Role of prostaglandin D₂ receptor DP as a suppressor of tumor hyperpermeability and angiogenesis in vivo

Abstract

Although COX-dependent production of prostaglandins (PGs) is known to be crucial for tumor angiogenesis and growth, the role of PGD₂ remains virtually unknown. Here we show that PGD₂ receptor (DP) deficiency enhances tumor progression accompanied by abnormal vascular expansion. In tumors, angiogenic endothelial cells highly express DP receptor, and its deficiency accelerates vascular leakage and angiogenesis. Administration of a synthetic DP agonist, BW245C, markedly suppresses tumor growth as well as tumor hyperpermeability in WT mice, but not in DP-deficient mice. In a corneal angiogenesis assay and a modified Miles assay, host DP deficiency potentiates angiogenesis and vascular hyperpermeability under COX-2-active situation, whereas exogenous administration of BW245C strongly inhibits both angiogenic properties in WT mice. In an in vitro assay, BW245C does not affect endothelial migration and tube formation, processes that are necessary for angiogenesis; however, it strongly improves endothelial barrier function via an increase in intracellular cAMP production. Our results identify PGD₂/DP receptor as a new regulator of tumor vascular permeability, indicating DP agonism may be exploited as a potential therapy for the treatment of cancer.

Angiogenesis, the growth of new blood vessels from preexisting vasculature, is crucial for both tumor progression and metastasis (1). VEGF is a well-recognized angiogenic cytokine that promotes tumor angiogenesis and growth. VEGF also potently induces vascular leakage (2, 3). The resulting extravasation of plasma proteins through the microvasculature provides a provisional matrix to sequester growth factors and support endothelial and tumor cell growth.

Numerous epidemiological, laboratory animal, and clinical studies have provided evidence that non-steroidal anti-inflammatory drugs that inhibit COX and prostaglandin (PG) synthesis can significantly reduce the risk of cancer development (4, 5). Two isoforms of COX have been characterized, COX-1 and COX-2. COX-1 is expressed constitutively in various tissues, whereas COX-2 is inducible by mitogens, cytokines, and tumor promoters. Selective COX-2 inhibitors and COX-2 gene disruption in mice suppress tumor progression (6, 7), suggesting PGs promote aspects of tumor growth. The main PGs responsible for tumor progression are being explored and PGE₂ has been documented to promote tumor growth and angiogenesis (8–10).

PGD₂ is another COX metabolite produced by activated mast cells, macrophages, and Th2 cells. Its biological actions are mediated through the G protein-coupled receptor, named DP (11, 12). Although there is evidence that PGD₂-DP signaling occurs in various inflammatory diseases including asthma (13), the role of PGD₂ in tumor growth remains unknown. Because various blood and immune cells possess the capacity to generate PGD₂, we hypothesized that PGD₂ produced by infiltrating cells modulates aspects of tumorigenesis, thus affecting tumor progression. In the present study, we investigated whether DP gene disruption and DP agonism influence tumor angiogenesis and progression in mice.

Results

DP Receptor Signal Exerts a Suppressive Effect on Tumor Growth.

