We have examined the effects of modulating nitric oxide (NO) levels on osteoclast-mediated bone resorption in vitro and the effects of nitric oxide synthase (NOS) inhibitors on bone mineral density in vivo. Diaphorase-based histochemical staining for NOS activity of bone sections or highly enriched osteoclast cultures suggested that osteoclasts exhibit substantial NOS activity that may account for basal NO production. Chicken osteoclasts were cultured for 36 hr on bovine bone slices in the presence or absence of the NOgenerating agent sodium nitroprusside or the NOS inhibitors N-nitro-L-arginine methyl ester and aminoguanidine. Nitroprusside markedly decreased the number of bone pits and the average pit area in comparison with control cultures. On the other hand, NOS inhibition by N-nitro-L-arginine methyl ester or aminoguanidine dramatically increased the number of bone pits and the average resorption area per pit. In a model of osteoporosis, aminoguanidine potentiated the loss of bone mineral density in ovariectomized rats. Aminoguanidine also caused a loss of bone mineral density in the sham-operated rats. Inhibition of NOS activity in vitro and in vivo resulted in an apparent potentiation of osteoclast activity. These findings suggest that endogenous NO production in osteoclast cultures may regulate resorption activity. The modulation of NOS and NO levels by cells within the bone microenvironment may be a sensitive mechanism for local control of osteoclast bone resorption.Bone-remodeling disorders such as osteoporosis, osteoarthritis, and periodontal disease are frequently associated with perturbations in the interplay between local and systemic bone-remodeling regulatory pathways. Inflammatory cytokines and arachidonic acid derivatives have been implicated as intercellular messengers involved in humoral-mediated and local osteopenia (1, 2). Postmenopausal bone loss associated with diminished estrogen levels is correlated with increased levels of interleukin 1 and stromal cell-derived interleukin 6, cytokines known to stimulate osteoclast activity and development (3, 4). Estrogen also directly inhibits osteoclast-mediated resorption (5,6) Howard (13) reported that NO-generating compounds may increase cGMP levels in isolated chicken osteoclasts. Furthermore, sodium nitroprusside (SNP) has been shown to inhibit the parathyroid hormone or 1,25-(OH)2-vitamin D3 stimulation of resorption in the 19-day fetal rat limb resorption assay system, with concomitant increases in cGMP (14).The current study was designed to investigate the role of NO in both an isolated in vitro avian osteoclast system and an in vivo rat osteoporosis model system using a NOgenerating agent and selective NOS inhibitors. These findings demonstrate that NO regulates osteoclast bone-resorption activity in vitro and in vivo and that similar effects are seen in birds and mammals.MATERIALS AND METHODS Animals. Three-month-old female Sprague-Dawley rats (250-300 g) from Charles River Breeding Laboratories were used in all in vivo experim...

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1 Endotoxin E. Coli lipopolysaccharide (LPS)-treatment in conscious, restrained rats increased plasma and urinary prostaglandin (PG) and nitric oxide (NO) production. Inducible cyclo-oxygenase (COX-2) and nitric oxide synthase (iNOS) expression accounted for the LPS-induced PG and NO release since the glucocorticoid, dexamethasone inhibited both effects. Thus, LPS (4 mg kg-1) increased the plasma levels of nitrite/nitrate from 14 ± 1 to 84 ± 7 JM within 3 h and this rise was inhibited to 35 ± 1 flM by dexamethasone. Levels of 6-keto PGFI,, in the plasma were below the detection limit of the assay (<0.2 ng ml-'). However, 3 h after the injection of LPS these levels rose to 2.6 ± 0.2 ng ml' and to 0.7 ± 0.01 ng ml' after LPS in rats that received dexamethasone. 2 The induced enzymes were inhibited in vivo with selective COX and NOS inhibitors. Furthermore, NOS inhibitors, that did not affect COX activity in vitro markedly suppressed PG production in the LPS-treated animals. For instance, the LPS-induced increased in plasma nitrite/nitrate and 6-keto PGFIe.at 3 h was decreased to 18 ± 2 iM and 0.5 ± 0.02 ng ml-', 23 ± 1 ilM and 0.7 ± 0.01 ng ml-', 29 ± 2 LLM and 1 ± 0.01 ng ml-' in rats treated with LPS in the presence of the NOS inhibitors NG-monomethyl-Larginine, N'-nitro arginine methyl ester and aminoguanidine, respectively.3 The intravenous infusion of the NO donors sodium nitroprusside (SNP) or glyceryl trinitrate (GTN) increased prostaglandin production in normal animals (for instance urinary PGE2 excretion was increased from 96 ± 10 to 576 ± 12 pg min-' and 400 ± 24 pg min-' in the presence of GTN or SNP respectively). 4 Proteinuria was measured in order to evaluate the roles of NO and PG in renal damage associated with the in vivo injection of LPS. Interestingly, dexamethasone and the NOS inhibitors attenuated proteinuria in the LPS-treated rats. The COX inhibitors had no effect. It therefore appears that NO and not PG contributes to the LPS-induced renal damage; these findings support the potential use of NOS inhibitors in the treatment of renal inflammation. 5 This study demonstrates the regulatory contribution of NO on the in vivo production of prostanoids and suggests that in inflammatory diseases that are driven by both NO and the prostaglandins, NOS inhibitors may act to reduce inflammation by the dual inhibition of cytotoxic NO and pro-inflammatory PG.

