Expression of NOS and VEGF in feline mammary tumours and their correlation with angiogenesis

The Veterinary Journal 192 (2012) 338–344

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Expression of NOS and VEGF in feline mammary tumours and their correlation with angiogenesis M.S. Islam a, M. Matsumoto a,⇑, R. Hidaka a, N. Miyoshi b, N. Yasuda b a b

Laboratory of Veterinary Anatomy, Department of Veterinary Medicine, Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan Laboratory of Veterinary Pathology, Department of Veterinary Medicine, Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan

a r t i c l e

i n f o

Article history: Accepted 30 August 2011

Keywords: Angiogenesis Feline mammary tumours Immunohistochemistry iNOS Tumour grade VEGF

a b s t r a c t In order to define the role of nitric oxide (NO) in feline mammary tumours, the expression of endothelial or inducible nitric oxide synthase (e/iNOS) and vascular endothelial growth factor (VEGF), and their relationship with angiogenesis, was investigated in 23 feline mammary tumours (two hyperplastic, 19 adenocarcinoma, one osteosarcoma and one squamous cell carcinoma) by immunohistochemistry. Tumour angiogenesis was assessed by CD31 immunostaining and was expressed as microvessel density (MVD). In general, iNOS immunoreactivity was localised in tumour cells and occasionally in stromal myofibroblasts, whereas eNOS and VEGF were localised in the cytoplasm of tumour epithelial cells and endothelium. In malignancy, expression of iNOS increased from well- to less-differentiated phenotypes (Grades 1–3) and was significantly higher in G3 and G2 when compared with G1 cases. However, increasing eNOS expression was limited only in hyperplastic lesions and showed no significant changes among the grades. In addition, expression of iNOS was positively correlated with VEGF and MVD in feline mammary tumours and both measures were significantly greater in less differentiated phenotypes (P < 0.05). In conclusion, the expression of NOS isoforms in feline mammary tumours depended on tumour grade, and the positive correlations between iNOS and angiogenic markers suggests that iNOS synthesised by tumour cells promotes tumour growth. Thus, iNOS can be used as an important immunohistochemical marker to determine the degree of malignancy and prognosis of feline mammary carcinoma. Ó 2011 Elsevier Ltd. All rights reserved.

Introduction NO is a gaseous free radical that plays a vital role as a cell signalling molecule in the vascular, nervous and immune systems (Moncada et al., 1991). It is synthesised as a byproduct of the reaction that converts the amino acid L-arginine to L-citrulline, which is catalysed by NOS. The NOS isoenzymes are classified into two groups, namely: (1) calcium-dependent, constitutively expressed NOS enzymes, such as endothelial NOS (eNOS) and neuronal NOS (nNOS); and (2) calcium-independent inducible NOS (iNOS), which is induced in the presence of inflammatory cytokines or bacterial endotoxins (Forstermann et al., 1994). Under physiological conditions, NO produced by constitutive NOS in mammary epithelium acts as an important mediator of post-pubertal mammary gland development (Islam et al., 2009) and plays a role in vascular permeability and regulation of blood flow (Lacasse et al., 1996). Under inductive conditions, high levels of NO synthesised by iNOS can mediate anti-bacterial and anti-tumour functions (Anggard, 1994). Over the past decade, NO generated from e/iNOS been implicated in carcinogenesis and tumour progression (Lala and ⇑ Corresponding author. Tel.: +81 99 285 8711. E-mail address: [email protected] (M. Matsumoto). 1090-0233/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2011.08.032

Chakrabarty, 2001; Xu et al., 2002; Fukumura et al., 2006). NO mediates tumour blood flow through its effects on tumour angiogenesis, vascular tone and vascular permeability, partly via its interaction with VEGF (Van Buren et al., 2006). Various human studies have shown that tumour cell-derived NO can both promote and inhibit tumour progression resulting from its dual action on apoptosis and angiogenesis (Jenkins et al., 1995; Vakkala et al., 2000a; Aaltoma et al., 2001). Overwhelming evidence suggests that overexpression of iNOS in mammary tumours is correlated with angiogenesis and degree of malignancy (Thomsen et al., 1995; Vakkala et al., 2000a; Bulut et al., 2005; Loibl et al., 2005). In veterinary oncology, NO and NOS expression in clinical species remain largely unexplored. However, experimental animal model studies have established that tumour-derived NO promotes mammary tumour growth and metastasis by enhancing the invasive, angiogenic and migratory capacities of tumour cells (Jadeski et al., 2002; Ellies et al., 2003). Despite these studies, the roles of NO and NOS in the development of feline mammary tumours, which is considered to be the third most common neoplasm in feline species (Giménez et al., 2010), remain uninvestigated. The aim of the present study is to investigate the localisation of eNOS, iNOS and VEGF in feline mammary tumours and to clarify the correlations between these proteins and MVD.

