buffer (25 mM Tris-HCl, pH 7.8, 1 mM CaCl2/MgCl2) and counted in a Gamma Counter (Packard Auto 5650, Meriden, CT).

In vivo studies
Animals. Mice and rats were obtained from Taconic (Germantown, NY) and were housed in temperature-controlled rooms with 12-h light, 12-h dark cycles and with free access to tap water and a commercial diet (Rodent Laboratory Chow 5001, Ralston-Purina, St. Louis, MO). Surgical procedures were performed in animals anesthetized with an ip injection of ketamine hydrochloride and acepromazine maleate (75 and 2.5 mg/kg body weight, respectively). All studies were conducted in accordance with the Institutional Laboratory Animal Care Guide for the care and use of Laboratory Animals (1996).

Protocols
A. Mouse studies
Study 1: Female Swiss-Webster mice, 10–11 weeks old, were allocated to four groups (n = 10 each). After body weight measurements, three groups underwent bilateral OVX via dorsal approach and the fourth group sham-OVX. After surgery, echistatin solution or vehicle (0.9% NaCl) was infused continuously via osmotic minipumps (Model 2002, Alza Corp., Palo Alto, CA) according to the following regimen: sham/vehicle, OVX/vehicle, OVX/echistatin at 0.12 µg/h·g body weight (~17 nmol/day per mouse), and OVX/echistatin at 0.36 µg/h·g (~50 nmol/day). The minipumps were implanted sc in the backs of the mice after sufficient preincubation periods in sterile isotonic saline as suggested by the manufacturer. The echistatin dosages were chosen based on inhibitory effects on PTH-induced elevation of serum calcium in TPTX mice (29). The higher dose corresponds to the approximate IC50 of the acute echistatin effect in the TPTX model. The lower dose of echistatin, one third of the higher dose, was ineffective in the TPTX model. Using the echistatin binding assay, we confirmed that the echistatin solution was stable in osmotic minipumps in vitro at room temperature for up to 14 days.

Study 2: The protocol was essentially the same as in Study 1 except for the following. The number of mice was 8 for the OVX groups and 10 for the sham/vehicle group. Osmotic minipumps were replaced after 2 weeks and infusions were continued for an additional 2 weeks (total 4 weeks).

At the end of each study, blood was obtained by cardiac puncture under anesthesia with CO2 inhalation. After euthanasia and body weight measurements, left femora and tibiae were dissected free from soft tissue and fixed in 70% ethanol for histomorphometric analysis. Right femora were removed intact for length measurements. Uterine wet weight was measured to confirm successful OVX. Lung, liver, kidney, spleen, and right tibia were removed from four animals per group in Study 1 and placed in 10% formalin for standard histopathological examinations.

B. Rat study
Six-month-old virgin Sprague-Dawley rats (average weight, ~300 g) were randomized into sham/vehicle (n = 9), OVX/vehicle (n = 8), and OVX/echistatin (n = 6) groups. After OVX or sham operation, animals received echistatin (0.26 µg/h·g) or vehicle infusion via osmotic minipumps (model: 2ML1) as described in the mouse studies. Infusion was continued for 4 weeks by replacing osmotic minipumps weekly. Blood was obtained via retro-orbital sinus at 1 and 14 days after the start of infusion and by cardiac puncture at the time the rats were euthanized. Fluorochrome double labeling was performed with sc calcein, 25 mg/kg body weight, 11 days and 1 day before euthanasia. At 28 days post surgery animals were killed, and bilateral femora and tibiae were excised and stored in 70% ethanol. Right femora were used for length and ash weight measurements, and left femora for bone mineral content and bone mineral density (BMD) measurements. Tibiae were used for histomorphometric analysis. Uterine wet weight was determined to confirm successful OVX.

Measurements. Biochemical analyses of serum: Serum calcium and phosphate were measured by colorimetric methods using commercial kits (Sigma Chemical Co., St. Louis, MO). Echistatin concentrations in serum of treated animals were determined by competitive binding assay as described above (see In vitro studies). Briefly, the binding assay was performed in the presence of serial dilution of echistatin (10-11 to 10-8 M) or serum samples (1:50 to 1:6250) from echistatin-treated animals. Because the serum dilution curve was parallel to the dilution curve of echistatin solution, serum concentrations of echistatin were calculated using the echistatin dilution standard curve. The detection limit of echistatin in serum was approximately 10 nM.

