A Cell-Penetrant Manganese Superoxide Dismutase (MnSOD) Mimic Is Able To Complement MnSOD and Exerts an Antiinflammatory Effect on Cellular and Animal Models of Inflammatory Bowel Diseases.

Inorganic complexes are increasingly used for biological and medicinal applications, and the question of the cell penetration and distribution of metallodrugs is key to understanding their biological activity. Oxidative stress is known to be involved in inflammation and in inflammatory bowel diseases for which antioxidative defenses are weakened. We report here the study of the manganese complex Mn1 mimicking superoxide dismutase (SOD), a protein involved in cell protection against oxidative stress, using an approach in inorganic cellular chemistry combining the investigation of Mn1 intracellular speciation using mass spectrometry and of its quantification and distribution using electron paramagnetic resonance and spatially resolved X-ray fluorescence with evaluation of its biological activity. More precisely, we have looked for and found the MS signature of Mn1 in cell lysates and quantified the overall manganese content. Intestinal epithelial cells activated by bacterial lipopolysaccharide were taken as a cellular model of oxidative stress and inflammation. DNBS-induced colitis in mice was used to investigate Mn1 activity in vivo. Mn1 exerts an intracellular antiinflammatory activity, remains at least partially coordinated, with diffuse distribution over the whole cell, and functionally complements mitochondrial MnSOD.

High-resolution mass spectrometry and partial de novo sequencing constitute a useful approach for determining the profile of chemokine secretion following the stimulation of human intestinal epithelial cells.

RATIONALE: Intestinal epithelial cells (IEC) secrete many chemokines in response to proinflammatory stimuli. We investigated their role in the mucosal inflammatory response in the intestine, by developing a non-targeted approach for analyzing the profile of peptides secreted by stimulated IEC, based on differential mass spectrometry analysis. METHODS: Lipopolysaccharide (LPS) was incubated with IEC as a proinflammatory stimulus. Differential peptidomic analysis was then carried out, comparing the profiles of IEC with and without LPS stimulation. A mass spectrometry procedure was developed, based on a liquid chromatography/tandem mass spectrometry (LC/MS/MS) approach without enzymatic pretreatment of the peptides. Partial de novo sequencing was carried out by Fourier transform ion cyclotron resonance (FTICR), and the native peptides in the culture media were identified. RESULTS: A major ion (m/z 7862.51) detected after stimulation was identified as GRO alpha and a minor ion (m/z 8918.17) was identified as IL-8. ELISA-based comparisons gave results consistent with those obtained by MS. Surprisingly, GRO alpha was secreted in amounts 5 to 15 times higher than those for IL-8 in our cellular model. The truncated form of IL-8, resulting from activation, was detected and distinguished from the native peptide by MS, whereas this was not possible with enzyme-linked immunosorbent assay (ELISA). CONCLUSIONS: Mass spectrometric analysis of culture media can be used to identify the principal peptides produced in response to the stimulation of IEC, and their metabolites. Mass spectrometry provides a comprehensive view of the chemokines and peptides potentially involved in gut inflammation, making it possible to identify the most appropriate peptides for further quantification.

Connecting dysbiosis, bile-acid dysmetabolism and gut inflammation in inflammatory bowel diseases.

OBJECTIVE: Gut microbiota metabolises bile acids (BA). As dysbiosis has been reported in inflammatory bowel diseases (IBD), we aim to investigate the impact of IBD-associated dysbiosis on BA metabolism and its influence on the epithelial cell inflammation response. DESIGN: Faecal and serum BA rates, expressed as a proportion of total BA, were assessed by high-performance liquid chromatography tandem mass spectrometry in colonic IBD patients (42) and healthy subjects (29). The faecal microbiota composition was assessed by quantitative real-time PCR. Using BA profiles and microbiota composition, cluster formation between groups was generated by ranking models. The faecal BA profiles in germ-free and conventional mice were compared. Direct enzymatic activities of BA biotransformation were measured in faeces. The impact of BA on the inflammatory response was investigated in vitro using Caco-2 cells stimulated by IL-1ß. RESULTS: IBD-associated dysbiosis was characterised by a decrease in the ratio between Faecalibacterium prausntizii and Escherichia coli. Faecal-conjugated BA rates were significantly higher in active IBD, whereas, secondary BA rates were significantly lower. Interestingly, active IBD patients exhibited higher levels of faecal 3-OH-sulphated BA. The deconjugation, transformation and desulphation activities of the microbiota were impaired in IBD patients. In vitro, secondary BA exerted anti-inflammatory effects, but sulphation of secondary BAs abolished their anti-inflammatory properties. CONCLUSIONS: Impaired microbiota enzymatic activity observed in IBD-associated dysbiosis leads to modifications in the luminal BA pool composition. Altered BA transformation in the gut lumen can erase the anti-inflammatory effects of some BA species on gut epithelial cells and could participate in the chronic inflammation loop of IBD.

