Agonist-induced capping of adhesion proteins and microparticle shedding in cultures of human renal microvascular endothelial cells.
Capping and release of membranous, small (< 1.5 microm) endothelial microparticles were quantified by immunofluorescence microscopy and flow cytometry after treatment of cultures of human renal microvascular endothelial cells with agonists tumor necrosis factor-alpha (TNF-alpha) or mitomycin C. For constitutive marker CD31, both agonist-treated attached, monolayer, and detached, free endothelial cells formed caps and released microparticles. TNF-alpha and mitomycin C induced dissimilar appearing CD31-containing caps after 3 h, followed by endothelial microparticle release after 6 h. The degree of capping correlated with increasing counts of released microparticles. For lymphokine-inducible CD54, TNF-alpha also induced CD54-containing caps and microparticle release, but mitomycin C failed to induce the expression of either entity. Neither capping nor microparticle release caused by TNF-alpha was part of an apoptotic pathway that involved caspase 3. Mitomycin C treatment of endothelial cells caused capping and microparticle release with a time course similar to TNF-alpha induction for 15 to 24 h, but assays for caspase 3 were positive, confirming the apoptotic action of mitomycin C. Membrane capping and microparticle release from endothelial cells are a convenient experimental model for studying protein movement, release of microparticles, and their possible biological significance.
Jy
Wenche
W
Department of Medicine, University of Miami School of Medicine, Florida 33101, USA.
Jimenez
Joaquin J
JJ
Mauro
Lucia M
LM
Ahn
Yeon S
YS
Newton
Kenneth R
KR
Mendez
Armando J
AJ
Arnold
Patricia I
PI
Schultz
Duane R
DR
D000911:Antibodies, Monoclonal; D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases / Q000032:analysis; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002457:Cell Extracts; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis / Q000378:metabolism; D007668:Kidney / Q000098:blood supply / Q000166:cytology*; D008833:Microcirculation; D008856:Microscopy, Fluorescence; D008863:Microspheres; D016685:Mitomycin / Q000494:pharmacology*; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000096:biosynthesis / Q000378:metabolism; D021381:Protein Transport; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D015815:Cell Adhesion Molecules; D002457:Cell Extracts; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1; D016685:Mitomycin; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases