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Said SI. Vasoactive Intestinal Polypeptide (VIP) in lung function and disease 1991. Nihon Kyobu Shikkan Gakkai Zasshi. 1991;29(12):1525-31. PMID: 1687288.
]. VIP mediates its functions through the G protein-coupled receptors VPAC1 (VIP receptor type 1) or VPAC2 (VIP receptor type 2) [[4]Iwasaki M Akiba Y Kaunitz JD. Recent advances in vasoactive intestinal peptide physiology and pathophysiology: Focus on the gastrointestinal system [version 1; peer review: 4 approved].]. VIP is expressed in the central and peripheral nervous systems as well as in the digestive, respiratory, reproductive and cardiovascular systems as a neurotransmitter or neuromodulator [4Iwasaki M Akiba Y Kaunitz JD. Recent advances in vasoactive intestinal peptide physiology and pathophysiology: Focus on the gastrointestinal system [version 1; peer review: 4 approved]., 5Umetsu Y Tenno T Goda N Shirakawa M Ikegami T Hiroaki H. Structural difference of Vasoactive Intestinal Peptide in two distinct membrane-mimicking environments.]. Additionally, it has potent anti-inflammatory, bronchodilatory and immunomodulatory functions and plays a crucial role in maintaining clean airways through mucociliary clearance, contributing to local innate defense by stimulating the movement of water and chloride across intestinal and tracheobronchial epithelia [3Said SI. Vasoactive Intestinal Polypeptide (VIP) in lung function and disease 1991. Nihon Kyobu Shikkan Gakkai Zasshi. 1991;29(12):1525-31. PMID: 1687288.
, 6VIP as a modulator of lung inflammation and airway constriction., 7Vasoactive Intestinal Polypeptide (VIP) in Asthma., 8The Vasoactive Intestinal Peptide gene is a key modulator of pulmonary vascular remodeling and inflammation., 9Heinz-Erian P Dey RD Flux M Said SI. Deficient Vasoactive Intestinal Peptide innervation in the sweat glands of cystic fibrosis patients.]. Previous data from Chappe and colleagues have demonstrated that chronic exposure to VIP is needed to maintain functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels at the cell surface of airways and intestinal epithelium, as well as normal exocrine tissue morphology [10Alcolado NG Conrad DJ Poroca D Li M Alshafie W Chappe FG et al.Cystic fibrosis Transmembrane Conductance Regulator dysfunction in VIP knockout mice., 11Rafferty S Alcolado N Norez C Chappe F Pelzer S Becq F et al.Rescue of functional F508del Cystic Fibrosis Transmembrane Conductance Regulator by Vasoactive Intestinal Peptide in the human nasal epithelial cell line JME/CF15., 12VIP as a corrector of CFTR trafficking and membrane stability.., 13Alcolado N Conrad DJ Rafferty S Chappe FG Chappe VM. VIP-dependent increase in F508del-CFTR membrane localization is mediated by PKCε., 14Alshafie W Chappe FG Li M Anini Y Chappe VM. VIP regulates CFTR membrane expression and function in Calu-3 cells by increasing its interaction with NHERF1 and P-ERM in a VPAC1- and PKCε-dependent manner.]. Mutations in the CFTR gene cause cystic fibrosis, the most common fatal, chronic autosomal recessive disease in European and North American populations. Worldwide, 1 in 2,500 children are born with CF, and approximately 100,000 people are affected with the disease [[15]Environmental scan of cystic fibrosis research worldwide.]. While the primary cause of morbidity and mortality is progressive obstructive lung disease, CF is a multifactorial disorder as CFTR is expressed in multiple organs. In addition to the lungs, it affects the gastrointestinal tract, the liver, the pancreas and the reproductive system [[16]Kelsey R Manderson Koivula FN McClenaghan NH Kelly C Cystic Fibrosis–Related Diabetes: pathophysiology and therapeutic challenges.].As the life expectancy of patients with CF is improving, CF-related diabetes (CFRD) is becoming the most prevalent CF co-morbidity, increasing the mortality rate by 6-fold in comparison to CF patients without diabetes [[17]Brennan AL Geddes DM Gyi KM Baker EH. Clinical importance of Cystic Fibrosis-Related Diabetes.]. CFRD impacts approximately 2% of children, 19% of adolescents, and 50% of adults over the age of 30 with CF and appears to affect females slightly more than males [18Gibson-Corley KN Meyerholz DK Engelhardt JF. Pancreatic pathophysiology in cystic fibrosis., 19Laguna TA Nathan BM Moran A. Managing diabetes in cystic fibrosis.]. Usually, CFRD is not detected before the age of 10, as the disease onset is thought to produce an asymptomatic phenotype [18Gibson-Corley KN Meyerholz DK Engelhardt JF. Pancreatic pathophysiology in cystic fibrosis., 20Management of endocrine disease: Cystic Fibrosis-Related Diabetes: Novel pathogenic insights opening new therapeutic avenues., 21Konrad K Thon A Fritsch M Fröhlich-Reiterer E Lilienthal E Wudy SA et al.Comparison of cystic fibrosis-related diabetes with type 1 diabetes based on a German/Austrian pediatric diabetes registry.]. CFRD is a complex disease that does not fall into the type 1 diabetes (T1D) or T2D categories, and it is also referred to as pancreatogenic (type 3c) diabetes [[22]Rickels LG and MR. Pancreatogenic (Type 3c) Diabetes. Pancreapedia Exocrine Pancreas Knowl Base 2015. doi:10.3998/PANC.2015.35.
