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Biomedical subjects

I R Garrett

Publications and source records attributed to I R Garrett.

At least 19 recordsLinked to original sources
PubMed2006

Transdermal application of lovastatin to rats causes profound increases in bone formation and plasma concentrations.

INTRODUCTION: Statins are drugs that inhibit HMG Co-A reductase and have been shown to enhance bone formation in vitro and in vivo in rodents. However, the statins currently used for cholesterol-lowering have been selected for their capacity to target the liver where their effects on cholesterol synthesis are mediated and they undergo first pass metabolism. When given in lipid-lowering doses, these agents do not likely reach sufficient blood concentrations to reliably cause substantial increases in bone formation in humans. Moreover, statins are inactivated by cytochrome P450 enzymes, resulting in even less peripheral distribution of the biologically active moieties beyond the liver. METHOD: To investigate whether an alternate method of administration might produce beneficial effects on bone formation, we administered lovastatin by dermal application to rats to circumvent the first-pass effects of the gut wall and liver. RESULTS: We found that the statin blood levels measured by HMG Co-A reductase activity were higher, maintained longer and less variable following transdermal application than those following oral administration. Also the increased circulating statin levels were associated with significantly enhanced biological effects on bone. After only 5 days of administration of transdermal lovastatin to rats, there was a 30-60% increase in trabecular bone volume, and 4 weeks later, we observed more than a 150% increase in bone formation rates. There was also a significant increase in serum osteocalcin, a marker of bone formation. We also found that lovastatin administered transdermally produces these profound effects at doses in the range of 1% of the oral dose, without any evidence of the hepatotoxicity or myotoxicity that can occur following oral statin administration. Several doses (0.01-5 mg kg(-1) day(-1)) and dosage schedules were examined, and collectively the data strongly suggest a powerful anabolic effect but with an unusually flat dose-response curve. CONCLUSION: These results show transdermal application of statins produces greater beneficial effects on bone formation than oral administration does.

Administration, Cutaneous↗
PubMed2005

Inhibition of osteoporosis in autoimmune disease prone MRL/Mpj-Fas(lpr) mice by N-3 fatty acids.

OBJECTIVE: Rheumatoid arthritis (RA) is a systemic autoimmune inflammatory disease involving the breakdown of cartilage and juxta-articular bone, which is often accompanied by decreased bone mineral density (BMD) and increased risk of fracture. Anti-inflammatory omega-3 fatty acids may prevent arthritis and bone loss in MRL/lpr mice model of arthritis and in humans. METHODS: In this study, the effect of long term feeding of 10% dietary n-3 (fish oil (FO)) and n-6 (corn oil (CO)) fatty acids begun at 6 weeks of age on bone mineral density (BMD) in different bone regions in an MRL/lpr female mouse model of RA was measured at 6, 9, and 12 months of age by dual energy x-ray absorptiometry (DEXA). After sacrificing the mice at 12 months of age, antioxidant enzyme activities were measured in spleen, mRNA for receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG) was measured by RT-PCR in lymph nodes, and synovitis was measured in leg joints. RESULTS: At 6, 9 and 12 months of age, BMD was significantly higher (p < 0.05) in distal femur, proximal tibia, and lumbar spine of FO fed mice than those of CO fed mice. Spleen catalase (CAT) and superoxide dismutase (SOD) activities were also significantly higher (p < 0.01) in FO fed mice than in CO fed mice. Histology of knee joints revealed mild synovitis in CO fed mice, which was not present in FO fed mice. RT-PCR analysis of lymph nodes revealed decreased RANKL mRNA (p < 0.001) expression and enhanced OPG mRNA expression (p < 0.01) in FO fed mice compared to CO fed mice. CONCLUSIONS: These results suggest beneficial effects of long-term FO feeding in maintaining higher BMD and lower synovitis in this mouse model. These beneficial effects may be due, in part, to increased activity of antioxidant enzymes, decreased expression of RANKL, and increased expression of OPG in FO fed mice thereby altering the RANKL/OPG ratio. These significant beneficial effects on BMD suggest that FO may serve as an effective dietary supplement to prevent BMD loss in patients with RA.

Absorptiometry, Photon↗
PubMed2004

MEPE has the properties of an osteoblastic phosphatonin and minhibin.