Initially, we examined tumor growth in PGD₂ receptor DP-deficient mice (DP^−/−). As shown in Fig. 1A, Lewis lung carcinoma (LLC) cells implanted onto the backs of DP^−/− mice grew faster than those implanted onto WT mice, and thus decreased their survival rate (Fig. 1B). Although similar concentrations of PGD₂ were detected in these tumors (WT, 1072.4 ± 54.4 pg/g; DP^−/−, 1,200.7 ± 80.4 pg/g on day 14, n = 4–5), their levels were much higher than those in normal intact tissues (lung, 98.1 ± 9.3 pg/g; liver, 102.1 ± 13.1 pg/g, n = 4 each; P < 0.05 with tumor on WT mice), implying a substantial role of PGD₂ in tumor growth. Next, we administered either a DP receptor blocker, BWA868C (3 mg/kg i.p., twice a day), or a DP agonist, BW245C (50 μg/kg i.p., twice a day), into WT mice and our findings were consistent with the idea that DP blockage accelerates tumor growth (Fig. 1C) and shortens lifespan (Fig. 1D). Importantly, the anti-tumor effects of BW245C were abrogated by host DP deficiency (final tumor volume on day 14: DP^−/−, 1,608.6 ± 106.8 mm³; DP^−/−+BW245C, 1,581.6 ± 125.3 mm³, n = 5 each), showing that the effects of BW245C were mediated via the DP receptor. During the treatment periods, vehicle, BWA868C, or BW245C did not influence normal bodily functions and behavior including weight gain, appetite, or grooming behavior. Based on the data showing that the implanted LLC cells grow faster in DP^−/− mice and that BW245C exhibits no detectable anti-proliferative effects on LLC [supporting information (SI) Fig. S1A], we hypothesized that the effect of DP activity on tumor growth may be dependent on the response of host cells that are deficient in DP receptor, e.g., immune and vascular cells, and not directly on tumor cells.

Fig. 1.

(A and B) Tumors implanted into DP^−/− mice have increased tumor growth rates and decreased survival rates. LLCs were injected into WT or DP^−/− mice, and tumor growth and survival were monitored (n = 25 each, *, P < 0.05 vs. WT). (C and D) DP agonism (BW245C) suppressed tumor growth and extended life span. Four days after LLC implantation (quoted as day 0), mice were injected i.p. with saline solution, 50 μg/kg of BW245C, or 3 mg/kg BWA868C twice a day (n = 10, *, P < 0.05 vs. vehicle treatment).

DP Receptor Signal Modulates Environment for Tumor Growth.

To further analyze the effect of DP deficiency on tumor growth, we examined end-point tumors (day 14 after implantation) histologically. H&E staining of tumor cross sections from WT mice revealed the commonly observed pockets of necrosis spread throughout the tumor mass (arrowheads in Fig. 2A) in contrast to those seen in DP^−/− mice, which had smaller necrotic areas (summarized in Fig. 2B). Also, tumor sections from BW245C-treated mice had larger areas of necrosis (Fig. 2A Right, Inset) throughout the tumor mass than those from vehicle-treated mice. As tumor growth is the net result of tumor cell proliferation and apoptosis, we next determined tumor cell proliferation by Ki-67 immunohistochemical staining on tumor tissue sections. As shown in Fig. 2C, neither DP deficiency nor treatment with BW245C affected the number of proliferating Ki-67-positive cells (summarized in Fig. 2D). In contrast, by using the TUNEL cell death detection assay, we found that tumors grown in a DP-deficient environment had decreased TUNEL-positive apoptotic cells (representative pictures are in Fig. 2E and summarized in Fig. 2F), whereas activation of DP receptor via the BW245C agonist significantly increased the number of apoptotic cells. These studies suggest that host DP deficiency leads to a favorable environment for tumor growth by limiting tumor cell death, whereas stimulation of the DP receptor, either on host or cancer cells themselves, can lead to increased cell death, thus explaining decreased tumor growth.

Fig. 2.

DP deficiency decreased and BW245C increased necrotic/apoptotic regions in implanted tumors without affecting tumor proliferation. Tumors from WT, DP^−/−, or BW245C-treated WT mice were excised on day 14 and processed for immunostaining. Representative microscopic images of LLC tumors are shown here on day 14. (A) H&E staining, (Scale bar, 200 μm.) (C) Ki67 immunostaining. (Scale bar, 10 μm.) (E) TUNEL labeling. (Scale bar, 50 μm.) Ki67 immunostaining (brown) was counterstained with hematoxylin (blue). TUNEL was counterstained with DAPI (blue) for nuclear labeling. Pictures are randomly taken from four fields each at a magnification of ×100 (H&E) or ×200 (Ki67 and TUNEL) from eight independent sections. Necrotic areas in the tumors were quantified relative to total pixel density (B). The number of Ki67-positive or TUNEL-positive cells were normalized to a total nuclei number (D and F, respectively; n = 6 each).

