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Nitric Oxide Synthase, Cyclooxygenase 2, and Vascular Endothelial Growth Factor in the Angiogenesis of Non-Small Cell Lung Carcinoma

Laboratory of Human Carcinogenesis [A. J. M., J. A. W., M. A. K., H. R., C. C. H.], Laboratory of Pathology [L. A. L.], Genetic Epidemiology Branch [N. E. C.], National Cancer Institute, NIH, Bethesda, Maryland 20892; Armed Forces Institute of Pathology, Washington, DC 20306 [W. D. T.]; and Mayo Clinic, Rochester, Minnesota 55905 [H. T., P. P., V. T., J. J.].

	ABSTRACT

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We have investigated the hypothesis that nitric oxide synthase (NOS2), cyclooxygenase-2 (COX2), and vascular endothelial growth factor (VEGF) protein levels individually demonstrate a direct correlation with microvessel density (MVD) and clinical outcome in human non-small cell lung cancer (NSCLC). Furthermore, we hypothesized that MVD may explain the propensity of certain histological lung cancer subtypes for early metastasis via a hematological route. Immunohistochemically, we studied the protein expression levels of NOS2, COX2, and VEGF and MVD by counting CD31-reactive blood vessels (BVs) in 106 surgically resected NSCLC specimens. NOS2, COX2, and VEGF immunoreactivity were observed in 48, 48, and 58%, respectively, of the study subjects, and their levels correlated with MVD at the tumor-stromal interphase (P 0.001). More adenocarcinomas and large cell carcinomas displayed overexpression of NOS2 when compared with squamous cell carcinoma (SCC; r = 0.44; P < 0.001). NOS2 and COX2 levels were found to correlate positively with VEGF status (r = 0.44; P < 0.001, 0.01, and 0.03, respectively). These results attest to the significant interaction of these factors in the angiogenesis of NSCLC. Although neither angiogenic factors nor MVD correlated with patient survival, the latter correlated with tumor clinical stage in both squamous (SCC; 73 BVs/mm²) and non-SCC (78 BVs/mm²) tumors. These results indicate that angiogenesis is a complex process that involves multiple factors including NOS2, COX2, and VEGF. Furthermore, the role of angiogenesis in the biology of various histological lung cancer types may be different. The complexity of angiogenesis may explain the modest results observed in antiangiogenesis therapy that target a single protein.

	INTRODUCTION

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Angiogenesis is essential for tumor growth in vivo (1) . Cytokines and growth factors, such as TGF² -ß, TGF-, platelet-derived growth factor, basic fibroblast growth factor, and VEGF, are known to promote angiogenesis (2, 3, 4, 5) . Although the up-regulation of these factors may be related, in part, to the host immune system, oncogenes, e.g., K-ras (6) and tumor suppressor genes, e.g., p53 (7 , 8) , can regulate the expression of these angiogenic proteins.

Angiogenesis is a complex process where several proteins and enzymatic pathways converge. COX2, a catalyst in prostaglandin synthesis from arachidonic acid, contributes to the regulation of angiogenesis by various genes, including platelet-derived growth factor, VEGF, fibroblast growth factor-, and TGF-ß. Using selective inhibitors of COX2, Tsujii et al. (9) were able to block the expression of several angiogenic factors including VEGF. Furthermore, Celecoxib (NS398), a specific COX2 inhibitor, has been shown to inhibit the growth of 97% of colon cancer cells by reducing the number of hotspots in the tumor-stromal bed (10) .