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M.S. Islam et al. / The Veterinary Journal 192 (2012) 338–344 Table 2 Primary antibodies used in this study.

Materials and methods Samples and histological examination Biopsy specimens from 23 female cats referred for diagnostic purposes to the Laboratory of Veterinary Pathology from external veterinary clinics or internally from the Veterinary Teaching Hospital of the Kagoshima University, Japan, between 2008 and 2009 were studied. Tissues were fixed in 10% neutral buffered formalin and were embedded in paraffin. Sections (4 lm) were stained with Hematoxylin and Eosin (HE) for histological examination and classified according to the World Health Organisation (WHO) criteria for classification of feline mammary lesions (Misdorp et al., 1999) (Table 1). Histological grading of carcinomas was performed according to the Elston and Ellis scoring system (1998), which is based on three morphological features: tubule formation, nuclear pleomorphism and mitotic index. There were 2 Grade-1 (G1), 10 Grade-2 (G2) and 9 Grade-3 tumours (G3).

Antibodiesa

Suppliers

Dilution

eNOS iNOS VEGF aSMA CD31

Affinity Bioreagents; Golden, CO Affinity Bioreagents; Golden, CO Santa Cruz Biotechnology, Inc., CA Neomarkers, Fremont, CA Santa Cruz Biotechnology, Inc., CA

1:100 Ready to use 1:400 1:300 1:2,000

a All primary antibodies were rabbit polyclonal antibodies, except CD31 (goat polyclonal).

Results Animals

Immunohistochemistry Serial sections of neoplasms were immunohistochemically examined by the avidin–biotin-peroxidase complex (ABC) procedure (Vectastain Elite ABC Kit; Vector Laboratories). Primary antibodies used in this study are summarised in Table 2. For antigen retrieval, deparaffinised sections were microwave-pretreated in 10 mM citrate buffer (pH 6.0) and endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 30 min. All sections were incubated with primary antibody at 4 °C overnight, and with the biotinylated secondary antibody for 30 min at room temperature. Immunoreactivity was then detected by diaminobenzidine (DAB), followed by counterstaining with Mayer’s hematoxylin. Positive reactions were observed as brown precipitates. Negative controls included incubation with matched IgG (Dakocytomation, Glostrup, Denmark) or PBS instead of primary antibody and did not show any reactivity (data not shown). In addition, to characterise stromal myofibroblast cell, alpha smooth muscle actin (aSMA) immunohistochemistry was performed on the same serial sections. It should be noted that nature of samples (formalin-fixed) did not permit further analysis of mammary tissues using quantitative RT-PCR/western blotting.

The mean age of cats was 10.93 ± 3.17 years (range, 7–16 years). Most cats were of mixed breed with a past history of ovariectomy.

Evaluation of immunohistochemical staining

iNOS was diffusely detected in the cytoplasm of the tumour epithelium and occasionally in stromal myofibroblast and endothelium. Positive immunostaining was observed in 100% of carcinomas. The staining pattern was heterogeneous; strongly positive cells were near areas exhibiting little or no staining. In hyperplastic lesions, low numbers of tumour cells showed weak immunoreactivity (Figs. 1, B1). However, IRS was significantly higher in G2 and G3 tumours when compared with G1 tumours (P < 0.05).

Expression levels for eNOS, iNOS and VEGF in tumour epithelial cells were scored semi-quantitatively based on the staining intensity and percentage of stained tumour cells using immunoreactive score (IRS). Briefly, IRS = SI (staining intensity)  PP (percentage of positive cells). SI for each antibody was assigned to four grades: 0 = no staining; 1 = weak; 2 = moderate; and 3 = strong. The number of positive cells was subjectively evaluated in the most stained area of each tissue; a minimum of 500 cells were counted, and PP was scored as 1–4 as follows: 1: <25%; 2: 25–50%; 3: 50–75%; and 4: >75% of cells showing cytoplasmic positivity. Myofibroblasts visualised by aSMA expression were assessed semiquantitatively using a simplified scale based on the percentage of myofibroblasts in the population of tumour stromal cells (0: no myofibroblasts; 1: <10% of myofibroblasts in tumour stroma; 2: 10–30% of myofibroblasts in tumour stroma; and 3: >30% of myofibroblasts in tumour stroma). MVD was assessed as described previously (Martin et al., 1997). Briefly, the areas of the highest vascular density (hot spots) in the CD31-immunostained sections were marked under low magnification. After individualisation of hot spots, three adjacent, non-overlapping fields from each section were selected at 200 (20 objective and 10 ocular) magnification. MVD was quantified as the mean vessel count obtained from the average vascular density of three fields from each tumour.

