Wim van Duijn

CuZn superoxide dismutase (SOD1) accumulates in vacuolated mitochondria in transgenic mice expressing amyotrophic lateral sclerosis-linked SOD1 mutations

Abstract Cytosolic Cu/Zn superoxide dismutase (SOD1) is a ubiquitous small cytosolic metalloenzyme, which catalyses the conversion of superoxide anion to hydrogen peroxide. Mutations in the SOD1 gene cause a familial form of amyotrophic lateral sclerosis (fALS). The mechanism by which mutant SOD1s cause the degeneration of motor neurons is not understood. Transgenic mice expressing multiple copies of fALS-mutant SOD1s develop an ALS-like motor neuron disease. Vacuolar degeneration of mitochondria has been identified as the main pathological feature associated with motor neuron death and paralysis in several lines of fALS-SOD1 mice. Using confocal and electron microscopy we show that mutant SOD1 is present at a high concentration in vacuolated mitochondria, where it colocalises with cytochrome c. Mutant SOD1 is also present in mildly swollen mitochondria prior to the appearance of vacuoles, suggesting that the leakage or translocation of mutant human SOD1 in mitochondria may be the primary event triggering their further degeneration. Vacuolated mitochondria containing SOD1 also occur in transgenic mice expressing a high concentration of wild-type human SOD1. In sum, our data suggest that both fALS-mutant and wild-type SOD1 may cross the mitochondrial outer membrane, and by doing so induce the degeneration of these mitochondria.

Imbalanced secondary mucosal antioxidant response in inflammatory bowel disease

Intestinal mucosal damage in the inflammatory bowel diseases (IBD) Crohns disease (CD) and ulcerative colitis (UC) involves reactive oxygen metabolites (ROMs). ROMs are neutralized by endogenous antioxidant enzymes in a carefully balanced two‐step pathway. Superoxide dismutases (SODs) convert superoxide anion to hydrogen peroxide (H2O2), which is subsequently neutralized to water by catalase (CAT) or glutathione peroxidase (GPO). Remarkably changed expression levels of the three isoforms of SOD in paired non‐inflamed and inflamed mucosae from CD and UC patients have been previously reported in comparison to normal control mucosa. Most notable was the strong up‐regulation of Mn‐SOD in inflamed epithelium. It was hypothesized that in order to provide optimal protection against ROM‐mediated damage, these changes should be coordinately counterbalanced by an increased H2O2‐neutralizing capacity. Therefore, the same tissue samples were used to assess the levels, activities, and/or localization of the most prominent mucosal H2O2‐related antioxidants CAT, GPO, glutathione (GSH), myeloperoxidase (MPO), and metallothionein (MT). Quantitative measurements showed that in both CD and UC patients, intestinal inflammation was associated with increased activities of CAT, GPO, and MPO, whereas the mucosal GSH content was unaffected and the concentration of MT was decreased. Despite this overall increase in mucosal H2O2‐metabolizing enzyme capacity, immunohistochemical analysis revealed a differentially disturbed antioxidant balance in IBD epithelium and lamina propria. In the lamina propria, the risk of direct H2O2‐mediated damage seemed to be restrained by the increasing numbers of CAT‐ and MPO‐positive monocytes/macrophages and neutrophils that infiltrated the inflamed areas. On the other hand, MPO overexpression might increase the lamina propria levels of hypochlorous acid, a stable ROM with multiple pro‐inflammatory effects. In the epithelium, the number of cells that expressed CAT remained unchanged during inflammation and GPO was found in only a very low and constant number of epithelial cells. In addition, the inflamed epithelium displayed decreased expression of the hydroxyl radical (OH•) scavenger MT. In view of the high epithelial SOD levels in inflamed IBD epithelium, it is speculated that the efficient removal of excess H2O2 is hampered in these cells, thereby increasing not only the risk of detrimental effects of H2O2 directly, but also those of its extremely reactive derivatives such as OH•. Taken together, the results suggest an imbalanced and inefficient endogenous antioxidant response in the intestinal mucosa of IBD patients, which may contribute to both the pathogenesis and the perpetuation of the inflammatory processes. Copyright

VEGF release by MMP-9 mediated heparan sulphate cleavage induces colorectal cancer angiogenesis

Angiogenesis is crucial for the progression of colorectal carcinomas in which the bioavailability of Vascular Endothelial Growth Factor (VEGF) plays a major role. VEGF bioavailability is regulated by proteolytic release or cleavage. In colorectal cancer patients, we observed a significant correlation between circulating VEGF and tumour tissue Matrix Metalloproteinase-9 (MMP-9) levels but not with MMP-2. Therefore, we evaluated the role of MMP-9 in regulating VEGF bioavailability and subsequent angiogenesis in 3-dimensional human cell culture models. MMP-9 treatment released VEGF dose-dependently from HT29 colon carcinoma spheroids, comparable to heparitinase, a known mediator of VEGF release. Conditioned medium from human neutrophils, containing high amounts of active MMP-9, released VEGF comparable to recombinant MMP-9, in contrast to myofibroblast medium. MMP-9 treated spheroids showed decreased extracellular levels of heparan sulphates, required for VEGF binding to the matrix, whereas the levels in the medium were increased. Western blot analysis revealed that VEGF(165) is the major isoform released by MMP-9 treatment. In vitro experiments indicated that MMP-9 is not capable to cleave VEGF(165) into smaller isoforms, like plasmin does. These data suggested that MMP-9 mediates release rather than the cleavage of larger VEGF isoforms. Medium from MMP-9 treated HT29 spheroids induced endothelial cell sprouting in an angiogenesis assay, comparable to the effect of recombinant VEGF(165). Anti-VEGF antibody treatment resulted in a strongly reduced number of sprouts. In conclusion, we have shown that neutrophil-derived MMP-9 is able to release biologically active VEGF(165) from the ECM of colon cancer cells by the cleavage of heparan sulphates.

Differential mucosal expression of three superoxide dismutase isoforms in inflammatory bowel disease.

Mucosal tissue damage and dysfunction in chronic inflammatory bowel disease (IBD) are partly caused by an enduring exposure to excessive amounts of reactive oxygen metabolites (ROMs). Although the three human isoforms of superoxide dismutase (SOD), copper/zinc (Cu/Zn)‐SOD, manganese (Mn)‐SOD, and extracellular (EC)‐SOD, form the primary endogenous defence against ROMs, their expression levels and cellular localization in IBD mucosa are largely unknown. The present study used enzyme‐linked immunosorbent assays (ELISAs), spectrophotometric activity assays, and immunohistochemistry to evaluate the protein concentration, enzymatic activity, and distribution of Cu/Zn‐, Mn‐, and EC‐SOD in paired inflamed and non‐inflamed mucosal resection specimens of patients with Crohns disease (CD) or ulcerative colitis (UC) and compared these with the levels obtained in normal control mucosa. Gut mucosal SOD isoform expression was found to be differentially affected in IBD patients, without major differences between CD and UC. A marked step‐wise increase in Mn‐SOD protein levels was observed in non‐inflamed and inflamed IBD mucosae, whereas the Cu/Zn‐SOD content decreased with inflammation. EC‐SOD was only found in low amounts, which tended to be decreased in IBD patients. Immunohistochemical evaluation confirmed these observations. Mn‐SOD and Cu/Zn‐SOD were both predominantly expressed in intestinal epithelial cells and the percentage of epithelial cells positive for Mn‐SOD was considerably increased in IBD, whereas epithelial Cu/Zn‐SOD expression was much less affected. Within the lamina propria, SOD expression was much lower. Cu/Zn‐SOD and Mn‐SOD were prominently present in neutrophils and macrophages, and EC‐SOD was mainly localized in small vessels, stromal cells, and neutrophils. The percentage of lamina propria cells positive for Cu/Zn‐, Mn‐, or EC‐SOD was not affected by inflammation. Enzyme activity measurements showed consistent results for Cu/Zn‐SOD and EC‐SOD, but the activity of Mn‐SOD did not concordantly increase with the immunological assessments, which may indicate that a proportion of the Mn‐SOD in IBD is present in an enzymatically inactive form. This study reveals remarkable changes in the expression levels of the three SOD isoforms in IBD, particularly in the epithelium. Disturbances in the carefully orchestrated mucosal antioxidant cascade may contribute to the induction and perpetuation of intestinal inflammation in IBD, and may have important implications for the development of antioxidant treatment of IBD patients. Copyright

