Enhanced Expression of Tie2, Its Ligand Angiopoietin-1, Vascular Endothelial Growth Factor, and CD31 in Human Non-Small Cell Lung Carcinomas1

2506 Vol. 5, 2506–2510, September 1999 Clinical Cancer Research

Makoto Takahama, Masahiro Tsutsumi, INTRODUCTION Toshifumi Tsujiuchi, Kunimoto Nezu, Lung cancer is the leading cause of cancer-related death of 3 Keiji Kushibe, Shigeki Taniguchi, males in both Japan and the United States (1). Surgical resection remains the most effective treatment for NSCLC,4 al- Yashige Kotake, and Yoichi Konishi2 though, at the time of diagnosis, the majority of patients present Department of Oncological Pathology, Cancer Center [M. Ta., M. Ts., with advanced-stage disease that is no longer amenable to T. T., Y. Kon.], Department of Surgery III [M. Ta., K. N., K. K., S. T.], Nara Medical University, Kashihara, Nara 634-8521, Japan, curative therapy (2). Indeed, a large percentage of patients and Department of Free Radical Biology and Aging Research undergoing surgical resection ultimately die of recurrent Program, Oklahoma Medical Research Foundation, Oklahoma, NSCLC, due to the presence of occult metastatic sites at the time Oklahoma 73104 [Y. Kot.] of diagnosis (3). Attempts to improve this situation are focused on eliminating the causes, preventing the disease in high-risk groups, diagnosing lesions at an early curable stage, and devel- ABSTRACT oping new adjuvant and neoadjuvant protocols. Each of these Expression of angiogenesis-associated genes was com- strategies has met with limited success. Recently, there have pared in 32 primary non-small cell lung carcinoma samples been intense studies of molecular abnormalities involving dom- (14 adenocarcinomas, 17 squamous cell carcinomas, and 1 inant and recessive oncogenes in clinically evident lung cancers large cell carcinoma) and paired adjacent noncancerous (4–6), targeting biomarkers for early detection, due to chemo- lung tissues using a multiprobe RNase protection assay. therapeutic response and gene therapy. Changes in angiogenic Levels of Tie2, angiopoietin (Ang)-1, vascular endothelial factors that contribute to NSCLC progression, some as inde- growth factor (VEGF), and CD31 mRNAs were higher in pendent predictors of prognosis, are particularly important in cancers than in adjacent noncancerous tissues, in contrast to this context (7–9). the fms-like tyrosine kinase (Flt)-1, Flt-4, Tie1, thrombin Tie1 and Tie2 (tek) are members of the endothelial cell- receptor, endoglin, and VEGF-C, for which no differences specific receptor tyrosine kinase family (10, 11), essential for were evident. Overexpression did not seem to differ with formation of the embryonic vasculature (12). They are up- histological type and pathological stage. Significant positive regulated in the vascular endothelium of metastatic melanomas correlations were found between mRNA expression of Ang-1 (13), brain tumors (14) and mammary carcinomas (15). A ligand and those of Tie2 and CD31, and that of VEGF and those of for the Tie1 receptor has not yet been identified, but ligands for Flt-1 and CD31. These findings suggest that Ang-1 and Tie2, Ang-1, and Ang-2 have recently been shown to be essen- VEGF are important angiogenic factors in human non-small tial for normal vascular development in the mouse (16, 17). cell lung carcinomas. Whether Tie2 and Ang-1 might be up-regulated in vessels of lung neoplasms has not been addressed, to our knowledge. The recently developed multiprobe RPA has advantages for simul- taneous investigation of mRNA expression of several genes. In the present study, it was used to examine the expression of 10 angiogenesis-associated factors in human NSCLC. In addition to Tie1, Tie2, and Ang-1, VEGF, a well-established angiogenic Received 4/6/99; revised 7/6/99; accepted 7/8/99. factor (18, 19), and its receptors VEGF receptor-1/Flt-1 and The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked VEGF receptor-2/fetal liver tyrosine kinase-1 were included advertisement in accordance with 18 U.S.C. Section 1734 solely to because they have been reported to be up-regulated in human (7, indicate this fact. 8, 20) and rat (21) NSCLCs. CD31 was also studied as a marker 1 Supported in part by Grants-in-Aid for Cancer Research (designated for vascularity (22). 7-1 and 8-2 to Y. Kon. and 10-4 to T. T.) and for Scientific Research Expenses for Health and Welfare Programs, 2nd-Term Comprehensive 10-Year Strategy for Cancer Control, Cancer Prevention, from the Ministry of Health and Welfare of Japan (to Y. Kon.), and by Grant- in-Aid 08264108 (to Y. Kon.) for Scientific Research in Priority Areas, Cancer Research, from the Ministry of Education, Science, Sports and 3 Ministry of Health and Welfare of Japan. Statistics and other data, Culture of Japan. http://mhw.go.jp/toukei/sibouritsu/mokuji.html (in Japanese). 2 To whom requests for reprints should be addressed, at the Department 4 The abbreviations used are: NSCLC, non-small cell lung carcinoma; of Oncological Pathology, Cancer Center, Nara Medical University, 840 Ang, angiopoietin; Flt, fms-like tyrosine kinase; GAPDH, glyceralde- Shijo-cho, Kashihara, Nara 634-8521, Japan. Phone: 81-744-22-3051, hyde 3-phosphate dehydrogenase; PSL, phosphostimulating lumines- ext. 2574; Fax: 81-744-25-7308; E-mail: [email protected] cence; RPA, RNase protection assay; VEGF, vascular endothelial u.ac.jp. growth factor.

Table 1 Histological types and pathological stages of NSCLC No. of cases examined Pathological stagea Histology Male (aged, yr) Female (aged, yr) IA IB IIA IIB IIIA IIIB Adenocarcinoma 8 (44–74) 6 (54–76) 4 3 2 2 2 1 Squamous cell carcinoma 16 (60–81) 1 (63) 2 9 0 0 6 0 Large cell carcinoma 1 (63) 0 0 1 0 0 0 0 Total 25 7 6 13 2 2 8 1 a Pathological stage was determined according to the general rules for clinical and pathological recording of Union International Contre le Cancer (23).

MATERIALS AND METHODS Patients and Tissue Samples. Primary NSCLC tissues obtained from 32 patients undergoing surgery in Nara Medical University Hospital between April 1998 and December 1998 were frozen in liquid nitrogen and stored at Ϫ80°C until use. Data for histological types and details for sex, age distribution, and pathological stages are summarized in Table 1. The histology and pathological stage were classified according to General Rules for Clinical and Pathological Recording of Union Inter- national Contre le Cancer (23). Noncancerous tissues adjacent to tumors were also frozen in liquid nitrogen and stored at Ϫ80°C. Total RNA was extracted from each sample using an ISOGENE kit (Nippon Gene, Toyama, Japan), as described previously (21). RPA for Angiogenesis-associated mRNA. A panel of angiogenesis-associated mRNA species were detected using a multiprobe protection assay system (RiboQuant, PharMingen, San Diego, CA). Radiolabeled probes were synthesized from DNA templates containing a T7 RNA polymerase promoter (PharMingen), transcribed in the presence of 100 ␮Ci [␣-32P] dCTP to yield radioactive probes of defined sizes. Probes were hybridized with 5 ␮g of total RNA, then samples were treated with RNase A and T1 to digest single-stranded RNA. Intact double-stranded RNA hybrids were resolved on 5% polyacryl- amide/7 M urea gels at 50 W for 70 min. Dried gels were Fig. 1 Representative autoradiogram with multiprobe RPA. Increased analyzed for band locations and PSL values with a BAS 1000 expression of Tie2, CD31, Ang-1, and VEGF is apparent in lung Phospho Imaging Analyzer (Fuji Photo Film Co., Ltd., Minato- cancerous tissues compared with levels in adjacent noncancerous tis- ku, Tokyo, Japan). Within each sample, the intensity of each sues. Lanes assigned are as: Cases 30, 32, and 33, squamous cell angiogenesis-associated mRNA band was divided by the sum of carcinomas; Case 31, large cell carcinoma; Case 34, adenocarcinoma. ϩ Ca, cancerous tissue; N, noncancerous tissue; TR, thrombin receptor; the L32 GAPDH bands. The size of each band was analyzed Endg, endoglin; L32, L32 rRNA. in terms of its migration distance against a plotted standard curve of migration distance versus log nucleotide length for each undigested probe (RiboQuant, Instruction manual, 5th edi- tion, April 1998, PharMingen). The resulting value for each of significant departure from linearity for each regression func- mRNA species was then expressed as a percentage of the tion was confirmed doubly by a Runs test and a test using the average for the parameter. Finally, dried gels were redeveloped ANOVA table. overnight by traditional autoradiography. Statistics. Statistical analyses were performed using a RESULTS personal computer, basically as described previously elsewhere The results of the multiprobe RPA analysis are depicted in (24). To assess the statistical significance of differences in Fig. 1. The range of PSL values was from 7.29–979.3, therefore, angiogenesis-associated mRNA species between cancer and all values quantified could be analyzed in the linear range. The noncancer lung specimens, the Mann-Whitney nonparametric PSL values for angiogenesis-associated gene expression are test was applied. To assess relationships between two specified summarized in Table 2. Four angiogenesis-associated gene factors, Pearson’s correlation coefficients were obtained with products, Tie2, Ang-1, CD31, and VEGF were increased in linear trends also determined using the data generated in the cancers, the average magnitudes being 1.3-fold for Tie2, 3.9- ANOVA. Significant differences from zero of the slope of each fold for Ang-1, 1.5-fold for CD31, and 1.4-fold for VEGF. regression function were assessed using ANOVA tables. Lack Expression of VEGF-C mRNA was not detected in any of the

Table 2 PSL values for gene expression in 32 paired cases of NSCLCs and adjacent noncancerous tissues Gene expressiona Flt-1 Flt-4 Tie1 TRb Tie2 CD31 Endg Ang-1 VEGF Ca 0.36 Ϯ 0.28 0.31 Ϯ 0.27 0.36 Ϯ 0.28 0.35 Ϯ 0.26 0.35 Ϯ 0.25c 0.83 Ϯ 0.29d 0.58 Ϯ 0.24 0.33 Ϯ 0.25e 0.89 Ϯ 0.33d N 0.33 Ϯ 0.26 0.26 Ϯ 0.24 0.28 Ϯ 0.26 0.33 Ϯ 0.23 0.27 Ϯ 0.26 0.56 Ϯ 0.28 0.65 Ϯ 1.21 0.085 Ϯ 0.070 0.62 Ϯ 0.23 a Gene expression after normalization for L32ϩ GAPDH levels. b TR, thrombin receptor; Endg, endoglin; Ca, carcinoma; N, adjacent noncancerous lung tissue. c P Ͻ 0.05. d P Ͻ 0.01. e P Ͻ 0.001. Quantitative comparisons of paired carcinomas and adjacent noncancerous lung tissues by Mann-Whitney test.

Fig. 2 Correlations between Ang-1 mRNA expression and those of Tie2 mRNA (A) and CD31 mRNA (B). A, the difference from zero of the slope of the regression function was significant (r ϭ 0.98, P Ͻ Fig. 3 Correlations between VEGF mRNA expression and those of 0.0001) with no significant departure from linearity [Y ϭ (1.03) ϫ CD31 mRNA (A) and Flt-1 mRNA (B). A, the difference from zero of Ϫ0.01]. B, the difference from zero of the slope of the regression the slope of the regression function was significant (r ϭ 0.43, P ϭ function was significant (r ϭ 0.47, P Ͻ 0.0001), but there was signif- 0.0003) with no significant departure from linearity [Y ϭ (0.60) ϫ a icant departure from linearity (P Ͻ 0.0001). Normalized for the ϩ3.24]. B, the difference from zero of the slope of the regression L32ϩGAPDH values. function was significant (r ϭ 0.79, P Ͻ 0.0001), but there was significant departure from linearity (P ϭ 0.019). a Normalized for the L32ϩGAPDH values.

samples examined. Correlations between Ang-1 mRNA and Tie2 mRNA, and Ang-1 mRNA and CD31 mRNA are shown in Fig. 2, A and B, respectively. A clear relationship was observed VEGF mRNA and Flt-1 mRNA expression was also significant ϭ Ͻ between Ang-1 mRNA and Tie2 mRNA expression (r ϭ 0.98, statistically (r 0.79, P 0.0001), there was again a signifi- ϭ P Ͻ 0.0001). The correlation coefficient between Ang-1 and cant departure from linearity (P 0.019). Overexpression of CD31 mRNA expression was also significant statistically (r ϭ Tie2, Ang-1, CD31, and VEGF and the correlations among Tie2 0.47, P Ͻ 0.0001), but with a significant departure from line- and Ang-1 or VEGF and CD31 did not differ with the histolog- arity (P Ͻ 0.0001). Correlations between VEGF mRNA and ical type or pathological stage of NSCLCs. CD31 mRNA, and VEGF mRNA and Flt-1 mRNA are shown in Fig. 3, A and B, respectively. A clear relationship was observed DISCUSSION between VEGF mRNA and CD31 mRNA expression (r ϭ 0.43, Angiogenesis, the sprouting of new blood vessels from P ϭ 0.0003). Although the correlation coefficient between preexisting ones, is a complex process involving the interactions

of a magnitude of angiogenesis-associated genes (25–28). It is pressor gene at 17p13.3, distal to p53, in the pathogenesis of lung well known that angiogenic factors contribute to development cancers. Oncogene, 17: 2095–2100, 1998. and progression of solid tumors (26, 29). The present finding of 5. Tomizawa, Y., Nakajima, T., Kohno, T., Saito, R., Yamaguchi, N., overexpression and correlation of mRNA levels of both Tie2 and Yokota, J. Clinicopathological significance of Fhit protein expression in stage I non-small cell lung carcinoma. Cancer Res., 58: 5478– and Ang-1 in NSCLC is indicative of activation of the Tie2/ 5483, 1998. Ang-1 system (16). The results for VEGF and CD31 are further 6. Mao, L., Lee, J. S., Kurie, J. M., Fan, Y. H., Lippman, S. M., Lee, suggestive of angiogenesis in NSCLC (18, 22). J. J., Ro, J. Y., Broxson, A., Yu, R., Morice, R. C., Kemp, B. L., Khuri, Angiopoietins (Ang-1 and Ang-2) constitute a novel family F. R., Walsh, G. L., Hittelman, W. N., and Hong, W. K. Clonal genetic of endothelial growth factors that are ligands for Tie2 (9–12, 14, alterations in the lungs of current and former smokers. J. Natl. Cancer 30). Whereas Ang-1 can stimulate endothelial sprouting in vitro Inst., 89: 857–862, 1997. (31), however, exposure of Tie2 in cultured endothelial cells to 7. Ohta, Y., Endo, Y., Tanaka, M., Shimizu, J., Oda, M., Hayashi, Y., and Watanabe, Y. Significance of vascular endothelial growth factor either Ang-1 or Ang-2, unlike the case with other endothelial messenger RNA expression in primary lung cancer. Clin. Cancer Res., growth factors, does not produce a mitogenic response (9). 2: 1411–1416, 1996. Unfortunately, we could not analyze the expression of Ang-2 8. Takanami, I., Tanaka, F., Hashizume, T., and Kodaira, S. Vascular because of the lack of an appropriate probe, but this warrants endothelial growth factor and its receptor correlate with angiogenesis further investigation. and survival in pulmonary adenocarcinoma. Anticancer Res., 17: 2811– 2814, 1997. VEGF, first detected as a factor inducing microvascular 9. Mattern, J., Koomagi, R., and Volm, M. Vascular endothelial growth permeability (32) and extravasation of various proteins to me- factor expression and angiogenesis in non-small cell lung carcinomas. tastases, also acts as an endothelial cell mitogen (33). Previ- Int. J. Oncol., 6: 1059–1062, 1995. ously, we reported frequent expression of VEGF detected by 10. Schnurch, H., and Risau, W. Expression of tie-2, a member of a immunohistochemistry in human NSCLCs (34). The present novel family of receptor tyrosine kinases, in the endothelial lineage. results, thus, provide confirmation at the mRNA level with a Development (Camb.), 119: 957–968, 1993. molecular biological technique. 11. Maisonpierre, P. C., Goldforb, M., Yancopoulos, G. D., and Gao, G. The realization that multiple pathways are likely to be Distinct rat genes with related profiles of expression define a TIE receptor tyrosine kinase family. Oncogene, 8: 1631–1637, 1993. required for the assembly of a functional vasculature has 12. Sato, T. N., Tozawa, Y., Deutsch, U., Wolburg-Buchholz, K., broad implications for therapeutic modulation. For example, Fujiwara, Y., Gendron-Maguire, M., Gridley, T., Wolburg, H., Risau, it has already been shown that blocking either the Tie2/Ang-1 W., and Quin, Y. Distinct roles of the receptor tyrosine kinases Tie-1 pathway or the VEGF pathway can inhibit tumor angiogen- and Tie-2 in blood vessel formation. Nature (Lond.), 376: 70–74, esis (35, 36). Because Tie2/Ang-1 and VEGF function at 1995. different stages of vascular development, it seems likely that 13. Kaipaimen, A., Vlaykova, T., Hatva, E., Bohling, T., Jekunen, A., Pyrhonen, S., and Alitalo, K. Enhanced expression of the tie receptor inhibiting both pathways may be more effective than inhib- tyrosine kinase messenger RNA in the vascular endothelium of meta- iting each individually (37). Moreover, our results suggest static melanomas. Cancer Res., 54: 6571–6577, 1994. the possibility that antibody therapy against each of these 14. Hatva, E., Kaipaimen, A., Jaaskelainen, J., Paetau, A., Haltia, M., may improve the outcome with this otherwise generally re- and Alitalo, K. Expression of endothelial cell-specific receptor tyrosine fractory cancer. kinases and growth factors in human brain tumors. Am. J. Pathol., 146: 368–378, 1995. 15. Peters, K. G., Coogan, A., Berry, D., Marks, J., Iglehart, J. D., ACKNOWLEDGMENTS Kontos, C. D., Rao, P., Sankar, S., and Trogan, E. Expression of Tie2/Tek in breast tumor vasculature provides a new marker for eval- We express our gratitude to Dr. Malcolm Moore for English uation of tumor angiogenesis. Br. J. Cancer, 77: 51–56, 1998. correction and careful reading of this manuscript; to Megumi Inagaki- 16. Davis, S., Aldrich, T. H., Jones, P. F., Acheson, A., Compton, D. L., Yamaguchi, Hiroko Masuda, and Sachiko Nakai for technical assist- Jain, V., Ryan, T. E., Bruno, J., Radziejewski, C., Maisonpierre, P. C., ance; and to Yumi Horikawa, Rie Maeda, Nami Makimura, and Yuko and Yancopoulos, G. D. Isolation of angiopoietin-1, a ligand for the Yoshinaka for editorial assistance. TIE2 receptor, by secretion-trap expression cloning. Cell, 87: 1161– 1169, 1996. 17. Maisonpierre, P. C., Suri, C., Jones, P. F., Bartunkova, S., Wiegand, REFERENCES S. J., Radziejewski, C., Compton, D., McClain, J., Aldrich, T. H., 1. Landis, S. H., Murray, T., Bolden, A., and Wingo, P. A. Cancer Papadopoulos, N., Daly, T. J., Davis, S., Sato, T. N., and Yancopoulos, statistics, 1998 (published erratum appears in CA Cancer J. Clin., 48: G. D. Angiopoitin-2, a natural antagonist for Tie2 that disrupts in vivo 192, 1998). CA Cancer J. Clin., 48: 6–29, 1998. angiogenesis. Science (Washington DC), 277: 55–60, 1997. 2. Ahrendt, S. A., Chow, J. T., Xu, L-H., Yang, S. C., Eisenberger, 18. Hanahan, D., and Folkman, J. Patterns and emerging mecha- C. F., Esteller, M., Herman, J. G., Wu, L., Decker, P. A., Jen, J., and nisms of the angiogenic switch during tumorigenesis. Cell, 86: Sidransky, D. Molecular detection of tumor cells in bronchoalveolar 353–364, 1996. lavage fluid from patients with early stage lung cancer. J. Natl. Cancer 19. Folkman, J. Antiangiogenic gene therapy. Proc. Natl. Sci. USA, 95: Inst., 91: 332–339, 1999. 9064–9066, 1998. 3. Naruke, T., Goya, T., Tsuchiya, R., and Suemasu, K. Prognosis 20. Mattern, J., Koomagi, R., and Volm, M. Vascular endothelial and survival in resected lung carcinoma based on the new interna- growth factor expression and angiogenesis in non-small cell lung car- tional staging system (published erratum appears in J. Thorac. Car- cinomas. Int. J. Oncol., 6: 1059–1062, 1995. diovasc. Surg., 97: 350, 1989). J. Thorac. Cardiovasc. Surg., 96: 21. Takahama, M., Tsutsumi, M., Tsujiuchi, T., Kido, A., Sakitani, H., 440–447, 1988. Iki, K., Taniguchi, S., Kitamura, S., and Konishi, Y. Expression of 4. Konishi, H., Takahashi, T., Kozaki, K., Yatabe, Y., Mitsudomi, T., vascular endothelial growth factor and its receptors during lung carci- Fujii, Y., Sugiura, T., Matsuda, H., Takahashi, T., and Takahashi, T. nogenesis by N-nitrosobis(2-hydroxypropyl)amine in rats. Mol. Carci- Detailed deletion mapping suggests the involvement of a tumor sup- nogenesis, 24: 287–293, 1999.

