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Molecular Biology and Genetics

Cyclooxygenase 2- and Prostaglandin E2 Receptor EP2-dependent Angiogenesis in ApcΔ716 Mouse Intestinal Polyps

Hiroshi Seno, Masanobu Oshima, Tomo-o Ishikawa, Hiroko Oshima, Kazuaki Takaku, Tsutomu Chiba, Shuh Narumiya and Makoto M. Taketo
DOI:  Published January 2002

Abstract

To investigate angiogenesis during intestinal polyp development, we determined the microvessel density (MVD) in polyps of Apc knockout (ApcΔ716) mice, a model for human familial adenomatous polyposis. We scored MVD also in several compound mutants carrying ApcΔ716, namely, mice with an additional mutation in Smad4, in which the polyps progress into invasive adenocarcinomas; mice with a cyclooxygenase (COX)-2 gene (Ptgs2) mutation, in which adenoma growth is suppressed; and mice with prostaglandin E2 EP receptor gene mutations. In both simple ApcΔ716 and compound ApcΔ716 Smad4 mutants, MVD increased in a polyp size-dependent manner only in the polyps expanded beyond a threshold of about 1 mm in diameter. These results indicate that tumor angiogenesis is stimulated only after tumors grow to a certain size, and this angiogenic switch is common to both benign adenomas and malignant adenocarcinomas. In ApcΔ716 polyposis attenuated by the COX-2 gene mutation, in contrast, MVD did not increase even in polyps larger than 1 mm. The same phenomenon was observed in the compound mutant mice with ApcΔ716 and the EP2 receptor gene mutations, but not in other EP compound mutants. We also immunohistochemically studied COX-2 and angiogenic factors, vascular endothelial growth factor and basic fibroblast growth factor. Interestingly, expression of these proteins was also increased in polyps larger than 1 mm. These results suggest that, in both benign and malignant mouse intestinal tumors, stromal expression of COX-2 results in elevated prostaglandin E2 levels that stimulate cell surface receptor EP2, followed by induction of vascular endothelial growth factor that causes tumor angiogenesis.

INTRODUCTION

Angiogenesis is one of the key mechanisms that support tumor development (1, 2, 3) . In addition to observations that angiogenesis occurs in clinical tumors, many transplantation experiments in animals demonstrated a strong association between angiogenesis and tumor growth as well as metastasis. After the seminal proposal by Folkman that blocking angiogenesis could be a strategy to arrest tumor growth (4) , intensive searches were taken by his and other groups for pro- and antiangiogenic molecules (5) . At the same time, studies of several tumor models in transgenic mice revealed that induction of angiogenesis is a discrete component of the tumor phenotype, one that is often activated during the early preneoplastic stages in the development of tumor (5) . Namely, in each model of islet cell carcinoma (6) , dermal fibrosarcoma (7) , and epidermal squamous cell carcinoma (5) , an angiogenic switch could be visualized during early stages preceding the appearance of solid tumors. Different from vasculogenesis in embryonic development, tumor vessels develop by sprouting or intussusception from preexisting vessels (3) . In addition, tumor vessels lack protective mechanisms such as functional perivascular cells (8) . Moreover, tumor vessel wall is not always formed by a homogeneous layer of endothelial cells. Instead, it may be lined with only cancer cells or a mosaic of cancer and endothelial cells (9) . At the same time, tumor vasculature is highly disorganized, vessels are tortuous and dilated, with uneven diameter, excessive branching, and shunts. Their walls have numerous “openings,” causing a high vascular permeability (9) . These observations indicate that tumor vessels are structurally and functionally abnormal, not only in their quantitative density but also in their qualitative characteristics. In fact, studies of the expression phage libraries yielded homing peptides that preferentially recognize tumor vessels (10) . Likewise, comparison of gene expression patterns of blood vessels derived from normal mucosa and human colorectal cancer led to identification of genes specifically expressed in the tumor endothelium (11) .

To determine the role of angiogenesis in colorectal carcinogenesis, we have investigated our mouse models of colon cancer (Fig. 1)⇓ . We earlier constructed Apc3 knockout (ApcΔ716) mice that develop numerous benign polyps in the intestines (12) and can be used as a model for human FAP. After Apc LOH, a microadenoma is initiated as an outpocketing pouch in a single intestinal crypt and develops into a polyp adenoma (13) . We also constructed a compound mutant strain that carried ApcΔ716 and Smad4 mutations and demonstrated that the mice develop very invasive adenocarcinomas that are much larger than the ApcΔ716 polyp adenomas (14) . On the other hand, in another compound mutant strain with ApcΔ716 and the COX-2 gene (Ptgs2) mutations, the polyp number and size are reduced dramatically (15) , establishing the rationale for treating colonic polyposis in FAP with COX-2 inhibitors (16 , 17) .

