Henrike Lenzen

LDL Cholesterol Upregulates Synthesis of Asymmetrical Dimethylarginine in Human Endothelial Cells: Involvement of S-Adenosylmethionine–Dependent Methyltransferases

Asymmetrical dimethylarginine (ADMA) is an endogenous nitric oxide synthase inhibitor. It is formed by protein arginine N-methyltransferases (PRMTs), which utilize S-adenosylmethionine as methyl group donor. ADMA plasma concentration is elevated in hypercholesterolemia, leading to endothelial dysfunction and producing proatherogenic changes of endothelial cell function. Four different isoforms of human PRMTs have been identified. Because the release of ADMA from human endothelial cells is increased in the presence of native or oxidized LDL cholesterol, we investigated the potential involvement of PRMT activity and gene expression in this effect. We found that the production of ADMA by human endothelial cells is upregulated in the presence of methionine or homocysteine and inhibited by either of the methyltransferase inhibitors S-adenosylhomocysteine, adenosine dialdehyde, or cycloleucine. This effect is specific for ADMA but not symmetrical dimethylarginine. The upregulation of ADMA release by native and oxidized LDL is abolished by S-adenosylhomocysteine and by the antioxidant pyrrollidine dithiocarbamate. Furthermore, a methyl-(14)C label is transferred from S-adenosylmethionine to ADMA but not symmetrical dimethylarginine, in human endothelial cells. The expression of PRMTs is upregulated in the presence of native or oxidized LDL. Our data suggest that the production of ADMA by human endothelial cells is regulated by S-adenosylmethionine-dependent methyltransferases. This activity is upregulated by LDL cholesterol, which may be due in part to the enhanced gene expression of PRMTs. In concentrations reached by stimulation of methyltransferases (5 to 50 micromol/L), ADMA significantly inhibited the formation of (15)N-nitrite from L-[guanidino-(15)N(2)]arginine. These findings suggest a novel mechanism by which ADMA concentration is elevated in hypercholesterolemia, leading to endothelial dysfunction and atherosclerosis.

Urinary 8-iso-Prostaglandin F2α as a Risk Marker in Patients With Coronary Heart Disease A Matched Case-Control Study

Background—Oxidative stress is involved in the pathophysiology of atherosclerosis, diabetes mellitus, hypertension, obesity, and cigarette smoking, all of these being risk factors for coronary heart disease (CHD). We tested the hypothesis that risk factors of CHD are associated with abundant systemic oxidative stress. Methods and Results—We conducted a case-control study with 93 CHD patients and 93 control subjects frequency-matched by age and sex. Urinary excretion of the F2-isoprostane 8-iso-prostaglandin (PG) F2&agr; and its major urinary metabolite, 2,3-dinor-5,6-dihydro-8-iso-PGF2&agr;, were measured by gas chromatography–tandem mass spectrometry. Body mass index, systolic blood pressure, and C-reactive protein were elevated in CHD patients (P <0.01). Urinary 8-iso-PGF2&agr; and 2,3-dinor-5,6-dihydro-8-iso-PGF2&agr; also differed, from 77 (interquartile range, 61–101) to 139 (93–231) pmol/mmol creatinine and from 120 (91–151) to 193 (140–275) pmol/mmol in control subjects and case subjects, respectively (P <0.001). 8-iso-PGF2&agr; and its metabolite were highly correlated (Spearman’s &rgr;=0.664, P <0.001). HDL cholesterol was diminished in CHD patients (P <0.001). All of these characteristics predicted CHD in univariate analysis. In a multivariate model, the odds ratios were increased only for 8-iso-PGF2&agr; (≥131 pmol/mmol, P <0.001) and C-reactive protein (>3 mg/L, P <0.01), ie, by 30.8 (95% CI, 7.7–124) and 7.2 (1.9–27.6), respectively. 8-iso-PGF2&agr; was found to be a novel marker in addition to known risk factors of CHD, ie, diabetes mellitus, hypercholesterolemia, hypertension, and smoking. Urinary excretion of 8-iso-PGF2&agr; correlated with the number of risk factors for all subjects (P <0.001 for trend). Conclusions—8-iso-PGF2&agr; is a sensitive and independent risk marker of CHD.

