Portal de Periódicos da CAPES

HIF1-alpha Regulates Acinar Cell Function and Response to Injury in Mouse Pancreas

2018; Elsevier BV; Volume: 154; Issue: 6 Linguagem: Inglês

10.1053/j.gastro.2018.01.037

1528-0012

Min-Jung Park, Sapna Iyer, Xiang Xue, Juliana Bragazzi Cunha, Shufang Gu, David S. Moons, Steven W. Pipe, John A. Williams, Diane M. Simeone, Yatrik M. Shah, M. Bishr Omary,

Pancreatic function and diabetes

We investigated whether intrapancreatic coagulation, with deposition of the fibrinogen-γ dimer (Fib-γD) and hypoxia, affect the severity of acute pancreatitis (AP) in mice. Pancreata of mice with AP induced by administration of cerulein or by L-arginine, or from patients with pancreatitis, had increased deposition of Fib-γD compared with control pancreata. Heparin administration protected mice from cerulein-induced AP and prevented Fib-γD formation. Cerulein administration resulted in activation and stabilization of hypoxia-inducible factor-1α (HIF1α) in pancreata of oxygen-dependent degradation domain–luciferase HIF1α reporter mice. Cerulein also led to induction of genes regulated by HIF1α, including Vegfa and Ero1a, before evidence of Fib-γD deposition or histologic features of AP. Expression of tissue factor, which is regulated by vascular endothelial growth factor, also increased following cerulein administration. Mice with acinar cell–specific disruption of Hif1a (Hif1aAc–/–) developed spontaneous endoplasmic reticulum stress and less severe AP, but did not accumulate Fib-γD following administration of cerulein. Feeding mice increased pancreatic expression of HIF1α, indicating a physiologic role in the exocrine pancreas. Therefore, HIF1α has bifunctional roles, in exocrine pancreas homeostasis and progression of AP that is promoted by intrapancreatic coagulation. We investigated whether intrapancreatic coagulation, with deposition of the fibrinogen-γ dimer (Fib-γD) and hypoxia, affect the severity of acute pancreatitis (AP) in mice. Pancreata of mice with AP induced by administration of cerulein or by L-arginine, or from patients with pancreatitis, had increased deposition of Fib-γD compared with control pancreata. Heparin administration protected mice from cerulein-induced AP and prevented Fib-γD formation. Cerulein administration resulted in activation and stabilization of hypoxia-inducible factor-1α (HIF1α) in pancreata of oxygen-dependent degradation domain–luciferase HIF1α reporter mice. Cerulein also led to induction of genes regulated by HIF1α, including Vegfa and Ero1a, before evidence of Fib-γD deposition or histologic features of AP. Expression of tissue factor, which is regulated by vascular endothelial growth factor, also increased following cerulein administration. Mice with acinar cell–specific disruption of Hif1a (Hif1aAc–/–) developed spontaneous endoplasmic reticulum stress and less severe AP, but did not accumulate Fib-γD following administration of cerulein. Feeding mice increased pancreatic expression of HIF1α, indicating a physiologic role in the exocrine pancreas. Therefore, HIF1α has bifunctional roles, in exocrine pancreas homeostasis and progression of AP that is promoted by intrapancreatic coagulation. Editor's NotesBackground and ContextThe role of intra-parenchymal coagulation, as assessed by fibrinogen dimer (Fib-γD) deposition, hypoxia, and hypoxia inducible factor-1α (HIF1α) in modulating the progression of pancreatitis is not known.New FindingsIntra-pancreatic coagulation is augmented during experimental pancreatitis and reversed by heparin, with upregulation of HIF1α early during pancreatitis. Feeding induces HIF1α, while its acinar cell-specific deficiency causes ER stress but protects from pancreatitis.LimitationsThe mechanism of feeding-associated HIF1α induction remains to be determined.ImpactThe study demonstrates novel associations of HIF1-alpha with normal pancreas function and with progression of acute pancreatitis. It also links