HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fosslien, E. Search for Related Content PubMed PubMed Citation Articles by Fosslien, E.

Molecular Pathology of Cyclooxygenase-2 in Cancer-induced Angiogenesis

Address correspondence to Egil Fosslien, M.D., Department of Pathology (M/C 847), College of Medicine, University of Illinois at Chicago, 1819 West Polk Street, Chicago, IL 60612, USA; tel 312 996 7323; fax 312 996 7586; e-mail: efosslie{at}uic.edu.

	Abstract

Top Abstract Introduction Vasculogenesis and Angiogenesis The Malignant Tumor Molecular Pathology Regulation Discussion and Hypothesis Diagnosis and Therapy Conclusions References

 
Cancer-induced angiogenesis is the result of increased expression of angiogenic factors, or decreased expression of anti-angiogenic factors, or a combination of both events. For instance, in colon cancer, the malignant cells, the stromal fibroblasts, and the endothelial cells all exhibit strong staining for cyclooxygenase-2 (COX-2), the rate-controlling enzyme in prostaglandin (PG) synthesis. In various cancer tissues, vascular endothelial growth factor (VEGF) and transforming growth factorß (TGF-ß) co-localize with COX-2. Strong COX-2 and VEGF expression is highly correlated with increased tumor microvascular density (MCD); new vessels proliferate in areas of the tumor that express COX-2. Moreover, high MVD is a predictor of poor prognosis in breast and cervical cancers. COX-2 and VEGF expression are elevated in breast and prostate cancer tissues and their cell-lines. In vitro, PGE2 induces VEGF. Supernatants of cultured cells from breast, prostate, and squamous cell cancers contain angiogenic proteins such as COX-2 and VEGF that induce in vitro angiogenesis. A selective COX-2 inhibitor, NS-398, restores tumor cell apoptosis, reduces microvascular density, and reduces tumor growth of PC-3 prostate carcinoma cells xenografted into nude mice. The COX-2 produced by a malignant tumor and COX-2 produced by the surrounding host tissue both contribute to new vessel formation, which explains how selective COX-2 inhibition reduces tumor growth where the tumor COX-2 gene has been silenced by methylation.

(received 6 June 2001; accepted 8 July 2001)

Keywords: cancer, angiogenesis, microvascular density, COX-2, PGE2, VEGF, TGF-ß, iNOS, TSP-1, p53, hypoxia, NSAIDs, celecoxib, rofecoxib

	Introduction

Top Abstract Introduction Vasculogenesis and Angiogenesis The Malignant Tumor Molecular Pathology Regulation Discussion and Hypothesis Diagnosis and Therapy Conclusions References

 
Angiogenesis, the formation of new vessels from existing vessels, is an important feature of embryogenesis, inflammation, and cancer growth and metastasis [1]. Chronic inflammation is a risk factor for cancer [2], but the exact reason why is unknown. At the site of inflammation, cyclooxygenase-2 (COX-2) is the rate-limiting enzyme in the synthesis of pro-inflammatory and angiogenic prostaglandins (PG) such as PGE2, which induces metalloproteinases (MMP) and vascular endothelial growth factor (VEGF) [3,4]. The antiphlogistic and analgesic effects of nonsteroidal anti-inflammatory drugs (NSAIDs) are largely due to their inhibition of COX-2 [5].

A variety of human malignancies overexpress COX-2 and prostaglandin [6] along with VEGF and transforming growth factor-beta (TGF-ß). For instance, in colorectal carcinoma strong COX-2 expression, as evidenced by immunostaining, is highly correlated with tumor microvascular density: a large number of small vessels form around the areas that express COX-2 [7]. Furthermore, the expression of COX-2, TGF-ß, and VEGF in the same areas of the tumor suggests their coordinated expression in the cancer-induced angiogenesis.

Tumor invasion into the local tissue and tumor growth at the site of metastasis are preceded by tumor-induced proliferation of an abundantly vascular stroma, for instance in breast cancer [8]. For such tumors, anti-angiogenesis therapy is an encouraging new approach. COX-2, the angiogenic molecules associated with it, and their interrelated signaling pathways in malignant tumors are therefore of major interest in understanding the tumor-induced angiogenesis. Studying the expression of COX-2 in cancer tissues and its role in the growth of malignant tumors is important because NSAIDs might help to prevent cancer [9]. Furthermore, selective COX-2 inhibitors are available that block the effects of COX-2 expression but spare the expression of COX-1 [5,10].

	Vasculogenesis and Angiogenesis

Top Abstract Introduction Vasculogenesis and Angiogenesis The Malignant Tumor Molecular Pathology Regulation Discussion and Hypothesis Diagnosis and Therapy Conclusions References

 
A. The yolk sac  During implantation, trophoblast invasion, vasculogenesis, and angiogenesis are essential for the formation of the placenta. The human trophoblast secretes metalloproteinases, and metalloproteinase inhibitors can abort in vitro invasion. In contrast to the invasion by a malignant tumor, the invasion by the trophoblast is stringently controlled. However, the controlling factors are unknown [11]. Many angiogenic factors that are highly expressed in malignant tumors are essential for blastocyst implantation and yolk sac vasculogenesis, for formation of new vessels from stem cells, and for yolk sac angiogenesis (Fig. 1) [12,13]. Vasculogenesis requires the development of the hemangioblast, the precursor for hematopoesis and vessel development, into the endothelial cell. A multitude of endothelial cells form the capillaries, which mature into veins and arteries during angiogenesis.

View larger version (27K): [in this window] [in a new window]   Fig. 1. Cyclooxygenase (COX-2), prostaglandin (PG), and transforming growth factor (TGF)-ß are involved in vascular development and vascular pathology from vasculogenesis and angiogenesis, to atherosclerosis and cancer. Lack of maternal COX-2 or its inhibition by a nonsteroidal anti-inflammatory drug (NSAID) prevents implantation. Lack of TGF-ß1 is lethal due to faulty vasculogenesis and angiogenesis during embryogenesis with a penetration of about 50%. Lack of the TGF-ß receptor component endoglin (ENG) prevents proper angiogenesis. Disruption of the PGE2 receptor EP4 gene prevents closure of the ductus arteriosus. COX-2 and TGF-ß co-localize in atherosclerotic lesions and in carcinogen-induced colon carcinomas, suggesting coordinated expression. In the cancer, COX-2 induces new vessel formation via PGE2 that induces vascular endothelial growth factor (VEGF). In some poorly differentiated malignancies, vasculogenesis is also induced. A human cancer tissue may overexpress one or more of the proteins indicated in the highlighted boxes. Text in italics indicates an agent used in vitro.

For successful implantation, the blastocyst must induce the expression of maternal COX-2 in the uterus [15]. Studies of the COX-2 null mouse prove the vital importance of the COX-2 expression; complete failure of implantation renders the mice sterile. Half of the COX-2 knockout mice develop cardiac fibrosis and all develop renal dysplasia [16].

In the mouse, disruption of the gene for TGF-ß1, a strong inducer of COX-2, causes defective vessel formation and may cause death. However, there are dosage and mouse-strain effects [17,18] that modulate the severity of this gene disruption. One-half of TGF-ß1(-/-) and one quarter of TGF-ß(+/-) mice died at about 1.5 wk post-conception because of failing endothelial cell differentiation [17].

VEGF can regulate vascular permeability and is an important mediator of vasculogenesis and angiogenesis [19]. VEGF signals are conveyed via the VEGF receptors Fli-1 and Flk-1. Fli-1 is expressed in the hemangioblast. Disruption of the Flk-1 gene is lethal in mice at about day 9 of gestation, due to lack of hematopoesis and vasculogenesis. Normally, Fli-1 is strongly expressed during physiological vasculogenesis and angiogenesis in embryos and is re-expressed during tumor-induced angiogenesis [20].

B. Vascular remodeling  COX-2 and prostaglandins are evidently involved in vascular remodeling. For example, rapid and selective physiological vascular remodeling occurs during closure of the ductus arteriosus and there may be a difference in response of the prenatal and postnatal ductus.

Firstly, a lack of the PGE2 receptor EP4 in the EP4-null mice causes the ductus to remain open. By comparison, indomethacin administered during pregnancy may cause premature closure of the ductus arteriosus. In fetal lambs, the COX-2 is expressed in the endothelium of the ductus arteriosis, while COX-1 is expressed in both the endothelium and the muscular layers [21].

Secondly, the inhibition of prostaglandin synthesis by indomethacin constricts the fetal ductus arteriosus in normal mice, but not in EP4(-/-) mice, which do not express the prostaglandin EP4 receptor. Such knockout mice generally die shortly after birth. They (as well as the few that survive for a longer time) have a patent ductus arteriosus (Fig. 1) [22].

In mice, a lack of either COX-1 or COX-2 or both does not cause premature closure of the ductus arteriosus in utero. Furthermore, lack of COX-1 does not affect postnatal life. In contrast, about one third of COX-2 null mice and almost 80% of COX-2(-/-) + COX-1(+/-) mice die within 48 hr after birth. Importantly, the absence of both COX isoforms causes postnatal death within 12 hr [23]. This suggests that maternal prostaglandins alone can maintain intrauterine patency of the ductus arteriosus. During closure of the ductus arteriosus, the expression of the 3 TGF-ß isoforms increases significantly. Over the 10 postnatal days, it increases from the very low levels before birth. The highest levels are seen in the neo-intima and the outer muscle media, but TGF-ß is not detected in vascular endothelial cells [24].

With advancing age, there is diffuse intimal thickening (DIT) of the elastic arteries (Fig. 1) [25,26] and there is evidence that TGF-ß is involved in vascular remodeling. A comparison of 30-mo-old versus 6- mo-old Fisher rats revealed significant increases of TGF-ß and MMP-2 in the intima, which was 5-fold thicker in the older rats [27].

C. Inflammation  Chronic inflammation is a risk factor for a variety of human malignancies [2]; many of the angiogenic proteins expressed in cancer tissues are the same as those expressed at sites of inflammation [5]. In vitro studies and animal models of inflammatory angiogenesis show that COX-2 plays a pivotal role in new vessel formation at sites of inflammation. For instance, COX-2 is strongly expressed and produces pro-inflammatory prostaglandins such as PGE2. Besides, PGE2 can induce VEGF, for instance in synovial fibroblasts in vitro. The signaling occurs via the prostaglandin EP2 receptor and protein kinase A pathways [4].

Angiogenesis is an essential part of the formation of granulation tissue [28]. In an animal model of wound healing, application of neutralizing anti-VEGF antibody to a post-surgical wound demonstrated that VEGF is an essential angiogenic signal transducer in the formation of wound granulation tissue. Neutralizing anti-VEGF antibody reduced granulation tissue VEGF levels and new vessel formation [29].

COX-2 and VEGF are both expressed in rat sponge-implant-angiogenesis [30]. COX-2 is located in the endothelial cells of the new vessels that are formed in the sponge-granuloma tissue [31]. The experimental angiogenesis can be blocked by either a non-selective NSAID, indomethacin, or by selective COX-2 inhibitors, NS-398 or JTE-522, as well as by VEGF anti-sense oligonucleotides [30]. VEGF-induced angiogenesis in a mouse cornea model of angiogenesis can be inhibited by NS-398 or by cyclosporin-A administered systematically. Importantly, PGE2 restores corneal angiogenesis even in the presence of these inhibitors [32].

In a rat model of pleurisy, carrageenan-treated rats express elevated levels of COX-2 in the lung. Treatment of rats with ip melatonin prior to the carrageenan administration inhibits the COX-2 expression [33]. High dosages of melantonin were used in this study, ranging from 12.5 to 50 mg/kg. However, it is remarkable that melatonin can inhibit COX-2 expression.

	The Malignant Tumor

Top Abstract Introduction Vasculogenesis and Angiogenesis The Malignant Tumor Molecular Pathology Regulation Discussion and Hypothesis Diagnosis and Therapy Conclusions References

 
A. Cancer-induced angiogenesis  The pathological growth of human cancers resembles in several ways the physiological growth of the implanting blastocyst. Both need to evade the immune defenses of the host and secure a vascular supply in order to grow [34,35]. To induce new vessel formation, the cancer must induce the expression of angiogenic growth factors and their receptors and regulate the expression of proteolytic enzymes and adhesion molecules [36]. In a manner similar to the blastocyst, the malignant tumor forces the host tissue to grant it a supply of oxygen and nutrients that enables the tumor expansion, which otherwise is confined to 1–2 mm by the maximum oxygen diffusion limit. Progression from limited tumor growth to invasion and metastases requires degradation of the extracellular matrix by proteolysis by metalloproteases (MMP, matrixins), induction of angiogenesis, tumor cell migration, and modification of cell adhesion [37,38].

COX-2 is important because it induces metalloproteinase and strongly induces VEGF, the common angiogenic factor, which thereupon directs tumor-induced angiogenesis [39]. Together, these factors contribute to tumor growth, invasion, and metastasis. Magnetic resonance imaging (MRI) shows that tumor-induced angiogenesis follows a common pattern, leading to a well-perfused tumor periphery but a residual necrotic core. Host vessels enter the periphery of the tumor, and the perfusion front moves toward the center of the tumor [40].

