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Annexin A1 Is a Key Modulator of Mesenchymal Stromal Cell–Mediated Improvements in Islet Function

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Annexin A1 Is a Key Modulator of Mesenchymal Stromal Cell–Mediated Improvements in Islet Function Diabetes Volume 65, January 2016 129 Chloe L. Rackham, Andreia E. Vargas, Ross G. Hawkes, Stefan Amisten, Shanta J. Persaud, Amazon L.F. Austin, Aileen J.F. King, and Peter M. Jones Annexin A1 Is a Key Modulator of Mesenchymal Stromal Cell–Mediated Improvements in Islet Function Diabetes 2016;65:129–139 | DOI: 10.2337/db15-0990 We have previously demonstrated that coculture of been shown to influence the graft niche by modifying the islets with mesenchymal stromal cells (MSCs) enhanced responses of host immune, endothelial, or progenitor islet insulin secretory capacity in vitro, correlating with cells to reduce inflammatory or immune responses (1,2) IMMUNOLOGY AND TRANSPLANTATION improved graft function in vivo. To identify factors that and to improve graft revascularization (3–5). However, contribute to MSC-mediated improvements in islet func- we (6–8) and others (4,9–11) have demonstrated that tion, we have used an unbiased quantitative RT-PCR MSCs also have direct effects on donor islet cells to screening approach to identify MSC-derived peptide improve their survival and secretory function. Thus, we ligands of G-protein–coupled receptors that are expressed previously used direct contact coculture of islets with by islets cells. We demonstrated high expression of annexin A1 (ANXA1) mRNA by MSCs and confirmed MSCs derived from kidney (6) or adipose tissue (7) to expression at the protein level in lysates and MSC- enhance glucose-stimulated insulin secretion (GSIS) in conditioned media by Western blot analysis and ELISA. vitro and demonstrated that this results in superior in Preculturing islets with exogenous ANXA1 enhanced vivo function for islet-alone grafts at the experimental glucose-stimulated insulin secretion (GSIS), thereby mim- renal subcapsular (6) and clinically preferred intraportal icking the beneficial influence of MSC preculture in vitro. transplantation site (7). Small interfering RNA–mediated knockdown of ANXA1 in Previous studies suggest that the beneficial effect of MSCs reduced their capacity to potentiate GSIS. MSCs MSCs on islet function is at least partly mediated by 2 2 derived from ANXA1 / mice had no functional capac- soluble bioactive molecules (12), so we have now applied ity to enhance GSIS, in contrast to wild-type controls. a nonbiased screening approach to identify novel MSC- Preculturing islets with ANXA1 had modest effects on derived secretory products that may influence islet func- their capacity to regulate blood glucose in streptozotocin- tion. We have based the current screen on our recent induced diabetic mice, indicating that additional MSC- demonstration that islets express 293 different G-protein– derived factors are required to fully mimic the beneficial coupled receptors (GPCRs) that are known to be acti- effects of MSC preculture in vivo. These findings demon- vated by more than 250 identified ligands (13). In this strate the feasibility of harnessing the MSC secretome as adefined, noncellular strategy to improve the efficiency study, we have used a quantitative (q)RT-PCR approach of clinical islet transplantation protocols. to focus on the 131 peptides or proteins that have been identified previously as ligands for islet GPCRs to iden- tify MSC-derived molecules that may contribute to MSC- Thereisagrowingbodyofevidencethatmesenchymal mediated improvements in islet function. This strategy stromal cells (MSCs) can enhance the functional sur- has identified therapeutic factors that may be used in a vival of islet grafts after transplantation, offering a noncellular approach to direct defined modifications to potential therapeutic method for improving the out- islet transplantation protocols, ensuring reproducible comes of islet transplantation as a therapy for type 1 improvements in islet function and thus improved islet diabetes. A number of MSC-derived trophic factors have transplantation outcomes. Diabetes Research Group, Division of Diabetes & Nutritional Sciences, School of This article contains Supplementary Data online at http://diabetes Medicine, King’s College London, London, U.K. .diabetesjournals.org/lookup/suppl/doi:10.2337/db15-0990/-/DC1. Corresponding author: Peter M. Jones, [email protected]. © 2016 by the American Diabetes Association. Readers may use this article as fi Received 19 July 2015 and accepted 7 October 2015. long as the work is properly cited, the use is educational and not for pro t, and the work is not altered. 