Bile acid profiling in human biological samples: comparison of extraction procedures and application to normal and cholestatic patients.

The role of bile acids in cell metabolism, membrane biology and cell signaling is increasingly recognized, thus making necessary a robust and versatile technique to extract, separate and quantify a large concentration range of these numerous molecular species. HPLC-MS/MS analysis provides the highest sensitivity to detect and identify bile acids. However, due to their large chemical diversity, extraction methods are critical and quite difficult to optimize, as shown by a survey of the literature. This paper compares the performances of four bile acid extraction protocols applied to either liquid (serum, urine, bile) or solid (stool) samples. Acetonitrile was found to be the best solvent for deproteinizing liquid samples and NaOH the best one for stool extraction. These optimized extraction procedures allowed us to quantitate as much as 27 distinct bile acids including sulfated species in a unique 30 min HPLC run, including both hydrophilic and hydrophobic species with a high efficiency. Tandem MS provided a non ambiguous identification of each metabolite with a good sensitivity (LOQ below 20 nmol/l except for THDCA and TLCA). After validation, these methods, successfully applied to a group of 39 control patients, detected 14 different species in serum in the range of 30-800 nmol/l, 11 species in urine in the range of 20-200 nmol/l and 25 species in stool in the range of 0.4-2000 nmol/g. The clinical interest of this method has been then validated on cholestatic patients. The proposed protocols seem suitable for profiling bile acids in routine analysis.

C-reactive protein and transesophageal echocardiographic markers of thromboembolism in patients with atrial fibrillation.

BACKGROUND: To determine whether C-reactive protein (CRP) in combination with a stroke risk stratification scheme can help in identifying transesophageal echocardiographic (TEE) markers of thromboembolism such as left atrial (LA)/left atrial appendage (LAA) thrombus, severe LA/LAA spontaneous echocardiographic contrast (SEC), and aortic plaque ≥ 4 mm. METHODS: Transthoracic echocardiography, TEE, and CRP measurement were performed at admission in 178 patients with non-valvular atrial fibrillation not receiving oral anticoagulant therapy. Patients were classified as at low, moderate, or high risk of thromboembolism based on seven clinical risk stratification schemes (SPAF, CHADS(2), Framingham, Birmingham/NICE, ACC/AHA/ESC 2006 guidelines, ACCP 2008, CHA(2)DS(2)VASc). RESULTS: Severe LA/LAA SEC, LA/LAA thrombus, and aortic plaque ≥ 4 mm were present in 6.2%, 6.7%, and 10.1% of patients, respectively. The combination of CRP with a cut-off value of 3.4 mg/L with the Birmingham/Nice or ACC/AHA/ESC 2006 risk score, led to a negative predictive value of 100% in low-risk patients and 91% in moderate-risk patients. For the detection of severe LA/LAA SEC or thrombus, a good discrimination (area under curve ≥ 0.70) using only clinical risk markers was observed for all classifications except for the Framingham and CHADS(2) risk scores. The addition of CRP did not improve the detection of LA/LAA SEC or thrombus, or of severe LA/LAA SEC, thrombus, or aortic plaque. CONCLUSION: The combination of clinical risk markers and CRP can help to exclude the presence of the TEE markers LA/LAA SEC or LA/LAA thrombus, particularly in patients classified at low or moderate risk of stroke.