B. COX-2 expression in cancer tissues  Enhanced COX-2 expression has been demonstrated in numerous types of cancer cells and tissues, such as colorectal cancer [5]. For example, COX-2 expression was increased in all but 2 of 20 lung cancers, 14 of 20 colon adenocarcinomas, and 11 of 20 breast tumors, but not in epithelial cells of the corresponding non-tumor tissue. Expression of COX-2 was minimal in the stromal cells of the tumors and in normal tissues [41]. Furthermore, immunohistochemical expression of COX-2 was significantly increased in malignant cells of all but 1 of 13 cervical carcinomas, compared to non-tumor cervical tissue [42].

High expression levels of COX-2 may foretell a poor prognosis; for instance, in 24 patients with cervical carcinoma who had been treated with radiation, increased expression of COX-2 was associated with shorter survival [43]. In patients with gastric cancer, the tissue COX-2 levels were significantly higher in invasive versus non-invasive tumors [44]. A study of 63 patients with colorectal cancer showed that high COX-2 expression correlated with tumor recurrence, and particularly with hematogenous spread of the tumors [45]. The follow-up period in this study (6 to 98 mo, average 60 mo) was significantly longer than in earlier studies that did not show correlation between COX-2 expression and survival. The Kaplan-Meyer curves clearly showed longer survival for patients with low expression of COX-2 in their tumor tissues [45].

Prostatic tissue cancer cells showed significantly elevated, mainly intracytoplasmic, expression of COX-2 compared to the COX-2 expressed along the cell membrane in benign prostatic hyperplasia. The intensity of staining of COX-2 was significantly higher in poorly-differentiated versus well-differentiated prostate cancers and correlated with the Gleason grade [46]. Other studies corroborate these findings. For instance, significant elevations of COX-2 protein and mRNA were demonstrated in prostate cancer cells by immunostaining and RT-PCR in a series of 28 carcinomas, compared to controls (8 cases of benign prostatic hyperplasia and 8 prostate samples that were free of tumor) [47]. In another series of prostatic carcinoma, all but 4 of 31 cancers showed intense and uniform immunostaining for COX-2 [48]. COX-2 was expressed in all but 2 of 28 primary skin melanomas (16 showing moderate to strong expression), but not in 4 benign nevi [49]. All except 1 of 29 retinoblastomas overexpressed COX-2 [50].

In a study of 100 patients with colon cancer, those with COX-2-expressing tumors had significantly shorter survival time than those with COX-2-negative tumors; moreover, correlation was noted between COX-2 expression and microvascular density [7]. Immunostaining for CD-34 to show the microvascular density of the tumors revealed that a large number of small vessels had formed around areas that expressed COX-2 [7]. In addition, a study of paraffin-embedded tumor tissue from 120 patients with breast cancer showed that a high tumor MVD correlated with the histological grade and with poor survival [51]. In another study of patients with breast cancer, detecting increased MVD of axillary lymph nodes by iv digital subtraction angiography predicted lymph node metastases [52], suggesting that MVD may be reliably determined using non-invasive techniques.

These findings in malignant tumors suggest that high COX-2 expression is associated with tumor-induced angiogenesis, invasion, and metastases.

C. VEGF in cancer tissues  Enhanced expression of VEGF has been demonstrated in, for instance, ovarian [53], breast [54], renal [55,56], and esophageal [57] cancers and in malignant melanomas [58]. The malignant cells in breast cancers showed variable expression of VEGF, but no VEGF expression was found in epithelial cells in areas of breasts that were free of tumor [54]. VEGF expression was increased in 55 prostate cancers compared to 5 cases of prostatic adenomas and 20 normal prostate samples; elevation of VEGF correlated with tumor MVD, tumor grade, and stage [59]. In 85 patients that underwent surgical resection of stage I non-small cell lung cancers, high tumor MVD and poor prognosis correlated with high levels of VEGF expression [60].

Immunostaining of VEGF might be an indicator of aggressiveness in serous ovarian tumors. Firstly, VEGF immunostaining of tumor tissue was significantly greater in 32 invasive tumors, compared to16 borderline and 10 benign ovarian tumors. Secondly, there was significant correlation between VEGF elevation and metalloproteinase-2 (MMP-2) elevation [53]. Moreover, in 44 cases of hepatocellular carcinoma, the microvascular density correlated significantly with the expression levels of VEGF mRNA. Poorly encapsulated tumors showed higher levels than well-encapsulated tumors. Interestingly, serum VEGF concentration was significantly higher in patients with tumors, compared to patients with benign liver disease [61].

However, high MVD does not always indicate a poor prognosis, particularly in cases with extensive tumor necrosis. For instance, in a series of 49 localized renal cell carcinomas, MVD was inversely correlated with the magnitude of tumor necrosis; low MVD and extensive necrosis predicted a poor outcome [62]. And, in a study of 69 patients with stage I-II non-small-cell lung carcinomas, the cancer cells, stromal fibroblasts, and endothelial cells in the tumors all exhibited strong VEGF staining. However, the level of tumor microvascular density by CD34 immunostaining in these cases correlated neither with the tumor levels of VEGF and its receptors, nor with the patients’ survival [63]. Determining the levels of VEGF and its receptors flt-1 and KDR/flk-1 by immunostaining in 35 adenocarcinomas, 6 papillary serous carcinomas, and 6 carcinosarcomas was ineffective in predicting metastases, recurrence, or survival [64]. On the other hand, immunostaining for VEGF may be of use to differentiate malignant melanomas from benign nevi. Cytoplasmic VEGF was found in almost half of 45 malignant melanomas and the intensity of immunostaining was related to the Clark level. In contrast, VEGF was not detected in benign nevi [58].

D. Haptoglobin  It has been reported that the multifunctional haptoglobin molecule is angiogenic (for a review see [5]). Haptoglobin expression is regulated by dexamethasone and by IL-6, which is induced by PGE2 [65]. Whether haptoglobin induces VEGF is unknown. Haptoglobin expression has been detected in human breast carcinoma tissue extracts and cell lines [66,67]. Furthermore, in vitro cell lines established from squamous cell and ovarian cancers and from embryonic lung secrete a multiprotein complex that has subunits with sequence homology to the ß chain of haptoglobin [68].

E. Vascular patterns in malignant tumors  Although tumor-induced angiogenesis resembles the angiogenesis observed at sites of inflammation, there are important differences between physiological angiogenesis and cancer-induced angiogenesis. In addition to increased microvascular density in and around many tumors, the pattern of neoplasia-induced vascularization differs from normal vascular patterns. Measuring and evaluating this difference might have significant diagnostic and prognostic value. To illustrate, in uveal melanomas, the presence of parallel vessels with cross-linking indicates a poor prognosis [69]. Moreover, the fractal character of tumor-induced new vessel proliferation reflects a higher degree of irregular branching and vessel distribution, compared to physiological angiogenesis. This difference is probably caused by more variable local distribution of vascular growth factors within the tumor, owing to the inherent genetic instability and progressive molecular diversity of malignant cells [70]. Caution is advised when extensive necrosis is present. In the renal carcinoma cases mentioned above, the microvascular complexity as evidenced by the fractal dimension of vessels visualized with CD34 staining in the tumors correlated inversely with the extent of tumor necrosis, which predicted a poor outcome [62].

F. Cancer cell supernatants  Supernatants of cultured cells from several types of cancer contain angiogenic factors, such as fibroblast growth factor-2 (FGF-2) in prostate cancer [71] and TGF-ß, PGE2, and VEGF in squamous cell cancer [72]. Supernatants of cultured cells from head and neck squamous cell cancer, breast cancer, and liver cancer secrete PGE2, VEGF, and TGF-ß and induce in vitro angiogenesis [72–74]. VEGF expression is elevated in the cancer tissue and cell lines derived from breast tumors [54,75]. Prostaglandin E2 induces VEGF in a variety of cell lines and VEGF expression strongly stimulates angiogenesis in vitro. As examples, PGE2 induces VEGF in cell lines derived from the rat osteoblasts [76] and Müller cells [3], as well as human rheumatoid synovial fibroblasts [4] and monocytes [77].

Metalloproteases play an important role in tumor cell invasion by proteolysis of the extracellular matrix. As a case in point, cultured prostate cancer cells release proMMP-2, proMMP-9, and MMP-9 into the medium and invade through Matrigel. The factor release and tumor cell invasion can be inhibited by a PLA2 inhibitor, 4-bromophenacyl bromide, and by a selective COX-2 inhibitor, NS-398. Inhibition of lipoxygenase with esculetin does not affect MMP secretion. On the other hand, the tumor cell invasion can be restored by PGE2 supplementation. These experiments indicate that cycloxygenase-2 induces metalloproteinases (MMP) via PGE2 [78].

G. Tumor xenograft-induced angiogenesis  Experiments have shown that COX-2 and VEGF are essential angiogenic proteins that determine the growth of various xenografted human cancer cells. In addition, such experiments have corroborated the sequential propagation of the angiogenic signal from COX-2 via PGE2 to VEGF. The tumor-induced COX-2 expression in the host tissue located adjacent to the malignant tumor adds to the angiogenesis induced by the COX-2 produced by the cancer cells, and both enhance tumor growth.

The significance of host COX-2 expression in cancer growth is illustrated by the proliferation of Lewis lung cancer cells xenografted into the COX-2(-/-) mouse. The xenograft growth is significantly reduced compared to that in the wild-type mouse. Expression of VEGF by host COX-2 null fibroblasts is reduced >90% and is comparable to the reduced VEGF expression by wild-type murine fibroblasts following treatment with a selective COX-2 inhibitor [79].

The importance of VEGF in tumor-induced angiogenesis is evidenced by the effect of neutralizing anti-VEGF antibody in nude mice xenografted with tumor. The antibody treatment completely inhibits the angiogenesis by xenografted cell lines derived from human rhabdomyosarcoma and prostate cancer. The tumors become dormant after the initial angiogenesis-independent growth phase [80], thus indicating the critical importance of tumor-induced new vessel formation in the tumor growth.

	Molecular Pathology

Top Abstract Introduction Vasculogenesis and Angiogenesis The Malignant Tumor Molecular Pathology Regulation Discussion and Hypothesis Diagnosis and Therapy Conclusions References

 
A. Angiogenic factors  1. Cyclooxygenase and carcinogenesis  Transgenic overexpression of either COX-1 or COX-2 can transform cells [81,82]. No sequence aberration of the COX-2 gene has been reported in malignant cells. A great variety of cancer cells and tissues overexpress COX-2 and prostaglandins that reduce cancer cell apoptosis [6] and induce angiogenesis via VEGF. NSAIDs inhibit the growth of such cancers and the anti-neoplastic effects of the NSAIDs are partly due to COX-2 inhibition [5].

On the other hand, neoplastic transformation appears to be independent of cyclooxygenase. Thus, Haras or SV40 cause in vitro transformation of fibroblasts that lack COX-1 or COX-2, or both. By comparison, when the genes for either or both enzyme isoforms are present, the corresponding protein is highly expressed in the transformed cells. These results suggest that cyclooxygenases play a role in tumorigenesis at a later step [83].

Several oncogenes and growth factors can induce COX-2 expression. In cultured CaCo-2 colon carcinoma cells, COX-2 is upregulated by insulin-like growth factor (IGF)-II via the IGF-I receptor [84]. Besides, TGF-, epidermal growth factor (EGF), TGF-ß, and hepatocyte growth factor (HGF) can all induce COX-2 [5,6].

2. Prostaglandin and carcinogenesis  Prostaglandin E2 induces VEGF and basic fibroblast growth factor (bFGF) [3]. There is evidence that bFGF might contribute to cancer-induced new vessel formation. For example, when co-cultured with bovine aortic endothelial cells, 4 human glioma cell lines that expressed high levels of bFGF mRNA induced the endothelial cells to form tubes. The tube formation was blocked by supplementation with anti-bFGF antibody. Moreover, 3 human glioma cell lines with low expression of bFGF failed to induce tube formation [85].

PGE2 induces the expression of metalloproteinases by a multistep process involving NF-kappaB. It was recently suggested that PGE2 might enhance the invasive potential of colorectal carcinoma through activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt/PKB) pathway. PGE2 treatment of LS-174 human colorectal carcinoma cells increased their motility and altered their shape [86]. Prostaglandin E2 suppresses both cellular and humoral immunity [2,87] (reviewed in [6]). Inhibition of cyclooxygenase with NSAIDs restores the immune reactivity [5,88].

3. VEGF and carcinogenesis  Vascular endothelial cell growth factor (VEGF) is the primary member of a family of growth factors. VEGF and its homologues can convey regulatory signals via receptors KDR/Flk-1, Flt-1, and Flt-4 [89] (Fig. 2). However, VEGF and its homologues have different effects on different cells. For instance, the orf paracoxvirus encodes the VEGF-E homologue [90] that causes orf disease in sheep and is associated with vascular proliferation at the site of infection in human erythema multiforme [91,92].

View larger version (22K): [in this window] [in a new window]   Fig. 2. Molecular pathology of the role of cyclooxygenase (COX-2) in cancer-induced angiogenesis. Soluble (s) and cytoplasmic (c) phospholipase (sPLA2, cPLA2) convert membrane phospholipids (MPL) to arachidonic acid (AA), which is converted by COX-1 and COX-2 to prostaglandins (PG). PGE2 induces the stromal fibroblast (SF) to produce the hepatocyte growth factor (HGF), which induces the genes for COX-2 and cPLA located at chromosome region 1q25. Also, PGE2 induces vascular endothelial growth factor (VEGF) that controls angiogenesis via its receptors flt-1 and flk-1. Thrombospondin (TSP)-1 inhibits VEGF expression. Numbers in boxes: 1: Non-selective NSAID; 2: selective COX-2 inhibitor; 3: iNOS inhibitor; 4: soluble TGF-ß type II receptor; 5: anti-receptor antibody; 6: anti-PGE2-antibody.

