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Biomedical subjects

G A Rodan

Publications and source records attributed to G A Rodan.

At least 73 records · Page 4Linked to original sources
PubMed1996

Effect of alendronate on fracture healing and bone remodeling in dogs.

To examine the effect of alendronate (4-amino-1-hydroxybutylidene bisphosphonate) on fracture repair, the drug was given to mature beagle dogs orogastrically at 2 mg/kg/day for 9 weeks preceding fracture. 16 weeks after fracture, or both before and after fracture (25 weeks). A transverse mid-diaphyseal fracture of the right radius was surgically induced and was stabilized by external coaptation splinting. Fracture healing and bone remodeling were evaluated by radiography, gross and histological examination, and bone histomorphometry. The mechanical properties of the fracture callus were determined by a four-point bending test. Radiographs and gross and microscopic examination demonstrated normal bone healing at the fracture site in all dogs. In dogs that received alendronate during the fracture healing period, at 16 weeks the calluses were approximately 2-3 times larger than those in dogs that received a placebo during the healing period. This is consistent with slower callus bone remodeling, an expected pharmacological effect of the compound. Bone histomorphometry demonstrated that treatment with alendronate did not inhibit bone formation or mineralization. Mechanical testing showed that the ultimate load at failure and the flexural rigidity of both the fractured and contralateral intact bone were unaffected by treatment with alendronate. Therefore, in this study, treatment with alendronate before or during fracture healing, or both, resulted in no adverse effects on the union, strength, or mineralization of bone in mature beagle dogs.

Alendronate↗
PubMed1996

Identification of fatty acid methyl ester as naturally occurring transcriptional regulators of the members of the peroxisome proliferator-activated receptor family.

The nuclear hormone receptors NUC-1 (PPAR delta) and PPAR alpha are members of the peroxisome proliferator-activated receptor (PPAR) family. The members of this receptor family are activated by agents that stimulate peroxisome proliferation, free fatty acids, prostaglandin 12 metabolites, and agents considered for the therapy of insulin-independent diabetes mellitus. To identify putative physiological agents that activate NUC-1, we tested the ability of acetone extracts of various rat tissues to activate the transcription of an MMTV-luciferase reporter gene, via a GR/NUC-1 hybrid receptor. GR/NUC-1 contains the ligand binding region of the NUC-1 receptor and the DNA binding domain of the glucocorticoid receptor. Using this assay, we found stimulatory activity in the pancreas, which upon purification and characterization was identified as methyl-palmitate, known to be enriched in pancreatic lipids. In addition, we determined that ethyl esters of palmitic and oleic acids are also potent activators of this receptor. Thus, fatty acid ester formation may control the cellular concentrations of fatty acids, and acyl-ester formation may play a role in the control of metabolic pathways and the activation of the PPAR.

Animals↗
PubMed1996

Comparison of the distribution of 3H-alendronate and 3H-etidronate in rat and mouse bones.

