Donald R. Senger

Vascular permeability factor (VPF, VEGF) in tumor biology

SummaryVascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), is a multifunctional cytokine expressed and secreted at high levels by many tumor cells of animal and human origin. As secreted by tumor cells, VPF/VEGF is a 34–42 kDa heparin-binding, dimeric, disulfide-bonded glycoprotein that acts directly on endothelial cells (EC) by way of specific receptors to activate phospholipase C and induce [Ca2+]i transients. Two high affinity VPF/VEGF receptors, both tyrosine kinases, have thus far been described. VPF/VEGF is likely to have a number of important roles in tumor biology related, but not limited to, the process of tumor angiogenesis. As a potent permeability factor, VPF/VEGF promotes extravasation of plasma fibrinogen, leading to fibrin deposition which alters the tumor extracellular matrix. This matrix promotes the ingrowth of macrophages, fibroblasts, and endothelial cells. Moreover, VPF/VEGF is a selective endothelial cell (EC) growth factorin vitro, and it presumably stimulates EC proliferationin vivo. Furthermore, VPF/VEGF has been found in animal and human tumor effusions by immunoassay and by functional assays and very likely accounts for the induction of malignant ascites. In addition to its role in tumors, VPF/VEGF has recently been found to have a role in wound healing and its expression by activated macrophages suggests that it probably also participates in certain types of chronic inflammation. VPF/VEGF is expressed in normal development and in certain normal adult organs, notably kidney, heart, adrenal gland and lung. Its functions in normal adult tissues are under investigation.

Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in breast cancer

Solid tumors must induce a vascular stroma to grow beyond a minimal size, and the intensity of the angiogenic response has been correlated with prognosis in breast cancer patients. Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), is a secreted protein that has been implicated in tumor-associated angiogenesis. Vascular permeability factor directly stimulates endothelial cell growth and also increases microvascular permeability, leading to the extravasation of plasma proteins, which alter the extracellular matrix in a manner that promotes angiogenesis. To determine whether VPF has a role in breast cancer, we used in situ hybridization to study VPF mRNA expression in normal breast tissue (13 specimens), comedo-type ductal carcinoma in situ (DCIS) (four specimens), infiltrating ductal carcinoma (12 specimens), infiltrating lobular carcinoma (two specimens), metastatic ductal carcinoma (three specimens) and metastatic lobular carcinoma (one specimen). Vascular permeability factor mRNA was expressed at a low level by normal duct epithelium but was expressed at high levels in tumor cells in all cases of comedo-type DCIS, infiltrating ductal carcinoma, and metastatic ductal carcinoma. In contrast, VPF mRNA was not expressed at high levels in infiltrating lobular carcinoma. We also used in situ hybridization to study the expression of two recently described endothelial cell surface VPF receptors, flt-1 and kdr. Vascular permeability factor receptor mRNA was strongly expressed in endothelial cells of small vessels adjacent to malignant tumor cells in DCIS, infiltrating ductal carcinoma, and metastatic ductal carcinoma. In contrast, no definite labeling for receptor mRNA was detected in infiltrating lobular carcinoma or nonmalignant breast tissue. The intense expression of VPF mRNA by breast carcinoma cells and of VPF receptor mRNA by endothelial cells of adjacent small blood vessels provides strong evidence linking VPF expression to the angiogenesis associated with comedo-type DCIS, infiltrating ductal, and metastatic ductal breast carcinoma.