VEGF stimulates the activation of cPLA2 and the release of arachidonic acid by human umbilical vein endothelial cells in vitro [95]. VEGF induces COX-1 but not COX-2 in cultured endothelial cells; it has been suggested that COX-1 has a physiological role in maintenance of vascular structures [96]. Such a feedback loop might help to maintain a basal level of VEGF expression.

4. Aging and carcinogenesis  With advancing age, both angiogenic and anti-angiogenic changes have been observed. In vitro studies suggest that as the generation of reactive oxygen species (ROS) increases with advancing age, there is increased expression of COX-2. The ROS reduces IkappaB expression and enhances the expression of NF-kappaB, a strong COX-2 inducer [97]. On the other hand, as the oxidative stress increases with age, the mitochondrial function may decline [98,99], and there is some evidence that decline of entothelial energy metabolism may inhibit angiogenesis [100].

B. Anti-angiogenic proteins  Physiological angiogenesis is the result of a dynamic balance between angiogenic and anti-angiogenic factors [101]. Orderly angiogenesis is essential for the endometrial cycle, for inflammation, and for the granulation tissue of wound repair [29]. Deranged expression of anti-angiogenic factors caused by viruses or by loss of function of tumor suppressor genes has been linked to various human malignancies. Because they can counterbalance the angiogenic effects of COX-2, a few anti-angiogenic factors are discussed below.

1. Thrombospondin-1 (TSP-1)  Reports on the role of the glycoprotein TSP-1 in carcinogenesis are controversial. Some reports indicate that thrombospondin inhibits angiogenesis in vitro, while other reports state the opposite. Some studies find elevated levels of TSP-1 in cancer tissues, while other studies report decreased levels. However, as explained below, the strongest evidence suggests that TSP-1 is a major anti-angiogenic protein. Most importantly, transgenic expression of TSP-1 can limit tumor angiogenesis and growth. For instance, injection of an adenovirus containing TSP-1 plasmid into pre-established tumors reduces tumor-induced angiogenesis, tumor growth, and metastatic spread. The overexpression of TSP-1 in squamous carcinoma cell lines inhibits their in vivo growth. Depending upon the cell-line used, overexpression of TSP-1 either prevents xenograft growth or, when a tumor develops, it exhibits marked central necrosis [102].

Cholangiocarcinoma is a tumor with low vascularity compared to hepatocellular carcinoma. When tumor tissues were compared to the matching non-tumor tissue, there were significant differences in the ratio of TSP-1 to VEGF expression in these two types of tumor. Cholangiocarcinomas showed much higher TSP-1 and lower VEGF expression than hepatocellular carcinomas [103]. These findings seem to corroborate that TSP-1 can inhibit VEGF expression and reduce tumor-induced angiogenesis. On the other hand, TSP-1 was strongly expressed and the expression levels were associated with high microvascular density in 87 of 98 pancreatic carcinomas. It was proposed that TSP-1 expression played a principal role in the new vessel formation and the spread of these tumors [104]. However, in vitro, thrombospondin-1 inhibits endothelial cell migration and tube formation. The inhibitory signal is transmitted through CD36, an endothelial cell transmembrane glycoprotein and is completely dependent on its expression [105]. As discussed below, both TSP-1 and CD36 are downregulated by the platelet endothelial cell adhesion molecule (PECAM) [106].

In vitro assays and tissue culture experiments indicate that TSP-1 may also inhibit angiogenesis by inhibiting MMP activity [107]. Almost half of 99 malignant melanomas expressed mutant p53 and significantly lower TSP-1 levels and higher microvessel counts than the tumors with the wild-type p53. Besides, the presence of mutated p53 was significantly more frequent and the TSP-1 levels significantly lower in the tumor metastases than in the primary tumors [108]. In contrast, immunostaining for p53 and TSP-1 in a series of 65 colon cancer tissues showed accumulation of p53 in 42 cases with reciprocal expression of TSP-1 in the areas that expressed p53, suggesting that p53 can suppress TSP-1 in vivo [109].

Surprisingly, a TSP-1 plasmid expression vector inserted into a human prostate-carcinoma cell-line failed to reduce the growth of the cancer cells in vitro. However, when the cells were transfected along with a liposomal agent and then xenografted into nude mice, there was reduced microvessel density and extensive necrosis in the tumors compared to xenografted but non-transfected tumor cells [101].

Mevalonate induces the expression of TSP-1 (Fig. 3). As a case in point, incubation of cultured human vascular smooth muscle cells for 24 hr with lovastatin at concentrations as low as 1 µM/L significantly inhibited TSP-1 expression. The expression of TSP-1 was restored by co-incubation with mevalonate [110].

View larger version (21K): [in this window] [in a new window]   Fig. 3. Thrombospondin (TSP-1) activates the transforming growth factor beta (TGF-ß). The statin-type of cholesterol lowering drugs lower the synthesis of mevalonate, which reduces the stimulation of TSP-1 synthesis and thereby increases angiogenesis by reducing the inhibition of VEGF. The reduction in mevalonate synthesis also decreases the synthesis of coenzyme (Co) Q10, which reduces mitochondrial synthesis of adenosine triphosphate (ATP) that reduces endothelial cell proliferation.

3. Angiostatin  Angiostatin can diminish vascular density and branching. For instance, in the quail chorioallantoic membrane assay, it reduced angiogenesis by almost 70% compared to the normal rate of developmental angiogenesis [112]. There is evidence that angiostatin affects angiogenesis by inhibiting the surface F₀F₁-ATPase activity of endothelial cells, thereby reducing endothelial cell metabolism [100].

C. Biphasic growth factors  Biphasic growth factors are characterized by an ability to induce tubulogenesis (ie, the formation of tubular structures) in vitro. Several are morphogens that are intimately involved in vasculogenesis and angiogenesis. The present author speculates that a biphasic growth factor is involved in establishing the curvature of a blood vessel wall and thereby determines the vessel diameter [113]. Several biphasic morphogens that are expressed during embryogenesis are re-expressed during inflammation and repair, and cancer growth, invasion, and metastasis. The important biphasic morphogens in connection with COX-2 and cancer-induced angiogenesis are TGF-ß, TGF-, and PECAM,

1. TGF-ß  Transforming growth factor ß (TGF-ß) induces COX-2 via the TGF-ß type 2 receptor (TGFBR2) [5,6]. It is secreted as a latent protein and converted to a 25 kDa active form. There is some evidence that TSP-1 can bring about this activation in vitro [114]. Other investigators found no evidence of such activation in vitro [115]. However, supernatants from glioma cell lines contain high levels of TSP-1 that can activate TGF-ß [116].

Because TGF-ß is a bimodal growth factor, a protein that activates it would appear to have bimodal properties as well. Assuming that TSP-1 can activate latent TGF-ß in certain cells, this might explain the observation that TSP-1 at low concentrations stimulated the growth of cultured bovine endothelial cells while it inhibited their growth at higher concentrations [117]. In addition, activation of TGF-ß might explain how TSP-1 enhances tumor invasion by breast cancer cells in vitro [118]. This impression is supported by in vitro experiments using pancreatic tumor cells; supplementation by either TSP-1 or TGF-ß produced upregulation of tumor cell invasion that was completely reversed by antibodies against either urokinase plasminogen activator or its receptor [119].

TGF-ß is of interest because it is abundantly expressed in the same areas as COX-2 in colon carcinoma tissues. This observation suggests a close collaboration in the process of carcinogenesis [120] especially because TGF-ß1 has been described as a powerful regulator of COX-2 expression [121]. Most important, from an etiologic point of view, deregulation of TGF-ß1 expression may be an early event in colon carcinogenesis [122]. Aberrations in TGF-receptors, such as repression of the expression of the TGF-type II receptors, have been shown to contribute to carcinogenesis [123].

There is evidence that the different isoforms of TGF-ß may have varying roles in carcinogenesis, and that their effects may differ in different types of malignancies. To cite an instance, in skin cancers, TGF-ß1 was found to be associated with highly differentiated tumors, TGF-ß3 was associated with tumor stroma growth and angiogenesis, and TGF-ß2 with invading, very malignant tumor cells [124]. Yet, in prior studies in xenografted nude mice, expression of TGF-ß2 was higher than that of the ß1 isoform in the poorly invasive DU145 prostate carcinoma cell line compared to the highly metastatic PC-3M prostate carcinoma cell line [125].

TGF-ß modulates angiogenesis by regulating vascular endothelial cell proliferation and migration. Besides, it affects the extracellular matrix and the synthesis of adhesion molecules [36]. TGF-ßstimulates smooth muscle cells at low concentration and inhibits them at high concentration [126,127]. It has similar effects on capillary lumen formation induced in vitro by bFGF or VEGF [128]. The TGF-ß signals via type I (ALK-1), type II, and type III (endoglin) receptors and Smad proteins, which regulate genes involved in angiogenesis [129,130].

Participation of TGF-ß in angiogenesis is corroborated by results from the mouse corneal model of inflammation induced by silver nitrate. When mice were injected into the femoral muscle with an adenovirus that expressed a soluble ligand binding part of the TGF-ß type II receptor, the corneal angiogenesis was significantly reduced when compared to control mice [131].

The TGFBR2(-/-) mouse dies about 10 days post-coitus due to defects in yolk sac vasculogenesis [129] that closely resemble those that occur in the TGF-ß1 null mouse [17]. In like manner, mice lacking the TGF-ßType I receptor (TGFBR1) die of defective yolk sac and placental vascular development. Cultured vascular endothelial cells from such mice show defective fibronectin production and abnormal migration, but enhanced proliferation, compared to control cells from wild-type mice [132].

Another example of the importance of TGF-ßand its receptors in proper angiogenesis is illustrated by mutations of the TGF-ß1 and TGF-ß3 binding protein endoglin (ENG, CD105) and ALK-1 genes. Such mutations are responsible for the vascular malformations seen in hereditary hemorrhagic teleangiectasia (HHT) types 1 and 2 respectively (Fig. 1) [130,133].

Endoglin is an essential factor for angiogenesis during embryogenesis. It is expressed on the surface of endothelial cells. Lack of endoglin does not affect vasculogenesis but it is essential for normal vascular maturation as part of angiogenesis. This is evident in endoglin null mice, which die by gestational day 11.5 from defective development of vascular smooth cells and aborted endothelial remodeling [133]. These experiments confirm that TGF-ß signaling through its receptors plays an important role in the regulation of angiogenesis. The importance of TGF-ß signaling in cancer-induced angiogenesis is further validated by a report that elevated levels of endoglin in breast cancer tissues predict a high risk of developing metastatic disease. Strong upregulation was detected by immunostaining in the tissues of 92 breast cancer patients [134].

Endoglin is highly expressed in some malignant tumors. It might find use as a tumor marker because the protein was detected in the plasma of patients who had early breast cancer. Analysis of plasmas obtained before any treatment was given revealed that patients who had high plasma levels of endoglin had a high risk of developing metastatic disease [134]. Also, the endothelial-specific expression of endoglin favors its use as an endothelial cell specific marker. As an example, in paraffin-embedded cervical cancer tissues immunostaining for endoglin was more sensitive than immunostaining factor VIII for visualizing capillaries and was a better predictor than factor VIII for lymph node metastases [135].

2. TGF-  TGF- induces COX-2 by binding to the EGF receptor (EGFR) [136,137]. Supplementation with TGF- of cultured cervical cancer cells corroborates these findings. TGF- supplementation markedly increased COX-2 expression [42]. Presence of EGFR, but not of TGF-, in 86 breast cancers was associated with poor prognosis. However, intensity of TGF- immunostaining reflected the extent of angiogenesis in the tumor [137].

3. PECAM  Platelet endothelial cell adhesion molecule (PECAM-1) has a bimodal effect, stimulating endothelial cell morphogenesis at low concentrations and inhibiting it at high concentrations. PECAM is found at sites of cell-to-cell contact in polyoma middle T-transformed mouse brain endothelial cells. These cells express high levels of PECAM-1 that inhibit the expression of TSP-1 and TSP-1 receptor CD36 and form hemangiomas in the mouse [138]. The bimodal character of PECAM may be related to the activation of latent TGF-ß by TSP-1, which regulates the formation of active, bimodal TGF-ß.

	Regulation

Top Abstract Introduction Vasculogenesis and Angiogenesis The Malignant Tumor Molecular Pathology Regulation Discussion and Hypothesis Diagnosis and Therapy Conclusions References

 
A. Hypoxia  In vitro, hypoxia induces COX-2, increases PGE2 synthesis, elevates the VEGF level, and induces angiogenesis. The selective COX-2 inhibitor NS-398 can block this process. Hypoxia induced by cobalt chloride markedly increased COX-2 and upregulated VEGF via PGE2 in the cultured, highly invasive PC-3ML prostate-carcinoma cell line. Inhibition of the VEGF expression using the selective COX-2 inhibitor NS-398 was overruled by administration of exogenous PGE2 [139]. In vitro, hypoxia typically induces VEGF expression in several different human cell types [140]. One study found that hypoxia increases the expression of TSP-1 [141], which would inhibit VEGF expression. However, recent data show that hypoxia suppresses TSP-1 expression and stimulates VEGF expression [140].