Femoral length and ash weight: Femora were measured for bicondylar length (anatomical length) with a micrometer and were ashed at 700 C for 24 h using a muffle furnace. The bone ash was weighed and weight/length (mg/mm) was calculated.

Ex vivo measurements of BMD by dual energy x-ray absorptiometry (DEXA): De-fleshed femora were immersed in a 2 cm depth of 70% ethanol and scanned using a Lunar DPX instrument (Lunar Radiation Corp., Madison, WI) with a small animal software application in the high resolution mode. In addition to whole femora, the areas measured were a distal region representing 25% of the total length, which contains significant amounts of cancellous bone, and the diaphysis, which represents 50% of the femoral length, mostly consisting of cortical bone. The precision for DEXA scanning was 1.3% (coefficient of variation) for 10 measurements of the same bone following repositioning of both the scanner and the bone.

Bone histomorphometry: Bone samples were dehydrated through increasing concentrations of ethanol and processed undecalcified in methylmethacrylate using a Hypercenter XP2 automatic tissue processor (Shandon, Pittsburgh, PA). Longitudinal frontal sections, 5 or 10 µm thick, were obtained using a Polycut S microtome (Reichert-Jung, Heidelberg, Germany). The 5-µm sections were stained either with Goldner or Masson’s trichrome, 10-µm sections were unstained. Histomorphometric analysis was carried out using a semiautomated image analysis system (Bioquant IV, R & M Biometrics, Nashville, TN) without knowledge of group allocation. Cancellous bone volume and cellular parameters were measured in stained sections, and fluorochrome-based indices of bone formation were measured in unstained sections in a mean tissue area of 6.0 mm2 beginning 1 mm below the epiphyseal growth plate. The data were presented according to Parfitt et al. (30).

Statistics
Results are expressed as the mean ± SEM. Statistical analyses of the data were performed using the statistical package Statview (Abacus Concepts Inc., Berkeley, CA) on a Macintosh computer. Differences between treatment groups were tested by one-way ANOVA and Fisher’s protected least significant difference (PLSD). Paired t tests were used to compare the data between two different time points of the same group. P values less than 0.05 at a 95% confidence level were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Purification of {alpha}vß3 integrin from human placenta and its binding to 125I-echistatin
Figure 1AGo shows the elution profile of the placental extract off the GRGDSPK affinity chromatography column. The pooled eluate fractions 5–18 were cross-linked with 125I-echistatin (1700 Ci/mmol) with 0.2 mM bis(sulfosuccinimidyl) suberate (BS3) in the presence or absence of 1 µM unlabeled echistatin and immunoprecipitated with the antihuman {alpha}vß3 antibody, which yielded on SDS-PAGE gels two proteins of apparent molecular mass of 130 and 110 kDa corresponding to {alpha}v- and ß3-subunits, respectively (Fig. 1BGo). Figure 2Go shows the displacement (IC50 = 0.5 nM) of the 125I-echistatin radioligand bound to the pooled fractions 5–18 by increasing concentrations of unlabeled echistatin.



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  		Figure 1. Purification (A) and characterization (B) of purified integrin. A, Placental extract prepared as described inMaterials and Methods was chromatographed on a GRGDSPK-Sepharose column. The column was eluted with HBS buffer containing 100 mM octylglucoside, 20 mM EDTA and 1 mM PMSF as described in Materials and Methods. Fractions were collected into tubes containing 25 mM CaCl2/MgCl2. The activity of the fractions were determined by 125I-echistatin binding. Fractions (5 to 18) were then pooled. B, The pooled fractions obtained from the placental extract by GRGDSPK-sepharose affinity chromatography were cross-linked with 125I-echistatin with 0.2 mM bis(sulfosuccinimidyl) suberate (BS)3 in the presence or absence of 1 µM unlabeled echistatin and immunoprecipitated with antihuman {alpha}vß3 polyclonal antibodies. Two protein bands of 130 and 110 kDa on 7.5% SDS-PAGE under reducing conditions correspond to {alpha}v- and ß3-subunits.

