Cytosolic calcium changes in endothelial cells induced by a protein product of human gliomas containing vascular permeability factor activity

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✓ A vascular permeability factor (VPF) derived from serum-free conditioned medium of cultured human malignant gliomas (HG-VPF) has been described previously. The rapid kinetics of HG-VPF activity in an in vivo assay of vascular permeability suggests a direct action upon the vascular endothelial cell. To determine whether HG-VPF was capable of inducing a physiologically significant alteration in isolated endothelial cells, cytosolic calcium [Ca++]i was measured in vitro in these cells before and after their exposure to media containing this substance. This was accomplished by preloading cultured endothelial cells with a fluorescent intracellular Ca++ probe fura-2/AM.

It was found that HG-VPF induced a rapid and transient elevation of [Ca++]i in normal endothelial cells derived from human umbilical vein, bovine adrenal medulla, bovine pulmonary artery, and rat brain. This effect was inhibited by chelating extracellular calcium [Ca++]e with ethyleneglycol-bis (β-aminoethylether)-N,N″ -tetra-acetic acid (EGTA), indicating that the HG-VPF-induced response resulted from the influx of extracellular calcium. The addition of cations that act as nonspecific calcium channel blockers (Li+, Co++, Mn++, La+++) completely inhibited VPF activity, further supporting the role of [Ca++]e influx. The HG-VPF activity was not, however, blocked by verapamil, a calcium antagonist that appears to be specific for voltage-gated calcium channels. Furthermore, exposure of endothelial cells to 120 mM [K+]e did not result in a calcium transient. Coincubation of endothelial cells with dexamethasone inhibited HG-VPF-induced rises in [Ca++]i, while having no effect upon cyclic nucleotide-induced changes in calcium.

The present studies indicate that vascular extravasation induced by human glioma-derived VPF may be mediated by a direct action upon vascular endothelial cells. Furthermore, the observed dexamethasone-induced inhibition of this process suggests a specific cellular action for corticosteroids. This, together with previous observations that dexamethasone suppresses both the production of VPF by tumor cells in vitro and its permeability-inducing activity in vivo, may explain the efficacy of glucocorticoids in the treatment of neoplastic vasogenic brain edema.

Finally, studies with a polycationic peptide (protamine) known to induce blood-brain barrier disruption in vivo revealed similar effects upon endothelial cytosolic calcium levels. As HG-VPF is a positively charged macromolecule, a common interaction between these substances and the negatively charged endothelial cell surface in the induction of permeability is suggested. Nonspecific cross-linking of charged groups of the endothelial glycocalyx and specific HG-VPF receptor binding are both valid mechanisms of HG-VPF-mediated calcium changes. Their potential relevance in the setting of microvascular permeability is discussed.

Article Information

Address reprint requests to: Gregory R. Criscuolo, M.D., Attn. Mrs. Lynn Schofield, National Institutes of Health, Surgical Neurology Branch — NINDS, 9000 Rockville Pike, Building 10, Room 5D-37, Bethesda, Maryland 20892.

© AANS, except where prohibited by US copyright law.

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Figures

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    Induction of cytosolic calcium transients in various endothelial cell lines by partially purified human glial tumor-derived vascular permeability factor (HG-VPF). The relative degree of intracellular calcium change induced by a standard HG-VPF stimulus is indicated in parentheses. All graphic data were obtained by computerized tracing of original hard-copy data after reassignment of y-axis values by conversion to absolute Ca++ concentrations. BAMEC = bovine adrenal medullary endothelial cells; BPAEC = bovine pulmonary artery endothelial cells; RBMVEC = rat brain microvessel endothelial cells; HUVEC = human umbilical vein endothelial cells.

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    Left: Induction of cytosolic calcium ion transients by protamine. Right: Inhibition of polycation-induced calcium ion flux in endothelial cells by the nonspecific calcium channel blockers Co++ and Mn++.

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    Dose-response relationship of cytosolic calcium transients induced by human glioma-derived vascular permeability factor (VPF) in human umbilical vein endothelial cells. Fold concentration is expressed as multiples of a standard solution containing 0.25 mg/ml (1x) of partially purified lyophilized glioma-conditioned medium dissolved in Dulbecco's phosphate-buffered saline (2.5 mg/ml = 10x, 5.0 mg/ml = 20x). See Fig. 1 for origin of data.

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    Failure of verapamil to inhibit human glioma-derived vascular permeability factor (VPF)-induced calcium transients. Prior exposure of endothelial cells to 10−6 M verapamil did not diminish cytosolic calcium changes induced by VPF. Calcium transients were not induced by exposure of endothelial cells to 120 mM KCl. This suggests that VPF-induced influx of [Ca++]e occurs by non-voltage-gated calcium ion channels. See Fig. 1 for origin of data.

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    Inhibition of human glioma-derived vascular permeability factor (VPF)-induced calcium ion transients by 2 mM Li+ and Co++ cations. Similar results were obtained with 2 mM concentrations of Mn++ and La++ cations (data not shown). Nonspecific cationic calcium channel blockers appear to inhibit VPF-induced intracellular calcium transients. This suggests that influx of extracellular calcium via non-voltage-gated membranous channels may be the primary event in the VPF-elicited response. See Fig. 1 for origin of data.

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    Inhibition of human glioma-derived vascular permeability factor (VPF)-induced cytosolic calcium ion transients in endothelial cells incubated 2 hours with 10 µM dexamethasone. See Fig. 1 for origin of data.

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