Alendronate and etidronate are bisphosphonates used clinically to treat diseases associated with increased bone resorption. Etidronate is less potent and was reported to cause osteomalacia. This study examines if differences in distribution of alendronate and etidronate in the skeleton can explain differences in efficacy and in effects on mineralization between the two drugs. Eight-day old rat pups were injected s.c. with 3H-alendronate or 3H-etidronate both at either 1.3 mumol/kg or at their respective pharmacological effective doses in the growing rat of 0.12 mumol/kg for alendronate and 72.8 mumol/kg for etidronate. Twelve hours after administration at 1.3 mumol/kg both drugs showed a three- to fourfold higher localization on osteoclast vs. osteoblast surface. At the pharmacologically effective doses, 3H-alendronate labeled eightfold more osteoclast surface than osteoblast surface. In contrast, 3H-etidronate labeled approximately equal fractions of osteoclast and osteoblast surface. When similar doses of 3H-etidronate and 3H-alendronate (0.24 mumol/kg 3H-etidronate vs. 0.20 mumol/kg 3H-alendronate; 1.5 mumol/kg 3H-etidronate vs. 1.2 mumol/kg 3H-alendronate; and 14.6 mumol/kg 3H-etidronate vs. 12.0 mumol/kg 3 H-alendronate) were injected intravenously into adult mice at similar specific activities, 3H-etidronate labeled 1.5-2.5 times more osteoclast surface than 3-H-alendronate, but 3 to 15 times more osteoblast surface. Consequently, the ratio between the fraction of labeled osteoclast surface and the fraction of labeled osteoblast surface ranged for 3H-alendronate from 9 to 24, whereas for 3H-etidronate the range was from 4 to 7, due to more extensive labeling of osteoblast surface by 3H-etidronate. In a third experiment, we confirmed in adult mice the previous observation made in rat pups that normal bone formation occurs over alendronate-covered bone surfaces, and found that it occurred over etidronate-covered surfaces as well. Forty nine days after s.c. administration of alendronate at 0.12 mumol/kg or etidronate at 1.3 mumol/kg or 55.3 mumol/kg into adult mice bone formed over drug label. The distance from incorporated label to bone surface for both drugs (12.7 microns for alendronate and 8.7 and 9.2 microns for etidronate) was similar to wall width (defined by cement line) in controls (10.6 microns). In conclusion, alendronate, especially at pharmacologically active doses, shows higher uptake on resorption vs. formation surfaces than etidronate. The extent of bone formation on surfaces containing alendronate or etidronate is similar and is comparable to the "wall width" in controls.

Alendronate↗
PubMed1996

Long-term safety of the aminobisphosphonate alendronate in adult dogs. II. Histomorphometric analysis of the L5 vertebrae.

Osteoporosis is a major health problem. Bisphosphonates are synthetic compounds that are taken up preferentially by the skeleton and suppress osteoclast-mediated bone resorption. Alendronate (ALN) is an aminobisphosphonate used in the treatment of diseases characterized by increased bone resorption including osteoporosis. The purpose of this study was to rule out possible adverse effects of the chronic administration of ALN on the quality of bone. Thirty-one 20- to 21-month-old beagles (n = 7-8 per group, females and males) were treated p.o. for 157 weeks with ALN at 0.00, 0.25, 0.5 or 1.00 mg/kg/day and labeled in vivo with oxytetracycline and calcein. Undecalcified bone sections obtained from the fifth lumbar vertebra were evaluated by static and dynamic histomorphometry. Cortical or cancellous bone volume and indices of architecture were not affected by treatment. Osteoid thickness and mineralization lag time were not changed by treatment. Tissue level bone turnover was inhibited in a dose-dependent manner in males. The results indicate that long-term ALN administration at up to 5 times the dose used for treatment of osteoporosis in clinical trials causes no abnormalities in bone remodeling or bone structure.

Alendronate↗
PubMed1995

Isolation and characterization of highly enriched, prefusion mouse osteoclastic cells.

This study describes the isolation and characterization of highly enriched mammalian osteoclast precursors, released by the "disintegrin" echistatin, from an osteoclast formation culture. Incubation of a 6-day coculture of mouse bone marrow cells and mouse osteoblastic cells (MB1.8) with echistatin (30 nM), an RGD-containing snake venom, for 20 min yielded an 88-95% pure population of tartrate-resistant acid phosphatase-positive cells, 1.5 x 10(5) cells per 150 cm2 culture dish. These cells were mostly mononucleated and based on the following characteristics are considered to be prefusion osteoclasts (pOC cells): (i) presence of calcitonin (CT) receptors documented by 125I-sCT autoradiography and cAMP generation in response to salmon calcitonin; (ii) expression of mRNAs for alpha v beta 3 integrin, osteopontin, 92-kDa type IV collagenase (matrix metalloproteinase 9), carbonic anhydrase II, OC-2 (an "osteoclastic" cysteine proteinase), and protein tyrosine phosphatase epsilon; and (iii) high level expression of pp60c-src protein. The pOC cells resorb bone (form "pits" on bone slices) but only in the presence of osteoblastic MB1.8 cells and 1,25(OH)2D3. Resorption was inhibited by CT. In conclusion, we describe a rapid, reproducible procedure to isolate virtually pure mammalian prefusion osteoclasts, which should help in the study of osteoclast formation, composition, and function.

