Increased microvascular permeability in mice lacking Epac1 (Rapgef3) Kopperud, R. K.; Rygh, C. Brekke; Karlsen, T. V.; Krakstad, C.; Kleppe, R.; Hoivik, E. A.; Bakke, M.; Tenstad, O.; Selheim, F.; Lidén, Å.; Madsen, Lise; Pavlin, T.; Taxt, T.; Kristiansen, Karsten; Curry, F. -R. E.; Reed, R. K.; Døskeland, S. O. Published in: Acta Physiologica DOI: 10.1111/apha.12697 Publication date: 2017 Document version Publisher's PDF, also known as Version of record Document license: CC BY-NC-ND Citation for published version (APA): Kopperud, R. K., Rygh, C. B., Karlsen, T. V., Krakstad, C., Kleppe, R., Hoivik, E. A., Bakke, M., Tenstad, O., Selheim, F., Lidén, Å., Madsen, L., Pavlin, T., Taxt, T., Kristiansen, K., Curry, F. -R. E., Reed, R. K., & Døskeland, S. O. (2017). Increased microvascular permeability in mice lacking Epac1 (Rapgef3). Acta Physiologica, 219(2), 441-452. https://doi.org/10.1111/apha.12697 Download date: 30. Sep. 2021 Acta Physiol 2017, 219, 441–452 Increased microvascular permeability in mice lacking Epac1 (Rapgef3) † § R. K. Kopperud,1,2,*, C. Brekke Rygh,1,*, T. V. Karlsen,1 C. Krakstad,1 R. Kleppe,1 † ¶ ‡ E. A. Hoivik,1, M. Bakke,1 O. Tenstad,1 F. Selheim,1 A. Liden, 1, L. Madsen,1,3, T. Pavlin,1 T. Taxt,1 K. Kristiansen,3 F.-R. E. Curry,4 R. K. Reed1,2 and S. O. Døskeland1 1 Department of Biomedicine, University of Bergen, Bergen, Norway 2 Centre for Cancer Biomarkers (CCBIO), University of Bergen, Bergen, Norway 3 Department of Biology, University of Copenhagen, Copenhagen, Denmark 4 Department of Physiology and Membrane Biology, School of Medicine, University of California, Davis, CA, USA Received 18 February 2016, Abstract revision requested 15 March Aim: Maintenance of the blood and extracellular volume requires tight con- 2016, trol of endothelial macromolecule permeability, which is regulated by cAMP revision received 12 April 2016, signalling. This study probes the role of the cAMP mediators rap guanine accepted 14 April 2016 Correspondence: S. O. nucleotide exchange factor 3 and 4 (Epac1 and Epac2) for in vivo control of Døskeland, MD, PhD, Jonas Lies microvascular macromolecule permeability under basal conditions. À À À À vei 91, N-5009 Bergen, Norway. Methods: Epac1 / and Epac2 / C57BL/6J mice were produced and E-mail: [email protected] compared with wild-type mice for transvascular flux of radio-labelled albumin in skin, adipose tissue, intestine, heart and skeletal muscle. The See Editorial Commentary: transvascular leakage was also studied by dynamic contrast-enhanced Waschke, J. Epac1 – a tonic stabilizer of the endothelial magnetic resonance imaging (DCE-MRI) using the MRI contrast agent barrier. Acta Physiol (Oxf) 219, Gadomer-17 as probe. À/À 339–340. Results: Epac1 mice had constitutively increased transvascular macro- molecule transport, indicating Epac1-dependent restriction of baseline per- À À *These authors contributed meability. In addition, Epac1 / mice showed little or no enhancement of equally to this work. † vascular permeability in response to atrial natriuretic peptide (ANP), Present address: Department of À/À Clinical Sciences, University of whether probed with labelled albumin or Gadomer-17. Epac2 and Bergen, Bergen, Norway wild-type mice had similar basal and ANP-stimulated clearances. ‡ À/À Present address: National Ultrastructure analysis revealed that Epac1 microvascular interendothe- Institute of Nutrition Research lial junctions had constitutively less junctional complex. (NIFES), Bergen, Norway § Conclusion: Epac1 exerts a tonic inhibition of in vivo basal microvascular Present address: Bergen Univer- permeability. The loss of this tonic action increases baseline permeability, sity College, Bergen, Norway ¶ Present address: Galderma presumably by reducing the interendothelial permeability resistance. Part Nordic AB, Uppsala, Sweden of the action of ANP to increase permeability in wild-type microvessels may involve inhibition of the basal Epac1-dependent activity. The copyright line for this article Keywords atrial natriuretic peptide, cAMP, endothelial junction, Epac was changed on 11th August, 2016 deletion (mouse), microvascular permeability (in vivo), Rapgef. after original online publication. The primary aim of the study was to investigate cAMP-dependent protein kinase (PKA) only, but the whether the cAMP-stimulated Rap activator Epac likely involvement of Epac was demonstrated using contributes to the tonic control of microvessel base- Epac-activating cAMP analogues (Christensen et al., line permeability in major organs of the mouse. The 2003) that lowered the permeability of monolayers of well-recognized endothelial