 


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  		Figure 2. Competition of 125I-echistatin bound to purified {alpha}vß3 by increasing concentrations of unlabeled echistatin. The pooled fractions (~10 µg) described as in Fig. 1AGo were incubated with 30 pM 125I-echistatin in binding buffer (25 mM Tris-HCl, pH 7.8, 1 mM CaCl2/MgCl2) for 1 h in the presence of increasing concentrations of unlabeled echistatin. The percentage of radioactivity bound was calculated and plotted vs. unlabeled echistatin concentrations, after separation of bound from unbound echistatin.

 
Effects of echistatin in OVX mice
OVX significantly increased body weight and decreased uterine weight in both the 2-week (Table 1Go) and 4-week (data not shown) studies. Echistatin treatment had no effect on body weight nor uterine weight changes. Femoral length was slightly greater (P < 0.05) in OVX echistatin-treated mice than in sham control mice in the 2-week study (Table 1Go) and was not statistically different among groups in the 4-week study (data not shown). Serum calcium concentrations (mg/dl) were not different among the four groups in the 2-week (data not shown) and the 4-week studies [sham/vehicle = 9.58 ± 0.12, OVX/vehicle = 9.66 ± 0.13, OVX/echistatin (0.12 µg/h·g) = 9.71 ± 0.20, OVX/echistatin (0.36 µg/h·g) = 9.92 ± 0.12]. The serum phosphate concentrations (mg/dl) in the 4-week study were 9.29 ± 0.32, 10.24 ± 0.34, 9.76 ± 0.46, and 10.87 ± 0.59 for sham/vehicle, OVX/vehicle, OVX/echistatin lower and higher doses, respectively. Although the phosphate concentrations were higher in all three OVX groups compared with control group, the difference was statistically significant only in animals receiving the higher dose of echistatin.


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  		Table 1. Changes in body weight, uterine weight and femoral length of mice 2 weeks after OVX and echistatin treatment

 
The effects of OVX and echistatin treatment on cancellous bone volume and architecture at the distal femur are shown in Figs. 3Go and 4Go. In both the 2- and 4-week studies, cancellous bone volume decreased in OVX/vehicle mice significantly (by 37% and 39%, respectively) relative to sham/vehicle controls. This resulted from significant decreases in trabecular number and trabecular thickness. The higher echistatin dose completely prevented the OVX-induced decrease in cancellous bone volume, trabecular number, and thickness in both studies (Figs. 3Go and 4Go). The lower dose was as effective as the higher dose in the 2-week study (Fig. 3Go) but not in the 4-week study (Fig. 4Go). Figure 5Go shows representative photomicrographs of the distal femur of sham-operated, OVX, and OVX mice treated with 0.36 µg/h·g echistatin for 4 weeks. The findings in the proximal tibiae were essentially the same as in the distal femora (data not shown).



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  		Figure 3. Cancellous bone volume (A), trabecular number (B), and trabecular thickness (C) in the distal femora of mice 2 weeks after OVX and echistatin treatment. Closed column, Sham-operated group treated with vehicle. Open column, OVX group treated with vehicle. Hatched column, OVX group infused with lower dose (0.12 µg/h·g) of echistatin. Cross-hatched column, OVX group infused with higher dose (0.36 µg/h·g) of echistatin. *, Significantly (P < 0.05) lower than sham/vehicle group. **, Significantly higher than OVX/vehicle group.

 


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  		Figure 4. Cancellous bone volume (A), trabecular number (B), and trabecular thickness (C) in the distal femora of mice 4 weeks after OVX and echistatin treatment. Closed column, sham-operated group treated with vehicle. Open column, OVX group treated with vehicle. Hatched column, OVX group infused with lower dose (0.12 µg/h·g) of echistatin. Cross-hatched column, OVX group infused with higher dose (0.36 µg/h·g) of echistatin. *, Significantly (P < 0.05) lower than sham/vehicle group. **, Significantly higher than OVX/vehicle group.