Current smoking differentially affects blood mononuclear cells from patients with Crohn's disease and ulcerative colitis: relevance to its adverse role in the disease.

BACKGROUND: Epidemiologic data suggest that smoking increases the risk and the severity of Crohn's disease (CD), although it may protect patients with ulcerative colitis (UC). To investigate this paradox, we evaluated the effect of cigarette smoke in the function of blood mononuclear cells from healthy subjects and patients with CD or UC in flare up. METHODS: The production of mediators associated with inflammation but also with protective functions was evaluated by enzyme-linked immunosorbent assay (ELISA) and enzyme immunoassay (EIA), following either in vivo or in vitro exposure to cigarette smoke. RESULTS: We found that mononuclear cells from smokers with CD were functionally impaired. These cells secreted lower levels of chemokines and cytokines as compared with nonsmoker counterparts, whereas healthy smokers or smokers with UC were not affected. Similar findings were noted after in vitro exposure to cigarette smoke extract. In addition, cells from patients with CD who smoke presented a defective sensitivity to antiinflammatory or antioxidant protection, and particularly synthesized lower levels of cytoprotective Hsp70. The effects observed were not due to diminished cell viability. Our experiments suggest that cigarette smoke-related responses were largely dependent on oxidative stress generated, and not on the nicotine component. CONCLUSIONS: Overall, our data point out the presence of biological differences between blood mononuclear cells from patients with CD and UC toward cigarette smoke that might support its opposite role in both diseases.

Regulation of colon cancer cell proliferation and migration by MD-2 activity.

Evidence suggests that signalling through lipopolysaccharide (LPS) has a significant role in the development of gastrointestinal malignancies. We previously demonstrated the critical role of myeloid differentiation (MD)-2, the essential co-receptor of LPS, for induction of cyclooxygenase (Cox)-2 in intestinal epithelial cells. Cyclooxigenase-2 was suggested to play a key role in colorectal cancer through the effects of prostaglandin (PG) E(2) generated. We, therefore, addressed the role of MD-2 in several parameters related to malignancy, namely cell proliferation and migration, using colon cancer cells (HT-29). We found that overexpression of MD-2 confers a significantly greater proliferation and migration capacity to these cells. MD-2-dependent proliferation and migration appeared independent of Cox-2 activity but was reduced by endothelial growth factor receptor (EGFR) neutralizing antibodies as well as by pharmacological inhibition of EGFR tyrosine phosphorylation. We propose that MD-2 overexpression contributes to tumour aggressiveness via a Cox-2-independent excessive EGFR signalling. Moreover, MD-2 expression levels were higher in tissue from patients with colorectal cancer as compared with paired control colorectal mucosa. Our data attest to a role of MD-2 activity in colon cancer epithelial cell proliferation and migration, which may be important in the general correlation between innate immune response, chronic inflammation, and cancer.

Sera from patients with Crohn's disease break bacterial lipopolysaccharide tolerance of human intestinal epithelial cells via MD-2 activity.

Myeloid differentiation (MD)-2 is linked to the cell surface as a Toll-like receptor (TLR) 4-bound protein though may also function as a soluble receptor to enable the lipopolysaccharide (LPS)-driven response. We recently demonstrated the importance of MD-2 either as a cell-associated or as a soluble receptor in the control of intestinal epithelial cell response toward LPS. High levels of circulating MD-2 were recently proposed as a risk factor for infectious/ inflammatory diseases as septic shock. We hypothesized that MD-2 might be present in sera from patients with inflammatory bowel disease and have pathogenic consequences. We analysed MD-2 activity in sera from patients with inflammatory bowel disease or from healthy subjects. We measured MD-2 activity as the capacity to mediate LPS-driven stimulation of intestinal epithelial cells (HT29). We found that sera from patients with inflammatory bowel disease, particularly Crohn's disease, endowed HT29 cells with a markedly higher LPS-dependent stimulating capacity as compared to sera from healthy subjects. The effect of sera was specific for LPS activation and was reduced in the presence of anti-MD-2, and anti-TLR4 antibodies. We conclude that sera from patients with inflammatory bowel disease might contain increased MD-2. This might result in higher local availability of the protein leading to a loss of tolerance toward gut microbiota.