Matrix extracellular phosphoglycoprotein (MEPE) is expressed exclusively in osteoblasts, osteocytes and odontoblasts with markedly elevated expression found in X-linked hypophosphatemic rickets (Hyp) osteoblasts and in oncogenic hypophosphatemic osteomalacia (OHO) tumors. Because these syndromes are associated with abnormalities in mineralization and renal phosphate excretion, we examined the effects of insect-expressed full-length human-MEPE (Hu-MEPE) on serum and urinary phosphate in vivo, (33)PO(4) uptake in renal proximal tubule cultures and mineralization of osteoblast cultures. Dose-dependent hypophosphatemia and hyperphosphaturia occurred in mice following intraperitoneal (IP) administration of Hu-MEPE (up to 400 microg kg(-1) 31 h(-1)), similar to mice given the phosphaturic hormone PTH (80 microg kg(-1) 31 h(-1)). Also the fractional excretion of phosphate (FEP) was stimulated by MEPE [65.0% (P < 0.001)] and PTH groups [53.3% (P < 0.001)] relative to the vehicle group [28.7% (SEM 3.97)]. In addition, Hu-MEPE significantly inhibited (33)PO(4) uptake in primary human proximal tubule renal cells (RPTEC) and a human renal cell line (Hu-CL8) in vitro (V(max) 53.4% inhibition; K(m) 27.4 ng/ml, and V(max) 9.1% inhibition; K(m) 23.8 ng/ml, respectively). Moreover, Hu-MEPE dose dependently (50-800 ng/ml) inhibited BMP2-mediated mineralization of a murine osteoblast cell line (2T3) in vitro. Inhibition of mineralization was localized to a small (2 kDa) cathepsin B released carboxy-terminal MEPE peptide (protease-resistant) containing the acidic serine-aspartate-rich motif (ASARM peptide). We conclude that MEPE promotes renal phosphate excretion and modulates mineralization.

Amino Acid Sequence↗
PubMed2003

Selective inhibitors of the osteoblast proteasome stimulate bone formation in vivo and in vitro.

We have found that the ubiquitin-proteasome pathway exerts exquisite control of osteoblast differentiation and bone formation in vitro and in vivo in rodents. Structurally different inhibitors that bind to specific catalytic beta subunits of the 20S proteasome stimulated bone formation in bone organ cultures in concentrations as low as 10 nM. When administered systemically to mice, the proteasome inhibitors epoxomicin and proteasome inhibitor-1 increased bone volume and bone formation rates over 70% after only 5 days of treatment. Since the ubiquitin-proteasome pathway has been shown to modulate expression of the Drosophila homologue of the bone morphogenetic protein-2 and -4 (BMP-2 and BMP-4) genes, we examined the effects of noggin, an endogenous inhibitor of BMP-2 and BMP-4 on bone formation stimulated by these compounds and found that it was abrogated. These compounds increased BMP-2 but not BMP-4 or BMP-6 mRNA expression in osteoblastic cells, suggesting that BMP-2 was responsible for the observed bone formation that was inhibited by noggin. We show proteasome inhibitors regulate BMP-2 gene expression at least in part through inhibiting the proteolytic processing of Gli3 protein. Our results suggest that the ubiquitin-proteasome machinery regulates osteoblast differentiation and bone formation and that inhibition of specific components of this system may be useful therapeutically in common diseases of bone loss.

Animals↗
PubMed2001

Statins and bone formation.

