Reamed versus nonreamed intramedullary nailing of lower extremity long bone fractures: a systematic overview and meta-analysis

Structured abstract from an article published on the Journal of Orthopaedic Trauma which aims to determine the effect of of reamed versus nonreamed intramedullary (IM) nailing of lower extremity long bone fractures, on the rates of nonunion, implant failure, malunion, compartment syndrome, pulmonary embolus and infection.

Stimulation of osteoclast differentiation in vitro by mouse oncostatin M, leukaemia inhibitory factor, cardiotrophin-1 and interleukin 6: synergy with dexamethasone.

The role of oncostatin M in bone metabolism is not clearly defined, and the actions of mouse oncostatin M (mOSM) on osteoclast development has not been previously determined. We therefore examined the ability of recombinant mOSM to stimulate osteoclast formation and activity using cocultures of murine calvaria and bone marrow cells, and compared the responses to other members of the interleukin 6 family of cytokines including mouse leukaemia inhibitory factor (LIF), cardiotrophin-1 (CT-1) and IL-6. Mouse OSM, LIF and CT-1 strongly induced the formation of tartrate resistant acid phosphatase positive (TRAP(+)) multinucleated cells (MNC) in a dose-dependent fashion. OSM, LIF or CT-1 also elevated the number and size of resorptive pits when cocultures were added to smooth cortical bone slices, indicating enhancement of osteoclast activity. The activity of OSM was reduced by indomethacin (10(-8)-10(-6) M), whereas addition of dexamethasone (DEX) at 10(-7)-10(-5) M synergistically enhanced OSM-induced numbers of TRAP(+)MNC. DEX (10(-7) M) costimulation also synergistically enhanced TRAP(+)cell numbers of LIF, and CT-1 treated cocultures. IL-6 had no activity alone, but further enhanced TRAP(+)cell formation in mOSM or DEX (10(-7) M) treated cocultures. When added to mouse calvarial osteoblast cultures, mOSM induced secretion of IL-6 protein and elevation of mRNA whereas LIF or CT-1 did not. IL-6 mRNA levels and protein secretion were reduced in osteoblasts by costimulation with DEX. These results show that mouse OSM, LIF and CT-1 induce osteoclast differentiation and activation, that DEX synergizes with each in this activity, and that mouse OSM induces responses in osteoblasts that are not shown by LIF or CT-1. Collectively these data suggest an important role of these cytokines in osteoporosis caused by high levels of corticosteroid.

New insights into the size and stoichiometry of the plasminogen activator inhibitor type-1.vitronectin complex.

Plasminogen activator inhibitor-type 1 (PAI-1) is the primary inhibitor of endogenous plasminogen activators that generate plasmin in the vicinity of a thrombus to initiate thrombolysis, or in the pericellular region of cells to facilitate migration and/or tissue remodeling. It has been shown that the physiologically relevant form of PAI-1 is in a complex with the abundant plasma glycoprotein, vitronectin. The interaction between vitronectin and PAI-1 is important for stabilizing the inhibitor in a reactive conformation. Although the complex is clearly significant, information is vague regarding the composition of the complex and consequences of its formation on the distribution and activity of vitronectin in vivo. Most studies have assumed a 1:1 interaction between the two proteins, but this has not been demonstrated experimentally and is a matter of some controversy since more than one PAI-1-binding site has been proposed within the sequence of vitronectin. To address this issue, competition studies using monoclonal antibodies specific for separate epitopes confirmed that the two distinct PAI-1-binding sites present on vitronectin can be occupied simultaneously. Analytical ultracentrifugation was used also for a rigorous analysis of the composition and sizes of complexes formed from purified vitronectin and PAI-1. The predominant associating species observed was high in molecular weight (M(r) approximately 320,000), demonstrating that self-association of vitronectin occurs upon interaction with PAI-1. Moreover, the size of this higher order complex indicates that two molecules of PAI-1 bind per vitronectin molecule. Binding of PAI-1 to vitronectin and association into higher order complexes is proposed to facilitate interaction with macromolecules on surfaces.

Type 1 plasminogen activator inhibitor binds to fibrin via vitronectin.