Animals↗
PubMed1995

Comparison of alendronate and sodium fluoride effects on cancellous and cortical bone in minipigs. A one-year study.

Fluoride stimulates trabecular bone formation, whereas bisphosphonates reduce bone resorption and turnover. Fracture prevention has not been convincingly demonstrated for either treatment so far. We compared the effects of 1-yr treatment of 9-mo-old minipigs with sodium fluoride (NaF, 2 mg/kg/d p.o.) or alendronate (ALN, 4 amino-1-hydroxybutylidene bisphosphonate monosodium, 1 mg/kg/d p.o.) on the biomechanical and histomorphometric properties of pig bones. As expected, NaF increased and ALN decreased bone turnover, but in these normal animals neither changed mean bone volume. NaF reduced the strength of cancellous bone from the L4 vertebra, relative to control animals, and the stiffness (resistance to deformation) of the femora, relative to the ALN group. In the ALN-treated animals, there was a strong positive correlation between bone strength and L5 cancellous bone volume, but no such correlation was observed in the NaF group. Furthermore, the modulus (resistance to deformation of the tissue) was inversely related to NaF content and there was a relative decrease in bone strength above 0.25 mg NaF/g bone. Moreover, within the range of changes measured in this study, there was an inverse correlation between bone turnover, estimated as the percentage of osteoid surface, and modulus. These findings have relevant implications regarding the use of these agents for osteoporosis therapy.

Alendronate↗
PubMed1995

Acid extrusion is induced by osteoclast attachment to bone. Inhibition by alendronate and calcitonin.

Acid extrusion is essential for osteoclast (OC) activity. We examined Na+ and HCO3(-)-independent H+ extrusion in rat- and mouse OCs by measuring intracellular pH (pHi) changes, with the pHi indicator BCECF (biscarboxyethyl-5-(6) carboxyfluorescein) after H+ loading with an ammonium pulse. 90% of OCs attached to glass do not possess HCO3- and Na(+)-independent H(+)-extrusion (rate of pHi recovery = 0.043 +/- 0.007 (SEM) pH U/min, n = 26). In contrast, in OCs attached to bone, the pHi recovery rate is 0.228 +/- 0.011 pHi U/min, n = 25. OCs on bone also possess a NH(4+)-permeable pathway not seen on glass. The bone-induced H+ extrusion was inhibited by salmon calcitonin (10(-8) M, for 2 h), and was not present after pretreating the bone slices with the aminobisphosphonate alendronate (ALN). At ALN levels of 0.22 nmol/mm2 bone, H+ extrusion was virtually absent 12 h after cell seeding (0.004 +/- 0.002 pH U/min) and approximately 50% inhibition was observed at 0.022 pmol ALN/mm2 bone. The Na(+)-independent H+ extrusion was not inhibited by bafilomycin A1 (up to 10(-7) M), although a bafilomycin A1 (10(-8) M)-sensitive H+ pump was present in membrane vesicles isolated from these osteoclasts. These findings indicate that Na(+)-independent acid extrusion is stimulated by osteoclast attachment to bone and is virtually absent when bone is preincubated with ALN, or when osteoclasts are treated with salmon calcitonin.

Alendronate↗
PubMed1995

Endogenous calcitonin attenuates parathyroid hormone-induced cancellous bone loss in the rat.