The main therapy needed most in the bone field is an anabolic agent for the treatment of osteoporosis. Current drugs on the market, which included bisphosphonates, calcitonin, estrogen and related compounds, vitamin D analogues trabecular microarchitecture. Therefore, it would be desirable to have a satisfactory and universally and iprifalvone, are essentially bone resorption inhibitors that mainly act to stabilize bone mass. Patients with established osteoporosis have lost more than 50% of their bone mass at critical sites in the skeleton, and more over have marked disruption of acceptable drug that would stimulate new bone formation and correct this disturbance of trabecular microarchitecture characteristic of established osteoporosis. Recently inhibitors of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, which controls the first step in the biosynthesis of cholesterol, have been shown to stimulate bone formation in rodents both in vitro and in vivo. The effect is associated with an increased expression of the bone morphogenetic protein-2 (BMP-2) gene in bone cells. These statins drugs are widely used agents for lowering cholesterol and reducing heart attacks, however they are also known to elicit numerous pleiotropic effects including inhibition of proliferation and migration of smooth muscle cells, inhibition of tumor growth and anti-inflammatory activity. Some of these effects have been attributed to not only to the reduction of cholesterol synthesis by inhibition of the HMG-CoA reductase enzyme but also by the concurrent reduction in downstream metabolites of the mevalonate pathway such as mevalonate, farnesyl pyrophosphate and geranylgeranyl pyrophosphate. The findings that statins are capable of increasing bone formation and bone mass in rodents suggests a potential new action for the statins, which may be beneficial in patients with established osteoporosis where marked bone loss has occurred. Recent clinical data suggests that they may reduce the risk of fracture in patients taking these drugs. However, their precise role can only be determined by appropriate randomized clinical trials, which demonstrate their efficacy in this regard in patients.

Animals↗
PubMed1999

Ibandronate reduces osteolytic lesions but not tumor burden in a murine model of myeloma bone disease.

We determined the effects of the potent bisphosphonate ibandronate in a murine model of human myeloma bone disease. In this model, bone lesions typical of the human disease develop in mice following inoculation of myeloma cells via the tail vein. Treatment with ibandronate (4 micrograms per mouse per day) significantly reduced the occurrence of osteolytic bone lesions in myeloma-bearing mice. However, ibandronate did not prevent the mice from developing hindlimb paralysis and did not produce a detectable effect on survival. There was no significant effect of ibandronate on total myeloma cell burden, as assessed by morphometric measurements of myeloma cells in the bone marrow, liver, and spleen, or by measurement of serum IgG2b levels. These results support clinical findings that bisphosphonates may be useful for the treatment of myeloma-associated bone destruction, but suggest that other therapies are also required to reduce tumor growth.

Animals↗
PubMed1999

Systemic administration of acidic fibroblast growth factor (FGF-1) prevents bone loss and increases new bone formation in ovariectomized rats.

There are no universally accepted agents that will substantially increase bone mass in osteoporotic patients. A number of peptides important in normal bone formation, such as members of the transforming growth factor-beta superfamily, are not satisfactory for this purpose either because their beneficial effects are predominantly local or there is systemic toxicity associated with their administration. We have examined the effects of exogenous fibroblast growth factor-1 and -2 (FGF-1 and FGF-2) on bone in vivo, since FGFs have been shown recently to be essential for normal skeletal development. FGF-1 was injected daily (0.2 mg/kg intravenously) for 28 days into the tail vein of adult female rats immediately following and 6 months after sham operation or ovariectomy (OVX). In rats treated immediately post-OVX, OVX produced more than a 30% decrease in tibial bone density, which was prevented by FGF-1 and estrogen. However, FGF-1 also had an anabolic effect. In sham-operated rats, FGF-1 increased bone density to 2-fold, whereas estrogen had no effect. In rats 6 months post-OVX, severe bone loss and disruption of trabecular microarchitecture occurred similar to that seen in patients with severe osteoporosis. In these rats, administration of FGF-1 induced extensive new woven bone formation with new trabecular-like structures filling much of the marrow spaces, and bone density in the tibial metaphysis increased 3-fold. FGF-1 and FGF-2 were also administered subcutaneously over the calvaria of mice in doses of 2-2000 microg/day for 3 days and shown to produce substantial increases in bone formation when examined morphologically. Thus, we conclude that both local and systemic FGF-1 increases new bone formation and bone density, and systemic FGF-1 also appears to restore bone microarchitecture and prevent bone loss associated with estrogen-withdrawal.

Animals↗
PubMed1998

5-Lipoxygenase metabolites inhibit bone formation in vitro.