 


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  		Figure 5. Photomicrographs of representative distal femora from sham-operated (Sham-op) and OVX mice treated with vehicle and OVX mice treated with echistatin (OVX + Echi) for 4 weeks. Longitudinal sections were stained with Masson’s trichrome and photographed with a Nikon Microphot image microscope. The bone trabeculae are stained blue, and the marrow stroma is stained red. Scale bar, 1 mm.

 
The mean osteoclast surface, mineralizing surface, and bone formation rate measured only in the 4-week study were approximately 50% higher in OVX/vehicle mice than sham/vehicle mice (data not shown). These differences did not achieve statistical significance, possibly due to wide intergroup variations, previously seen in other mouse studies (31, 32). The morphology of osteoclasts in echistatin-treated mice showed no detectable differences at the light microscopic level from untreated animals.

Echistatin administration for up to 4 weeks was not associated with any overt signs of toxicity. No abnormal bleeding was seen in echistatin-treated mice during the study. At the time of euthanasia, pleural and peritoneal cavities and the major internal organs were macroscopically normal. Standard microscopic examination of lung, liver, kidney, spleen, and tibial bone marrow revealed no abnormalities.

Effects of echistatin in OVX rats
As in mouse studies, echistatin treatment did not affect the OVX-induced increase in body weight and the decrease in uterine weight (data not shown). Femoral length tended to be greater (not statistically significant) in OVX rats with or without echistatin treatment than in sham rats (Table 2Go). Femoral ash weight corrected for length was significantly lower (by 12%) in OVX/vehicle rats than in sham/vehicle controls, whereas in OVX/echistatin it was not significantly different from the sham/vehicle group. BMD of the whole femora as well as the distal femora, covering 25% of femoral length, were also lower in OVX/vehicle and were maintained at control levels by echistatin (Table 2Go). BMD of the midshaft of the femur was not statistically different among the three groups. Calculation of bone mineral content yielded similar findings to BMD (data not shown).


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  		Table 2. Changes in femoral lengths, ash weights, and bone mineral densities in rats 4 weeks after OVX and echistatin treatment

 
Cancellous bone volume, trabecular number, and trabecular thickness decreased significantly in OVX/vehicle rats compared with sham/vehicle controls (Fig. 6Go). Echistatin infused at 0.26 µg/h·g for 4 weeks prevented over 50% of the OVX-induced bone loss: the mean bone volume of OVX/vehicle and of OVX/echistatin-treated rats was 66% and 86% of sham/vehicle controls, respectively. The bone volume of OVX/echistatin rats was not significantly different from controls, nor from OVX/vehicle rats (Fig. 6AGo). Echistatin also prevented by about 50% the OVX-induced decrease in trabecular number (not significantly different from control or OVX/vehicle) (Fig. 6BGo), and it completely prevented the decrease in trabecular thickness (Fig. 6CGo). The osteoclast surface showed a tendency to increase in the OVX/vehicle group compared with sham/vehicle group and was significantly higher in the OVX/echistatin group (Table 3Go). Osteoclasts in echistatin-treated rats appeared morphologically normal and were not detached from the bone surface (data not shown). OVX caused a significant increase in the mineralizing surface and bone formation rate, indicative of increased bone turnover (Table 3Go). Echistatin treatment normalized these bone formation indices and did not suppress them below control levels. The mineral apposition rate was comparable in all groups. Figure 7Go shows photomicrographs of trabecular bone area in the proximal tibia of sham-operated, OVX, and OVX animals treated with echistatin (0.26 µg/h·g) for 4 weeks.



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  		Figure 6. Cancellous bone volume [A], trabecular number [B], and trabecular thickness [C] in the proximal tibiae of rats 4 weeks after OVX and echistatin treatment. Closed column, sham-operated group treated with vehicle. Open column, OVX group treated with vehicle. Hatched column, OVX group infused with echistatin at 0.26 µg/h·g. *, Significantly (P < 0.05) lower than sham/vehicle group. **, Significantly higher than OVX/vehicle group.

 

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  		Table 3. Changes in histomorphometric indices in rats 4 weeks after OVX and echistatin treatment

 


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  		Figure 7. Photomicrographs of representative proximal tibiae from sham-operated (Sham-op) and OVX rats treated with vehicle and echistatin (OVX + Echi) for 4 weeks. Longitudinal sections were stained with Masson’s trichrome and photographed with a Nikon Microphot image microscope. The bone trabeculae are stained blue, and the marrow stroma is stained red. Scale bar, 1 mm.