Fig. 1.
Fig. 1.

Schematic drawing of three mouse models for human colon polyposis and cancer. The ApcΔ716 mice carry a knockout mutation in one of the Apc alleles and develop numerous adenomatous polyps in the intestines after loss of the remaining Apc allele; LOH (12) . In this model for human FAP, a nascent polyp is formed from an outpocketing pouch of the proliferative zone crypt epithelium, expanding into the inner (lacteal) side of a villus (13) . In A, when such a nascent polyp expands, COX-2 is induced in the polyp stroma and plays an essential role in further growth. In B, disruption of the COX-2 gene (Ptgs2) or inhibition of COX-2 by a specific inhibitor suppresses the tumor growth (15) . In C, on the other hand, introduction into the ApcΔ716 mice of an additional knockout mutation of Smad4, a gene in the transforming growth factor β signaling, quickly converts the benign adenoma into a rapidly growing and invasive adenocarcinoma (14) . Blue, normal villous epithelium; yellow, normal crypt epithelium; pink, tumor (adenoma or adenocarcinoma) epithelium; black, COX-2-expressing stromal cells; white, the activated fibroblasts; purple, the submucosal smooth muscle layer.

Here we show the results of the MVD determinations (18) in the course of intestinal tumor growth, focusing on the difference in angiogenesis between benign adenomas with or without COX-2 expression, and malignant adenocarcinomas. We also present the results of angiogenic gene expression studies in these mouse tumors, as well as the effects of mutations in the PGE2 receptors.

MATERIALS AND METHODS

Animals.

Constructions of ApcΔ716, Smad4, and Ptgs2 knockout mice, and ApcΔ716 compound mutants with Smad4 and Ptgs2, respectively, were described previously (12, 13, 14, 15) . Construction of PGE2 receptor gene knockout mice was also described (19) . Using five mice for each Ptgs2 genotype, we examined 314, 116, and 51 intestinal polyps from the ApcΔ716 Ptgs2(+/+), ApcΔ716 Ptgs2(+/−), and ApcΔ716 Ptgs2(−/−) mice, respectively. We also examined 33 polyps from eight cis-ApcΔ716 Smad4 mice; 5 polyps larger than 1 mm of 28 polyps from three ApcΔ716 EP2(−/−) mice; 21polyps from five ApcΔ716 EP1(−/−) mice; and 13 polyps from four ApcΔ716 EP3(−/−) mice.

Tissue Preparation and Immunohistochemistry.

Small intestinal sections were prepared as described previously (13, 14, 15) . They were blocked with 10% normal goat or rabbit serum, and incubated for 2 h at room temperature with polyclonal anti-vWF rabbit serum (1:500; DAKO, Copenhagen, Denmark); polyclonal anti-vascular VEGF goat serum (1:300; R&D Systems, Minneapolis, MN) after a 0.1% trypsin pretreatment; or polyclonal anti-COX-2 goat serum (1:300; Santa Cruz Biotechnology, Santa Cruz, CA). For immunostainings of bFGF, the sections were incubated with anti-bFGF rabbit serum (1:2000; Chemicon International, Temecula, CA) for 12 h at 4°C. The immune complex was visualized using Vectastain Elite kit (Vector Laboratories, Burlingame, CA) according to the manufacturer’s protocol. Sensitivities and specificities of the antibodies had been verified previously (18 , 20, 21, 22) . Sections incubated with the normal rabbit or goat serum served as negative controls.

Determination of MVD.

MVD was determined as described previously (18) . Briefly, the microscopic field that contained the highest number of capillaries was chosen for each sample by an initial scan at a low-magnification (×100). Then, the vessels were counted in high-magnification fields (×400).

Statistical Analysis.

Nonrepeated measures ANOVA, Dunnett’s test, unpaired Student’s t tests, and χ2 test were used for comparison of the data sets, and Ps smaller than 0.01 were considered as statistically significant.

RESULTS

Correlation of MVD with Intestinal Polyp Size.

