Hein W. Verspaget

Genome-wide meta-analysis increases to 71 the number of confirmed Crohn's disease susceptibility loci

We undertook a meta-analysis of six Crohns disease genome-wide association studies (GWAS) comprising 6,333 affected individuals (cases) and 15,056 controls and followed up the top association signals in 15,694 cases, 14,026 controls and 414 parent-offspring trios. We identified 30 new susceptibility loci meeting genome-wide significance (P < 5 × 10−8). A series of in silico analyses highlighted particular genes within these loci and, together with manual curation, implicated functionally interesting candidate genes including SMAD3, ERAP2, IL10, IL2RA, TYK2, FUT2, DNMT3A, DENND1B, BACH2 and TAGAP. Combined with previously confirmed loci, these results identify 71 distinct loci with genome-wide significant evidence for association with Crohns disease.

Oxidative stress as a pathogenic factor in inflammatory bowel disease — radicals or ridiculous?

Virtually all inflammatory mediators investigated to date seem to be dysregulated in the inflamed intestinal mucosa of patients with inflammatory bowel disease. However, which of these are actually involved in the initiation and perpetuation of intestinal tissue damage is still not fully understood. Amongst these mediators are the reactive oxygen metabolites, produced in large amounts by the massively infiltrating leucocytes. These reactive oxygen metabolites are believed to constitute a major tissue‐destructive force and may contribute significantly to the pathogenesis of inflammatory bowel disease.

Intestinal oxidative damage in inflammatory bowel disease: semi-quantification, localization, and association with mucosal antioxidants

Intestinal inflammation is accompanied by excessive production of reactive oxygen and nitrogen metabolites. In order to counteract their harmful effects, the intestinal mucosa contains an extensive system of antioxidants. It has previously been shown that the levels of and the balance between the most important antioxidants are seriously impaired within the intestinal mucosa from inflammatory bowel disease (IBD) patients compared with normal mucosa. The present study investigated the consequences of this antioxidative imbalance by evaluating parameters of oxidative stress‐related mucosal damage in the same tissue samples. The extent of apoptosis, peroxynitrite‐mediated protein nitration (3‐NT), and lipid peroxidation were assessed in relation to the expression of nitric oxide synthase (NOS) and the superoxide‐producing enzyme xanthine oxidase (XO). In addition, bi‐ and multi‐variate regression analyses were performed to associate these parameters with the levels of the antioxidants assessed previously. Apoptotic cell death was visualized by TUNEL staining in luminal epithelium of normal controls, and in IBD additionally in the inflammatory infiltrate and in deeper parts of the crypts, but its frequency was unrelated to the severity of inflammation. In Crohns disease (CD), epithelial apoptosis levels were strongly associated with the expression of XO, implying a role for this enzyme in the regulation of epithelial cell homeostasis, although its levels were unaffected by intestinal inflammation and were comparable to those in normal control mucosa. 3‐NT immunoreactivity was substantially increased in luminal crypt cells, neutrophils, and mononuclear cells in the inflamed mucosa of ulcerative colitis (UC) patients. The inflamed IBD luminal epithelium, but not the inflammatory cells, also contained increased amounts of NOS. The immunoreactivity of both 3‐NT and NOS was significantly higher in UC than in CD. Unexpectedly, the increased 3‐NT expression in UC was associated with neutrophilic myeloperoxidase and not with NOS, which suggests that 3‐NT is formed in areas with a dense neutrophilic infiltrate via a peroxynitrite‐independent oxidation pathway. Lipid peroxidation, as estimated by the malondialdehyde (MDA) concentration, was elevated in both the inflamed CD and the inflamed UC mucosa, and was identified in the luminal epithelium using a histochemical technique. In CD, lipid peroxidation was independently associated with the concentration of metallothionein and with Mn‐superoxide dismutase activity, suggesting the involvement of hydroxyl radicals and superoxide anions. In UC, however, the amount of MDA was associated with epithelial catalase expression and neutrophilic myeloperoxidase activity, suggesting a hydrogen peroxide‐ and/or hypochlorous acid‐mediated mechanism. The present study underlines the importance of oxidative stress in the pathogenesis of IBD and provides clues regarding the (anti)oxidants involved which indicate that this process evolves through diverging pathways in CD and UC. Copyright

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.

Role of Tumor Necrosis Factor-α and Interleukin-10 Gene Polymorphisms in Irritable Bowel Syndrome

OBJECTIVES:Imbalances in the genetically controlled pro- and anti-inflammatory cytokine production may promote ongoing low-grade inflammation after an acute gastroenteritis, and subsequently, irritable bowel syndrome (IBS) (post-infectious IBS, PI-IBS). We studied gene promoter single nucleotide polymorphisms (SNPs) of tumor necrosis factor-α (TNF-α, pro-inflammatory) and interleukin-10 (IL-10, anti-inflammatory) in IBS patients and controls.METHODS:DNA was extracted from peripheral blood leucocytes of 111 IBS patients and 162 healthy controls. Genotype and allele frequencies were assessed by analyzing SNPs at position −308 (TNF-α) and −1082 and −819 (IL-10).RESULTS:Homozygous high producers for TNF-α (A/A) were rare (overall prevalence 2.6%). The heterozygous TNF-α genotype (G/A, high producer) was significantly more prevalent in IBS compared to controls (41%vs 26%, p = 0.02). More patients (41%) than controls (30%) were positive for the A allele (p = 0.044; odds ratio (OR) 1.68, 95% confidence interval (CI) 1.01–2.79), with a similar trend for diarrhea (54%) versus constipation and alternating subtypes (<33%, p = 0.079), but not for subgroups according to a history of acute gastroenteritis. IL-10 genotypes were similarly distributed in patients and controls for both SNPs. Possession of a high producer TNF-α and a low producer IL-10 genotype were significantly more prevalent in IBS (9%) versus controls (3%, p = 0.035; OR 3.11, 95% CI 1.03–9.36) and in diarrhea (20%) compared to other IBS subtypes (<4%, p = 0.026).CONCLUSIONS:Our results support the emerging hypothesis that genetically determined immune activity plays a role in the pathophysiology of IBS. Future studies in larger, clinically relevant, IBS subgroups are warranted to establish definite associations with cytokine gene polymorphisms.