]. It constitutes a special, separate category that has features of both T1D and T2D, with specific pathophysiological differences to support its own distinct classification [[23]Hart NJ Aramandla R Poffenberger G Fayolle C Thames AH Bautista A et al.Cystic Fibrosis-Related Diabetes is caused by islet loss and inflammation.]. CFRD is characterized by decreased insulin secretion due to a loss of beta cell mass and a progressive increase in inflammation [[18]Gibson-Corley KN Meyerholz DK Engelhardt JF. Pancreatic pathophysiology in cystic fibrosis.]. Some of the most common risk factors that can predispose patients to CFRD are the severe CFTR genotypes (class I, II and III CFTR gene mutations), modifier genes, a family background of T2D and pancreatic insufficiency [20Management of endocrine disease: Cystic Fibrosis-Related Diabetes: Novel pathogenic insights opening new therapeutic avenues., 23Hart NJ Aramandla R Poffenberger G Fayolle C Thames AH Bautista A et al.Cystic Fibrosis-Related Diabetes is caused by islet loss and inflammation., 24Granados A Chan CL Ode KL Moheet A Moran A Holl R. Cystic Fibrosis Related Diabetes: Pathophysiology, screening and diagnosis., 25Blackman SM Commander CW Watson C Arcara KM Strug LJ Stonebraker JR et al.Genetic modifiers of Cystic Fibrosis-Related Diabetes., 26Moran A Becker D Casella SJ Gottlieb PA Kirkman MS Marshall BC et al.Epidemiology, pathophysiology, and prognostic implications of Cystic Fibrosis-Related Diabetes: A technical review., 27Konrad K Scheuing N Badenhoop K Borkenstein MH Gohlke B Schöfl C et al.Cystic Fibrosis-Related Diabetes compared with type 1 and type 2 diabetes in adults.].VIP is known to augment insulin secretion in the presence of glucose [4Iwasaki M Akiba Y Kaunitz JD. Recent advances in vasoactive intestinal peptide physiology and pathophysiology: Focus on the gastrointestinal system [version 1; peer review: 4 approved]., 28Sanlioglu AD Karacay B Balci MK Griffith TS Sanlioglu S. Therapeutic potential of VIP vs PACAP in diabetes.]. Through VPAC2, which is highly expressed in the pancreatic islets, VIP activates adenylate cyclase (AC) to increase intracellular cAMP levels, which triggers the activation of protein kinase A (PKA) and intracellular cAMP sensors EPAC. This results in membrane depolarization due to the closure of ATP-dependent K+ channels and calcium (Ca2+) entry into cells. The influx of calcium into the cells initiates insulin secretion after glucose stimulation [[29]Role of VIP and PACAP in islet function.]. While numerous studies utilizing genetically altered mice models have examined the therapeutic ability of VIP and VPAC receptor agonists to modulate glucose-induced insulin secretion in T2D, to our knowledge, none have investigated the role of VIP or VPACs in CFRD [28Sanlioglu AD Karacay B Balci MK Griffith TS Sanlioglu S. Therapeutic potential of VIP vs PACAP in diabetes., 30Persson-Sjögren S Forsgren S Lindström P. Vasoactive Intestinal Polypeptide and Pituitary Adenylate Cyclase Activating Polypeptide: Effects on insulin release in isolated mouse islets in relation to metabolic status and age., 31Tsutsumi M Claus TH Liang Y Li Y Yang L Zhu J et al.A potent and highly selective VPAC2 agonist enhances glucose-induced insulin release and glucose disposal: A potential therapy for type 2 diabetes., 32Triggering and amplifying pathways of regulation of insulin secretion by glucose.]