Myeloid, but not pancreatic, RelA/p65 is required for fibrosis in a mouse model of chronic pancreatitis.

BACKGROUND & AIMS: Little is known about how transcription factors might regulate pathogenesis of chronic pancreatitis (CP). We analyzed the in vivo role of RelA/p65, a component of the transcription factor nuclear factor (NF)-κB, in different cell types during development of CP in mice. METHODS: RelA/p65 was functionally inactivated in the pancreas (relaΔpanc), in myeloid cells (relaΔmye), or both (relaΔpanc,Δmye) compartments using the Cre-loxP strategy. Experimental CP was induced with repetitive injections of cerulein over 6 weeks. Pancreata were investigated histologically and biochemically. We created an in vitro coculture assay of pancreatic stellate cells (PSC) and macrophages and performed gene arrays from pancreata and macrophages with functionally inactivated RelA/p65. Tissue samples from patients with CP were analyzed for matrix metalloproteinase (MMP) 10 expression. RESULTS: In contrast to their relaF/F littermates, relaΔpanc displayed typical signs of CP after long-term stimulation with cerulein. Numerous macrophages and activated α-smooth muscle actin (SMA)-positive PSCs were detected. Additional inactivation of RelA/p65 in myeloid cells (relaΔpanc,Δmye) attenuated fibrosis. In vitro, RelA/p65-deficient, lipopolysaccharide (LPS)-stimulated macrophages degraded fibronectin in cocultured PSCs. Using gene expression analysis, MMP-10 was identified as a candidate for this process. Recombinant MMP-10 degraded fibronectin in LPS-stimulated PSCs. In tissue samples from patients with CP, MMP-10 was up-regulated in myeloid cells. CONCLUSIONS: RelA/p65 functions in myeloid cells to promote pathogenesis of CP. In acinar cells, RelA/p65 protects against chronic inflammation, whereas myeloid RelA/p65 promotes fibrogenesis. In macrophage, MMP-10 functions as a RelA/p65-dependent, potentially antifibrogenic factor during progression of CP.

Assessment of the protective effects of oral tocotrienols in arginine chronic-like pancreatitis.

Tocotrienols exhibit anti-inflammatory properties over macrophages and promote cytotoxicity in activated pancreatic stellate cells, suggesting that they may limit chronic pancreatitis progression. We aimed to quantitate the effect of oral tocotrienols on a rat model of chronic pancreatic injury. Chronic-like pancreatitis was induced by repeated arginine pancreatitis. Palm oil tocotrienol-rich fraction (TRF) was given by gavage before and after pancreatitis inductions. Amylase and hydroxyproline were determined in pancreatic homogenates; collagen, fibronectin, α-smooth muscle actin (SMA), glial fibrillary acidic protein (GFAP), and phosphorylated Smad3 were assessed by Western blotting. Transforming growth factor (TGF)-ß1 was measured in plasma. Morphological assessment included light microscopy, fibrosis area fraction, and collagen network fractal analysis. Arginine pancreatitis induced pancreatic atrophy and increased hydroxyproline that ameliorated after TRF. Arginine increased TGF-ß1 (185 ± 40 vs. 15 ± 2 ng/ml; P <0.01) that was blunted by TRF (53 ± 19; P < 0.01). TRF reduced protease and Smad3 activation, collagen, and fibronectin. α-SMA increased and GFAP diminished in arginine pancreatitis, consistent with long-term stellate cell activation, and TRF reverted these changes to basal. Arginine pancreatitis increased fibrosis area fraction (4.5 ± 0.3% vs. 0.2 ± 0.2%), collagen network complexity (fractal dimension 1.52 ± 0.03 vs. 1.42 ± 0.01; P < 0.001), and inhomogeneity (lacunarity 0.63 ± 0.03 vs. 0.40 ± 0.02; P < 0.001), which were all reduced by TRF (1.3 ± 0.4%, 1.43 ± 0.02%, and 0.51 ± 0.03%, respectively; P < 0.01). Best correlation coefficients were obtained when comparing fibrosis area fraction with lacunarity (r = 0.88) and both parameters with pancreatic weight (r = -0.91 and -0.79, respectively). TRF administered only before pancreatitis best, but not fully, recapitulated the beneficial effects of TRF. Tocotrienols improve quantitative measures of chronic pancreatic damage. They may be of benefit in human chronic pancreatitis.

Tocotrienols have potent antifibrogenic effects in human intestinal fibroblasts.