Induction of VEGF, despite a lack of hypoxia, occurs in von Hippel-Landau disease (VHL), which is caused by mutations in the VHL tumor suppressor gene. The mutations lead to constitutional instead of hypoxia-regulated expression of hypoxia inducible factor (HIF) that induces VEGF. Patients with VHL often develop hemangioblastomas, which exhibit increased levels of VEGF. [142,143].

B. Tumor suppressor protein p53  The tumor suppressor protein p53 inhibits COX-2 expression but stimulates TSP-1 expression [144]. Transfection experiments have demonstrated that p53 can stimulate the promoter sequences of the TSP-1 gene in fibroblasts. The preponderance of evidence suggests that thrombospondin (TSP-1) is strongly anti-angiogenic, inhibiting the expression of VEGF. Thus, loss of function of the tumor suppressor protein p53, which normally stimulates TSP-1, might contribute to cancer-induced angiogenesis. Wild-type p53 inhibits angiogenesis by stimulating TSP-1 synthesis. Mutation or loss of p53 results in lack of TSP-1 inhibition of angiogenesis. Tissue studies corroborate these findings. Almost half of 99 malignant melanomas expressed mutant p53 and showed significantly lower TSP-1 levels and higher microvessel counts, compared to melanomas with wild-type p53. Moreover, presence of mutated p53 was more frequent and TSP-1 levels were lower in the tumor metastases than in the primary tumors [145].

High VEGF protein levels in tumor cytosol correlated with poor prognosis, based on enzyme immunoassays in 224 estrogen-receptor-positive primary breast cancer patients who received adjuvant tamoxifen. Higher VEGF expression was correlated with the presence of the mutations of the tumor-suppressor protein p53 [146]. In a geographical area with high risk for esophageal cancer, 48 of 74 esophageal carcinoma patients carried one or more point mutations of p53 in their tumor; the presence of p53 mutation correlated significantly with high COX-2 expression in the tumor [147].

C. Viruses  Human cytomegalovirus (CMV) inactivates wild-type p53 of diverse cell types resulting in loss of inhibition of COX-2 expression and enhanced angiogenesis. Furthermore, CMV increases tumor-induced angiogenesis independently of p53 expression by depressing TSP-1 expression. For instance, infecting p53-defective astrocytoma cells with CMV showed that the virus significantly reduced the TSP-1 mRNA and protein levels. Supplementation with CMV anti-sense oligonucleotides fully blocked the abolition of TSP-1 expression [144].

Human papilloma virus (HPV) is associated with angiogenesis in cervical cancer. One study examined 230 cervical tissues from 6 groups of 31 to 43 subjects, including a control group and 5 groups with tumors that ranged from low grade, intraepithelial lesions to poorly differentiated squamous cell carcinomas. There was good correlation between the extent of new vessel formation and the degree of histological abnormality. Importantly, the amount of angiogenesis determined by CD34 immunostaining was associated with the presence of HPV types 16 and 18 [148].

The E6 oncoprotein of the human papilloma virus (HPV)-16 upregulates the VEGF gene via a promoter region that contains 4 Sp-1 sites. VEGF is highly expressed in carcinomas of the cervix, with high levels in cells that are HPV-16 positive [149].

The latent membrane protein (LMP-1) of the Epstein-Barr virus induces COX-2 via NF-kappB. COX-2 is often expressed in preinvasive nasopharyngeal lesions that harbor the virus and in nasopharyngeal carcinomas. In vitro studies suggest that EBV, present in over two-thirds of such tumors, contributes to VEGF expression and tumor-induced angiogenesis [150].

D. Regulation of COX-2 expression  Several oncogenes and growth factors can induce COX-2 (Fig. 2) (for review, see [6]). For example, EGF induces COX-2. TGF-ß enhances COX-2 expression induced by EGF both in mink lung and rat intestinal epithelial cells but not in a mink lung cell line that lacks the TGF-ß type I receptor. Inhibition of the EGF receptor tyrosine kinase activity with AG1478 entirely abolished the expression of COX-2 induced either by EGF alone or by a combination of the two growth factors [151]. A prior report from the same laboratory documented that TGF-ß strongly induces COX-2 and that COX-2 can participate in the inhibitory signaling pathway of TGF-ß. In other studies the in vitro inhibitory part of the biphasic effects of TGF-ßvia COX-2 and PGE2 was blocked by indomethacin and restored by supplementation with PGE2 [152].

Oncostatin, a member of the IL-6 family of cytokines, is a potent growth factor for Kaposi’s sarcoma. It is a strong inducer of COX-2 expression in cultured human aortic smooth muscle cells [153]. The angiogenic effect of oncostatin depends upon the cell type. It was angiogenic in vitro for dermal microvascular endothelial cells but not for macrovascular, human umbilical vein endothelial cells. It induced COX-2 in the former, but not in the latter, and the angiogenic effect was nullified by selective inhibition of COX-2 [154]. Oncostatin was angiogenic in vivo in the rabbit corneal model, but it slightly inhibited calf pulmonary artery endothelial cells proliferation in vitro [155], suggesting that it might be a bimodal angiogenic factor.

	Discussion and Hypothesis

Top Abstract Introduction Vasculogenesis and Angiogenesis The Malignant Tumor Molecular Pathology Regulation Discussion and Hypothesis Diagnosis and Therapy Conclusions References

 
The experimental findings that have been discussed show that caution is needed in interpreting the sometimes contradictory data. There are clearly differences in the ways different cell types respond to various cytokines and growth factors. The following hypothesis attempts to explain some of these differences. The present hypothesis differs from the angiogenic balance theory that considers that a balance between TSP-1 and VEGF expression determines angiogenesis. Rather, the present hypothesis proposes that cancer-induced angiogenesis is determined mainly by the differential signal input that controls VEGF expression (Figs. 4 and 5), which in turn governs angiogenesis. In this view, the VEGF system is comparable to an operational amplifier, with negative feedback via VEGF induction of TSP-1, which inhibits VEGF, (Fig. 5). Evidence that such a negative feedback-loop exists was found in the murine model of retinal new vessel formation [156]. The importance of the negative feedback is that it may ensure a linear response of the VEGF-mediated angiogenesis. The amount of new vessel formation responds to the magnitude of the differential input to COX-2 and TSP-1. All angiogenic and anti-angiogenic signals that regulate the VEGF system are important, but only the difference between the stimulating and inhibiting signals determines the amount of VEGF expression and angiogenesis.

View larger version (22K): [in this window] [in a new window]   Fig. 4. Evidence-based and hypothetical illustration of regulation and cancer-induced deregulation of angiogenesis. The cancer distorts the physiological balance between angiogenic and anti-angiogenic factors that regulate the expression of vascular endothelial growth factor (VEGF). COX-2 increases VEGF and metalloproteinases (MMP) and thrombospondin (TSP-1) inhibits VEGF and MMP expression. Overexpression of COX-2 by cancer cells increases VEGF and MMP expression, inhibits apoptosis, and promotes angiogenesis, invasion, and metastatic spread. Loss of function of the tumor suppressor protein p53 increases tumor-induced angiogenesis by reducing TSP-1 and increasing COX-2 expression, resulting in reduced inhibition and increased stimulation of VEGF by TSP-1 and COX-2 respectively. Ctomegalovirus (CMV) inhibits TSP-1 expression, which decreases the inhibition of VEGF expression. Human papilloma virus (HVP) contains a VEGF-like segment. Both viruses induce angiogenesis in the cervical cancer.

View larger version (13K): [in this window] [in a new window]   Fig. 5. Simplified diagram of the angiogenesis control system. Triangles represent enzymes or growth factors. Resistors symbolize receptors or drugs. Imputs A-D control angiogensis via COX-2 (stimulation) or TSP-1 (inhibition) of VEGF, the output of which induces its stimultor TSP-1, which via its receptor CD36 inhibits VEGF, thus forming a negative feedback loop for VEGF. Nonsteroidal inflammatory drugs (NSAIDs) inhibit the stimulatory signal (PGE2). Examples of signal imputs: A: mevalonate, p53; B: CMV; C: p53; D: TGF-ß; EBV.

Furthermore, this hypothesis may explain why loss of the tumor-suppressor protein p53 function is so significant to tumor growth. Such loss not only enhances COX-2 but in addition reduces TSP-1 expression, which together greatly increase VEGF protein expression, angiogenesis, tumor invasion, and metastasis.

In most cases of cervical cancer, p53 is not mutated, but rather is elevated [148]. One might speculate that p53 expression could be induced in vivo in response to a malignant tumor, to defend against tumor-induced angiogenesis in an attempt to combat tumor growth, invasion, and metastasis. A better understanding of the pathophysiology involved in the growth of a specific tumor might be obtained by determining the levels of both angiogenic and anti-angiogenic factors that affect VEGF expression, and not assaying only VEGF.

Finally, the role of interference of energy metabolism in the inhibition of angiogenesis is most interesting. Angiostatin inhibits the surface F₀F₁-ATPase activity of endothelial cells [100]. In comparison, the cholesterol-lowering statin drugs inhibit coenzyme Q10 synthesis and thereby mitochondrial oxidative phosphorylation and ATP generation [99]. Such statins might thereby inhibit angiogenesis,, and at the same time enhance angiogenesis by reducing TSP-1 expression.

	Diagnosis and Therapy

Top Abstract Introduction Vasculogenesis and Angiogenesis The Malignant Tumor Molecular Pathology Regulation Discussion and Hypothesis Diagnosis and Therapy Conclusions References

 
A. Tumor markers  Several proteins that are angiogenesis regulators have been detected in blood and some are elevated in patients with cancer. However, their potential use to detect or monitor post-operative patients for tumor recurrence needs clarification [157,158].

Tumor vascular density is a prognostic factor for a variety of cancers. It reflects the importance of the vascular supply to the tumor growth. Furthermore, the fractal character of tumor-induced vessel formation exhibits differences compared to the fractal character of physiological new vessel formation. Tumor-induced vessels follow a more irregular course, probably due to inherent regional genetic differences in the tumor cell population. Magnetic resonance imaging can detect alterations in vessel permeability and permits in vivo evaluation of experimental tumor-induced angiogenesis. In the future it might be used in clinical studies to follow the reduction in cancer-induced angiogenesis that is achieved by COX-2 inhibition [159,160].

B. Anticancer therapy with NSAIDs  1. Non-selective NSAIDs  Many epidemiological studies, a large number of in vitro and animal experiments, and several clinical studies have demonstrated that non-selective NSAIDs can restrain the development and growth of different types of cancer [5]. Non-selective NSAIDs (nsNSAIDs) differ in their ability to inhibit the two known isoforms of cyclooxygenase (COX) [5], but most of them significantly inhibit COX-1 in platelets. Especially after prolonged use, they can cause bleeding and gastrointestinal ulceration and uncouple mitochondria [161].

Some anti-neoplastic effects of NSAIDs might be independent of cyclooxygenase inhibition. For instance, NSAIDs suppress colony formation on soft agar by transformed fibroblasts that either possess or lack COX-1 or COX-2 [83]. These anti-neoplastic effects might be due, in part, to the mitochondrial uncoupling that is common to many non-selective NSAIDs.

2. Selective COX-2 inhibitors  NSAIDs that inhibit COX-2 restore tumor cell apoptosis in vitro and reduce in vivo COX-2-tumor-induced angiogenesis. Furthermore, COX-2 inhibition lowers the synthesis of metalloproteinases and reduces matrix proteolysis. Also, as noted above, by inhibiting the COX-2 synthesis induced by the tumor in the adjacent host tissue, such agents obstruct the growth of xenografted tumors in which COX-2 has been silenced by methylation [79,162]. These selective COX-2 inhibitors might have great potential for treatment of human cancer. In comparison to nonselective NSAIDs, selective COX-2 inhibitors have fewer gastrointestinal side effects, do not interfere with platelet function, and do not uncouple mitochondria. Selective COX-2 inhibitors have shown significant anti-cancer effects in a variety of animal tumor xenograft models. They inhibit the growth and spread of the malignant cells [5,6].

The US Food and Drug Administration (FDA) has approved celecoxib and rofecoxib for treatment of rheumatoid arthritis and osteoarthritis respectively. A review of >50 clinical studies involving 13,000 patients and lasting 12 wk to 2 yr concluded that these drugs are well-tolerated [163]. Twice daily doses of celecoxib at 200 mg [164] and 400 mg [165] have been well tolerated by arthritis patients. It is encouraging that selective COX-2 inhibitors might prevent the development of cancer in patients with premalignant conditions such as adenomatous polyposis of the colon. A 6-mo clinical trial demonstrated that celecoxib, 400 mg twice daily (30 patients), but not 100 mg twice a day (32 patients) significantly reduced the number of polyps, compared to 15 patients given placebo [166].