Abnormal transbilayer distribution of phospholipids in red blood cell membranes in schizophrenia.

Abnormalities in membrane lipids have been repeatedly reported in patients with schizophrenia. These abnormalities include decreased phosphatidylethanolamine (PE) and n-3 and n-6 polyunsaturated fatty acids in peripheral and brain cell membranes. The present study investigates the hypothesis of an overrepresentation of PE in the external leaflet of the red blood cell (RBC) membrane in patients with schizophrenia. The assumption was that this modification of PE asymmetrical distribution could explain the reported lipid membrane abnormalities. Phosphatidylethanolamine located in the external leaflet was specifically labeled in RBC membranes from 65 medicated patients with schizophrenia and 38 healthy controls. Labeled (external) and non-labeled (internal) PE and their respective fatty acid composition were analyzed by mass spectrometry. A significant increase in the percentage of external leaflet PE was found in RBC membranes in 63.1% of the patients. In this subgroup, a significant depletion of n-3 and n-6 polyunsaturated fatty acids from internally located PE was also observed. Age, sex and antipsychotic treatment were not associated with the transbilayer membrane distribution of PE. Potential mechanisms underlying these abnormalities may involve membrane phospholipid transporters or degradative enzymes involved in phospholipid metabolism. The anomaly described could characterize a subgroup among patients with schizophrenia.

MD-2 controls bacterial lipopolysaccharide hyporesponsiveness in human intestinal epithelial cells.

Intestinal epithelial cells (IEC) have adapted to the presence of commensal bacteria through a state of tolerance that involves a limited response to lipopolysaccharide (LPS). Low or absent expression of two LPS receptor molecules, the myeloid differentiation (MD)-2 receptor, and toll-like receptor (TLR)4 was suggested to underlie LPS tolerance in IEC. In the present study we performed transfections of TLR4 and MD-2 alone or combined in different IEC lines derived from intestinal cancer (Caco-2, HT-29, and SW837). We found that LPS responsiveness increased more than 100-fold when IEC were transfected with MD-2 alone, but not TLR4. The release of interleukin (IL)-8, but also the expression of cyclooxygenase (Cox-)2 and the related secretion of prostaglandin (PG)E2 were coordinately stimulated by LPS in IEC transfected with MD-2 alone. Supernatants collected from MD-2-transfected IEC supported LPS activation of naïve HT-29, providing additional support to the concept that MD-2 alone endows IEC with LPS responsiveness. LPS responsiveness detected at concentrations as low as 110 pg/ml, and maximal values obtained by 10 ng/ml were clearly beyond those evoked by classical stimuli as IL-1beta. In polarized cells, apical LPS stimulation was markedly more efficient than basolateral. Our data contradict previous opinion that both TLR4 and MD-2 limit IEC response to LPS, and emphasize the prominent role of MD-2 in intestinal immune responses to Gram-negative bacteria.

Secretory phospholipase A(2) activity and release kinetics of vascular tissue remodelling biomarkers after coronary artery bypass grafting with and without cardiopulmonary bypass.

BACKGROUND: Cardiopulmonary bypass (CPB) has long been recognised as a main cause of a postoperative complex systemic inflammatory response after coronary artery bypass grafting (CABG). METHODS: We determined the kinetics of peripheral blood release of the novel inflammatory biomarkers secretory phospholipase A(2) (sPLA(2)), matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) during the first 6 days following surgery in 16 patients undergoing CABG with (on-pump, n=9) or without (off-pump, n=7) CPB. Kinetic curves for these markers were compared to those of the well-known inflammatory parameters C-reactive protein (CRP) and fibrinogen. RESULTS: sPLA(2) activity exhibited a maximum value on day 2, then decreased until day 6 for both groups and in a similar manner as CRP levels. On the other hand, elevation of plasma levels of both MMP-9 and TIMP-1 occurred as early as on day 1 and remained at this level until day 6. No significant difference in kinetic characteristics (peak value, area under the curve, initial slope) between CABG with and without CPB was observed. CONCLUSIONS: These data show that the off- and on-pump groups did not show significantly different kinetics for the releases of all biomarkers studied, including sPLA(2) and biomarkers of the MMP-TIMP network. The off-pump procedure may therefore lead to global surgical trauma as important as CPB in terms of the systemic inflammatory process and matrix proteolysis pathway activation.