The leukotrienes and peptido-leukotrienes are 5-lipoxygenase (5-LO) metabolites of arachidonic acid that appear to have unique effects on bone, distinct from those of the prostaglandins. Application of exogenous leukotrienes in vitro and in vivo results in increased osteoclast formation and bone resorption. While 5-LO metabolites of arachidonic acid clearly stimulate osteoclastic bone resorption, little is known concerning their effects on osteoblastic bone formation. We examined the effects of the 5-LO metabolites 5-HETE, the leukotriene LTB4 and, as representative of the peptido-leukotrienes, LTD4 on the formation of mineralized nodules of fetal rat calvarial cells in the presence of dexamethasone and recombinant human bone morphogenetic protein-2 (rhBMP-2). We also examined the effects of these 5-LO metabolites on alkaline phosphatase activity and cell proliferation in these cultures and the effects of 5-HETE and LTB4 on cultured explants of neonatal murine calvariae. We found that the bone-forming capacity of osteoblasts was impaired when cells were cultured in the presence of 5-LO metabolites. These data indicate that metabolites of the 5-LO pathway are negative regulators of bone formation. The continued presence of these metabolites in the bone environment might account, in part, for the bone loss associated with chronic inflammatory conditions.

Alkaline Phosphatase↗
PubMed1997

A murine model of human myeloma bone disease.

Myeloma causes a devastating and unique form of osteolytic bone disease. Although osteoclast activation is responsible for bone destruction, the precise mechanisms by which myeloma cells increase osteoclast activity have not been defined. An animal model of human myeloma bone disease would help in clarification of these mechanisms. Multiple myeloma occurs spontaneously in aging C57 BL/KaLwRij mice and has all of the features of the disease in humans, including the characteristic bone lesions. The disease can be induced in normal C57 BL/KaLwRij mice by inoculation of fresh marrow-derived cells from mice with myeloma, but this model is difficult to study because of variability in the number of myeloma cells in marrow-derived preparations. To develop a better animal model of human myeloma bone disease, we have established and subcloned a cell line from this murine myeloma and found that it causes osteolytic bone lesions in mice characteristic of human myeloma bone disease. The cell line produces interleukin-6, but grows independent of exogenous interleukin-6. Mice inoculated intravenously with the cultured cells predictably develop an identical disease to the mice injected intravenously with fresh bone-marrow-derived myeloma cells, including monoclonal gammopathy and radiologic bone lesions. We found that some of the mice became hypercalcemic, and the bone lesions are characterized by increased osteoclast activity. We found identical results when we inoculated Nu/Bg/XID mice with cultured murine myeloma cells. Because we can inoculate mice with precise numbers of cells and predict accurately when the mice will develop bone lesions, become hypercalcemic, and die, this should be a convenient model for determining the mechanisms by which the myeloma cells cause osteoclast activation in this model of human myeloma bone disease.

Animals↗
PubMed1993

Interleukin-1 receptor antagonist inhibits the hypercalcemia mediated by interleukin-1.

Recently, the effects of interleukin-1 (IL-1) on bone resorption in organ culture have been shown to be inhibited by an interleukin-1 receptor antagonist (IL-1RA), a novel monocyte cytokine in the IL-1 family. IL-1RA, which binds to IL-1 receptors and inhibits many of the effects of IL-1 alpha and beta, has been purified, cloned, and expressed. We used IL-1RA to investigate its effects on calcium homeostasis in vivo. After confirming that IL-1RA completely inhibited the effects of IL-1 on bone resorption in organ cultures, we tested the effects of IL-1RA on hypercalcemia mediated by IL-1 in normal mice and found that prolonged hypercalcemia provoked by IL-1 was completely inhibited by IL-1RA. The initial transient decrease in blood ionized calcium observed following an injection of IL-1 was also abrogated. IL-1RA had no effect alone on blood ionized calcium or on hypercalcemia mediated by parathyroid hormone (PTH) or PTH-related protein (PTHrP). These data suggest that antagonists to the IL-1 receptor may provide a useful therapeutic approach to osteoclastic bone resorption and hypercalcemia that is IL-1 dependent.

Animals↗
PubMed1993

Evidence that interleukin-1 mediates its effects on bone resorption via the 80 kilodalton interleukin-1 receptor.

