Purpose: Vascular endothelial growth factor (VEGF), angiopoietins (Ang-1 and Ang-2), and their receptor Tie2 are critically involved in both normal and pathologic angiogenesis. The aim of this study was to explore the role of Ang-1, Ang-2, VEGF, and Tie2 in the development and progression of bladder cancer as well as to examine their prognostic value in this tumor type.

Experimental Design: Tumor samples of 113 bladder cancer patients, normal bladder epithelium of 5 noncancer patients, and two low-grade (UMUC3 and RT4) and two high-grade (J82 and T24) bladder cancer cell lines were analyzed by quantitative real-time PCR. The expression data were analyzed performing Wilcoxon rank-sum and Kaplan-Meier log-rank tests as well as univariate Cox analyses and Cox proportional hazards regression model.

Results: In tissues of noninvasive bladder tumors, Ang-1 expression was significantly lower (P < 0.001), whereas VEGF expression was significantly higher (P = 0.031) than in normal bladder tissue. These findings were also confirmed at the protein level by immunohistochemistry. In contrast, Tie2 and Ang-2 abundance in tumor did not differ significantly from that in normal bladder tissue. Multivariate analysis identified Ang-2 as a strong and independent predictor of tumor recurrence [hazard ratio (HR), 10.18; 95% confidence interval (95% CI), 2.69-38.49; P < 0.001] and Tie2 expression as an independent favorable prognostic factor for both metastasis (HR, 0.31; 95% CI, 0.11-0.89; P = 0.029) and disease-specific survival (HR, 0.25; 95% CI, 0.10-0.62; P = 0.003).

Conclusions: These data show the strongest change in expression of VEGF and Ang-1 in superficial bladder cancer in comparison with normal bladder epithelium and the invasive tumor stages. The prognostic significance of Ang-2 and Tie2 underlines the essential role of angiopoietins-Tie2 system in progression of bladder cancer.

Translational Relevance

In the present work, we show an “angiogenic switch” regarding the expression of VEGF and angiopietins-Tie2 system in early superficial stages of bladder cancer. This is characterized by increased expression of proangiogenic factors VEGF and Ang-2 and concurrently by a dramatic decrease of Ang-1 expression, particularly in tumor stage Ta. The clinical relevance of these findings is studied on over 100 bladder cancer specimens with a long (up to 189 months) follow-up period. We identified Ang-2 as a strong and independent predictor of disease recurrence and showed the predictive potential of Tie2 expression for bladder cancer metastasis and disease-specific survival. These data suggest that determination of the tissue expression level of the mentioned angiogenic factors might serve as a useful tool in the clinical assessment of these tumors and in determining which patient could benefit from a more aggressive therapy or from an additional targeted antiangiogenic treatment.

Angiogenesis, the formation of new vessels from the existing ones, is essential for the growth and progression of solid tumors. This process begins when the tumor colony expands to a size where simple diffusion of oxygen and nutrients are insufficient. The subsequent hypoxia results in a switch in the net balance between activators and inhibitors of angiogenesis toward the activators (1).

Angiopoietins (Ang-1, Ang-2, Ang-3, and Ang-4) comprise a family of angiogenic modulators that all bind with equal affinity to Tie2 tyrosine kinase receptor, which is primarily expressed by endothelial cells (2, 3). Ang-1 stabilizes new vessels leading to vascular maturation by promoting interaction between endothelial cells and covering mural cells such as pericytes or smooth muscle cells (2, 4, 5). In contrast, Ang-2 acts as a destabilization signal, mostly prior vessel sprouting or regression (2). In presence of vascular endothelial growth factor (VEGF), Ang-2 potentiates angiogenesis, whereas, in the absence of VEGF, the vessels may regress (2, 6). Hypoxia initiates a change in Ang-1/Ang-2 ratio in favor of Ang-2 and results in vessel destabilization (7). VEGF is also induced by hypoxia and can drive the destabilized vessels toward angiogenesis. Different expression pattern of Ang-1, Ang-2, VEGF, and Tie2 in normal and tumor tissues was found in several tumors such as breast, prostate, colon, lung, and ovarian cancers (811).

In the last few years, antiangiogenic therapy became a promising strategy of cancer treatment. A large number of antiangiogenic agents are currently being investigated in clinical trials.7

The advances on this field lay claim to new markers to measure the biological effects of targeted agents and to identify patients most likely to benefit from treatment (12, 13).

In relation to bladder cancer, there are only two publications dealing with VEGF, Ang-1, and Ang-2 with conflicting results, whereas Tie2 was not yet analyzed in bladder cancer (14, 15). The aim of this study was to explore the role of Ang-1, Ang-2, VEGF, and Tie2 in the development and progression of bladder cancer as well as to examine the prognostic value of these angiogenic markers. Therefore, we analyzed a large number of bladder tumors with a long follow-up period by real-time PCR. Furthermore, we defined the gene expressions of these angiogenic markers in two low-stage, low-grade (UMUC3 and RT4) and two high-grade (J82 and T24) bladder cancer cell lines.

Clinical samples. We have used tumor tissue samples from patients who underwent surgical treatment for bladder cancer in the Department of Urology of the University Hospital of Essen between 1990 and 1996. The criteria for enrollment were histopathologic diagnosis of transitional cell carcinoma of the bladder, no history of other tumor, no chemotherapy before surgery, availability of sufficient tumor sample, and the potential to follow-up. The Ethics Committee of the University Hospital of Essen approved the study protocol. We assessed smoking habits at the time of hospitalization (yes versus no), but data on smoking history or pack-years were not routinely available. Initially, all tumors were reclassified according to the WHO classification of urothelial neoplasm of the year 2004 (16). Afterwards, adequacy of stored samples (−80°C) of respective cases was evaluated by frozen sections. In case sufficient tumor sample was available, RNA isolation was done. The five normal bladder epithelia we used as controls originated from an open enucleation in cases of benign prostatic hyperplasia.

In cases of five specimens, we could not detect any PCR products by the five primer pairs we used. Insufficient PCRs with a high SD (>0.5) between parallel measurements were repeated. In some cases (one case of VEGF, eight cases of Ang-1, and five cases of Tie2), there was not enough RNA to perform this. These cases were marked as “not informative” reactions and were not further analyzed.

Cell lines and culture conditions. The four cell lines used in this study (UMUC3, RT4, J82, and T24) were purchased from the American Tissue Culture Collection. All cells were maintained as monolayer cultures at 37°C and 5% CO2. T24 and RT4 were grown in McCoy's medium (Invitrogen), J82 in MEM (Invitrogen), and UMUC3 in MEM-α (Invitrogen). All media were supplemented with 10% FCS (Invitrogen), 100 units/mL penicillin, and 100 μg/mL streptomycin (Invitrogen).

RNA isolation and cDNA synthesis. Before RNA isolation, tissue sections from each biopsy were stained with H&E and reviewed by a pathologist to define the tumor type and stage. Only biopsies containing ≥50% tumor cells were selected for further analysis. After homogenization by Ultra-Turrax T25 (Janke & Kunkel) in QIAzol reagent (Qiagen), RNA isolation was done according to the manufacturer's instructions. The isolated RNA was further purified using the RNeasy Mini kit (Qiagen). To avoid DNA contamination, DNA was digested with RNase-free DNase Set (Qiagen) as recommended by the supplier. Total RNA was quantified using an UV spectrophotometer (Peqlab ND-1000; Erlangen) at A260, and the quality and integrity of samples were assessed on a 1.5% agarose gel. RNA was reverse transcribed in a final volume of 20 μL containing 200 ng RNA, 1× reverse transcription buffer, 0.5 mmol/L deoxynucleotriphophates, 1.8 μmol/L oligo(dT), 10 units RNase inhibitor, and 40 units Omniscript RTase. The cDNA synthesis was done at 37°C for 60 min.

TaqMan two-step reverse transcription-PCR assay. Quantitative real-time PCR was done using 96-well optic plates on an ABI Prism 7500 Sequence Detection System (Applied Biosystems). To provide high reproducibility, we used the predeveloped TaqMan Gene Expression Assay (Applied Biosystems). The assay IDs were as follows: Ang-1, Hs00919202_m1; Ang-2, Hs01048042_m1; VEGF, Hs00900055_m1; Tie2, Hs00945144_m1; and TATA box-binding protein, 4333769. The expressions were related to Universal Human Reference RNA (Stratagene), which composed of pooled RNA from 10 human cell lines. This allows a reliable laboratory-to-laboratory comparison of gene expression data independently of the actually used control sample.

Choosing the right housekeeping gene as an endogenous control is essential in data analysis of real-time PCR. This issue was investigated by Ohl et al. and found that the most commonly used endogenous control GAPDH is not stably expressed in bladder cancer. They found TATA box-binding protein and succinate dehydrogenase complex, subunit A, the most appropriate endogenous control in bladder caner (17). Therefore, we used TATA box-binding protein to normalize our target genes expression.

PCRs contained 2.5 μL cDNA, 1× TaqMan Gene Expression Assay (containing primers and probe), and 1× TaqMan Universal PCR Master Mix (Applied Biosystems) in a 25 μL volume. The thermal cycling conditions were as follows: 95°C for 10 min to activate the AmpliTaq Gold enzyme followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Each sample was tested in duplicate. The threshold cycle (Ct) was determined on the amplification linear area of the target and control genes. The normalized quantity of the target gene was obtained by subtracting Ct for the internal control (TATA box-binding protein) from the Ct for the target gene (ΔCt sample). The same calculation was done in Human Reference RNA (ΔCt reference). Then, ΔΔCt was calculated as the difference of these values (ΔΔCt = ΔCt sample - ΔCt reference). Finally, the result was expressed as 2-ΔΔCt (18).

Immunohistochemistry. Immunohistochemical staining was done on sections obtained from formalin-fixed and paraffin-embedded tissue blocks of bladder cancer of different tumor stages (n = 15). In paraffin sections of some blocks (n = 5), tissue areas with normal transitional epithelium and those with superficial and muscle invasive tumor stages were identified side by side in the same section by histopathologic evaluation. Immunohistochemical staining was done according to the procedure described previously (1921). Immunostaining was carried out using rabbit polyclonal antibodies against VEGF (final dilution, 1:200; Santa Cruz Biotechnology) and Tie2 (final dilution, 1:100; Santa Cruz Biotechnology) and goat polyclonal antibodies against Ang-1 (final dilution, 1:50; Santa Cruz Biotechnology) and Ang-2 (final dilution, 1:50; Santa Cruz Biotechnology). For the visualization of the specific immunostaining, a modified nickel-enhanced glucose oxidase method of the development of peroxidase activity was used. Controls were done as follows: (a) Primary and/or secondary antibodies were replaced by PBS. (b) Instead of the primary antibody, sections were incubated with normal rabbit or goat serum (Sigma) at dilutions in the range of 0.1% and 0.01%. Sections were counterstained with calcium red to visualize the whole tissue structure.

Statistical analysis. For statistical analysis, the nonparametric two-sided Wilcoxon rank-sum test (Mann-Whitney test) for paired group comparisons was applied. Univariate recurrence-free, metastasis-free, and disease-specific survival analysis was done using both Kaplan-Meier log-rank test and univariate Cox analysis. For multiple analysis, the Cox proportional hazards regression model was used. We applied the Spearman's ρ test to analyze the correlations between the mRNA expressions of angiogenic factors. In all tests, a P value of at least 0.05 was considered to be statistically significant. All statistical analyses were done with SPSS software package, version 14.0.

Clinical background. The main characteristics of the patients are given in Table 1. The median follow-up period was 34 months with a maximum of 189 months. Sixty-seven percent of patients with noninvasive (Ta/T1) bladder cancer had a recurrence after surgery. The median time to first recurrence was 5 months. Twenty-eight of 65 patients with invasive transitional cell carcinomas of urinary bladder developed metastasis and 8 patients had already known metastasis at the time of surgical treatment.

Table 1.

Patients characteristics and gene expression

VEGF
Ang-1
Ang-2
Tie2
Median (range)PMedian (range)PMedian (range)PMedian (range)P
Age, 71.6 (36-96), y         
    ≤65 (n = 51) 9.11 (0.77-76.11) 0.195 0.30 (0.005-3.34) 0.01 509 (68-3,373) 0.089 1.46 (0.11-14.47) 0.110 
    >65 (n = 62) 6.5 (0.17-101.83)  0.13 (0.001-1.21)  336 (7-3,072)  0.86 (0.04-12.64)  
Gender         
    Male (n = 88) 8.86 (0.17-101.83) 0.334 0.29 (0.01-2.01) 0.164 386 (7-3,373) 0.82 1.59 (0.19-6.66) 0.245 
    Female (n = 25) 7.23 (0.42-82.71)  0.17 (0.001-3.34)  401 (42-3,126)  0.97 (0.04-14.47)  
Stage         
    Ta (n = 31) 25.41 (1.06-78.25)  0.07 (0.001-3.34)  948 (110-3,126)  0.86 (0.27-14.47)  
    T1 (n = 17) 8.11 (1.19-101.83)  0.10 (0.01-0.85)  322 (68-2,427)  0.76 (0.16-11.47)  
    T2 (n = 13) 7.08 (1.03-51.09)  0.22 (0.02-2.43)  316 (113-1,783)  1.47 (0.04-10.09)  
    T3 (n = 39) 4.52 (0.77-76.11)  0.31 (0.01-2.11)  326 (20-3,009)  1.77 (0.23-12.64)  
    T4 (n = 13) 4.89 (0.17-42.96)  0.36 (0.005-2.01)  189 (7-3,373)  1.06 (0.18-6.66)  
    Noninvasive (n = 48) 13.55 (1.1 -101.83) <0.001 0.31 (0.01-2.11) <0.001 671 (68-3,126) 0.001 0.84 (0.16-14.47) 0.071 
    Invasive (n = 65) 4.92 (0.17-76.11)  0.36 (0.005-2.01)  285 (7-3,373)  1.49 (0.04-12.64)  
Grade         
    1 (n = 17) 18.32 (1.06-101.83)  0.05 (0.005-3.34)  626 (110-2,427)  0.84 (0.16-14.47)  
    2 (n = 40) 9.04 (1.03-82.71)  0.11 (0.001-1.21)  426 (20-3,126)  0.90 (0.29-7.67)  
    3 (n = 56) 5.98 (0.17-76.11)  0.36 (0.005-2.43)  336 (7-3,373)  1.57 (0.04-12.64)  
    Low-grade (grade 1-2; n = 57) 10.85 (1.03-101.83) 0.023 0.09 (0.001-3.34) <0.001 519 (20-3,126) 0.075 0.86 (0.16-14.47) 0.064 
    High-grade (grade 3; n = 56) 5.98 (0.17-76.11)  0.36 (0.005-2.43)  335 (7-3,373)  1.57 (0.04-12.64)  
Primer (n = 63) 8.65 (0.42-82.71) 0.926 0.16 (0.001-2.01) 0.697 369 (20-3,373) 0.300 1.39 (0.04-12.64) 0.936 
Recurrent (n = 50) 7.83 (0.17-101.83)  0.24 (0.005-3.34)  541 (7-3,126)  0.91 (0.16-14.47)  
Smoking         
    Yes (n = 49) 6.50 (0.42-82.71) 0.728 0.15 (0.005-2.01) 0.247 317 (30-3,373) 0.353 0.94 (0.04-11.47) 0.195 
    No (n = 54) 7.39 (0.17-101.83)  0.28 (0.005-3.34)  481 (7-3,009)  1.58 (0.11-14.47)  
    Unknown (n = 10)         
Control (n = 5) 2.12 (0.98-7.89) 0.031 27.85 (6.89-46.53) <0.001 530 (215-1,704) 0.51 1.96 (1.40-5.22) 0.106 
Tumor (n = 113) 8.08 (0.17-101.83)  0.2 (0.001-3.34)  396 (7-3,373)  1.20 (0.04-14.47)  
VEGF
Ang-1
Ang-2
Tie2
Median (range)PMedian (range)PMedian (range)PMedian (range)P