Effect of the anti‐tumor necrosis factor‐α antibody infliximab on the ex vivo mucosal matrix metalloproteinase–proteolytic phenotype in inflammatory bowel disease

Background Previous studies have shown an upregulation of matrix metalloproteinases (MMPs) in intestinal tissue of patients with inflammatory bowel disease (IBD) and significant clinical improvement after administration of the anti‐TNF‐&agr; antibody infliximab. The aims of our study were to determine expression and secretion of MMP‐1, ‐2, ‐3, ‐9, and their inhibitors TIMP‐1, ‐2 by IBD versus control intestinal mucosa ex vivo and to assess the regulatory capacity by infliximab of the proteolytic phenotype. Methods Intestinal mucosal explants from 20 IBD and 15 control patients were cultured with or without infliximab and/or the T‐cell activator pokeweed mitogen (PWM). Explants and culture supernatants were analyzed for MMPs, TIMPs, and TNF‐&agr; protein, activity and/or mRNA levels. All patients were genotyped for functional TNF‐&agr;, MMP, and TIMP single nucleotide polymorphism (SNP) loci. Results Expression of MMP and TIMP protein/activity in basal medium was higher in IBD versus control explants. Dependent on genotype at SNP loci, infliximab downregulated MMP‐1, ‐3, and ‐9 relative to TIMP‐1 and ‐2 and also decreased MMP‐1 and ‐3 activities, while PWM enhanced these levels, partly counteracted again by infliximab. The expression of MMP‐2 relative to TIMP did not change by treatment with infliximab and/or PWM. Conclusions The high expression of MMPs in patients with IBD suggests a role for these proteinases in the pathogenesis of this disease. Infliximab seems to induce a genotype‐associated matrix protective phenotype, which may contribute to the observed therapeutic efficacy of this drug in IBD, particularly at the mucosal surface. (Inflamm Bowel Dis 2007)

Infliximab treatment influences the serological expression of matrix metalloproteinase (MMP)-2 and -9 in Crohn's disease.

Background Matrix metalloproteinases (MMPs) are actively involved in the pathogenesis of Crohns disease (CD). We assessed the effect of the anti‐tumor necrosis factor‐&agr; (TNF‐&agr;) monoclonal antibody infliximab on the in vitro and in vivo expression of MMP‐2 and MMP‐9 in CD. Methods Infliximab‐treated fistulizing (n = 10) or active disease (n = 7) CD patients, from an in‐house study, and fistulizing CD patients (n = 42) and active CD patients (n = 24) from 2 placebo controlled studies were evaluated for serum MMP levels and clinical response. Biopsies were evaluated immunohistochemically for the MMPs. Whole blood cultures stimulated with lipopolysaccharide (LPS)/infliximab were evaluated for MMP mRNA and protein levels. Results Serum MMP‐2 levels in CD patients increased during follow‐up, similarly in responders and nonresponders, by infliximab. Immunohistochemistry showed no clear MMP‐2 change in biopsies. Serum MMP‐9 levels, however, showed a consistent pattern of decrease in most CD patients, particularly in those responding, and MMP‐9‐positive polymorphonuclear leukocytes in biopsies also decreased by infliximab. LPS stimulation of whole blood increased the MMP‐9 levels in plasma significantly in CD patients and controls, but infliximab had no effect on the secretion. Long‐term LPS stimulation raised leukocyte MMP‐9 mRNA levels 16‐fold and infliximab inhibited this induction by 80%. Conclusions Infliximab treatment increases MMP‐2 and decreases MMP‐9 in serum of patients with CD, the latter also in the intestine, which extends and confirms our previous ex vivo explants observations. However, these changes were not strictly associated with the response to treatment. The enhanced leukocyte MMP‐9 expression in CD seems to be regulated by TNF‐&agr;. (Inflamm Bowel Dis 2007)

Eradication of Helicobacter pylori infection favourably affects altered gastric mucosal MMP-9 levels.