HIF1α with intra-pancreatic coagulation/Fib-γD formation, and pancreatitis, with therapeutic implications. The role of intra-parenchymal coagulation, as assessed by fibrinogen dimer (Fib-γD) deposition, hypoxia, and hypoxia inducible factor-1α (HIF1α) in modulating the progression of pancreatitis is not known. Intra-pancreatic coagulation is augmented during experimental pancreatitis and reversed by heparin, with upregulation of HIF1α early during pancreatitis. Feeding induces HIF1α, while its acinar cell-specific deficiency causes ER stress but protects from pancreatitis. The mechanism of feeding-associated HIF1α induction remains to be determined. The study demonstrates novel associations of HIF1-alpha with normal pancreas function and with progression of acute pancreatitis. It also links HIF1α with intra-pancreatic coagulation/Fib-γD formation, and pancreatitis, with therapeutic implications. Systemic alterations in coagulation are associated with complications from acute pancreatitis (AP), and are one of the reasons for the high mortality rate of AP.1Kakafika A. Papadopoulos V. Mimidis K. et al.Coagulation, platelets, and acute pancreatitis.Pancreas. 2007; 34: 15-20Crossref PubMed Scopus (53) Google Scholar, 2Lisman T. Porte R.J. Activation and regulation of hemostasis in acute liver failure and acute pancreatitis.Semin Thromb Hemost. 2010; 36: 437-443Crossref PubMed Scopus (26) Google Scholar Fibrinogen, a major coagulation protein, is composed of a dimer of 3 polypeptide chains (α,β,γ), of which γ-chains form protruded structures and contain sites allowing interaction with other factors such as clotting factors and cytokines, including vascular endothelial growth factor (VEGF) and fibroblast growth factor-2.3Mosesson M.W. Fibrinogen gamma chain functions.J Thromb Haemost. 2003; 1: 231-238Crossref PubMed Scopus (77) Google Scholar, 4Farrell D.H. Pathophysiologic roles of the fibrinogen gamma chain.Curr Opin Hematol. 2004; 11: 151-155Crossref PubMed Scopus (55) Google Scholar Notably, insoluble fibrinogen-γ dimers (Fib-γD) deposit in liver during acute liver injury in mice and humans and are an early marker of tissue damage5Weerasinghe S.V. Moons D.S. Altshuler P.J. et al.Fibrinogen-gamma proteolysis and solubility dynamics during apoptotic mouse liver injury: heparin prevents and treats liver damage.Hepatology. 2011; 53: 1323-1332Crossref PubMed Scopus (28) Google Scholar; however, no other tissues were assessed and the underlying mechanism is poorly understood. To investigate whether intraparenchymal coagulation occurs during AP, pancreatitis was induced in mice by cerulein administration. As expected, histologic and serologic changes were noted, including interstitial edema, intracellular vacuoles and inflammatory infiltration, and elevated serum amylase (Supplementary Figure 1A). Notably, Fib-γD was readily detectable in the insoluble protein fractions from the pancreata, and crosslinked fibrin was dramatically increased without changes in serum D-dimers, in parallel with severity of the AP (Figure 1A and B; Supplementary Figure 1B). Examination of early time points after cerulein administration showed that Fib-γD begins to accumulate in the early stage of AP (Figure 1C; Supplementary Figure 1C). Another AP mouse model, induced by L-arginine, also showed elevated Fib-γD and crosslinked fibrin (Supplementary Figure 2). The Fib-γD forms at the earliest stages when serum amylase is either normal or just beginning to increase in both AP models, before obvious histopathologic alterations. Importantly, Fib-γD was observed in human surgical pancreata samples from patients with pancreatitis (Supplementary Figure 3). Unlike the cerulein or L-arginine models, choline-deficient ethionine-supplemented diet-induced AP did not lead to Fib-γD formation or fibrin crosslinking despite significant pancreatic injury (Supplementary Figure 4A–C). This is likely because the choline-deficient ethionine-supplemented