Interleukin-1 (IL-1) mediates its effects through two distinct receptors, one of 80 kilodaltons (80 kD) present in athymic lymphocytes and fibroblasts, and one of 60 kD present in cells of the monocyte-macrophage lineage. A novel monocyte cytokine in the IL-1 family which binds to both the 80 and the 60 kD receptors has been purified, cloned, and expressed. As the interleukin-1 receptor antagonist (IL-1ra) has been shown to inhibit bone resorption in organ culture, it is not clear whether these effects are mediated through the 80 or the 60 kD receptor. Recently, neutralizing antibodies (35F5) have been developed to the 80 kD receptor which inhibit IL-1 effects mediated through this receptor. To determine the importance of the 80 kD receptor to IL-1-mediated bone resorption, we used the neutralizing antibodies (35F5) to the 80 kD receptor to determine if they inhibited bone resorption stimulated by IL-1 in bone organ cultures. The 35F5 antibody blocked bone-resorbing activity due to IL-1 completely, and also blocked control or "endogenous" bone-resorbing activity present in murine bone organ cultures incubated in control media. The 35F5 antibody had no effect on bone resorption mediated by tumor necrosis factor (TNF), or parathyroid hormone (PTH). These data suggest that the availability of the 80 kD IL-1 receptor is required for osteoclastic bone resorption mediated by IL-1.

Animals↗
PubMed1991

Chinese hamster ovarian cells transfected with the murine interleukin-6 gene cause hypercalcemia as well as cachexia, leukocytosis and thrombocytosis in tumor-bearing nude mice.

The effects of interleukin-6 (IL-6) in vivo were assessed by inoculating Chinese hamster ovarian (CHO) cells which were transfected with the murine IL-6 gene in nude mice. Nude mice bearing CHO cells expressing IL-6 developed hypercalcemia. Tumor-bearing mice also showed increases in white cell count, platelet count, and decreases in body weight. In nude mice carrying CHO tumors which had not been transfected with the IL-6 gene, there were no changes in these parameters. These results suggest that increased circulating concentrations of IL-6 in patients with malignant disease may contribute to a number of paraneoplastic syndromes including hypercalcemia, cachexia, leukocytosis and thrombocytosis.

Animals↗
PubMed1990

Induction of bone resorbing activity by normal and rheumatoid arthritis T cells.

We studied the role of T cells in the production of osteoclast activating factor (OAF) using anti-CD3 MAb as a specific T cell activator. OAF activity was totally inhibited by anti-IL-1 beta. Peripheral blood mononuclear cells (PBMNC) from normal subjects produced more OAF than did PBMNC from rheumatoid arthritis (RA) patients. Mononuclear cells (MNC) from RA synovial fluid produced less OAF than did RA peripheral blood. We conclude that (i) specific T cell stimulation induces OAF production which may be attributed to interleukin 1 (IL-1) activity, (ii) the inhibition by anti-IL-1 beta of OAF production following anti-CD3 stimulation suggests that the T cell signal is being transmitted to the macrophage, and (iii) RA MNC are deficient in T cell-mediated OAF production.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1990

Oxygen-derived free radicals stimulate osteoclastic bone resorption in rodent bone in vitro and in vivo.

The mechanisms by which bone resorbing osteoclasts form and are activated by hormones are poorly understood. We show here that the generation of oxygen-derived free radicals in cultured bone is associated with the formation of new osteoclasts and enhanced bone resorption, identical to the effects seen when bones are treated with hormones such as parathyroid hormone (PTH) and interleukin 1 (IL-1). When free oxygen radicals were generated adjacent to bone surfaces in vivo, osteoclasts were also formed. PTH and IL-1-stimulated bone resorption was inhibited by both natural and recombinant superoxide dismutase, an enzyme that depletes tissues of superoxide anions. We used the marker nitroblue tetrazolium (NBT) to identify the cells that were responsible for free radical production in resorbing bones. NBT staining was detected only in osteoclasts in cultures of resorbing bones. NBT staining in osteoclasts was decreased in bones coincubated with calcitonin, an inhibitor of bone resorption. We also found that isolated avian osteoclasts stained positively for NBT. NBT staining in isolated osteoclasts was increased when the cells were incubated with bone particles, to which they attach. We confirmed the formation of superoxide anion in isolated avian osteoclasts using ferricytochrome c reduction as a method of detection. The reduction of ferricytochrome c in isolated osteoclasts was inhibited by superoxide dismutase. Our results suggest that oxygen-derived free radicals, and particularly the superoxide anion, are intermediaries in the formation and activation of osteoclasts.