. In a recent study, we demonstrated that VIP content is 50% lower in the lungs, sweat glands and small intestine of C57Bl/6 mice homozygous for the F508del-CFTR mutation compared to wild-type (WT) mice. This deficiency results from a reduction in VIPergic and cholinergic innervation starting at a young age, before signs of CF disease are observed [[33]Semaniakou A Brothers S Gould G Zahiremani M Paton J Chappe F et al.Disrupted local innervation results in less VIP expression in CF mice tissues.]. In the present study, we assessed whether there were changes in VIP abundance and innervation of the pancreas at different stages of CF disease progression that could affect insulin and glucagon secretion and contribute to CFRD development. We used 8- and 17-week-old CF mice homozygous for the F508del-CFTR mutation, the most common disease-causing mutation found in patients with CF. This mutation is associated with a higher risk of CFRD development. Our data show that reduction in VIP levels results in reduced innervation of the pancreas, affecting insulin and glucagon secretion and potentially contributing to the development of CFRD.2. Materials and methods2.1 ChemicalsAntibodies are described in Table 1; Normal mouse blocking serum was from Santa Cruz Biotechnology (ImmunoCruz goat ABC Staining System; sc-2023); Normal rabbit blocking serum was from Santa Cruz Biotechnology (ImmunoCruz goat ABC Staining System; sc-2018); Normal donkey serum (D9663) was from Sigma (St. Louis, MO); Normal goat blocking serum was from Vector Laboratories (Cat. No. NC9270494); Avidin-Biotin complex was from Santa Cruz (ImmunoCruz ABC kit; sc-516216); DAB (3, 3-diaminobenzidine) HRP Substrate Kit (ab64238) was from Abcam; Enzyme-Linked-Immunosorbent Assay kit for murine VIP was from Cloud-Clone Corp. (Cat. No. CEA380Mu; Houston-TX); Enzyme-Linked-Immunosorbent Assay kit for murine insulin was from Millipore (Cat. No. EZRMI-13K); Enzyme-Linked-Immunosorbent Assay kit for murine glucagon (MyBioSource; San Diego; MBS740756); Cayman's glucose colorimetric assay kit was from Cayman Chemical (Cat. No. 10009582); Vectashield Mounting Media for fluorescence was from Vector Laboratories (Cat. No. 101098-042). Protease inhibitor cocktail tablets were from Roche (complete, Mini; Cat. No. 11 836 153 001). Other chemicals were from Sigma and of the highest grade available.Table 1Summary of antibodies and dilutions used.
2.2 MiceMale 8- and 17-week-old WT C57Bl/6 mice and C57Bl/6 mice homozygous for the F508del mutation (C57Bl/6 homozygous Cftrtm1Kth ΔF508) were obtained from Dr. Craig Hodges at Case Western Reserve University (Ohio, USA). Mice were housed in an Allentown IVC rack with irradiated corncob bedding, Prolab autoclaved RMH 3500 feed and municipal (chlorinated tap) water with Colyte added. Mice were evaluated daily during routine health checks. If they showed signs of distress or gastrointestinal complications, they were weighed, given subcutaneous fluids if needed and monitored more frequently. If signs of distress did not resolve (i.e. the obstruction did not pass), they were euthanized. All experimental procedures were in accordance with the principles of the Canadian Council on Animal Care (CCAC) and according to the National Institutes of Health Guide to the Care and Use of Experimental Animals. Protocols were approved by Dalhousie University Animal Care and Use ethic committee.
