Abstract
The class II α-isoform of phosphatidylinositol 3-kinase (PI3K-C2α) is localized in endosomes, the trans-Golgi network and clathrin-coated vesicles; however, its functional role is not well understood. Global or endothelial-cell–specific deficiency of PI3K-C2α resulted in embryonic lethality caused by defects in sprouting angiogenesis and vascular maturation. PI3K-C2α knockdown in endothelial cells resulted in a decrease in the number of PI3-phosphate–enriched endosomes, impaired endosomal trafficking, defective delivery of VE-cadherin to endothelial cell junctions and defective junction assembly. PI3K-C2α knockdown also impaired endothelial cell signaling, including vascular endothelial growth factor receptor internalization and endosomal RhoA activation. Together, the effects of PI3K-C2α knockdown led to defective endothelial cell migration, proliferation, tube formation and barrier integrity. Endothelial PI3K-C2α deficiency in vivo suppressed postischemic and tumor angiogenesis and diminished vascular barrier function with a greatly augmented susceptibility to anaphylaxis and a higher incidence of dissecting aortic aneurysm formation in response to angiotensin II infusion. Thus, PI3K-C2α has a crucial role in vascular formation and barrier integrity and represents a new therapeutic target for vascular disease.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Adams, R.H. & Alitalo, K. Molecular regulation of angiogenesis and lymphangiogenesis. Nat. Rev. Mol. Cell Biol. 8, 464–478 (2007).
Coultas, L. et al. Endothelial cells and VEGF in vascular development. Nature 438, 937–945 (2005).
Andrae, J. et al. Role of platelet-derived growth factor in physiology and medicine. Genes Dev. 22, 1276–1312 (2008).
Mehta, D. & Malik, A.B. Signaling mechanisms regulating endothelial permeability. Physiol. Rev. 86, 279–367 (2006).
Dejana, E., Tournier-Lasserve, E. & Weinstein, B.M. The control of vascular integrity by endothelial cell junctions: molecular basis and pathological implications. Dev. Cell 16, 209–221 (2009).
Yoshimura, K. et al. Regression of abdominal aortic aneurysm by inhibition of c-Jun N-terminal kinase. Nat. Med. 11, 1330–1338 (2005).
Satoh, K. et al. Cyclophilin A enhances vascular oxidative stress and the development of angiotensin II–induced aortic aneurysms. Nat. Med. 15, 649–656 (2009).
Ferrara, N. & Kerbel, R.S. Angiogenesis as a therapeutic target. Nature 438, 967–974 (2005).
Carmeliet, P. Angiogenesis in life, disease and medicine. Nature 438, 932–936 (2005).
Engelman, J.A. et al. The evolution of phosphatidylinositol 3-kinases as regulator of growth and metabolism. Nat. Rev. Genet. 7, 606–619 (2006).
Takenawa, T. & Suetsugu, S. The WASP-WAVE protein network: connecting the membrane to the cytoskeleton. Nat. Rev. Mol. Cell Biol. 8, 37–48 (2007).
Vanhaesebroeck, B. et al. The emerging mechanisms of isoform-specific PI3K signaling. Nat. Rev. Mol. Cell Biol. 11, 329–341 (2010).
Graupera, M. et al. Angiogenesis selectively requires the p110α isoform of PI3K to control endothelial cell migration. Nature 453, 662–666 (2008).
Yuan, T.L. & Cantley, L.C. PI3K pathway alterations in cancer: variations on a theme. Oncogene 27, 5497–5510 (2008).
Funderburk, S.F. et al. The Beclin 1–Vps34 complex at the crossroads of autophagy and beyond. Trends Cell Biol. 20, 355–362 (2010).
Falasca, M. et al. The role of phosphoinositide 3-kinase C2α in insulin signaling. J. Biol. Chem. 282, 28226–28236 (2007).
Linassier, C. et al. Molecular cloning and biochemical characterization of a Drosophila phosphatidylinositol-specific phosphoinositide 3-kinase. Biochem. J. 321, 849–856 (1997).