Integrins expressed on endothelial cells modulate cell migration and survival during angiogenesis. Integrins expressed on carcinoma cells potentiate metastasis by facilitating invasion and movement across blood vessels. We describe the activities of two synthetic low-molecular-weight peptidomimetics of the ligand amino acid sequence arg-gly-asp (RGD) in integrin-based functional assays in vitro. We also evaluate efficacy and potential mechanisms of action in models of both spontaneous and experimental metastasis. Broad-spectrum potency against the family of alpha v subunit-containing integrins was observed, with significantly less potency against alpha5beta1 and alpha(IIb)beta3. Both endothelial and tumor cell migration mediated by alpha(v)beta3 was inhibited, whereas proliferation of endothelial cells but not tumor cells was diminished. Continuous infusion of compound by minipumps or oral administration twice daily significantly reduced metastatic tumor burden in the lungs of mice despite no reduction in growth of 435/HAL primary tumors, and only a slight reduction in tumor cells detected in circulating blood. Delaying treatment in this model until after extensive dissemination of tumor cells to the lungs had occurred, and after primary tumor resection, still produced significant efficacy. Conversely, administration of the agent for only the first 18 h after tumor-cell inoculation into the tail vein also resulted in decreased metastases observed after several weeks. These data suggest these compounds or their relatives have potential to interfere with both early and late steps of metastasis involving tumor and endothelial cell functions. Furthermore, the metastatic process can be effectively inhibited independently of primary tumor growth using integrin antagonists.

Objective. To demonstrate that the novel highly selective matrix metalloproteinase 13 (MMP-13) inhibitor PF152 reduces joint lesions in adult dogs with osteoarthritis (OA) and decreases biomarkers of cartilage degradation.Methods. The potency and selectivity of PF152 were evaluated in vitro using 16 MMPs, TACE, and ADAMTS-4 and ADAMTS-5, as well as ex vivo in human cartilage explants. In vivo effects were evaluated at 3 concentrations in mature beagles with partial medial meniscectomy. Gross and histologic changes in the femorotibial joints were evaluated using various measures of cartilage degeneration. Biomarkers of cartilage turnover were examined in serum, urine, or synovial fluid. Results were analyzed individually and in combination using multivariate analysis.Results. The potent and selective MMP-13 inhibitor PF152 decreased human cartilage degradation ex vivo in a dose-dependent manner. PF152 treatment of dogs with OA reduced cartilage lesions and decreased biomarkers of type II collagen (type II collagen neoepitope) and aggrecan (peptides ending in ARGN or AGEG) degradation. The dose required for significant inhibition varied with the measure used, but multivariate analysis of 6 gross and histologic measures indicated that all doses differed significantly from vehicle but not from each other. Combined analysis of cartilage degradation markers showed similar results.Conclusion. This highly selective MMP-13 inhibitor exhibits chondroprotective effects in mature animals. Biomarkers of cartilage degradation, when evaluated in combination, parallel the joint structural changes induced by the MMP-13 inhibitor. These data support the potential therapeutic value of selective MMP-13 inhibitors and the use of a set of appropriate biomarkers to predict efficacy in OA clinical trials.Osteoarthritis (OA) is a chronic degenerative joint disease affecting primarily aged or injured joints. The disease is characterized by an imbalance between cartilage synthesis and degradation, with increased breakdown of matrix components leading to proteoglycan loss and cartilage fibrillation, eventually resulting in severe cartilage defects. These changes are irreversible, and the only treatment other than palliative symptom control is total joint replacement. Therefore, the discovery of a disease-modifying osteoarthritis drug (DMOAD) would fill a large unmet medical need.