diet-induced injury also causes prominent liver damage with hemorrhage earlier than development of pancreatitis (Supplementary Figure 4D and E). Notably, administration of heparin as a potential therapy after initiation of cerulein-medicated injury alleviated the extent of pancreatic injury and prevented Fib-γD formation and fibrin crosslink-formation (Figure 1D; Supplementary Figure 5), supporting a beneficial effect of heparin in improving the resolution of AP. Coagulation is accomplished by activation of the intrinsic and extrinsic pathways. As an essential and terminal blood-clotting factor in the intrinsic coagulation pathway, the effect of factor VIII (FVIII) on Fib-γD formation was evaluated. During cerulein-induced AP, FVIII activity was elevated but FVIII-deficient mice had similar levels of Fib-γD deposition during AP (Supplementary Figure 6), indicating that Fib-γD formation is not directly related to the intrinsic coagulation pathway. We hypothesized that the enhanced intrapancreatic coagulation during AP causes hypoxia. Indeed, cerulein administration resulted in activation of hypoxia-inducible factor-alpha (HIF1α) in pancreata of oxygen-dependent degradation domain–luciferase HIF1α reporter mice, and promoted HIF1α stabilization with induction of HIF1α transcriptional targets such as Vegfa and Ero1a within 2 hours of cerulein administration and before evidence of Fib-γD deposition or histologic AP (Figure 1E–G; Supplementary Figure 7). Importantly, the HIF1α target, VEGF, is a well-known factor that binds to fibrinogen and regulates cell proliferation.6Sahni A. Francis C.W. Vascular endothelial growth factor binds to fibrinogen and fibrin and stimulates endothelial cell proliferation.Blood. 2000; 96: 3772-3778Crossref PubMed Google Scholar, 7Verheul H.M. van Erp K. Homs M.Y. et al.The relationship of vascular endothelial growth factor and coagulation factor (fibrin and fibrinogen) expression in clear cell renal cell carcinoma.Urology. 2010; 75: 608-614Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar Consistent with this, mRNA and protein levels of the extrinsic initiator of coagulation, tissue factor (TF), increased (Figure 1F and G), consistent with previously known TF induction by VEGF.8Mechtcheriakova D. Wlachos A. Holzmüller H. et al.Vascular endothelial cell growth factor-induced tissue factor expression in endothelial cells is mediated by EGR-1.Blood. 1999; 93: 3811-3823Crossref PubMed Google Scholar, 9Zucker S. Mirza H. Conner C.E. et al.Vascular endothelial growth factor induces tissue factor and matrix metalloproteinase production in endothelial cells: conversion of prothrombin to thrombin results in progelatinase A activation and cell proliferation.Int J Cancer. 1998; 75: 780-786Crossref PubMed Scopus (262) Google Scholar These findings suggest a feed-forward cycle, in which the HIF1α-VEGF-TF cascade not only induces intrapancreatic coagulation, but this clotting, in turn, further enhances HIF1α signaling during AP. The observation of early activation of HIF1α signaling, before Fib-γD formation, led us to hypothesize that HIF1α signaling contributes directly to Fib-γD formation rather than being an output of coagulation. Indeed, acinar cell–specific HIF1α deficiency (Hif1aAc−/−) prevented cerulein-induced Fib-γD accumulation and ameliorated the histopathologic abnormalities and amylase release (Figure 2A and B; Supplementary Figure 8), thereby suggesting an upstream regulatory tissue hemostasis role of HIF1α during AP. Pancreatic HIF1α deficiency led to several pancreatic alterations, including increased vacuolization, degranulation, and endoplasmic reticulum dilation (Figure 2B and C; Supplementary Figure 8 and 9A), induction of endoplasmic reticulum stress proteins including GRP78 and CHOP, and alterations in autophagy-related proteins (p62, ATGs) (Supplementary Figure 9B). Pancreatic infiltration of leukocytes and cell death were elevated in Hif1aAc−/− mice