22. Yoshiji, H., Kuriyama, S., Yoshii, J., Yamazaki, M., Kikukawa, M., 31. Koblizek, T. I., Weiss, C., Yancopoulos, G. D., Deutsch, U., and Tsujinoue, H., Nakatani, T., and Fukui, H. Vascular endothelial growth Risau, W. Angiopoietin-1 induces sprouting angiogenesis in vitro. Curr. factor tightly regulates in vivo development of murine hepatocellular Biol., 8: 529–532, 1998. carcinoma cells. Hepatology, 28: 1489–1496, 1998. 32. Senger, D. R., Galli, S. J., Dvorak, A., M., Perruzzi, C. A., Harvey, 23. UICC. TNM Classification of Malignant Tumors, Ed. 5, pp. 93–97. V. S., and Dvorak, H. F. Tumor cells secrete a vascular permeability New York: Wiley-Liss, 1997. factor that promotes accumulation of ascites fluid. Science (Washington 24. Nakae, D., Kobayashi, Y., Akai, H., Andoh, N., Satoh, H., DC), 219: 983–985, 1983. Ohashi, K., Tsutsumi, M., and Konishi, Y. Involvement of 8-hydrox- 33. Leung, D. W., Cachianes, G., Kuang, W. J., Goeddel, D. V., and yguanine formation in the initiation of rat liver carcinogenesis by low Ferrara, N. Vascular endothelial growth factor is a secreted angiogenic dose levels of. N-nitrosodiethylamine. Cancer Res., 57: 1281–1287, mitogen. Science (Washington DC), 246: 1306–1309, 1989. 1997. 34. Takahama, M., Tsutsumi, M., Tsujiuchi, T., Kido, A., Okajima, E., 25. Hanahan, D. Signaling vascular morphogenesis and maintenance. Nezu, K., Tojo, T., Kushibe, K., Kitamura, S., and Konishi, Y. Frequent Science (Washington DC), 277: 48–50, 1997. expression of the vascular endothelial growth factor in human non-small 26. Folkman, J., and D’Amore, P. A. Blood vessel formation: what is its cell lung cancers. Jpn. J. Clin. Oncol., 27: 176–181, 1998. molecular basis? Cell, 87: 1153–1155, 1996. 35. Lin, P., Polverini, P., Dewhirst, M., Shan, S., Rao, P. S., and Peters, 27. Klagsbrun, M., and D’Amore, P. A. Regulation of angiogenesis. K. G. Inhibition of tumor angiogenesis using a soluble receptor estab- Annu. Rev. Physiol., 53: 217–239, 1991. lishes a role for Tie-2 in pathologic vascular growth. J. Clin. Invest., 28. Folkman, J. Tumor angiogenesis. In: J. Mendelsohn, P. Howley, 100: 2072–2078, 1997. L. A. Liotta, and M. Israel (eds.), The Molecular Basis of Cancer, pp. 36. Kim, K. J., Li, B., Winer, J., Armanini, M., Gillett, N., Phillips, 206–232. Philadelphia: W. B. Saunders, 1995. H. S., and Ferrara, N. Inhibition of vascular endothelial growth factor- 29. Craft, P. S., and Harris, A. L. Clinical prognostic significance of induced angiogenesis suppresses tumor growth in vivo. Nature (Lond.), tumor angiogenesis. Ann. Oncol., 5: 305–311, 1994. 362: 841–844, 1993. 30. Sato, T. N., Qin, Y., Kozak, C. A., and Audus, K. L. tie-1 and tie-2 37. Asahara, T., Chen, D., Takahashi, T., Fujikawa, K., Kearney, M., define another class of putative receptor tyrosine kinase genes expressed Magner, M., Yancopoulos, G. D., and Isner, J. M. Tie2 receptor ligands, in early embryonic vascular system. Proc. Natl. Acad. Sci. USA, 90: angiopoietin-1 and angiopoietin-2, modulate VEGF-induced postnatal 9355–9358, 1993. neovascularization. Circ. Res., 83: 233–240, 1998.

Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 1999 American Association for Cancer Research. Enhanced Expression of Tie2, Its Ligand Angiopoietin-1, Vascular Endothelial Growth Factor, and CD31 in Human Non-Small Cell Lung Carcinomas

Makoto Takahama, Masahiro Tsutsumi, Toshifumi Tsujiuchi, et al.

Clin Cancer Res 1999;5:2506-2510.

Updated version Access the most recent version of this article at: http://clincancerres.aacrjournals.org/content/5/9/2506

Cited articles This article cites 31 articles, 12 of which you can access for free at: http://clincancerres.aacrjournals.org/content/5/9/2506.full#ref-list-1

Citing articles This article has been cited by 9 HighWire-hosted articles. Access the articles at: http://clincancerres.aacrjournals.org/content/5/9/2506.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/5/9/2506. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.