Expression of eNOS in feline mammary tumours (Figs. 1, A1–A4) eNOS expression in feline neoplastic mammary tissues was localised in the cytoplasm of tumour cells. As expected, blood vessel endothelia were always strongly positive and were used as an internal positive control. IRS was highest in two hyperplastic lesions (Figs. 1, A1), while a few (5/21) malignant lesions were positive for eNOS with low IRS. Expression of iNOS in feline mammary tumours (Figs. 1, B1–B4)

Expression of VEGF in feline mammary tumours (Figs. 1, C1–C4) VEGF expression was detected diffusely or focally in the cytoplasm of tumour cells and weakly in endothelial cells. Positive immunostaining was detected in 90.5% (19/21) of carcinomas. Hyperplastic lesions did not show any reactivity (Figs. 1, C1). G1 lesions showed weak reactivity, while marked granular staining patterns were observed in G2 and G3 (Figs. 1, C3 and C4), and IRS was significantly higher in G3 than G1 tumours (P < 0.05).

Statistical analysis

Expression of CD31 in feline mammary tumours (Figs. 2, A1–A4) Expression of eNOS, iNOS, VEGF, aSMA and CD31 was compared within the groups and between the groups by one-way ANOVA followed by Tukey’s post hoc test. Correlations between eNOS, iNOS, VEGF, aSMA, MVD and tumour grade were compared by the linear regression test. SPSS 16.0 for Windows was employed for all statistical analyses and P < 0.05 was considered to indicate statistical significance. Table 1 Classification of feline mammary tumours used in this study. Histological type Non-neoplastic Lobular hyperplasia Malignant Tubulo-papillary Solid carcinoma Cribriform Osteosarcoma Squamous cell carcinoma

The number of positively labeled endothelial cells/mm2 was higher in malignant (27.0 ± 3.9) than in hyperplastic lesions (8.2 ± 1.2) (P < 0.01). Among the malignant tumours, the number of labeled endothelial cells increased from G1 (10.8 ± 0.8) to G2 (28.9 ± 2.9) (P > 0.05) and from G2 to G3 (41.3 ± 4.5) (P > 0.05). There were significant differences in periductal MVD between G1 and G3 (P < 0.05).

No.

Expression of aSMA in feline mammary tumours (Figs. 2, B1–B4) 2 15 3 1 1 1

In hyperplastic lesions, aSMA expression was detected in the acinar myoepithelia, but aSMA-reactive stromal myofibroblasts were absent in hyperplastic lesions (Figs. 2, B1), while myoepithelia were absent in malignant lesions (Figs. 2, B2–B4). aSMA was also detected in the walls of mascularised vessels in all specimens examined.

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eNOS

iNOS

VEGF

HE

B1

C1

D1

A2

B2

C2

D2

A3

B3

C3

D3

A4

B4

C4

D4

Tubulo-papillary carcinoma (G3)

Tubulo-papillary carcinoma (G2)

Solid carcinoma (G1)

Lobular hyperplasia

A1

Fig. 1. Immunohistochemical (IHC) expression of eNOS, iNOS, VEGF in feline mammary lesions. Row 1 shows IHC of eNOS (A1), iNOS (B1) and VEGF (C1) in hyperplastic lesions. Row 2 shows IHC of eNOS (A2), iNOS (B2) and VEGF (C2) in solid carcinoma (Grade 1). Row 3 shows IHC of eNOS (A3), iNOS (B3) and VEGF (C3) in tubulo-papillary carcinoma (Grade 2). Row 4 shows IHC of eNOS (A4), iNOS (B4) and VEGF (C4) in tubulo-papillary carcinoma (Grade 3). ABC method. Column 4 (D1–D4) shows corresponding serial tissue sections. HE. Bar = 25 lm.

Among the carcinomas, stromal fibroblast proliferation gradually increased from G1 (1.4 ± 0.5) to G2 (2.6 ± 0.4) (P < 0.05) and from G2 to G3 (2.8 ± 0.4) (P > 0.05). There were significant differences in aSMA between G1 and G3 (P < 0.01).

Correlation between eNOS, iNOS, VEGF, MVD and tumour grade Among the malignant tumours, expression of iNOS, VEGF, and MVD increased progressively as the carcinomas proceeded from

M.S. Islam et al. / The Veterinary Journal 192 (2012) 338–344

CD31

341

SMA B1

A2

B2

A3

B3

A4

B4

Solid carcinoma (G3)

Solid carcinoma (G2)

Tubulo-papillary carcinoma (G1)

Lobular hyperplasia

A1

Fig. 2. Immunohistochemical (IHC) expressions of CD31 and aSMA in feline mammary lesions. Endothelial cells were identified by IHC for CD31 (Left column) and stromal myofibroblast by IHC of aSMA (Right column). Row 1, lobular hyperplasia; row 2, tubulo-papillary carcinoma (Grade-1); row 3, solid carcinoma (Grade-2); and row 4, solid carcinoma (Grade-3). ABC method. Bar = 50 lm.

well-differentiated (G1) to less-differentiated phenotypes (G3) (Fig. 3). Pair-wise correlation coefficient test with tumour grade revealed an association with tumourigenesis (Fig. 4). However, eNOS was negatively correlated with grade.

expression was positively correlated with VEGF expression (Fig. 5). On the other hand, 76.2% of malignant lesions were negative for eNOS and positive for VEGF. eNOS was negatively correlated with VEGF expression (Fig. 5).

Correlation between eNOS, iNOS and VEGF

Discussion

Most carcinomas had a positive score on staining with antiiNOS and anti-VEGF antibodies. Only 9.5% of lesions were positive for iNOS and negative for VEGF. Among feline carcinomas, iNOS

To the best of our knowledge, this is the first study demonstrating the localisation of NOS isoforms in feline mammary tumours. We showed that the most predominant isoform of NOS in feline

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60

20 eNOS

iNOS

VEGF

MVD

18 50 *

14

40

12 * *

10

30

8 20 6 4

MVD (ccapillaary deensity//mm2) M

Immunnoreacctive score ((IRS) s

16

10

2 ND

0

0

Hyperplasia

Grade 1

Grade 2

Grade 3

iNOS

100

eNOS positive cells (%)

iNOS positive cells (%)

Fig. 3. Changes in expression of iNOS, eNOS, VEGF and MVD in feline mammary lesions. The right secondary axis shows MVD and left axis shows the IRS for eNOS, iNOS and VEGF. Each bar represents the mean ± SE. ND: not detected. Changes in expression of iNOS, VEGF and MVD were significant when compared with G1 cases. P < 0.05.

80 60 40

r = 0.88 P<0.05

20 0

0

1

2

3

eNOS

100 80 60

r = - 0.68 P<0.05

40 20 0

0

1

VEGF

100

60 40

r = 0.86 P<0.05

20 0

1

3

MVD

100

80

0

2

Grade

capillary density

VEGF positive cells (%)

Grade

2

80

40 20 0

3

r = 0.73 P<0.05

60

0

1

Grade

2

3

Grade

VEGF and iNOS

100

eNOS positive cells (%)

iNOS positive cells (%)

Fig. 4. Relationships between iNOS, eNOS, VEGF, MVD and grades in feline mammary tumours.

80 60 r = 0.82 P<0.05

40 20 0

0

20

40

60

80

VEGF positive cells (%)

100

VEGF and eNOS

100 80 60

r =-0.66 P<0.05

40 20 0 0

20

40

60

80

100

VEGF positive cells (%)

Fig. 5. Relationships between iNOS, eNOS and VEGF in feline mammary tumours of different grade. s: Grade 0; h: Grade 1; D: Grade 2; : Grade 3.

