Live dynamic imaging of caveolae pumping targeted antibody rapidly and specifically across endothelium in the lung.
Journal: 2007/April - Nature Biotechnology
ISSN: 1087-0156
Abstract:
How effectively and quickly endothelial caveolae can transcytose in vivo is unknown, yet critical for understanding their function and potential clinical utility. Here we use quantitative proteomics to identify aminopeptidase P (APP) concentrated in caveolae of lung endothelium. Electron microscopy confirms this and shows that APP antibody targets nanoparticles to caveolae. Dynamic intravital fluorescence microscopy reveals that targeted caveolae operate effectively as pumps, moving antibody within seconds from blood across endothelium into lung tissue, even against a concentration gradient. This active transcytosis requires normal caveolin-1 expression. Whole body gamma-scintigraphic imaging shows rapid, specific delivery into lung well beyond that achieved by standard vascular targeting. This caveolar trafficking in vivo may underscore a key physiological mechanism for selective transvascular exchange and may provide an enhanced delivery system for imaging agents, drugs, gene-therapy vectors and nanomedicines. 'In vivo proteomic imaging' as described here integrates organellar proteomics with multiple imaging techniques to identify an accessible target space that includes the transvascular pumping space of the caveola.
Relations:
Content
Citations
(93)
References
(47)
Grants
(14)
Chemicals
(4)
Organisms
(3)
Processes
(1)
Anatomy
(3)
Similar articles
Articles by the same authors
Discussion board
Nat Biotechnol 25(3): 327-337

Live dynamic imaging of caveolae pumping targeted antibody rapidly and specifically across endothelium in the lung

+2 authors
Sidney Kimmel Cancer Center, 10905 Road to the Cure, San Diego, California 92121, USA
EE Department, University of California, Los Angeles, Box 951594, Los Angeles, California 90095, USA
Gamma Medica, Inc., 19355 Business Center Drive, Suite #18, Northridge, California 91324, USA
Departments of Medicine, Radiology & Pathology, University of Alabama at Birmingham, 1808 Seventh Avenue South, Birmingham, Alabama 35294, USA
Correspondence should be addressed to J.E.S. (gro.ccks@reztinhcsj).

Abstract

How effectively and quickly endothelial caveolae can transcytose in vivo is unknown, yet critical for understanding their function and potential clinical utility. Here we use quantitative proteomics to identify aminopeptidase P (APP) concentrated in caveolae of lung endothelium. Electron microscopy confirms this and shows that APP antibody targets nanoparticles to caveolae. Dynamic intravital fluorescence microscopy reveals that targeted caveolae operate effectively as pumps, moving antibody within seconds from blood across endothelium into lung tissue, even against a concentration gradient. This active transcytosis requires normal caveolin-1 expression. Whole body γ-scintigraphic imaging shows rapid, specific delivery into lung well beyond that achieved by standard vascular targeting. This caveolar trafficking in vivo may underscore a key physiological mechanism for selective transvascular exchange and may provide an enhanced delivery system for imaging agents, drugs, gene-therapy vectors and nanomedicines. ‘In vivo proteomic imaging’ as described here integrates organellar proteomics with multiple imaging techniques to identify an accessible target space that includes the transvascular pumping space of the caveola.

Abstract

Caveolae are caveolin-coated, omega-shaped plasmalemmal invaginations 60–70 nm in diameter that bud from the plasma membrane in a dynamin and GTP-dependent manner12. They are especially abundant in vascular endothelia, where they function in endocytosis and transcytosis to traffic select macromolecules and to maintain tissue homeostasis. Caveolin knockout mice exhibit poor endothelial cell barrier function with compensatory tissue disruption and edema, particularly evident in the lung34. The study of trafficking by caveolae has been hampered by a lack of caveolae-specific probes. This is especially true for the caveolae of endothelial cells, which in cell culture exhibit phenotypic drift, including altered protein expression56 and a greater than tenfold decrease in caveolae density7. Studies of caveolae trafficking in many types of cultured cells have suggested that caveolae mediate endocytosis at a much slower rate than that observed for clathrin-mediated trafficking (1–2 h versus 5–10 min)810. Caveolae have even been described as static structures that do not constitutively traffic cargo1113. In vivo data on caveolae trafficking are conspicuously lacking. Electron microscopy (EM) has provided static images supporting transendothelial transport1416, but usually in situ with probes that are not specific for caveolae1718.