Type 1 plasminogen activator inhibitor (PAI-1), the primary inhibitor of tissue-type plasminogen activator (t-PA), circulates as a complex with the abundant plasma glycoprotein, vitronectin. This interaction stabilizes the inhibitor in its active conformation In this report, the effects of vitronectin on the interactions of PAI-1 with fibrin clots were studied. Confocal microscopic imaging of platelet-poor plasma clots reveals that essentially all fibrin-associated PAI-1 colocalizes with fibrin-bound vitronectin. Moreover, formation of platelet-poor plasma clots in the presence of polyclonal antibodies specific for vitronectin attenuated the inhibitory effects of PAI-1 on t-PA-mediated fibrinolysis. Addition of vitronectin during clot formation markedly potentiates PAI-1-mediated inhibition of lysis of (125)I-labeled fibrin clots by t-PA. This effect is dependent on direct binding interactions of vitronectin with fibrin. There is no significant effect of fibrin-associated vitronectin on fibrinolysis in the absence of PAI-1. The binding of PAI-1 to fibrin clots formed in the absence of vitronectin was characterized by a low affinity (K(d) approximately 3.5 micrometer) and rapid loss of PAI-1 inhibitory activity over time. In contrast, a high affinity and stabilization of PAI-1 activity characterized the cooperative binding of PAI-1 to fibrin formed in the presence of vitronectin. These findings indicate that plasma PAI-1.vitronectin complexes can be localized to the surface of fibrin clots; by this localization, they may modulate fibrinolysis and clot reorganization.

Reamed versus nonreamed intramedullary nailing of lower extremity long bone fractures: a systematic overview and meta-analysis.

OBJECTIVE: To determine the effect of reamed versus nonreamed intramedullary (IM) nailing of lower extremity long bone fractures on the rates of nonunion, implant failure, malunion, compartment syndrome, pulmonary embolus, and infection. DESIGN: Quantitative systematic review of prospective, randomized controlled trials. DATA IDENTIFICATION: MEDLINE and SCISEARCH computer searches provided lists of published randomized clinical trials from 1969 to 1998. Extensive hand searches of major orthopaedic journals, bibliographies of major orthopaedic texts, and personal files identified additional studies. STUDY SELECTION AND DATA EXTRACTION: Of 676 citations initially identified, sixty proved potentially eligible, of which four published and five unpublished randomized trials met all eligibility criteria. Each of three investigators assessed study quality and abstracted relevant data. RESULTS: The pooled relative risk of reamed versus nonreamed nails (nine trials, n = 646 patients) was 0.33 [95% confidence interval (CI), 0.16 to 0.68; p = 0.004]. The absolute risk difference in nonunion rates with reamed IM nailing was 7.0 percent (95% CI, 1 to 11 percent). Thus, one nonunion could be prevented for every fourteen patients treated with reamed IM nailing [number needed to treat (NNT) = 14.28]. The risk ratios for secondary outcome measures were: implant failure, 0.30 (95% CI, 0.16 to 0.58; p < 0.001); malunion, 1.06 (95% CI, 0.32 to 3.57); pulmonary embolus, 1.10 (95% CI, 0.26 to 4.76); compartment syndrome, 0.45 (95% CI, 0.13 to 1.56); and infection, 0.98 (95% CI, 0.21 to 4.76). Sensitivity analyses suggested that reported rates of nonunion and implant failure were higher in studies of lower quality. The type of long bone fractured (tibia or femur), the degree of soft tissue injury (open or closed), study quality, and whether a study was published or unpublished did not significantly alter the relative risk of nonunion between reamed and nonreamed IM nailing. CONCLUSIONS: There is evidence from a pooled analysis of randomized trials that reamed IM nailing of lower extremity long bone fractures significantly reduces rates of nonunion and implant failure in comparison with nonreamed nailing.

High and low pressure irrigation in contaminated wounds with exposed bone.