Although calcitonin (CT) treatment has been shown to prevent bone loss in estrogen-deficient states, the function of endogenous CT in bone metabolism is not clearly established. To test the hypothesis that endogenous CT has a role in bone conservation, we compared the bone-resorbing effect of exogenous PTH between CT-deficient [thyroparathyroidectomized (TPTX)] and CT-sufficient [parathyroidectomized (PTX)] rats. Studies were carried out with two doses (30 and 40 pmol/h) of bovine PTH-(1-34) to examine dose responsiveness and with or without T4 replacement in TPTX rats to exclude the influence of thyroid function on the results. Sham-operated control rats received vehicle. At comparable hypercalcemia (mean +/- SEM, 13.6 +/- 0.8 vs. 12.7 +/- 1.0 mg/dl) after 3 days of sc infusion of 30 pmol/h PTH, serum CT levels were significantly (P < 0.05) higher in PTX rats (66.0 +/- 8.0 pg/ml) than in TPTX rats (17.7 +/- 4.3). CT-deficient TPTX rats showed a significant cancellous bone loss in the proximal tibia [bone volume (BV/TV), 4.2 +/- 1.0%] compared with control rats 10.4 +/- 1.2%) In contrast, there was no bone loss in CT-sufficient PTX rats (BV/TV, 10.9 +/- 0.5%). A similar difference in the serum CT level and more marked difference in BV/TV (0.9 +/- 0.3% vs. 8.1 +/- 1.3%) were observed between TPTX and PTX rats infused with 40 pmol/h PTH. The magnitude of bone loss in TPTX rats was not different between T4-supplemented and nonsupplemented groups. Unlike cancellous bone, the PTH-induced decrease in the cortical thickness of the tibia was comparable in TPTX and PTX rats. The extent of increase in serum osteocalcin after PTH infusion was not different between TPTX and PTX groups. These results indicate that in the rat, endogenous CT has a protective effect against PTH-stimulated cancellous bone loss, but not cortical bone loss.

Animals↗
PubMed1995

Identification of a rat osteocalcin promoter 3',5'-cyclic adenosine monophosphate response region containing two PuGGTCA steroid hormone receptor binding motifs.

In MC3T3-E1 mouse osteoblastic cells, the adenylate cyclase activator forskolin increases osteocalcin (OC) mRNA levels. We have analyzed the effects of forskolin and 8-Br cAMP on the transcriptional activity of the rat OC promoter (with luciferase reporter) in MC3T3-E1 cells. Both forskolin and 8-Br cAMP activate the rat OC promoter 2- to 5-fold. By 5' deletion analysis, we have mapped the cAMP response to the region -121 to -92. The 48-base pair rat OC promoter region -121 to -74 (hence denoted ROCRR) can confer cAMP responsiveness to an unresponsive heterologous minimal promoter. Crude nuclear extracts prepared from MC3T3-E1 cells form three complexes with the ROCRR by gel shift analysis. No specific change in nuclear factor binding in response to cellular forskolin treatment could be demonstrated. Intriguingly, two nuclear factor complexes bound to the ROCRR also recognized the thyroid hormone response element palindrome (AGGTCATGACCT) but did not bind the classic cAMP (TGACGTCA) or glucocorticoid (AGAACANNNTGTTCT) response elements. The rat OC promoter possesses two directly repeated PuGGTCA steroid hormone response element hexamer motifs (bottom strand) in the region -114 to -93 within the ROCRR, separated by a 10 nucleotide spacer. Oligos encoding the individual rat OC hexamer sites compete for the ROCRR DNA:protein complexes recognized by the thyroid hormone response element palindrome. Removal of the up-stream hexamer site by 5' deletion (-121 to -100) in the context of the native OC promoter abrogates cAMP responsiveness. Taken together, these data suggest that this novel rat OC cAMP response region assembles a protein:DNA complex containing member(s) of the steroid hormone receptor superfamily. Transcriptional activity, but not DNA binding, is regulated by cAMP.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

The role of prostaglandins in bone formation.

