Heparin-binding protein targeted to mitochondrial compartments protects endothelial cells from apoptosis
A. Maria Olofsson, … , Werner Müller-Esterl, Evy Lundgren-Åkerlund
A. Maria Olofsson, … , Werner Müller-Esterl, Evy Lundgren-Åkerlund
Published October 1, 1999
Citation Information: J Clin Invest. 1999;104(7):885-894. https://doi.org/10.1172/JCI6671.
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Article

Heparin-binding protein targeted to mitochondrial compartments protects endothelial cells from apoptosis

Abstract

Neutrophil-borne heparin-binding protein (HBP) is a multifunctional protein involved in the progression of inflammation. HBP is stored in neutrophil granules and released upon stimulation of the cells in proximity to endothelial cells. HBP affects endothelial cells in multiple ways; however, the molecular and cellular mechanisms underlying the interaction of HBP with these cells are unknown. Affinity isolation and enzymatic degradation demonstrated that HBP released from human neutrophils binds to endothelial cell-surface proteoglycans, such as syndecans and glypican. Flow cytometry indicated that a significant fraction of proteoglycan-bound HBP is taken up by the endothelial cells, and we used radiolabeled HBP to determine the internalization rate of surface-bound HBP. Confocal and electron microscopy revealed that internalized HBP is targeted to perinuclear compartments of endothelial cells, where it colocalizes with mitochondria. Western blotting of isolated mitochondria from HBP-treated endothelial cells showed that HBP is present in 2 forms — 28 and 22 kDa. Internalized HBP markedly reduced growth factor deprivation–induced caspase-3 activation and protected endothelial cells from apoptosis, suggesting that uptake and intracellular routing of exogenous HBP to mitochondria contributes to the sustained viability of endothelial cells in the context of locally activated neutrophils.

Authors

A. Maria Olofsson, Mikael Vestberg, Heiko Herwald, Jørgen Rygaard, Guido David, Karl-E. Arfors, Viggo Linde, Hans Flodgaard, Jürgen Dedio, Werner Müller-Esterl, Evy Lundgren-Åkerlund

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Figure 2

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Internalization of HBP by HUVECs. (a) HUVECs were incubated with 50 μg/m...
Internalization of HBP by HUVECs. (a) HUVECs were incubated with 50 μg/mL of HBP for the indicated periods of time. The cells were washed, fixed in 1% formaldehyde, permeabilized, and stained with mAb 2F23C3 to human HBP, followed by an FITC-labeled anti-mouse Ig (from goat); they were then subjected to flow cytometry. Negative controls were stained excluding the primary antibody. Background staining was subtracted. The data represent mean fluorescence intensity normalized for HBP binding at 24 hours and are given as means ± SD. (b) HUVECs were incubated with 0.65 nM of 125I-labeled HBP at 37°C. At the indicated times, the radioactivity present on the cell surface (squares) or in the interior of the cell (diamonds) was quantified. Data represent means ± SD of 3 independent experiments, each done in duplicate. (c) HUVECs were incubated with 50 μg/mL HBP for 30 minutes at 37°C, washed, fixed, permeabilized, incubated with mAb 2F23C3 and FITC–anti-mouse Ig, and analyzed by flow cytometry (top bar). To test for specificity, cells were incubated with buffer alone (Control) at 4°C or with a mixture of 50 μg/mL HBP and 100 μg/mL heparin. Alternatively, cells were incubated for 60 minutes with 50 mM NH4Cl, 1 nM cycloheximide, or 1 μM cytochalasin D before adding 50 μg/mL HBP. Data represent mean fluorescence intensity normalized for HBP binding at 37°C (=100%) and are given as means ± SD (n = 5).