BACKGROUND: Irrigation and debridement are essential in the initial management of traumatic wounds. The relative efficicacy of low pressure irrigation compared with high pressure irrigation remains unclear. AIMS: The purpose of this study was to examine the time dependent efficacy of both high and low pressure lavage in removing adherent bacteria from traumatic wounds with exposed bone. METHODS: Using an in-vivo model, we created bilateral Staphylococcus aureus contaminated femoral wounds in 30 mice. The wounds were incubated for 1 to 10 hours and lavaged with either high (70 psi) or low pressure lavage (1-2 psi). The mean colony forming units of bacteria were compared between groups at each time point. RESULTS: High and low pressure lavage were effective in removing adherent bacteria from contaminated wounds up to 8 hours and 4 hours, respectively. High pressure lavage was more effective than low pressure at every time point. CONCLUSION: The efficacy of low pressure lavage in wounds with exposed bone is questionable when irrigation is delayed beyond 4 hours.

A histomorphometric evaluation of heparin-induced bone loss after discontinuation of heparin treatment in rats.

Although it is well established that long-term heparin therapy causes osteoporosis, it is unknown whether heparin-induced bone loss is reversible when heparin treatment is stopped. To address this question, we randomized rats to once daily subcutaneous injections of either unfractionated heparin (1.0 U/g or 0.5 U/g) or saline for 28 days and then followed the rats for an additional 28 days off treatment. Based on histomorphometric analysis of the distal third of the right femur proximal to the epiphyseal growth plate, 1.0 U/g heparin caused a 30% loss in cancellous bone volume over the first 28 days. This was accompanied by a 137% increase in osteoclast surface and a 60% decrease in both osteoblast and osteoid surface. One month after cessation of heparin treatment, no recovery in these parameters was observed. Similarly, serum levels of alkaline phosphatase, a biochemical marker of bone formation, which continued to decrease over the course of heparin treatment, showed no signs of recovery in the subsequent 28 days off treatment. To explore the mechanism responsible for the prolonged effect of heparin on bone, we repeated the experiment giving 125I-labeled heparin in place of unlabeled heparin. 125I-labeled heparin was found to accumulate in bone during the course of its administration, and be retained in bone for at least 56 days after stopping heparin treatment. These findings suggest that heparin-induced osteoporosis is not rapidly reversible because heparin is sequestered in bone for an extended period.

High and low pressure pulsatile lavage of contaminated tibial fractures: an in vitro study of bacterial adherence and bone damage.

OBJECTIVE: This study was designed to examine the effect of pulsatile irrigation on microscopic bone architecture and its time-dependent efficacy in removing adherent slime-producing bacteria from cortical bone. DESIGN: Using an in vitro model, ten-millimeter transverse cut sections from five human tibiae were contaminated with Staphylococcus aureus and subjected to either high pressure pulsatile lavage (HPPL; seventy pounds per square inch, normal saline) or low pressure pulsatile lavage (LPPL; fourteen pounds per square inch, normal saline) or served as controls. Alteration of bony architecture was quantified by using a previously described ordinal scale and histomorphometric analysis of each transverse cut section of tibia. To assess the time-dependent effectiveness of pulsatile lavage in removing adherent bacteria from bone, ten-millimeter transverse cut sections from ten canine tibiae were contaminated with S. aureus and subjected to high or low pressure pulsatile lavage immediately or after one, three, or six hours. Scanning electron microscopy and bacterial cultures were used to assess the removal of adherent bacteria. RESULTS: HPPL resulted in significantly greater macroscopic damage than was seen with LPPL or in controls (ANOVA, p < 0.001). Histomorphometry revealed that HPPL was associated with significantly larger and more numerous fissures or defects in the cortical bone when compared with low pressure irrigation (p < 0.001). However, high and low pressure lavage were associated with similar degrees of periosteal separation from the cortical bone surface (p = 0.87). Both high and low pressure lavage were effective in removing adherent bacteria from bone at three hours irrigation delay, but only high pressure lavage removed adherent bacteria from bone at six hours delay. CONCLUSION: In this in vitro study, compared with HPPL, LPPL led to less structural damage and was equally effective in removing bacteria within three hours debridement delay; however, the efficacy of LPPL at six hours debridement delay is questionable. This finding may have clinical significance in the development of infection following open tibial fractures.