2.3 Tissue preparationMice were humanely euthanized by intraperitoneal overdose of sodium pentobarbital before tissue collection. For sectioning, tissues were immediately fixed in 10% formalin buffer. Tissues were then embedded in paraffin blocks before longitudinal sectioning into 5μm-thin sections for histology with haematoxylin and eosin (H & E) staining [[34]Tissue processing and hematoxylin and eosin staining.] or immunohistochemistry (IHC) experiments as described below. Tissue embedding and sectioning were performed at the Histology & Research Services (HRS) Laboratory, Pathology Department, Faculty of Medicine, Dalhousie University. For protein analysis, tissues were flash frozen in liquid nitrogen before storage at -80°C for later use.2.4 Immunohistochemistry and microscopy imagingThin tissue sections (5μm) annealed to microscopy slides were deparaffinized in xylene and rehydrated prior to a 35min heat-induced antigen retrieval. Next, the slides were washed twice for 5 min each in phosphate-buffered saline (PBS) and incubated in 1% peroxidase blocking solution for 5 min. After another 10 min washing step with PBS, the slides were incubated in normal mouse blocking serum for 1h at RT in a humidified chamber, then overnight with the primary antibody for VIP (1:100), insulin (1:50), or ubiquitin C-terminal hydrolase 1 (PGP 9.5; 1:200). The next day, tissue slides were washed three times for 5 min each with PBS and then incubated for 30 min at room temperature (RT) with a biotinylated anti-rabbit secondary antibody for VIP and PGP 9.5 (1:200; ABC ImmunoCruz staining system) or for 1-hour with anti-guinea pig secondary antibody for insulin (1:500; Jackson ImmunoResearch). VIP, insulin and PGP 9.5 signals were revealed with a DAB HRP peroxidase substrate and slides were counterstained with haematoxylin. After dehydration, a xylene-based Permount was used to mount a coverslip. Both wild-type and CF tissues were tested simultaneously. Optimal signal intensity revealed by DAB staining were initially set in wild-type tissues for every primary antibody. Same DAB staining time were used for CF and wild-type tissues. Slides were observed under light microscopy with 10X, 25X, 40X and 63X objectives. Images were taken with a Zeiss Axiocam HRC colour camera mounted on an Axioplan II microscope. For the semi-quantification of DAB signal intensity, a scoring system of images was used by two blinded investigators using an arbitrary scale: 0 = no signal; 0.5 = minimal signal intensity; 1 = low intensity; 1.5 = some signal; 2 = moderate signal intensity; 2.5 = strong signal intensity; and 3 = intense signal. Colour balance, contrast, and brightness of whole images were adjusted for consistency within tissue sections. Experiments were repeated at least twice for each tissue sample and three to five mice in each age group were used. An average of 8 to 15 images from each tissue section was used for the semi-quantification of the DAB signal.
2.5 PGP 9.5 Immunohistochemistry signal quantificationTo measure PGP 9.5 DAB signal intensity in both the endocrine and exocrine pancreas, we used 5 to 10 images containing 1 or 2 islets per image, for every mouse in each age group (8- and 17-week-old). The signal raw integrated intensity function of Image J software was used according to recommended protocols.
2.6 Immunofluorescent stainingParaffin sections of pancreatic tissues, after the deparaffinization and rehydration steps as mentioned above, were rinsed three times in Tris-Buffered Saline (TBS), next incubated in TBS for 5 min and afterwards in blocking solution for 20 min at RT. The tissues were then covered with the glucagon primary antibody (1:50) and stored overnight at 4°C in a humidity chamber. The next day, the slides were washed with TBS three times for 5 min each to remove the primary antibody and then incubated with Alexa Fluor 594 secondary antibody overnight (1:200). Before mounting the tissue with Vectashield Mounting Media for fluorescence, slides were washed three times for 5 min each with TBS in the dark to remove the secondary antibody. Afterwards, the slides were stored at -20°C until they were viewed with a Zeiss LSM 710 confocal microscope.
2.7 Glucagon immunofluorescence signal quatification5-10 images containing 1 to 3 islets per image, were used for every mouse in each age group (8- and 17- week-old). We used the raw integrated signal intensity function of the Image J software. Each islet area (μm2) was defined using the ROI Manager option.
2.8 Random glucose measurementsPlasma, from all mice in each age group, was used to estimate random glucose level using the Cayman's Glucose Colorimetric assay kit, a high-sensitivity enzymatic assay for precise glucose quantification. All mice were fed the same regular diet (Prolab RMH 3500) and plasma was collected early in the morning. We used intra-cardiac puncture to collect blood by aspiration with a fine needle filled with 2% EDTA. Blood collected was placed into a microcentrifuge tube with 100μl 2% EDTA and centrifuged for 10 min at 2,500rpm at 4°C. The supernatant was collected, aliquoted and stored it at -80°C until further use.
2.9 Enzyme-linked immunosorbent assay (ELISA)Flash-frozen pancreatic tissue samples from C57Bl/6 male 8- and 17-week-old, wild-type and F508del homozygous CF mice were homogenized in RIPA buffer containing protease inhibitors, and lysates were tested using mouse ELISA kits with high sensitivity and high specificity for VIP (Cloud-Clone Corporation, competitive inhibition; CEA380Mu; Intra-assay coefficient < 10%; Inter-assay coefficient < 12%), insulin (Millipore Mouse Insulin ELISA kit; EZRMI-13K; Intra-assay coefficient: 0.9-8.4%, Inter-assay coefficient: 6.0-17.9%) and glucagon (MyBioSource, competitive inhibition; MBS740756; Intra-assay coefficient < 10%; Inter-assay coefficient < 10%) according to the manufacturers’ instructions. VIP, insulin and glucagon concentrations were normalized to total protein content measured with a standard Bradford assay.
