1, January 1991 Copyright © American Association of Pathologists
American Journal of Pathology, Vol. 138, No.
Tumor-secreted Vascular Permeability Factor Increases Cytosolic Ca2+ and von Willebrand Factor Release in Human Endothelial Cells
Tommy A. Brock,* Harold F. Dvorak,t and Donald R. Sengert From the Hypertension Program,* Division of Cardiovascular Diseases, Department of Medicine, University ofAlabama at Birmingham, Birmingham, Alabama; and the Departments of Pathology,j
Beth Israel Hospital and Harvard Medical School, and the Charles A. Dana Research Institute, Beth Israel Hospital, Boston, Massachusetts
Vascular permeability factor (VPF), a tumor-secreted heparin-binding protein (Mr 38, 000), is responsible for increased vessel permeability and fluid accumulation associated with tumor growth. Vascular permeability factor also promotes the growth of human umbilical vein endothelial cells (EC) and bovine pulmonary ECs in vitro. It is shown for the first time that guinea pig VPF (half-
maximal and maximal dose 0.4 and 22 pmol/l (picomolar), respectively), as well as human VPF, are potent stimulifor human ECs resulting in [Ca2+Ji increases (maximal three- to fourfold) and inositol trisphosphate (IP3) formation. Unlike the maximal responses to thrombin and histamine, the [Ca2"J1 response to a maximal VPF dose was preceded by a characteristic 10- to 15-second delay. Guinea pig VPF also selectively increased [Ca2+Ji in cultured aortic and pulmonary artery ECs, but not aortic smooth muscle cells, human fibroblasts, or neutrophils. Affinity-purified rabbit antibody (raised to a synthetic peptide representing VPF N-terminal amino acids 1 to 24) adsorbed all vessel permeability-increasing activity, EC growth-promoting activity, and specifically all activity responsible for increasing EC [Ca2+]j. Similar to other mediators that increase [Ca2+Ji in cultured ECs, VPF also induced a 200% increase in von Willebrand factor release. Together these data indicate that VPF acts directly on ECs and that rapid cellular events in its in vivo/in vitro actions are likely to involve phospholipase C activation, [Ca2+J1 increase, and von
Willebrandfactor release. (AmjPathol 1991, 138: 213-221)
A variety of human and rodent tumor cell lines, including carcinomas,'12 sarcomas, 23glioblastomas,3 and a monocytic leukemia,4 secrete a novel, potent vascular permeability factor (VPF). Vascular permeabililty factor is detectable readily in tumor ascites fluids but not in normal serum or plasma.1 Experiments using a neutralizing antibody against VPF in tumor-bearing animals have shown that VPF is responsible largely for the increased vessel permeability and fluid accumulation associated with tumor growth.' Recently a pituitary-derived protein (called vascular endothelial growth factor or folliculostellate-derived growth factor), which is very similar or identical to VPF, was purified.5'6 This protein is a growth factor that is specific for vascular endothelial cells (EC). Consistent with the mitogenic effects of this pituitary-derived protein, tumor VPF also stimulates EC proliferation,47 thus raising the possibility that VPF contributes to tumor-elicited angiogenesis. Structural studies on highly purified VPF, as well as studies involving metabolic labeling and antibody precipitation, have indicated that VPF consists of two disulfidebonded polypeptides with a heterogeneous aggregate Mr ranging from 34,000 to 42,000.8 Although the individual disulfide-bonded VPF polypeptides appear to have identical N-termini, tumor cells synthesize several structurally different species,8 the functional significance of which remain to be determined. Recently several similar but distinct VPF cDNA clones were isolated and sequenced, suggesting that VPF mRNA may be alternatively spliced.9'0 Interestingly the reported VPF cDNA sequences show a low but significant homology with the A and B chains of platelet-derived growth factor (PDGF), 15% and 18%, respectively, but not with acidic or basic fibroblast growth factor, transforming growth factor-a, epidermal growth factor, or platelet-derived EC growth factor.9'10 Supported by grants HL 41180 from the National Heart, Lung and Blood Institute, a Grant-in-Aid from the American Heart Association, Alabama affiliate (to Tommy A. Brock), and CA 43967 (to Donald R. Senger) and CA 40624 (to Harold F. Dvorak) from the National Cancer Institute. Accepted for publication September 6, 1990. Address reprint requests to Tommy A. Brock, PhD, Hypertension Program, 1046 Zeigler Building, University of Alabama at Birmingham, Birmingham, AL 35294.
