Tibial bone versican content decreases with zoledronate treatment in adult mice.

UNLABELLED: In bone remodeling, the expression and turnover of the proteoglycans versican and aggrecan are poorly understood. We report changes in adult mouse bone contents of versican and aggrecan associated with both age and treatment with the drug zoledronate. The data may have implications for experimental animal models of osteoporosis and related conditions. INTRODUCTION: Versican and aggrecan are large, aggregating proteoglycans involved in skeletal development, but little is known about their roles in bone remodeling. The purpose of this study was to investigate versican and aggrecan contents in adult mouse bones, and changes in their contents in response to the bisphosphonate zoledronate (ZOL). METHODS: Mice (9 weeks old) were treated with 125 µg/kg ZOL or vehicle for 3 or 15 weeks. Versican and aggrecan were isolated from tibial bones for Western blotting, automated integrated densitometry, and analysis (two-way ANOVA, α = 0.05). RESULTS: In ZOL-treated mouse bones, compared to vehicle, 340 and 60 kDa versican content decreased significantly, and 100 and 60 kDa aggrecan content decreased significantly (drug effect). In 24-week-old mouse bones, compared to 12 weeks, statistically significant decreases were observed in 340, 80, 60, and 11 kDa versican, and in 100, 70, and 40 kDa aggrecan (age effect). There was a statistically significant ZOL-age interaction for 330 kDa aggrecan. CONCLUSION: This is the first study to assess physiological versican and aggrecan adaptations in adult mammalian bone tissue, in the presence and absence of ZOL. We observed large decreases in some versican and aggrecan species from 12 to 24 weeks. We also observed decreases in several versican and aggrecan species in the presence of ZOL. This indicates that bone proteoglycan expression and turnover may be important in bone remodeling.

Differential cellular synthesis of insulin-like growth factor binding protein-1 (IGFBP-1) and IGFBP-3 within human liver.

The two major insulin-like growth factor-binding protein (IGFBP) species in the human circulation, IGFBP-1 and IGFBP-3, are synthesized in large amounts by liver. To determine which hepatic cell populations in human liver were responsible for the synthesis and release of these IGFBPs, we 1) performed in situ hybridization with specific complementary RNA (RNA) probes for human IGFBP-1 or-3 or performed immunohistochemical analysis to reveal the sites of messenger RNA (mRNA) presence and peptide translation, respectively, in sections of normal liver derived from organ donors; and 2) examined the release of IGFBP species by Western ligand and immunoblots of medium conditioned by isolated cultures of hepatocytes, lipocytes, and Kupffer cells. In situ hybridization showed that IGFBP-1 mRNA was distributed widely among the parenchymal cell population, which also showed immunohistochemical staining for IGFBP-1 peptide. Conversely, IGFBP-3 mRNA and immunoreactive peptide were mainly localized to Kupffer cells, which were positively identified by immunoreactivity with antiserum against the glycoprotein marker, CD68. Isolated hepatocytes released two species of IGFBP of 28 and 30-32 kilodaltons, which were recognized immunologically as IGFBP-1. Isolated Kupffer cells released only a 43- to 46-kilodalton IGFBP immunologically recognized as IGFBP-3. Lipocyte cultures released no detectable IGFBP species. The results suggest that IGFBP-1 and IGFBP-3 are derived from separate cell populations in human liver.

Measurement of serum electrolytes and phosphate after sodium phosphate colonoscopy bowel preparation: an evaluation.

Our purpose was to observe the effects of sodium phosphate (NaP) colonoscopy preparation on serum electrolytes, phosphate, and calcium and to identify factors associated with any adverse effects. In an unselected group of 100 consecutive patients attending for out patient colonoscopy, 45% of patients had raised serum phosphate, which was positively correlated with creatinine and age. There was a negative association of phosphate with calcium; 16% of patients had hypocalcemia and 26% had hypokalemia. Patients taking ACE inhibitors, AT2 antagonists, or diuretics were associated with hyperphosphatemia. Significant electrolyte and metabolic disturbance from colonoscopy preparation has been shown with NaP preparation, without overt clinical effects. We recommend that elderly patients and those with significant comorbidity have their electrolytes and calcium measured, and diuretics and ACE inhibitors stopped, before NaP administration. Endoscopy units should be alert for patients who might be suffering from electrolyte disturbance postpreparation and be prepared to measure their electrolytes.

