Angiotensin type 1a receptor signaling-dependent induction of vascular endothelial growth factor in stroma is relevant to tumor-associated angiogenesis and tumor growth

Mamoru Fujita1,2, Izumi Hayashi1, Shohei Yamashina3, Akiyoshi Fukamizu4, Moritoshi Itoman2 and Masataka Majima1,*

1 Department of Pharmacology, 2 Department of Orthopaedic Surgery and 3 Department of Anatomy, Kitasato University School of Medicine and 1 Department of Molecular Pharmacology, Graduate School of Medical Sciences, Kitasato 1-15-1, Sagamihara, Kanagawa 228-8555, Japan and 4 Center for Tsukuba Advanced Research Alliance, Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan

* To whom correspondence should be addressed at: Department of Pharmacology, Kitasato University School of Medicine, Kitasato 1-15-1, Sagamihara, Kanagawa 228-8555, Japan. Tel: +81 42 778 8822; Fax: +81 42 778 7604; Email: en3m-mjm{at}asahi-net.or.jp


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Angiotensin II is a multi-functional bioactive peptide and recent reports have suggested that angiotensin II is a proangiogenic growth factor. A retrospective cohort study revealed that angiotensin converting enzyme inhibitors decreased cancer risk, however, the precise mechanism is unknown. We hypothesized that endogenous angiotensin II plays a crucial role in tumor-associated angiogenesis. Tumors implanted in the subcutaneous tissue of wild-type mice developed intensive angiogenesis with vascular endothelial growth factor (VEGF) induction in tumor stroma. AT1a receptor (AT1a-R), but not AT1b receptor or AT2 receptor was expressed in tumor stroma and systemic administration of an AT1-R antagonist reduced tumor-associated angiogenesis and VEGF expression in tumor stroma. Angiotensin II up-regulates VEGF expression through the pathway including protein kinase C, AP-1 and NF-{kappa}B in fibroblasts, the major cellular component of tumor stroma. VEGF is a major determinant of tumor-associated angiogenesis in the present model, since angiogenesis was markedly reduced by either a VEGF neutralizing antibody or a VEGF receptor kinase inhibitor. Compared with the wild-type, tumor-associated angiogenesis was reduced in AT1a-R null mice, with reduced expression of VEGF in the stroma, and this reduction in AT1a-R null mice was not inhibited by an AT1-R antagonist. These suggest that host stromal VEGF induction by AT1a-R signaling is a key regulator of tumor-associated angiogenesis and tumor growth. AT1a-R signaling blockade may be a novel and effective therapeutic strategy against cancers.

Abbreviations: ACE, angiotensin I-converting enzyme; Ang II, angiotensin II; AT1-R, AT1 receptor; AT2-R, AT2 receptor; DMEM, Dulbecco's modified Eagle's medium; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFX, bisindolylmaleimide; Hb, hemoglobin; MVD, microvessel density; MVA, microvessel area; PDTC, pyrrolidine dithiocarbamate; S-180, Sarcoma 180; VEGF, vascular endothelial growth factor


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Angiogenesis, the formation of new capillary blood vessels, is recognized as an important mechanism in tumor development (1,2). Substantial increases in blood supply are required in order to provide the nutrients and oxygen for tumor development and the mechanisms for promotion of angiogenesis are believed to be activated in the early stages of tumor development (3). Inhibition of angiogenesis limits tumor expansion to less than a few millimeters in diameter. An analysis of the precise mechanism which underlies angiogenesis is important and prevention of angiogenesis could be a useful strategy for tumor treatment (1,2). It appears that an excessive vascular supply including a supply of growth factors must facilitate tumor development.

Angiotensin II (Ang II) is a multi-functional bioactive peptide. One important role of this peptide is regulation of blood pressure and blood flow by modification of vascular tone. Recently many reports have suggested that Ang II has a significant role as a growth factor (46). Several in vitro studies have shown that Ang II promotes proliferation, migration and growth factor synthesis in several types of vascular cells, including smooth muscle cells (79) and pericytes (10,11), suggesting a probable role in vascular remodeling. Other studies have also investigated the angiogenic effects of exogenous Ang II in several in vivo angiogenesis models (1215). In spite of these previous studies, the role of locally generated Ang II in tumor-associated angiogenesis, however, remains to be elucidated. Many pathophysiolosical activities of Ang II are known to be mediated by seven transmembrane receptors, and two major subtypes of Ang II receptor, termed AT1 receptor (AT1-R) and AT2 receptor (AT2-R), have been identified, the former having the subtypes AT1a-R and AT1b-R (16). A retrospective cohort study revealed that long-term inhibition of angiotensin I-converting enzyme (ACE), which cleaves angiotensin I to form Ang II, decreased the incidence of fatal malignant tumors when compared with other anti-hypertensive drugs (17). Some clinical studies (18,19) have tried to treat patients with ACE inhibitors in combination with other anticancer agents, but the precise involvement of Ang II was not examined. Previously we reported that endogenous Ang II stimulates angiogenesis in a sponge implantation model, which characteristically induces chronic and proliferative inflammation around the implants with the induction of a potent proangiogenic factor, vascular endothelial growth factor (VEGF) (6). Initiation of tumor-associated angiogenesis was observable in the stroma around the tumor and the angiogenic responses in the granulation tissues in the sponge implantation model can mimic tumor-associated angiogenesis (20). Although such evidence, which suggests that Ang II contributes to tumor-associated angiogenesis, is accumulating, the precise mechanisms underlying Ang II-induced facilitation of tumor-associated angiogenesis have not yet been determined. In the present experiment we have used AT1a-R knockout (AT1a–/–) mice (21), which we developed, together with pharmacological tools and clarified the site of action of Ang II and the mechanisms of Ang II receptor signaling to facilitate tumor-associated angiogenesis. The present results may provide a novel and effective strategy to treat malignant tumors.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tumor implantation model
Male ICR mice (8 weeks old) were obtained from SLC (Hamamatsu, Japan). AT1a–/– mice and their wild-type counterparts (male, 8 weeks old) were developed as reported previously (21). All mice were housed at a controlled humidity of 60 ± 5% and a temperature of 25 ± 1°C, with a 12 h light/dark cycle. All animal experiments were performed in accordance with the guidelines for animal experiments of Kitasato University School of Medicine.

Murine Sarcoma 180 (S-180) cells (20,22) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Gibco BRL Life Technologies, Rockville, MD). The cells were washed in phosphate-buffered saline and were suspended in the same solution at a density of 1 x 107 cells/ml and 100 µl of the resulting suspension was injected into the subcutaneous tissue of male ICR mice. In a separate experiment, Lewis lung carcinoma cells (20,22) were s.c. injected into AT1a–/– and wild-type mice. An AT1-R antagonist, TCV-116 (Takeda Chemical Industries, Osaka, Japan) (23), an ACE inhibitor, lisinopril (Shionogi Phamaceutical, Osaka, Japan), or a VEGF receptor (KDR/VEGFR-2) tyrosine kinase inhibitor, ZD6474 (AstraZeneca, Cheshire, UK) (24), were orally administrated daily. In some mice a neutralizing antibody against mouse VEGF (Genzyme) was administered topically in the vicinity of the tumors formed in mice (once a day, 10 µg/site/day) (20). For control mice a non-immune IgG fraction (Genzyme) was administered topically (20). Microvessel density (MVD) and microvessel area (MVA) as markers of tumor-associated angiogenesis were determined following our previous report (20,22). Some sample tumor tissues, including stroma, were assayed for hemoglobin (Hb) content (20).

Immunohistochemistry
Tissue was immediately fixed with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4), dehydrated with a graded series of ethanol solutions and embedded in paraffin. Sections (4 µm thickness) were prepared from the paraffin-embedded tissue and mounted on glass slides. After removal of the paraffin with xylene, the slides were then placed in cold (4°C) acetone. The sections were subjected to either hematoxylin and eosin staining or immunostaining. For immunostaining the sections were first exposed to dilute normal horse serum and then incubated with either rat antiserum to mouse CD-31 (Pharmingen, San Diego, CA) (20), to identify endothelial cells of the microvessels, rabbit antiserum to mouse VEGF (Santa Cruz Biotechnology, Santa Cruz, CA) (20) or rabbit antiserum to mouse AT1-R (Santa Cruz) (20). Immune complexes were detected with a Vectastain ABC kit (Vector, Burlingame, CA).