permeability lowering human embryonic vascular endothelial cells and atten- effect of cAMP was first thought to be mediated by uated the response to inflammatory conditions such © 2016 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society, doi: 10.1111/apha.12697 441 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. Microvessel permeability in Epac1KO mice · R K Kopperud et al. Acta Physiol 2017, 219, 441–452 as exposure to thrombin (Cullere et al. 2005). A role et al. 2010, Lin et al. 2011), and also shown that it is of Epac1 and its target, the small GTPase Rap, to attenuated when cAMP is elevated, and proposed that modulate inflammation-associated endothelial hyper- ANP might increase permeability via decreased Epac permeability has been confirmed in numerous in vitro activity (Lin et al. 2011). We show here that Epac1- studies and noted in some in situ perfusion studies of deficient mice, but not Epac2-deficient mice have an accessible microvessels, although the relative roles of attenuated response to ANP. PKA, cGMP-dependent protein kinase (PKG) and Epac in different organ vascular beds and under dif- Materials and methods ferent experimental conditions still remain uncertain (for recent reviews, see Spindler et al. 2010, Surapi- Ethical approval sitchat & Beavo 2011, Kuhn 2012, Curry & Adam- son 2013). The animal experiments were approved by the Norwe- The role of Epac to maintain stable baseline perme- gian Animal Research Authority and conducted accord- ability in intact microvessels under non-inflammatory ing to the European Convention for the Protection of conditions is less well studied. A major reason is the Vertebrates Used for Scientific Purposes, Norway. lack of ways to reliably inhibit Epac and other cAMP- dependent pathways pharmacologically in vivo (Spind- Materials ler & Waschke 2011), in part due to rapid metabolism and renal clearance of cAMP analogues used to modu- The anti-Epac1 antibody was kindly provided by late Epac and PKA activity (Kleppe et al. 2015). J. Bos, University of Utrecht, the Netherlands. The À À In this study, Epac-deficient mice (Epac1 / or anti-Epac2 antibody (5B1) was from Cell Signaling À À Epac2 / ) were generated to elucidate the potential technology (www.cellsignal.com). The anti-CD31 anti- in vivo role of Epac in regulation of baseline macro- body (DIA-310) was from Dianova, Hamburg, Ger- molecule flux across microvascular endothelium. many. ANP and rolipram were from Sigma-Aldrich À À À À Others have reported that Epac1 / and Epac2 / mice (St. Louis, MO, USA). Gadomer-17 was from in vivo À À have only subtle phenotypes. Epac2 / mice have a Contrast GmbH, Schering Pharma (Berlin, Germany). slightly perturbed pathway of pancreatic beta cell insu- Human serum albumin (HSA) was labelled with 131I À À lin exocytosis (Shibasaki et al. 2007), while Epac1 / or 125I (from Perkin Elmer, Waltham, MA, USA) by mice have been reported to gain slightly more (Kai et al. the iodogen method, as previously described (Curry 2013) or slightly less (Yan et al. 2013) body weight on et al. 2010). Any free iodide was removed by spinning both low- and high-fat diets, and be less susceptible to twice on a 3-kDa cut-off column (Microcon filter; rickettsial invasion (Gong et al. 2013). Millipore, Bedford, MA, USA) prior to experiments. We report here an increased basal microvascular The labelled HSA eluted like native HSA upon anionic transvascular albumin flux in skin, skeletal muscle and exchange and high-resolution size exclusion chro- À À À À intestines of Epac1 / , but not Epac2 / mice. matography with online gamma radiation detection in Dynamic contrast-enhanced magnetic resonance imag- series with UV detector. Low molecular weight ing (DCE-MRI) studies of the masseter muscle using radioactivity accounted for <0.3% of the total activity. Gadomer-17 demonstrated transvascular flux of the À À À À high molecular weight MR contrast agent, which con- Generation of Epac1 / and Epac2 / knock-out À À À À À À firmed that Epac1 / mice had constitutively increased models. Epac1 / and Epac2 / mice were generated microvascular permeability to macromolecules. Trans- by genomic deletion of exons 7–10 in Rapgef3 (encod- mission electron microscopy was used to compare the ing Epac1) and exons 12–13 of Rapgef4 (encoding ultrastructure of the endothelial junctions in masseter Epac2) encoding the cAMP-binding domains of both À À muscle biopsies from basal state in Wt and Epac1 / proteins (Fig. 1a,d) by use of the Cre–LoxP system. An mice. They revealed decrease of junctional complex in additional frame shift codon was introduced in exon À À the interendothelial