The MVD is a well-established parameter of angiogenesis in vivo (18) . To determine MVD in early-stage polyps, we immunostained polyp sections with an antibody for vWF and scored the stained vessels in the mutant mice that we established earlier (Fig. 1)⇓ . The ApcΔ716 heterozygous mutant is a mouse model for FAP (12) , whereas the polyps in the cis-compound ApcΔ716 Smad4 heterozygotes progress to adenocarcinomas with marked submucosal invasion and desmoplasia (14) . On the other hand, introduction of a COX-2 gene (Ptgs2) mutation into the ApcΔ716 mice dramatically attenuates intestinal polyposis (15) .

In polyps smaller than 1 mm, small numbers of stained vessels were found in mice of all of the genotypes examined (Fig. 2A⇓ , and data not shown). In contrast, in larger polyps of the ApcΔ716 and cis-ApcΔ716 Smad4 mice, abundant vessels were found in the tumor stroma near the luminal surface (Fig. 2B⇓ , and data not shown). These results indicate that angiogenesis is stimulated only after the polyps expand to about 1 mm in diameter (see next paragraph). Furthermore, some vessels with relatively large lumens (often >50 μm) were found at the base of the polyps larger than 1 mm (Fig. 2C)⇓ , although such large vessels were not prominent in smaller polyps nor in the normal mucosa (Fig. 2D)⇓ . To characterize these vessels, we then immunostained the sections with an antibody for αSMA, a marker for periendothelial cells. It has been reported that newly formed tumor vessels might not have periendothelial cells (8) . The small vessels near the polyp luminal surface were not stained for αSMA (Fig. 2E)⇓ , although they were strongly positive for vWF (Fig. 2B)⇓ . In contrast, most vessels near the polyp base (Fig. 2C)⇓ were strongly stained for αSMA (Fig. 2F)⇓ . These data suggest that the small vessels near the luminal surface are newly formed vessels, whereas the vessels near the polyp base are mature arterioles or venules expanded from preexisting vessels. In other words, two vascular changes take place in polyp formation: genuine angiogenesis at the luminal side, and expansion of preexisting vessels at the polyp base (see below). The increase in MVD was contributed essentially by that of the angiogenic vessels on the luminal side of the polyp, and they reached about 65% of the total vessels in large polyps.

Fig. 2.
Fig. 2.

Immunostaining of microvessels with antibodies for vWF or αSMA in the small intestinal polyps of the ApcΔ716 mice. A, section with a polyp smaller than 1 mm in diameter (∗) stained for vWF. B, luminal side of a large polyp stained for vWF. C, basal region of the same polyp shown in B and E stained for vWF. D, microvessels stained for vWF among the normal crypts. E, section adjoining to that shown in B stained for αSMA. F, section adjoining to that shown in C stained for αSMA. Arrows, microvessels; scale bars, 50 μm.

The relationship of MVD with the polyp size is summarized as a histogram in Fig. 3A⇓ . The mean MVD was approximately 18 in the polyps smaller than 1 mm, without any significant difference from that in the normal intestinal mucosa. In larger polyps of ApcΔ716 and cis-ApcΔ716 Smad4 mice, on the other hand, MVD was significantly higher than in the normal mucosa, correlating with the polyp size. Interestingly, however, there was no significant difference in MVD between the polyps of the ApcΔ716 polyps and the cis-ApcΔ716 Smad4 compound mutant. In polyps larger than 3 mm, MVD was ∼29, i.e., 1.6 times higher than that in the nascent polyps (<0.5 mm). It is worth noting that, in the cis-ApcΔ716 Smad4 compound mutant, most polyps larger than 3 mm were invasive adenocarcinomas (14) . In contrast, MVD in the ApcΔ716 Ptgs2(+/−) mice was not significantly higher even in the small number of polyps larger than 1 mm. In the ApcΔ716 Ptgs2(−/−) mice, polyps larger than 1 mm were scarcely seen, and we could not determine their MVD in a statistically meaningful manner. These results suggest that polyps in the 1∼2-mm size class could not expand further because of the lack of MVD increase and indicate that COX-2 plays a key role in angiogenesis in these mouse intestinal tumors.

Fig. 3.
Fig. 3.

The polyp MVDs. A, MVD in the simple ApcΔ716, and compound ApcΔ716 Ptgs2(+/−), ApcΔ716 Ptgs2(−/−), and cis-ApcΔ716 Smad4 mutant mice. (∗, P < 0.01). B, MVD in the compound ApcΔ716 EP1(−/−) and ApcΔ716 EP2(−/−) mutant polyps compared with those in simple ApcΔ716 (∗, P < 0.01). In the ApcΔ716 Ptgs2(−/−) mice, polyps larger than 1 mm (†) were scarce, whereas in the ApcΔ716 Ptgs2(+/−) and ApcΔ716 EP2(−/−) mice, polyps larger than 2 mm (‡) were rare, and MVD could not be evaluated, accordingly.