Predictors of in-hospital mortality following operative management of hip fractures.

OBJECTIVE: To identify predictors of in-hospital mortality and hospital stay following hip fractures. DESIGN: Retrospective cohort study of 185 consecutive patients. SETTING: Tertiary Care University Hospital. PARTICIPANTS: Individuals requiring operative treatment of a proximal femoral fracture excluding those individuals < 50 years old, with femoral head or subtrochanteric fractures, and significant co-morbidity. OUTCOMES: In-hospital mortality and length of hospital stay (days). RESULTS: 116 patients met the inclusion criteria. Predictors of in-hospital mortality from logistic regression analysis included male gender (odds ratio with 95% CI: 5.5, 1.5-20.5), admission from a long term care facility (5.5, 1.4-22.6), age greater than 90 years (4.5, 0.9-22.1), and living at home with support (0.2, 0.03-0.9). Predictors of hospital stay from multivariate regression analysis in order of magnitude included presence of a post-operative complication (odds ratio with 95% CI: 14.1, 4.7-44), living at home with support (3.4, 1.3-8.9) and older age (> 85 years) (2.7, 1.0-7.3). Moreover, confusion, urinary tract infections and decubitus ulcers accounted for greater than 50% of all complications encountered. A trend between the number of positive predictors and length of hospital stay was observed. CONCLUSIONS: Elderly individuals admitted from a long term care facility are at high risk of mortality following operative fixation of hip fractures. Early recognition and aggressive management of post-operative complications such as confusion, urinary tract infections and decubitus ulcers through careful patient monitoring may decrease hospital stays in those that survive.

Homocysteine-dependent alterations in mitochondrial gene expression, function and structure. Homocysteine and H2O2 act synergistically to enhance mitochondrial damage.

Mitochondrial abnormalities have been identified in hepatocytes of patients with hyperhomocysteinemia and in endothelial cells from the aortas of rats with diet-induced hyperhomocysteinemia. However, the mechanism by which homocysteine affects mitochondria is unknown. In this report, homocysteine-induced expression of the mitochondrial electron transport chain gene, cytochrome c oxidase III/ATPase 6,8 (CO3/ATPase 6,8), was identified in a human megakaryocytic cell line DAMI using mRNA differential display. Steady-state mRNA levels of CO3/ATPase 6,8, as well as other mitochondrial transcripts, were increased in DAMI cells by homocysteine in a concentration- and time-dependent manner. Despite an increase in mitochondrial RNA levels and changes in mitochondrial ultrastructure, no effect on either cell growth or mitochondrial respiration rates was observed in DAMI cells exposed to homocysteine at concentrations up to 1 mM. In contrast, 1 mM homocysteine in the presence of Cu2+, which is known to generate H2O2, significantly decreased mitochondrial RNA levels, caused gross morphological changes in mitochondrial ultrastructure, and inhibited both cell growth and mitochondrial respiration rates. However, precursors of cellular glutathione and preexposure to heat shock blocked the decrease in mitochondrial RNA levels caused by homocysteine and Cu2+. The observations that (i) homocysteine and H2O2, but not H2O2 alone, caused a decrease in mitochondrial RNA levels, (ii) intracellular levels of H2O2 were significantly increased in the presence of homocysteine and Cu2+, and (iii) catalase, but not free radical scavengers, prevented a decrease in mitochondrial RNA levels, provide evidence that homocysteine and H2O2 act synergistically to cause mitochondrial damage. Furthermore, our findings suggest that intracellular glutathione and heat shock proteins play a role in protecting mitochondria against the adverse effects elicited by homocysteine and H2O2.

The effects of standard and low molecular weight heparin on bone nodule formation in vitro.