The patient’s physician should carefully supervise any selective COX-2 therapy. For example, it has been advised that while gastrointestinal bleeding is not a problem in the use of the selective COX-2 inhibitors, the possible development of dyspepsia and renal problems should be carefully monitored [167]. Renal side effects might appear due to inhibition of COX-2 in the macula densa and medullary interstitial cells [168]. Second, because of the role COX-2 plays in implantation, COX-2 inhibition is not advisable in women who desire to conceive. Pregnant women should not use NSAIDs that inhibit COX-2 because the enzyme prevents the premature closure of the fetal ductus arteriosus [169]. Third, because the statin type of cholesterol-lowering drugs also lowers the synthesis of mevalonate and thereby the synthesis of the anti-angiogenic protein TSP-1, it might be reasonable to re-evaluate the need for statin therapy in patients with cancer. Fourth, analysis of 3 studies involving >17,000 rheumatoid arthritis patients showed an increase of thromboembolic events (eg, myocardial infarction , stroke) in patients who took rofecoxib 50 mg daily, compared to those who took celecoxib 400 mg twice daily plus low dose aspirin [170].

	Conclusions

Top Abstract Introduction Vasculogenesis and Angiogenesis The Malignant Tumor Molecular Pathology Regulation Discussion and Hypothesis Diagnosis and Therapy Conclusions References

 
High expression levels of cyclooxygenase (COX-2), vascular endothelial growth factor (VEGF), and metalloproteinase (MMP), and low thrombospondin (TSP-1) expression in cancer tissues is associated with high tumor vascular density (MVD), a prognostic factor in human malignancies that predicts aggressive growth, invasion, and metastases. The human cancer derails the normal balance between angiogenic (COX-2) and anti-angiogenic factors (TSP-1) that control the expression of VEGF, the pivotal mediator of new vessel formation. By expressing COX-2, which via PGE2 induces VEGF, the solid cancer forces its host to provide a vascular supply, which enables the tumor to grow beyond 1–2 mm.

The importance of COX-2 in angiogenesis is evidenced by its role in implantation-induced new vessel formation. The trophoblast of the implanting blastocyst induces maternal COX-2 expression, which is essential for the placental vasculogenesis and angiogenesis that are required for successful implantation. In granulation tissue at the sites of inflammation, and in a variety of solid tumors, COX-2 is re-expressed.

Chronic inflammation is a risk factor for cancer. The exact reason is not known, but in vitro, transgenic overexpression of COX-2 is capable of transforming cells. On the other hand, COX-2 negative cells can also be transformed. No gene abnormality of COX-2 in human cancer has been reported, but COX-2 can be induced by a variety of oncogenes, cytokines, growth factors, and carcinogens – for example, benzo[a]pyrene in tobacco smoke. In a large variety of human cancers, COX-2 is upregulated, producing eicosanoids such as PGE2, which induces VEGF and thereby angiogenesis. By inhibiting COX-2 in experimental cancers that overexpress this enzyme, the balance between angiogenic and anti-angiogenic signals, which control VEGF expression and angiogenesis, can be restored, causing tumor necrosis and tumor regression to a small, dormant state.

	References

Top Abstract Introduction Vasculogenesis and Angiogenesis The Malignant Tumor Molecular Pathology Regulation Discussion and Hypothesis Diagnosis and Therapy Conclusions References

 