Pathogenesis of tumor stroma generation: a critical role for leaky blood vessels and fibrin deposition

Tumor stroma formation results from the interaction of tumor cells and their products with the host and certain of its normal defense mechanisms, particularly the clotting and fibrinolytic systems. It is a process in which tumor cells render local venules and veins hyperpermeable with the result that fibrinogen and other proteins extravasate and clot, forming an extravascular crosslinked fibrin gel. Coagulation is mediated by an interaction between extravasated plasma clotting factors and tumor-associated and perhaps other tissue procoagulants. Parallel activation of the fibrinolytic system leads to substantial fibrin turnover, but fibrin nonetheless accumulates in amounts, variable from tumor to tumor, that are sufficient to provide a provisional stroma. This provisional stroma imposes on tumor cells a structure that persists even as tumor cells multiply and as the fibrin provisional stroma is replaced by mature connective tissue. The provisional fibrin stroma also serves to regulate the influx of macrophages, and perhaps other inflammatory cells, but at the same time, and in ways that are not fully understood, facilitates the inward migration of new blood vessels and fibroblasts, integral components of mature tumor stroma. Ascites tumors differ from solid tumors in that fibrin gel is not ordinarily deposited in body cavities and, as a result, there is no provisional stroma to impose an initial structure. Tumor stroma generation resembles the process of wound healing in many respects. However, it differs in the mechanism of its initiation, and in the apparent lack of a role for platelets. It also differs fundamentally in that invading tumor cells continually render new vessels hyperpermeable to plasma, thus perpetuating the cycle of extravascular fibrin deposition. In this sense, tumors behave as wounds that do not heal. Largely neglected in this review has been discussion of the numerous cytokines, mitogens, and growth factors that are widely believed to play important roles in tumor angiogenesis and wound healing; i.e., PDGF, FGF, EGF, TGF alpha, TGF beta, TNF, interferons, etc. This omission has been intentional, and for two reasons. First, these cytokines have already received considerable attention [100,123-128]. Second, it is not yet clear how closely the actions of these molecules, as described in vitro, relate to their functions in vivo. At present we are deluged with a surfeit of factors that have the capacity to induce new blood vessel formation in angiogenesis assays; these factors include not only peptides but lipids and even ions [126,129-131].(ABSTRACT TRUNCATED AT 400 WORDS)

Vascular Permeability Factor/Vascular Endothelial Growth Factor: An Important Mediator of Angiogenesis in Malignancy and Inflammation

Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), is a multifunctional cytokine that is overexpressed in many transplantable animal and autochtonous human cancers, in healing wounds, and in chronic inflammatory disorders such as psoriasis and rheumatoid arthritis. All of these entities are characterized by angiogenesis, altered extracellular matrix, and variable degrees of hypoxia. In addition, two VPF/VEGF receptors, flt-1 and kdr, are overexpressed by endothelial cells that line the microvessels that supply these tumors/inflammatory reactions. On the basis of these and other data, we have proposed a model of angiogenesis in which VPF/VEGF plays a central role: this model is applicable to tumors and also to the angiogenesis that occurs in non-neoplastic processes.

Vascular Permeability Factor, Fibrin, and the Pathogenesis of Tumor Stroma Formationa

An association between malignancy and abnormal hemostasis has been appreciated for more than a hundred years.1.2 A broad spectrum of clinically significant hemostatic abnormalities (eg., migratory thrombophlebitis, hemorrhage, disseminated intravascular coagulation) afflicts as many as 15% of cancer patients, and hemostatic complications are the second most common cause of mortality in cancer. Only recently, however, has it been recognized that, in addition to a proclivity to systemic coagulation, cancer patients activate the clotting system locally and extravasdarly, depositing fibrin gels in solid tumors that serve as a provisional stroma and that have a role in the induction of mature tumor stroma. The Irish pathologist, R.A.Q. OMeara, was the first to propose in the late 1950s that fibrin was deposited in solid tumors; however, his work was inconclusive because the techniques then available fbr identifylng fibrin in tissues were primitive and relatively nonspecific. The first solid evidence that fibrinogen-related proteins (FRP) were localized in solid tumors came fiom studies which showed that systemically administered fibrinogen, and also antifibrinogen antibodies, were selectively concentrated in animal and human tumors.2 Thereafter, seved groups demonstrated FRP in rat and human neoplasms by immunofluorescence. For some time there was considerable reluctance to accept even these findings. Valid questions were raised as to the specificity of the antibodies used, the generality of the observations, and the possibility ofartifict, arising from accumulation of FRP either postmortem or as tissues were being removed surg~cally. More recent work has W l y laid these doubts to rest.2 Moreover, immunohistochemical, electron microscopic, and, in some instances, biochemical studies

Purification of a human milk protein closely similar to tumor-secreted phosphoproteins and osteopontin

A wide variety of rodent and human tumor cells secrete antigenically related phosphoproteins with molecular weights (Mr) of approximately 58,000 (hamster), 62,000 (rat, mouse), 67,000 (human) (Senger, D.R. and Perruzzi, C.A. (1985) Cancer Res. 45, 5818-5823). Expression of these phosphoproteins is transformation-related; tumor cells produce at least 10-fold or more of this protein as compared to their normal or untransformed counterparts. N-terminal and internal sequences derived from the rat tumor-secreted phosphoprotein indicate that it is identical to rat osteopontin, a bone protein with an Arg-Gly-Asp cell-binding sequence (Oldberg, A., Franzen, A. and Heinegard, D. (1986) Proc. Natl. Acad. Sci. USA 83, 8819-8823). Antibody raised to the Mr 62,000 rat tumor-secreted phosphoprotein was found to bind Mr 75,000 and Mr 35,000 components of human milk, indicating that milk contains antigenically related proteins. The Mr 75,000 protein, which is present in human milk at concentrations ranging from 3 to 10 micrograms/ml, has been purified to homogeneity. The Mr 35,000 component is apparently derived from the Mr 75,000 protein by proteolytic cleavage, and this cleavage also occurs in vitro in the presence of thrombin. N-terminal and internal amino acid sequences were derived from the Mr 75,000 milk protein and found to be similar (12/21 residues) to N-terminal and internal sequences derived from the rat tumor-secreted phosphoprotein and osteopontin. Moreover, sequence derived from the N-terminus of the human milk protein is identical to that of human bone sialoprotein I (the likely human homolog of rat osteopontin) (Fisher, L.W., Hawkins, G.R., Tuross, N. and Termine, J.D. (1987) J. Biol. Chem. 262, 9702-9708).

CELL MIGRATION PROMOTED BY A POTENT GRGDS-CONTAINING THROMBIN-CLEAVAGE FRAGMENT OF OSTEOPONTIN

Osteopontin (OPN) is a secreted adhesive glycoprotein with a gly-arg-gly-asp-ser (GRGDS) cell binding domain. Several independent studies have suggested that OPN functions in tumor growth and metastasis, and one likely possibility is that OPN facilitates tumor invasion by promoting tumor cell migration. Consistent with this hypothesis, immobilized OPN promoted concentration-dependent tumor cell migration (i.e., haptotaxis) in modified Boyden chambers. In particular, cleavage of OPN by thrombin, which likely occurs in the tumor microenvironment, resulted in enhancement of OPNs haptotactic activity; and assays performed with purified preparations of the two individual OPN thrombin-cleavage fragments demonstrated that all detectable activity was associated with the GRGDS-containing fragment. In contrast to the activity of both OPN and its GRGDS-containing fragment in promoting haptotaxis, neither of these proteins in solution promoted chemotaxis, indicating that each must be immobilized to promote cell migration. In haptotaxis assays, antibody LM609 to integrin alpha v beta 3 blocked > 80% cell migration towards the GRGDS-containing OPN fragment, implicating alpha v beta 3 as its principal functional receptor. In comparison with equimolar quantities of other adhesive proteins, the GRGDS-containing OPN thrombin-cleavage fragment was not only > 2-fold more effective than intact OPN at promoting haptotaxis, but also > 8-fold and > 6-fold more effective than fibrinogen and vitronectin, respectively, indicating that this OPN fragment is highly active relative to other alpha v beta 3 ligands.

Ultrastructural localization of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) to the abluminal plasma membrane and vesiculovacuolar organelles of tumor microvascular endothelium.

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a cytokine secreted by many animal and human tumors, activated macrophages, keratinocytes, rheumatoid synovial cells, embryonic tissues, and by cultured epithelial and mesenchymal cell lines. It acts selectively on vascular endothelial cells to increase their permeability to circulating macromolecules and to stimulate their replication. Although not detectably expressed by vascular cells in the human and animal tumors we have studied, VPF/VEGF accumulates in the microvessels supplying tumors and certain inflammatory reactions in which VPF/VEGF is also overexpressed. Light microscopic immunohistochemistry lacked the resolution necessary to localize VPF/VEGF precisely in such vessels. Therefore, we used a pre-embedding immunocytochemical method to localize VPF/VEGF at the ultrastructural level in the new blood vessels that are elicited in the peritoneal walls of mice bearing a transplantable mouse ascites tumor of ovarian origin. Intense immunostaining for VPF/VEGF was observed on the abluminal plasma membrane of tumor-associated microvascular endothelial cells and in vesiculovacuolar organelles (VVOs) present in these same endothelial cells. (VVOs are recently described cytoplasmic organelles present in tumor vascular endothelium that provide an important pathway for extravasation of circulating macromolecules.) In contrast to labeling of the abluminal plasma membrane and VVO vesicles and vacuoles, endothelial cytoplasmic organelles, such as multivesicular bodies and Weibel-Palade bodies, and the underlying basal lamina, did not stain with antibodies to VPF/VEGF. The distribution of VPF/VEGF here described corresponds to that anticipated for high-affinity VFP/VEGF receptors, although binding of VPF/VEGF to other endothelial cell surface structures, such as plasma membrane proteoglycans, is also a possibility.

Structural requirements for dimerization, glycosylation, secretion, and biological function of VPF/VEGF

Vascular permeability factor (VPF) also known as vascular endothelial growth factor (VEGF), is a dimeric protein that affects endothelial cell (EC) and vascular functions including enhancement of microvascular permeability and stimulation of EC growth. To investigate the structural features of VPF/VEGF necessary for efficient dimerization, secretion, and biological activities, we employed site-directed mutagenesis with a Cos-1 cell expression system. Several cysteine residues essential for VPF dimerization were identified by mutation analysis of the Cys-25, Cys-56, and Cys-67 residues. Mutant VPF isoforms lacking either of these cysteines were secreted as monomers and were completely inactive in both vascular permeability and endothelial cell mitotic assays. VPF Cys-145 mutant protein was efficiently secreted as a glycosylated, dimeric polypeptide, but had a reduction in biological activities. The site of N-linked glycosylation was directly identified as Asn-74, which, when mutated produced an inefficiently secreted dimeric protein without post-translational glycosylation, yet maintained full vascular permeability activity. Finally, we found that one VPF mutant isoform Cys-101 was not secreted and this mutant functioned as a dominant-negative suppressor of wild-type VPF secretion as demonstrated by co-expression assays in Cos-1 cells.

Glycosylation is essential for efficient secretion but not for permeability-enhancing activity of vascular permeability factor (vascular endothelial growth factor)

The hyperpermeability of the microvasculature supplying solid tumors is largely attributable to a heterodimeric Mr 34,000-43,000 tumor-secreted protein, vascular permeability factor. Upon reduction, the vascular permeability factor secreted by line 10 tumor cells is resolved by SDS-PAGE into 3 discrete bands of Mr 24,000, 19,500, and 15,000. We demonstrate here that line 10 vascular permeability factor is an N-linked glycoprotein. Nonglycosylated vascular permeability factor migrates on reduced SDS-PAGE as two bands of Mr 20,000 and 15,000. Pulse-chase studies demonstrated that all three chains of native vascular permeability factor were secreted rapidly following synthesis and at equal rates, with a cellular half-retention time of approximately 37 min. When glycosylation was prevented by tunicamycin, individual bands of nonglycosylated vascular permeability factor were also secreted at equivalent rates, but much more slowly (approximately 60 min) than native glycoprotein. Both glycosylated and nonglycosylated forms of vascular permeability factor were equally potent at increasing dermal vessel permeability.