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AJPJanuary
1991, Vol. 138, No. 1
Growth factor and hormone receptor stimulation of many cell types are associated with a phosphoinositidespecific phospholipase C (PLC) activation and cytosolic free Ca2+ ([Ca2+]i) increase.1" Classical agonists that increase vascular permeability, such as histamine and bradykinin, also influence many biologically relevant parameters in ECs by inducing rapid [Ca2+]j changes,12 eg, cytoskeletal rearrangement,"4 von Willebrand factor (vWF) release,13 PGI2 formation, and endothelial-derived relaxing factor release.12 Vascular permeability factor, like bradykinin and histamine, increases vessel permeability within minutes.1 Other proinflammatory mediators that act directly on ECs, such as interleukin 1 or tumor necrosis factor, induce their effects within hours15 and do not stimulate rapid [Ca2+]j increases (T. Brock, unpublished observations). In the current study, we used an antibody that was raised to a synthetic peptide corresponding to VPF Nterminal amino acids 1 to 248 to demonstrate unequivocally that VPF-stimulated increases in [Ca2+]i and phospholipase C activity are quite different from those of thrombin and histamine in human umbilical vein ECs. The findings reported here also suggest that tumor-secreted VPF is a potent stimulus for vWF release. Portions of these data have been reported in abstract form."6
Materials and Methods Cell Culture Primary cultures of human ECs (HEC) were established from pooled human umbilical veins and were propagated (passages 2 to 4) as described previously.17 Medium 199 was supplemented with 20% fetal bovine serum (FBS), 100 ug/ml porcine heparin, 50 ,ug/ml endothelial cell growth factor, and 100 ,ug/ml each of penicillin and streptomycin. The cell culture conditions for other cell types were 10% calf serum /Dulbecco's Modified Eagle's medium (DMEM)-bovine aortic ECs and rat aortic smooth muscle cells; 10% FBS/medium 199-porcine and bovine pulmonary artery ECs; and 10% FBS/DMEM-human dermal and guinea pig lung fibroblasts.
tibody (Ab) raised to a synthetic peptide corresponding to VPF N-terminal amino acids 1 to 24.8 Antibody was affinity purified using this peptide coupled to Sepharose, followed by elution with glycine (0.1 mol/l [molar], pH = 3.0). This Ab was shown previously to adsorb specifically both radiolabeled VPF and vessel permeability-increasing activity, as well as to stain VPF on Western blots.8 Normal rabbit IgG and an affinity-purified Ab raised to an unrelated 20-amino acid synthetic peptide served as controls. For immunoadsorption studies, antibodies first were bound to protein A Sepharose beads. Neither the control anti-peptide Ab nor the control IgGs adsorbed any of the VPF-associated activities.
Measurement of Ca2+-sensitive Fura-2 Fluorescence Fura-2, a Ca2+-sensitive fluorescent dye,18 was used to monitor changes in [Ca2+]i in EC suspensions as described by Brock and Capasso.17 [Ca2]j was calculated using the formula [Ca2+]i = Kd(R-Rmnn)/(Rma,-R)* (EGTA3w/ DIG3w), where R was the fluorescence ratio within the cell, Kd was 224 nmol/l (nanomolar),'5 EGTA3BQ was the fluorescence signal (380 nmol/l) in the presence of 10 mmol/ (millimolar) EGTA (pH = 10), and DIG3w was the fluorescence signal following digitonin addition in the presence of 1.5 mmol/l CaCI2. Fluorescence tracings depicting [Ca2+]i recordings are typical tracings and are representative of multiple experiments. Data are summarized as mean ± standard error of the mean and analyzed using an unpaired Student's t-test.
Inositol Phosphate Separation Human endothelial cells grown in 35-mm culture dishes were incubated with [3H]myoinositol (155,Ci/ml, 48 hours) in Medium 199 supplemented with 20% fetal calf serum. Inositol phosphates were separated as previously described.17 Data are summarized as mean ± standard error of the mean.
VPF Isolation and Specific Immunoadsorption
von Willebrand Secretion
Experiments were performed with highly purified VPF that was prepared from guinea pig line 10 tumor cells as previously described.8 In addition, certain experiments were performed with human VPF purified from a human tumor cell line, MNNG-HOS.2 To demonstrate unequivocally that the observed biologic activities were associated specifically with VPF, experiments were performed using an an-
Human endothelial cell cultures were washed three times with Hanks' balanced salt solution buffered with 10 mmol/ HEPES (pH = 7.4) and then incubated in 1 ml of the same solution for 15 minutes in the absence of agonist. Next buffer containing agonist was added and the supernatants were collected after 15 minutes. von Willebrand factor was quantitated with a 'sandwich' enzyme-linked
Cytosolic Ca2+ and vWF in VPF-stimulated HEC
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AJPJanuary 1991, Vol. 138, No. 1
level, n = 11) reached a maximum in response to 22 pmol/l VPF. Peak [Ca2+]i increases in response to 22 pmol/ VPF were as large as those due to 30 ,umol/l histamine but smaller than that seen in response to 3 U/ml thrombin."7 Even at maximal VPF doses (.22 pmol/l), there was a significant delay (18 ± 1.4 seconds, n = 11; P
American Journal of Pathology, Vol. 138, No.
Tumor-secreted Vascular Permeability Factor Increases Cytosolic Ca2+ and von Willebrand Factor Release in Human Endothelial Cells
Tommy A. Brock,* Harold F. Dvorak,t and Donald R. Sengert From the Hypertension Program,* Division of Cardiovascular Diseases, Department of Medicine, University ofAlabama at Birmingham, Birmingham, Alabama; and the Departments of Pathology,j
Beth Israel Hospital and Harvard Medical School, and the Charles A. Dana Research Institute, Beth Israel Hospital, Boston, Massachusetts
Vascular permeability factor (VPF), a tumor-secreted heparin-binding protein (Mr 38, 000), is responsible for increased vessel permeability and fluid accumulation associated with tumor growth. Vascular permeability factor also promotes the growth of human umbilical vein endothelial cells (EC) and bovine pulmonary ECs in vitro. It is shown for the first time that guinea pig VPF (half-
maximal and maximal dose 0.4 and 22 pmol/l (picomolar), respectively), as well as human VPF, are potent stimulifor human ECs resulting in [Ca2+Ji increases (maximal three- to fourfold) and inositol trisphosphate (IP3) formation. Unlike the maximal responses to thrombin and histamine, the [Ca2"J1 response to a maximal VPF dose was preceded by a characteristic 10- to 15-second delay. Guinea pig VPF also selectively increased [Ca2+Ji in cultured aortic and pulmonary artery ECs, but not aortic smooth muscle cells, human fibroblasts, or neutrophils. Affinity-purified rabbit antibody (raised to a synthetic peptide representing VPF N-terminal amino acids 1 to 24) adsorbed all vessel permeability-increasing activity, EC growth-promoting activity, and specifically all activity responsible for increasing EC [Ca2+]j. Similar to other mediators that increase [Ca2+Ji in cultured ECs, VPF also induced a 200% increase in von Willebrand factor release. Together these data indicate that VPF acts directly on ECs and that rapid cellular events in its in vivo/in vitro actions are likely to involve phospholipase C activation, [Ca2+J1 increase, and von
Willebrandfactor release. (AmjPathol 1991, 138: 213-221)
A variety of human and rodent tumor cell lines, including carcinomas,'12 sarcomas, 23glioblastomas,3 and a monocytic leukemia,4 secrete a novel, potent vascular permeability factor (VPF). Vascular permeabililty factor is detectable readily in tumor ascites fluids but not in normal serum or plasma.1 Experiments using a neutralizing antibody against VPF in tumor-bearing animals have shown that VPF is responsible largely for the increased vessel permeability and fluid accumulation associated with tumor growth.' Recently a pituitary-derived protein (called vascular endothelial growth factor or folliculostellate-derived growth factor), which is very similar or identical to VPF, was purified.5'6 This protein is a growth factor that is specific for vascular endothelial cells (EC). Consistent with the mitogenic effects of this pituitary-derived protein, tumor VPF also stimulates EC proliferation,47 thus raising the possibility that VPF contributes to tumor-elicited angiogenesis. Structural studies on highly purified VPF, as well as studies involving metabolic labeling and antibody precipitation, have indicated that VPF consists of two disulfidebonded polypeptides with a heterogeneous aggregate Mr ranging from 34,000 to 42,000.8 Although the individual disulfide-bonded VPF polypeptides appear to have identical N-termini, tumor cells synthesize several structurally different species,8 the functional significance of which remain to be determined. Recently several similar but distinct VPF cDNA clones were isolated and sequenced, suggesting that VPF mRNA may be alternatively spliced.9'0 Interestingly the reported VPF cDNA sequences show a low but significant homology with the A and B chains of platelet-derived growth factor (PDGF), 15% and 18%, respectively, but not with acidic or basic fibroblast growth factor, transforming growth factor-a, epidermal growth factor, or platelet-derived EC growth factor.9'10 Supported by grants HL 41180 from the National Heart, Lung and Blood Institute, a Grant-in-Aid from the American Heart Association, Alabama affiliate (to Tommy A. Brock), and CA 43967 (to Donald R. Senger) and CA 40624 (to Harold F. Dvorak) from the National Cancer Institute. Accepted for publication September 6, 1990. Address reprint requests to Tommy A. Brock, PhD, Hypertension Program, 1046 Zeigler Building, University of Alabama at Birmingham, Birmingham, AL 35294.
213
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Brock, Dvorak, and Senger
AJPJanuary
1991, Vol. 138, No. 1
Growth factor and hormone receptor stimulation of many cell types are associated with a phosphoinositidespecific phospholipase C (PLC) activation and cytosolic free Ca2+ ([Ca2+]i) increase.1" Classical agonists that increase vascular permeability, such as histamine and bradykinin, also influence many biologically relevant parameters in ECs by inducing rapid [Ca2+]j changes,12 eg, cytoskeletal rearrangement,"4 von Willebrand factor (vWF) release,13 PGI2 formation, and endothelial-derived relaxing factor release.12 Vascular permeability factor, like bradykinin and histamine, increases vessel permeability within minutes.1 Other proinflammatory mediators that act directly on ECs, such as interleukin 1 or tumor necrosis factor, induce their effects within hours15 and do not stimulate rapid [Ca2+]j increases (T. Brock, unpublished observations). In the current study, we used an antibody that was raised to a synthetic peptide corresponding to VPF Nterminal amino acids 1 to 248 to demonstrate unequivocally that VPF-stimulated increases in [Ca2+]i and phospholipase C activity are quite different from those of thrombin and histamine in human umbilical vein ECs. The findings reported here also suggest that tumor-secreted VPF is a potent stimulus for vWF release. Portions of these data have been reported in abstract form."6
Materials and Methods Cell Culture Primary cultures of human ECs (HEC) were established from pooled human umbilical veins and were propagated (passages 2 to 4) as described previously.17 Medium 199 was supplemented with 20% fetal bovine serum (FBS), 100 ug/ml porcine heparin, 50 ,ug/ml endothelial cell growth factor, and 100 ,ug/ml each of penicillin and streptomycin. The cell culture conditions for other cell types were 10% calf serum /Dulbecco's Modified Eagle's medium (DMEM)-bovine aortic ECs and rat aortic smooth muscle cells; 10% FBS/medium 199-porcine and bovine pulmonary artery ECs; and 10% FBS/DMEM-human dermal and guinea pig lung fibroblasts.