DP Protein Is Localized in Tumor Endothelial Cells.

By using double immunofluorescent staining, we found that the DP receptor (green fluorescence) is highly expressed in tumor endothelial cells (labeled by platelet endothelial cell adhesion molecule-1 [PECAM-1], red fluorescence) on WT mice but not DP^−/− mice (Fig. 3A). Whereas an equivalent amount of mRNA expression of DP receptor was detected regardless of tumor growth stage (days 6–14; Fig. S1B), their expression levels were higher than those in normal intact tissues (i.e., lung and liver). These data suggest continuous involvement of DP receptor in tumor growth. Furthermore, abundant DP mRNA expression was detected in the endothelium-intact—but not in endothelium-denuded—vascular tissue (e.g., mouse aorta, pulmonary artery, and carotid artery), and in isolated endothelial cell lines (Fig. S1 C and D, respectively). There is another G protein-coupled receptor, CRTH2, which mediates PGD₂-indcued bioactivities. As shown in Fig. S1E, mRNA expressions of CRTH2 were detected in vascular cells and growing tumor.

Fig. 3.

(A) DP receptor localized in tumor-vascular endothelial cells. Sections were double-labeled for DP receptor (green) and vascular endothelium (PECAM-1, red), and counterstained with DAPI (blue; n = 5). (Scale bar, 20 μm.) (B–D) DP deficiency accelerated, and daily DP agonism suppressed fibrinogen deposition and angiogenesis in tumors. Excised tumors were subjected to double immunostaining for PECAM-1 (green) and fibrinogen (red), and then counterstained with DAPI (blue). (Scale bar, 50 μm in B.) To quantify the angiogenic areas in the tumors, PECAM-1 staining was quantified relative to total pixel density (C). Fibrinogen deposition was normalized to total PECAM-1-positive pixel density (D; n = 6 each). (E) DP deficiency and DP agonism on tumor permeability. Mice bearing LLC tumors (on day 10 after the LLC implantation) were treated with vehicle (10 min), BW245C (50 μg/kg; i.v., 10 min). Tumors were excised and the Evans blue dye content was quantified (n = 6–8; *, P < 0.05 vs. WT and †, P < 0.05 vs. vehicle treatment).

DP Receptor Signal Modulates Tumor Vascular Leakage and Tumorigenesis.

We examined the effect of DP deficiency or receptor activation on tumor endothelial cells. Similar to what has often been observed in solid tumors (1, 14), the LLC implanted in WT mice exhibits substantial neovascularization (PECAM-1-positive staining; Fig. 3B, Upper) and vascular leakage (fibrinogen deposition; Fig. 3C, Middle). Tumor sections from DP^−/− mice revealed increased tumor angiogenesis, marked by increased PECAM-1-positive blood vessels per unit area (Fig. 3C). This correlated positively with its increase in tumor growth, whereas treatment with the DP agonist BW245C in WT mice had the opposite effect. In addition, tumor sections from DP^−/− mice showed increased vascular leakage compared with WT mice, measured as the ratio of fibrinogen to PECAM-1 immunopositivity (1.44 fold increase from WT; Fig. 3D). In comparison with the angiogenic vasculature in WT mice, the tumor vasculature of DP^−/− mice exhibited reduced tubule length and more branch points but less smooth muscle coverage (representative pictures in Fig. S1F; summarized in Fig. S1G), indicative of a less mature vasculature (1, 15). Thus, our data support the concept that endogenous PGD₂-DP signaling may reduce vascular leakage, angiogenesis, and tumor growth.

To examine if DP signal can directly and acutely influence tumor vascular leakage, extravasation of Evans blue dye was monitored as a measure of albumin leakage. We infused Evans blue intravenously into mice bearing equal-sized tumors and the amount of dye extravasation was quantified spectrophotometrically. Fig. 3E demonstrates that tumors implanted in DP^−/− mice had increased vascular permeability compared with those in WT mice. This observation is in line with the increased fibrinogen deposition found in end-point tumors implanted into DP^−/− mice, supporting the idea that DP receptor is required for constitutive maintenance of tumor vascular barrier function. In tumors implanted onto WT mice, administration of the DP agonist BW245C significantly reduced Evans blue extravasation into tumors compared with vehicle-treated mice.