Another angiogenesis modulator is NOS2, which appears to exert a direct effect on several angiogenic factors such as VEGF. Through the depletion of intracellular iron, the expression of VEGF is activated (11) . These observations were further confirmed by Ambs et al. (12) , who reported higher VEGF protein and mRNA levels in NOS2-expressing cells when compared with the control vector-containing cell lines. These levels were reduced when an inhibitor was added to the culture (12) . NOS2 can induce COX2 through the overexpression of nuclear factor-B and its dimer subunit, p60/p65, which enters the nucleus and induces NOS2 and COX2 among other genes (13) . Although VEGF levels appear to be mediated primarily by hypoxia-induced factor-1 (7) , there is evidence to suggest that both NOS2 and COX2 may play a role in the signaling pathway that leads to its overexpression. NOS2 is known to function as an up-regulator of VEGF-regulated kinases and mitogen-activated protein kinases (14) . In contrast, wild-type p53 exerts a significant influence on the process through the induction of thrombospondin-1 (15) , which down-regulates NOS2, COX2, and VEGF directly (15) , or though the activation of pro-TGF-ß to active TGF-ß, a major suppressor of these protein levels (16) . In addition, p53 trans-suppresses COX2 levels directly (17) . Our previous work has shown that wild-type p53 may play a more central role in the regulation of angiogenesis through its direct control loop of NOS2 and VEGF (8 , 12 , 18) .

Tumor angiogenesis has received attention as a plausible candidate in relation to prognosis in NSCLC. Several studies have found that VEGF, VEGF receptors, and MVD have a direct correlation with prognosis (2 , 19) , node-free intervals, and relapse- and recurrence-free periods (20) . However, other studies were not as conclusive (21 , 22) . The discrepancy in these results can be attributed to several variables, such as tumor histology and the type of vascular marker used to measure MVD.

In a study of 108 NSCLC samples, where tissues were stained with CD34 and factor VIII, Yano et al. (19) concluded that MVD, as measured by CD34 and VEGF levels, correlated with survival, postoperative recurrence, and metastasis in ADC. No such correlation was found when MVD was measured by factor VIII (23) . Giatromanolaki et al. (24) found a positive correlation with CD31 MVD but not with factor VIII. In a separate study of 87 NSCLC samples, Shijubo et al. (25) reported that the levels of VEGF, osteopontin, and MVD were higher and correlated with a poor clinical outcome in ADC patients in comparison with SCC. The discrepancy as to the role of angiogenesis and patient survival may be related to the morphology of BVs and the type of VEGF isoform present. Data from a study of 500 NSCLC samples showed that neoangiogenesis could differ in its morphological appearance. Three patterns were characterized by the destruction of lung parenchyma and the production of new BVs. The fourth pattern, which was called alveolar and presented in 16% of the tumors, showed a lack of parenchymal destruction and the absence of both tumor-associated stroma invasion and neoangiogenesis (26) . Furthermore, VEGF forms four isoforms involving alternative splicing, resulting in amino acid sequences ranging from 121 to 206. Isoform expressions were measured on normal and transformed lung and colon tissues. Shorter peptides (121 and 165) were found at higher levels in malignant tissues, whereas longer isoforms were observed in normal tissues. These observations suggest that during malignant transformation, a switch takes place to a more active bioavailable and diffusible form of VEGF through alternative splicing (27) .

We have studied the levels of NOS2, COX2, and VEGF protein levels in 106 surgically resected NSCLC tumors and correlated these levels with MVD. Furthermore, we investigated the effect of these markers on tumor size, histology, and clinical outcome.

	MATERIALS AND METHODS

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Patient Population.
One hundred and six patients with surgically resected NSCLC tumors were included in this study, all of which were collected prospectively from the Mayo Clinic (Rochester, MN) from 1992 to 1993. The epidemiological data including demographics, family history, occupational exposure, medical history, and histopathological data on these patients have been reported previously (28) . The patients included 62 males and 44 females. All but 4 patients were cigarette smokers, with an average pack-year history of 63.1 for men and 41.5 for women. Histologically, 55 cases (52%) had ADCs, 29 had SCCs (27%), and 22 had LCCs (21%). The clinical stages are summarized in Table 1 . The tumor status (T) was available on all patients. In addition, the lymph node status and the clinical stage were known for all but 2 and 1 patient, respectively.

Statistical Analysis.
Statistical analysis was performed using SPSS version 10 (Chicago, IL). The Spearman rank-order correlation coefficient was used to assess the relation among VEGF, NOS2, COX2 (using the combined score of intensity and distribution), MVD, and other continuous variables. Associations among a variety of variables, including, gender, tumor histology, smoking history, and family history of malignancy, were evaluated using the ² test for heterogeneity or Fisher’s exact test as appropriate. Kaplan-Meier analysis was used to assess the relation of disease-free survival to overexpression using the log-rank test. Student’s t test was used to compare the continuous variables including age at diagnosis and lifetime smoking dose expressed in pack-years, by the nominally classified VEGF, NOS2, and COX2. Associations were considered statistically significant if the two-tailed P was <0.05.