Prostaglandins of the E series have been shown to be effective inducers of bone formation in vivo. In this study, the effects of PGE2 were evaluated in vivo using subcutaneous administration (3 mg/kg/d for 25 days) to ovariectomized rats or local application in the marrow cavity of tibiae of rats using biodegradable implants (0.13, 1.4 and 32 microg released over 8 days). Systemic treatment of rats with PGE2 stimulated cancellous bone formation in the metaphysis of the proximal tibiae as well as endocortical bone formation and de novo trabecular bone formation in the marrow cavity. Local delivery of PGE2 increased cancellous bone volume in the secondary spongiosa and cortical thickness (at 32 microg). Comparisons of prostanoid effects in vitro, in a bone-derived cell line, showed that PGF2alpha was a better stimulator of DNA synthesis than PGE2. PGF2alpha increased the steady state levels of IGF-I receptor mRNA while PGE2 increased IGF-I expression. Although the mechanism of bone formation by PGE2 is not known at this time, it is clear that PGE2 has powerful local anabolic effects on bone formation in vivo possibly by mediating responses to signals such as changes in mechanical force.

Animals↗
PubMed1995

Expression and regulation of vascular endothelial growth factor in osteoblasts.

Bone formation is linked closely to angiogenesis. Because prostaglandin E2 (PGE2) is a potent stimulator of bone formation, its effects were evaluated on vascular endothelial growth factor, a secreted endothelial cell-specific mitogen, and a potent angiogenic protein. Prostaglandin E2 increased vascular endothelial growth factor protein in conditioned media of osteoblastic RCT-3 cells within 3 hours. Prostaglandin E2 also increased the steady-state levels of vascular endothelial growth factor mRNA in a dose-dependent manner. The increased expression of vascular endothelial growth factor mRNA produced by PGE2 was rapid (maximal at 1 hour) and was enhanced by the protein synthesis inhibitor cycloheximide (5 micrograms/ml). The increase in vascular endothelial growth factor mRNA by PGE2 was inhibited strongly by pretreatment for 3 hours with dexamethasone (10(-7) M). Stimulation of vascular endothelial growth factor by PGE2 and its suppression by dexamethasone implicate the involvement of vascular endothelial growth factor in bone metabolism.

Animals↗
PubMed1994

NER, a new member of the gene family encoding the human steroid hormone nuclear receptor.

NER, a new member of the steroid hormone nuclear receptor (NR)-encoding gene family, was isolated from a human osteosarcoma SAOS/B10 cell line cDNA library. NER codes for a polypeptide of 461 amino acids which contains the conserved sequences of the DNA-binding and ligand-binding domains of typical steroid hormone NR. It has highest homology with the retinoic acid receptors: 55% at the DNA-binding domain and 38-40% at the ligand-binding domain. A single transcript of 2.3 kb was detected in all cells and tissues tested. Although no ligand was identified for NER-I, its wide distribution may indicate that this novel steroid hormone NR may play a basic role in cell function.

Amino Acid Sequence↗
PubMed1994

Interleukin-6 deficient mice are protected from bone loss caused by estrogen depletion.

Interleukin-6 (IL-6) is a multifunctional cytokine whose circulating levels are under physiological conditions below detection, but whose production is rapidly and strongly induced by several pathological and inflammatory stimuli. IL-6 has been implicated in a number of cell functions connected to immunity and hematopoiesis. Recently, it has been proposed to act as a stimulator of osteoclast formation and activity, in particular following estrogen depletion. The purpose of this study was to gain additional insights into the role of IL-6 during development, as well as in physiological and pathological conditions. We report here that IL-6 deficient mice generated by gene targeting are viable and do not present any evident phenotypic abnormality. However, analysis of bone metabolism revealed a specific bone phenotype. IL-6 deficient female mice have a normal amount of trabecular bone, but higher rates of bone turnover than control littermates. Estrogen deficiency induced by ovariectomy causes in wild type animals a significant loss of bone mass together with an increase in bone turnover rates. Strikingly, ovariectomy does not induce any change in either bone mass or bone remodeling rates in the IL-6 deficient mice. These findings indicate that IL-6 plays an important role in the local regulation of bone turnover and, at least in mice, appears to be essential for the bone loss caused by estrogen deficiency.