The effect of pharmacological manipulation of atriopeptin (AP) activity on sodium excretion and blood pressure was examined in the rat aortovenocaval (A-V) fistula model of cardiac failure. Introduction of an A-V shunt led to a marked and sustained elevation of plasma AP immunoreactivity and urinary cGMP levels. Further elevation ofplasma AP levels by infusion of exogenous peptide induced modest increases in urinary sodium and cGMP excretion and a decrease in blood pressure but these responses were significantly attenuated compared to sham-operated animals. In contrast, lowdose infusion of M+B 22948 (a cGMP phosphodiesterase inhibitor) or thiorphan [a neutral endopeptidase (membrane metallo-endopeptidase, EC 3.4.24.11) inhibitor] induced a natriuresis in A-V fistula rats, which exceeded that seen in control animals given these compounds and matched the peak natriuresis produced in sham-operated animals by high doses of AP. In the doses used, these compounds had little effect on blood pressure. The greater renal efficacy of M+B 22948 in A-V fistula rats is consistent with postreceptor facilitation of AP activity. The effect of thiorphan on sodium excretion was accompanied by a pronounced increase in urinary cGMP and AP iminunoreactivity excretion (and was attenuated by anti-AP monoclonal antibody) but could not be explained solely in terms of an increase in circulating AP levels. It is proposed that thiorphan allows filtered AP to reach renal tubule sites that are normally inaccessible to the peptide and are thus protected from down-regulation by high circulating AP levels. The implication of these observations for patients in cardiac failure is the potential for using pharmacological agents to maximize the response to endogenous AP without compromising cardiac function.The cardiac atria synthesize and store a peptide with natriuretic-diuretic and vasorelaxant properties, known as atriopeptin (AP) or atrial natriuretic factor ], which is thought to participate in the regulation of blood volume and blood pressure (1, 2). Raised plasma AP immunoreactivity (APir) levels have been reported in patients with congestive cardiac failure and the increase appears to correlate with the severity of the disease (3, 4). It is likely that the increase in plasma AP concentration represents a compensatory response, lessening cardiac workload by facilitating sodium and water excretion and reducing peripheral vascular resistance. This interpretation has stimulated studies to evaluate the therapeutic value of further increases in plasma AP levels in patients with heart failure. Studies in which the peptide has been infused have shown an enhancement of sodium and water excretion and an improvement in ventricular performance, as judged by changes in atrial and ventricular pressure and stroke volume (5-7). Unfortunately, the short half-life of the peptide, the necessity for parenteral administration, and occasional excessive drops in blood pressure limit the usefulness of the peptide itself as a therapeutic agent.An alternative...

The healing response to implanted biomedical materials involves varying degrees and stages of inflammation and healing which in some cases leads to device failure. In this article, we describe synthetic methods and in vivo results of a novel surface treatment for biomedical materials involving covalent conjugation of a low molecular weight superoxide dismutase mimic (SODm), which imparts anti-inflammatory character to the material. SODm investigated in this study are a new class of anti-inflammatory drugs consisting of a Mn(II) complex of a macrocyclic polyamine ring that catalyze the dismutation of superoxide at rates equivalent to that of native enzyme. The SODms were covalently linked to small disks of ultra-high molecular weight polyethylene, poly(etherurethane urea), and tantalum metal at two concentrations and implanted in a subcutaneous rat implant model for 3, 7, 14, and 28 days. Histological examination of the implant tissue performed at 3 and 28 days revealed striking anti-inflammatory effects on both acute and chronic inflammatory responses. At 3 days, the formation of a neutrophil-rich acute inflammatory infiltrate seen in control implants was inhibited for all three materials treated with SODm. At 28 days, foreign body giant cell formation (number of FBGCs per field) and fibrous capsule formation (mean thickness of implant capsule) were also significantly inhibited over untreated control implants. A mechanism based on our current understanding of superoxide as an inflammatory mediator at implanted biomedical materials is proposed.