RT-PCR
RNA was prepared using the Trizol protocol (Gibco). A sample of RNA was extracted from the tissue according to the manufacturer's instructions. Single-stranded cDNA was synthesized from 250 µg total RNA using 0.4 µg oligo-p(dT)15 primer and 4 U AMV reverse transcriptase (Roche Diagnostics, Basel, Switzerland). PCR was performed in 20 µl of 20 mM Tris–HCl (pH 8.7) containing 10 mM KCl, 5 mM (NH4)2SO4, 1.5 mM MgCl2, 0.2 mM dNTP mix, 0.5 µM forward and reverse primers and 0.5 U Taq DNA polymerase (Qiagen). Complementary DNA from 25 ng total RNA was used as the template. The amplification protocol comprised 40 cycles of 30 s at 94°C, 45 s at 50°C and 45 s at 72°C. The reaction mixtures were subsequently applied to a 2% agarose gel and the amplified products were stained with ethidium bromide. The oligonucleotide primers were as follows: for AT1a-R, 5'-GCATCATCTTTGTGGTGGG-3' (sense) and 5'-ATCAGCACATCCAGGAATG-3' (antisense) (690 bp); for AT1b-R, 5'-GCATCATCTTTGTGGTGGG-3' (sense) and 5'-ATGAGCACATCCAGAAAAC-3' (antisense) (690 bp); for AT2-R, 5'-ATGCTCAGTGGTCTGCTGG-3' (sense) and 5'-AACACAGCTGTTGGTGAATCC-3' (antisense) (328 bp); for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 5'-CCCTTATTGACCTCAACTACATGGT-3' (sense) and 5'-GAGGGGCCATCCACAGTCTTCTG-3' (antisense) (470 bp). The primers for VEGF were 5'-AACCATGAACTTTCTGCTCT-3' (sense) and 5'-CCG AAACCCTGAGGAGCTC-3' (antisense) (720 bp).

ELISA for VEGF
Tumor tissues were removed from the mice 2 weeks after implantation and samples for ELISA were prepared as described previously (25). After the protein concentration was equalized, the VEGF level was measured with an ELISA kit (R&D Systems, Minneapolis, MN) (25). The samples were measured in duplicate and averaged. VEGF levels were expressed as pg/mg wet tissue.

Fibroblast preparation
Circular sponge discs, 5 mm thick x 1.3 cm in diameter, weighing 14.2 ± 0.1 mg, were prepared from a polyurethane foam sheet. The sponge discs were implanted into the subcutaneous tissue of the back of mice under light ether anesthesia. Granulation tissue developed around the sponge. Fibroblasts were cultured from granulation tissue (20,26). Granuloma fibroblasts (Mac-1/CD3 double negative cells) (20) were seeded into 35 mm culture dishes and incubated for 24 h. The cells were washed twice with serum-free DMEM and incubated in serum-free DMEM containing 10 nM Ang II. To examine the involvement of protein kinase C, 10 µM H7 (Sigma-Aldrich, St Louis, MO) or 10 µM bisindolylmaleimide (GFX) (Calbiochem-Novabiochem, San Diego, CA) was added. To test the involvement of NF-{kappa}B and AP-1, 100 µM pyrrolidine dithiocarbamate (PDTC) (Sigma-Aldrich) and 10 µM curcumin (Sigma-Aldrich), respectively, were added to the medium. The cultured cells were treated with each inhibitor for 1 h and then treated 10 nM Ang II concomitant with each inhibitor for 4 h. Using the acid guanidinium thiocyanate–phenol–chloroform extraction method (27), total RNA was extracted. RT-PCR was then performed to purify poly(A)+ RNA using an mRNA Capture kit. VEGF mRNA levels were expressed as a densitometric ratio (VEGF mRNA/ß-actin mRNA).

Statistical analysis
Values are expressed as means ± SEM. ANOVA was used to evaluate the significance of differences with a post hoc test. A P value <0.05 was regarded as significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Expression of AT receptors in tumor tissues
To investigate the mechanism of Ang II-induced tumor-associated angiogenesis, we used ICR mice injected with S-180 cells into the subcutaneous tissue of the flank. The expression of AT receptors were first examined (Figure 1).



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Fig. 1. Expression of AT receptors in tumor tissues. (a) Expression of Ang II receptors in tumor and surrounding stromal tissues in Sarcoma 180-bearing mice (lane a). Tumor and stromal tissues were isolated 14 days after tumor implantation and total RNA was prepared and subjected to RT-PCR analysis of VEGF mRNA. AT1a-R, but not AT1b-R or AT2-R, was detected. Expression of Ang II receptors in mouse adrenal glands was determined as a positive control (lane b). (b) Immunohistochemical localization of AT1-R in tumors and surrounding stromal tissues. Tumor tissues excised with encapsulating stromal tissues in mice treated with vehicle were stained with AT1-R antibody. Scale bar 50 µm. (c) High power field of part of the stromal tissues in (b). Immunoreactive AT1-R was detected on fibroblast-like cells (red arrows).