Previously, we demonstrated in a rat model of heparin-induced osteoporosis that low molecular weight heparin (LMWH) produces less bone loss than unfractionated heparin, and that only heparin increases osteoclast number and activity. In contrast, both heparin and LMWH were found to decrease osteoblast function to a similar extent, possibly because at the doses tested both agents produced maximal inhibition. To examine the relative effects of heparin and LMWH on osteoblast function more closely we used an in vitro bone nodule assay, together with measurements of alkaline phosphatase (ALP) activity. Both agents inhibited bone nodule formation and ALP activity in a concentration-dependent manner, but 6 to 8-fold higher concentrations of LMWH were required to achieve equivalent effects. The effect of heparin on osteoblast function was both chain-length and negative charge-dependent because the ability of defined heparin fragments to inhibit nodule formation correlated with their molecular weight (r = 0.98), and N-desulfated heparin was less inhibitory than heparin. In contrast, the effect of heparin on osteoblast function was pentasaccharide-independent because heparin with low affinity for antithrombin had similar activity to heparin with high antithrombin activity. These findings help to explain mounting clinical evidence that the risk of osteoporosis is lower with LMWH than with heparin.

A histomorphometric comparison of the effects of heparin and low-molecular-weight heparin on cancellous bone in rats.

Long-term heparin treatment causes osteoporosis through, an as yet, undefined mechanism. To investigate this phenomenon and to determine the relative benefits of low-molecular-weight heparin (LMWH) use, we treated rats with once daily subcutaneous injections of either unfractionated heparin (1.0 U/g or 0.5 U/g), the LMWH, Tinzaparin (1.0 U/g or 0.5 U/g), or placebo (saline) for a period of 32 days. The effects on bone were then compared both histomorphometrically and biochemically by measuring urinary type I collagen cross-linked pyridinoline (PYD) and serum alkaline phosphatase, markers of bone resorption and formation, respectively. Histomorphometric analysis of the distal third of the right femur, in the region proximal to the epiphyseal growth plate, demonstrated that both heparin and LMWH decrease cancellous bone volume in a dose-dependent fashion, but that heparin causes significantly more cancellous bone loss than does LMWH. Although both heparin and LMWH decrease osteoblast and osteoid surface to a similar extent, only heparin increases osteoclast surface. In support of these histomorphometric findings, biochemical markers of bone turnover demonstrated that both heparin and LMWH treatment produce a dose-dependent decrease in serum alkaline phosphatase, consistent with reduced bone formation, whereas only heparin causes a transient increase in urinary PYD, consistent with an increase in bone resorption. Based on these observations, we conclude that heparin decreases cancellous bone volume both by decreasing the rate of bone formation and increasing the rate of bone resorption. In contrast, LMWH, causes less osteopenia than heparin because it only decreases the rate of bone formation.

Histomorphometric analysis of the effects of standard heparin on trabecular bone in vivo.

Long-term heparin treatment causes osteoporosis through an as yet undefined mechanism. To investigate this phenomenon, we treated rats with once daily subcutaneous injections of heparin (in doses ranging from 0.25 to 1.0 U/g) or saline for 8 to 32 days and monitored the effects on bone both histomorphometrically and by serial measurements of urinary type 1 collagen cross linked-pyridinoline (PYD) and serum alkaline phosphatase, markers of bone resorption and formation, respectively. Histomorphometric analysis of the distal third of the right femur in the region proximal to the epiphyseal growth plate showed that heparin induces both a time- and dose-dependent decreased in trabecular bone volume, with the majority of trabecular bone loss occurring within the first 8 days of treatment. Thus, heparin doses of 1.0 U/g/d resulted in a 32% loss of trabecular bone. Heparin-treated rats also showed a 37% decrease in osteoblast surface as well as a 75% decrease in osteoid surface. In contrast, heparin treatment had the opposite effect on osteoclast surface, which was 43% higher in heparin-treated rats, as compared with that in control rats. Biochemical markers of bone turnover showed that heparin treatment produced a dose-dependent decrease in serum alkaline phosphatase and a transient increase in urinary PYD, thus confirming the histomorphometric data. Based on these observations, we conclude that heparin decreases trabecular bone volume both by decreasing the rate of bone formation and increasing the rate of bone resorption.

Differential mechanisms targeting type 1 plasminogen activator inhibitor and vitronectin into the storage granules of a human megakaryocytic cell line.