COX-2 Effects on Angiogenesis in ApcΔ716 Polyps Are Mediated by PGE2 Receptor EP2.

The direct metabolite of arachidonic acid by COX-2 is prostaglandin H2, which is subsequently metabolized to various prostanoids by converting enzymes. Among them, PGE2 appears to be the major prostanoid responsible for polyposis, because its level is significantly elevated in the human FAP polyps (23) . Four PGE2 receptor subtypes, EP1, EP2, EP3, and EP4 have been identified and cloned (24) . Although all these receptors are coupled with G proteins, the intracellular effects by activation of these receptors are diverse. It has been reported that EP1 increases the cytoplasmic Ca2+ concentration (25) . Whereas EP2 and EP4 increase the cAMP level, EP3 decreases it (25) . By crossing ApcΔ716 knockout mice with the respective EP receptor gene knockout mice (19) , we recently demonstrated that the compound mutation with the EP2 gene homozygosity caused a significant suppression of intestinal polyposis; a phenotype very similar to that of the Apc and Ptgs2 compound mutation (26) . To determine whether this suppression of intestinal polyposis was caused by angiogenesis attenuation, we scored MVD in the polyps of the compound mutants between the ApcΔ716 and EP receptor gene knockouts for EP1, EP2, and EP3, respectively. Because most EP4 gene homozygotes are neonatally lethal (19) , its compound mutants with ApcΔ716 could not be investigated.

In the polyps smaller than 1 mm, MVD was essentially the same in all three EP compound mutant strains (Fig. 3B⇓ , and data not shown). As in the compound mutants with Ptgs2(+/−) (Fig. 3A)⇓ , however, the polyps between 1 and 2 mm in the EP2(−/−) compound mutant exhibited a significantly lower MVD than in the simple ApcΔ716 polyps (Fig. 3B)⇓ . This MVD was essentially at the same level as that of the normal mucosa and the polyps smaller than 1 mm. Polyps larger than 2 mm were very rare and could not be evaluated in the EP2 (−/−) compound mutant. On the other hand, the polyp MVD in the compound mutants with the EP1(−/−) or EP3 (−/−) did not show any significant difference from that in the simple ApcΔ716 polyps in any size classes (Fig. 3B⇓ , and data not shown). These results strongly indicate that the suppression of polyp angiogenesis in the COX-2 gene mutant polyps is caused by the lack of the PGE2 signaling through the EP2 receptor. In other words, intestinal polyposis in the simple ApcΔ716 heterozygotes is stimulated by the COX-2-induced PGE2 signal that is transduced through the EP2 receptor to cause angiogenesis. It is worth noting, however, that the extent of angiogenesis suppression in the ApcΔ716 EP2(−/−) polyps was similar to that in the ApcΔ716 Ptgs2 (+/−) polyps, and that the ApcΔ716 Ptgs2 (−/−) mice produced few polyps larger than 1 mm. These results collectively suggest that COX-2 plays some additional roles in polyp formation other than the EP2-mediated angiogenesis (see “Discussion”).

Induction of COX-2 in Polyps Larger Than 1 mm in Diameter.

To further investigate the role of COX-2 in angiogenesis, we determined COX-2 expression by immunostaining the intestinal polyps in the ApcΔ716 mice. Polyps smaller than 1 mm showed little COX-2 expression (Fig. 4A)⇓ . In contrast, >75% of the polyps larger than 1 mm expressed COX-2 on the luminal side. As we reported previously (15 , 22 , 27 , 28) , this expression was found exclusively in the polyp stromal cells but not in the adenoma epithelium (Fig. 4, B and C)⇓ . These results are summarized as a histogram in Fig. 4J⇓ . Like the higher MVD, COX-2 expression at significant levels was found only in the polyps larger than 1 mm in diameter (compare Fig. 4J⇓ with Fig. 3A⇓ ), and the proportion of COX-2-stained polyps was significantly higher in the polyps that were larger than 1 mm in diameter (P < 0.01).

Fig. 4.
Fig. 4.

Immunohistochemistry of COX-2, VEGF, and bFGF in the ApcΔ716 mouse intestinal polyps. A–C, staining for COX-2 in polyps; smaller than 1 mm in diameter (∗ in A), and larger than 1 mm (B and C), respectively. D–F, staining for VEGF in polyps; smaller than 1 mm (∗ in D), and larger than 1 mm (E and F), respectively. G-I, staining for bFGF in polyps; smaller than 1 mm (∗ in G), and larger than 1 mm (H and I), respectively. G, arrowheads, weak staining in the stroma of the normal mucosa. C, F, and I show higher magnifications of B, E, and H, respectively. Arrows in B, E, and H, immunostained cells; scale bars, 50 μm. J, COX-2 staining in the ApcΔ716 polyps classified by size. K, VEGF and bFGF stainings in the ApcΔ716 polyps classified by size. L, VEGF and bFGF expressions compared between the COX-2 gene (Ptgs2) wild-type- and heterozygous-ApcΔ716 mouse polyps.

Induction of VEGF and bFGF in a Polyp Size- and COX-2-dependent Manner.

COX-2 has been implicated in the regulation of two angiogenic factors, VEGF and bFGF (29 , 30) . To investigate their roles in the intestinal polyp formation, we analyzed these factors by immunohistochemistry in the ApcΔ716 and ApcΔ716 Ptgs2(+/−) mouse polyps. In the ApcΔ716 mouse, polyps <1 mm in diameter showed little VEGF staining (Fig. 4D)⇓ . However, like COX-2, VEGF was expressed strongly in the stromal cells on the luminal side of the polyps larger than 1 mm (Fig. 4, E and F)⇓ . Although some immunostaining of bFGF was detected in polyps larger than 1 mm, it was found essentially in the stroma at the polyp base (Fig. 4, H and I)⇓ where mature vessels were found (Fig. 2, E and F)⇓ . Weak staining of bFGF was detected also in the stroma of the normal portions of the intestinal mucosa (Fig. 4G⇓ , arrowheads), as reported previously (21) . These results are consistent with the interpretation that VEGF is responsible for polyp angiogenesis, whereas bFGF plays a role in the expansion and maintenance of the preexisting vessels. As summarized in Fig. 4K⇓ , the proportions of the VEGF- and bFGF-expressing polyps, respectively, increased in a size-dependent manner. To investigate the effects of COX-2 on the expression of VEGF and bFGF, we then analyzed these factors in the ApcΔ716 Ptgs2(+/−) mouse intestinal polyps. Although the polyp number and size decreased in the compound mutant, we chose the size class between 1 and 2 mm, because no difference in MVD was detected in the polyps smaller than 1 mm as described above (Fig. 3)⇓ . As shown in Fig. 4L⇓ , the proportions of stained polyps decreased from 90% to 36% for VEGF, and from 80% to 37% for bFGF, respectively, when the COX-2 heterozygous mutation was introduced.

DISCUSSION

Because of the clinical importance, tumor angiogenesis has been investigated essentially in malignant tumors (5) . Many reports show that extensive angiogenesis occurs in the malignant stage of tumor development (1, 2, 3, 4, 5, 6 , 31) . In several transgenic mouse tumor models, however, it has been demonstrated that induction of angiogenesis is a discrete component of the tumor phenotype, one that is often activated during early preneoplastic stages in the development of tumor (5) . Accordingly, we have investigated several mouse models for colon tumorigenesis (Fig. 1)⇓ . As in the earlier transgenic mouse models, the angiogenic switch is turned on at an early stage in our intestinal tumorigenesis models also. Our immunohistochemical analysis showed that VEGF was expressed in the same polyp stromal subregion in which MVD was increased and COX-2 and cytosolic phospholipase A2 were also expressed (26) . Several reports suggest that COX-2 regulates angiogenesis, in part through the induction of VEGF and bFGF (29 , 30) . In our mouse models, the proportion of VEGF-positive polyps was much smaller in the ApcΔ716 Ptgs2(+/−) mice than in the simple ApcΔ716 heterozygotes. Thus, COX-2 appears to regulate angiogenesis through the VEGF induction in the ApcΔ716 polyps, and this induction is dependent on the PGE2 signal that is transduced through the EP2 receptor. It is established that EP2 receptor is coupled with G protein Gs and elevates the cytoplasmic cAMP level (24 , 25) . Because no cAMP responsive elements (CRE) have been found in the 1.2-kb region of the mouse VEGF promoter sequence (32) , it is likely that VEGF expression is induced indirectly by increased cAMP level through protein kinase A (33) . This is consistent with a report that Ptgs2(−/−) mouse fibroblasts produce decreased levels of VEGF (34) . In contrast to VEGF, bFGF was expressed in a different stromal subregion from that of the COX-2 expression, namely, at the polyp base where vessels with relatively large lumens express periendothelial marker αSMA (Fig. 2, E and F⇓ , and Fig. 4, H and I⇓ ). Accordingly, it is conceivable that paracrine factors other than COX-2-induced PGE2 regulate bFGF expression, and that bFGF plays a role in the expansion of preexisting arterioles and venules. This interpretation is consistent with a recent report that VEGF affects the initiation of tumor angiogenesis, whereas bFGF plays a role in both the onset and the maintenance of angiogenesis (35) . It is worth noting that, in endothelial cells, hypoxia induces VEGF expression through hypoxia-inducible factor-1α (36) , and COX-2 appears to be involved in the process as well (37) .