One of the major challenges in delivering imaging agents, drugs, nanoparticles or gene therapies to specific tissues of the body is overcoming endothelial and epithelial cell barriers that prevent entry into tissue compartments171924. For example, the treatment of multiple genetic and acquired diseases of the lung, such as cystic fibrosis, lung cancer, pulmonary fibrosis, pulmonary hypertension and acute respiratory distress syndrome, could benefit from a means of delivering agents across the endothelial barrier to the cells deeper in the tissue192527. Vascular targeting is directed towards the accessible endothelial cell surface of blood vessels feeding the tissue rather than relatively inaccessible sites located on cells inside the tissue101718242831. Agents injected into the blood have direct and almost immediate exposure to the vascular endothelial cell surface, including its caveolae141732. Whether proteins with sufficient tissue specificity exist at this critical blood-tissue interface is unclear33, however, and rapid tissue-specific targeting with high blood extraction has seldom been attained and validated in vivo14243436.

It has been hypothesized17182932 that targeting caveolae may improve specific delivery into select tissues beyond standard vascular targeting by providing a transcellular trafficking pathway that overcomes normal barriers. Here we use an in vivo proteomic mapping and imaging strategy to discover and validate targets in lung endothelial caveolae as useful for achieving tissue-specific targeting. We develop and characterize antibody probes to lung endothelial cell–surface proteins and use small-animal imaging techniques to provide a dynamic, sensitive and quantitative visualization of tissue-specific vascular targeting and transendothelial transport in vivo.

Footnotes

Note: Supplementary information is available on the Nature Biotechnology website.

AUTHOR CONTRIBUTIONS

P.O., Figures 1a,c, ,2,2, ,3,3, ,4,4, ,6,6, Supplementary Figures 2b,c, 3b–d, 4, 5, subcellular fractionation, immunoblotting, lentiviral gene silencing siRNA, animal surgical procedures, intravital microscopy imaging and analysis, fluorophore and I labeling of proteins, A-SPECT and X-SPECT imaging, Supplementary Videos 1–6; P.B., Figures 2, ,3,3, ,4,4, ,5,5, intravital microscopy and analysis, Supplementary Videos 1, 2; H.W., Figure 1b, c, all electron microscopy imaging and analysis; Y.L., Supplementary Figures 1 and 2a, mass spectrometry (MS) and analysis of MS data; B.J.B., Figures 4, ,5,5, intravital microscopy image processing, custom MatLab scripting; A.C., Figure 7a–c, X-SPECT imaging, radiolabeling, Supplementary Figure 5; K.I., Figure 6a, bj, k, m, Supplementary Videos 3–6; K.R.Z., Figure 7d–f; R.B., Figure 1c, gold particle preparation; J.E.T., Figure 1a, Supplementary Figures 3a, b and 5, monoclonal antibody production, screening and characterization; J.E.S., manuscript, experiment design and result analysis.

COMPETING INTERESTS STATEMENT

The authors declare that they have no competing financial interests.