	RESULTS

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The status of NOS2, COX2, VEGF, and MVD are summarized in relation to demographic, histopathological, and clinical data in Table 1 . Of 106 cases, 51 tumors showed anti-NOS2 immunoreactivity of 2 or higher (48%; Fig.1 A). Fifty-one (48%) samples demonstrated a score of 2 or higher for anti-COX2 (Fig. 1 B), and 61 tumors (58%) showed anti-VEGF scores of 2 or higher (Fig. 1 C). Both NOS2 and COX2 levels showed a positive correlation with VEGF overexpression (P < 0.001 and P < 0.03, respectively). NOS2 levels of overexpression were higher in certain histological subtypes, such as LCCs (16 of 22; 73%) and ADCs (28 of 55; 51%). NOS2 overexpression in SCC was uncommon and only seen in 7 of 29 samples (24%; P < 0.002). However, increased expression of COX2 or VEGF did not show a preference to any histological subtypes of tumor. Increased levels of VEGF were seen in 34 ADCs (62%), 13 SCCs (45%), and 14 LCCs (64%). COX2 overexpression was present in 26 ADCs (47%), 14 SCCs (52%), and 11 LCCs (50%). In addition, elevated VEGF levels were found in 39 (76%) and 36 (71%) of all tumors.

View larger version (117K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Angiogenic proteins in a NSCLC case. A, NOS2 expression in a female patient with ADC. Reactivity is seen in the cytoplasm. This case received a score of 2. B, COX2 status. In addition to tumor cell reactivity with anti-COX2 MAb, adjacent mononuclear inflammatory cells show the enzyme expression as well. C, represents the VEGF status. Intense cytoplasmic staining is observed in tumor cells. Reactivity also is present in intratumoral blood vessels. x200. CD31 immunoreactivity in two samples with different BV density. High BV count is seen in D as compared with low vascularity in E. x100.

	DISCUSSION

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The data presented here highlights three significant findings: (a) NOS2, COX2, and VEGF exhibit overexpression in 50% or more of the tumors; (b) increased levels of NOS2, COX2, and VEGF are directly correlated with MVD, as measured by the number of CD31-reactive BVs at the tumor-stromal interphase; and (c) the incidence of NOS2 overexpression was more common in ADC and LCC tumors when compared with SCCs. Although the NOS2 and COX2 status have been investigated in several neoplastic and preneoplastic conditions, little work has been done on the lung. In a separate work and on a limited number of samples, Ambs et al. (8) were able to detect increased levels of NOS2 in NSCLC samples. Furthermore, Fujimoto et al. (29) in a study of 72 primary NSCLC samples were able to detect high levels of NOS2 more frequently in ADCs than other histotypes. In five of eight cases with high NOS2 overexpression, a p53 transversion, G:C to T:A, was identified, suggesting that NOS2 may play a key role in the carcinogenesis of certain histological types of lung cancer (30) . Although our data showed increased overexpression of NOS2 in ADC and LCC histological subtypes, we were not able to correlate with p53 GT transversions that were published earlier by our group (28) . On the other hand, the ADC subtype presents with several unique features both clinically and biologically. The tendency to predominate in women, and individuals with no known history of tobacco exposure (31) , and the tendency for early hematological dissemination suggests a different carcinogenic pathway, perhaps involving endocrine factors (32) . Our work and others have shown a difference in frequency and spectra of p53 mutations in various NSCLC histological subtypes. Overall, p53 mutations were 2.5 higher in SCCs with greater propensity for GT transversions than those observed in ADCs (33 , 34) . The MVD in our cohorts of tumors may explain the higher incidence of early metastasis observed in the course of ADCs, because the mean MVD in ADCs was 79/mm² , compared with SCCs, with a mean of 64 (P < 0.05).