without a significant change in fibrosis (Supplementary Figure 9C–F). Also, isolated acini from Hif1aAc−/− mice were susceptible to cerulein despite the basally damaged pancreas (Supplementary Figure 9G and H), suggesting that HIF1α deficiency alleviates cerulein-induced severe AP at least in part through preventing coagulation, rather than basal damage preventing traditional pancreatitis responses. The decrease in amylase was observed in Hif1aAc−/− pancreata without alterations in other pancreatic enzymes, whereas gene expressions of amylase, lipase, and elastase were decreased, implying potential direct or indirect regulation by HIF1α (Figure 2D; Supplementary Figure 10). Additional evidence for the importance of HIF1α in normal pancreas function is the finding that refeeding oxygen-dependent degradation domain–luciferase mice after fasting triggers marked up-regulation of HIF1α, possibly through activation of Akt–mammalian target of rapamycin signaling (Figure 2E and F; Supplementary Figure 11). Several prior findings lend support to our observations. For example, spontaneous pancreatitis and decreased tissue amylase have been reported in several acinar cell–specific autophagy-related proteins in null mice.10Antonucci L. Fagman J.B. Kim J.Y. et al.Basal autophagy maintains pancreatic acinar cell homeostasis and protein synthesis and prevents ER stress.Proc Natl Acad Sci U S A. 2015; 112: E6166-E6174Crossref PubMed Scopus (153) Google Scholar, 11Diakopoulos K.N. Lesina M. Wörmann S. et al.Impaired autophagy induces chronic atrophic pancreatitis in mice via sex- and nutrition-dependent processes.Gastroenterology. 2015; 148: 626-638.e17Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar Similarly, Elastase-Cre–mediated Atg5−/− mice showed normal morphology during basal conditions but protection from cerulein-induced injury.12Hashimoto D. Ohmuraya M. Hirota M. et al.Involvement of autophagy in trypsinogen activation within the pancreatic acinar cells.J Cell Biol. 2008; 181: 1065-1072Crossref PubMed Scopus (183) Google Scholar In addition, the Human Protein Atlas database shows moderate expression of HIF1α in normal pancreas, particularly in exocrine cells,13Uhlen M. Fagerberg L. Hallström B.M. et al.Proteomics. Tissue-based map of the human proteome.Science. 2015; 347: 1260419Crossref PubMed Scopus (7240) Google Scholar thereby suggesting a fundamental role of HIF1α during normal pancreatic exocrine function (Figure 2G). Although a link between HIF1α and amylase regulation remains to be investigated, there is strong evidence for the involvement of HIF1α in insulin secretion in β-cells and the regulation of glucose metabolism genes, including Glut2, G6pi, Aldob, and Hnf4a, in a hypoxia-independent manner.14Cheng K. Ho K. Stokes R. et al.Hypoxia-inducible factor-1alpha regulates beta cell function in mouse and human islets.J Clin Invest. 2010; 120: 2171-2183Crossref PubMed Scopus (168) Google Scholar Coagulation abnormalities during AP lead to severe complications in some patients, ranging from localized intravascular thrombosis to disseminated intravascular coagulation.15Maeda K. Hirota M. Ichihara A. et al.Applicability of disseminated intravascular coagulation parameters in the assessment of the severity of acute pancreatitis.Pancreas. 2006; 32: 87-92Crossref PubMed Scopus (77) Google Scholar The findings herein demonstrate that Fib-γD formation is an early event during AP and is observed in mouse and human pancreata. Our results show novel functions of HIF1α in promoting coagulation during AP through HIF1α-VEGF-TF cascade, and in the response of the exocrine pancreas to normal physiologic stimulation (Figure 2G). We thank Bradley Nelson and Maria-Dolors Sans-Gili for excellent technical support in the electron microscopy experiments and in acini isolation, respectively. All experiments with mice were carried out as approved by the University Committee on Use and Care of Animals at the University of Michigan. FVB/N mice were obtained from Jackson Laboratory (Bar Harbor, ME). Cerulein- or choline-deficient ethione-supplemented diet-induced acute pancreatitis (AP) models were carried out using FVB/N mice as described.1Iyer S. Park M.J. Moons D. et al.Clusterin and Pycr1 alterations associate with strain and model differences in susceptibility to experimental pancreatitis.Biochem Biophys Res Commun. 