Cytoplasmic phospholipase A2 expression in human colon adenocarcinoma is correlated with cyclooxygenase-2 expression and contributes to prostaglandin E2 production.

Cytoplasmic phospholipase A2 (cPLA2) has a key role in prostaglandin production. The role of cPLA2 in intestinal tumorigenesis has been suggested, however, contradictory data are found in the literature. We evaluated cPLA2 and cyclooxygenase-2 (COX-2) protein expression in 65 colon carcinomas by immunohistochemistry, and in eight colorectal cancer cell lines by Western Blot. PGE2 production was evaluated by enzyme-immunoassay in the cell lines. We demonstrate that cPLA2 is overexpressed in approximately 50% of colon cancers and cell lines. cPLA2 expression is correlated with COX-2 expression. Both cPLA2 and COX-2 expressions are important in regard to PGE2 production. Our data suggest that cPLA2 might be involved in colon tumor development.

Circulating secretory phospholipase A2 activity predicts recurrent events in patients with severe acute coronary syndromes.

OBJECTIVES: The purpose of this study was to determine the prognostic value of circulating secretory phospholipase A2 (sPLA2) activity in patients with acute coronary syndromes (ACS). BACKGROUND: The plasma level of type IIA sPLA2 is a risk factor for coronary artery disease (CAD) and is associated with adverse outcomes in patients with stable CAD. The prognostic impact of sPLA2 in patients with ACS is unknown. METHODS: Secretory phospholipase A2 antigen levels and activity were measured in plasma samples of 446 patients with ACS, obtained at the time of enrollment. RESULTS: Baseline sPLA2 activity was associated with the risk of death and myocardial infarction (MI). The unadjusted rate of death and MI increased in a stepwise fashion with increasing tertiles of sPLA2 activity (p < 0.0001). The association remained significant in the subgroup of patients who had MI with ST-segment elevation (p = 0.014) and the subgroup of patients who had unstable angina or non-ST-segment elevation MI (p < 0.002). After adjustment for clinical and biological variables, the hazard ratios for the combined end point of death or MI in the third tertile of sPLA2 compared with the first and second tertiles was 3.08 (95% confidence interval, 1.37 to 6.91, p = 0.006). CONCLUSIONS: A single measurement of plasma sPLA2 activity at the time of enrollment provides strong independent information to predict recurrent events in patients with ACS.

Cytoplasmic phospholipase A2 alpha overexpression in stromal cells is correlated with angiogenesis in human colorectal cancer.

In colorectal cancer, cyclooxygenase-2 (COX-2) overexpression in stromal cells induces angiogenesis through EP2 prostaglandin E2 receptor signaling. Cytoplasmic phospholipase A2 (PLA2) alpha preferentially hydrolyses arachidonic acid, which is the limiting substrate for prostaglandin production, from membrane phospholipids. We therefore investigated a possible relationship between cytoplasmic PLA2 and COX-2 overexpression in stromal cells, angiogenesis and microsatellite instability in 48 human colorectal adenocarcinomas. Cytoplasmic PLA2 and COX-2 expression in stromal cells and vascular endothelial growth factor (VEGF) expression in tumor cells were evaluated by immunohistochemistry. Microvessel density was assessed in 10 x 400 fields after CD31 staining. Microsatellite instability was evaluated by PCR and immunohistochemistry. A total of 16 tumors had microsatellite instability. We found an overexpression of cytoplasmic PLA2 in superficial stromal cells. These cells corresponded to fibroblasts and myofibroblasts. There was an association between the number of cytoplasmic PLA2 and COX-2-expressing cells (P=0.006). Cytoplasmic PLA2-positive stromal cells usually also expressed COX-2. A high number of cytoplasmic PLA2-positive stromal cells was correlated with a high microvessel density (P=0.002), a strong VEGF (P=0.01) and the absence of microsatellite instability (P=0.001). The coordinate overexpression of cytoplasmic PLA2 and COX-2 in stromal cells could lead to an important prostaglandin production. These results suggest that cytoplasmic PLA2 overexpression in these cells regulates COX-induced angiogenesis probably by providing arachidonic acid, which is the limiting factor for prostaglandin production. The lower number of cytoplasmic PLA2-positive stromal cells in carcinomas with microsatellite instability could be related to their lower microvessel density and VEGF expression.