Type 1 plasminogen activator inhibitor (PAI-1) and its cofactor vitronectin (Vn) are stored within the alpha-granules of platelets. The two possible sources for their biosynthetic origin are endogenous synthesis in megakaryocytes or endocytosis from plasma. Using ultrastructural and confocal laser scanning microscopic (CLSM) image analysis, we observed that treatment of Dami cells, a human megakaryocytic cell line, with phorbol myristate acetate (PMA) induces the accumulation of PAI-1 and Vn in intracellular storage vacuoles that contain other alpha-granule proteins such as von Willebrand factor. To examine evidence for biosynthesis of PAI-1 and Vn by Dami cells, we immunoprecipitated PAI-1 and Vn from the conditioned media of cells biosynthetically radiolabeled with 35S-methionine in the presence or absence of PMA. In contrast to Hep G2 cells, which synthesize both PAI-1 and Vn, only 35S-PAI-1 was recovered from PMA-treated Dami cells. Reverse transcription-PCR analysis of RNA extracted from resting and PMA-treated Dami cells confirmed that PAI-1 mRNA expression was detectable at low levels in resting cells and induced by PMA treatment. In contrast, Vn mRNA was not detected. We examined binding and internalization (endocytosis) of PAI-1 and Vn by Dami cells using biotinylated analogs (b-PAI-1 and b-Vn). Flow cytometry analysis indicated that the binding of b-Vn to Dami cells was dose-dependent, saturable, and specific for multimeric forms of Vn. Cells were incubated at 4 degrees C or 37 degrees C and endocytosis of b-Vn was shown by probing electrophoretically fractionated cell lysates with 125I-labeled streptavidin. Only cells incubated at 37 degrees C internalized b-Vn. CLSM image analysis confirmed that the b-Vn was internalized and that it colocalized with PAI-1 in storage granules. The binding of b-Vn to cells was inhibited by the presence of PAI-1, and there was no evidence of specific b-PAI-1 binding or uptake to resting or PMA-treated cells. These data suggest that accumulation of PAI-1 in Dami cell storage granules is due to endogenous synthesis and that the accumulation of Vn is due to endocytosis of serum-derived Vn.

The effects of low molecular weight and standard heparin on calcium loss from fetal rat calvaria.

Osteoporosis is a well-recognized complication of long-term heparin use. However, the mechanisms by which heparin can influence bone metabolism are unclear. We report here that unfractionated heparin stimulates the process of bone resorption and that the low molecular weight heparins (LMWHs), enoxaparin, fragmin, logiparin, and ardeparin produce significantly less calcium loss than unfractionated heparin. To assess calcium loss from bone, we quantified the release of 45Ca into the culture medium of fetal rat calvaria. 45Ca release was increased in a dose-dependent manner by the addition of either unfractionated heparin or the LMWHs; but more than 50-fold higher LMWH concentrations were required to obtain an equivalent effect to unfractionated heparin. Thus, at concentration > or = 2 micrograms/mL (0.35 anti-Xa units/mL), unfractionated heparin stimulated 45Ca release 1.53 +/- 0.06 fold. 45Ca release was increased to a similar extent by the addition of either 10(-7) mol/L parathyroid hormone (PTH) or 10(-6) mol/L 1,25 dihydroxyvitamin D3 (1,25 Vit D3). In contrast to unfractionated heparin, LMWH concentrations > or = 100 micrograms/mL (> or = 14.0 anti-Xa units/mL) were required before maximum isotope release was observed. At concentrations well above therapeutic levels, the LMWHs stimulated 45Ca release by only 1.25 /+- 0.01-fold. Heparins with high and low antithrombin III affinities stimulated 45Ca release equally well. Both size and sulfation were found to be major determinants of heparin's ability to promote isotope release. Thus, the ability of defined heparin fragments to stimulate 45Ca release correlated with their molecular weight, and after N-desulfation the ability of heparin to induce isotope release was greatly diminished. Dermatan sulfate had no effect on 45Ca release. We conclude that size and sulfation are major determinants of heparin's ability to promote bone resorption and that the risk of heparin-induced osteoporosis may be reduced by the use of LMWH preparations.

Quantification and morphologic demonstration of reactive oxygen species produced by Walker 256 tumor cells in vitro and during metastasis in vivo.