The Role of DP Receptor on VEGF- or IL-1β-Induced Corneal Angiogenesis.

Tumor angiogenesis is a consequence of complex interactions effected by a set of growth factors and cytokines. IL-1β is a principal inducer of inflammation mediated by COX-2, multiple prostanoids, chemokines, and angiogenic mediators including VEGF (16). Previous studies reported that IL-1β promotes COX-2-dependent angiogenesis and carcinoma growth (17, 18). Other groups separately provided evidence that COX-2 activation was required for VEGF production and tumor angiogenesis (7, 8). Thus, we examined the role of the PGD₂-DP signaling pathway on IL-1β- and VEGF-induced corneal angiogenesis. As shown Fig. 4A, hydron pellets containing VEGF (100 ng) or IL-1β (30 ng) were implanted into corneas. Corneal neovasculature extended from the limbus toward the pellets in WT and DP^−/− animals (Fig. 4B), but responses to both cytokines were augmented in corneas of DP^−/− mice. Notably, whereas DP^−/− corneas exhibited an ≈1.3-fold increase in VEGF-induced angiogenesis compared with WT, IL-1β stimulation resulted in a more than twofold increase in the DP^−/− mice (Fig. 4B). Given that the COX-2 inhibitor CAY10404 (5 μl of 0.37 ng/ml twice a day) prevented IL-1β-induced neovascularization in WT and DP^−/− mice, endogenous PGD₂-DP signaling appears to promote angiogenesis when COX-2 is induced. As shown in Fig. 4 C and D, exogenous administration of the DP agonist BW245C (5 μl of 0.37 ng/ml twice a day) greatly suppressed both VEGF- and IL-1β-induced angiogenesis. The inhibitory actions by DP agonism on angiogenesis may explain why the DP agonist BW245C retarded tumor growth.

Fig. 4.

DP receptor modulates VEGF- and IL-1β-induced angiogenesis. Hydron pellets containing VEGF (100 ng) or IL-1β (30 ng) were implanted into the corneas of C57B/6 (WT or DP^−/−) or BalbC mice. Five microliters of 0.37 ng/ml BW245C or CAY10404 (CAY) was dropped onto the mice corneas twice daily. On day 6, corneal neovasculature was photographed (A and C) and areas were quantified (B and D; n = 9–12; *, P < 0.05 vs. WT or vehicle and †, P < 0.05 with IL-1β-treated mice corneas).

The Role DP Receptor on VEGF- or IL-1β-Mediated Vascular Permeability In Vivo.

To further define the potential role of the endogenous DP receptor in endothelial barrier protection, VEGF- and IL-1β-mediated Evans blue dye extravasation was assessed in a modified Miles assay. As seen in Fig. 5A and summarized in Fig. 5B, the amount of dye extravasation caused by local administration with VEGF (30 ng, 5 min before dye injection) was identical in WT and DP^−/− mice, whereas the IL-1β-mediated (10 ng, 1 h before dye injection) dye extravasation was more abundant in DP^−/− mice compared with WT mice. This result was blocked by the COX-2 inhibitor CAY10404 (100 μg/kg i.p. 30 min before IL-1β treatment). Exogenous DP stimulation by BW245C (50 μg/kg i.p. 10 min before VEGF or IL-1β treatment) significantly inhibited both VEGF- and IL-1β-induced Evans blue extravasation (Fig. 5C; summarized in Fig. 5D). As DP^−/− mice exhibited greater hyperpermeability when treated with IL-1β (Fig. 5B), DP antagonism (BWA868C, 3 mg/kg, 30 min before IL-1β injection) also enhanced IL-1β-induced vascular leakage while, at the same time, it was ineffective in augmenting VEGF-mediated vascular permeability. These results suggest that endogenous PGD₂-DP signaling mediates inflammation-linked vascular leakage.