One of the major arachidonic acid metabolites by COX-2 that is elevated in FAP patients is PGE2, implicating its role in polyposis (23) . We recently demonstrated that the cell surface PGE2 receptor EP2 is strongly induced in the ApcΔ716 polyps. Disruption of the EP2 gene in the ApcΔ716 mice reduces the polyp number and size significantly, indicating that the COX-2 signal for polyposis is transduced through EP2 (26) . Because signaling through EP2 increases intracellular cAMP level, and because the COX-2 gene is under the control of cAMP (38) , EP2 is responsible for up-regulation of the COX-2 gene through a positive feedback mechanism by PGE2 (26) . At the same time, we also demonstrated that a basement membrane protein laminin α2, which is secreted from the stromal cells, is strongly up-regulated in the ApcΔ716 polyps through EP2. These results collectively indicate that COX-2 induction plays a dual role in intestinal polyp development: angiogenesis and basement membrane protein biosynthesis.

The results of our MVD analysis suggest that tumor angiogenesis is stimulated not necessarily by malignant transformation but rather by other factors, such as tissue remodeling or hypoxia during tumor enlargement. Recently, it was reported that reexpression of SMAD4 in a SMAD4-negative pancreatic cancer cell line restores slower tumor transplant growth, and that such a change is caused by suppression of angiogenesis rather than the recovery of transforming growth factor β-mediated growth inhibition (39) . Interestingly, this SMAD4-dependent angiogenic change is seen in larger vessels rather than in capillaries, which is inconsistent with our cis-ApcΔ716 Smad4 compound mutant data. It is possible that the pancreatic cancer cell line in the other report contains additional mutations.

In conclusion, MVD increased in a tumor size-dependent manner in both benign and malignant intestinal tumorigenesis models. Tumor angiogenesis is regulated by COX-2 and PGE2 receptor EP2 expressed in the tumor stromal cells rather than by malignant transformation of the adenoma epithelium.

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 Supported by grants from the Ministry of Education, Culture, Sports, Science and Technology and the Organization of Pharmaceutical Safety and Research, Japan.

  • ↵2 To whom requests for reprints should be addressed, at Department of Pharmacology, Kyoto University Graduate School of Medicine, Yoshida-Konoé-cho, Sakyo-ku, Kyoto 606-8501, Japan. Phone: 81-75-753-4391; Fax: 81-75-753-4402; E-mail: taketo{at}mfour.med.kyoto-u.ac.jp

  • ↵3 The abbreviations used are: Apc, adenomatous polyposis coli (gene); αSMA, α-smooth muscle actin; bFGF, basic fibroblast growth factor; cAMP, cyclic AMP; COX-2, cyclooxygenase 2; FAP, familial adenomatous polyposis; LOH, loss of heterozygosity; PGE2, prostaglandin E2; VEGF, vascular endothelial growth factor; vWF, von Willebrand factor; MVD, microvessel density.

  • Received July 16, 2001.
  • Accepted November 12, 2001.

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Cancer Research: 62 (2)
January 2002
Volume 62, Issue 2

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Cyclooxygenase 2- and Prostaglandin E2 Receptor EP2-dependent Angiogenesis in ApcΔ716 Mouse Intestinal Polyps
Hiroshi Seno, Masanobu Oshima, Tomo-o Ishikawa, Hiroko Oshima, Kazuaki Takaku, Tsutomu Chiba, Shuh Narumiya and Makoto M. Taketo
Cancer Res January 15 2002 (62) (2) 506-511;
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