 
Echistatin levels in serum
The mean serum level of echistatin at the time of euthanasia was roughly proportional to the dose infused: 28 nM in mice infused with 0.36 µg/h·g of echistatin, approximately 20 nM in rats infused with 0.26 µg/h·g and undetectable (limit of detection is 10 nM, therefore <10 nM) in mice given the lower dose of 0.12 µg/h·g. Variation in serum echistatin concentration during infusion was examined in rats at two time points. It was found that the concentration was higher 1 day after the start of infusion than 14 days later, 53. 7 ± 2.7 vs. 22.1 ± 2.1 nM. The latter value measured at the end of 2 weeks was not different from that measured at the time of euthanasia (at the end of 4 weeks). These results indicate that serum echistatin concentrations of 20 nM or more effectively inhibited bone resorption.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The osteoclast antigen that Horton et al. (31) identified by raising monoclonal antibodies against osteoclastoma and subsequently showed to inhibit osteoclast activity in vitro, was identified as the vitronectin receptor {alpha}vß3 (23). {alpha}vß3 has since been found in osteoclasts from all species examined (13, 14, 15, 16). Moreover, not only {alpha}vß3 antibodies but also ligands that bind with high affinity to {alpha}vß3, such as echistatin or kistrin, were shown to inhibit osteoclast activity in vitro and PTH dependent rise in serum calcium in vivo (17, 19, 20, 32). The object of this study was to evaluate the effect of echistatin in models of estrogen deficiency in mice and rats, at the level of the bone tissue.

Echistatin inhibition of estrogen deficiency bone loss was clearly documented by several means in all three experiments reported in this study. In mice, echistatin administered at a dose of 0.36 µg/h·g for 2 or 4 weeks was fully effective in the prevention of OVX-induced cancellous bone loss. Echistatin at 0.12 µg/h·g was as effective as 0.36 µg/h·g in the 2-week study but was ineffective in the 4-week study. Although the reason for the difference in echistatin efficacy at the lower dose in the two studies is unclear, this could occur at a nonsaturating dose when the dose-response curve is steep. The mouse is not a widely used model for estrogen deficiency bone loss, but several studies have documented a reduction in the bone volume of mice in the secondary spongiosa of the femur following OVX (33, 34). Although differences in bone mass and bone turnover between mouse strains have been recently reported (35), previous studies have shown that the Swiss Webster mice, used in the present study, lose bone after OVX (36).

The concentration of echistatin that prevented bone loss in ovariectomized animals (20–50 nM), is somewhat lower than that required to block PTH-dependent calcium mobilization. This could reflect the chronic, milder, osteoclast stimulation caused by estrogen deficiency, compared with the acute intensive osteoclast activation by PTH. The concentrations effective in vivo are, however, higher than the apparent Kd for echistatin binding to {alpha}vß3 in vitro, possibly due to echistatin protein binding or to barriers for echistatin access to its target in vivo.

To carry out a more extensive evaluation and obtain more definitive information, we examined the effect of echistatin on estrogen deficiency bone loss in the well established model of OVX rats. Bone mineral density measurements of the various sites of femora showed that echistatin prevented bone loss. Ash weight measurements also demonstrated a protective effect of echistatin against OVX-induced bone loss. These findings were confirmed by histomorphometry of cancellous bone volume in the secondary spongiosa of the tibia. Moreover, measurement of mineralizing surface, an estimate of bone formation and thus bone turnover, showed the expected 4-fold increase in OVX animals. In echistatin-treated OVX rats, mineralizing surface as well as bone formation rate decreased to levels that were not significantly different from controls. This reduction in bone turnover is fully consistent with a mode of action of echistatin based on inhibition of osteoclastic bone resorption, similar to that of estrogen (37), alendronate (38), or other inhibitors of bone resorption.

