Sandeep Choudhary

Oxidant-induced intestinal barrier disruption and its prevention by growth factors in a human colonic cell line: role of the microtubule cytoskeleton.

Reactive oxygen metabolites (ROM) are increased in the inflamed mucosa of inflammatory bowel disease (IBD) and may contribute to loss of intestinal barrier function in this disorder. Growth factors (GF) are protective. But the mechanisms of disruption and protection remain elusive. In the present investigation, we hypothesized that the microtubules (a critical cytoskeletal element) play a key role in the molecular mechanism of intestinal barrier dysfunction induced by ROM and in GF-mediated protection. Utilizing monolayers of a human colonic cell line (Caco-2), we evaluated the effects of ROM (H(2)O(2) or HOCl), in the presence or absence of GF (epidermal growth factor [EGF]; transforming growth factor-alpha [TGF-alpha]), on intestinal barrier function, tubulin (microtubule structural protein), and microtubule stability. Monolayers were also processed for two highly sensitive western immunoblots: fractionated polymerized tubulin (S2; an index of stability); monomeric tubulin (S1; an index of disruption) to detect the oxidation and disassembly/assembly of tubulin. ROM exposure led to a significant increase in the oxidation of tubulin, decrease in the stable S2 polymerized tubulin, and increase in the unstable S1 monomeric tubulin. In concert, each ROM in a dose dependent manner damaged the microtubule cytoskeleton and disrupted barrier function. GF pretreatment not only increased the S2 stable tubulin and decreased tubulin oxidation but also, concomitantly, prevented the disruption of microtubules and loss of barrier function in monolayers exposed to ROM. Antibody against the GF-receptor and inhibitors of GF-receptor tyrosine kinase abolished GF protection, indicating the involvement of epidermal growth factor receptor (EGFR) signaling pathway. As predicted, colchicine, an inhibitor of microtubule assembly, caused barrier dysfunction and prevented GF protection whereas taxol, a microtubule-stabilizing agent, mimicked the protective effects of GF. Thus, organization and stability of the microtubule cytoskeleton appears to be critical to both oxidant-induced mucosal barrier dysfunction and protection of intestinal barrier mediated by GF. Therefore, microtubules may be useful targets for development of drugs for the treatment of IBD.

Successful and sustained treatment of chronic radiation proctitis with antioxidant vitamins E and C

OBJECTIVE:Chronic radiation proctitis, a common sequelae of pelvic radiation, is characterized by obliteration of the submucosal vasculature with subsequent ischemia and reperfusion injury. Oxidative stress is thought to be a major mechanism in radiation proctitis. Therefore, antioxidants (vitamins E and C) may be beneficial.METHODS:Twenty consecutive symptomatic outpatients with endoscopically documented radiation proctitis seen in a single gastroenterology clinic were given a combination of vitamin E (400 IU tid) and vitamin C (500 mg tid). Previous radiation therapy was given for prostatic (n = 10) or gynecological (n = 10) malignancies. These patients presented with one or more of the following symptoms: rectal bleeding, rectal pain, diarrhea, or fecal urgency. Using a questionnaire, these symptoms were rated by the patients in terms of their severity (grade 0–4) and frequency (grade 0–4) before and after treatment with vitamins E and C. A symptom index was calculated by the addition of the severity and frequency scores (8 = most symptomatic). The lifestyle impact of the symptoms was also assessed by questionnaire grading from 0 (no effect on daily activity) to 4 (afraid to leave home). Among these 20 patients, 10 patients who received vitamins E and C for 1 yr were assessed again to determine whether their initial responses were sustained.RESULTS:There was a significant (p < 0.05; Wilcoxon rank) improvement in the symptom index (before treatment vs after treatment with vitamins E and C) for bleeding (median score: 4 vs 0), diarrhea (median score: 5 vs 0), and urgency (median score: 6 vs 3). Patients with rectal pain did not improve significantly. Bleeding resolved in four of 11 patients, diarrhea resolved in eight of 16 patients, fecal urgency resolved in three of 16 patients, and rectal pain resolved in two of six patients. Lifestyle improved in 13 patients, including seven patients who reported a return to normal. Two of the patients with no improvement in their daily symptoms also had radiation ileitis. All 10 patients who underwent a second follow-up interview reported sustained improvement in their symptoms 1 yr later.CONCLUSION:A substantial number of patients with radiation proctitis seem to benefit from antioxidant therapy. A double-blind placebo-controlled trial is needed to confirm this open-labeled pilot study.

