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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: Patricia Masarachia, Department of Bone Biology and Osteoporosis Research, Merck Research Laboratories, Sumneytown Pike, West Point, Pennsylvania 19486. E-mail: patricia_masarachia{at}merck.com
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Integrins are heterodimeric transmembrane glycoproteins that mediate cell-cell and cell-matrix interactions. Ligand binding to integrins initiates signal transduction pathways involved in cytoskeletal rearrangements associated with cell activation, adhesion, and motility as well as growth and differentiation (5, 6). Several integrins recognize the arginine-glycine-aspartic acid (RGD) moiety in extracellular matrix (ECM) proteins such as fibronectin or vitronectin (7).
The vitronectin receptor 
vß3 is the
predominant integrin expressed in osteoclasts (8, 9, 10). Additional
integrins present in osteoclasts include
vß1, 2ß1,
3ß1 and 5ß1
(11, 12, 13, 14). RGD peptides, vß3 antibodies, an
RGD peptide mimetic and echistatin, a member of the RGD-containing
disintegrin family, were shown to inhibit bone resorption, osteoclast
formation, attachment, and spreading in vitro (9, 15, 16, 17, 18, 19).
In vivo, anti-ß3 antibodies, echistatin, and
another disintegrin, kistrin, inhibited calcium mobilization,
presumably via inhibition of bone resorption (20, 21, 22). Recently, a
synthetic RGD peptide mimetic,
ß-[2[[5-[(aminoiminomethyl)amino]-1-oxopentyl]amino]-1-oxoethyl]amino-3-pyridinepropanoic
acid, bistrifluoracetate, also inhibited calcium mobilization in
parathyroidectomized rats and the loss of bone density in
ovariectomized rats (19).
None of these studies have provided direct histological evidence for inhibition of bone resorption. The mode of action of integrin ligands is also not fully known. The objectives of this study were to: 1) examine, using histomorphometry, if echistatin can protect against bone loss caused by secondary hyperparathyroidism (2°HPT); 2) determine whether echistatin localizes to osteoclasts in vivo; and 3) examine, using electron microscopy, if there are morphological changes in osteoclasts caused by echistatin treatment. We found that echistatin localizes to osteoclasts in vivo, inhibits cancellous bone turnover, reduces bone loss, increases osteoclast number, and does not detectably alter osteoclast morphology.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Infusion of echistatin in thyroparathyroidectomized (TPTX’d) mice, as
previously described in rats (20), was used to determine the effective
dose that inhibits the acute calcemic response to exogenous PTH.
Briefly, male BALB/C mice (Taconic Farms, Germantown, NY), 9 weeks old,
averaging 20 g, were TPTX’d and were placed the next day on a low
calcium diet (calcium, < 0.01%; phosphorus, 0.4%; ICN Biochemicals,
Cleveland, OH). Two days following TPTX, mice were infused for 6 h
with bovine PTH (bPTH (1–34) amide (Bachem, King of Prussia, PA) at
0.006 nmol/mouse·h via an sc implanted osmotic minipump (Alzet 1003D,
Alza Corp., Palo Alto, CA) either with echistatin (6 or 30
µg/kg·min doses) in the test groups (n = 7 and 10,
respectively) or with vehicle in the control group (n = 10). Serum
calcium measurements were made from blood samples drawn from the tail
vein at four time points: 1) before TPTX (baseline); 2) 24 h after
TPTX; 3) before PTH infusion; and 4) at the end of PTH infusion. Serum
calcium measurements were performed by atomic absorption
spectrophotometry using a Perkin-Elmer model 2380 AA spectrophotometer
(Foster City, CA). As shown in Fig. 1
, 30
µg/kg·min was determined to be an effective dose of echistatin. At
the end of PTH infusion, animals were euthanized by CO2
inhalation. Serum echistatin levels were obtained by an
125I-echistatin competitive receptor binding assay (23).
Briefly, cell extracts (25 µg) prepared from HEK 293 cells
overexpressing vß3 were incubated with 30
pM 125I-echistatin in a binding buffer
consisting of 100 mM Tris-HCl, 100 mM NaCl, 1
mM CaCl2/MgCl2, pH 7.8, in the
presence of serial dilutions of tested serum. The integrin bound
echistatin was separated from the unbound by filtration through a
Skatron Cell Harvester (Skatron 11021, Sterling, VA) onto filters
preequilibrated with 1.5% polyethyleneamine. Filters were washed in 25
mM Tris-HCl, 1 mM
CaCl2/MgCl2, pH 7.8 and counted in a Packard
Auto Gamma 5650 Counter (Meriden, CT). Serum levels, estimated by using
an echistatin displacement curve, averaged at time the mice were
euthanized, 77.8 ± 70.4 and 152.2 ± 98.0 nM,
for the 6 and 30 µg/kg·min dosages, respectively.
