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 μm) 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-α) or mitomycin C. For constitutive marker CD31, both agonist-treated attached, monolayer, and detached, free endothelial cells formed caps and released microparticles. TNF-α 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-α 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-α 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-α 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.