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

Matrix Metalloproteinase-14 (MT1-MMP)–Mediated Endoglin Shedding Inhibits Tumor Angiogenesis

Endoglin is a transforming growth factor-beta coreceptor with a crucial role in angiogenesis. A soluble form of endoglin is present in the circulation, but the role of soluble endoglin (sEndoglin) is poorly understood. In addition, the endoglin shedding mechanism is not known. Therefore, we examined the role of sEndoglin in tumor angiogenesis and the mechanism by which the extracellular domain of endoglin is released from the membrane.In colorectal cancer specimens, we observed high endothelial endoglin protein expression, accompanied with slightly lower sEndoglin levels in the circulation, compared with healthy controls. In vitro analysis using endothelial sprouting assays revealed that sEndoglin reduced spontaneous and vascular endothelial growth factor-induced endothelial sprouting. Human umbilical vascular endothelial cells were found to secrete high levels of sEndoglin. Endoglin shedding was inhibited by matrix metalloproteinase (MMP) inhibitors and MMP-14 short hairpin RNA, indicating MMP-14 as the major endoglin shedding protease. Coexpression of endoglin and membrane-bound MMP-14 led to a strong increase in sEndoglin levels. Endoglin shedding required a direct interaction between endoglin and membrane-localized MMP-14. Using cleavage site mutants, we determined that MMP-14 cleaved endoglin at a site in close proximity to the transmembrane domain. Taken together, this study shows that MMP-14 mediates endoglin shedding, which may regulate the angiogenic potential of endothelial cells in the (colorectal) tumor microenvironment.

Pretreatment with interferon-γ enhances the therapeutic activity of mesenchymal stromal cells in animal models of colitis

Mesenchymal stromal cells (MSCs) are currently under investigation for the treatment of inflammatory disorders, including Crohns disease. MSCs are pluripotent cells with immunosuppressive properties. Recent data suggest that resting MSCs do not have significant immunomodulatory activity, but that the immunosuppressive function of MSCs has to be elicited by interferon‐γ (IFN‐γ). In this article, we assessed the effects of IFN‐γ prestimulation of MSCs (IMSCs) on their immunosuppressive properties in vitro and in vivo. To this end, we pretreated MSCs with IFN‐γ and assessed their therapeutic effects in dextran sodium sulfate (DSS)‐ and trinitrobenzene sulfonate (TNBS)‐induced colitis in mice. We found that mice treated with IMSCs (but not MSCs) showed a significantly attenuated development of DSS‐induced colitis. Furthermore, IMSCs alleviated symptoms of TNBS‐induced colitis. IMSC‐treated mice displayed an increase in body weight, lower colitis scores, and better survival rates compared with untreated mice. In addition, serum amyloid A protein levels and local proinflammatory cytokine levels in colonic tissues were significantly suppressed after administration of IMSC. We also observed that IMSCs showed greater migration potential than unstimulated MSCs to sites within the inflamed intestine. In conclusion, we show that prestimulation of MSCs with IFN‐γ enhances their capacity to inhibit Th1 inflammatory responses, resulting in diminished mucosal damage in experimental colitis. These data demonstrate that IFN‐γ activation of MSCs increases their immunosuppresive capacities and importantly, their therapeutic efficacy in vivo. STEM CELLS 2011;29:1549–1558

Molecular prediction of disease risk and severity in a large Dutch Crohn's disease cohort

Background: Crohn’s disease and ulcerative colitis have a complex genetic background. We assessed the risk for both the development and severity of the disease by combining information from genetic variants associated with inflammatory bowel disease (IBD). Methods: We studied 2804 patients (1684 with Crohn’s disease and 1120 with ulcerative colitis) and 1350 controls from seven university hospitals. Details of the phenotype were available for 1600 patients with Crohn’s disease and for 800 with ulcerative colitis. Genetic association for disease susceptibility was tested for the nucleotide-binding and oligomerisation domain 2 gene (NOD2), the IBD5 locus, the Drosophila discs large homologue 5 and autophagy-related 16-like 1 genes (DLG5 and ATG16L1) and the interleukin 23 receptor gene (IL23R). Interaction analysis was performed for Crohn’s disease using the most associated single nucleotide polymorphism (SNP) for each locus. Odds ratios were calculated in an ordinal regression analysis with the number of risk alleles as an independent variable to analyse disease development and severity. Results: Association with Crohn’s disease was confirmed for NOD2, IBD5, DLG5, ATG16L1 and IL23R. Patients with Crohn’s disease carry more risk alleles than controls (p = 3.85 × 10−22). Individuals carrying an increasing number of risk alleles have an increasing risk for Crohn’s disease, consistent with an independent effects multiplicative model (trend analysis p = 4.25 × 10−23). Patients with Crohn’s disease with a more severe disease course, operations or an age of onset below 40 years have more risk alleles compared to non-stricturing, non-penetrating behaviour (p = 0.0008), no operations (p = 0.02) or age of onset above 40 years (p = 0.028). Conclusion: Crohn’s disease is a multigenic disorder. An increase in the number of risk alleles is associated with an increased risk for the development of Crohn’s disease and with a more severe disease course. Combining information from the known common risk polymorphisms may enable clinicians to predict the course of Crohn’s disease.

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.