Fig. 5.

The DP receptor regulates vascular permeability. VEGF (30 ng, 5 min) or IL-1β (10 ng, 60 min) was administered to DP^−/− and WT mice intradermally on the ear. Evans blue dye (30 mg/kg) was injected and circulated for 30 min. Evans blue dye was extracted and the content was determined. (Representative pictures in A and summary in B, n = 7 each; *, P < 0.05 vs. WT and †, P < 0.05 vs. IL-1β single treatment). Mice were treated with the agents intravenously according to the following protocols: CAY10404 (CAY), 100 μg/kg, 30 min; BW245C, 50 μg/kg, 10 min; BWA868C, 3 mg/kg, 30 min. Then, after VEGF (30 ng, 5 min) or IL-1β (10 ng, 60 min) administration, Evans blue dye (30 mg/kg) was injected and circulated for 30 min. (Representative picture in C and summary in D, n = 7 each; *, P < 0.05 vs. vehicle treatment and †, P < 0.05 vs. BW245C single treatment).

In vivo, the tumor vascular barrier function is determined by several factors, including systemic and microvascular hemodynamics and permeability. Intraperitoneal administration of BW245C only slightly decreased systemic blood pressure when administered in a 50 μg/kg bolus (77.8 ± 6.2 mmHg 5 min after administration vs. 83.6 ± 4.1 mmHg in vehicle-treated mice; n = 4 per group).

DP Receptor Agonism Strongly Suppresses Endothelial Permeability, but Does Not Influence Endothelial Proliferation, Migration, or Tube Formation.

To see if DP agonism directly affected endothelial cell junctions, we performed an in vitro permeability assay. Confluent monolayers of bovine aortic endothelial cell (BAEC) plated into trans-well plates had minimal FITC-dextran flux across the monolayer under non-stimulated condition, whereas administration of VEGF greatly increased FITC-dextran flux into the lower chamber in a dose-dependent manner (Fig. 6A). Pretreatment with both BW245C (0.1–1 μM, 5 min) and PGD₂ (0.1–1 μM, 5 min) minimized VEGF (10 ng/ml)-mediated changes in barrier function. The effect of BW245C was reversed by the DP antagonist BWA868C (10 μM, 30 min before BW245C), as well as by DP siRNA. Previous reports have shown that DP receptor activation leads to Gs-mediated increases in intracellular cAMP (11). Elevation in intracellular cAMP tightens endothelial junctions via cAMP-dependent protein kinase (PKA)-dependent pathway (19, 20) and/or an independent pathway including Epac-dependent signaling (21). As seen in Fig. 6A, exogenous administration of a cell-permeable cAMP analogue, db-cAMP (1–10 μM), also counteracted VEGF-induced dextran leakage. Conversely, a potent PKA inhibitor, KT5720 (10 μM, 30 min), only partially attenuated BW245C-induced barrier protection. These results suggest cAMP is indispensable for barrier protection through the mechanism of DP agonism, whereas PKA might only be a partial downstream mediator of cAMP.

Fig. 6.

(A) DP agonism diminished in vitro EC permeability via the cAMP-PKA pathway. Confluent monolayers of BAECs were pretreated with the following test agents before the stimulation: BW245C and PGD₂, 1 μM, 5 min; db-cAMP, 10–100 nM, 10 min; BWA868C, 5 μM; and KT5720, 10 μM, 30 min. After VEGF stimulation (10 ng/ml, 20 min), 1% FITC-dextran was added on the upper chamber and incubated for 10 min. The amount of FITC-dextran fluxed into the lower chamber was measured. For DP knock-down, DP receptor-targeted, or control, siRNA was transfected into BAECs 48 h before the experiments (n = 5–6; *, P < 0.05 vs. 10 ng/ml VEGF treatment and †, P < 0.05 vs. VEGF+BW245C treatment). (B and C) DP agonism did not affect in vitro endothelial tube formation. BAECs were sandwiched between gelatinized collagen and incubated. After a 4-day incubation, images were taken from four areas in one gel, and tube length was quantified. (B) Representative picture. (Scale bar, 35 μm.) (C) Summary (n = 5–6; *, P < 0.05 vs. vehicle). (D and E) DP receptor stimulation increased cAMP production in endothelial layer. BAECs and mouse thoracic aortas with (E+) or without (E-) endothelium were stimulated with specified test agents (1 μM PGD₂, PGE₂, PGI₂, BW245C, and 1–10 μM forskolin for 5 min; 5 μM BWA868C for 10 min before BW245C treatment) and assayed for intracellular cAMP concentration (n = 4 each; *, P < 0.05 vs. BW245C treatment and †, P < 0.05 vs. endothelium intact aorta).