Footnotes

References

  • 1. Schnitzer JE, Oh P, McIntosh DPRole of GTP hydrolysis in fission of caveolae directly from plasma membranes. Science [publisher’s erratum appears in Science 1996 Nov 15;274(5290):1069] 1996;274:239–242.[PubMed][Google Scholar]
  • 2. Oh P, McIntosh DP, Schnitzer JEDynamin at the neck of caveolae mediates their budding to form transport vesicles by GTP-driven fission from the plasma membrane of endothelium. J Cell Biol. 1998;141:101–114.[Google Scholar]
  • 3. Razani B, et al Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities. J Biol Chem. 2001;276:38121–38138.[PubMed][Google Scholar]
  • 4. Drab M, et al Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice. Science. 2001;293:2449–2452.[PubMed][Google Scholar]
  • 5. Durr E, et al Direct proteomic mapping of the lung microvascular endothelial cell surface in vivo and in cell culture. Nat Biotechnol. 2004;22:985–992.[PubMed][Google Scholar]
  • 6. Oh P, et al Subtractive proteomic mapping of the endothelial surface in lung and solid tumours for tissue-specific therapy. Nature. 2004;429:629–635.[PubMed][Google Scholar]
  • 7. Schnitzer JE, Carley WW, Palade GESpecific albumin binding to microvascular endothelium in culture. Am J Physiol. 1988;254:H425–H437.[PubMed][Google Scholar]
  • 8. Parton RGUltrastructural localization of gangliosides; GM1 is concentrated in caveolae. J Histochem Cytochem. 1994;42:155–166.[PubMed][Google Scholar]
  • 9. Pelkmans L, Kartenback J, Helenius ACaveolar endocytosis of Simian virus 40 reveals a novel two-step vesicular transport pathway to the ER. Nat Cell Biol. 2001;3:473–483.[PubMed][Google Scholar]
  • 10. Schnitzer JE, Oh P, Pinney E, Allard JFilipin-sensitive caveolae-mediated transport in endothelium: reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules. J Cell Biol. 1994;127:1217–1232.[Google Scholar]
  • 11. Thomsen P, Roepstorff K, Stahlhut M, van Deurs BCaveolae are highly immobile plasma membrane microdomains, which are not involved in constitutive endocytic trafficking. Mol Biol Cell. 2002;13:238–250.[Google Scholar]
  • 12. Severs NJCaveolae: static inpocketings of the plasma membrane, dynamic vesicles or plain artifact? J Cell Sci. 1988;90:341–348.[PubMed][Google Scholar]
  • 13. Bundgaard M, Frokjaer-Jensen J, Crone CEndothelial plasmalemmal vesicles as elements in a system of branching invaginations from the cell surface. Proc Natl Acad Sci USA. 1979;76:6439–6442.[Google Scholar]
  • 14. McIntosh DP, Tan XY, Oh P, Schnitzer JETargeting endothelium and its dynamic caveolae for tissue-specific transcytosis in vivo: A pathway to overcome cell barriers to drug and gene delivery. Proc Natl Acad Sci USA. 2002;99:1996–2001.[Google Scholar]
  • 15. Simionescu M, Simionescu N, Palade GEMorphometric data on the endothelium of blood capillaries. J Cell Biol. 1974;60:128–152.[Google Scholar]
  • 16. Simionescu M, Simionescu N. Endothelial transport of macromolecules: transcytosis and endocytosis. A look from cell biology. Cell Biol Rev. 1991;25:1–78.[PubMed]
  • 17. Carver LA, Schnitzer JECaveolae: mining little caves for new cancer targets. Nat Rev Cancer. 2003;3:571–581.[PubMed][Google Scholar]
  • 18. Schnitzer JEUpdate on the cellular and molecular basis of capillary permeability. Trends Cardiovasc Med. 1993;3:124–130.[PubMed][Google Scholar]
  • 19. Jain RKThe next frontier of molecular medicine: delivery of therapeutics. Nat Med. 1998;4:655–657.[PubMed][Google Scholar]