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Serum echistatin values at the time the mice were euthanized ranged between 63 and 160 nM, with a mean value of 112 ± 31 nM (mean ±SD). Because echistatin half-life in the circulation is very short (t1/2 = 5 min, unpublished observations), small changes in the infusion/delivery rates from the minipumps just before sampling, possibly due to animal movement, can cause variations in serum echistatin concentrations. However, these variations may even out over time, as judged by the efficacy of the drug among animals, assessed by histology.
Femurs and tibiae were dissected free and denuded of soft tissue. Bone ash weights/mm were measured in isolated femurs as described previously (24). Tibiae were processed in methyl methacrylate for histomorphometric analysis (25). Measurements were made in an area spanning between 0.5 and 2.5 mm below the growth plate using a BioQuant IV image analysis system (BioQuant, Nashville, TN). The complete secondary spongiosa and half of the primary spongiosa in one section from each tibia/animal were measured. Section depth was matched for each bone for the maximum area of analysis. Data were collected without knowledge of group allocation, and measurements were repeated and confirmed by a second independent observer. The following parameters were measured: trabecular bone volume (TB/BV, %), expressed as the percentage area of trabecular bone; osteoid surface, expressed as percentage of trabecular surface (OS/BS, %); multinucleated osteoclast surface, expressed as percent of trabecular surface (OcS/BS, %); mean osteoclast length, measured as the length in contact with the bone surface; osteoclast number per mm of trabecular bone surface (Oc#/TbS, #/mm); osteoclast number per mm2 trabecular area (Oc#/TbA, #/mm2); and osteoclast number per mm2 tissue area (OC#/TsA, #/mm2). Total trabecular thickness (TTB.Th, µm), total trabecular number (TTB.N, #/mm), and total trabecular spacing (TTB.Sp, µm) were derived from primary measurements of areas and perimeters according to Parfitt et al. (26). Means and SD were computed for each variable for each treatment group. Differences between treatment groups were tested by one-way ANOVA using the statistical package Statview (Macintosh, Apple Computer, Cupertino, CA).
Electron microscopy
Mice were treated with echistatin as described above and tissues
were isolated for light and electron microscopy. As described above,
mice were divided into three groups: controls (n = 4), Ca(-)/veh
(n = 9) and Ca(-)/ echistatin (n = 5). At the end of
infusion, animals were deeply anaesthetized with 0.08 mg/g ketamine and
0.004 mg/g xylazine, bled through the tail vein for serum echistatin
assay, and perfused through the heart with 4% paraformaldehyde and
2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.3.
Echistatin serum levels at the time the mice were euthanized ranged
from 170 to 750 nM and averaged 341 ± 195
nM. One tibia was processed in methyl methacrylate for
histomorphometry. For electron microscopy, the contralateral distal
femurs and proximal tibiae from some animals were immersed overnight in
the same perfusate fixative at 4 C. Bones were postfixed in 1%
OsO4 and 1.5% potassium ferricyanide, en bloc
stained in uranyl acetate, dehydrated in a graded ethanol series and
embedded in Epon through propylene oxide. Thin sections (0.1 µm),
stained with uranyl acetate and lead citrate, were photographed in a
Phillips CM12 electron microscope (Mahwah, NJ). Profiles of osteoclasts
were photographed in an area of cancellous bone in the distal femur
extending from the lower edge of the growth plate to approximately 1.0
mm below and extending for a width of approximately 0.5 mm, which
included both primary and secondary spongiosa. Profiles of osteoclasts
were categorized into three groups: 1) polarized osteoclasts with a
ruffled border and clear zones; 2) osteoclasts with clear zone or clear
zone-like areas only [profiles in this category may belong to active
osteoclasts whose clear zone only is in the plane of section or to
osteoclasts in the process of migrating (27)]; and 3) nonpolarized
profiles lacking specialized attachment or resorption structures. These
cells were identified as osteoclasts if mitochondria were abundant and
had densely staining matrix; cytoplasmic ground substance stained
darker than other cells and rough endoplasmic reticulum was of medium
length. Examples of each group are shown in Fig. 2.