2.10 StatisticsResults are reported as means ± SE. Statistical differences between groups were calculated by the one-way ANOVA. p < 0.05 was considered statistically significant. N represents the number of independent experiments.
2.11 Study limitationsTwo limitations should be acknowledged, the small number of mice in each age group and the use of only male C57Bl/6 mice. Although small, due to the poor survival rate post-weaning and the difficulty in maintaining these mice beyond 8 weeks of age, our success with maintaining 17-week-old CF animals for this study is remarkable. The current study was performed with males only, therefore it is not known yet whether females also exhibit the same defects. Future studies in female F508del homozygous mice will further delineate our findings as human studies have shown that CFRD is more common in female patients.
4. DiscussionThe role of VIP in human diseases such as CF, asthma, chronic obstructive pulmonary disease and diabetes is a matter of investigation [10Alcolado NG Conrad DJ Poroca D Li M Alshafie W Chappe FG et al.Cystic fibrosis Transmembrane Conductance Regulator dysfunction in VIP knockout mice., 42Gene deletion of VIP leads to increased mortality associated with progressive right ventricular hypertrophy., 43Szema AM Forsyth E Ying B Hamidi SA Chen JJ Hwang S et al.NFATc3 and VIP in idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease., 44Asnicar MA Köster A Heiman ML Tinsley F Smith DP Galbreath E et al.Vasoactive Intestinal Polypeptide/Pituitary Adenylate Cyclase-Activating Peptide Receptor 2 deficiency in mice results in growth retardation and increased basal metabolic rate.]. A study by Alcolado et al. who used a VIP-KO mouse model that developed a CF-like phenotype, demonstrated that treatment with VIP corrected the lymphocyte aggregation, increased airway secretion, alveolar thickening and edema in the lung, as well as the goblet cell hyperplasia and tissue alterations in the duodenum found in the absence of VIP, with the post-treatment phenotype being similar to those of WT mice. In vitro, addition of VIP to the cell media regulated the defective CFTR internalization rate and increased membrane stabilization in the apical membrane of the human bronchial serous cell line Calu-3 [13Alcolado N Conrad DJ Rafferty S Chappe FG Chappe VM. VIP-dependent increase in F508del-CFTR membrane localization is mediated by PKCε., 14Alshafie W Chappe FG Li M Anini Y Chappe VM. VIP regulates CFTR membrane expression and function in Calu-3 cells by increasing its interaction with NHERF1 and P-ERM in a VPAC1- and PKCε-dependent manner., 45Chappe F Loewen ME Hanrahan JW Chappe V. Vasoactive Intestinal Peptide increases Cystic Fibrosis Transmembrane Conductance Regulator levels in the apical membrane of Calu-3 cells through a protein kinase C-dependent mechanism.]. In the immune system, the critical role of VIP is illustrated in autoimmune diseases in which VIP can skew the pro-inflammatory immune response to an anti-inflammatory response, down-regulating the expression of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), IL-12 and IL-1β and enhancing IL-10 production, blocking Th1 cells and consequently preventing Th2 production as well as mediating the functions of Th1/Th17 cells [[46]Ganea D Hooper KM Kong W. The neuropeptide Vasoactive Intestinal Peptide: Direct effects on immune cells and involvement in inflammatory and autoimmune diseases.].VIP therapeutic actions have been tested in many chronic inflammatory and autoimmune diseases, including diabetes [47Jimeno R Gomariz RP Gutiérrez-Cañas I Martínez C Juarranz Y Leceta J. New insights into the role of VIP on the ratio of T-cell subsets during the development of autoimmune diabetes., 48Rosignoli F Torroba M Juarranz Y García-Gómez M Martinez C Gomariz RP et al.VIP and tolerance induction in autoimmunity.], sepsis [[49]Delgado M Gomariz RP Martinez C Abad C Leceta J. Anti-inflammatory properties of the type 1 and type 2 Vasoactive Intestinal Peptide receptors: role in lethal endotoxic shock.], Crohn's disease [[50]Gonzalez-Rey E Delgado M. Therapeutic treatment of experimental colitis with regulatory dendritic cells generated with vasoactive intestinal peptide.] and multiple sclerosis [[51]Cobo M Anderson P Benabdellah K Toscano MG Muñoz P García-Pérez A et al.Mesenchymal stem cells expressing Vasoactive Intestinal Peptide ameliorate symptoms in a model of chronic multiple sclerosis.]. Specifically, VIP treatment in non-obese diabetic mice prevented them from developing T1D through the activation of T cells, suppression of Th1 cytokines and up-regulation of IL-10 synthesis [[48]
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