 
Fourteen days after S-180 cell implantation, solid tumors were apparent and tumor tissues, including capsular stromal tissues, were isolated. Expression of AT1a-R mRNA, but not of that for AT1b-R or AT2-R, was detected in tumor and stroma (Figure 1a) by RT-PCR amplification. Expression of AT1a-R mRNA was not detected in cultured S-180 cells (data not shown), suggesting that the detectable AT1a-R mRNA was derived from the stromal tissues. In fact, immunohistochemical localization of AT1-R was faint in tumor tissues, but predominant staining was found in surrounding stromal tissues (Figure 1b and c). Immunoreactive AT1 was localized not only on the cell membrane but also in the cytoplasm of stromal cells. This may not be due to a non-specific reaction, since immunoreactive AT1a-R was reported to be localized in the cytoplasm beneath the plasma membrane and in endosome-like granules as well as Golgi lamellae and outer nuclear membranes (28,29). We previously confirmed that the main component of the stromal tissues were Mac-1/CD3-negative fibroblast-like cells (20) and these AT1a-R expressing cells in stroma may be fibroblasts.

Effects of an AT1-R antagonist and an ACE inhibitor on tumor-associated angiogenesis and tumor growth
Tumors treated with vehicle for 14 days were well developed, whereas tumor growth was significantly reduced in a dose-dependent manner by daily oral administration of TCV-116 or lisinopril (Figure 2a and b). Using samples isolated from tumors and the surrounding stromal tissue, tumor-associated angiogenesis was examined as MVD and MVA, which correlate with neovascularization, as indicated by immunohistochemical analysis. Both TCV-116 and lisinopril reduced MVD and MVA significantly (Figure 2c and d). These two agents caused essentially the same degree of inhibition of tumor growth and tumor-associated angiogenesis. In contrast, Ang II does not stimulate S-180 cell proliferation directly in vitro (Figure 2e).



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Fig. 2. Effects of the Ang II AT1-R inhibitor TCV-116 and the ACE inhibitor lisinopril on tumor growth and tumor-associated angiogenesis. (a) Typical appearance of tumors. A suspension of Sarcoma 180 (S-180) cells was injected into the subcutaneous tissue of ICR mice. TCV-116 or lisinopril was administered orally at a dose of 100 mg/kg/day from the day of cell implantation and continued throughout the 14 day experimental period. Tumors were then dissected and photographed. (b) Tumor growth was evaluated by tumor weight. The dose-dependent decreases represent the effects of the agents. The dissected tumors were weighed. These two agents suppressed tumor weight in a dose-dependent manner. Tumor growth was substantially suppressed by oral administration of these two agents at doses of 100 mg/kg/day. Data are expressed as means ± SEM; *P < 0.05; **P < 0.01 versus vehicle-treated mice (n = 5) (ANOVA). (c and d) At the end of the 14 day experiment the MVD (per mm2) and MVA (%) of tumor tissue, as markers of angiogenesis, were determined by immunohistochemical examination with the image analysis software NIH Image (NIH Research Service Branch). Error bars indicate SEM; *P < 0.05 versus vehicle-treated group (n = 5) (ANOVA). (e) Effect of Ang II on proliferation of cultured S-180 cells in vitro. Each datum point is the mean ± SEM cell count from three wells.

 
The roles of VEGF in tumor-associated angiogenesis and up-regulation of expression of VEGF by AT1a signaling
An antibody against VEGF, an important mediator of angiogenesis, inhibited growth of S-180 tumors (Figure 3a, left). The Hb content of the tumor tissues, which appeared well correlated with tumor neovascularization upon histological examination (20), was also determined in the present study and was reduced by the anti-VEGF antibody (Figure 3a, right). Furthermore, oral administration of the low molecular weight VEGF receptor (KDR/VEGFR-2) tyrosine kinase inhibitor ZD6474 also significantly reduced tumor growth and the Hb content of the tumor (Figure 3b). In these experiments neutralization or blockade of VEGF function reduced both tumor growth and tumor-associated angiogenesis.



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Fig. 3. Roles of VEGF in tumor-associated angiogenesis and up-regulation of expression of VEGF by AT1a signaling. (a) Effect of VEGF antibody. Tumor growth was assessed by tumor weight (left). Tumor-associated angiogenesis was assessed by hemoglobin (Hb) content (right). Non-immune control IgG or antibody specific for VEGF (10 µg/tumor/day) was injected topically. The error bars represent SEM; **P < 0.01 compared with mice receiving control IgG (n = 6) (ANOVA). (b) Effect of VEGF receptor kinase inhibitor. Tumor growth (left) and tumor-associated angiogenesis (right) were evaluated after oral administration of the VEGF receptor kinase inhibitor ZD6474 (100 mg/kg/day). Data are means ± SEM; **P < 0.01 versus vehicle-treated mice (n = 10 for vehicle-treated mice; n = 6 for ZD6474-treated mice) (ANOVA). (c) Analysis of VEGF expression in mice treated with vehicle, TCV-116 or lisinopril (100 mg/kg/day) using RT-PCR. VEGF expression was suppressed by treatment with each of these two agents. (d) Immunohistochemical expression of VEGF in tumor and stromal tissues. Expression of VEGF was apparent in the surrounding stromal tissue. Also, VEGF staining was more intense in mice treated with vehicle (left) than in mice treated with TCV-116 or lisinopril (center and right). Scale bar 50 µm. (e) VEGF protein level in the tumor. VEGF level was measured with an ELISA kit. Data are expressed as means ± SEM; **P < 0.01 versus vehicle-treated mice (n = 5) (ANOVA). (f and g) Effects of blockade of intracellular signaling on Ang II-induced VEGF mRNA expression in fibroblasts in vitro. Effects of the protein kinase C inhibitors H7 and GFX, the NF-{kappa}B activation inhibitor PDTC and the AP-1 inhibitor curcumin were tested. Data are means ± SEM values of four independent experiments.

 
RT-PCR analysis revealed that expression of VEGF mRNA was reduced in mice treated with TCV-116 or lisinopril in comparison with mice treated with vehicle (Figure 3c). Immunohistochemical studies revealed that VEGF-expressing cells were mainly observed in capsular stromal tissues (Figure 3d, left). VEGF in these fibroblast-like cells seemed to be co-localized with AT1a-R (Figure 1b and c). The intensity of VEGF expression in immunohistochemical specimens was attenuated in mice treated with TCV-116 or lisinopril (Figure 3d, center and right), somewhat as in the RT-PCR analysis of VEGF mRNA (Figure 3c). According to the immunohistochemical study of AT1-R and VEGF, stromal tissues around the tumor may play a significant role in tumor growth and tumor-associated angiogenesis, since blockade of VEGF activity or VEGF receptor signaling significantly reduced tumor growth and tumor-associated angiogenesis (Figure 3a and b).

VEGF levels in the tumor tissues with stroma were determined by specific ELISA. The levels of VEGF in the mice treated with TCV-116 or lisinopril were significantly lower than those in the vehicle-treated mice (Figure 3e).

To test whether Ang II directly induces VEGF in the stromal tissues, isolated fibroblasts taken from granulation tissues of sponge implantation models, which were developed for mechanistic analysis of angiogenesis in vivo (20,3034), were cultured and stimulated with Ang II. Expression of VEGF was significantly increased in fibroblasts stimulated with 10 nM Ang II, compared with that in the vehicle-treated group. Ang II-mediated up-regulation of VEGF in fibroblasts was inhibited by H7 and GFX, which are protein kinase C inhibitors (Figure 3f). An inhibitor of NF-{kappa}B activation, PDTC, and an AP-1 inhibitor, curcumin, also inhibited up-regulation of VEGF mRNA (Figure 3g). These results taken together suggest that Ang II up-regulates VEGF expression through the pathway including protein kinase C, AP-1 and NF-{kappa}B in fibroblasts.

Reduced tumor-associated angiogenesis and tumor growth with concomitant reduction of VEGF expression in AT1a–/– mice
To verify the importance of host stromal AT1a-R signaling, we further used AT1a–/– mice developed by us (21) and their wild-type counterparts, as controls. In this series of experiments we used another tumor cell line, Lewis lung carcinoma cells, which are syngeneic for C57BL/6 mice. The tumors in wild-type mice showed a marked red color. In contrast, those formed in AT1a–/– mice had a pale appearance, as if the level of angiogenesis was low (Figure 4a). The results for tumor volume (35) revealed that Lewis lung carcinomas grew gradually in the subcutaneous tissues, but tumors grew more rapidly in wild-type than in AT1a–/– mice (Figure 4b). Tumor weight determined 15 days after implantation was also significantly reduced in AT1a–/– mice (Figure 4c). Immunohistochemical localization of CD-31 can be used to identify endothelial cells in the microvessels and clearly indicated that marked neovascularization was more frequently present at the margins of the tumor tissues in wild-type than in AT1a–/– mice (Figure 4d). Both MVD and MVA, parameters of angiogenesis, were significantly reduced in AT1a–/– mice, compared with wild-type mice (Figure 4e and f).



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Fig. 4. Tumor growth, tumor-associated angiogenesis and VEGF expression in AT1a–/– mice. (a) Typical appearance of tumors. Tumors formed 15 days after subcutaneous injection of Lewis lung carcinoma cells into wild-type and AT1a–/– mice. (b) Time courses of tumor volume in wild-type and AT1a–/– mice and AT1a–/– mice treated with the AT1-R antagonist TCV-116 by oral administration (100 mg/kg/day). Data are means ± SEM; **P < 0.01 versus vehicle-treated mice (n = 5) (ANOVA). Tumor volume was suppressed in AT1a–/– mice with or without the AT1-R antagonist TCV-116 from 9 days after tumor implantation. (c) Tumor growth was determined in wild-type and AT1a–/– mice and AT1a–/– mice treated with TCV-116 15 days after tumor implantation. Error bars indicate SEM; **P < 0.01 versus vehicle-treated group (n = 5) (ANOVA). (d) Immunohistochemical expression of endothelial cells and vessel lumens stained with CD-31 antibody in wild-type (left) and AT1a–/– mice (right). (e and f) Tumor-associated angiogenesis was determined by immunohistochemical expression. MVD (per mm2) and MVA (%). Data are expressed as means ± SEM; **P < 0.01 versus vehicle-treated mice (n = 5) (ANOVA). (g) RT-PCR expression of VEGF, AT1a-R, AT1b-R and AT2-R in tumor and surrounding stroma tissues in wild-type and AT1a–/– mice and AT1a–/– mice treated with TCV-116. (h) Immunohistochemical localization of VEGF in tumors and surrounding stromal tissues. VEGF staining was more intense in wild-type (left) than in AT1a–/– mice (right). (i) VEGF protein level in the tumor. Expression of VEGF protein level in the tumor was determined using an ELISA kit. Data are expressed as means ± SEM; **P < 0.01 versus vehicle-treated mice (n = 5) (ANOVA).

 
Furthermore, systemic administration of a sufficient dose of TCV-116, which blocks AT1-R in tumor cells, if present, and stroma cells, did not exhibit any inhibitory effects on tumor volume, tumor weight or tumor-associated angiogenesis in AT1a–/– mice (Figure 4b, c, e and f). Intense VEGF mRNA expression was observed by RT-PCR in the encapsulating stromal tissues excised from wild-type mice. In AT1a–/– mice the level of VEGF mRNA in the sample obtained from tumor cells together with stromal cells was significantly suppressed, compared with that in wild-type mice. Systemic administration of TCV-116 did not suppress expression of VEGF in AT1a–/– mice (Figure 4g). The localization of VEGF expression evaluated by immunohistochemical examination was mainly observed in capsular stromal tissue in wild-type mice (Figure 4h, left). Expression of VEGF in surrounding stromal tissues was markedly suppressed in AT1a–/– mice (Figure 4h, right). As shown in Figure 4i, VEGF levels in the tumor tissues with stroma in AT1a–/– mice were significantly lower than those in their wild-type counterparts.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Evidence that Ang II contributes to proliferation, migration and growth factor synthesis in several types of vascular cells and has a probable role in vascular remodeling has been accumulating (711). We have previously reported that implantation of sponges into subucutaneous tissues resulted in increased formation of Ang II and that the locally generated Ang II had a proangiogenic activity (6,30,31). This model has characteristics of proliferative inflammation and can mimic the angiogenesis in tumor stroma (20,3234). Although other evidence suggesting that Ang II contributes to angiogenesis is also accumulating (1215), the precise mechanisms underlying Ang II-induced facilitation of tumor-associated angiogenesis have not yet been fully determined. In the present experiment we used AT1a–/–mice (21), which we have developed, together with pharmacological tools and clarified the sites of action of Ang II, and the mechanisms of Ang II receptor signaling to facilitate tumor-associated angiogenesis. When tumor cells are implanted as in the present study, proangiogenic factors together with activators of the renin–angiotensin system or renin-like activity may be secreted from tumor cells and may cooperate during tumor angiogenesis. Angiotensin I may be generated through the action of renin-like activity secreted from tumor and/or stromal cells.