  • 20. Herschman HRMolecular imaging: looking at problems, seeing solutions. Science. 2003;302:605–608.[PubMed][Google Scholar]
  • 21. Rudin M, Weissleder RMolecular imaging in drug discovery and development. Nat Rev Drug Discov. 2003;2:123–131.[PubMed][Google Scholar]
  • 22. Tomlinson ETheory and practice of site-specific drug delivery. Adv Drug Deliv Rev. 1987;1:87–198.[PubMed][Google Scholar]
  • 23. Dvorak HF, Nagy JA, Dvorak AMStructure of solid tumors and their vasculature: implications for therapy with monoclonal antibodies. Cancer Cells. 1991;3:77–85.[PubMed][Google Scholar]
  • 24. Schnitzer JEVascular targeting as a strategy for cancer therapy. N Engl J Med. 1998;339:472–474.[PubMed][Google Scholar]
  • 25. Jenkins RG, McAnulty RJ, Hart SL, Laurent GJ. Pulmonary gene therapy. Realistic hope for the future, or false dawn in the promised land? Monaldi Arch Chest Dis. 2003;59:17–24.[PubMed]
  • 26. Courrier HM, Butz N, Vandamme TFPulmonary drug delivery systems: recent developments and prospects. Crit Rev Ther Drug Carrier Syst. 2002;19:425–498.[PubMed][Google Scholar]
  • 27. Kozower BD, et al Immunotargeting of catalase to the pulmonary endothelium alleviates oxidative stress and reduces acute lung transplantation injury. Nat Biotechnol. 2003;21:392–398.[PubMed][Google Scholar]
  • 28. Burrows FJ, Thorpe PEVascular targeting–a new approach to the therapy of solid tumors. Pharmacol Ther. 1994;64:155–174.[PubMed][Google Scholar]
  • 29. Schnitzer JECaveolae: from basic trafficking mechanisms to targeting transcytosis for tissue-specific drug and gene delivery in vivo. Adv Drug Deliv Rev. 2001;49:265–280.[PubMed][Google Scholar]
  • 30. Carver LA, Schnitzer JE. Tissue-specific pharmacodelivery and overcoming key cell barriers in vivo: Vascular targeting of caveolae. In: Muzykantov V, Torchilin B, editors. Biomedical Aspects of Drug Targeting. Kluwer Academic Publishers; Boston: 2002. pp. 107–128. [PubMed]
  • 31. Denekamp JVasculature as a target for tumour therapy. Progr Appli Microcirc. 1984;4:28–38.[PubMed][Google Scholar]
  • 32. Schnitzer JE. The endothelial cell surface and caveolae in health and disease. In: Born G, Shwartz CJ, editors. Vascular Endothelium: Physiology, Pathology, and Therapeutic Opportunities. Schattauer; Stuttgart: 1997. pp. 77–95. [PubMed]
  • 33. Carver LA, Schnitzer JE. Proteomic mapping of endothelium and vascular targeting in vivo. In: Aird W, editor. Endothelial Biomedicine. 2007. pp. 881–897. In press. [PubMed]
  • 34. Pasqualini R, Ruoslahti EOrgan targeting in vivo using phage display peptide libraries. Nature. 1996;380:364–366.[PubMed][Google Scholar]
  • 35. Muzykantov VR, et al Streptavidin facilitates internalization and pulmonary targeting of an anti-endothelial cell antibody (platelet-endothelial cell adhesion molecule 1): a strategy for vascular immunotargeting of drugs. Proc Natl Acad Sci USA. 1999;96:2379–2384.[Google Scholar]
  • 36. Hughes BJ, Kennel SK, Lee R, Huang LMonoclonal antibody targeting of liposomes to mouse lung in vivo.Cancer Res. 1989;49:6214–6220.[PubMed][Google Scholar]
  • 37. Schnitzer JE, McIntosh DP, Dvorak AM, Liu J, Oh PSeparation of caveolae from associated microdomains of GPI-anchored proteins. Science. 1995;269:1435–1439.[PubMed][Google Scholar]