Novel Antioxidants Zolimid and AEOL11201 Ameliorate Colitis in Rats

The mechanism of tissue damage in ulcerative colitis (UC) is unknown. However, recent evidence suggests that reactive oxygen species (ROS) are critical mediators of inflammation, and tissue damage in UC and antioxidants could be beneficial in the treatment of UC. Our aim was to evaluate the effects of two new antioxidants, Zolimid and AEOL11201 on experimental colitis. Antioxidants or vehicle were given to rats for five days after induction of colitis by intrarectal administration of 4% acetic acid. Severity of colitis was assessed on day 5. Zolimid and AEOL11201 significantly improved acetic acid-induced colitis. Both Zolimid and AEOL11201 significantly decreased the severity of diarrhea, and severity of macroscopic and histological changes in the colon. Both agents also significantly decreased colonic MPO levels. In conclusion, Zolimid and AEOL11201 are effective antiinflammatory agents in an animal model of colitis. Further studies are needed to evaluate their beneficial therapeutic effects in patients with UC.

Critical Role of the Atypical λ Isoform of Protein Kinase C (PKC-λ) in Oxidant-Induced Disruption of the Microtubule Cytoskeleton and Barrier Function of Intestinal Epithelium

Oxidant injury to epithelial cells and gut barrier disruption are key factors in the pathogenesis of inflammatory bowel disease. Studying monolayers of intestinal (Caco-2) cells, we reported that oxidants disrupt the cytoskeleton and cause barrier dysfunction (hyperpermeability). Because the λ isoform of protein kinase C (PKC-λ), an atypical diacylglycerol-independent isozyme, is abundant in parental (wild type) Caco-2 cells and is translocated to the particulate fractions upon oxidant exposure, we hypothesized that PKC-λ is critical to oxidative injury to the assembly and architecture of cytoskeleton and the intestinal barrier function. To this end, Caco-2 cells were transfected with an inducible plasmid, a tetracycline-responsive system, to create novel clones stably overexpressing native PKC-λ. Other cells were transfected with a dominant-negative plasmid to stably inhibit the activity of native PKC-λ. Cells were exposed to oxidant (H2O2) ± modulators. Parental Caco-2 cells were treated similarly. We then monitored barrier function (fluorescein sulfonic acid clearance), microtubule cytoskeletal stability (confocal microscopy, immunoblotting), subcellular distribution of PKC-λ (immunofluorescence, immunoblotting, immunoprecipitation), and PKC-λ isoform activity (in vitro kinase assay). Monolayers were also processed to assess alterations in tubulin assembly, polymerized tubulin (S2, an index of cytoskeletal integrity), and monomeric tubulin (S1, an index of cytoskeletal disassembly) (polyacrylamide gel electrophoresis fractionation and immunoblotting. In parental cells, oxidant caused: 1) translocation of PKC-λ from the cytosol to the particulate (membrane + cytoskeletal) fractions, 2) activation of native PKC-λ, 3) tubulin pool instability (increased monomeric S1 and decreased polymerized S2), 4) disruption of cytoskeletal architecture, and 5) barrier dysfunction (hyperpermeability). In transfected clones, overexpression of the atypical (74 kDa) PKC-λ isoform by itself (∼3.2-fold increase) led to oxidant-like disruptive effects, including cytoskeletal and barrier hyperpermeability. Overexpressed PKC-λ was mostly found in particulate cell fractions (with a smaller cytosolic distribution) indicating its activation. Disruption by PKC-λ overexpression was also potentiated by oxidant challenge. Stable inactivation of endogenous PKC-λ (∼99.6%) by a dominant-negative protected against all measures of oxidant-induced disruption. We conclude that: 1) oxidant induces disruption of epithelial barrier integrity by disassembling the cytoskeleton, in large part, through the activation of PKC-λ isoform; and 2) activation of PKC-λ by itself appears to be sufficient for disruption of cellular cytoskeleton and monolayer barrier permeability. The unique ability to mediate an oxidant-like injury and cytoskeletal depolymerization and instability is a novel mechanism not previously attributed to the atypical subfamily of PKC isoforms.