2017; 82: 1346-1352Crossref Scopus (4) Google Scholar, 2Lerch M.M. Gorelick F.S. Models of acute and chronic pancreatitis.Gastroenterology. 2013; 144: 1180-1193Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar For the heparin experiment, 8-week old male mice were fasted for 14 hours then injected intraperitoneally (IP) 7 times with saline or cerulein (50 μg/kg mouse weight [Sigma, Burlington, MA]) hourly. In addition, 8 mg/kg low molecular weight heparin (Enoxaparin [Amphastar Pharmaceuticals, Rancho Cucamonga, CA]) was co-administered 2 times at the 5-hour and 10-hour post first injection of saline or cerulein. For L-arginine (L-Arg)–induced AP model,3Dawra R. Sharif R. Phillips P. et al.Development of a new mouse model of acute pancreatitis induced by administration of L-arginine.Am J Physiol Gastrointest Liver Physiol. 2007; 292: G1009-G1018Crossref PubMed Scopus (146) Google Scholar FVB/N mice were injected (twice, at time 0 and 1 hour later) IP with 12% L-Arg (pH 7), at a dose of 4 g/kg body weight. The oxygen-dependent degradation domain–luciferase (ODD-luc) mice were described previously.4Safran M. Kim W.Y. O'Connell F. et al.Mouse model for noninvasive imaging of HIF prolyl hydroxylase activity: assessment of an oral agent that stimulates erythropoietin production.Proc Natl Acad Sci U S A. 2006; 103: 105-110Crossref PubMed Scopus (250) Google Scholar Hif1a-floxed mice (C57BL/6 background) containing the loxP site5Xue X. Ramakrishnan S. Anderson E. et al.Endothelial PAS domain protein 1 activates the inflammatory response in the intestinal epithelium to promote colitis in mice.Gastroenterology. 2013; 145: 831-841Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar were crossed with elastase-creERT2 mice (C57BL/6 background) followed by Cre induction by daily administration of tamoxifen (3 mg/40 g of body weight) dissolved in corn oil for 3 days to generate acinar cell–specific HIF1α-null mice (Hif1aAC−/−). After an additional 3 days from the last administration, mice were fasted overnight, then given saline or cerulein IP. There was no difference in the results between sexes (based on preliminary experiments we carried out, not shown; all mice used for the cerulein and L-Arg experiments were 8–10 weeks old) except for the choline-deficient ethione-supplemented diet-induced AP model in that 4-week old female mice were used. Factor VIII–null mice (C57BL/6 background; 8-week-old males) were kindly provided by Dr Haig H. Kazazian.6Bi L. Lawler A.M. Antonarakis S.E. et al.Targeted disruption of the mouse factor VIII gene produces a model of haemophilia A.Nat Genet. 1995; 10: 119-121Crossref PubMed Scopus (513) Google Scholar Acini were isolated as described7Williams J.A. Isolation of rodent pancreatic acinar cells and acini by collagenase digestion.Pancreapedia: Exocrine Pancreas Knowledge Base. 2010; (Available from: https://www.pancreapedia.org/tools/methods/isolation-of-rodent-pancreatic-acinar-cells-and-acini-by-collagenase-digestion. Accessed March 30, 2018.)https://doi.org/10.3998/panc.2010.18Crossref Google Scholar with minor modifications. Briefly, 8-week-old Hif1aF/F and Hif1aAC−/− mice were killed by CO2 asphyxiation, and collected pancreata were washed and trimmed quickly. Each pancreas was injected with 5 mL of digestion solution containing 0.025% bovine serum albumin, 0.001% trypsin inhibitor, and 0.001% collagenase (SERVA) in Dulbecco's modified Eagle's medium with a 27-gauge needle until well distended, then incubated in the digestion solution with shaking (120 rpm, 37°C, 10 minutes). The incubated solution was removed, and the pancreas was incubated in 5 mL of fresh digestion solution for an additional 45 minutes under the same conditions. The digested pancreas-containing solution was resuspended 10 times to disperse the acini, and the suspension was filtered through a 250-μm nylon mesh (Lab Pak, Buffalo, NY). After preincubation at 37°C for 30 minutes, the acini were incubated with cerulein (0, 3, 10, 30, 100, or 300 pM) for 30 minutes, then the culture media were subjected to measurement of amylase or to immune blotting. Amylase levels were measured using Phadebas reagent (Magle Life Sciences, Cambridge, MA) as recommended by the supplier. For histological analysis, pancreata were fixed in 10% neutral buffered formalin (NBF) and the paraffin sections were stained with hematoxylin and eosin. Histological scoring was done using the following criteria: edema 1 to 3 scale (0, absent or rare; 1, involving interlobular space; 2, involving intralobular space; 3, involving the entire pancreatic acinus); necrosis (0, absent; 1, focal [<5%]; 2, sublobular [ 20%]); vacuoles, inflammation, and hemorrhage (0, absent; 1, mild; 2, moderate; 3, severe). Scores were assigned in a blind fashion without knowledge of the genotype of the pancreata in the histology sections. For Picrosirius Red staining, deparaffinized pancreas sections were dipped in 0.1% Picrosirius Red solution (1 hour, 22°C), followed by washes 2 times with 0.5% acetic acid. The stained area was quantified using ImageJ software (National Institutes of Health, Bethesda, MD).8Schneider C.A. Rasband W.S. Eliceiri K.W. NIH Image to ImageJ: 25 years of image analysis.Nat Methods. 2012; 9: 671-675Crossref PubMed Scopus (34967) Google Scholar Human pancreatic surgical specimens were obtained using a human subjects–approved protocol that provides access to such samples without any patient identifiers. Nonidentifier patient information is included in Supplemental Table 1. The normal pancreas specimen was obtained from the nonpancreatitis segment of the pancreas from patient 1. Peripheral blood was collected from the retro-orbital venous plexus of mice and anticoagulated with 3.8% sodium citrate. Plasma was separated by centrifugation (2000 × g). Factor VIII activity was measured by a 2-stage chromogenic method using the COAMATIC assay kit (Chromogenix, Diapharma Group, Inc, West Chester, OH) according to the manufacturer's instructions. The calibration standard included with this kit is assayed according to the Fourth International World Health Organization standard. The factor VIII plasma standard was normal pooled plasma from George King Biomedical (Overland Park, KS). The enzyme-linked immunosorbent assay (ELISA) for serum D-dimer and tissue vascular endothelial growth factor were carried out using the colorimetric mouse D-dimer ELISA kit (NeoScientific, Cambridge, MA) and mouse vascular endothelial growth factor ELISA kit (R&D Systems, Minneapolis, MN), respectively, as recommended by the suppliers. ODD-luc reporter mice were administered cerulein to induce AP. Mice were then injected with 50 mg/kg luciferin and imaged to assess HIF1α activity. For the fasting/refeeding experiments, liquid diet (Dyets, Inc, Bethlehem, PA) was administered by oral gavage of mice fasted for 14 hours. After 10 minutes, mice were anesthetized using IP injection of pentobarbital or by inhalation of isoflurane, then imaged after another 5 minutes using the Xenogen IVIS 200 bioluminescence system (Xenogen Corporation, Hopkinton, MA). Total pancreatic lysates were prepared by homogenizing the pancreata in sodium dodecyl sulfate–containing sample buffer. The preparation of insoluble proteins was described previously.9Snider N.T. Omary M.B. Assays for posttranslational modifications of intermediate filament proteins.Methods Enzymol. 