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mammary carcinoma is iNOS, which is positively associated with angiogenic markers (VEGF and MVD) and tumour grade. Although hyperplastic lesions strongly expressed eNOS, this expression was not correlated with tumour grade and angiogenesis. The present data indicate that iNOS expression in tumour cells is related to tumour angiogenesis and has predictive value with regard to the progression of feline mammary carcinoma. NO and NOS activity has been extensively demonstrated in breast cancer. Thomsen et al. (1995) first reported that iNOS expression was localised predominantly within tumour-associated macrophages and eNOS was present in vascular endothelial cells in human breast cancer. In contrast, recent studies (Tschugguel et al., 1996, 1999; Vakkala et al., 2000a; Loibl et al., 2002; Bulut et al., 2005; Loibl et al., 2005) have shown that both isoforms are localised primarily in tumour cells, with iNOS occasionally being detected in stromal cells, such as myofibroblasts, endothelium and immune cells. In feline mammary carcinoma, we also confirmed that both isoforms are mainly localised in tumour cells, with iNOS occasionally being seen in stromal myofibroblasts. The role of NOS, particularly iNOS, in tumourigenesis is well accepted in several established human tumours, but is still poorly understood in breast cancer due to differing or completely opposing findings in the literature. Most authors have reported the presence of iNOS in carcinoma and higher iNOS expression in malignant tissue than in benign breast tissues (Thomsen et al., 1995; Reveneau et al., 1999; Vakkala et al., 2000a; Loibl et al., 2002, 2005). A positive correlation between iNOS and tumour grade has also been reported, although two studies (Reveneau et al., 1999; Loibl et al., 2002) have reported a negative correlation. These discrepancies may be explained by the fact that NO has both facilitatory and inhibitory roles in tumour biology, depending on localisation and type of NOS isoform, concentration and duration of NO exposure and cellular sensitivity to NO (Fukumura et al., 2006). Although iNOS expression has been reported extensively in breast cancer, some studies have reported that along with iNOS, eNOS is also expressed in tumour cells and is related to breast lesions. Tschugguel et al. (1996) first reported eNOS localisation in the pre-neoplastic cells of apocrine metaplasia in the breast, and that it disappears during neoplastic transformation. In contrast, other studies subsequently reported eNOS in malignancies (Martin et al., 2000; Vakkala et al., 2000b; Loibl et al., 2002). In feline mammary lesions, strong eNOS expression was limited only in hyperplastic lesions (2/2), whereas a few (5/21) carcinomatous lesions were positive for eNOS. We postulated that the decrease in eNOS expression in malignancies may act as a trigger for the induction of large amounts of iNOS and NO expression in higher grades, ultimately increasing the aggressiveness of feline mammary carcinomas. It has been well established that angiogenesis, the formation of neovessels, is essential for tumour growth and metastasis, and the process is regulated by multiple factors secreted by tumour cells and their surrounding host stromal cells (Folkman and Shing, 1992). Among these factors, VEGF is regarded as a key factor in angiogenesis (Ferrara et al., 2003), which is physiologically upregulated in pregnancy-associated mammary glands (Pepper et al., 2000; Islam et al., 2010) and canine mammary tumours (Restucci et al., 2002). In the present study, VEGF was overexpressed in feline mammary tumours and was found to be related to tumour grade and angiogenesis (MVD), which agrees with a previous study in canines (Restucci et al., 2002). A similar finding was previously reported for feline mammary tumours (Millanta et al., 2002), but the authors did not find a correlation between VEGF and tumour angiogenesis. This discrepancy may have been due to differences in the antibody used for localisation and identification of endothelial cells. In our study, we used anti-CD31 antibody to localise

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endothelial cells, which is the most sensitive among the various endothelial cell markers, and is considered to be a panendothelial marker (Horak et al., 1992). To our knowledge, this is the first report of a positive correlation between VEGF and tumour vascularity in the development of feline mammary tumourigenesis. In the present study, we also characterised and quantified stromal cell proliferation in different grades of feline mammary tumour using aSMA, which is regarded as an important prognostic factor in breast cancer (Yazhou et al., 2004; Yamashita et al., 2010). It has been shown that the differentiation of fibroblasts to myofibroblasts plays an important role in tumour invasion and metastasis via secretion of angiogenic factors (De-Wever and Mareei, 2003; Sugimoto et al., 2006). Moreover, stromal myofibroblast cells, which are also positive for iNOS, have an indirect angiogenic role in feline tumours; quantification of stromal fibroblast proliferation revealed significantly higher expression in poorly differentiated phenotypes, which is also in line with previous studies in humans. There is mounting evidence demonstrating an interaction between NO and VEGF in mediating angiogenesis and vascular permeability (Ziche et al., 1997; Ambs et al., 1998). It has been shown that hypoxia is a strong stimulus for both VEGF and iNOS expression in tumour cells (Shweiki et al., 1992; Melillo et al., 1995; Forsythe et al., 1996). NO stabilises hypoxia inducible factor-1-a (HIF-1-a), a major mediator of VEGF expression (Kimura and Esumi, 2003). Furthermore, several immunohistochemical studies have also revealed a link between high angiogenic activity or high VEGF and iNOS expression in human head and neck (Gallo et al., 1998), stomach (Song et al., 2002), colon (Cianchi et al., 2003), and breast (Vakkala et al., 2000a) tumours. The present study confirmed a similar link in feline mammary carcinomas where both iNOS and VEGF were overexpressed, and were significantly higher in less differentiated phenotypes, showing correlations with tumour MVD. At present, we lack clinico-pathological parameters, which is a major limitation. Apart from other prognostic parameters, histological grade is an independent prognostic factor (Seixas et al., 2011), which suggests that along with VEGF, iNOS expression may have some predictive value in the prognosis of feline mammary tumour progression. Although iNOS has a positive association with tumour growth and progression, further studies with a larger sample size are necessary in order to evaluate its prognostic value in feline mammary carcinoma.

Conclusions The present study showed the expression of NOS isoforms in feline mammary tumours depended on tumour grade, and the positive correlations between iNOS and angiogenic markers suggests that iNOS synthesised by tumour cells promotes tumour growth. Thus, iNOS can be used as an important immunohistochemical marker to determine the degree of malignancy and prognosis of feline mammary carcinoma.

Conflict of interest statement None of the authors has any financial or personal relationships that could inappropriately influence or bias the contents of the paper.

Acknowledgement This work was supported in part by a Grant from the Japan Society for the Promotion of Science (No. 21580366).

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References Aaltoma, S.H., Lipponen, P.K., Kosma, V.M., 2001. Inducible nitric oxide synthase (iNOS) expression and its prognostic value in prostate cancer. Anticancer Research 21, 3101–3106. Ambs, S., Merriam, W.G., Ogunfusika, M.O., Bennett, W.P., Ishibe, N., Hussain, S.P., Tzeng, E.E., Geller, D.A., Billiar, T.R., Harris, C.C., 1998. p53 and vascular endothelial growth factor regulate tumour growth of NOS2-expressing human carcinoma cells. Nature Medicine 4, 1371–1376. Anggard, E., 1994. Nitric oxide: Mediator, murderer, and medicine. Lancet 343, 1199–1206. Bulut, A.S., Erden, E., Sak, S.D., Doruk, H., Kursun, N., Dincol, D., 2005. Significance of inducible nitric oxide synthase in benign and malignant breast epithelium: An immunohistochemical study of 151 cases. Virchows Archiv 447, 24–30. Cianchi, F., Cortesini, C., Fantappiè, O., Messerini, L., Schiavone, N., Vannacci, A., Nistri, S., Sardi, I., Baroni, G., Marzocca, C., Perna, F., Mazzanti, R., Bechi, P., Masini, E., 2003. Inducible nitric oxide synthase expression in human colorectal cancer. American Journal of Pathology 162, 793–801. De-Wever, O., Mareei, M., 2003. Role of tissue stroma in cancer cell invasion. Journal of Pathology 200, 429–447. Ellies, L.G., Fishman, M., Hardison, J., Kleeman, J., Maglione, J.E., Manner, C.K., Cardiff, R.D., MacLeod, C.L., 2003. Mammary tumour latency is increased in mice lacking the inducible nitric oxide synthase. International Journal of Cancer 106, 1–7. Elston, C.W., Ellis, I.O., 1998. Assessment of histological grade. In: Elston, C.W., Ellis, I.O. (Eds.), Systemic Pathology – The Breast, Third Ed. Churchill and Livingstone, London, UK, pp. 365–384. Ferrara, N., Gerber, H.P., LeCouter, J., 2003. The biology of VEGF and its receptors. Nature Medicine 9, 669–676. Folkman, J., Shing, Y., 1992. Angiogenesis. Journal of Biological Chemistry 267, 10931–10934. Forstermann, U., Closs, E.I., Pollock, J.S., Nakane, M., Schwarz, P., Gath, I., Kleinert, H., 1994. Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions. Hypertension 23, 1121–1131. Forsythe, J.A., Jiang, B.H., Iyer, N.V., Agani, F., Leung, S.W., Koos, R.D., Semenza, G.L., 1996. Activation of vascular endothelial growth factor gene transcription by a hypoxia-induced factor. Molecular and Cellular Biology 16, 4604–4613. Fukumura, D., Kashiwagi, S., Jain, R.K., 2006. The role of nitric oxide in tumour progression. Nature Reviews Cancer 6, 521–534. Gallo, O., Masini, E., Morbidelli, L., Franchi, A., Fini-Storchi, I., Vergari, W.A., Ziche, M., 1998. Role of nitric oxide in angiogenesis and tumour progression in head and neck cancer. Journal of the National Cancer Institute 90, 587–596. Giménez, F., Hecht, S., Craig, L.E., Legendre, A.M., 2010. Early detection, aggressive therapy: optimizing the management of feline mammary masses. Journal of Feline Medicine and Surgery 12, 214–224. Horak, E.R., Leek, R., Klenk, N., LeJeune, S., Smith, K., Stuart, N., Greenall, M., Stepniewska, K., Harris, A.L., 1992. Angiogenesis, assessed by platelet/ endothelial cell adhesion molecule antibodies, as indicator of node metastases and survival in breast cancer. Lancet 340, 1120–1124. Islam, M.S., Matsumoto, M., Tsuchida, K., Oka, T., Kanouchi, H., Suzuki, S., 2009. Immunohistochemical localization of nitric oxide synthase (NOS) in mouse mammary gland during reproductive cycle. Journal of Veterinary Medical Science 71, 945–949. Islam, M.S., Matsumoto, M., Ishida, R., Oka, T., Kanouchi, H., 2010. Change in VEGF Expression in mouse mammary gland during reproductive cycle. Journal of Veterinary Medical Science 72, 1159–1163. Jadeski, L.C., Chakraborty, C., Lala, P.K., 2002. Role of nitric oxide in tumour progression with special reference to a murine breast cancer model. Canadian Journal of Physiology and Pharmacology 80, 125–135. Jenkins, D.C., Charles, I.G., Thomsen, L.L., Moss, D.W., Holmes, L.S., Baylis, S.A., Rhodes, P., Westmore, K., Emson, P.C., Moncada, S., 1995. Role of nitric oxide in tumour growth. Proceedings of the National Academy of Sciences of United States of America 92, 4392–4396. Kimura, H., Esumi, H., 2003. Reciprocal regulation between nitric oxide and vascular endothelial growth factor in angiogenesis. Acta Biochimica Polonica 50, 49–59. Lacasse, P., Farr, V.C., Davis, S.R., Prosser, C.G., 1996. Local secretion of nitric oxide and the control of mammary blood flow. Journal of Dairy Science 79, 1369– 1374. Lala, P.K., Chakrabarty, C., 2001. Role of nitric oxide in carcinogenesis and tumour progression. Lancet Oncology 2, 149–156. Loibl, S., von Minckwitz, G., Weber, S., Sinn, H.P., Schini-Kerth, V.B., Lobysheva, I., Nepveu, F., Wolf, G., Strebhardt, K., Kaufmann, M., 2002. Expression of endothelial and inducible nitric oxide synthase in benign and malignant lesions of the breast and measurement of nitric oxide using electron paramagnetic resonance spectroscopy. Cancer 95, 1191–1198. Loibl, S., Buck, A., Strank, C., von Minckwitz, G., Roller, M., Sinn, H.P., Schini-Kerth, V., Solbach, C., Strebhardt, K., Kaufmann, M., 2005. The role of early expression of inducible nitric oxide synthase in human breast cancer. European Journal of Cancer 41, 265–271.