BACKGROUND: Pulmonary endothelial damage can be caused by agents that generate oxidants, e.g., bleomycin, hyperoxia, neutrophils or x-irradiation. In animals with intravascular cancer cells, there is increased tumor cell arrest and the subsequent formation of metastatic tumors at the sites of such endothelial injury. We have previously shown that Walker 256 (W256) tumor cells, stimulated with phorbol esters (phorbol 12-myristate 13 acetate) or the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine, generate chemiluminescence that is inhibitable by catalase. Such activated cells can injure cultured endothelial monolayers. The purpose of the present study was to quantify and obtain morphological confirmation of the generation of reactive oxygen species by W256 cells in vitro, and to determine if this phenomenon could be morphologically detected in vivo during the metastatic process. EXPERIMENTAL DESIGN: The production of oxidants from W256 cells was quantitated in vitro by the scopoletin fluorescence assay, by a ferrithyiocyanate colorimetric assay (Thurman reaction), and confirmed morphologically, in vitro and in vivo, by the formation of cerium perhydroxide (Ce[OH]2OOH) deposits from cerium chloride (CeCl3). To demonstrate generation of reactive oxygen species in vivo, we examined W256 cells collected from the pulmonary circulation and at sites of spontaneous metastasis in the lung after intramuscular tumor transplantation, or cells arrested in the lungs after intravenous injection. The specificity of the CeCl3 reaction was confirmed by blocking in the presence of catalase. RESULTS: As measured by the loss in scopoletin fluorescence and by generation of ferrithiocyanate 5 x 10(6) activated W256 cells produced an equivalent of 18 nM of H2O2 per hour A. Ce-[OH]2OOH deposits were identified in vitro on the surface of W256 cells, and at points of attachment between W256 cells and cultured endothelial cell monolayers. In vivo, CeCl3-derived deposits were seen on circulating W256 cells and on W256 cells that had arrested in the lungs following the intravenous injection of activated or non-activated W256 cells, or in spontaneous pulmonary metastases which formed after intramuscular tumor inoculation. Pretreatment of tumor-bearing animals with phorbol 12-myristate 13 acetate increased the number of CeCl3-derived deposits more than 2 fold. Catalase inhibited the formation of the electron-dense deposits in vitro and in vivo. CONCLUSIONS: These data provide morphologic evidence that cancer cells can produce reactive oxygen species in vivo and suggest that free radicals might contribute to endothelial damage during the metastatic process.

Walker 256 tumor cell degradation of extracellular matrices involves a latent gelatinase activated by reactive oxygen species.

The invasion of blood vessel walls is a critical step in cancer metastasis, in which endothelial cells and their vascular basement membranes act as barriers to tumor cell passage. Here we report that Walker 256 carcinosarcoma (W256) cells degrade subendothelial matrices by a process involving both the generation of hydrogen peroxide and the secretion of a matrix metalloproteinase. As an assay of basement membrane degradation, [3H]proline-labeled subendothelial matrices were exposed to W256 cells in the presence or absence of the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). The release of [3H]proline, in the presence of 5 x 10(6) W256 cells, was increased from 49 +/- 2.5 to 64 +/- 2.2% by the addition of 10(-6) M fMLP. In the presence of fMLP-activated W256 cells, [3H]proline release was completely inhibited by the addition of 2000 units/ml catalase or by the metalloproteinase inhibitors 1,10-phenanthroline and EDTA at concentrations > or = 10 micrograms/ml. alpha 1-Antitrypsin or alpha 2-macroglobulin were without effect. Cell-free supernatants obtained from activated W256 cells were also able to promote basement membrane degradation. Electrophoresis of the cell-free supernatants from fMLP or PMA-activated W256 cells in gelatin-containing sodium dodecyl sulfate-polyacrylamide gels revealed a major band of gelatinolytic activity at 94 kDa. The 94-kDa band represented the activity of a latent gelatinase since incubation with 1 mM 4-aminophenylmercuric acetate (APMA; a known activator of latent metalloproteinases) resulted in the loss of gelatinolytic activity at 94 kDa and the appearance of five new bands of lower molecular weight (M(r) 86, 79, 74, 70, and 66 kDa). Two of these lower molecular weight bands (M(r) 86 and 66 kDa) were also detected in the absence of APMA, following 10-fold concentration of the cell-free supernatants. When the cell-free supernatants of phorbol myristate acetate-activated W256 cells (concentrated 10-fold) were incubated with increasing concentrations of hydrogen peroxide (35 to 70 mM), the band at 66 kDa demonstrated enhanced gelatinolytic activity. We suggest that W256 cells can secrete a latent metalloproteinase of molecular weight 94 kDa which, when activated by hydrogen peroxide, can degrade subendothelial matrices.