Molecular Mechanisms of Disturbed Electrolyte Transport in Intestinal Inflammation

Abstract:  Diarrhea is the hallmark of both ulcerative colitis (UC) and Crohns disease. Loss of resorptive area, destruction of epithelial cells, leaky tight junctions, and release of inflammatory mediators and products from immune cells that stimulate fluid secretion all have been implicated in the pathogenesis of inflammatory diarrhea. Very early studies in patients, however, have pinpointed the overwhelming transport abnormality in inflamed intestinal mucosa: a virtually complete loss of sodium resorptive capacity. Recently, tools have become available to study the molecular basis of disturbances in the major electrolyte transport systems during intestinal inflammation. This review gives a brief overview of the historical development of research related to electrolyte transport in inflammatory bowel disorders, focusing on the studies performed in humans, and highlights recent understanding of the molecular mechanisms that may help explain the origin of diarrhea in intestinal inflammation.

Biliary Strictures and Recurrence After Liver Transplantation for Primary Sclerosing Cholangitis: A Retrospective Multicenter Analysis

Liver transplantation (LT) is the only definitive treatment for patients with end‐stage liver disease due to primary sclerosing cholangitis (PSC), but a high rate of biliary strictures (BSs) and of recurrent primary sclerosing cholangitis (recPSC) has been reported. In this multicenter study, we analyzed a large patient cohort with a long follow‐up in order to evaluate the incidence of BS and recPSC, to assess the impact on survival after LT, and to identify risk factors. We collected clinical, surgical, and laboratory data and records on inflammatory bowel disease (IBD), immunosuppression, recipient and graft outcome, and biliary complications (based on cholangiography and histology) of all patients who underwent LT for PSC in 10 German transplant centers between January 1990 and December 2006; 335 patients (68.4% men; mean age, 38.9 years; 73.5% with IBD) underwent transplantation 8.8 years after PSC diagnosis with follow‐up for 98.8 months. The 1‐, 5‐, and 10‐year recipient and graft survival was 90.7%, 84.8%, 79.4% and 79.1%, 69.0%, 62.4%, respectively. BS was diagnosed in 36.1% after a mean time of 3.9 years, and recPSC was diagnosed in 20.3% after 4.6 years. Both entities had a significant impact on longterm graft and recipient survival. Independent risk factors for BS were donor age, ulcerative colitis, chronic ductopenic rejection, bilirubin, and international normalized ratio (INR) at LT. Independent risk factors for recPSC were donor age, IBD, and INR at LT. These variables were able to categorize patients into risk groups for BS and recPSC. In conclusion, BS and recPSC affect longterm graft and patient survival after LT for PSC. Donor age, IBD, and INR at LT are independent risk factors for BS and recPSC and allow for risk estimation depending on the recipient‐donor constellation. Liver Transpl 22:42‐52, 2016.

Low Risk of Hepatocellular Carcinoma in Patients With Primary Sclerosing Cholangitis With Cirrhosis

BACKGROUND & AIMS Primary sclerosing cholangitis (PSC) is associated with an increased risk of hepatobiliary malignancies. However, little is known about the incidence of hepatocellular carcinoma (HCC) among patients with PSC; current recommendations on screening these patients for HCC are conflicting. We investigated the risk of HCC in patients with PSC with cirrhosis. METHODS We performed a retrospective study of patients with well-defined PSC from 2 large-volume tertiary care centers in Germany; data were collected from periods of up to 33 years. Liver cirrhosis was based on histology results or the presence of ascites, esophageal varices, or transient elastography values greater than 14 kPa. Statistical analysis was performed using the Kaplan-Meier method and the Cox proportional hazard model. Data from 509 patients (67% male), with a total of 4202 patients-years, were included in the final analysis. RESULTS We identified 119 patients with cirrhosis. During 292 patient-years, none of these patients developed HCC. Most HCCs were identified incidentally at the time of liver transplantation. We therefore reviewed data on liver explants from 140 patients who underwent transplantation; none were found to contain HCC. In contrast to the low numbers of HCCs among patients with PSC, 35 patients developed cholangiocarcinoma, 3 patients developed gallbladder cancer, and 9 patients developed colorectal cancer. CONCLUSIONS Based on a retrospective analysis of more than 500 patients with PSC, we confirm their high risk for hepatobiliary malignancies. However, the risk of HCC, even among patients with cirrhosis, seems to be low--regular HCC surveillance may not be warranted.

