Endothelial cells internalize monoclonal antibody to angiotensin-converting enzyme

Vladimir R. Muzykantov, Elena Nikolaevna Atochina, Alice Kuo, Elliott S. Barnathan, Kathy Notarfrancesco, Henry Shuman, Chandra Dodia, Aron B. Fisher

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Abstract

We investigated the fate of MAb 9B9, a monoclonal antibody to angiotensin-converting enzyme (ACE), which binds to endothelium both in vitro and in vivo. Using cultured human umbilical vein endothelial cells (HUVEC) and isolated perfused rat lungs (IPL), we demonstrated specific and saturable binding of 125I-labeled MAb 9B9 at 4°C [affinity constant (Kd) = 20-50 nM, maximal number of binding sites (Bmax) = 1.5-3.0 × 105 sites/cell]. When 125I-MAb 9B9 was bound to HUVEC at 37°C, only 40% of cell-associated radioactivity was acid elutable, suggesting antibody internalization. This was confirmed by finding that 1) the amount of MAb 9B9 uptake at 37°C was higher than at 4°C both in HUVEC and IPL; 2) binding of 125I-labeled streptavidin with HUVEC and IPL pretreated with biotinylated MAb 9B9 (b-MAb 9B9) was diminished in a temperature- and time-dependent fashion at 37°C; and 3) b-MAb 9B9 bound to HUVEC at 37°C was found intracellularly by ultrastructural analysis using streptavidin gold. Intracellular 125I-MAb 9B9 was found in microsomal fractions of lung homogenate from IPL and after intravenous (iv) injections in rats. Degradation of internalized MAb 9B9 was minimal, since >90% of cell-associated 125I label remained precipitable by trichloracetic acid in HUVEC, IPL, and in vivo. Autoradiography of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of lung homogenates made as late as several days after iv injections of 125I-MAb 9B9 in rats demonstrated a predominant band above 140 kDa. These data indicate that endothelial cells either in vitro or in vivo internalize the ACE ligand MAb 9B9 without significant intracellular degradation. Therefore MAb 9B9 may be useful for selective intracellular delivery of drugs to the pulmonary vascular endothelium after systemic administration.

Original languageEnglish
JournalAmerican Journal of Physiology - Lung Cellular and Molecular Physiology
Volume270
Issue number5 14-5
Publication statusPublished - May 1996
Externally publishedYes

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Keywords

  • Angiotensin-converting enzyme turnover
  • Drug targeting
  • Lung
  • Pulmonary vasculature
  • Rats
  • Vascular biology

ASJC Scopus subject areas

  • Cell Biology
  • Physiology
  • Physiology (medical)
  • Pulmonary and Respiratory Medicine

Cite this

Muzykantov, V. R., Atochina, E. N., Kuo, A., Barnathan, E. S., Notarfrancesco, K., Shuman, H., ... Fisher, A. B. (1996). Endothelial cells internalize monoclonal antibody to angiotensin-converting enzyme. American Journal of Physiology - Lung Cellular and Molecular Physiology, 270(5 14-5).