Cyclooxygenase-2 and its role in colorectal cancer development.

Cyclooxygenase 2 (COX-2), also called prostaglandin endoperoxide synthase 2, is involved in colorectal tumor development. This review deals with particular questions raised in this field such as the mechanisms of COX-2 related tumor promotion, the role of the different types of cells (epithelial and interstitial) expressing COX-2, the factors that trigger COX-2 induction, and the clinical potential of selective COX-2 inhibitors to treat or prevent colorectal tumors. Several mechanisms of COX-2 related tumor promotion have been identified. Some are dependent on prostaglandin E(2) production (such as induction of cell proliferation, angiogenenis or local immunosuppression, inhibition of apoptosis, increase in cell motility) and others are not (such as carcinogen activation or malondialdehyde production). COX-2 expression has been demonstrated in epithelial cells of colorectal cancers and adenomas and also in interstitial cells. These cells correspond to macrophages and/or fibroblasts and endothelial cells. COX-2 expression in these interstitial cells participates in tumor development. Factors or events that trigger COX-2 expression include oncogene activation, antioncogene inactivation, cytokines, growth factors, some fatty acids, bile salts, and mucins. Finally, selective COX-2 inhibitors may be effective in preventing or treating colorectal adenomas or carcinomas. However, their real efficiency and the cost/benefit balance are currently evaluated, and no definite conclusion can be made at the moment.

Cytosolic phospholipase A2-p11 interaction controls arachidonic acid release as a function of epithelial cell confluence.

Madin-Darby canine kidney type II cells were shown to release low amounts of AA (arachidonic acid) and prostaglandin E2 in response to various stimuli when analysed after cell confluence. In contrast, non-confluent Madin-Darby canine kidney type II cells released much higher amounts of AA and prostaglandin E2. In both stationary and non-confluent cells, AA was released by type IV cPLA2 (cytosolic phospholipase A2), as shown by the use of specific inhibitors and by analysis of the profile of fatty acids released. This confluence-dependent cPLA2 activation was not due to a difference in expression, or in phosphorylation of the enzyme, or in the amount of its substrate. To find out the mechanism by which cPLA2 activation may be regulated as a function of cell confluence, immunofluorescence and co-immunoprecipitation experiments were performed using cPLA2, p11, a natural inhibitor of the enzyme, and annexin II, the natural ligand of p11. These three proteins were expressed at a constant level, regardless of the cell confluence. In contrast, whereas annexin II and cPLA2 interacted at a constant rate, p11 and cPLA2 interacted more strongly in stationary cells, thus indicating that cPLA2 activation is regulated by its accessibility to p11, independent of their expression level. Our results indicate that, in epithelial cells, the cell confluence, i.e. the establishment of cell-cell contacts, rather than cell proliferation directly controls cPLA2 activation by changing the stoichiometry of p11/cPLA2 interaction.

Expression of the molecular chaperone Hsp70 in detergent-resistant microdomains correlates with its membrane delivery and release.