Reduced sensitivity to intra-oesophageal acid in patients with reflux-induced strictures.

Patients presenting with benign reflux-induced oesophageal strictures often have no antecedent symptoms of reflux, which might be a consequence of decreased oesophageal sensitivity. This has been investigated in 36 patients with strictures and 30 patients with uncomplicated oesophagitis by means of a visual analogue scale (0 to 10) to assess preceding symptoms and acid perfusion tests to assess oesophageal sensitivity. Patients with strictures had lower symptom scores (median, 2.7) than those with uncomplicated oesophagitis (median, 4.85) and were more likely to have a negative acid perfusion test (18 of 28 compared with 6 of 23, respectively; p < 0.05). Furthermore, the volumes of acid perfused at the onset of symptoms in the stricture group (median, 80 ml) were greater than in the oesophagitis group (median, 40; p < 0.05). These results support the hypothesis that patients who develop reflux-induced strictures have decreased sensitivity to intra-oesophageal acid. This may be a factor in the pathogenesis of reflux-induced strictures.

Bleeding oesophageal varices associated with anabolic steroid use in an athlete.

A 30 year old bodybuilder who had been taking anabolic steroids for 18 months presented with bleeding oesophageal varices. Serious liver disease secondary to anabolic steroids including peliosis hepatis, nodular hyperplasia and malignant change is well recognized. We report what is, to our knowledge, the first case of bleeding oesophageal varices associated with the use of anabolic steroids.

Systemic arteriovenous malformations: a feature of advanced liver disease.

Arterial and arteriovenous abnormalities are reported in association with advanced liver disease, those most commonly recognised are spider naevi, pulmonary arteriovenous shunts, and generalised vasodilatation. The first two cases of peripheral systemic arteriovenous malformations in association with cirrhosis are reported. After liver transplantation in one of these patients the vascular malformation regressed. A review of published works shows that other vascular complications of advanced liver disease such as pulmonary shunts and generalised vasodilation also regress after orthotopic liver transplantation.

Reversible inhibition of albumin production by rat hepatocytes maintained on a laminin-rich gel (Engelbreth-Holm-Swarm) in response to secretory products of Kupffer cells and cytokines.

Decreased albumin synthesis by hepatocytes in liver injury is thought to occur in response to Kupffer cell-derived acute-phase cytokines. In this study we used hepatocytes maintained in a differentiated phenotype, by culture on a laminin-rich gel substratum (Engelbreth-Holm-Swarm matrix), to investigate the effects of Kupffer cell-conditioned medium and purified cytokines (interleukin-1, interleukin-6 and tumor necrosis factor-alpha) on albumin synthesis. Kupffer cell-conditioned medium caused a reversible decrease in albumin synthesis to 64.7% of control (p less than 0.01, Wilcoxon's rank sum test, n = 11) on day 2. Repeated doses caused further dose-dependent reversible responses. The same result was obtained when protease inhibitors (alpha 1-antitrypsin and alpha 2-macroglobulin) were added to Kupffer cell-conditioned medium (n = 3), thus eliminating the potential effect of matrix degradation. Pure interleukin-1, interleukin-6 and tumor necrosis factor-alpha also inhibited albumin synthesis (p less than 0.05, Wilcoxon's rank sum test, n = 5), interleukin-6 having the greatest effect. After exposure to interleukin-1 (30 U.ml-1) and tumor necrosis factor-alpha (300 U.ml-1), decreased albumin synthesis was followed by a rebound increase (n = 3). Our results support the hypothesis that reduced albumin synthesis in the acute-phase response is modulated by cytokines released from Kupffer cells. Moreover, our results suggest that hepatocytes may exhibit a compensatory increase in albumin synthesis after cytokine withdrawal. These findings may be of physiological importance in the recovery from injury and the acute-phase response in vivo.

Thrombotic thrombocytopenic purpura mimicking acute small bowel Crohn's disease.

A 39 year old woman presented with a short history of bloody diarrhoea. She subsequently developed microangiopathic haemolysis, platelet consumption, and renal impairment. Initial investigations suggested underlying Crohn's disease of the terminal ileum complicated by sepsis and disseminated intravascular coagulation. However, after resection of a perforated caecum and terminal ileum, the diagnosis of thrombotic thrombocytopenic purpura was made. There was weak serological evidence of yersinia infection, this may have caused the early localisation of the lesions to the terminal ileum. This is believed to be the first report of thrombotic thrombocytopenic purpura affecting the small bowel alone at presentation.