BACKGROUND: Excessive fibroblast expansion and extracellular matrix (ECM) deposition are key events for the development of bowel stenosis in Crohn's disease (CD) patients. Tocotrienols are vitamin E compounds with proven in vitro antifibrogenic effects on rat pancreatic fibroblasts. We aimed at investigating the effects of tocotrienols on human intestinal fibroblast (HIF) proliferation, apoptosis, autophagy, and synthesis of ECM. METHODS: HIF isolated from CD, ulcerative colitis (UC), and normal intestine were treated with tocotrienol-rich fraction (TRF) from palm oil. HIF proliferation was quantified by (3) H-thymidine incorporation, apoptosis was studied by DNA fragmentation, propidium iodide staining, caspase activation, and poly(ADP-ribose) polymerase cleavage, autophagy was analyzed by quantification of LC3 protein and identification of autophagic vesicles by immunofluorescence and production of ECM components was measured by Western blot. RESULTS: TRF significantly reduced HIF proliferation and prevented basic fibroblast growth factor-induced proliferation in CD and UC, but not control HIF. TRF enhanced HIF death by promoting apoptosis and autophagy. HIF apoptosis, but not autophagy, was prevented by the pan-caspase inhibitor zVAD-fmk, whereas both types of cell death were prevented when the mitochondrial permeability transition pore was blocked by cyclosporin A, demonstrating a key role of the mitochondria in these processes. TRF diminished procollagen type I and laminin γ-1 production by HIF. CONCLUSIONS: Tocotrienols exert multiple effects on HIF, reducing cell proliferation, enhancing programmed cell death through apoptosis and autophagy, and decreasing ECM production. Considering their in vitro antifibrogenic properties, tocotrienols could be useful to treat or prevent bowel fibrosis in CD patients.

Tocotrienols: balancing the mitochondrial crosstalk between apoptosis and autophagy.

Tocotrienols are a group of natural vitamin E compounds with patent antitumoral effects, mostly based on their ability to induce apoptosis in cancer cells. In activated pancreatic stellate cells (PSCs) we have determined that tocotrienols elicit a dramatic mitochondrial destabilization followed by initiation of non-necrotic forms of programmed cell death, namely apoptosis and autophagy. PSCs are the main cell type involved in the generation of pancreatic fibrosis, and their removal is critical to limit the fibrogenic process. Noteworthy, tocotrienol death-promoting actions are exclusively directed to activated PSCs, but not to their quiescent counterparts nor to terminally differentiated acinar cells. Here, we hypothesize that the transformed phenotype of PSCs may include "activated" mitochondria, which can be used by tocotrienols to trigger autophagic and apoptotic signaling. We propose that mitochondria are the cornerstone of cell sensitivity to tocotrienols, and suggest possible mechanisms, that may be interconnected, on how tocotrienols may govern mitochondrial death pathways.

Tocotrienols induce apoptosis and autophagy in rat pancreatic stellate cells through the mitochondrial death pathway.

BACKGROUND & AIMS: Selective removal of activated pancreatic stellate cells (PSCs) through induction of their own programmed death is a goal of therapeutic interest in patients with chronic pancreatitis. Here, we investigated the effects of tocotrienols on PSC death outcomes. METHODS: Activated and quiescent PSCs and acinar cells from rat pancreas were treated with vitamin E derivatives alpha-tocopherol; individual alpha-, beta-, gamma-, and delta-tocotrienols; and a tocotrienol rich fraction (TRF) from palm oil. RESULTS: TRF, but not alpha-tocopherol, reduced viability of activated PSC by setting up a full death program, independent of cell cycle regulation. Activated PSCs died both through apoptosis, as indicated by increased DNA fragmentation and caspase activation, and through autophagy, as denoted by the formation of autophagic vacuoles and LC3-II accumulation. In contrast to alpha-tocopherol, TRF caused an intense and sustained mitochondrial membrane depolarization and extensive cytochrome c release. Caspase inhibition with zVAD-fmk suppressed TRF-induced apoptosis but enhanced autophagy. However, mitochondrial permeability transition pore blockade with cyclosporin A completely abolished the deadly effects of TRF. beta-, gamma-, and delta-tocotrienol, but not alpha-tocotrienol nor alpha-tocopherol, reproduced TRF actions on activated PSCs. TRF death induction was restricted to activated PSCs because it did not cause apoptosis either in quiescent PSCs or in acinar cells. CONCLUSIONS: Tocotrienols selectively trigger activated pancreatic stellate cell death by targeting the mitochondrial permeability transition pore. Our findings unveil a novel potential for tocotrienols to ameliorate the fibrogenesis associated with chronic pancreatitis.