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The number of nuclei within each osteoclast profile were counted and the mean ± SD per group was calculated. Differences between groups were tested by one-way ANOVA.
Immunohistochemical localization of echistatin in mice with
2°HPT
In a third study, the same treatment protocol was used to
generate tissue for immunohistochemistry at the light and electron
microscopy levels, except that perfusion was with 4% paraformaldehyde
and 0.1% glutaraldehyde in 0.1 M cacodylate buffer. Serum
echistatin values at the time the mice were euthanized ranged from 93
nM to 150 nM and averaged 122 ± 28.5
nM. Femurs and tibiae were dissected free of soft tissue
and immersion fixed for 4 h at 4 C and then washed in 0.1
M cacodylate buffer overnight. Bones were decalcified in
4.13% (wt/vol) ethylenediaminetetra-acetic acid (EDTA) and 5%
polyvinylpyrrolidone (PVP), pH 7.2, for 3 weeks. Decalcified bones were
dehydrated in a graded ethanol series. The right femur and tibia were
infiltrated and embedded in LR White (The London Resin Co., Ltd.,
Electron Microscopy Sciences, Fort Washington, PA) under vacuum at 50 C
overnight. Contralateral bones were processed and embedded in
paraffin.
LR White sections for light microscopy immunolabeling were cut at 0.3 µm with a diamond Histoknife (Diatome, Electron Microscopy Sciences) on a Reichert-Jung Utracut E (Vienna, Austria; M.O.C., Inc., Valley Cottage, NY). Thin LR White sections (0.1 µm) for EM-immunolabeling were mounted on 200 mesh nickel grids.
Electron microscopy immunogold labeling
Grids containing bone sections were floated, in vapor-saturated
chambers, on drops of blocking solution consisting of 10% normal goat
serum, 2% BSA, and 0.05% Tween-20 in Primary Antibody Diluent
(BioMeda, Foster City, CA), for 15 min at room temperature. Grids were
then incubated overnight at 4 C or for 2 h at 37 C with rabbit
antiechistatin antibody diluted 1:100 in Primary Antibody Diluent with
0.5% BSA, 0.05% Tween-20. Grids were washed in 0.05 M
Tris, 2% NaCl, 0.1% Tween-20, pH 8.2, and then incubated in goat
antirabbit IgG conjugated to 10 or 20 nm gold (BioCell; Goldmark
Biologicals, Phillipsburg, NJ) for 1 h at room temperature.
Sections were rinsed in buffer (3x, 2 min each) and then in deionized
water (4x, 1 min each). Sections were stained with uranyl acetate and
photographed in a Phillips CM 12 electron microscope (Mahwah, NJ).
Negative controls included sections that were incubated with nonspecific rabbit serum instead of primary antibody or with secondary antibodies alone. Sections from nonechistatin treated animals were also processed. The labeling profile observed in negative controls was considered to be nonspecific.
Peroxidase immunolabeling of paraffin embedded bone sections
Using capillary gap technology described in detail previously
(28), 4-µm sections were deparaffinized, and blocked for endogenous
peroxidase with 1% H2O2 in methanol for 30
min. After washing in dH2O (3x, 2 min) and 1x Automation
buffer (Biomeda Corp., Foster City, CA) (3x, 2 min each), sections
were blocked with 10% normal goat serum, 4% BSA, and 1% Tween-20 in
Primary Antibody Diluting Buffer (Biomeda Corp., Foster City, CA) for
20 min, followed by incubation overnight at 4 C in antiechistatin
antiserum (1:500 dilution) diluted in blocking solution. After washing
for 20 min in 1x Automation Buffer, sections were next incubated in
biotinylated goat antirabbit IgG (1:100 dilution; Fisher Scientific,
Inc., Pittsburgh, PA) for 30 min at room temperature. After washing in
1x Automation buffer, sections were incubated for 30 min in diluted
avidin-biotin-peroxidase complex (ABC kit, Vector Labs, Burlingame,
CA), washed in Tris-buffered saline-Tween 20, and incubated in 0.05%
(wt/vol) diaminobenzidine tetrahydrochloride (DAB, Sigma Chemical Co.,
St. Louis, MO), 0.001% H2O2 in 0.05
M Tris buffer, pH 7.6 for 10 min. After washing in
dH2O, sections were counterstained with Gill’s hematoxylin
no.1 (Sigma) for 1.5 min, washed, dehydrated, and sealed with Permount
before being examined by light microscopy.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, the calcemic
response to exogenous PTH was completely blocked by 30 µg/kg·min
echistatin.