  • 38. Oh P, Schnitzer JE. Isolation and subfractionation of plasma membranes to purify caveolae separately from glycosyl-phospatidylinositol-anchored protein microdomains. In: Celis J, editor. Cell Biology: A laboratory handbook. Academic Press; 1998. pp. 34–46. [PubMed]
  • 39. Oh P, Schnitzer JEImmunoisolation of caveolae with high affinity antibody binding to the oligomeric caveolin cage: toward understanding the basis of purification. J Biol Chem. 1999;274:23144–23154.[PubMed][Google Scholar]
  • 40. Muro H, Shirasawa H, Maeda M, Nakamura S. Fc receptors of liver sinusoidal endothelium in normal rats and humans. A histology study with soluble immune complexes. Gastroenterology. 1987;93:1078–1085.[PubMed]
  • 41. Russell J, et al Iodination of annexin V for imaging apoptosis. J Nucl Med. 2002;43:671–677.[PubMed][Google Scholar]
  • 42. Chaurand P, Sanders ME, Jensen RA, Caprioli RMProteomics in diagnostic pathology: profiling and imaging proteins directly in tissue sections. Am J Pathol. 2004;165:1057–1068.[Google Scholar]
  • 43. Nichols BJ, et al Rapid cycling of lipid raft markers between the cell surface and Golgi complex. J Cell Biol. 2001;153:529–541.[Google Scholar]
  • 44. Porter GA, Bankston PWMaturation of myocardial capillaries in the fetal and neonatal rat: an ultrastructural study with a morphometric analysis of the vesicle populations. Am J Anat. 1987;178:116–125.[PubMed][Google Scholar]
  • 45. Schnitzer JE, Liu J, Oh PEndothelial caveolae have the molecular transport machinery for vesicle budding, docking, and fusion including VAMP, NSF, SNAP, annexins, and GTPases. J Biol Chem. 1995;270:14399–143404.[PubMed][Google Scholar]
  • 46. McIntosh DP, Oh P, Schnitzer JECaveolae require intact VAMP-2 for targeted transport in vascular endothelium. Am J Physiol. 1999;277:H2222–H2232.[PubMed][Google Scholar]
  • 47. Miller N, Vile RTargeted vectors for gene therapy. FASEB J. 1995;9:190–199.[PubMed][Google Scholar]
  • 48. Galanis E, Vile R, Russell SJDelivery systems intended for in vivo gene therapy of cancer: targeting and replication competent viral vectors. Crit Rev Oncol Hematol. 2001;38:177–192.[PubMed][Google Scholar]
  • 49. Bilbao G, Gomez-Navarro J, Curiel DTTargeted adenoviral vectors for cancer gene therapy. Adv Exp Med Biol. 1998;451:365–374.[PubMed][Google Scholar]
  • 50. Lehr HA, Leunig M, Menger MD, Nolte D, Messmer KDorsal skinfold chamber technique for intravital microscopy in nude mice. Am J Pathol. 1993;143:1055–1062.[Google Scholar]
  • 51. Alger GHAn adaptation of the transparent chamber technique to the mouse. J Natl Cancer Inst. 1943;4:1–11.[PubMed][Google Scholar]
  • 52. Frost GI, Borgstrom PReal time in vivo quantitation of tumor angiogenesis. Methods Mol Med. 2003;85:65–78.[PubMed][Google Scholar]
  • 53. Frost GI, et al Novel syngeneic pseudo-orthotopic prostate cancer model: vascular, mitotic and apoptotic responses to castration. Microvasc Res. 2005;69:1–9.[PubMed][Google Scholar]
  • 54. Moffat J, et al A Lentiviral RNAi Library for Human and Mouse Genes Applied to an Arrayed Viral High-Content Screen. Cell. 2006;124:1283–1298.[PubMed][Google Scholar]
  • 55. McElroy D, et al Performance evaluation of A-SPECT: A high resolution desktop pinhole SPECT system for imaging small animals. IEEE Trans Nucl Sci NS. 2002;49:2139–2147.[PubMed][Google Scholar]
Collaboration tool especially designed for Life Science professionals.Drag-and-drop any entity to your messages.