Accumulating evidence suggests that some heat shock proteins (Hsps), in particular the 72-kDa inducible Hsp70, associate to the cell membrane and might be secreted through an unknown mechanism to exert important functions in the immune response and signal transduction. We speculated that specialized structures named lipid rafts, known as important platforms for the delivery of proteins to the cell membrane, might be involved in the unknown mechanism ensuring membrane association and secretion of Hsp70. Lipid rafts are sphingolipid-cholesterol-rich structures that have been mainly characterized in polarized epithelial cells and can be isolated as detergent-resistant microdomains (DRMs). Analysis of soluble and DRM fractions prepared from unstressed Caco-2 epithelial cells revealed that Hsp70, and to a lesser extent calnexin, were present in DRM fractions. Increased expression of Hsps, through heat shock or by using drugs acting on protein trafficking or intracellular calcium level, induced an efficient translocation to DRM. We also found that Hsp70 was released by epithelial Caco-2 cells, and this release dramatically increased after heat shock. Drugs known to block the classical secretory pathway were unable to reduce Hsp70 release. By contrast, release of the protein was affected by the raft-disrupting drug methyl-beta-cyclodextrin. Our data suggest that lipid rafts are part of a mechanism ensuring the correct functions of Hsps and provide a rational explanation for the observed membrane association and release of Hsp70.

COX-2, inflammatory secreted PLA2, and cytoplasmic PLA2 protein expression in small bowel adenocarcinomas compared with colorectal adenocarcinomas.

Cyclooxygenase-2 (COX-2), human synovial inflammatory secreted phospholipase A2 (sPLA2) and cytoplasmic phospholipase A2 (cPLA2) are involved in eicosanoid production and also seem to participate in colorectal tumorigenesis. As there are no data regarding these enzymes in human small bowel tumors, we wanted to determine whether they were involved in human small bowel tumorigenesis, and whether their expression was different in small bowel compared to colorectal adenocarcinomas, as suggested by animal studies. We studied their protein expression by immunohistochemistry in 25 small bowel adenocarcinomas and compared it to 48 colorectal adenocarcinomas. Seventy-six percent of the small bowel and 88% of the colorectal adenocarcinomas had a moderate or strong COX-2 expression. Sixty-eight percent of the small bowel and 67% of the colorectal adenocarcinomas had a moderate or strong sPLA2 expression. Forty-eight percent of the small bowel and 35% of the colorectal adenocarcinomas had a moderate or strong cPLA2 expression. In conclusion, the increased expression of COX-2, sPLA2, and sometimes cPLA2 in both small bowel and colorectal adenocarcinomas is in accordance with the likely eicosanoid involvement in tumor development. The same pattern of protein expression found in both types of adenocarcinoma contradicts experimental results in mice. Moreover, our results strengthen the similarities between these two types of human cancer.

Mucinous colon carcinomas with microsatellite instability have a lower microvessel density and lower vascular endothelial growth factor expression.

We determined the association between cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF) expression, microvessel density (MVD) and microsatellite instability (MSI) or the histological type in colon adenocarcinomas. Sixty-six cases were studied, 28 MSI+ and 38 MSI-. MSI phenotype was determined using polymerase chain reaction. MVD was assessed after CD31 staining in ten x400 fields (0.96 mm(2)) in the most vascularized areas. VEGF and COX-2 expression were studied by means of immunohistochemistry. MVD positively correlated with the levels of VEGF expression (P=10(-4)) and also with the levels of COX-2 expression (P=0.007). MVD and VEGF expression were lower in MSI+ carcinomas (P=0.002 and P=0.03 respectively). When mucinous tumors were excluded from the statistical analysis, the association between low MVD, low VEGF and MSI status disappeared (P=0.5, P=1, respectively). MSI+ mucinous carcinomas had a lower MVD and VEGF expression than other MSI+ carcinomas (P=0.008 and P=0.004, respectively) and MSI- mucinous carcinomas (P=0.01 and P=0.001, respectively). COX-2 expression was lower in medullary carcinomas (P=0.001). In conclusion, mucinous MSI+ colon carcinomas represent a special group of colon adenocarcinomas relating to angiogenesis, with a lower MVD and VEGF expression than both MSI- mucinous carcinomas and MSI+ non-mucinous carcinomas. A low COX-2 expression could be related to the medullary phenotype. However, this has to be confirmed in a larger series. Finally, the low MVD of MSI+ mucinous colon adenocarcinomas could participate in their overall better prognosis.