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 1

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Release and uptake of HBP. (a) Release of HBP from activated human neutr...
Release and uptake of HBP. (a) Release of HBP from activated human neutrophils was followed after stimulating isolated PMNs with the indicated concentrations of PMA in the absence (gray bars) or presence (open bars) of HUVECs (ECs) cultured in 48-well plates. After removal of the cell suspension from the plate and centrifugation, the concentration of HBP in the cell-free supernatant was determined by ELISA. Experiments were done in triplicate; means ± SD are presented. (b) 35S-labeled HBP binding sites were isolated from whole lysates of HUVECs by affinity chromatography on HBP-streptavidin agarose. Fractions eluted at 300, 400, or 500 mM NaCl were incubated in the presence (+) or absence (–) of CABC (top) or HNO2 (bottom). Cleavage products were separated on agarose gels and viewed by phosphoimaging. (c) Affinity-purified HBP binding sites eluted at 300 mM NaCl (left) or whole HUVEC lysates (right) were incubated in the absence (–) or presence (+) of CABC and heparitinase. The cleavage products were separated by SDS-PAGE, electrotransferred to Zeta-Probe membranes, and probed by mAb 3G10. The relative positions of known endothelial proteoglycans of the heparan sulfate type are indicated by stars (from top to bottom): perlecan (>200 kDa), syndecan-3 (125 kDa), syndecan-1 (90 kDa), glypican (64 kDa), syndecan-2 (48 kDa), and syndecan-4 (35 kDa). The relative molecular masses of marker proteins are given on the left.