One possible mechanism which BW245C treatment could attenuate tumor growth is by direct anti-angiogenic effects (i.e., by blocking endothelial cell proliferation, migration, and/or organization). This could lead to the observed decrease in vascular permeability (i.e., decreased overall accumulation of Evans blue dye). To directly test this, we performed in vitro assays to monitor the effects of BW245C on BAECs. VEGF (50 ng/ml) or serum (2%) triggered robust endothelial cell migration (assessed by a modified Boyden chamber migration assay; see Fig. S1H), proliferation (indexed by BrdU uptake; see Fig. S1I), and in vitro tube formation in collagen gels (representative pictures in Fig. 6B; summarized in Fig. 6C). Whereas the angiogenic prostanoid PGE₂ potentiates endothelial cell proliferation, migration, and organization as previously described (22, 23), BW245C did not influence any of these angiogenic processes.

DP Agonism Strongly Increases Intracellular cAMP Concentration in the Endothelial Layer but Not in the Smooth Muscle Layer.

As with the DP receptor, other prostanoid receptors (PGE receptor, EPs and PGI₂ receptor, IP) are also coupled with Gs, acting through intracellular cAMP production. Finally, therefore, we compared the capacity of intracellular cAMP increases by these PGs (Fig. 6D). Of interest, 1 μM PGD₂ or 1 μM BW245C prominently increased intracellular cAMP in BAECs. The amount induced was much more than that stimulated by PGE₂ (1 μM), PGI₂ (1 μM), or 10 μM of forskolin. The effect of BW245C was abolished by the DP antagonist BWA868C (10 μM, 30 min before BW245C), as well as by DP siRNA. Given that this abundant cAMP elevation by BW245C or PGD₂ was not seen in endothelium-denuded intrapulmonary artery (Fig. 6E), endothelial DP receptor activation is assumed to be the main source of cAMP production in vascular tissue. This data are consistent with the distribution of DP mRNA levels in intact (E+) or endothelium-denuded (E-) arteries (see Fig. S1C). Although PGI₂ also increased cAMP in BAEC (Fig. 6D), it increased intracellular cAMP content in both endothelium denuded and intact arterial segments (Fig. 6E). This suggested IP receptor distribution both in endothelial and smooth muscle layers.

Discussion

In the present study, we showed that PGD₂ receptor DP is localized in endothelial cells in tumor vasculature and its deficiency accelerates vascular leakage and tumorigenesis. Furthermore, we demonstrated exogenous administration of a DP agonist strongly suppresses tumor expansion by a concomitant decrease in tumor vascularity.

Given that an elevated expression of COX-2 is observed in various malignancies including lung, esophageal, breast, head, and prostate cancer, investigators have focused their attention on the role of each prostanoid in tumor growth. Several groups have reported that the major prostanoids, PGE₂ and thromboxane A2 (TXA2), accelerate angiogenesis through their distinct receptors, EPs and TXA2 receptor. Amano et al. reported that host PGE₂-EP3 signal is a prerequisite for stromal vascular growth factor expression and tumor angiogenesis in lung carcinoma (10), whereas Kamiyama et al. suggested PGE₂-EP2 signal also accelerates tumor angiogenesis affecting endothelial motility in breast carcinoma (23). Furthermore, the other group reported that TXA2 receptor agonism promotes angiogenesis and its antagonism blocks IL-1β-induced corneal angiogenesis (17). These results indicating angiogenic properties of major prostanoids may partially explain the anti-tumor effects of non-steroidal anti-inflammatory drugs or COX-2 inhibitors. In this article, we provide data documenting the importance of the PGD₂-DP receptor signaling pathway as an endogenous negative regulator of vascular permeability and tumorigenesis. Moreover, DP agonism strongly prevented vascular leakage in implanted LLC tumors and in cultured endothelial cells.