Cancer cell interactions with injured or activated endothelium.

Blood vessels and lymphatics are the most important pathways for dissemination of cancer cells but the entry and exit of these cells into and from the vasculature requires that they pass through barriers formed by the endothelium and its basement membrane. This review summarizes evidence that this step in metastasis can be regulated by microenvironmental influences which alter the properties of this barrier. These phenomena can be attributed to both 'passive' and 'active' responses of the endothelium. The microvasculature is susceptible to perturbation from environmental agents, host cells and cancer cells. There is clinical and experimental evidence that this can upregulate the metastatic process. Using established animal models of pulmonary microvascular injury it has been shown that endothelial damage promotes the localization and metastasis of circulating cancer cells to the lung and that this effect is lost after endothelial repair. Oxidative stress is an effector of vascular damage in several of the experimental models. While endothelial cells appear to be directly susceptible to free radical attack, basement membranes are not. However, oxidative injury of endothelial cells causes release of proteases which can then degrade the basement membrane. This event is associated with generation of tumor cell chemoattractants and enhances cancer cell invasion of vascular basement membranes in vitro. Vascular endothelial cells are also susceptible to stimulation by systemic mediators including cytokines, thrombin, or endotoxin which induce a series of active responses in the vessel wall. These perturbed endothelial cells synthesize and express cell surface adhesion molecules which can interact with cancer cells. They also release chemoattractants which stimulate cancer cell motility. We postulate that such responses endow the vessel wall with the potential to act as a determinant of metastatic rate.

Endothelial cell damage by Walker carcinosarcoma cells is dependent on vitronectin receptor-mediated tumor cell adhesion.

The transport of cancer cells from blood vessels to extravascular tissue is a critical step in metastasis, where endothelial cells and the vascular basement membrane act as barriers to cell traffic. Because endothelial injury can facilitate the metastasis of intravascular cancer cells in vivo, the authors have studied in vitro the free-radical-mediated endothelial damage caused by the rat Walker 256 carcinosarcoma (W256) cell after stimulation with 10(-6) mol/l (molar) phorbol ester. Here the authors have examined the hypothesis that W256 cell-mediated endothelial injury is dependent on adhesion between the effector and target cells. Attachment of phorbol 12-myristate, 13-acetate (PMA)-stimulated W256 cells to endothelial monolayers was increased 1.8 +/- 0.1-fold and damage (3H-2-deoxyglucose release from labeled endothelium) 1.4 +/- 0.1-fold after 4-hour pretreatment of the endothelium with 10 ng/ml recombinant human interleukin-1 alpha (rIL-1 alpha). Under various assay conditions, the release of 3H-2-deoxyglucose correlated directly with tumor cell adhesion (r = 0.98, P less than 0.005). In the presence of a polyclonal anti-vitronectin receptor antiserum, adhesion of stimulated W256 cells to rIL-1 alpha-treated monolayers was inhibited by 39% +/- 2%, and 3H-2-deoxyglucose release was inhibited by 53% +/- 13%. Immunoblot analysis and immunofluorescence flow cytometry demonstrated that the endothelial cells but not the W256 cells expressed vitronectin receptor (VnR) on their cell surface. The surface expression of VnR by endothelial cells was increased 1.9 +/- 0.1-fold after 4 hours' incubation with rIL-1 alpha. The authors conclude that W256 cell-mediated endothelial damage is dependent on cell adhesion, which, in turn, is partly regulated by the expression of VnR on the endothelial cell surface.