We have shown that an AT1-R antagonist and an ACE inhibitor significantly suppressed tumor-associated angiogenesis and tumor growth (Figure 2a–d). These results suggest that locally produced Ang II induces tumor-associated angiogenesis. To clarify the mechanism of Ang II-induced tumor-associated angiogenesis, we first examined the expression of AT receptors (Figure 1). Expression of AT1a-R, but not of AT1b-R or AT2-R, was detected in tumor and stroma (Figure 1a). In contrast, AT1a-R mRNA was not detectable in cultured S-180 cells, although the results are not included in the present report. These suggest that the detected AT1a-R mRNA was derived from stromal tissues, not from tumor cells. In fact, immunohistochemical localization of AT1-R was faint in tumor tissues, but predominant staining was found in surrounding stromal tissues (Figure 1b and c). The antibody used here did not differentiate between AT1a-R and AT1b-R, however, previous RT-PCR analysis revealed that only expression of AT1a-R mRNA was detected (Figure 1a, lane a), so the expression of AT1-R by immunohistochemistry must be that of AT1a-R. The effect of exogenous Ang II was lacking in cultured tumor cell growth (Figure 2e), suggesting that the AT1-R relevant to tumor growth and angiogenesis was present in the tumor stroma. Since the main component of the stromal tissues was Mac-1/CD3-negative fibroblast-like cells (20), AT1a-R-expressing fibroblasts may be the site of action of endogenous Ang II.

VEGF is an important mediator of angiogenesis (3638) and the inhibitory effects of daily topical injections of neutralizing antibody specific for VEGF (20) on growth and angiogenesis of S-180 tumors (Figure 3a) suggests that tumor-associated angiogenesis and tumor growth in the presents study were highly dependent on VEGF induction. The results for oral administration of the low molecular weight VEGF receptor (KDR/VEGFR-2) tyrosine kinase inhibitor ZD6474 (24) also support a significant contribution of VEGF (Figure 3b). RT-PCR analysis together with an immunohistochemical study revealed that the expression of VEGF mRNA was reduced in mice treated with TCV-116 or lisinopril in comparison with mice treated with vehicle (Figure 3c and d). Since VEGF in these fibroblast-like cells seems to be co-localized with AT1a-R (Figure 1b and c), stromal fibroblast-like cells may play a significant role in tumor growth and tumor-associated angiogenesis through induction of VEGF.

This was confirmed in isolated fibroblasts stimulated with Ang II (Figure 3f and g). The increase in VEGF in fibroblasts stimulated with Ang II suggests that Ang II directly acts on the Ang II receptors on fibroblasts. Ang II-induced up-regulation of VEGF expression was reported to be mediated by the protein kinase C pathway in rat heart endothelial cells (39). In the present experiment the major signaling pathway of induction of VEGF in fibroblasts may be also a protein kinase C-dependent one, judging from the inhibitory effects of H7 and GFX, which are protein kinase C inhibitors (Figure 3f). An inhibitor of NF-{kappa}B activation, PDTC, and an AP-1 inhibitor, curcumin, also inhibited up-regulation of VEGF mRNA (Figure 3g). In fact, the mouse VEGF promoter contains a binding site for AP-1 and NF-{kappa}B in mice (40). Thus, AP-1/NF-{kappa}B may play a significant role in Ang II-induced up-regulation of VEGF. Overall, therefore, we can conclude that Ang II up-regulates VEGF expression through the pathway including protein kinase C, AP-1 and NF-{kappa}B in fibroblasts.

It is widely accepted that the host microenvironment and tumor–host interactions influence tumor development (41,42). AT1a-R signaling may be a novel determinant of the host microenvironment, which can facilitate tumor-associated angiogenesis and will be a target of tumor therapy. We further clarified the importance of host stromal AT1a-R signaling using AT1a–/– mice developed by us (21) and their wild-type counterparts, as controls. In this series of experiments we used another tumor cell line, Lewis lung carcinoma cells, which are syngeneic for C57BL/6 mice. Tumor growth and tumor-associated angiogenesis were significantly reduced in AT1a–/– mice (Figure 4a–f). Xenograft models using knockout mice are suitable to determine the contribution of host factors, which are null in the knockout mice (20,34). In spite of implantation of the same number of tumor cells, differences in tumor growth and tumor-associated angiogenesis were observed in these mice, strongly suggesting that AT1a-R in host cells, not in tumor cells, has a major, indeed a critical, role in tumor-induced angiogenesis and tumor growth. The systemic administration of an AT1-R antagonist which can block AT1-R in tumor and stroma cells did not exhibit any further inhibitory effects on tumor growth and tumor-associated angiogenesis in AT1a–/– mice (Figure 4b, c, e and f). These results obtained with an AT1-R antagonist together with the result that Ang II does not stimulate tumor cell proliferation directly in vitro (Figure 2e) confirm that host stromal AT1a-R signaling is important in tumor growth and tumor-associated angiogenesis. VEGF expression in stromal tissues in AT1a–/– mice was suppressed, compared with that in wild-type mice (Figure 4g and h). Administration of an AT1 antagonist did not suppress expression of VEGF in AT1a–/– mice (Figure 4g). These results using AT1a–/– mice indicate that tumor growth and tumor-associated angiogenesis are highly dependent on host stromal AT1a-R signaling, which can induce a potent proangiogenic factor, VEGF. AT1a-R signaling exhibits tumor landscaping effects (43) through the induction of VEGF in stroma.