After infusion of 30 µg/kg·min echistatin for 3 days into mice with
2°HPT, histomorphometric measurements in the corresponding groups
yielded similar values in the two experiments. Results from the pooled
analysis (N = 11–15 animals per group) are shown in Tables 1 and 2.
Compared with mice fed a normal diet, mice with 2°HPT had 36% lower
trabecular bone volume (BV/TV, Table 1). Trabecular bone volume of
echistatin treated animals was similar to that in normal controls and
significantly higher than in calcium deficient mice.
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, trabeculae were significantly thicker, more
numerous, and more closely spaced in both control and echistatin
treated animals as compared with mice on a calcium deficient diet.
Thus, the changes in cancellous bone architecture caused by a calcium
deficient diet were prevented by echistatin infusion. Femoral ash weight/length (mg/mm) were 1.70 ± 0.05 for the control group, 1.51 ± 0.06 for the Ca(-)/veh group, and 1.64 ± 0.04 for the Ca(-)/echistatin group. The 11% decrease in ash weight/mm in the Ca(-)/veh group relative to the control group was statistically significant. Ash weight/mm in the echistatin treated group was 9% higher than in the Ca(-)/veh group. This difference was not statistically significant.
Although serum calcium was lowest in the echistatin group, there were no statistically significant differences among the three groups at the time the mice were euthanized (control, 8.10 ± 0.22 mg/dl; Ca(-)/veh, 7.91 ± 0.21 mg/dl; and Ca(-)/echistatin, 7.85 ± 0.10 mg/dl).
Echistatin inhibits cancellous bone turnover and increases
osteoclast surface in mice with 2°HPT
Histomorphometric measurements of bone formation
surfaces (osteoid), percent osteoclast surface and osteoclast related
parameters are shown in Table 2. Percent osteoid surface increased in
Ca(-)/veh animals to 9.4% compared with 3.6% in controls and was
maintained at 3.2% in Ca(-)/echistatin animals. Thus, the increase in
cancellous bone turnover induced by a calcium deficient diet was
inhibited by echistatin.
The percent osteoclast surface was 5.5% in echistatin treated animals, vs. 3.6% in controls (P < 0.05), and 3.9% in Ca(-)/veh animals. Osteoclast number per tissue area (Oc#/TsA) was highest in echistatin treated animals. Increases were significant compared with Ca(-)/veh animals. There was also a trend for an increase in osteoclast number per trabecular surface and trabecular area in echistatin treated animals, about 1.6 times greater than in normal controls.
The mean length of contact with the bone surface for individual osteoclasts was 23.7 ± 2.5 µm for controls, 24.4 ± 11.0 µm for Ca(-)/veh animals, and 25.6 ± 7.0 µm for echistatin-treated animals, with no significant differences between groups. Because echistatin treatment did not reduce the number or size of osteoclasts on the bone surface, we proceeded to analyze osteoclast ultrastructure.
Osteoclast ultrastructure in echistatin treated mice appears
normal
Ultrastructural analysis of osteoclasts at the bone surface in
echistatin treated animals and control animals is shown in Table 3. ANOVA of osteoclast profiles showed
that the fraction of cells with each of the following characteristics
was not statistically different between groups: 1) cells with ruffled
border and clear zone (36–48%); 2) cells with clear zone or clear
zone-like attachment area only; and 3) cells without apparent polarity
(30–37%). Examples of each type are illustrated in Fig. 2. The
ultrastructural data thus show that echistatin, at levels that cause
inhibition of bone resorption, does not produce detectable changes in
osteoclast morphology.
On the average only one nucleus per osteoclast was detected at the ultrastructural level, and there were no differences between groups: values were 1.02 ± 0.31 nuclei per osteoclast for normal controls; 0.83 ± 0.14 for Ca(-)/veh mice, and 1.01 ± 0.18 Ca(-)/echistatin mice. The presence of multinucleation was not a requirement for identification of osteoclasts at the ultrastructural level.