1. Suh DY. Understanding angiogenesis and its clinical applications. Ann Clin Lab Sci 2000;30:227–238.[Abstract]
2. Eschwege P, de Ledinghen V, Camilli T, Kulkarni S, Dalbagni G, Droupy S, Jardin A, Benoit G, Weksler BB. Arachidonic acid and prostaglandins, inflammation and oncology. Presse Med 2001;30: 508–510.
3. Cheng T, Cao W, Wen R, Steinberg RH, LaVail MM. Prostaglandin E2 induces vascular endothelial growth factor and basic fibroblast growth factor mRNA expression in cultured rat Müller cells. Invest Ophthalmol Vis Sci 1998;39:581–591.[Abstract/Free Full Text]
4. Ben-Av P, Crofford LJ, Wilder RL, Hla T. Induction of vascular endothelial growth factor expression in synovial fibroblasts by prostaglandin E and interleukin-1: a potential mechanism for inflammatory angiogenesis. FEBS Lett 1995;372:83–87.[Medline]
5. Fosslien E. Biochemistry of cyclooxygenase (COX)-2 inhibitors and molecular pathology of COX-2 in neoplasia. Crit Rev Clin Lab Sci 2000;37:431–502.[Medline]
6. Fosslien E. Molecular pathology of cyclooxygenase-2 in neoplasia. Ann Clin Lab Sci 2000;30:3–21.[Abstract]
7. Masunaga R, Kohno H, Dhar DK, Ohno S, Shibakita M, Kinugasa S, Yoshimura H, Tachibana M, Kubota H, Nagasue N. Cyclooxygenase-2 expression correlates with tumor neovascularization and prognosis in human colorectal carcinoma patients. Clin Cancer Res 2000;6:4064–4068.[Abstract/Free Full Text]
8. Brown LF, Guidi AJ, Schnitt SJ, Van De Water L, Iruela-Arispe ML, Yeo TK, Tognazzi K, Dvorak HF. Vascular stroma formation in carcinoma in situ, invasive carcinoma, and metastatic carcinoma of the breast. Clin Cancer Res 1999;5:1041–1056.[Abstract/Free Full Text]
9. Williams CS, Mann M, DuBois RN. The role of cyclooxygenases in inflammation, cancer, and development. Oncogene 1999;18:7908–7916.[Medline]
10. Gately S. The contributions of cyclooxygenase-2 to tumor angiogenesis. Cancer Metastasis Rev 2000;19: 19–27.[Medline]
11. Bischof P, Campana A. A putative role for oncogenes in trophoblast invasion? Hum Reprod 2000;15 (Suppl 6):51–58.
12. Murray MJ, Lessey BA. Embryo implantation and tumor metastasis: common pathways of invasion and angiogenesis. Semin Reprod Endocrinol 1999;17: 275–290.[Medline]
13. Zoltowska A, Stepinski J, Lewko B, Zamorska B, Roszkiewicz A, Serkies K, Kruszewski WJ. Malformations of angiogenesis in the low differentiated human carcinomas. immunohistochemical study. Arch Immunol Ther Exp (Warsz) 2001;49:59–61.[Medline]
14. Smith SK. Angiogenesis and implantation. Ann Surg Oncol 2001;8:72–79.[Medline]
15. Fazleabas AT, Kim JJ, Srinivasan S, Donnelly KM, Brudney A, Jaffe RC. Implantation in the baboon: endometrial responses. Semin Reprod Endocrinol 1999;17:257–265.[Medline]
16. Dinchuk JE, Car BD, Focht RJ, Johnston JJ, Jaffee BD, Covington MB, Contel NR, Eng VM, Collins RJ, Czerniak PM, et al. Renal abnormalities and an altered inflammatory response in mice lacking cyclooxygenase II. Nature 1995;378:406–409.[Medline]
17. Dickson MC, Martin JS, Cousins FM, Kulkarni AB, Karlsson S, Akhurst RJ. Defective haematopoiesis and vasculogenesis in transforming growth factor-beta 1 knock out mice. Development 1995;121:1845–1854.[Abstract]
18. Kallapur S, Ormsby I, Doetschman T. Strain dependency of TGFbeta function during embryogenesis. Mol Reprod Dev 1999;52:341–349.[Medline]
19. Chung IB, Yelian FD, Zaher FM, Gonik B, Evans MI, Diamond MP, Svinarich DM. Expression and regulation of vascular endothelial growth factor in a first trimester trophoblast cell line. Placenta 2000; 21:320–324.[Medline]
20. Shalaby F, Rossant J, Yamaguchi TP, Gertsenstein M, Wu XF, Breitman ML, Schuh AC. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature 1995;376:62–66.[Medline]
21. Clyman RI, Hardy P, Waleh N, Chen YQ, Mauray F, Fouron JC, Chemtob S. Cyclooxygenase-2 plays a significant role in regulating the tone of the fetal lamb ductus arteriosus. Am J Physiol 1999;276 :R913–921.
22. Segi E. A study for functions of prostaglandin E receptor EP4 subtype by analysing knockout mice. Yakugaku Zasshi 2001;121:35–45.[Medline]
23. Loftin CD, Trivedi DB, Tiano HF, Clark JA, Lee CA, Epstein JA, Morham SG, Breyer MD, Nguyen M, Hawkins BM, Goulet JL, Smithies O, Koller BH, Langenbach R. Failure of ductus arteriosus closure and remodeling in neonatal mice deficient in cyclooxygenase-1 and cyclooxygenase-2. Proc Natl Acad Sci USA 2001;98:1059–1064.[Abstract/Free Full Text]
24. Tannenbaum JE, Waleh NS, Mauray F, Gold L, Perkett EA, Clyman RI. Transforming growth factor-beta protein and messenger RNA expression is increased in the closing ductus arteriosus. Pediatr Res 1996;39:427–434.[Medline]
25. Song J, Stastny J, Fosslien E, Robertson AL. Effect of aging on human protein composition. I. One-dimensional polyacrylamide gel electrophoretic analysis of tissue extracts. Exp Mol Path 1985; 43:233–241.[Medline]
26. Song J, Stastny J, Fosslien E, Robertson, AL. Effect of aging on human aortic protein composition. II. Two-dimensional polyacrylamide gel electrophoretic analysis of tissue extracts. Exp Mol Path 1985; 43:297–304.[Medline]
27. Li Z, Froehlich J, Galis ZS, Lakatta EG. Increased expression of matrix metalloproteinase-2 in the thickened intima of aged rats. Hypertension 1999; 33:116–123.[Abstract/Free Full Text]
28. Ghosh AK, Hirasawa N, Niki H, Ohuchi K. Cyclooxygenase-2-mediated angiogenesis in carrageenin-induced granulation tissue in rats. J Pharmacol Exp Ther 2000;295:802–809.[Abstract/Free Full Text]
29. Howdieshell TR, Callaway D, Webb WL, Gaines MD, Procter CD Jr, Sathyanarayana, Pollock JS, Brock TL, McNeil PL. Antibody neutralization of vascular endothelial growth factor inhibits wound granulation tissue formation. J Surg Res 2001;96: 173–182.[Medline]
30. Majima M, Hayashi I, Muramatsu M, Katada J, Yamashina S, Katori M. Cyclooxygenase-2 enhances basic fibroblast growth factor-induced angiogenesis through induction of vascular endothelial growth factor in rat sponge implants. Br J Pharmacol 2000; 130:641–649.[Medline]
31. Majima M, Isono M, Ikeda Y, Hayashi I, Hatanaka K, Harada Y, Katsumata O, Yamashina S, Katori M, Yamamoto S. Significant roles of inducible cyclooxygenase (COX)-2 in angiogenesis in rat sponge implants. Jpn J Pharmacol 1997;75:105–114.[Medline]
32. Hernandez GL, Volpert OV, Iniguez MA, Lorenzo E, Martinez-Martinez S, Grau R, Fresno M, Redondo JM. Selective inhibition of vascular endothelial growth factor-mediated angiogenesis by cyclosporin A: roles of the nuclear factor of activated T cells and cyclooxygenase 2. J Exp Med 2001;193:607–620.[Abstract/Free Full Text]
33. Cuzzocrea S, Costantino G, Mazzon E, Caputi AP. Regulation of prostaglandin production in carrageenan-induced pleurisy by melatonin. J Pineal Res 1999;27:9–14.[Medline]
34. Fosslien E. Escape from immunological surveillance in blastocyst implantation and cancer. Ann Clin Lab Sci 2000;30:111–112.
35. Saaristo A, Karpanen T, Alitalo K. Mechanisms of angiogenesis and their use in the inhibition of tumor growth and metastasis. Oncogene 2000;19:6122–6129.[Medline]
36. Li C, Guo B, Bernabeu C, Kumar S. Angiogenesis in breast cancer: the role of transforming growth factor beta and CD105. Microsc Res Tech 2001;52: 437–449.[Medline]
37. Price JT, Bonovich MT, Kohn EC. The biochemistry of cancer dissemination. Crit Rev Biochem Mol Biol 1997;32:175–253.[Medline]
38. John A, Tuszynski G The role of matrix metalloproteinases in tumor angiogenesis and tumor metastasis. Pathol Oncol Res 2001;7:14–23.[Medline]
39. Detmar M. Tumor angiogenesis. J Investig Dermatol Symp Proc 2000;5:20–23.[Medline]
40. Craciunescu OI, Das SK, Clegg ST. Dynamic contrast-enhanced MRI and fractal characteristics of percolation clusters in two-dimensional tumor blood perfusion. J Biomech Eng 1999;121:486.
41. Soslow RA, Dannenberg AJ, Rush D, Woerner BM, Khan KN, Masferrer J, Koki AT. COX-2 is expressed in human pulmonary, colonic, and mammary tumors. Cancer 2000;89:2637–2645.[Medline]
42. Kulkarni S, Rader JS, Zhang F, Liapis H, Koki AT, Masferrer JL, Subbaramaiah K, Dannenberg AJ. Cyclooxygenase-2 is overexpressed in human cervical cancer. Clin Cancer Res 2001;7:429–434.[Abstract/Free Full Text]
43. Gaffney DK, Holden J, Davis M, Zemmpolich K, Murphy KJ, Dodson M. Elevated cyclooxygenase-2 expression correlates with diminished survival in carcinoma of the cervix treated with radiotherapy. Int J Radiat Oncol Biol Phys 2001;49:1213–1217.[Medline]
44. Ohno R, Yoshinaga K, Fujita T, Hasegawa K, Iseki H, Tsunozaki H, Ichikawa W, Nihei Z, Sugihara K. Depth of invasion parallels increased cyclooxygenase-2 levels in patients with gastric carcinoma. Cancer 2001;91:1876–1881.[Medline]
45. Tomozawa S, Tsuno NH, Sunami E, Hatano K, Kitayama J, Osada T, Saito S, Tsuruo T, Shibata Y, Nagawa H. Cyclooxygenase-2 overexpression correlates with tumour recurrence, especially haematogenous metastasis, of colorectal cancer. Br J Cancer 2000;83:324–328.[Medline]
46. Madaan S, Abel PD, Chaudhary KS, Hewitt R, Stott MA, Stamp GW, Lalani EN. Cytoplasmic induction and over-expression of cyclooxygenase-2 in human prostate cancer: implications for prevention and treatment. BJU Int 2000;86:736–741.[Medline]
47. Yoshimura R, Sano H, Masuda C, Kawamura M, Tsubouchi Y, Chargui J, Yoshimura N, Hla T, Wada S. Expression of cyclooxygenase-2 in prostate carcinoma. Cancer 2000;89:589–596.[Medline]
48. Kirschenbaum A, Klausner AP, Lee R, Unger P, Yao S, Liu XH, Levine AC. Expression of cyclooxygenase-1 and cyclooxygenase-2 in the human prostate. Urology 2000;56:671–676.[Medline]
49. Denkert C, Kobel M, Berger S, Siegert A, Leclere A, Trefzer U, Hauptmann S. Expression of cyclooxygenase 2 in human malignant melanoma. Cancer Res 2001;61:303–308.[Abstract/Free Full Text]
50. Karim MM, Hayashi Y, Inoue M, Imai Y, Ito H, Yamamoto M. Cox-2 expression in retinoblastoma. Am J Ophthalmol 2000;129:398–401.[Medline]
51. Tas F, Yavuz E, Aydiner A, Saip P, Disci R, Iplikci A, Topuz E. Angiogenesis and p53 protein expression in breast cancer: prognostic roles and interrelationships. Am J Clin Oncol 2000;23:546–553.[Medline]
52. Shimizu T, Hino K, Tauchi K, Ansai Y, Tsukada K. Predication of axillary lymph node metastasis by intravenous digital subtraction angiography in breast cancer, its correlation with microvascular density. Breast Cancer Res Treat 2000;61:261–269.[Medline]
53. Garzetti GG, Ciavattini A, Lucarini G, Pugnaloni A, De Nictolis M, Amati S, Romanini C, Biagini G. Expression of vascular endothelial growth factor related to 72-kilodalton metalloproteinase immunostaining in patients with serous ovarian tumors. Cancer 1999;85:2219–2225.[Medline]
54. Yoshiji H, Gomez DE, Shibuya M, Thorgeirsson UP. Expression of vascular endothelial growth factor, its receptor, and other angiogenic factors in human breast cancer. Cancer Res 1996;56:2013–2016.[Abstract/Free Full Text]
55. Paradis V, Lagha NB, Zeimoura L, Blanchet P, Eschwege P, Ba N, Benoit G, Jardin A, Bedossa P. Expression of vascular endothelial growth factor in renal cell carcinomas. Virchows Arch 2000;436:351–356.[Medline]
56. Slaton JW, Inoue K, Perrotte P, El-Naggar AK, Swanson DA, Fidler IJ, Dinney CP. Expression levels of genes that regulate metastasis and angiogenesis correlate with advanced pathological stage of renal cell carcinoma. Am J Pathol 2001;158:735–743.[Abstract/Free Full Text]
57. Ogata Y, Harada Y, Fujii T, Yamana H, Fujita H, Shirouzu K. Immunohistochemical localization of vascular endothelial growth factor in esophageal cancer. Kurume Med J 1996;43:157–163.[Medline]
58. Bayer-Garner IB, Hough AJ Jr, Smoller BR. Vascular endothelial growth factor expression in malignant melanoma: prognostic versus diagnostic usefulness. Mod Pathol 1999;12:770–774.[Medline]
59. Strohmeyer D, Rossing C, Bauerfeind A, Kaufmann O, Schlechte H, Bartsch G, Loening S. Vascular endothelial growth factor and its correlation with angiogenesis and p53 expression in prostate cancer. Prostate 2000;45:216–224.[Medline]
60. Han H, Silverman JF, Santucci TS, Macherey RS, d’Amato TA, Tung MY, Weyant RJ, Landreneau RJ. Vascular endothelial growth factor expression in stage I non-small cell lung cancer correlates with neoangiogenesis and a poor prognosis. Ann Surg Oncol 2001; 8:72–79.
61. Li XM, Tang ZY, Qin LX, Zhou J, Sun HC. Serum vascular endothelial growth factor is a predictor of invasion and metastasis in hepatocellular carcinoma. J Exp Clin Cancer Res 1999;18:511–517.[Medline]
62. Sabo E, Boltenko A, Sova Y, Stein A, Kleinhaus S, Resnick MB. Microscopic analysis and significance of vascular architectural complexity in renal cell carcinoma. Clin Cancer Res 2001;7:533–537.[Abstract/Free Full Text]
63. Decaussin M, Sartelet H, Robert C, Moro D, Claraz C, Brambilla C, Brambilla E. Expression of vascular endothelial growth factor (VEGF) and its two receptors (VEGF-R1-Flt1 and VEGF-R2-Flk1/KDR) in non-small cell lung carcinomas (NSCLCs): correlation with angiogenesis and survival. J Pathol 1999;188:369–377.[Medline]
64. Fine BA, Valente PT, Feinstein GI, Dey T. VEGF, flt-1, and KDR/flk-1 as prognostic indicators in endometrial carcinoma. Gynecol Oncol 2000;76:33–39.[Medline]
65. Marinkovic S, Baumann H. Structure, hormonal regulation, and identification of the interleukin-6-and dexamethasone-responsive element of the rat haptoglobin gene. Mol Cell Biol 1990;10:1573–1583.[Abstract/Free Full Text]
66. Stastny J, Prasad R, Fosslien E. Tissue proteins in breast cancer, as studied by use of two-dimensional electrophoresis. Clin Chem 1984;30:1914–1918.[Abstract]
67. Nayak SK, Kakati S, Harvey SR, Malone CC, Cornforth AN, Dillman RO. Characterization of cancer cell lines established from two human metastatic breast cancers. In Vitro Cell Dev Biol Anim 2000;36:188–193.[Medline]
68. Harvey SR, Nayak SK, Markus G, Ouhammouch M, Hemperly JJ, Dillman RO, Doyle DJ. Cancer cells release a covalent complex containing disulfide-linked domains from urinary plasminogen activator, neural cell adhesion molecule, and haptoglobin alpha and beta chains. Arch Biochem Biophys 1997;345: 289–298.[Medline]
69. Mehaffey MG, Folberg R, Meyer M, Bentler SE, Hwang T, Woolson R, Moore KC. Relative importance of quantifying area and vascular patterns in uveal melanomas. Am J Ophthalmol 1997;123: 798–809.[Medline]
70. Gazit Y, Baish JW, Safabakhsh N, Leunig M, Baxter LT, Jain RK. Fractal characteristics of tumor vascular architecture during tumor growth and regression. Microcirculation 1997;4:395–402.[Medline]
71. Hepburn PJ, Griffiths K, Harper ME. Angiogenic factors expressed by human prostatic cell lines: effect on endothelial cell growth in vitro. Prostate 1997; 33:123–132.[Medline]
72. Benefield J, Petruzzelli GJ, Fowler S, Taitz A, Kalkanis J, Young MR. Regulation of the steps of angiogenesis by human head and neck squamous cell carcinomas. Invasion Metastasis 1996;16:291–301.[Medline]
73. Relf M, LeJeune S, Scott PA, Fox S, Smith K, Leek R, Moghaddam A, Whitehouse R, Bicknell R, Harris AL. Expression of the angiogenic factors vascular endothelial cell growth factor, acidic and basic fibroblast growth factor, tumor growth factor beta-1, platelet-derived endothelial cell growth factor, placenta growth factor, and pleiotrophin in human primary breast cancer and its relation to angiogenesis. Cancer Res 1997;57:963–969.[Abstract/Free Full Text]
74. Yamaguchi R, Yano H, Iemura A, Ogasawara S, Haramaki M, Kojiro M. Expression of vascular endothelial growth factor in human hepatocellular carcinoma. Hepatology 1998;28:68–77.[Medline]
75. Harris AL, Zhang H, Moghaddam A, Fox S, Scott P, Pattison A, Gatter K, Stratford I, Bicknell R. Breast cancer angiogenesis--new approaches to therapy via antiangiogenesis, hypoxic activated drugs, and vascular targeting. Breast Cancer Res Treat 1996; 38:97–108.[Medline]
76. Harada S, Nagy JA, Sullivan KA, Thomas KA, Endo N, Rodan GA, Rodan SB. Induction of vascular endothelial growth factor expression by prostaglandin E2 and E1 in osteoblasts. J Clin Invest 1994;93: 2490–2496.
77. Hoper MM, Voelkel NF, Bates TO, Allard JD, Horan M, Shepherd D, Tuder RM. Prostaglandins induce vascular endothelial growth factor in a human monocytic cell line and rat lungs via cAMP. Am J Respir Cell Mol Biol 1997;17:748–756.[Abstract/Free Full Text]
78. Attiga FA, Fernandez PM, Weeraratna AT, Manyak MJ, Patierno SR. Inhibitors of prostaglandin synthesis inhibit human prostate tumor cell invasiveness and reduce the release of matrix metalloproteinases. Cancer Res 2000;60:4629–4637.[Abstract/Free Full Text]
79. Williams CS, Tsujii M, Reese J, Dey SK, DuBois RN. Host cyclooxygenase-2 modulates carcinoma growth. J Clin Invest 2000;105:1589–1594.[Medline]
80. Borgstrom P, Bourdon MA, Hillan KJ, Sriramarao P, Ferrara N. Neutralizing anti-vascular endothelial growth factor antibody completely inhibits angiogenesis and growth of human prostate carcinoma micro tumors in vivo. Prostate 1998;35:1–10.[Medline]
81. Narko K, Ristimaki A, MacPhee M, Smith E, Haudenschild CC, Hla T. Tumorigenic transformation of immortalized ECV endothelial cells by cyclooxygenase-1 overexpression. J Biol Chem 1997; 272:21455–21460.[Abstract/Free Full Text]
82. Liu CH, Chang SH, Narko K, Trifan OC, Wu MT, Smith E, Haudenschild C, Lane TF, Hla T. Overexpression of cyclooxygenase-2 is sufficient to induce tumorigenesis in transgenic mice. J Biol Chem 2001;276:18563–18569.[Abstract/Free Full Text]
83. Zhang X, Morham SG, Langenbach R, Young DA. Malignant transformation and antineoplastic actions of nonsteroidal antiinflammatory drugs (NSAIDs) on cyclooxygenase-null embryo fibroblasts. J Exp Med 1999;190:451–459.[Abstract/Free Full Text]
84. Di Popolo A, Memoli A, Apicella A, Tuccillo C, di Palma A, Ricchi P, Acquaviva AM, Zarrilli R. IGF-II/IGF-I receptor pathway up-regulates COX-2 mRNA expression and PGE2 synthesis in Caco-2 human colon carcinoma cells. Oncogene 2000;19: 5517–5524.[Medline]
85. Abe T, Okamura K, Ono M, Kohno K, Mori T, Hori S, Kuwano M. Induction of vascular endothelial tubular morphogenesis by human glioma cells. A model system for tumor angiogenesis. J Clin Invest 1993;92:54–61.
86. Sheng H, Shao J, Washington MK, DuBois RN. Prostaglandin E2 increases growth and motility of colorectal carcinoma cells. J Biol Chem 2001;276: 18075–18081.[Abstract/Free Full Text]