Our angiogenesis data did not correlate with patients’ outcome and prognosis. This is not surprising in light of contradicting data on the role of angiogenesis, because biological and prognostic markers have been reported (19 , 22 , 25) . These conflicting results may be attributed to several variables. In a study of 108 NSCLC samples, Yano et al. (19) showed that MVD levels as measured by CD34 had a direct relation with survival; however, using the same tumor samples, similar results could not be observed with factor VIII. Similarly, CD31, the most widely used vascular marker, is known to recognize an epitope present in both mature and immature venules and capillaries. Recently, Kakolyris et al. (35) observed that the presence of immature newly formed BVs can serve as a better indicator for neoangiogenesis. However, more tumors with MVD of >100 BVs/mm² presented in higher clinical stages than those with MVD of <100 BVs/mm² (62% versus 44% with a P < 0.01). Furthermore, the results were more significant when tumors were separated based on their histologies (P < 0.001).

The variability of the results may be attributed to the histological type of lung cancer used in these studies. Some investigators have reported that MVD and VEGF and its receptor status are associated with ADCs (19 , 21) but not SCCs (34) , whereas others have suggested only SCC tumors showed such correlation (36) , and occasionally with a subgroup of SCC tumors (37) . The methodology to measure MVD (manual versus computer-assisted imaging) as well as the site where MVD is measured can contribute to the discrepancy of the role of angiogenesis and patients’ survival. Some investigators have used the number of BVs per high-power field as an index for MVD (38) , whereas others have used the number of hotspots at the tumor-stromal interphase (2) . Still, other factors for this controversy may be attributed to the epitope of anti-VEGF MAb used. Evidence of several different VEGF isoforms secreted by different NSCLC tumors has been reported (35) . The morphology of the new vessel formation may yet represent another variable factor, because only BVs that display host-stromal infiltration appear to correlate with poor outcome (27) . Finally, our cohort represented lung cancer patients that were good candidates for surgical resection. Those with T4 tumors or N3 were not included in this group. This clinical bias also may explain the lack of correlation of MVD and other angiogenic factors studied with patient survival or tumor behavior.

Angiogenesis appears to play a significant role in cancer progression and evolution. Thus, novel therapies have emerged that use this phenomenon as a target for cancer therapies. To date, more than three dozen clinical trials have been approved that target tumor angiogenic and antiangiogenic factors such as endostatin, VEGF, IFN-2a, and -2-macroglobulin, or tissue inhibitors of metalloproteases and pharmaceutically prepared agents such as selenium-based drugs, e.g., Thalidomide, Aplidine, and others (39, 40, 41, 42) . However, we have shown that COX2, NOS2, and VEGF levels correlate with the degree of MVD individually, and their levels appear to correlate with each other. Thus, it is likely that angiogenesis-based treatment protocols that target individual proteins will have modest yields and perhaps disappointing results.

	ACKNOWLEDGMENTS

 
We thank Dorothea Dudek for editorial assistance.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom requests for reprints should be addressed, at Laboratory of Human Carcinogenesis, National Cancer Institute, NIH, Building 37, Room 2C05, MSC 4255, Bethesda, MD 20892-4255. Phone: (301) 496-2048; Fax (301) 496-0497; E-mail: Curtis_Harris{at}nih.gov

² The abbreviations used are: TGF, transforming growth factor; VEGF, vascular endothelial growth factor; COX, cyclooxygenase; NOS2, nitric oxide synthase; NSCLC, non-small cell lung cancer; MVD, microvessel density; ADC, adenocarcinoma; LCC, large cell carcinoma; BV, blood vessel; MAb, monoclonal antibody.

Received 6/20/00; revised 8/18/00; accepted 9/13/00.