Animals↗
PubMed1990

A noncyclical analog of salmon calcitonin (N alpha-propionyl Di-Ala1,7,des-Leu19 sCT) retains full potency without inducing anorexia in rats.

A new analog of salmon calcitonin (N alpha-propionyl Di-Ala1,7,des-Leu19 sCT; RG-12851; here termed CTR), which lacks the ring structure of native calcitonin, was tested for biological activity in several in vitro and in vivo assay systems. The analog (CTR) and salmon calcitonin (sCT) stimulated kidney cell adenylate cyclase activity and inhibited bone resorption in organ cultures of fetal rat long bones with similar potencies and efficacies. Furthermore, CTR and sCT, at similar doses, induced comparable hypocalcemic responses in mice following sc injection or infusions. However, unlike sCT, CTR did not induce anorexia and weight loss in rats following sc injection. These data suggest that the ring structure of sCT may be important for the anorexigenic effect but is not required for effect on bone resorption or calcium homeostasis. Clinical studies appear warranted as, potentially, CTR might induce fewer side effects than does sCT.

Adenylyl Cyclases↗
PubMed1990

Inhibitory effects of the bone-derived growth factors osteoinductive factor and transforming growth factor-beta on isolated osteoclasts.

Demineralized bone matrix contains a number of growth factors for osteoblast-like cells. Two of these, the novel glycoprotein osteoinductive factor (OIF) and transforming growth factor-beta (TGF beta), act together to cause ectopic bone formation in vivo. Since OIF, like TGF beta, is likely released from bone when the matrix is resorbed, we examined the effects of homogeneous OIF and TGF beta on osteoclast function. Osteoclast function was tested in isolated avian osteoclasts and was measured in terms of tartrate-resistant acid phosphatase (TRAP) activity, oxygen-derived free radical production, and formation of characteristic resorption lacunae on slices of sperm whale dentine. OIF (50-100 ng/ml) inhibited the capacity of these osteoclasts to form lacunae whether assessed by the number of excavations per slice or by the total area resorbed. OIF (10-100 ng/ml) or TGF beta (10-20 ng/ml) caused a decrease in TRAP activity as well as a reduction in oxygen-derived free radical generation detected by nitroblue tetrazolium staining. TGF beta had no effect on the resorption capacity of isolated osteoclasts in concentrations that inhibited TRAP activity and nitroblue tetrazolium staining. These results suggest that growth regulatory factors, such as OIF and TGF beta, released during the resorption of bone may be endogenous inhibitors of continued osteoclastic activity. This cessation of osteoclast activity may be an essential preliminary step to the new bone formation that occurs at resorption sites during bone remodeling.

Acid Phosphatase↗
PubMed1990

Increased production of tumor necrosis factor by normal immune cells in a model of the humoral hypercalcemia of malignancy.

The rat Leydig cell tumor is a well characterized model of the humoral hypercalcemia of malignancy. The studies reported here were provoked by the observation that tumor-bearing rats become extremely cachectic and develop hypertriglyceridemia as they become hypercalcemic. Since the bone resorbing cytokine tumor necrosis factor (TNF)/cachectin is associated with cachexia and hypertriglyceridemia, we examined hypercalcemic tumor-bearing rats for evidence of increased TNF production using a TNF radioimmunoassay. We found that immunoreactive TNF was increased in the plasma of tumor-bearing rats. The increase in plasma TNF was comparable to that previously shown in hypercalcemic nude mice bearing Chinese hamster ovarian cell tumors transfected with the human TNF gene. There was no detectable TNF activity in tumor culture media which suggested that the tumor itself was not the source of excess TNF production. However, we found that tumor cell conditioned media enhanced the production of TNF activity by normal macrophages in vitro, indicating that increased TNF production in vivo may result from a tumor factor(s) which stimulates TNF production by normal immune cells. When TNF was added together with tumor products to organ cultures of fetal rat long bones, osteoclastic bone resorption was potentiated. These data are consistent with the concept that in this model of the humoral hypercalcemia of malignancy, increased TNF production by normal immune cells is increased, has systemic effects as suggested by cachexia and hypertriglyceridemia, and may work in concert with factors produced directly by tumor cells to overwhelm normal calcium homeostasis.

Animals↗