tibody (Ab) raised to a synthetic peptide corresponding to VPF N-terminal amino acids 1 to 24.8 Antibody was affinity purified using this peptide coupled to Sepharose, followed by elution with glycine (0.1 mol/l [molar], pH = 3.0). This Ab was shown previously to adsorb specifically both radiolabeled VPF and vessel permeability-increasing activity, as well as to stain VPF on Western blots.8 Normal rabbit IgG and an affinity-purified Ab raised to an unrelated 20-amino acid synthetic peptide served as controls. For immunoadsorption studies, antibodies first were bound to protein A Sepharose beads. Neither the control anti-peptide Ab nor the control IgGs adsorbed any of the VPF-associated activities.
Measurement of Ca2+-sensitive Fura-2 Fluorescence Fura-2, a Ca2+-sensitive fluorescent dye,18 was used to monitor changes in [Ca2+]i in EC suspensions as described by Brock and Capasso.17 [Ca2]j was calculated using the formula [Ca2+]i = Kd(R-Rmnn)/(Rma,-R)* (EGTA3w/ DIG3w), where R was the fluorescence ratio within the cell, Kd was 224 nmol/l (nanomolar),'5 EGTA3BQ was the fluorescence signal (380 nmol/l) in the presence of 10 mmol/ (millimolar) EGTA (pH = 10), and DIG3w was the fluorescence signal following digitonin addition in the presence of 1.5 mmol/l CaCI2. Fluorescence tracings depicting [Ca2+]i recordings are typical tracings and are representative of multiple experiments. Data are summarized as mean ± standard error of the mean and analyzed using an unpaired Student's t-test.
Inositol Phosphate Separation Human endothelial cells grown in 35-mm culture dishes were incubated with [3H]myoinositol (155,Ci/ml, 48 hours) in Medium 199 supplemented with 20% fetal calf serum. Inositol phosphates were separated as previously described.17 Data are summarized as mean ± standard error of the mean.
VPF Isolation and Specific Immunoadsorption
von Willebrand Secretion
Experiments were performed with highly purified VPF that was prepared from guinea pig line 10 tumor cells as previously described.8 In addition, certain experiments were performed with human VPF purified from a human tumor cell line, MNNG-HOS.2 To demonstrate unequivocally that the observed biologic activities were associated specifically with VPF, experiments were performed using an an-
Human endothelial cell cultures were washed three times with Hanks' balanced salt solution buffered with 10 mmol/ HEPES (pH = 7.4) and then incubated in 1 ml of the same solution for 15 minutes in the absence of agonist. Next buffer containing agonist was added and the supernatants were collected after 15 minutes. von Willebrand factor was quantitated with a 'sandwich' enzyme-linked
Cytosolic Ca2+ and vWF in VPF-stimulated HEC
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AJPJanuary 1991, Vol. 138, No. 1
level, n = 11) reached a maximum in response to 22 pmol/l VPF. Peak [Ca2+]i increases in response to 22 pmol/ VPF were as large as those due to 30 ,umol/l histamine but smaller than that seen in response to 3 U/ml thrombin."7 Even at maximal VPF doses (.22 pmol/l), there was a significant delay (18 ± 1.4 seconds, n = 11; P