Animals↗
PubMed1994

Good hope for making osteoporosis a disease of the past.

There is currently very strong cross-sectional and prospective epidemiological data showing that low bone mass is a major risk factor for osteoporotic fractures. The prevention of bone loss caused by estrogen deficiency, calcium and vitamin D deficiency or other causes could therefore significantly reduce the incidence of osteoporotic fractures. Increased understanding of the pathophysiology of the bone loss that commonly occurs after the fourth decade in life, and the ability to control it by hormone replacement therapy or pharmacological means, offer good hope for making osteoporosis, as we known it today, a disease of the past.

Aging↗
PubMed1994

Histomorphometrical analysis of the effects of the bisphosphonate alendronate on bone loss caused by experimental periodontitis in monkeys.

This study tested the efficacy of alendronate, a bisphosphate, in reducing alveolar bone loss caused by experimental periodontitis in cynomolgus monkeys. Periodontitis was initiated in adult monkeys by ligating mandibular molar teeth at the cementoenamel junction (CEJ) and subsequently inoculating the ligature with Porphyromonas (Bacteroides) gingivalis. Contralateral, homologous non-ligated teeth served as controls. Animals received, intravenously, either saline (placebo) or alendronate at 0.05 or 0.25 mg/kg every 2 weeks for 16 weeks. After the animals were sacrificed, coronal sections through mandibular molars were subjected to histomorphometrical analysis. No overt side-effects were observed in any of the animals participating in this study. In placebo-treated animals, ligation and inoculation resulted in significant bone loss both at the CEJ and at the furcation. Alendronate at 0.05 mg/kg significantly reduced bone loss associated with the experimental periodontitis at both sites. In contrast, the dose of 0.25 mg/kg was ineffective in attenuating alveolar bone loss in the furcation area and only slightly effective in preventing it at the CEJ area. The results of the histomorphometric analysis correlate closely with those of the radiographic analysis of the same experiment. These data indicate that alendronate could reduce the loss of alveolar support associated with periodontitis and suggest that bisphosphonates, by virtue of their significant inhibitory action on osteoclasts, may become a treatment modality in the battle against alveolar bone destruction during periodontal disease.

Alendronate↗
PubMed1994

Soluble alpha v beta 3-integrin ligands raise [Ca2+]i in rat osteoclasts and mouse-derived osteoclast-like cells.

We evaluated the possible involvement of intracellular Ca2+ concentration ([Ca2+]i) changes in the action of alpha v beta 3-ligands, known to regulate osteoclast function. Rat osteoclasts or mouse osteoclast-like cells, as examined by microfluorimetry and fura 2, showed a transient [Ca2+]i increase when perfused with (all 0.1 microM) vitronectin, osteopontin, polypeptide echistatin, fibronectin, and Arg-Gly-Asp-Asp and Arg-Gly-Asp-Ser peptides (10(-4) M) but not with laminin, collagen I, collagen IV, or [Ala24]echistatin, in which Ala was substituted for Arg in the Arg-Gly-Asp complex. The threshold for echistatin was 10 pM, the 50% effective concentration was 1 nM, and the median [Ca2+]i increase was 420 nM above the resting level (217 +/- 22 nM) at saturating concentration of 0.1 microM. Echistatin did not cause Mn2+ influx, and 10 microM nifedipine, 10 nM omega-conotoxin, 5 mM Ni2+, or Cd2+ did not prevent [Ca2+]i change. However, extracellular Ca2+ was needed for the [Ca2+]i increase, probably enabling ligand-integrin interaction. Polyclonal and monoclonal (LM609) antibody as well as depletion of [Ca2+]i stores with 5 microM thapsigargin and Ca(2+)-free medium abolished the [Ca2+]i increase, after restoring extracellular Ca2+. Furthermore, the LM609 antibody induced a Ca2+ signal in the presence or absence of extracellular Ca2+, suggesting that the alpha v beta 3-ligand interaction is mediated at least partially by Ca2+ mobilized from intracellular stores.

Animals↗