We found, in echistatin-treated animals, an increase in osteoclast surface, defined as bone surface covered by osteoclasts, suggesting that echistatin did not reduce osteoclast formation, recruitment, or attachment to bone. At the light microscopic level, osteoclasts were adjacent to the bone surface and appeared morphologically normal. Furthermore, in a separate study, electronmicroscopic examination of osteoclasts in echistatin-treated animals showed normal clear zones and ruffled borders (38a). However, since bone loss was prevented, echistatin clearly reduced osteoclast activity. While this manuscript was in preparation, Engleman et al., (21) reported that a peptidomimetic {alpha}vß3 antagonist blocked bone resorption in vitro and in vivo, and histological examination of the bones from ovariectomized rats suggested that the bone sparing effect was associated with a decrease in osteoclast number. The difference in effects on osteoclast number could be due to differences in the compound, the duration of treatment (4 weeks vs. 6) or the mechanism of action, related for example to signaling (39) or apoptosis (40).

Both echistatin, a 49-amino acid peptide, and the nonpeptide {alpha}vß3 antagonist act most likely on {alpha}vß3. Echistatin binds with high affinity (Kd = 0.5 nM) to the vitronectin receptor and was found by immunohistochemistry to colocalize in rat osteoclasts with the vitronectin receptor {alpha}vß3 (17). In addition to {alpha}vß3, mammalian osteoclasts express {alpha}2ß1, and {alpha}vß1 and recently ß1 integrins have been implicated in osteoclast function/bone resorption via type I collagen (41). 125I-Echistatin cross-linked to integrins, obtained by affinity purification from either human placenta or HEK293 cells transfected with {alpha}vß3, was quantitatively immunoprecipitated by antibodies against {alpha}vß3 or ß3 but not ß1 (data not shown). We have also observed that 125I-echistatin does not bind to HEK293 cells which express a variety of ß1 integrins (including {alpha}5ß1 and {alpha}vß1); however, it binds with high affinity to HEK293 cells transfected with {alpha}vß3 (data not shown). Most importantly, we recently confirmed the interaction of 125I-echistatin with {alpha}vß3 integrin in mouse osteoclasts by immunoprecipitating 125I-echistatin cross-linked to osteoclasts with {alpha}vß3 antibodies (42). Thus, although there is no definitive proof that the effects of echistatin observed in this study are due to its exclusive interaction with {alpha}vß3, most evidence suggests that this accounts for at least a major part of its effect. The RGD mimetic (21) also recognizes {alpha}vß1, which is expressed in mammalian osteoclasts (14), and {alpha}vß5, which has so far been reported only in avian osteoclast precursors (43), and its contribution to mammalian osteoclastic bone resorption remains unknown. Echistatin, also inhibits {alpha}vß5-mediated attachment to vitronectin, however, only at much higher concentrations (>1 µM) than {alpha}vß3-mediated attachment (<1 nM) (our unpublished observations).

Integrins not only mediate cellular adhesion but are also implicated in migration. {alpha}vß3 plays a role in the migration of many cell types including endothelial cells and HEK 293 cells transfected with {alpha}vß3 (39, 44). Cell migration is essential for many physiological and pathological processes such as thrombosis, restenosis, and cancer metastasis. Osteoclasts are highly migrating cells (45) and based on the localization of {alpha}vß3 in osteoclasts, it has been suggested that {alpha}vß3 may be involved in their migration (46). Treatment with echistatin could reduce osteoclast migration, which would be consistent with an increase in osteoclast surface in conjunction with decreased activity. Taken together, these findings in OVX mice and rats as well as the recent reports that two additional nonpeptide integrin ligands effectively inhibit PTH-dependent calcium mobilization and ovariectomy-induced bone loss (47, 48), indicate that integrins, probably {alpha}vß3, play a rate-limiting role in osteoclast function and could be targeted for therapeutic suppression of bone loss.


   Acknowledgments
 
We would like to thank Drs. V. Garsky and W. Herber for preparation of synthetic and recombinant echistatin, respectively, Dr. C. Peter for histopathological examination of the major organs and bones, Dr. R. Balena for her help in histomorphometric analyses and Mr. E. Opas for the synthesis of the GRGDSPK peptide.


   Footnotes
 
1 Present address: Third Department of Internal Medicine, National Defense Medical College, Tokorozawa, Saitama 359, Japan. Back

2 These authors have contributed equally to this work. Back

Received August 27, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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