Increased expression of vascular permeability factor (vascular ...

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of VPF and VPF receptor mRNA and the distribution of VPF protein in human renal cell carcinomas. Three transitional cell carcinomas were also examinedfor.
Amencan journal of Patbology, Vol. 143, No. 5, November 1993 Copyrfght © Amenican Society for Investigative Patbology

Short Communication Increased Expression of Vascular Permeability Factor (Vascular Endothelial Growth Factor) and Its Receptors in Kidney and Bladder Carcinomas

Lawrence F. Brown, Brygida Berse, Robert W. Jackman, Kathi Tognazzi, Eleanor J. Manseau, Harold F. Dvorak, and Donald R. Senger

Vascular permeability factor (VPF), also known as vascular endothelial growth factor, is a secretedprotein implicated in tumor-associated microvascular hyperpermeability and angiogenesis. Tumor ceUs in 11 of 12 renal cell carcinomas expressed high levels of VPF messenger RNA (mRNA) by in situ hybridization, the only exception being a case of the relatively avascular papillary variant. Expression was further accentuated adjacent to areas of necrosis. Both tumor cells and endothelial cells in smaU vessels adjacent to tumor stained stronglyfor VPF protein by immunohistochemistry. Endothelial ceUs did not express detectable VPF mRNA, but did express high levels of mRNA for the VPF receptors fit-i and KDR indicating that the endothelial ceU staining likely reflects binding of VPF secreted by adjacent tumor ceUs. Three transitional ceU carcinomas also labeled stronglyfor VPF mRNA. These data suggest an important rolefor VPF in the vascular biology of these two common human malignancies. (AmJ Pathol 1993, 143:1255-1262)

over, VPF, which is also known as vascular endothelial growth factor (VEGF), stimulates endothelial cell growth and angiogenesis.5-10 The VPF gene transcript is alternatively spliced,11 and it has been reported that the largest isoform remains cell associated, presumably due to its greater affinity for heparin-containing proteoglycans.12 VPF interacts with at least two specific tyrosine kinase receptor proteins found on endothelial cells, fit-1 and KDR.13,14 In response to VPF, free cytosolic calcium is increased in endothelial cells, probably via a phospholipase C-mediated mechanism.15 VPF is synthesized and secreted by a variety of cultured tumor cells, transplantable animal tumors, and certain primary brain tumors in man.2,5,16-24 Tumors must induce a vascular stroma to grow past a minimal size,25 and VPF may play an important role in the induction of this stroma in at least two ways: as an endothelial growth factor and/or by increasing microvascular permeability to plasma proteins, leading to alterations of the extracellular matrix including fibrin deposition. Consistent with an important role, monoclonal antibody to VPFNEGF has been shown to suppress growth and decrease the density of blood vessels in experimental tumors.26 Given this data, it is important to determine if VPF is expressed in primary human cancers, particularly in common carcinomas. In a previous study, Northern analysis of messenger (m)RNAs isolated from normal kidneys demonstrated VPF mRNA, but much higher levels of transcript were found in a renal cell carci-

Vascular permeability factor (VPF) is a potent inducer of microvascular hyperpermeability.1-3 On a molar basis, it increases microvascular permeability with a potency some 50,000 times that of histamine.4 More-

Supported by U.S. Public Health Service NIH grants CA50453 and CA58845 (to HFD) and CA43967 (to DRS). Accepted for publication July 18, 1993. Address reprint requests to Dr. Lawrence F. Brown, Department of Pathology, Beth Israel Hospital, Boston, MA 02215.

From the Departments of Pathology, Beth Israel Hospital

and Harvard Medical School, Boston, Massachusetts

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noma.24 In a study of normal kidneys, VPF message and protein were localized primarily to glomerular epithelial cells by in situ hybridization (ISH) and immunohistochemistry (IH).27 The primary goal of this study was to examine in greater detail the expression of VPF and VPF receptor mRNA and the distribution of VPF protein in human renal cell carcinomas. Three transitional cell carcinomas were also examined for VPF expression.

