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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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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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
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
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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- 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 vß3
integrin is highly expressed in osteoclasts from all species examined
(13, 14, 15, 16). Echistatin colocalizes with the integrin
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 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 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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vß3 but was
shown to cross-react weakly with vß5.
GRGDSPK peptide was synthesized using an automated peptide synthesizer
(Applied Biosystems 430A Peptide Synthesizer, Foster City, CA).
In vitro studies
Purification of integrin 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).
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 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 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
vß3. The binding of
125I-echistatin to purifed 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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vß3 integrin from
human placenta and its binding to 125I-echistatin
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 vß3
antibody, which yielded on SDS-PAGE gels two proteins of apparent
molecular mass of 130 and 110 kDa corresponding to v-
and ß3-subunits, respectively (Fig. 1B). Figure 2 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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) 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 1) 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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and 4. 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. 3 and 4). The lower dose was as effective as the higher
dose in the 2-week study (Fig. 3) but not in the 4-week study (Fig. 4).
Figure 5 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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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 2).
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 2). 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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). 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. 6A). Echistatin also
prevented by about 50% the OVX-induced decrease in trabecular number
(not significantly different from control or OVX/vehicle) (Fig. 6B),
and it completely prevented the decrease in trabecular thickness (Fig. 6C). 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 3). 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 3). 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 7 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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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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vß3 (23). vß3
has since been found in osteoclasts from all species examined (13, 14, 15, 16).
Moreover, not only vß3 antibodies but also
ligands that bind with high affinity to
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
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 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
vß3 antagonist act most likely on
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 vß3 (17). In
addition to vß3, mammalian osteoclasts
express 2ß1, and
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 vß3, was
quantitatively immunoprecipitated by antibodies against
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
5ß1 and vß1);
however, it binds with high affinity to HEK293 cells transfected with
vß3 (data not shown). Most importantly, we
recently confirmed the interaction of 125I-echistatin with
vß3 integrin in mouse osteoclasts by
immunoprecipitating 125I-echistatin cross-linked to
osteoclasts with 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 vß3, most evidence suggests that this
accounts for at least a major part of its effect. The RGD mimetic (21)
also recognizes vß1, which is expressed in
mammalian osteoclasts (14), and 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
vß5-mediated attachment to vitronectin,
however, only at much higher concentrations (>1 µM) than
vß3-mediated attachment (<1
nM) (our unpublished observations).
Integrins not only mediate cellular adhesion but are also implicated in
migration. vß3 plays a role in the
migration of many cell types including endothelial cells and HEK 293
cells transfected with 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 vß3 in osteoclasts, it
has been suggested that 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
vß3, play a rate-limiting role in
osteoclast function and could be targeted for therapeutic suppression
of bone loss.
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Acknowledgments |
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Footnotes |
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2 These authors have contributed equally to this work.
Received August 27, 1997.
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References |
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vß3,
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integrin subunits in rat osteoclasts in situ. J Bone
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vß3 antagonists promote tumor regression
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vß3 and
vß5 in a reciprocal manner during
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5ß1-mediated cell migration toward
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confocal laser scanning microscopy. J Cell Sci 104:663–670[Abstract]
vß3
ligand inhibits osteoclast activity in vitro and in
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Bone and Mineral Research, Seattle, WA, 1996, p S289 (Abstract)
vß3 vitronectin receptor. Program of the
Annual Meeting of the American Society for Bone and Mineral Research,
Seattle, WA, 1996, p S228 (Abstract)
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P. T. Lakkakorpi, A. J. Bett, L. Lipfert, G. A. Rodan, and L. T. Duong PYK2 Autophosphorylation, but Not Kinase Activity, Is Necessary for Adhesion-induced Association with c-Src, Osteoclast Spreading, and Bone Resorption J. Biol. Chem., March 21, 2003; 278(13): 11502 - 11512. [Abstract] [Full Text] [PDF]
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 S. J. Hoffman, J. Vasko-Moser, W. H. Miller, M. W. Lark, M. Gowen, and G. Stroup Rapid Inhibition of Thyroxine-Induced Bone Resorption in the Rat by an Orally Active Vitronectin Receptor Antagonist J. Pharmacol. Exp. Ther., July 1, 2002; 302(1): 205 - 211. [Abstract] [Full Text] [PDF]
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 G. A. Rodan and T. J. Martin Therapeutic Approaches to Bone Diseases Science, September 1, 2000; 289(5484): 1508 - 1514. [Abstract] [Full Text]
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M. W. Lark, G. B. Stroup, S. M. Hwang, I. E. James, D. J. Rieman, F. H. Drake, J. N. Bradbeer, A. Mathur, K. F. Erhard, K. A. Newlander, et al. Design and Characterization of Orally Active Arg-Gly-Asp Peptidomimetic Vitronectin Receptor Antagonist SB 265123 for Prevention of Bone Loss in Osteoporosis J. Pharmacol. Exp. Ther., November 1, 1999; 291(2): 612 - 617. [Abstract] [Full Text]
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H. Yoshitake, S. R. Rittling, D. T. Denhardt, and M. Noda Osteopontin-deficient mice are resistant to ovariectomy-induced bone resorption PNAS, July 6, 1999; 96(14): 8156 - 8160. [Abstract] [Full Text] [PDF]
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 I Nakamura, M. Pilkington, P. Lakkakorpi, L Lipfert, S. Sims, S. Dixon, G. Rodan, and L. Duong Role of alpha(v)beta(3) integrin in osteoclast migration and formation of the sealing zone J. Cell Sci., January 11, 1999; 112(22): 3985 - 3993. [Abstract] [PDF]
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I. Nakamura, H. Tanaka, G. A. Rodan, and L. T. Duong Echistatin Inhibits the Migration of Murine Prefusion Osteoclasts and the Formation of Multinucleated Osteoclast-Like Cells Endocrinology, December 1, 1998; 139(12): 5182 - 5193. [Abstract] [Full Text] [PDF]
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P. Masarachia, M. Yamamoto, C.-T. Leu, G. Rodan, and L. Duong Histomorphometric Evidence for Echistatin Inhibition of Bone Resorption in Mice with Secondary Hyperparathyroidism Endocrinology, March 1, 1998; 139(3): 1401 - 1410. [Abstract] [Full Text] [PDF]
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