Domin, J. et al. The class II phosphoinositide 3-kinase PI3K–C2α is concentrated in the trans-Golgi network and present in clathrin-coated vesicles. J. Biol. Chem. 275, 11943–11950 (2000).
El Sheikh, S.S. et al. Topographical expression of class IA and class II phosphoinositide 3-kinase enzymes in normal human tissues is consistent with a role in differentiation. BMC Clin. Pathol. 3, 4 (2003).
Yoshioka, K. et al. Ca2+-induced, Rho- and Rho kinase-dependent regulation of myosin phosphatase and contraction in isolated vascular smooth muscle cells. Mol. Pharmacol. 71, 912–920 (2007).
Traer, C.J. et al. Are class II phosphoinositide 3-kinases potential targets for anticancer therapies? Bull. Cancer 93, E53–E58 (2006).
Gaidarov, I. et al. Individual phosphoinositide 3-kinase C2α domain activities independently regulate clathrin function. J. Biol. Chem. 280, 40766–40772 (2005).
Falasca, M. & Maffucci, T. Role of class II phosphoinositide 3-kinase in cell signalling. Biochem. Soc. Trans. 35, 211–214 (2007).
Wang, Y. et al. Class II phosphoinositide 3-kinase α-isoform regulates Rho, myosin phosphatase and contraction in vascular smooth muscle. Biochem. J. 394, 581–592 (2006).
Harris, D.P. et al. Requirement for class II phosphoinositide 3-kinase C2α in maintenance of glomerular structure and function. Mol. Cell. Biol. 31, 63–80 (2011).
Moses, K.A. et al. Embryonic expression of an Nkx2–5/Cre gene using ROSA26 reporter mice. Genesis 31, 176–180 (2001).
Kisanuki, Y.Y. et al. Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo. Dev. Biol. 230, 230–242 (2001).
Wang, Y. et al. Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis. Nature 465, 483–486 (2010).
Pannekoek, W.J. et al. Cell-cell junction formation: the role of Rap1 and Rap1 guanine nucleotide exchange factors. Biochim. Biophys. Acta 1788, 790–796 (2009).
Gillooly, D.J. et al. Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells. EMBO J. 19, 4577–4588 (2000).
Lindmo, K. & Stenmark, H. Regulation of membrane traffic by phosphoinositide 3-kinase. J. Cell Sci. 119, 605–614 (2006).
Di Paolo, G. & De Camilli, P. Phosphoinositides in cell regulation and membrane dynamics. Nature 443, 651–657 (2006).
Bryan, B.A. et al. RhoA/ROCK signaling is essential for multiple aspects of VEGF-mediated angiogenesis. FASEB J. 24, 3186–3196 (2010).
Macia, E. et al. Dynasore, a cell-permeable inhibitor of dynamin. Dev. Cell 10, 839–850 (2006).
Cauwels, A. et al. Anaphylactic shock depends on PI3K and eNOS-derived NO. J. Clin. Invest. 116, 2244–2251 (2006).
Daugherty, A. & Cassis, L.A. Mouse models of abdominal aortic aneurysms. Arterioscler. Thromb. Vasc. Biol. 24, 429–434 (2004).
Di Gennaro, A. et al. Increased expression of leukotriene C4 synthase and predominant formation of cysteinyl-leukotrienes in human abdominal aortic aneurysm. Proc. Natl. Acad. Sci. USA 107, 21093–21097 (2010).
Wheeler, M. & Domin, J. The N-terminus of phosphoinositide 3-kinase–C2β regulates lipid kinase activity and binding to clathrin. J. Cell. Physiol. 206, 586–593 (2006).
Simonsen, A. & Tooze, S.A. Coordination of membrane events during autophagy by multiple class III PI3-kinase complexes. J. Cell Biol. 186, 773–782 (2009).
Johnson, E.E. et al. Gene silencing reveals a specific function of hVps34 phosphatidylinositol 3-kinase in late versus early endosomes. J. Cell Sci. 119, 1219–1232 (2006).
Doherty, G.J. & McMahon, H.T. Mechanisms of endocytosis. Annu. Rev. Biochem. 78, 31.1–31.46 (2009).