Expression of tissue inhibitor of metalloproteinases 1 and 2 is increased in fibrotic human liver.

BACKGROUND & AIMS: Tissue inhibitors of metalloproteinases may contribute to liver fibrosis by preventing remodeling of fibrillar collagens by interstitial collagenase. This hypothesis was investigated by comparing the relative expression of messenger RNA for interstitial collagenase, gelatinase A, and tissue inhibitor of metalloproteinases 1 and 2 in fibrotic and normal human liver. METHODS: Hepatic expression of metalloproteinases and their inhibitors was examined using ribonuclease protection assay, immunocytochemistry, and immunoassay. Lipocyte expression of tissue inhibitors of metalloproteinases was examined using Northern blotting and reverse zymography. RESULTS: Messenger RNA levels for tissue inhibitors of metalloproteinase 1 and 2 were elevated to 260%-526% of levels in normal liver in biliary atresia, primary biliary cirrhosis, and primary sclerosing cholangitis. In fibrotic livers, tissue inhibitor of metalloproteinase 1 protein was 367%-724% of normal. Gelatinase A messenger RNA level increased to 324%-430% of normal values in fibrotic liver, but interstitial collagenase messenger RNA level was not significantly altered. Normal human liver lipocytes activated by culture on plastic expressed messenger RNA and protein for tissue inhibitor of metalloproteinase 1 and 2. CONCLUSIONS: Increased expression of tissue inhibitors of metalloproteinases relative to interstitial collagenase may promote deposition of interstitial collagens in liver fibrosis. Our studies further suggest that lipocytes are an important source of tissue inhibitors of metalloproteinases in progressive liver fibrosis.

Rat hepatic stellate cell expression of alpha2-macroglobulin is a feature of cellular activation: implications for matrix remodelling in hepatic fibrosis.

1. Hepatic stellate cells are key mediators of hepatic fibrosis. We have studied hepatic stellate cell expression of the collagenase and general protease inhibitor alpha2-macroglobulin after activation in tissue culture and in response to certain cytokines. 2. Hepatic stellate cells isolated by Pronase-collagenase digestion were activated by culture on uncoated plastic. By Northern analysis hepatic stellate cells undergoing activation (5 days) expressed alpha2-macroglobulin mRNA and alpha2-macroglobulin could be immunolocalized to hepatic stellate cells from 5 to 15 days of culture. 3. By ELISA of cell culture supernatants hepatic stellate cell secretion of alpha2-macroglobulin was found to increase from 2. 78+/-1.13 ng x ml-1 x microgram-1 DNA per 24 h at 5 days of culture (n=8) to 13.55+/-4.64 ng x ml-1 x microgram-1 DNA per 24 h at 15 days of culture (n=7). Stimulation of hepatic stellate cells with interleukin-6 at 5 days caused a significant increase in alpha2-macroglobulin expression as did exposure to Kupffer-cell conditioned medium. However, exposure of hepatic stellate cells to interleukin-1, transforming growth factor-beta1 and tumour necrosis factor-alpha had no significant effect. 4. During profibrotic liver injury plasma alpha2-macroglobulin levels were found to increase to between 850% and 250% of the control value (100%) after bile duct ligation (72 h to 13 days respectively), and to 1166% and 1106% of the control value during progressive CCl4-induced fibrosis (24 h to 4 weeks respectively). 5. These data suggest that hepatic stellate cells are a potential source of the potent protease inhibitor alpha2-macroglobulin, expression of which may inhibit matrix remodelling during progressive fibrosis.

Long-term survival associated with metastatic small cell carcinoma of the esophagus treated by chemotherapy, autologous bone marrow transplantation, and adjuvant radiation therapy.