87. Plescia OJ, Smith AH, Grinwich K. Subversion of immune system by tumor cells and role of prostaglandins. Proc Natl Acad Sci USA 1975;72: 1848–1851.[Abstract/Free Full Text]
88. Grinwich KD, Plescia OJ. Tumor-mediated immunosuppression: prevention by inhibitors of prostaglandin synthesis. Prostaglandins 1977;14: 1175–1182.[Medline]
89. Shibuya M. Structure and function of VEGF/VEGF-receptor system involved in angiogenesis. Cell Struct Funct 2001;26:25–35.[Medline]
90. Clauss M. Molecular biology of the VEGF and the VEGF receptor family. Semin Thromb Hemost 2000;26:561–569.[Medline]
91. Mourtada I, Le Tourneur M, Chevrant-Breton J, Le Gall F. Human orf and erythema multiforme. Ann Dermatol Venereol 2000;127:397–399[Medline]
92. Savory LJ, Stacker SA, Fleming SB, Niven BE, Mercer AA. Viral vascular endothelial growth factor plays a critical role in orf virus infection. J Virol 2000;74: 10699–10706.[Abstract/Free Full Text]
93. Wei MH, Popescu NC, Lerman MI, Merrill MJ, Zimonjic DB. Localization of the human vascular endothelial growth factor gene, VEGF, at chromosome 6p12. Hum Genet 1996;97:794–797.[Medline]
94. De Juan C, Iniesta P, Cruces J, Sanchez A, Massa MJ, Gonzalez-Quevedo R, Torres AJ, Balibrea JL, Benito M. DNA amplification on chromosome 6p12 in non small cell lung cancer detected by arbitrarily primed polymerase chain reaction. Int J Cancer 1999; 84:344–349.[Medline]
95. Wheeler-Jones C, Abu-Ghazaleh R, Cospedal R, Houliston RA, Martin J, Zachary I. Vascular endothelial growth factor stimulates prostacyclin production and activation of cytosolic phospholipase A2 in endothelial cells via p42/p44 mitogen-activated protein kinase. FEBS Lett 1997;420:28–32.[Medline]
96. Bryant CE, Appleton I, Mitchell JA. Vascular endothelial growth factor upregulates constitutive cyclooxygenase 1 in primary bovine and human endothelial cells. Life Sci 1998;62:2195–2201.[Medline]
97. Kim HJ, Kim KW, Yu BP, Chung HY. The effect of age on cyclooxygenase-2 gene expression: NF-kappaB activation and IkappaBalpha degradation. Free Radic Biol Med 2000;28:683–692.[Medline]
98. Knight JA. The biochemistry of aging. Adv Clin Chem 2000;35:1–62.[Medline]
99. Fosslien E. Mitochondrial medicine–molecular pathology of defective oxidative phosphorylation. Ann Clin Lab Sci 2001;31:25–67.[Abstract/Free Full Text]
100. Moser TL, Kenan DJ, Ashley TA, Roy JA, Goodman MD, Misra UK, Cheek DJ, Pizzo SV. Endothelial cell surface F1-F0 ATP synthase is active in ATP synthesis and is inhibited by angiostatin. Proc Natl Acad Sci USA 2001;98:6656–6661.[Abstract/Free Full Text]
101. Jin RJ, Kwak C, Lee SG, Lee CH, Soo CG, Park MS, Lee E, Lee SE. The application of an anti-angiogenic gene (thrombospondin-1) in the treatment of human prostate cancer xenografts. Cancer Gene Ther 2000;7:1537–1542.[Medline]
102. Streit M, Velasco P, Brown LF, Skobe M, Richard L, Riccardi L, Lawler J, Detmar M. Overexpression of thrombospondin-1 decreases angiogenesis and inhibits the growth of human cutaneous squamous cell carcinomas. Am J Pathol 1999;155:441–452.[Abstract/Free Full Text]
103. Kawahara N, Ono M, Taguchi K, Okamoto M, Shimada M, Takenaka K, Hayashi K, Mosher DF, Sugimachi K, Tsuneyoshi M, Kuwano M. Enhanced expression of thrombospondin-1 and hypovascularity in human cholangiocarcinoma. Hepatology 1998;28:1512–1517.[Medline]
104. Kasper HU, Ebert M, Malfertheiner P, Roessner A, Kirkpatrick CJ, Wolf HK. Expression of thrombospondin-1 in pancreatic carcinoma: correlation with microvessel density. Virchows Arch 2001;438:116–120.[Medline]
105. Dawson DW, Pearce SF, Zhong R, Silverstein RL, Frazier WA, Bouck NP. CD36 mediates the in vitro inhibitory effects of thrombospondin-1 on endothelial cells. J Cell Biol 1997;138:707–717.[Abstract/Free Full Text]
106. Sheibani N, Frazier WA. Thrombospondin-1, PECAM-1, and regulation of angiogenesis. Histol Histopathol 1999;14:285–294.[Medline]
107. Bein K, Simons M. Thrombospondin type 1 repeats interact with matrix metalloproteinase 2. Regulation of metalloproteinase activity. J Biol Chem 2000;275: 32167–32173.[Abstract/Free Full Text]
108. Grant SW, Kyshtoobayeva AS, Kurosaki T, Jakowatz J, Fruehauf JP. Mutant p53 correlates with reduced expression of thrombospondin-1, increased angiogenesis, and metastatic progression in melanoma. Cancer Detect Prev 1998;22:185–194.[Medline]
109. Tokunaga T, Nakamura M, Oshika Y, Tsuchida T, Kazuno M, Fukushima Y, Kawai K, Abe Y, Kijima H, Yamazaki H, Tamaoki N, Ueyama Y. Alterations in tumour suppressor gene p53 correlate with inhibition of thrombospondin-1 gene expression in colon cancer cells. Virchows Arch 1998;433:415–418.[Medline]
110. Riessen R, Axel DI, Fenchel M, Herzog UU, Rossmann H, Karsch KR. Effect of HMG-CoA reductase inhibitors on extracellular matrix expression in human vascular smooth muscle cells. Basic Res Cardiol 1999;94:322–332.[Medline]
111. Li H, Lindenmeyer F, Grenet C, Opolon P, Menashi S, Soria C, Yeh P, Perricaudet M, Lu H. AdTIMP-2 inhibits tumor growth, angiogenesis, and metastasis, and prolongs survival in mice. Hum Gene Ther 2001;12:515–526.[Medline]
112. Parsons-Wingerter P, Lwai B, Yang MC, Elliott KE, Milaninia A, Redlitz A, Clark JI, Sage EH. A novel assay of angiogenesis in the quail chorioallantoic membrane: stimulation by bFGF and inhibition by angiostatin according to fractal dimension and grid intersection. Microvasc Res 1998;55:201–214.[Medline]
113. Fosslien E. The establishment and maintenance of curvature: a morphodynamic hypothesis. Med Hypothesis (submitted).
114. Murphy-Ullrich JE, Poczatek M. Activation of latent TGF-beta by thrombospondin-1: mechanisms and physiology. Cytokine Growth Factor Rev 2000;11: 59–69.[Medline]
115. Grainger DJ, Frow EK. Thrombospondin 1 does not activate transforming growth factor beta-1 in a chemically defined system or in smooth-muscle-cell cultures. Biochem J 2000;350:291–298.
116. Kawataki T, Naganuma H, Sasaki A, Yoshikawa H, Tasaka K, Nukui H. Correlation of thrombospondin-1 and transforming growth factor-beta expression with malignancy of glioma. Neuropathology 2000;20:161–169.[Medline]
117. Qian X, Wang TN, Rothman VL, Nicosia RF, Tuszynski GP. Thrombospondin-1 modulates angiogenesis in vitro by up-regulation of matrix metalloproteinase-9 in endothelial cells. Exp Cell Res 1997;235:403–412.[Medline]
118. Wang TN, Qian X, Granick MS, Solomon MP, Rothman VL, Berger DH, Tuszynski GP. Thrombospondin-1 (TSP-1) promotes the invasive properties of human breast cancer. J Surg Res 1996; 63:39–43.[Medline]
119. Bleuel K, Popp S, Fusenig NE, Stanbridge EJ, Boukamp P. Tumor suppression in human skin carcinoma cells by chromosome 15 transfer or thrombospondin-1 overexpression through halted tumor vascularization. Proc Natl Acad Sci USA 1999;96:2065–2070.[Abstract/Free Full Text]
120. Shao J, Sheng H, Aramandla R, Pereira MA, Lubet RA, Hawk E, Grogan L, Kirsch IR, Washington MK, Beauchamp RD, DuBois RN. Coordinate regulation of cyclooxygenase-2 and TGF-beta1 in replication error-positive colon cancer and azoxymethane-induced rat colonic tumors. Carcinogenesis 1999; 20:185–191.[Abstract/Free Full Text]
121. O’Mahony CA, Beauchamp RD, Albo D, Tsujii M, Sheng HM, Shao J, Dubois RN, Berger DH. Cyclooxygenase-2 alters transforming growth factor-beta 1 response during intestinal tumorigenesis. Surgery 1999;126:364–370.[Medline]
122. Cardillo MR, Yap E. TGF-beta1 in colonic neoplasia: a genetic molecular and immunohistochemical study. J Exp Clin Cancer Res 1997;16:281–288.[Medline]
123. Kim SJ, Im YH, Markowitz SD, Bang YJ. Molecular mechanisms of inactivation of TGF-beta receptors during carcinogenesis. Cytokine Growth Factor Rev 2000;11:159–168.[Medline]
124. Gold LI, Jussila T, Fusenig NE, Stenback F. TGF-beta isoforms are differentially expressed in increasing malignant grades of HaCaT keratinocytes, suggesting separate roles in skin carcinogenesis. J Pathol 2000;190:579–588.[Medline]
125. Connolly JM, Rose DP. Angiogenesis in two human prostate cancer cell lines with differing metastatic potential when growing as solid tumors in nude mice. J Urol 1998;160:932–936.[Medline]
126. Qiu L. Regulatory role of hormones and growth factors in the pathogenesis of human uterine leiomyomas (PhD Dissertation in Pathology). Graduate College, Health Sciences Center, University of Illinois, Chicago, 1995.
127. Fosslien E, Lin Q, Yamashiroya H. Human uterine leiomyoma cells: Growth responses to epidermal growth factor and transforming growth factor beta. Ann Clin Lab Sci 1997;7:318–319.
128. Pepper MS, Vassalli JD, Orci L, Montesano R. Biphasic effect of transforming growth factor-beta 1 on in vitro angiogenesis. Exp Cell Res 1993; 204:356–363.[Medline]
129. Oshima M, Oshima H, Taketo MM. TGF-beta receptor type II deficiency results in defects of yolk sac hematopoiesis and vasculogenesis. Dev Biol 1996;179:297–302.[Medline]
130. Azuma H. Genetic and molecular pathogenesis of hereditary hemorrhagic telangiectasia. J Med Invest 2000;47:81–90.[Medline]
131. Sakamoto T, Ueno H, Sonoda K, Hisatomi T, Shimizu K, Ohashi H, Inomata H. Blockade of TGF-beta by in vivo gene transfer of a soluble TGF-beta type II receptor in the muscle inhibits corneal opacification, edema and angiogenesis. Gene Ther 2000;7:1915–1924.[Medline]
132. Larsson J, Goumans MJ, Sjostrand LJ, van Rooijen MA, Ward D, Leveen P, Xu X, ten Dijke P, Mummery CL, Karlsson S. Abnormal angiogenesis but intact hematopoietic potential in TGF-beta type I receptor-deficient mice. EMBO J 2001;20:1663–1673.[Medline]
133. Li DY, Sorensen LK, Brooke BS, Urness LD, Davis EC, Taylor DG, Boak BB, Wendel DP. Defective angiogenesis in mice lacking endoglin. Science 1999; 284:1534–1537.[Abstract/Free Full Text]
134. Li C, Guo B, Wilson PB, Stewart A, Byrne G, Bundred N, Kumar S. Plasma levels of soluble CD105 correlate with metastasis in patients with breast cancer. Int J Cancer 2000;89:122–126.[Medline]
135. Brewer CA, Setterdahl JJ, Li MJ, Johnston JM, Mann JL, McAsey ME. Endoglin expression as a measure of microvessel density in cervical cancer. Obstet Gynecol 2000;96:224–228.[Medline]
136. DuBois RN, Awad J, Morrow J, Roberts LJ 2nd, Bishop PR. Regulation of eicosanoid production and mitogenesis in rat intestinal epithelial cells by transforming growth factor-alpha and phorbol ester. J Clin Invest 1994;93:493–498.
137. Soares R, Pereira MB, Silva C, Amendoeira I, Wagner R, Ferro J, Schmitt FC. Expression of TGF-alpha and EGFR in breast cancer and its relation to angiogenesis. Breast J 2000;6:171–177.[Medline]
138. Sheibani N, Frazier WA. Down-regulation of platelet endothelial cell adhesion molecule-1 results in thrombospondin-1 expression and concerted regulation of endothelial cell phenotype. Mol Biol Cell 1998;9:701–713.[Abstract/Free Full Text]
139. Liu XH, Kirschenbaum A, Yao S, Stearns ME, Holland JF, Claffey K, Levine AC. Upregulation of vascular endothelial growth factor by cobalt chloride-simulated hypoxia is mediated by persistent induction of cyclooxygenase-2 in a metastatic human prostate cancer cell line. Clin Exp Metastasis 1999; 17:687–694[Medline]
140. Laderoute KR, Alarcon RM, Brody MD, Calaoagan JM, Chen EY, Knapp AM, Yun Z, Denko NC, Giaccia AJ. Opposing effects of hypoxia on expression of the angiogenic inhibitor thrombospondin 1 and the angiogenic inducer vascular endothelial growth factor. Clin Cancer Res 2000; 6:2941–2950.[Abstract/Free Full Text]
141. Phelan MW, Forman LW, Perrine SP, Faller DV. Hypoxia increases thrombospondin-1 transcript and protein in cultured endothelial cells. J Lab Clin Med 1998;132:519–529.[Medline]
142. Harris AL. von Hippel-Lindau syndrome: target for anti-vascular endothelial growth factor (VEGF) receptor therapy. Oncol 2000;5(Suppl 1):32–36.
143. Krieg M, Haas R, Brauch H, Acker T, Flamme I, Plate KH. Up-regulation of hypoxia-inducible factors HIF-1alpha and HIF-2alpha under normoxic conditions in renal carcinoma cells by von Hippel-Lindau tumor suppressor gene loss of function. Oncogene 2000;19:5435–5443.[Medline]
144. Cinatl J Jr, Kotchetkov R, Scholz M, Cinatl J, Vogel JU, Driever PH, Doerr HW. Human cytomegalo-virus infection decreases expression of thrombospondin-1 independent of the tumor suppressor protein p53. Am J Pathol 1999;155:285–292.[Abstract/Free Full Text]
145. Dameron KM, Volpert OV, Tainsky MA, Bouck N. Control of angiogenesis in fibroblasts by p53 regulation of thrombospondin-1. Science 1994;265: 1582–1584.[Abstract/Free Full Text]
146. Linderholm BK, Lindahl T, Holmberg L, Klaar S, Lennerstrand J, Henriksson R, Bergh J. The expression of vascular endothelial growth factor correlates with mutant p53 and poor prognosis in human breast cancer. Cancer Res 2001;61:2256–2260.[Abstract/Free Full Text]
147. Biramijamal F, Allameh A, Mirbod P, Groene HJ, Koomagi R, Hollstein M. Unusual profile and high prevalence of p53 mutations in esophageal squamous cell carcinomas from northern Iran. Cancer Res 2001;61:3119–3123.[Abstract/Free Full Text]
148. Nair P, Gangadevi T, Jayaprakash PG, Nair MB, Nair MK, Pillai MR. Increased angiogenesis in the uterine cervix associated with human papillomavirus infection. Pathol Res Pract 1999;195:163–169.[Medline]
149. Lopez-Ocejo O, Viloria-Petit A, Bequet-Romero M, Mukhopadhyay D, Rak J, Kerbel RS. Oncogenes and tumor angiogenesis: the HPV-16 E6 oncoprotein activates the vascular endothelial growth factor (VEGF) gene promoter in a p53 independent manner. Oncogene 2000;19:4611–4620.[Medline]
150. Murono S, Inoue H, Tanabe T, Joab I, Yoshizaki T, Furukawa M, Pagano JS. Induction of cyclooxygenase-2 by Epstein-Barr virus latent membrane protein 1 is involved in vascular endothelial growth factor production in nasopharyngeal carcinoma cells. Proc Natl Acad Sci USA 2001;98:6905–6910.[Abstract/Free Full Text]
151. Saha D, Datta PK, Sheng H, Morrow JD, Wada M, Moses HL, Beauchamp RD. Synergistic induction of cyclooxygenase-2 by transforming growth factor-beta1 and epidermal growth factor inhibits apoptosis in epithelial cells. Neoplasia 1999;1:508–517.[Medline]
152. Shinar DM, Rodan GA. Biphasic effects of transforming growth factor-beta on the production of osteoclast-like cells in mouse bone marrow cultures: the role of prostaglandins in the generation of these cells.Endocrinology. 1990;126:3153–3158.[Abstract/Free Full Text]
153. Bernard C, Merval R, Lebret M, Delerive P, Dusanter-Fourt I, Lehoux S, Creminon C, Staels B, Maclouf J, Tedgui A. Oncostatin M induces interleukin-6 and cyclooxygenase-2 expression in human vascular smooth muscle cells : synergy with interleukin-1beta. Circ Res 1999;85:1124–1131.[Abstract/Free Full Text]
154. Pourtau J, Mirshahi F, Li H, Muraine M, Vincent L, Tedgui A, Vannier JP, Soria J, Vasse M, Soria C. Cyclooxygenase-2 activity is necessary for the angiogenic properties of oncostatin M. FEBS Lett. 1999:459:453–457.[Medline]
155. Vasse M, Pourtau J, Trochon V, Muraine M, Vannier JP, Lu H, Soria J, Soria C. Oncostatin M induces angiogenesis in vitro and in vivo. Arterioscler Thromb Vasc Biol 1999;19:1835–1842.[Abstract/Free Full Text]
156. Suzuma K, Takagi H, Otani A, Oh H, Honda Y. Expression of thrombospondin-1 in ischemia-induced retinal neovascularization. Am J Pathol 1999;154:343–354.[Abstract/Free Full Text]
157. Morelli D, Lazzerini D, Cazzaniga S, Squicciarini P, Bignami P, Maier JA, Sfondrini L, Menard S, Colnaghi MI, Balsari A. Evaluation of the balance between angiogenic and antiangiogenic circulating factors in patients with breast and gastrointestinal cancers. Clin Cancer Res 1998;4:1221–1225.[Abstract]
158. Kuroi K, Toi M. Circulating angiogenesis regulators in cancer patients. Int J Biol Markers 2001;16:5–26.[Medline]
159. Daldrup H, Shames DM, Wendland M, Okuhata Y, Link TM, Rosenau W, Lu Y, Brasch RC. Correlation of dynamic contrast-enhanced MR imaging with histologic tumor grade: comparison of macromolecular and small-molecular contrast media. Am J Roentgenol 1998;171:941–949.[Abstract/Free Full Text]
160. Turetschek K, Huber S, Floyd E, Helbich T, Roberts TP, Shames DM, Tarlo KS, Wendland MF, Brasch RC. MR imaging characterization of microvessels in experimental breast tumors by using a particulate contrast agent with histopathologic correlation. Radiology 2001;218:562–569.[Abstract/Free Full Text]
161. Fosslien E. Adverse effects of nonsteroidal anti-inflammatory drugs on the gastrointestinal system. Ann Clin Lab Sci 1998;28:67–81.[Abstract]
162. Toyota M, Shen L, Ohe-Toyota M, Hamilton SR, Sinicrope FA, Issa JP. Aberrant methylation of the cyclooxygenase 2 CpG island in colorectal tumors. Cancer Res 2000;60:4044–4048.[Abstract/Free Full Text]
163. Whelton A, Maurath CJ, Verburg KM, Geis GS. Renal safety and tolerability of celecoxib, a novel cyclooxygenase-2 inhibitor. Am J Ther 2000;7:159–175.[Medline]
164. Goldstein JL, Correa P, Zhao WW, Burr AM, Hubbard RC, Verburg KM, Geis GS. Reduced incidence of gastroduodenal ulcers with celecoxib, a novel cyclooxygenase-2 inhibitor, compared to naproxen in patients with arthritis. Am J Gastro-enterol 2001;96:1019–1027.[Medline]
165. Silverstein FE, Faich G, Goldstein JL, Simon LS, Pincus T, Whelton A, Makuch R, Eisen G, Agrawal NM, Stenson WF, Burr AM, Zhao WW, Kent JD, Lefkowith JB, Verburg KM, Geis GS. Gastrointestinal toxicity with celecoxib vs nonsteroidal anti-inflammatory drugs for osteoarthritis and rheumatoid arthritis: the CLASS study: A randomized controlled trial. (Celecoxib Long-term Arthritis Safety Study). JAMA 2000;284:1247–1255.[Abstract/Free Full Text]
166. Steinbach G, Lynch PM, Phillips RK, Wallace MH, Hawk E, Gordon GB, Wakabayashi N, Saunders B, Shen Y, Fujimura T, Su LK, Levin B. The effect of celecoxib, a cyclooxygenase-2 inhibitor, in familial adenomatous polyposis. N Engl J Med 2000; 29;342:1946–1952.
167. Emery P. Cyclooxygenase-2: a major therapeutic advance? Am J Med 2001;110:42S–45S.[Medline]
168. Breyer MD, Harris RC. Cyclooxygenase 2 and the kidney. Curr Opin Nephrol Hypertens 2001;10:89–98.[Medline]
169. Takahashi Y, Roman C, Chemtob S, Tse MM, Lin E, Heymann MA, Clyman RI. Cyclooxygenase-2 inhibitors constrict the fetal lamb ductus arteriosus both in vitro and in vivo. Am J Physiol Regul Integr Comp Physiol 2000;278:R1496–1505.[Abstract/Free Full Text]