Zoncu, R. et al. A phosphoinositide switch controls the maturation and signaling properties of APPL endosomes. Cell 136, 1110–1121 (2009).
Palamidessi, A. et al. Endocytic trafficking of Rac is required for the spatial restriction of signaling in cell migration. Cell 134, 135–147 (2008).
van Nieuw Amerongen, G.P. et al. Involvement of Rho kinase in endothelial barrier maintenance. Arterioscler. Thromb. Vasc. Biol. 27, 2332–2339 (2007).
Yamada, S. & Nelson, W.J. Localized zones of Rho and Rac activities drive initiation and expansion of epithelial cell-cell adhesion. J. Cell Biol. 178, 517–527 (2007).
Harris, T.J.C. & Tepass, U. Adherens junctions: from molecules to morphogenesis. Nat. Rev. Mol. Cell Biol. 11, 502–514 (2010).
Abraham, S. et al. VE-cadherin–mediated cell-cell interaction suppresses sprouting via signaling to MLC2 phosphorylation. Curr. Biol. 19, 668–674 (2009).
Noda, K. et al. Vascular endothelial-cadherin stabilizes at cell-cell junctions by anchoring to circumferential actin bundles through α- and β-catenins in cyclic AMP-Epac-Rap1 signal-activated endothelial cells. Mol. Biol. Cell 21, 584–596 (2010).
Webb, D.J., Parsons, J.T. & Horwits, A.F. Adhesion assembly, disassembly and turnover in migrating cells—over and over and over again. Nat. Cell Biol. 4, E97–E100 (2002).
Mitra, S.K., Hanson, D.A. & Schlaepfer, D.D. Focal adhesion kinase: in command and control of cell motility. Nat. Rev. Mol. Cell Biol 6, 56–68 (2005).
Shivas, J.M. et al. Polarity and endocytosis: reciprocal regulation. Trends Cell Biol. 20, 445–452 (2010).
Sörensen, I., Adams, R.H. & Gossler, A. Dll1-mediated Notch activation regulates endothelial identity in mouse fetal arteries. Blood 113, 5680–5688 (2009).
Bazigou, E. et al. Integrin-α9 is required for fibronectin matrix assembly during lymphatic valve morphogenesis. Dev. Cell 17, 175–186 (2009).
Takakura, N. et al. Critical role of the TIE2 endothelial cell receptor in the development of definitive hematopoiesis. Immunity 9, 677–686 (1998).
Benedito, R. et al. The Notch ligands Dll4 and Jagged1 have opposing effects on angiogenesis. Cell 137, 1124–1135 (2009).
Compagni, A. et al. Control of skeletal patterning by ephrinB1-EphB interactions. Dev. Cell 5, 217–230 (2003).
Oyama, O. et al. The lysophospholipid mediator sphingosine-1-phosphate promotes angiogenesis in vivo in ischemic hindlimbs of mice. Cardiovasc. Res. 78, 301–307 (2008).
Du, W. et al. S1P2, the G protein–coupled receptor for sphingosine-1-phosphate, negatively regulates tumor angiogenesis and tumor growth in vivo in mice. Cancer Res. 70, 772–781 (2010).
Deng, G.G. et al. Urokinase-type plasminogen activator plays a critical role in angiotensin II–induced abdominal aortic aneurysm. Circ. Res. 92, 510–517 (2003).
Okamoto, H. et al. Inhibitory regulation of Rac activation, membrane ruffling, and cell migration by the G protein-coupled sphingosine-1-phosphate receptor EDG5 but not EDG1 or EDG3. Mol. Cell. Biol. 20, 9247–9261 (2000).
Sasaki, T. et al. Function of PI3Kγ in thymocyte development, T cell activation, and neutrophil migration. Science 287, 1040–1046 (2000).