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				T. Chen, M. E. Rose, H. Hwang, R. G. Nines, and G. D. Stoner Black raspberries inhibit N-nitrosomethylbenzylamine (NMBA)-induced angiogenesis in rat esophagus parallel to the suppression of COX-2 and iNOS Carcinogenesis, November 1, 2006; 27(11): 2301 - 2307. [Abstract] [Full Text] [PDF]

				H. Tsubochi, N. Sato, M. Hiyama, M. Kaimori, S. Endo, Y. Sohara, and T. Imai Combined Analysis of Cyclooxygenase-2 Expression With p53 and Ki-67 in Nonsmall Cell Lung Cancer. Ann. Thorac. Surg., October 1, 2006; 82(4): 1198 - 1204. [Abstract] [Full Text] [PDF]

				M. Ding, R. Feng, S. Y. Wang, L. Bowman, Y. Lu, Y. Qian, V. Castranova, B.-H. Jiang, and X. Shi Cyanidin-3-glucoside, a Natural Product Derived from Blackberry, Exhibits Chemopreventive and Chemotherapeutic Activity J. Biol. Chem., June 23, 2006; 281(25): 17359 - 17368. [Abstract] [Full Text] [PDF]

				J. H. Byun, M. A. Lee, S. Y. Roh, B. Y. Shim, S. H. Hong, Y. H. Ko, S. J. Ko, I. S. Woo, J. H. Kang, Y. S. Hong, et al. Association between Cyclooxygenase-2 and Matrix Metalloproteinase-2 Expression in Non-Small Cell Lung Cancer Jpn. J. Clin. Oncol., May 1, 2006; 36(5): 263 - 268. [Abstract] [Full Text] [PDF]

				R. S. Pruthi, J. E. Derksen, D. Moore, C. C. Carson, G. Grigson, C. Watkins, and E. Wallen Phase II Trial of Celecoxib in Prostate-Specific Antigen Recurrent Prostate Cancer after Definitive Radiation Therapy or Radical Prostatectomy. Clin. Cancer Res., April 1, 2006; 12(7): 2172 - 2177. [Abstract] [Full Text] [PDF]

				C. R. Mulligan, A. D. Meram, C. D. Proctor, H. Wu, K. Zhu, and A. J. Marrogi Unlimited Access to Care: Effect on Racial Disparity and Prognostic Factors in Lung Cancer Cancer Epidemiol. Biomarkers Prev., January 1, 2006; 15(1): 25 - 31. [Abstract] [Full Text] [PDF]

				W. H. Sun, Y. L. Sun, R. N. Fang, Y. Shao, H. C. Xu, Q. P. Xue, G. X. Ding, and Y. L. Cheng Expression of Cyclooxygenase-2 and Matrix Metalloproteinase-9 in Gastric Carcinoma and its Correlation with Angiogenesis Jpn. J. Clin. Oncol., December 1, 2005; 35(12): 707 - 713. [Abstract] [Full Text] [PDF]

				J. Zhang, B. Peng, and X. Chen Expressions of Nuclear Factor {kappa}B, Inducible Nitric Oxide Synthase, and Vascular Endothelial Growth Factor in Adenoid Cystic Carcinoma of Salivary Glands: Correlations with the Angiogenesis and Clinical Outcome Clin. Cancer Res., October 15, 2005; 11(20): 7334 - 7343. [Abstract] [Full Text] [PDF]

				K. Vermaelen and R. Pauwels Pulmonary Dendritic Cells Am. J. Respir. Crit. Care Med., September 1, 2005; 172(5): 530 - 551. [Abstract] [Full Text] [PDF]

				L. D. Dwyer-Nield, M. C. Srebernak, B. S. Barrett, J. Ahn, P. Cosper, A. M. Meyer, L. R. Kisley, A. K. Bauer, D. C. Thompson, and A. M. Malkinson Cytokines differentially regulate the synthesis of prostanoid and nitric oxide mediators in tumorigenic versus non-tumorigenic mouse lung epithelial cell lines Carcinogenesis, July 1, 2005; 26(7): 1196 - 1206. [Abstract] [Full Text] [PDF]

				P. G. Phillips and L. M. Birnby Nitric oxide modulates caveolin-1 and matrix metalloproteinase-9 expression and distribution at the endothelial cell/tumor cell interface Am J Physiol Lung Cell Mol Physiol, May 1, 2004; 286(5): L1055 - L1065. [Abstract] [Full Text] [PDF]

				J. R. Brown and R. N. DuBois Cyclooxygenase-2 in Lung Carcinogenesis and Chemoprevention: Roger S. Mitchell Lecture Chest, May 1, 2004; 125(5_suppl): 134S - 140S. [Full Text] [PDF]