Downregulation of the NHE3-Binding PDZ-Adaptor Protein PDZK1 Expression during Cytokine-Induced Inflammation in Interleukin-10–Deficient Mice

Background Impaired salt and water absorption is an important feature in the pathogenesis of diarrhea in inflammatory bowel disease (IBD). We analyzed the expression of proinflammatory cytokines in the infiltrating immune cells and the function and expression of the Na+/H+ exchanger isoform 3 (NHE3) and its regulatory PDZ-adaptor proteins NHERF1, NHERF2, and PDZK1 in the colon of interleukin-10–deficient (IL-10−/−) mice. Methodology/Principal Findings Gene and protein expression were analyzed by real-time reverse transcription polymerase chain reaction (qRT-PCR), in situ RT-PCR, and immunohistochemistry. NHE3 activity was measured fluorometrically in apical enterocytes within isolated colonic crypts. Mice developed chronic colitis characterized by a typical immune cell infiltration composed of T-lymphocytes and macrophages, with high levels of gene and protein expression of the proinflammatory cytokines interleukin-1β and tumor necrosis factor-α. In parallel, inducible nitric oxide synthase expression was increased while procaspase 3 expression was unaffected. Interferon-γ expression remained low. Although acid-activated NHE3 activity was significantly decreased, the inflammatory process did not affect its gene and protein expression or its abundance and localization in the apical membrane. However, expression of the PDZ-adaptor proteins NHERF2 and PDZK1 was downregulated. NHERF1 expression was unchanged. In a comparative analysis we observed the PDZK1 downregulation also in the DSS (dextran sulphate sodium) model of colitis. Conclusions/Significance The impairment of the absorptive function of the inflamed colon in the IL-10−/−mouse, in spite of unaltered NHE3 expression and localization, is accompanied by the downregulation of the NHE3-regulatory PDZ adaptors NHERF2 and PDZK1. We propose that the downregulation of PDZ-adaptor proteins may be an important factor leading to NHE3 dysfunction and diarrhea in the course of the cytokine-mediated inflammatory process in these animal models of IBD.

Loss of Slc26a9 anion transporter alters intestinal electrolyte and HCO3- transport and reduces survival in CFTR-deficient mice

Slc26a9 is an anion transporter that is strongly expressed in the stomach and lung. Slc26a9 variants were recently found associated with a higher incidence of meconium ileus in cystic fibrosis (CF) infants, raising the question whether Slc26a9 is expressed in the intestine and what its functional role is. Slc26a9 messenger RNA (mRNA) was found highly expressed in the mucosae of the murine and human upper gastrointestinal tract, with an abrupt decrease in expression levels beyond the duodenum. Absence of SLC26a9 expression strongly increased the intestinally related mortality in cystic fibrosis transmembrane conductance regulator (CFTR)-deficient mice. Proximal duodenal JHCO3− and fluid secretion were reduced in the absence of Slc26a9 expression. In the proximal duodenum of young Slc26a9 KO mice, the glands and villi/crypts were elongated and proliferation was enhanced. This difference was lost with ageing, as were the alterations in fluid movement, whereas the reduction in JHCO3- remained. Laser dissection followed by qPCR suggested Slc26a9 expression to be crypt-predominant in the duodenum. In summary, deletion of Slc26a9 caused bicarbonate secretory and fluid absorptive changes in the proximal duodenal mucosa and increased the postweaning death rates in CFTR-deficient mice. Functional alterations in the duodenum were most prominent at young ages. We assume that the association of meconium ileus and Slc26a9 variants may be related to maldigestion and impaired downstream signaling caused by loss of upper GI tract digestive functions, aggravating the situation of lack of secretion and sticky mucus at the site of obstruction in CF intestine.