Acknowledgements
We thank K. Mitsumori for comments on the histological study. We thank N. Mochizuki and K. Ando for assistance with the FRET imaging analysis. We thank N. Furusawa, K. Sunagawa and E. Kaneko for assistance with live-cell imaging using a Yokogawa confocal microscope system. We also thank Y. Ohta and T. Murakawa for technical assistance and T. Hirose for administrative assistance. C2α complementary DNA was obtained from J. Domin (Imperial College London). GFP-2 × FYVE and mRFP-2 × FYVE expression vectors were obtained from H. Stenmark (Oslo University Hospital) and Y. Ohsumi (Tokyo Institute of Technology), respectively. VE-cadherin-GFP expression vectors were obtained from N. Mochizuki (National Cerebral and Cardiovascular Center). The pRaichu-RhoA probe was obtained from M. Matsuda (Kyoto University). GFP-RhoAAsn19 and GFP-RhoAVal14 expression vectors were obtained from F. Valderrama (King's College London). This work was supported in part by grants-in-aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology, the Japan Society for the Promotion of Science (to K. Yoshioka, N. Takuwa, Y.O. and Y.T.), the Honjin Foundation, the Mitsubishi Pharma Research Foundation and the SENSIN Medical Research Foundation (to K. Yoshioka).
Author information
Authors and Affiliations
Contributions
K. Yoshioka designed the experiments, performed characterization of the developmental and retinal angiogenesis of the conditional knockout mice and most of the in vitro studies and analyzed the data with assistance from N. Takuwa, Y.O., W.D., S.A., H.M., C.N., K.B., M.U., N. Takakura and O.M. K.A. and T.S. analyzed the cellular content of phosphoinositides. K. Yoshida performed in vivo angiogenenesis experiments with K. Yoshioka, performed tumor implantation and aneurysm experiments and interpreted the results. H.C. performed the anaphylaxis experiments. W.D. performed the in vivo permeability study. X.Q. and Y.O. performed and interpreted the results of the ischemic angiogenesis model. T.W. and S.I. performed and interpreted the results of electron microscopy. K.S., M.A., N. Takuwa, R.J.S., H.O. and R.H.A. generated mouse mutants. K. Yoshioka and Y.T. planned and supervised the experiments, arranged the figures and wrote the manuscript. M.A. and N. Takuwa participated in writing the manuscript (M.A. wrote part of the Online Methods, and N. Takuwa wrote the Abstract, Introduction and Results sections).
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–20, Supplementary Tables 1–7 and Supplementary Methods (PDF 5179 kb)
Supplementary Video 1
Time-lapse imaging of GFP-C2α and mRFP-2xFYVE in HUVEC. (MOV 1720 kb)
Supplementary Video 2
Trafficking of the intracellular GFP-2xFYVE-positive endosomes in control HUVEC. (MOV 1755 kb)
Supplementary Video 3
Trafficking of the intracellular GFP-2xFYVE-positive endosomes in C2α-depleted HUVEC. (MOV 1880 kb)
Supplementary Video 4
Trafficking of VE-cadherin-GFP in control HUVEC. (MOV 3672 kb)
Supplementary Video 5
Trafficking of VE-cadherin-GFP in C2α-depleted HUVEC. (MOV 2159 kb)
Supplementary Video 6
Trafficking of VE-cadherin-GFP in adeno-RhoAN19-infected HUVEC. (MOV 839 kb)
Supplementary Video 7
RhoA FRET imaging in control HUVEC. (MOV 1741 kb)
Supplementary Video 8
RhoA FRET imaging in C2α-depleted HUVEC. (MOV 1807 kb)
Rights and permissions
About this article
Cite this article
Yoshioka, K., Yoshida, K., Cui, H. et al. Endothelial PI3K-C2α, a class II PI3K, has an essential role in angiogenesis and vascular barrier function. Nat Med 18, 1560–1569 (2012). https://doi.org/10.1038/nm.2928
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nm.2928
This article is cited by
-
Beyond PI3Ks: targeting phosphoinositide kinases in disease
Nature Reviews Drug Discovery (2023)
-
Development of selective inhibitors of phosphatidylinositol 3-kinase C2α
Nature Chemical Biology (2023)
-
Investigating the histological and structural properties of tendon gel as an artificial biomaterial using the film model method in rabbits
Journal of Experimental Orthopaedics (2022)
-
Structural basis of phosphatidylinositol 3-kinase C2α function
Nature Structural & Molecular Biology (2022)
-
Trafficking in blood vessel development
Angiogenesis (2022)