Martin, L., Green, B., Renshaw, C., Lowe, D., Rudland, P., Leinster, S.J., Winstanley, J., 1997. Examining the technique of angiogenesis assessment in invasive breast cancer. British Journal of Cancer 76, 1046–1054. Martin, J.H., Begum, S., Alalami, O., Harrison, A., Scott, K.W., 2000. Endothelial nitric oxide synthase: Correlation with histologic grade, lymph node status and estrogen receptor expression in human breast cancer. Tumour Biology 21, 90– 97. Melillo, G., Musso, T., Sica, A., Taylor, L.S., Cox, G.W., Varesio, L., 1995. A hypoxiaresponsive element mediates a novel pathway of activation of the inducible nitric oxide synthase promoter. Journal of Experimental Medicine 182, 1683– 1693. Millanta, F., Lazzeri, G., Vannozzi, I., Viacava, P., Poli, A., 2002. Correlation of vascular endothelial growth factor expression to overall survival in feline invasive mammary carcinomas. Veterinary Pathology 39, 690–696. Misdorp, W., Else, W., Hellmen, E., Lipscomb, T.P., 1999. Histological classification of the mammary tumours of the dog and the cat. In: Schulman, F.Y. (Ed.), World Health Organization International Histological Classification of Tumours of the Domestic Animals, vol. 7. Armed Forces Institute of Pathology, Washington, DC, pp. 11–56 (2nd Series). Moncada, S., Palmer, R.M., Higgs, E.A., 1991. Nitric oxide: Physiology, pathophysiology, and pharmacology. Pharmacological Reviews 43, 109–142. Pepper, M.S., Baetens, D., Mandriota, S.J., Di Santa, C., Oikemus, S., Lane, T.F., Soriano, J.V., Montesano, R., Iruela-Arispe, M.L., 2000. Regulation of VEGF and VEGF receptor expression in the rodent mammary gland during pregnancy, lactation, and involution. Developmental Dynamics 218, 507–524. Restucci, B., Papparella, S., Maiolino, P., De Vico, G., 2002. Expression of vascular endothelial growth factor in canine mammary tumours. Veterinary Pathology 39, 488–493. Reveneau, S., Arnould, L., Jolimoy, G., Hilpert, S., Lejeune, P., Saint-Giorgio, V., Belichard, C., Jeannin, J.F., 1999. Nitric oxide synthase in human breast cancer is associated with tumour grade, proliferation rate, and expression of progesterone receptors. Laboratory Investigation 79, 1215–1225. Seixas, F., Palmeira, C., Pires, M.A., Bento, M.J., Lopes, C., 2011. Grade is an independent prognostic factor for feline mammary carcinomas: A clinicopathological and survival analysis. The Veterinary Journal 187, 65–71. Shweiki, D., Itin, A., Soffer, D., Keshet, E., 1992. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 359, 843–845. Song, Z.J., Gong, P., Wu, Y.E., 2002. Relationship between the expression of iNOS, VEGF, tumour angiogenesis and gastric cancer. World Journal of Gastroenterology 8, 591–595. Sugimoto, H., Mundel, T.M., Kieran, M.W., Kalluri, R., 2006. Identification of fibroblast heterogeneity in the tumour microenvironment. Cancer Biology & Therapy 5, 1640–1646. Thomsen, L.L., Miles, D.W., Happerfield, L., Bobrow, L.G., Knowles, R.G., Moncada, S., 1995. Nitric oxide synthase activity in human breast cancer. British Journal of Cancer 72, 41–44. Tschugguel, W., Knogler, W., Czerwenka, K., Mildner, M., Weninger, W., Zeillinger, R., Huber, J.C., 1996. Presence of endothelial calcium-dependent nitric oxide synthase in breast apocrine metaplasia. British Journal of Cancer 74, 1423– 1426. Tschugguel, W., Schneeberger, C., Unfried, G., Czerwenka, K., Weninger, W., Mildner, M., Gruber, D.M., Sator, M.O., Waldhör, T., Huber, J.C., 1999. Expression of inducible nitric oxide synthase in human breast cancer depends on tumour grade. Breast Cancer Research and Treatment 56, 145–151. Vakkala, M., Kahlos, K., Lakari, E., Pääkkö, P., Kinnula, V., Soini, Y., 2000a. Inducible nitric oxide synthase expression, apoptosis, and angiogenesis in in situ and invasive breast cancer. Clinical Cancer Research 6, 2408–2416. Vakkala, M., Pääkkö, P., Soini, Y., 2000b. eNOS expression is associated with the estrogen and progesterone receptor status in invasive breast carcinoma. International Journal of Oncology 17, 667–671. Van Buren 2nd., G., Camp, E.R., Yang, A.D., Gray, M.J., Fan, F., Somcio, R., Ellis, L.M., 2006. The role of nitric oxide in mediating tumour blood flow. Expert Opinion on Therapeutic Targets 10, 689–701. Xu, W., Liu, L.Z., Loizidou, M., Ahmed, M., Charles, I.G., 2002. The role of nitric oxide in cancer. Cell Research 123, 311–320. Yamashita, M., Ogawa, T., Zhang, X., Hanamura, N., Kashikura, Y., Takamura, M., Yoneda, M., Shiraishi, T., 2010. Role of stromal myofibroblasts in invasive breast cancer: stromal expression of alpha-smooth muscle actin correlates with worse clinical outcome. Breast Cancer. doi:10.1007/s12282-010-0234-5 (Epub ahead of print). Yazhou, C., Wenlv, S., Weidong, Z., Licun, W., 2004. Clinicopathological significance of stromal myofibroblasts in invasive ductal carcinoma of the breast. Tumour Biology 25, 290–295. Ziche, M., Morbidelli, L., Choudhuri, R., Zhang, H.T., Donnini, S., Granger, H.J., 1997. Nitric oxide synthase lies downstream from vascular endothelial growth factorinduced but not basic fibroblast growth factor-induced angiogenesis. Journal of Clinical Investigation 99, 2625–2634.