Localization of echistatin in the bones of treated mice
At the light microscopy level, in either paraffin or LR White
sections, echistatin was detected specifically in osteoclasts and
megakarocytes (Fig. 3). Echistatin
labeling in osteoclasts was concentrated on basolateral surfaces,
particularly the basolateral microvilli, and to a lesser extent on
apical surfaces. Echistatin was not observed in other cell types. No
cells associated with capillary walls and no marrow cells other than
megakaryocytes were labeled.
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, echistatin label in osteoclasts was observed on the basolateral
membrane, in structures resembling coated pits (Fig. 4B, arrowhead) and in large vesicles with matrices of varying
electron densities located in the basolateral cytoplasm of the
osteoclast. Additionally, echistatin label was observed concentrated in
small granules above the ruffled border and the clear zone and at low
levels in the ruffled border membranes (Fig. 4A). A few grains were
detected in the clear zone membrane at the bone interface but not
significantly above background (Fig 4D). On the other hand echistatin
was clearly detected at the lateral edge of the clear zone (Fig. 4A, large arrowhead). At the EM level, a few endothelial cells
and fibroblast-like cells near the osteoclasts were labeled with
antiechistatin antibody (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study provides histomorphometric evidence for echistatin inhibition of bone resorption, by measuring cancellous bone volume and other parameters in mice with 2°HPT. In this model, increased resorption, in response to a rise in endogenous PTH levels, resulted in 36% reduction in cancellous bone volume and an 11% reduction in ash weight/mm. The architecture of the cancellous bone also changed: trabecular thickness and number were reduced and trabecular spacing increased. In addition, the rate of bone turnover increased, as indicated by a 2.6-fold increase in percent osteoid surface. These changes were all prevented by echistatin treatment, consistent with echistatin inhibition of bone resorption.
The ash weight/mm in the echi-treated group had an intermediate value between the calcium deficient and replete groups. The serum calcium level in echi treated animals was slightly lower than in the calcium replete controls. The explanation for these observations is as follows. During calcium deficiency, serum calcium is maintained by bone resorption. Cancellous bone, having a higher turnover rate compared with cortical bone, was more susceptible to increased resorption and the inhibitory effect of echistatin was, therefore, easier seen in cancellous bone. However, 80% of the ash weight represents cortical bone, which is also a major PTH target. Changes in both ash weight and serum calcium levels thus most likely reflect PTH effects on cortical bone which was only partially protected by echistatin treatment. Circulating levels of PTH in mice were not measured because appropriate antibodies are not available (to our knowledge).
Disintegrins, such as echistatin, bind to several RGD dependent
integrins, including the fibrinogen receptor,
IIbß3, the vitronectin receptor
vß3 and the fibronectin receptor
5ß1. The predominant target of echistatin
in bone has been suggested to be vß3, the
integrin expressed abundantly in osteoclasts (8, 9, 10, 11, 12, 13, 29, 30, 31).
In vivo at the resolution level of this study, echistatin
was localized predominantly to megakaryocytes, osteoclasts and no other
bone or bone marrow cells. In osteoclasts, at the ultrastructural
level, echistatin was localized to the basolateral membrane and
intracellular vesicles, and the edge of the clear zone. Some grains
were present in the clear zone membrane adjacent to the bone interface
but not significantly above background and the seal of the clear zone
did not appear to be morphologically disrupted (Fig. 4D).
Echistatin was also occasionally detected on fibroblast-like cells
usually associated with osteoclasts and on a few blood vessel
endothelial cells, probably by binding to
vß3. Although,
vß3 is one of the major integrins
expressed by isolated vascular endothelial cells (32, 33), in
vivo it was detected rarely in endothelial cells in mature blood
vessels. However, its expression was reported to be highly elevated
during neovascularization (34, 35).
Echistatin binding to megakaryocytes is consistent with the abundance
of IIbß3 in these cells (36, 37, 38), although
vß3 is also present at much lower levels
(38, 39). This localization raises the possibility of megakaryocyte or
stromal cell involvement in the effect of echistatin on bone
resorption. This in vivo study does not exclude such
effects, but the localization of echistatin to osteoclasts and the
previously reported in vitro effects on isolated osteoclasts
(15) would favor a direct effect on osteoclasts.
The identity of intracellular vesicles that contain echistatin has not been established. They could be endosomal vesicles involved in integrin endocytosis, following ligand binding. The recent work by Salo et al. (40) demonstrates a transcytosolic flow of bone resorption products from the apical surface to the basolateral surface of the resorbing osteoclast, that could include integrins and their ligands.