Complication and mortality rate after percutaneous endoscopic gastrostomy are low and indication-dependent*

Abstract Objective. Percutaneous endoscopic gastrostomy (PEG) is often used for the feeding of patients with malnutrition due to dysphagia, and despite more than 30 years experience, numerous questions on its benefit remain. This was a prospective observational study to assess the safety of PEG. Material and methods. One hundred and nineteen patients mean age 63 years (21–91 years) who were admitted to the Hannover Medical School between November 2010 and March 2012 and had an indication for PEG according to the German guidelines were included. Primary endpoints were the following: reason for indication, date of in-hospital mortality after PEG insertion, death within 3 months after PEG placement, and complications. Results. Most patients (54.6%) received PEG for dysphagia caused by tumors and second (29.4%) for neurologic diseases with a minor proportion of dementia (3%). About 73% of our patients had no complications at all and only 10% suffered severe effects. We saw only 1 case of aspiration, which did not lead to pneumonia. The 30-day mortality was 10%, and no patient died as a result of the PEG procedure. Significantly more patients with neurologic disorders died within 24 weeks of PEG placement than tumor patients (60% versus 27.7%, respectively, p = 0.002, n = 100). Conclusion. It is important to select patients receiving PEG very carefully. The patients’ indications, their primary disease, and their capability for mental cooperation are essential. If these aspects are taken into account, PEG is a safe method with few mainly mild complications.

Successful treatment of cervical esophageal leakage by endoscopic-vacuum assisted closure therapy

AIM To evaluate the efficacy and safety of endoscopic-vacuum assisted closure (E-VAC) therapy in the treatment of cervical esophageal leakage. METHODS Between May and November 2012, three male patients who developed post-operative cervical esophageal leakage were treated with E-VAC therapy. One patient had undergone surgical excision of a pharyngo-cervical liposarcoma with partial esophageal resection, and the other two patients had received surgical treatment for symptomatic Zenkers diverticulum. Following endoscopic verification of the leakage, a trimmed polyurethane sponge was fixed to the distal end of a nasogastric silicone tube and endoscopically positioned into the wound cavity, and with decreasing cavity size the sponge was positioned intraluminally to cover the leak. Continuous suction was applied, and the vacuum drainage system was changed twice a week. RESULTS The initial E-VAC placement was technically successful for all three patients, and complete closure of the esophageal leak was achieved without any procedure-related complications. In all three patients, the insufficiencies were located either above or slightly below the upper esophageal sphincter. The median duration of the E-VAC drainage was 29 d (range: 19-49 d), with a median of seven sponge exchanges (range: 5-12 sponge exchanges). In addition, the E-VAC therapy reduced inflammatory markers to within normal range for all three patients. Two of the patients were immediately fitted with a percutaneous enteral gastric feeding tube with jejunal extension, and the third patient received parenteral feeding. All three patients showed normal swallow function and no evidence of stricture after completion of the E-VAC therapy. CONCLUSION E-VAC therapy for cervical esophageal leakage was well tolerated by patients. This safe and effective procedure may significantly reduce morbidity and mortality following cervical esophageal leakage.

Calprotectin in bile: a disease severity marker in patients with primary sclerosing cholangitis.

Goals: Our aim was to evaluate the diagnostic potential of calprotectin in serum and bile of patients with primary sclerosing cholangitis (PSC). Background: PSC is a chronic cholestatic liver disease of unknown etiology. It is characterized by progressive inflammation and fibrosis of the bile ducts leading to biliary cirrhosis and eventually liver failure. Reliable markers for disease activity and severity are still lacking. Subunits of calprotectin, a fecal marker of inflammation in inflammatory bowel disease, have been recently identified in bile. Study: Calprotectin was measured in patients with PSC (n=56), cholangiocarcinoma (CC) complicating PSC (CC/PSC) (n=13), CC (n=30), and bile duct stones in bile (n=38) and serum (n=73) by enzyme-linked immunosorbent assay in a cross-sectional study. PSC patients were categorized by the Mayo risk score (MRS) to characterize the disease severity. Results: Calprotectin is present in bile, and the median concentration was significantly higher in PSC patients (P<0.05). Stratification of PSC patients by MRS showed significantly elevated calprotectin levels in bile in the MRS-high group (P<0.05). Calprotectin and MRS correlated significantly (P<0.05). The presence or absence of inflammatory bowel disease in PSC patients did not alter calprotectin levels in bile. Serum AP and calprotectin in bile correlated significantly (P=0.013). No significant correlation was found for other liver-related parameters. In contrast, serum calprotectin levels were significantly higher in patients with CC, but there was no association with PSC or disease activity/severity. Conclusions: Calprotectin in bile is a promising disease marker in patients with PSC with a potential prognostic value.