There has been argument about multifaceted biological actions of PGD₂ as a pro-inflammatory or anti-inflammatory mediator (11). PGD₂ exerts its biological action via binding DP or CRTH2. Furthermore, PGD₂ is quickly metabolized into 15d-PGJ₂ which exhibit anti-inflammatory response via the activation of PPARγ. These distinct signal pathways on various target-cells and/or -tissues are assumed to complicate PGD₂-induced physiological responses. Although, we detected mRNA expression in vascular cells and LLC tumor, we did not focus the role of CRTH2 in tumorigenesis. Further investigations are needed to settle the argument.

It is widely recognized that solid tumors require angiogenesis to grow beyond a certain size. This process involves a wide range of soluble mediators including VEGF, fibroblast growth factor, angiopoietin, IL-1, and tumor necrosis factor. In an in vivo permeability assay (Fig. 5 A and B) and an angiogenesis assay (Fig. 4 A and B), we demonstrated that host DP deficiency has more influence on COX-2-dependent vascularity when compared to that induced by VEGF. Thus, the impact of endogenous PGD₂-DP signal in tumor growth is assumed to vary with COX-2 activity. The opposite effect was seen with exogenous DP receptor agonism. This showed robust suppression of the angiogenic response (Fig. 4 C and D and Fig. 5 C and D) and its effect was irrespective of host COX activity. These features may broaden the clinical application spectrum of DP agonism.

Previous reports have suggested that vascular leakage, one of the first angiogenic responses of the peripheral vasculature to VEGF, may modulate tumor progression (24). To illustrate this, an anti-permeability peptide, cavtratin (25), and TNP-470 (26) have both been shown to acutely suppress vascular leakage and subsequent tumor growth. Mechanistically, these agents interfere with VEGF-dependent nitric oxide production, loosening endothelial cell junctions. Unlike these pathways, we have proposed a potential strategy to protect the vascular barrier in tumors by increasing endothelial-specific cAMP production through the agonistic activation of DP receptor.

The reduced leakage in vivo led to decreased tumor angiogenesis and tumor growth. Because vascular leakage and angiogenesis are important pathophysiological features of various diseases including cancer, ischemic injury, and inflammation, our findings implicate the therapeutic potential of DP agonists to reduce the untoward consequences of enhanced vascular leakage.

Materials and Methods

DP-Deficient Mice and Tumor Implantation.

All experiments were approved by the institutional animal care and use committees of the University of Tokyo. DP-deficient mice were generated, back-crossed more than 10 generations to C57BL/6CrSlc mice, and bred as described (13), and respective control WT mice of the same generation were used (8–10 weeks old). LLC was donated by Yoshikazu Sugimoto (through the Riken BRC Cell Bank; Tsukuba, Japan). LLC cells (1 × 10⁶ cells in 100 μl) were injected s.c. on the backs of mice. Tumor volume was determined daily using a caliper and applying the formula to approximate the volume of a spheroid (0.52 × [width]² × [length]).

PGD₂ Enzyme Immunoassay.

Tumors were harvested and immediately frozen in liquid nitrogen. They were homogenized in ethanol containing 0.02% HCl. After centrifugation, ³H-labeled PGD₂ (New England Nuclear) were added as tracers for estimation of the recovery to the supernatant. The PGD₂ were extracted with ethyl acetate, and the samples were separated by HPLC. The quantification was performed with a PGD₂-MOX enzyme immunoassay kit (Cayman Chemicals).