Although this study clearly demonstrates that echistatin inhibited bone
resorption in vivo, its mode of action requires further
study. The increase in osteoclast number is consistent with lower
osteoclast efficiency that leads to feedback driven osteoclast
recruitment and suggests that echistatin did not reduce osteoclast
formation or attachment to bone. Ultrastructural examination provided
no morphological explanation for the lower osteoclast efficiency. There
was no flattening of the ruffled border observed in the inactive
osteoclasts of osteopetrotic src (-/-) mice (41) or
bisphosphonate-treated rats (42), and in 150–200 osteoclasts examined
in each treatment group the fraction of cells with ruffled border or
clear zone was similar. In vitro
vß3 antibodies or RGD containing peptides
cause osteoclast retraction and detachment (8, 9, 10, 13, 14, 15, 17, 18, 43, 44, 45, 46, 47, 48). However, much higher concentrations of echistatin were
required to block osteoclast attachment than to reduce osteoclast
activity, suggesting additional effects (15).
There is ample evidence for an active role of
vß3 in the migration of many cell types
(49, 50, 51, 52, 53). One possibility is impaired osteoclast migration which could
increase the number and decrease the relative efficiency of osteoclasts
on the bone surface. Further studies will test this assumption.
The increase in osteoclast number also raises the
question of osteoclast life span. It has been shown that osteoclasts
undergo apoptosis and that the interaction of
vß3 with its natural ligands in other cell
types prevents apoptosis (3, 54). RGD compounds and integrin blocking
antibodies have been shown to induce apoptosis in angiogenic
endothelial cells and in melanoma tumor cells (55, 56, 57, 58). In this study
no apparent apoptotic osteoclasts were detected by EM criteria in
osteoclast population sizes ranging from 149 to 286, which could have
been too small.
The findings presented here are obviously valid for the model used. We have also completed a 4-week study in ovariectomized rats treated with echistatin and observed an increase in osteoclast number (58a). The effect of other agents could be different. In an in vivo study showing maintenance of bone mineral density by an RGD peptide mimetic (19) in ovariectomized rats, osteoclast number decreased after 6 weeks of treatment. These findings suggest a different mechanism, related possibly to the different compound, animal model, or duration of treatment.
In conclusion, this study presents direct evidence at the bone level that echistatin inhibits bone resorption in a 2°HPT model and that echistatin binds to osteoclasts in vivo. We have also provided the first in vivo observations on osteoclast morphology at the EM level following echistatin treatment, which show no detectable effects on ultrastructure or polarity. Echistatin may interfere with subtle aspects of integrin mediated signal transduction (6, 59, 60, 61, 62, 63) important for osteoclastic activity or migration. Regardless of mechanism, direct evidence for echistatin inhibition of bone resorption in vivo makes integrin ligands attractive candidates for the treatment of diseases associated with increased bone resorption.
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Acknowledgments |
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Footnotes |
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Received June 9, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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vß3,
2ß1 and vß1
integrins. J Biol Chem 268:16737–16745vß3 is present in the clear zone
and contributes to cellular polarization. Cell Tissue Res 286:507–515[CrossRef][Medline]
vß3 integrin inhibits
bone resorption in vitro and prevents osteoporosis in
vivo. J Clin Invest 99:2284–2292[Medline]
vß3 integrin. Program of the 16th Annual
Meeting of The American Society for Bone and Mineral Research, Kansas
City, MO, 1994, p S247 (Abstract)
vß3 and
vß5 integrins with extracellular matrix
components control cell attachment and migration. Program of the 17th
Annual Meeting of The American Society for Bone and Mineral Research,
Baltimore, MD, p S164 (Abstract)
vß3 for angiogenesis.
Science 264:569–571vß3 integrins in
platelet protein trafficking. Blood 78:2603–2610vß3 integrin is
necessary for smooth muscle cells to migrate in response to
insulin-like growth factor I. Proc Natl Acad Sci USA 93:2482–2487vß3-integrin
ligands raise [Ca2+]i in rat osteoclasts and
mouse-derived osteoclast-like cells. Am J Physiol
266:C376–C381
vß3 by osteopontin stimulates
phosphatidylinositol 3-hydroxyl kinase activity. Endocrinology 136:2984–2992[Abstract]
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