Materials and Methods Thirteen nephrectomy specimens and two cystectomy specimens containing primary carcinomas were examined shortly after resection. Sections of tumor and adjacent normal tissue were fixed and processed for ISH and IH. Tissues were fixed in 4% paraformaldehyde in phosphate-buffered saline, pH 7.4, for 2 to 4 hours at 4 C and were then transferred to 30% sucrose in phosphate-buffered saline overnight at 4 C, frozen in OCT compound (Miles Diagnostics, Elkhart, IN) and stored at -70 C. ISH was performed on 6-p frozen sections. Details of ISH, selection, and preparation of the anti-sense singlestranded VPF RNA probe and its sense control have been described,27 the anti-sense probe hybridizes specifically with a region of VPF mRNA common to the four known VPF splicing variants.11,28 Polymerase chain reaction primers for the VPF receptors fit-1 and KDR were derived from sequences within the kinase insert regions,29'30 adding BamHl and EcoRI sites to assist in cloning. The following primers were synthesized by David Gonzales at Beth Israel Hospital using an Applied Biosystem machine: 1) for fit-1: UP 5' CTAGGATCCGTGACTTATTTTTTCTCAACAAGG 3' and DN 5' CTCGAATTCAGATCTTCCATAGTGATGGGCTC 3'; 2) for KDR: UP 5' CGTGGATCCACCAAAGGGGCACGATTCCGTC 3' and DN 5' CTCGAATTCTGTAACAGATGAGATGCTCCAAGG 3'. Pairs of fit-1 and KDR primers were used in the polymerase chain reaction hot start ampliwax technique according to Perkin Elmer Cetus (Norwalk, CT) protocol (35 cycles, annealing temperature, 60 C, extension for 2 minutes at 72 C). Human fetal liver complementary DNA from Clontech (Palo Alto, CA) was used as a template. The resulting products were precipitated with ethanol, digested with BamHl and EcoRI, electrophoresed on 1% agarose gels (FMC, Rockland, ME) and intercepted on NA45 (Schleicher and Schuell, Keene, NH) according to the manufacturer's instructions. Fragments were cloned into pGEM 3 (ProMega, Madison, WI), and

clones were prepared and sequenced (Sequenase 2, USB, Cleveland, OH). Correct sequence clones of fit-1 and of KDR (225 and 209 bp, respectively) were used to prepare 35S-labeled anti-sense and sense probes for ISH using SP6 and T7 polymerases and materials from Promega.24 Preparation of an affinity-purified rabbit antibody to the N-terminal peptide (amino acid residues nos. 1 to 26) of human VPF has been described.16,31 This anti-peptide antibody specifically binds VPF in enzyme-linked immunosorbent assays and on immunoblots, blocks VPF activity, and when linked to agarose selectively binds VPF from solution31; because of its specificity, it has become the antibody of choice for demonstrating VPF by IH.16'27 IH was performed with an avidin-biotin peroxidase conjugate protocol.16'27 Normal rabbit immunoglobulin G (IgG) diluted to an equivalent protein concentration was used as a control in place of the primary antibody.

Results VPF mRNA Expression in Kidney and Bladder Tumors Sections of tumor and grossly normal kidney were studied from 13 nephrectomy specimens. Histologically, 1 1 cases were typical renal cell carcinomas, composed predominantly of clear cells and cells with granular cytoplasm, one of the 11 cases also displayed focal spindle cell differentiation. Of the two other cases examined, one was a renal cell carcinoma with a predominantly papillary histology, the other was a transitional cell carcinoma arising in the renal pelvis. Two high-grade invasive transitional cell carcinomas of the bladder were also studied. In all 11 cases of typical renal cell carcinoma (Figure 1, A and B) and in the three transitional cell carcinomas (Figure 1, G and H), the tumor cells labeled strongly for VPF mRNA by ISH, including the area of spindle cell differentiation in the one renal cell carcinoma that exhibited this growth pattern focally. Labeling was intense throughout and virtually all tumor cells were labeled. However, labeling to an even greater extent was observed adjacent to areas of overt tumor necrosis. In contrast to typical renal cell carcinoma, unequivocal labeling for VPF mRNA was not detected in the single papillary adenocarcinoma studied by ISH. Of interest, rare stromal cells in the transitional cell carcinomas also labeled for

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Figure 1. Bright-field (A and C) and dark-field (B and D) photomicrographs of ISH performed on renal cell carcinoma uith VPF anti-sense (A and B) or sense (C and D) riboprobes. Note intense specific labeling of tumor cells (A and B) and low background levels (C and D). Bnight-field (E) and dark-field (F) photomicrographs of ISHperformed on normal kidney with VPF anti-sense probe demonstrating louw-level labeling of kidney ttibtule (arrouw). Bright-field (G) and dark-field (H) photomicrographs of ISH performed on transitional cell carcinoma uwith VPF anti-sense probe demonstrating intense labelinig of tuimor cells. (288X; scale bar = 30 p).

VPFNEGF mRNA. For comparison, sections of grossly normal kidneys were studied in all 13 cases. As previously reported, glomerular epithelial cells labeled strongly for VPF mRNA but only focal low-level labeling was found in the tubular epithelium (Figure 1, E and F) from which renal cell carcinomas are thought to originate. Thus, the strong labeling for VPF mRNA in renal cell carcinomas was far in excess of that seen in normal tubular epithelium. In all cases, back-

ground levels were low, and no specific cellular labeling was seen with control sense probes (Figure 1, C and D).

Detection of VPF in Kidney Tumors by Immunohistochemistry Immunohistochemistry for VPF was performed on five representative renal cell carcinomas and corresponding sections of normal kidney with affinity-

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purified anti-VPF peptide antibody. In each case, antibody stained both the malignant cells and the numerous small blood vessels in the minimal stroma that separated nests of malignant cells (Figure 2, A

and C). In contrast, staining of blood vessels was not observed in sections of normal kidney, but antibody stained glomerular epithelial cells. No specific staining was seen in tumors or normal kidney when

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V . Figure 2. Low (A and B) and high-power (C and D) photomicrographs illustrating IH staining of a renal cell carcinoma with affinity-purified anti-VPFpeptide antibody (A and C) or control IgG (B and D). Both tumor cells (a typical nest of cells is labeled T in C and D) and blood vessels (labeled uith a V in the lumen in C and D) stain with anti-VPF but not control antibodies. A and B 107X; scale bar = 112 Y. C and D 279X; scale bar= 40,u.

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control IgG was substituted for the primary antibody (Figure 2, B and D).

Expression of mRNA Encoding the VPF/ VEGF Receptors fit- 1 and KDR in Kidney Tumors and Normal Kidney Sections of three typical renal cell carcinomas and corresponding normal kidneys were studied by ISH for expression of mRNA for the VPFNEGF receptors fit-1 and KDR. In all three carcinomas, endothelial cells of small tumor vessels labeled strongly for the mRNAs encoding each receptor (Figure 3, A to D). In contrast, no definite labeling for receptors was seen over endothelial cells in small vessels in normal kidney, although a suggestion of very weak labeling was observed over glomeruli in some sections. The level of labeling was too low to permit identification of a specific type of glomerular cellemitting signal. In all cases, background levels

were low, and no specific cellular labeling was observed with sense probe controls.

Discussion As judged by ISH, the malignant epithelial cells of the 11 typical renal carcinomas expressed markedly higher levels of VPF mRNA compared to normal renal tubular epithelium. In contrast, a single case of renal cell carcinoma with pure papillary architecture did not label for VPF mRNA. This confirms our previous report of another case of papillary renal cell carcinoma that did not express VPF mRNA by Northern analysis.24 Of interest, renal cell carcinomas of the papillary type are reported to be relatively avascular as compared to other renal cell carcinomas and have been reported to have a better prognosis.3233 Similar to the typical renal cell carcinomas, a transitional cell carcinoma arising in the renal pelvis and two invasive transitional cell carcinomas of the bladder also labeled strongly for VPF mRNA.

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Figure 3. Bright-field (A and C) and dark-field (B and D) photomicrographs of ISH performed on renal cell carcinomas with anti-sense riboprobes to the VPFIVEGF receptors KDR (A and B) andflt-1 (C and D) demonstrating labeling of endothelial cells in small vessels (vessel is outlined by arrows in A and B and labeled with a V in the lumen in C and D) adjacent to carcinoma cells (288x; scale bar = 30 ,u).

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Tumor cell labeling for VPF mRNA was strong throughout the renal and transitional cell carcinomas, but was intensified additionally in zones of tumor immediately adjacent to zones of overt necrosis. Hypoxia has been reported to increase expression of VPF mRNA by cultured cell lines and increased VPF expression has also been correlated with areas of necrosis in certain brain tumors.19'20 Hypoxia, however, is unlikely to be the only factor that regulates VPF expression in either tumors or normal kidney. In the kidney and bladder carcinomas studied, VPF expression was strong, even in well-vascularized areas far removed from zones of necrosis. Moreover, in previous studies, VPF expression in transformed cells was much higher than in their nontransformed counterparts, even though culture conditions were identical, indicating that increased VPF expression was associated with the transformed phenotype rather than exclusively with environmental factors such as hypoxia.2 17'24 Finally, epithelial cells of the normal renal glomerulus express VPF mRNA,27 a finding that cannot be explained by hypoxia because kidney is among the most well-vascularized organs in the body. It is likely that the high levels of VPF expressed by renal cell and transitional cell carcinomas is important to their biology. Tumors must induce a vascular stroma if they are to grow beyond a minimal size,25 and VPF may play an important role in the induction of such stroma by acting in several ways. VPF is an endothelial cell growth factor6'7'10 and may directly stimulate the growth of new blood vessels. VPF also acts on endothelial cells to increase microvascular permeability,1' 24 resulting in the extravasation of plasma proteins, including fibrinogen, into the extravascular space. Extravasated fibrinogen clots to form fibrin, and other proteins, such as fibronectin, may be incorporated into the fibrin clot.34 This clot provides a provisional matrix into which fibroblasts, endothelial cells, macrophages, and other cells migrate, eventually transforming it into vascularized connective tissue.35'36 This same series of events takes place in healing wounds37 and can also be initiated by implanting fibrin into the subcutaneous space.38 VPF is also likely to affect the vascular tumor stroma in other ways, because it has been reported to induce expression of plasminogen activator, plasminogen activator inhibitor,39 interstitial collagenase,40 and procoagulant activity41 by endothelial cells; in addition, VPF provokes the release of von Willebrand factor from endothelial cells.15

Small blood vessels in the tumor stroma labeled strongly for mRNA encoding the VPF receptors fit-1 and KDR. In addition, the same vessels stained for VPF protein by IH. In contrast, no VPF mRNA was detected in endothelial cells. Therefore, it seems likely that VPF is synthesized by tumor cells and released into tumor stroma where it is bound by receptors (fit-1 and KDR) on endothelial cells that line small vessels. In contrast, small vessels did not stain for VPF protein in normal kidney. We noted possible very weak glomerular labeling for transcripts encoding the VPF receptors fit-1 and KDR by ISH, but labeling was not observed in stromal blood vessels in the normal renal cortex or medulla. Therefore, our data indicate that mRNA-encoding VPF receptors is significantly overexpressed by endothelial cells of tumor blood vessels when compared to endothelial cells of normal kidney. Consistent with the findings reported here, IH staining for VPF protein has been described previously in small blood vessels in guinea pig bile duct carcinomas and human brain tumors,16 19 and fit-1 mRNA has been demonstrated in endothelial cells in glioblastomas in the central nervous system.19 In summary, VPF mRNA and protein are strongly expressed in renal cell carcinomas and transitional cell carcinomas. Moreover, in contrast to endothelial cells in normal kidney, endothelial cells lining tumor blood vessels strongly expressed mRNAs for the VPF receptors fit-1 and KDR and stained strongly for VPF protein. As an endothelial cell growth factor, potent inducer of microvascular permeability, and regulator of endothelial cell gene expression, VPF is likely to play an important role in the vascular biology of these common human tumors.

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4. Senger DR, Connolly DT, Van De Water L, Feder J, Dvorak HF: Purification and NH2-terminal amino acid sequence of guinea pig tumor-secreted vascular permeability factor. Cancer Res 1990, 50:1774-1778 5. Conn G, Bayne ML, Soderman DD, Kwok PW, Sullivan KA, Palisi TM, Hope DA, Thomas KA: Amino acid and cDNA sequences of a vascular endothelial cell mitogen that is homologous to platelet-derived growth factor. Proc NatI Acad Sci 1990, 87:2628-2632 6. Ferrara N, Henzel WJ: Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem Biophys Res Comm 1989, 161:851-858 7. Gospodarowicz D, Abraham JA, Schilling J: Isolation and characterization of a vascular endothelial cell mitogen produced by pituitary-derived folliculo stellate cells. Proc Natl Acad Sci 1989, 86:7311-7315 8. Keck PJ, Hauser SD, Krivi G, Sanzo K, Warren T, Feder J, Connolly DT: Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science 1989, 246:1309 9. Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N: Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 1989, 246:1306 10. Connolly DT, Heuvelman DM, Nelson R, Olander JV, Eppley BL, Delfino JJ, Siegel NR, Leimgruber RM, Feder J: Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. J Clin Invest 1989, 84:1470-1478 11. Tischer E, Mitchell R, Hartman T, Silva M, Gospodarowicz D, Fiddes JC, Abraham JA: The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. J Biol Chem 1991, 266:11947-11954 12. Houck KA, Leung DW, Rowland AM, Winer J, Ferrara N: Dual regulation of vascular endothelial growth factor bioavailability by genetic and proteolytic mechanisms. J Biol Chem 1992, 267:26031-26037 13. Terman BI, Dougher Vermazen M, Carrion ME, Dimitrov D, Armellino DC, Gospodarowicz D, Bohlen P: Identification of the KDR tyrosine kinase as a receptor for vascular endothelial growth factor. Biochem Biophys Res Comm 1992, 187:1579-1586 14. de Vries C, Escobedo JA, Ueno H, Houck K, Ferrara N, Williams LT: The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor. Science 1992, 255:989-991 15. Brock TA, Dvorak HF, Senger DR: Tumor-secreted vascular permeability factor increases cytosolic Ca2+ and von Willebrand factor release in human endothelial cells. Am J Pathol 1991, 138:213-221 16. Dvorak HF, Sioussat TM, Brown LF, Berse B, Nagy JA, Sotrel A, Manseau EJ, Van De Water L, Senger DR: Distribution of vascular permeability factor (vascular endothelial growth factor) in tumors: concentration in tumor blood vessels. J Exp Med 1991, 174:1275-1278

17. Senger DR, Perruzzi CA, Feder J, Dvorak HF: A highly conserved vascular permeability factor secreted by a variety of human and rodent tumor cell lines. Cancer Res 1986, 46:5629-5632 18. Berkman RA, Merrill MJ, Reinhold WC, Monacci WT, Saxena A, Clark WC, Robertson JT, Ali IU, Oldfield EH: Expression of the vascular permeability factor/ vascular endothelial growth factor gene in central nervous system tumors. J Clin Invest 1993, 91:153-159 19. Plate KH, Breier G, Weich HA, Risau W: Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature 1992, 359:845-848 20. Shweiki D, Itin A, Soffer D, Keshet E: Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 1992, 359:843845 21. Connolly DT, Olander JV, Heuvelman D, Nelson R, Monsell R, Siegel N, Haymore BL, Leimgruber R, Feder J: Human vascular permeability factor. Isolation from U937 cells. J Biol Chem 1989, 264:20017-20024 22. Myoken Y, Kayada Y, Okamoto T, Kan M, Sato GH, Sato JD: Vascular endothelial cell growth factor (VEGF) produced by A-431 human epidermoid carcinoma cells and identification of VEGF membrane binding sites. Proc NatI Acad Sci USA 1991, 88:5819-5823 23. Roberts WG, Hasan T: Tumor-associated vascular permeability factor/vascular endothelial growth factor influences photosensitizer uptake. Cancer Res 1993, 53:153-157 24. Berse B, Brown LF, Van De Water L, Dvorak HF, Senger DR: Vascular permeability factor (vascular endothelial growth factor) gene is expressed differentially in normal tissues, macrophages, and tumors. Mol Biol Cell 1992, 3:211-220 25. Folkman J, Shing Y: Angiogenesis. J Biol Chem 1992, 267:10931-10934 26. Kim KJ, Li B, Winer J, Armanini M, Gillett N, Phillips HS, Ferrara N: Inhibition of vascular endothelial growth factor induced angiogenesis suppresses tumour growth in vivo. Nature 1993, 362:841-844 27. Brown LF, Berse B, Tognazzi K, Manseau EJ, Van De Water L, Senger D, Dvorak H, Rosen S: Vascular permeability factor mRNA and protein expression in human kidney. Kid Int 1992, 42:1457-1461 28. Houck KA, Ferrara N, Winer J, Cachianes G, Li B, Leung DW: The vascular endothelial growth factor family: identification of a fourth molecular species and characterization of alternative splicing of RNA. Mol Endocrinol 1991, 5:1806-1814 29. Terman BI, Carrion ME, Kovacs E, Rasmussen BA, Eddy RL, Shows TB: Identification of a new endothelial cell growth factor receptor tyrosine kinase. Oncogene 1991, 6:1677-1683 30. Shibuya M, Yamaguchi S, Yamane A, Ikeda T, Tojo A, Matsushime H, Sato M: Nucleotide sequence and expression of a novel receptor-type tyrosine kinase gene

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Dvorak HF, Van De Water L: Expression of vascular permeability factor (vascular endothelial growth factor) by epidermal keratinocytes during wound healing. J Exp Med 1992, 176:1375-1379 Dvorak HF, Harvey VS, Estrella P, Brown LF, McDonagh J, Dvorak AM: Fibrin containing gels induce angiogenesis. Implications for tumor stroma generation and wound healing. Lab Invest 1987, 57:673-686 Pepper MS, Ferrara N, Orci L, Montesano R: Vascular endothelial growth factor (VEGF) induces plasminogen activators and plasminogen activator inhibitor-1 in microvascular endothelial cells. Biochem Biophys Res Comm 1991, 181:902-906 Unemori EN, Ferrara N, Bauer EA, Amento EP: Vascular endothelial growth factor induces interstitial collagenase expression in human endothelial cells. J Cell Physiol 1992, 153:557-562 Clauss M, Gerlach M, Gerlach H, Brett J, Wang F, Familletti PC, Pan Y-CE, Olander JV, Connolly DT, Stern D: Vascular permeability factor: a tumor-derived polypeptide that induces endothelial cell and monocyte procoagulant activity, and promotes monocyte migration. J Exp Med 1990, 172:1535-1545