				F. Cianchi, C. Cortesini, O. Fantappie, L. Messerini, I. Sardi, N. Lasagna, F. Perna, V. Fabbroni, A. Di Felice, G. Perigli, et al. Cyclooxygenase-2 Activation Mediates the Proangiogenic Effect of Nitric Oxide in Colorectal Cancer Clin. Cancer Res., April 15, 2004; 10(8): 2694 - 2704. [Abstract] [Full Text] [PDF]

				J. A. Crowell, V. E. Steele, C. C. Sigman, and J. R. Fay Is Inducible Nitric Oxide Synthase a Target for Chemoprevention? Mol. Cancer Ther., August 1, 2003; 2(8): 815 - 823. [Abstract] [Full Text] [PDF]

				F. Cianchi, C. Cortesini, O. Fantappie, L. Messerini, N. Schiavone, A. Vannacci, S. Nistri, I. Sardi, G. Baroni, C. Marzocca, et al. Inducible Nitric Oxide Synthase Expression in Human Colorectal Cancer: Correlation with Tumor Angiogenesis Am. J. Pathol., March 1, 2003; 162(3): 793 - 801. [Abstract] [Full Text] [PDF]

				S. Ekmekcioglu, J. A. Ellerhorst, J. B. Mumm, M. Zheng, L. Broemeling, V. G. Prieto, A. L. Stewart, A. M. Mhashilkar, S. Chada, and E. A. Grimm Negative Association of Melanoma Differentiation-associated Gene (mda-7) and Inducible Nitric Oxide Synthase (iNOS) in Human Melanoma: MDA-7 Regulates iNOS Expression in Melanoma Cells Mol. Cancer Ther., January 1, 2003; 2(1): 9 - 17. [Abstract] [Full Text] [PDF]

				L. R. Kisley, B. S. Barrett, A. K. Bauer, L. D. Dwyer-Nield, B. Barthel, A. M. Meyer, D. C. Thompson, and A. M. Malkinson Genetic Ablation of Inducible Nitric Oxide Synthase Decreases Mouse Lung Tumorigenesis Cancer Res., December 1, 2002; 62(23): 6850 - 6856. [Abstract] [Full Text] [PDF]

				L. Hlatky, P. Hahnfeldt, and J. Folkman Clinical Application of Antiangiogenic Therapy: Microvessel Density, What It Does and Doesn't Tell Us J Natl Cancer Inst, June 19, 2002; 94(12): 883 - 893. [Full Text] [PDF]

				G. Davies, L.-A. Martin, N. Sacks, and M. Dowsett Cyclooxygenase-2 (COX-2), aromatase and breast cancer: a possible role for COX-2 inhibitors in breast cancer chemoprevention Ann. Onc., May 1, 2002; 13(5): 669 - 678. [Abstract] [Full Text] [PDF]

				J. M. Wallace Nutritional and Botanical Modulation of the Inflammatory Cascade--Eicosanoids, Cyclooxygenases, and Lipoxygenases-- As an Adjunct in Cancer Therapy Integr Cancer Ther, March 1, 2002; 1(1): 7 - 37. [Abstract] [PDF]

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				S. A. Nicholson, N. T. Okby, M. A. Khan, J. A. Welsh, M. G. McMenamin, W. D. Travis, J. R. Jett, H. D. Tazelaar, V. Trastek, P. C. Pairolero, et al. Alterations of p14ARF, p53, and p73 Genes Involved in the E2F-1-mediated Apoptotic Pathways in Non-Small Cell Lung Carcinoma Cancer Res., July 1, 2001; 61(14): 5636 - 5643. [Abstract] [Full Text] [PDF]

Md. A. Rahman, D. K. Dhar, E. Yamaguchi, S. Maruyama, T. Sato, H. Hayashi, T. Ono, A. Yamanoi, H. Kohno, and N. Nagasue Coexpression of Inducible Nitric Oxide Synthase and COX-2 in Hepatocellular Carcinoma and Surrounding Liver: Possible Involvement of COX-2 in the Angiogenesis of Hepatitis C Virus-positive Cases Clin. Cancer Res., May 1, 2001; 7(5): 1325 - 1332. [Abstract] [Full Text]