We previously reported that growth and angiogenesis in mice implanted with NFSA fibrosarcoma were significantly reduced with TCV-116 and an ACE inhibitor, lisinopril (22). Blockade of AT1a signaling in mice was also reported to reduce the growth of B16-F1 melanoma (44). The latter report (44) indicated that tumor-associated macrophages around the tumors express VEGF and that their reduced infiltration may be related to the reduced angiogenesis (44). However, the reduction in macrophage infiltration was by 30% at best, which was well correlated with the reduction in tissue VEGF levels in the stroma (44). This suggests that cellular components other than tumor-associated macrophages that express AT1a-R may be relevant to the reduced VEGF levels. We have shown that isolated fibroblasts express AT1a-R (Figure 1b and c) and that Ang II increases VEGF mRNA levels in subcutaneous fibroblasts (Figure 3f and g). Stromal fibroblasts may be derived from bone marrow (45) and/or from tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes (46). Thus, the maturity of stromal cells may be related to the cell types that generate VEGF to facilitate tumor-associated angiogenesis.

In conclusion, as Figure 5 shows, the present study clearly shows that host stromal VEGF induction mediated by AT1a-R signaling is a key regulator of tumor-associated angiogenesis and tumor growth. AT1a-R, but not AT1b-R and AT2-R, is expressed in the stroma, but not in tumor cells. AP-1 and NF-{kappa}B, transcription factors linked to AT1a-R stimulation, are responsible for the induction of VEGF in stromal fibroblasts. Up-regulated VEGF certainly has a proangiogenic action and facilitates tumor growth in this model, judging from the effects of an anti-VEGF antibody and a VEGF receptor kinase inhibitor. Blockade of AT1a-R signaling by an AT1-R antagonist is effective in preventing tumor growth and tumor-associated angiogenesis. This effect is attributable to a reduction in VEGF in the stroma. In comparison with the wild-type, tumor-associated angiogenesis, together with expression of VEGF, was reduced in AT1a–/– mice, in which an AT1-R antagonist did not inhibit tumor-associated angiogenesis, since no functional AT1a-R is present on tumor cells. These results indicate that host stromal VEGF up-regulation via AT1a-R signaling is a key regulator of tumor-associated angiogenesis and that blockade of AT1a-R signaling may become a novel strategy for fighting cancers that cause >550 000 deaths annually in the USA (47).



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Fig. 5. Schemas of the major signaling pathway of Ang II to induce VEGF expression and tumor-associated angiogenesis. AT1a-R, but not AT1b-R and AT2-R, was expressed in the stroma, but not in tumor cells. AT1a-R signaling on the stromal cells was relevant to the induction of the potent proangiogenic growth factor VEGF in stromal cells. VEGF induces angiogenesis and tumor growth. Inhibition of AT1-R signaling by a selective antagonist suppresses the induction of VEGF and leads to a reduction in tumor growth and tumor-associated angiogenesis.

 

    Acknowledgments
 
We thank Michiko Ogino and Osamu Katsumata for technical assistance. We express our thanks to Mr C.W.P.Reynolds for correcting the English of this manuscript. This work was supported by a grant from the Integrative Research Program of the Graduate School of Medical Sciences, Kitasato University and by a Parents' Association Grant of Kitasato University School of Medicine and was also supported by research grants (15390084, 16022256 and 16659067), by a ‘High-tech Research Center’ grant and by a grant from The 21st Century COE Program, from the Ministry of Education, Culture, Sports, Science and Technology (MEXT).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received December 10, 2003; revised September 3, 2004; accepted October 26, 2004.