This article has been cited by other articles:

				E. Zattra, C. Coleman, S. Arad, E. Helms, D. Levine, E. Bord, A. Guillaume, M. El-Hajahmad, E. Zwart, H. van Steeg, et al. Polypodium leucotomos Extract Decreases UV-Induced Cox-2 Expression and Inflammation, Enhances DNA Repair, and Decreases Mutagenesis in Hairless Mice Am. J. Pathol., November 1, 2009; 175(5): 1952 - 1961. [Abstract] [Full Text] [PDF]

				C. Xie, B. Liang, M. Xue, A. S.P. Lin, A. Loiselle, E. M. Schwarz, R. E. Guldberg, R. J. O'Keefe, and X. Zhang Rescue of Impaired Fracture Healing in COX-2-/- Mice via Activation of Prostaglandin E2 Receptor Subtype 4 Am. J. Pathol., August 1, 2009; 175(2): 772 - 785. [Abstract] [Full Text] [PDF]

				A. K. Bauer and E. A. Rondini Review Paper: The Role of Inflammation in Mouse Pulmonary Neoplasia Veterinary Pathology, May 1, 2009; 46(3): 369 - 390. [Abstract] [Full Text] [PDF]

				M. Sahin, E. Sahin, and S. Gumuslu Cyclooxygenase-2 in Cancer and Angiogenesis Angiology, April 1, 2009; 60(2): 242 - 253. [Abstract] [PDF]

				P. Pakneshan, A. E. Birsner, I. Adini, C. M. Becker, and R. J. D'Amato Differential Suppression of Vascular Permeability and Corneal Angiogenesis by Nonsteroidal Anti-inflammatory Drugs Invest. Ophthalmol. Vis. Sci., September 1, 2008; 49(9): 3909 - 3913. [Abstract] [Full Text] [PDF]

				S. A. Danovi, H. H. Wong, and N. R. Lemoine Targeted therapies for pancreatic cancer Br. Med. Bull., September 1, 2008; 87(1): 97 - 130. [Abstract] [Full Text] [PDF]

				M Branca, C Giorgi, D Santini, L Di Bonito, M Ciotti, A Benedetto, P Paba, S Costa, D Bonifacio, P Di Bonito, et al. Aberrant expression of VEGF-C is related to grade of cervical intraepithelial neoplasia (CIN) and high risk HPV, but does not predict virus clearance after treatment of CIN or prognosis of cervical cancer J. Clin. Pathol., January 1, 2006; 59(1): 40 - 47. [Abstract] [Full Text] [PDF]

				V. Marwaha, Y.-H. Chen, E. Helms, S. Arad, H. Inoue, E. Bord, R. Kishore, R. D. Sarkissian, B. A. Gilchrest, and D. A. Goukassian T-oligo Treatment Decreases Constitutive and UVB-induced COX-2 Levels through p53- and NF{kappa}B-dependent Repression of the COX-2 Promoter J. Biol. Chem., September 16, 2005; 280(37): 32379 - 32388. [Abstract] [Full Text] [PDF]

				A. Pozzi, I. Macias-Perez, T. Abair, S. Wei, Y. Su, R. Zent, J. R. Falck, and J. H. Capdevila Characterization of 5,6- and 8,9-Epoxyeicosatrienoic Acids (5,6- and 8,9-EET) as Potent in Vivo Angiogenic Lipids J. Biol. Chem., July 22, 2005; 280(29): 27138 - 27146. [Abstract] [Full Text] [PDF]

				D. A. Heller, C. A. Clifford, M. H. Goldschmidt, D. E. Holt, M. J. Manfredi, and K. U. Sorenmo Assessment of Cyclooxygenase-2 Expression in Canine Hemangiosarcoma, Histiocytic Sarcoma, and Mast Cell Tumor Veterinary Pathology, May 1, 2005; 42(3): 350 - 353. [Abstract] [Full Text] [PDF]

				J. N.M. Ilsley, M. Nakanishi, C. Flynn, G. S. Belinsky, S. De Guise, J. N. Adib, R. T. Dobrowsky, J. V. Bonventre, and D. W. Rosenberg Cytoplasmic Phospholipase A2 Deletion Enhances Colon Tumorigenesis Cancer Res., April 1, 2005; 65(7): 2636 - 2643. [Abstract] [Full Text] [PDF]

				C. A. Martey, S. J. Pollock, C. K. Turner, K. M. A. O'Reilly, C. J. Baglole, R. P. Phipps, and P. J. Sime Cigarette smoke induces cyclooxygenase-2 and microsomal prostaglandin E2 synthase in human lung fibroblasts: implications for lung inflammation and cancer Am J Physiol Lung Cell Mol Physiol, November 1, 2004; 287(5): L981 - L991. [Abstract] [Full Text] [PDF]

				T. W. Davis, J. M. O'Neal, M. D. Pagel, B. S. Zweifel, P. P. Mehta, D. M. Heuvelman, and J. L. Masferrer Synergy between Celecoxib and Radiotherapy Results from Inhibition of Cyclooxygenase-2-Derived Prostaglandin E2, a Survival Factor for Tumor and Associated Vasculature Cancer Res., January 1, 2004; 64(1): 279 - 285. [Abstract] [Full Text] [PDF]

				G. Wu, A. P. Mannam, J. Wu, S. Kirbis, J.-L. Shie, C. Chen, R. J. Laham, F. W. Sellke, and J. Li Hypoxia induces myocyte-dependent COX-2 regulation in endothelial cells: role of VEGF Am J Physiol Heart Circ Physiol, December 1, 2003; 285(6): H2420 - H2429. [Abstract] [Full Text] [PDF]

				C. M. Ardies Inflammation as Cause for Scar Cancers of the Lung Integr Cancer Ther, September 1, 2003; 2(3): 238 - 246. [Abstract] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fosslien, E. Search for Related Content PubMed PubMed Citation Articles by Fosslien, E.