130 MSC-Derived Annexin A1 Improves Islet Function Diabetes Volume 65, January 2016 RESEARCH DESIGN AND METHODS Small Interfering RNA–Mediated Knockdown of ANXA1 Identification of MSC-Derived Islet GPCR Ligands Expression in MSCs Total RNA was extracted from cultured adherent mouse SMARTpool ON-TARGETplus small interfering (si)RNA fi MSC monolayers using RNeasy mini kits and RNase-free (50 nmol/L; Thermo Fisher Scienti c Biosciences, GmbH) DNase sets (Qiagen, Manchester, U.K.) and reverse directed against mouse ANXA1 was transfected into MSCs transcribed into cDNAs using an Applied Biosystems for 24 h in the presence of DharmaFECT 1 transfection fi high-capacity reverse transcription kit (Life Technolo- reagent (Thermo Fisher Scienti c Biosciences, GmbH), gies, Ltd., Paisley, U.K.). Pooled biological replicates of using a siRNA-to-DharmaFECT ratio of 2:1. Control MSCs mouse adipose- or kidney-derived MSC cDNAs were were transfected with ON-TARGETplus Non-targeting screened by qRT-PCR for a total of 131 potential Control siRNA. Transfected MSCs were cultured in candidate ligands of known islet GPCRs (13) to create antibiotic-free DMEM supplemented with 10% (vol/vol) a mRNA expression profile of MSC-encoded ligands spe- FCS for the initial 24 h after transfection. siRNAs were cific to islet GPCRs. Melt curve analysis was performed removed after 24 h, and MSCs were cultured alone or after amplification, and qPCR reactions showing posi- direct contact islet-MSC cocultures were set up. MSC tive melt curves were further analyzed by agarose gel lysates were collected at 24, 48, and 72 h after trans- electrophoresis to confirm that the qPCR product was fection for assessment of ANXA1 mRNA. ANXA1 protein the appropriate size. qRT-PCR of MSC biological repli- expression was assessed by Western blotting using a cate cDNAs was performed using QuantiTect primers rabbit anti-ANXA1 monoclonal antibody (1:1,000 dilu- (Qiagen). Relative expression of mRNAs was determined tion; Cell Signaling Technology, Hertfordshire, U.K.), with fi after normalization against GAPDH as an internal ref- equal loading con rmed by immunoprobing with a rabbit ΔΔ erence and calculated by the 2- Ct method. To allow monoclonal anti-GAPDH antibody (1:5,000 dilution; Cell discrimination between different levels of expression, Signaling Technology). MSCs were disrupted in RIPA lysis each ligand mRNA was classified by its Ct value relative buffer (Sigma-Aldrich), total protein was measured using to GAPDH-Ct 18 as high (Ct ,26), medium (Ct 26–30), a bicinchoninic acid assay, and 20 mg protein was loaded or low (Ct .30) levels of expression. onto 12% SDS gels (Life Technologies Ltd.). Islet Secretory Function In Vitro Islet Isolation and Culture Insulin secretion in vitro was assessed in static incuba- Mouseisletswereisolatedbycollagenase digestion (1 mg/mL; tions of isolated islets. Islets were preincubated for 2 h in type XI; Sigma-Aldrich, Poole, U.K.), followed by density RPMI containing 36 mg/dL glucose. Groups of three islets gradient separation (Histopaque-1077; Sigma-Aldrich). were transferred into 1.5-mL Eppendorf tubes and in- After washing with RPMI-1640, islets were picked into cubated at 37°C in a bicarbonate-buffered physiological groups of 100 for culture alone, with MSCs, or with re- salt solution, containing 2 mmol/L CaCl2 and 0.5 mg/mL combinant annexin A1 (ANXA1) (R&D Systems, Abingdon, BSA (14) and either 36 or 360 mg/dL glucose. Samples of U.K.) for 3 days, unless otherwise specified. For MSC pre- the incubation medium were taken after 1 h and stored at culture, we used a direct-contact monolayer configuration, 220°C until assayed for insulin content using in-house as previously described (6,7). Media was changed after radioimmunoassay (15,16). 2 days for all culture groups and ANXA1 replenished in Islet Apoptosis In Vitro the ANXA1 preculture group. To assess caspase 3/7 activity, luciferase activity de- – Measurement of ANXA1 Expression and Release pendent on caspase 3/7 mediated generation of lucifer- ase substrate was measured (Promega, Southampton, To confirm the expression of ANXA1 protein in MSC U.K.). Islets were precultured alone or with 5 nmol/L lysates and to determine whether ANXA1 is released ANXA1 for 72 h. For the final 20 h of the culture intotheMSCculturemedia,MSCswereseededinto period, half of the islets in each culture dish were ex- Nunclon 35-mm petri dishes to mimic the experimen- posed to mixed cytokines (50 units/mL interleukin-1b, tal setup used for our direct contact islet-MSC pre- 1,000 units/mL interferon-g, and 1,000 units/mL tumor culture configuration (6,7). After 72 h, MSCs were necrosis factor-a; PeproTech, London, U.K.) and half of trypsinized and resuspended in ice-cold PBS supple- the islets served as controls without cytokines. Islets were mented with cOmplete ULTRA Mini protease inhibi- picked into groups of five islets per well, and Caspase-Glo tors (Roche Diagnostics, Burgess Hill, U.K.) and then 3/7 reagent (Promega) was added. Light emission was sonicated. The MSC-conditioned media (CM) from each detected after 1 h using a Turner BioSystems Veritas petri dish was also collected and concentrated 312 using 10,000 nominal molecular weight limit Amicon Ultra luminometer (Promega). 0.5-mL centrifugal filters (Merck Millipore, Middlesex, Experimental Animals U.K.). Control samples were the MSC culture media Male CD1 mice (Charles River Laboratories, Margate, U.K.), alone, which was also concentrated 312. ANXA1 was aged 8–12 weeks, were used as islet donors for all in vitro measured in MSC-lysates and -CM using an ELISA kit investigations.
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