A 44-year-old woman had small cell carcinoma of the esophagus complicated by liver and lymph node metastases. She was treated with aggressive combination chemotherapy, followed by autologous bone marrow transplantation and adjuvant radiation therapy. (The authors believe this to be the first use of autologous bone marrow transplantation for treatment of this condition.) This regimen resulted in apparent complete regression of the disease as documented by computed tomography and endoscopic study. Three years later, she again experienced general malaise and was found to have extensive recurrent disease in the lung, bone, and liver. Her condition deteriorated rapidly, and she died within 1 month. A review of the literature reveals that this patient survived longer than any others who have had this rare but aggressive tumor. The authors suggest that this form of therapy should be considered for future patients.

Kupffer cell-derived 95-kd type IV collagenase/gelatinase B: characterization and expression in cultured cells.

Release of 92-kd type IV collagenase/gelatinase, also known as gelatinase B, by inflammatory and tumor cells is increasingly recognized and is believed to facilitate cellular migration across basement membranes. It has been implicated in the pathogenesis of many diseases, but little is known of its cellular origin(s) and function in liver. In this study we have demonstrated synthesis and release of gelatinase B by human and rat Kupffer cells in primary culture. Northern analysis of RNA extracted from Kupffer cells stimulated with phorbol ester demonstrated a 2.8 kb transcript for gelatinase B. Immunoblotting and zymography of serum-free Kupffer cell-conditioned media demonstrated extracellular release of immunoreactive enzyme and gelatinase activity, Mr 92,000 (95,000 from rat cells). The organomercurial 4-aminophenyl mercuric acetate (APMA) activated the enzyme in vitro, indicating secretion primarily as a proenzyme. Stimulation of Kupffer cells by phorbol ester markedly induced gelatinase B release, which was inhibited by cycloheximide. In contrast, cycloheximide had no effect on constitutive secretion in culture, suggesting that there is some intracellular storage. Kupffer cell-derived gelatinase B was also partially purified and characterized. After separation by gelatin sepharose and gel filtration chromatogrpahy, gelatin-degrading activities of 95, 88, 75, and 65 kd were detected, the three lower-molecular-weight species probably representing activated forms. Enzyme activity was inhibited by ethyl-enediaminetetra-acetic acid (EDTA), but not by serine- and thiol-protease inhibitors, and was restored by zinc. Activity was also inhibited by tissue inhibitor of metalloproteinase-1 (TIMP-1) and alpha-2 macroglobulin. The partially purified enzyme rapidly degraded denatured collagens (gelatin) as well as native types III, IV, and V collagens, but had no activity against casein, types I and VI collagens.

Tissue inhibitor of metalloproteinase-1 messenger RNA expression is enhanced relative to interstitial collagenase messenger RNA in experimental liver injury and fibrosis.

Liver fibrosis results from a relative imbalance between synthesis and degradation of matrix proteins. We have previously described release of the protein collagenase inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), by culture-activated human hepatic stellate cells (HSCs). In this study, we have investigated the relative expression of TIMP-1 and interstitial collagenase in culture-activated rat HSCs and rat models of liver injury and fibrosis. The complementary DNA (cDNA) for rat TIMP-1 was obtained by homology polymerase chain reaction (PCR) and sequenced. By Northern analysis using this probe, TIMP-1 messenger RNA (mRNA) expression was up-regulated with HSC activation by culture on plastic as defined by cellular expression of procollagen-1. Interstitial collagenase mRNA was expressed in early 1. Interstitial collagenase mRNA was expressed in early culture (<4 days) but became undetectable in more activated cells (7-21 days). By activity assay of serum-free cell-conditioned media, TIMP-1 was found to be released in increasingly concentrations with duration of culture on plastic. Expression of TIMP-1 interstitial collagenase, and procollagen-1 mRNAs were studied in rat models of biliary and parenchymal injury (bile duct ligation and CC14 administration) by ribonuclease protein assay. TIMP-1 mRNA expression was increased at 6, 24 hours, and 3 days after bile duct ligation and was also shown to rise in acute CC14 liver injury and remain elevated as the liver became fibrotic. TIMP-1 expression preceded procollagen-1 expression in both models. In contrasts, interstitial collagenase mRNA levels remained similar to control values throughout both models of liver injury. Total cellular RNA from hepatocytes, HSCs, and kupffer cells freshly isolated from livers after acute CC14 injury was subjected to Northern analysis. TIMP-1 transcripts were observed in nonparenchymal cells only. We suggest that increased expression of TIMP-1 relative to interstitial collagenase by HSCs may promote progression of liver fibrosis in these rat models by preventing degradation of secreted collagens.