2016; 568: 113-138Crossref PubMed Scopus (16) Google Scholar Lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. The blots were incubated with primary antibodies (information on antibodies is included in Supplemental Table 2) and processed using standard protocols. Total RNA was extracted using RNAeasy kit (Qiagen, Valencia, CA), and 1 μg RNA was reverse transcribed to complementary DNA using TaqMan reverse-transcriptase kit (Applied Biosystems, Foster City, CA). Quantitative polymerase chain reaction was done using Brilliant SYBR Green Master Mix (Bio-Rad, Hercules, CA) and Eppendorf MasterCycler RealPlex (Thermo Fisher Scientific, Waltham, MA). Primer information is included in Supplementary Table 3. Pancreata were embedded in optimal cutting temperature compound (Thermo Fisher Scientific), and cut into 5-μm sections. For crosslinked fibrin staining, sections were fixed with 2% acetic acid in 10% NBF (30 minutes), then rinsed with phosphate-buffered saline (PBS) and blocked with 10% goat serum in PBS. Sections were incubated with antibodies to: fibrinogen-γ (1:100; Abcam, Cambridge, UK), CD31 (1:100; BD Biosciences, San Jose, CA), HIF1 (1:100, Santa Cruz Biotechnology, Dallas, TX), CD45 (1:200; BD Biosciences), α-smooth muscle actin (1:50; Sigma-Aldrich, St Louis, MO) (4°C, overnight). Sections were mounted in ProLong Gold antifade reagent that included 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific) to visualize the nuclei. For amylase staining, sections were fixed with 10% NBF (10 minutes) and amylase antibodies (1:100; Santa Cruz Biotechnology) were used. Samples were analyzed by Axio Imager.M2 Microscope (Carl Zeiss, Oberkochen, Germany). Ultrastructural analysis of mouse pancreata was carried out similar to what has been described.10Toivola D.M. Nakamichi I. Strnad P. et al.Keratin overexpression levels correlate with the extent of spontaneous pancreatic injury.Am J Pathol. 2008; 172: 882-892Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar In brief, tissues were excised, minced into small pieces, and fixed in a solution of 2% paraformaldehyde and 2% glutaraldehyde in PBS for 2 hours at room temperature. After washing in PBS, the tissue samples were post fixed in osmium tetroxide for 45 minutes at room temperature. Dehydration of the samples was accomplished by transferring the samples through a series of graded ethanol and then 100% propylene oxide. The tissue was then infiltrated by transferring the samples into increasing concentrations of Epon to propylene oxide solutions: 1:3, 1:1, 3:1, then 100% Epon and finally embedded. Sections were cut with a Leica EM UC7 ultramicrotome (Leica, Wetzlar, Germany), stained for 15 minutes with 7% (saturated) aqueous uranyl acetate, washed, stained with lead citrate, and examined with a JEOL JEM 1400 plus transmission electron microscope (JEOL USA, Peabody, MA). Epon sections were also subjected to toluidine blue staining (stained for 15 seconds with 0.1% solution), then analyzed by an Axio Imager.M2 Microscope (Carl Zeiss). Myeloperoxidase activity was assayed as previously described. To measure myeloperoxidase activity of pancreas or lung, small pieces of tissues were homogenized in 20 mM sodium phosphate buffer (pH 7.4) and centrifuged at 13,000g for 10 minutes at 4°C. The pellet was resuspended in 0.5% cetrimonium bromide in phosphate buffer (50 mM, pH 6.0), then frozen and thawed 4 times followed by centrifugation (10 minutes, 13,000g, 4°C). The supernatant was mixed with the same volume of 3,3′,5,5′-tetramethylbenzidine using a 96-well plate and incubated at 37°C for 2 minutes. The reaction was stopped by addition of 2N H2SO4, then measurement of the absorbance (405 nm) and normalization of the values to tissue DNA content. Statistical analysis was performed using analysis of variance or t test and the GraphPad Prism 6 (GraphPad, La Jolla, CA) statistical software. A P < .05 was considered statistically significant. Download .pdf (1.96 MB) Help with pdf files Supplementary Tables 1–3 and Supplementary Figures 1–11