Immunofluorescence and TUNEL Assay.

Paraffin-embedded sections were used for H&E and Ki67 immunostaining. Cryosections were used for all other immunostaining. The primary antibodies used were DP (1:1,000, Cayman Chemicals), CD31 (1:1000, BD Biosciences), and fibrinogen (1:1,000, Santa Cruz Biotechnology). For apoptotic cell labeling, we applied the TUNEL assay kit (Roche) according to the manufacturer's instructions.

Tumor Permeability and Modified Miles Assay.

As a tumor permeability assay, LLC tumors were grown to ≈800–1,000 mm³ (10 days after implantation) on the backs of mice. After the mice were injected i.p. with test agents following each regimen, Evans blue (30 mg/kg) was injected through the tail vein and circulated for 30 min. For the modified Miles assay, male C57B/6 (WT and DP^−/−) or BalbC mice were used. After treatment with test agents, animals were anesthetized with ketamine/xylazine and VEGF (30 ng), IL-1β (10 ng), or saline solution was injected intradermally (30 μl total) into the dorsal ear skin before Evans blue dye (30 mg/kg) injection and circulation for 30 min. For both assays, animals were killed and perfused with 0.5% paraformaldehyde before tissues were excised, dried, and weighed. Evans blue dye was extracted in formamide and its content was quantified by reading at 610 nm in a spectrophotometer.

Corneal Micropocket Assay and Quantification of Corneal Neovascularization.

Hydron pellets (0.3 μl) containing VEGF (100 ng/pellet) or IL-1ß (30 ng/pellet) were prepared and implanted in mice corneas. BW245C (dissolved in saline solution at 0.37 ng/ml; 5 μl) was administered onto the test animal's eye twice a day. On day 6, the mice were anesthetized and their corneal vessels photographed. Areas of corneal neovascularization were analyzed using the ImageJ 1.37 software package and expressed in mm². The neovascular area was determined by subtracting non-stimulated vascular area from vascular area.

Measurement of Endothelial Permeability and siRNA Preparation.

BAECs were isolated from bovine thoracic aortas. Confluent monolayers of BAEC formed on the transwell inserts with 8 μm pores (Falcon; BD) and serum-starved. Test agents were treated to the upper and lower chamber following each protocol, and then VEGF was added to the upper chamber for 20 min. FITC-dextran (1%) was added to the upper chamber and the entire chamber was incubated for 10 min. Aliquots (100 μl) were then retrieved from the lower chamber and FITC concentrations measured using a fluorescence spectrophotometer. For siRNA transfection, we purchased siRNA targeted against bovine DP receptor (target sequence 5′-AAC ATG GAA TCC AGT CTA TAA-3′ and 5′-CAC GTC GGT GGA GAA GGG CAA-3′) and negative control siRNA (target sequence 5′-GCG CGC UUU GUA GGA UUC G-3′) from Qiagen. BAECs were transfected with siRNA (30 nM) using Lipofectamine 2000 (0.15% vol/vol), following the manufacturer's protocols. The cells were then incubated for 48 h to form a monolayer and serum-starved before the assay.

In Vitro Angiogenesis Assay.

Collagen gel was prepared by mixing type I collagen (Nitta Gelatin) solution with MEM and neutralizing buffer. Collagen solution (500 μl) was transferred to each well of 24-well plates and gelatinized. BAECs were plated onto the base layer at a density of 2 × 10⁵ cells/well. Additional collagen solution (500 μl/well) was layered onto the cells at and gelatinized. Media containing 2% FBS was added to each well, and 5 days later cultures were photographed for evaluation of tube formation and the mean length of tube-like structure was quantified in four fields per group.

Measurement of cAMP Content.

BAECs and dissected mouse aortas were stimulated by different treatment agents and immediately homogenized in 6% trichloroacetic acid solution. After centrifugation, the supernatants were applied to a cAMP enzyme-immunoassay system (Amersham Pharmacia).

Data Representation.

All data are shown as mean ± SEM.