Background:  Helicobacter pylori gastritis is recognized as an important pathogenetic factor in peptic ulcer disease and gastric carcinogenesis, and is accompanied by strongly enhanced gastric mucosal matrix metalloproteinase‐9 (MMP‐9) levels.

Effect of partial gastrectomy on serum anti-Helicobacter pylori immunoglobulins in peptic ulcer patients

Since biliary enterogastric reflux is suggested to eradicate gastric infection withHelicobacter pylori (HP), we have investigated in a prospective randomized study the effect of partial gastrectomy with either Billroth II or Roux-en-Y anastomosis on infection with HP as assessed by the titers of IgG and IgA antibodies against HP in serum. These antibodies were measured by ELISA in serum of 22 patients before and at 10 days and 6, 15, and 24 months after either Billroth II (N=11) or Roux-en-Y (N=11) gastrectomy for peptic ulcer. All patients had HP demonstrated in their preoperative endoscopic gastric biopsies. The preoperative serum IgA antibodies against HP (anti-HP IgA) were increased in 20 of the 22 patients (range 0.21–1.69) while the IgG antibodies (anti-HP IgG) were increased in all 22 patients (range 0.38–1.31). Four of the Billroth II patients had clearance of HP from gastric biopsies accompanied by rapid and pronounced decrease of anti-HP IgA. In contrast, the patients with Roux-en-Y gastrectomy and the Billroth II patients with persistent HP infection had no change in anti-HP IgA after surgery. Anti-HP IgG showed variable results in the four patients without gastric HP infection and was not affected by gastrectomy in the patients with persistent HP infection. We concluded that serum anti-HP IgA, but not anti-HP IgG, is helpful in identifying those patients in whom HP is no longer demonstrable after Billroth II gastrectomy. Gastrectomy with Roux-en-Y anastomosis had no effect on gastric HP infection.

Copeptin as an Indicator of Hemodynamic Derangement and Prognosis in Liver Cirrhosis

Background Advanced liver cirrhosis is associated with systemic hemodynamic derangement leading to the development of severe complications associated with increased mortality. Copeptin is a stable cleavage product of the precursor of arginine vasopressin, a key-regulator in hemodynamic homeostasis. Copeptin is currently considered a reliable prognostic marker in a wide variety of diseases other than cirrhosis. The present study aimed to assess copeptin, both experimentally and clinically, as a potential biomarker of hemodynamic derangement and to evaluate its prognostic significance in cirrhosis. Materials and Methods Two studies were executed: 1) in 18 thioacetamide-induced cirrhotic rats and 5 control rats, plasma copeptin and hemodynamic measurements were performed, 2) in 61 cirrhotic patients, serum copeptin concentration was measured in samples collected at time of registration at the waiting list for liver transplantation. In 46 patients, also a second copeptin measurement was performed during follow-up while registered at the waiting list for liver transplantation. To determine the association of serum copeptin and clinical data with outcome, Cox proportional hazard regression analysis and Kaplan Meier analysis were performed. Results Plasma copeptin concentration was significantly higher in cirrhotic rats than in controls (1.6 ± 0.5 vs. 0.9 ± 0.1 pmol/L, p< 0.01) and was negatively correlated to the mean arterial blood pressure (r = -0.574, p = 0.013). In cirrhotic patients, serum copeptin concentration was high [11.0 (5.2–24.0) pmol/L] and increased significantly during the time of registration at the waiting list for liver transplantation. MELD and MELD-sodium score were significantly correlated to serum copeptin [MELD: (r = 0.33, p = 0.01), MELD-sodium: (r = 0.29, p = 0.02)], also at time of the second copeptin measurement [MELD and MELD-sodium: r = 0.39, p< 0.01]. In cirrhotic humans, serum copeptin concentration was significantly associated with outcome, independently of the MELD and MELD-sodium score. Patients with a low serum copeptin concentration at time of registration at the liver transplant waiting list had significantly better transplant-free survival rates at 3, 6 and 12 months of follow-up as compared to those with a high serum copeptin concentration (Log-rank: p< 0.01, p< 0.01 and p = 0.02 respectively). Conclusions Circulating copeptin levels are elevated in rats and humans with cirrhosis. Copeptin is independently associated with outcome in cirrhotic patients awaiting liver transplantation.