Age, 71.6 (36-96), y         
    ≤65 (n = 51) 9.11 (0.77-76.11) 0.195 0.30 (0.005-3.34) 0.01 509 (68-3,373) 0.089 1.46 (0.11-14.47) 0.110 
    >65 (n = 62) 6.5 (0.17-101.83)  0.13 (0.001-1.21)  336 (7-3,072)  0.86 (0.04-12.64)  
Gender         
    Male (n = 88) 8.86 (0.17-101.83) 0.334 0.29 (0.01-2.01) 0.164 386 (7-3,373) 0.82 1.59 (0.19-6.66) 0.245 
    Female (n = 25) 7.23 (0.42-82.71)  0.17 (0.001-3.34)  401 (42-3,126)  0.97 (0.04-14.47)  
Stage         
    Ta (n = 31) 25.41 (1.06-78.25)  0.07 (0.001-3.34)  948 (110-3,126)  0.86 (0.27-14.47)  
    T1 (n = 17) 8.11 (1.19-101.83)  0.10 (0.01-0.85)  322 (68-2,427)  0.76 (0.16-11.47)  
    T2 (n = 13) 7.08 (1.03-51.09)  0.22 (0.02-2.43)  316 (113-1,783)  1.47 (0.04-10.09)  
    T3 (n = 39) 4.52 (0.77-76.11)  0.31 (0.01-2.11)  326 (20-3,009)  1.77 (0.23-12.64)  
    T4 (n = 13) 4.89 (0.17-42.96)  0.36 (0.005-2.01)  189 (7-3,373)  1.06 (0.18-6.66)  
    Noninvasive (n = 48) 13.55 (1.1 -101.83) <0.001 0.31 (0.01-2.11) <0.001 671 (68-3,126) 0.001 0.84 (0.16-14.47) 0.071 
    Invasive (n = 65) 4.92 (0.17-76.11)  0.36 (0.005-2.01)  285 (7-3,373)  1.49 (0.04-12.64)  
Grade         
    1 (n = 17) 18.32 (1.06-101.83)  0.05 (0.005-3.34)  626 (110-2,427)  0.84 (0.16-14.47)  
    2 (n = 40) 9.04 (1.03-82.71)  0.11 (0.001-1.21)  426 (20-3,126)  0.90 (0.29-7.67)  
    3 (n = 56) 5.98 (0.17-76.11)  0.36 (0.005-2.43)  336 (7-3,373)  1.57 (0.04-12.64)  
    Low-grade (grade 1-2; n = 57) 10.85 (1.03-101.83) 0.023 0.09 (0.001-3.34) <0.001 519 (20-3,126) 0.075 0.86 (0.16-14.47) 0.064 
    High-grade (grade 3; n = 56) 5.98 (0.17-76.11)  0.36 (0.005-2.43)  335 (7-3,373)  1.57 (0.04-12.64)  
Primer (n = 63) 8.65 (0.42-82.71) 0.926 0.16 (0.001-2.01) 0.697 369 (20-3,373) 0.300 1.39 (0.04-12.64) 0.936 
Recurrent (n = 50) 7.83 (0.17-101.83)  0.24 (0.005-3.34)  541 (7-3,126)  0.91 (0.16-14.47)  
Smoking         
    Yes (n = 49) 6.50 (0.42-82.71) 0.728 0.15 (0.005-2.01) 0.247 317 (30-3,373) 0.353 0.94 (0.04-11.47) 0.195 
    No (n = 54) 7.39 (0.17-101.83)  0.28 (0.005-3.34)  481 (7-3,009)  1.58 (0.11-14.47)  
    Unknown (n = 10)         
Control (n = 5) 2.12 (0.98-7.89) 0.031 27.85 (6.89-46.53) <0.001 530 (215-1,704) 0.51 1.96 (1.40-5.22) 0.106 
Tumor (n = 113) 8.08 (0.17-101.83)  0.2 (0.001-3.34)  396 (7-3,373)  1.20 (0.04-14.47)  

Angiogenic gene expression in bladder cancer cells. Supplementary Table S4 summarizes the VEGF, Ang-1, Ang-2, and Tie2 expression in UMUC3, RT4, J82, and T24 cells. Low-grade tumor cells (UMUC3 and RT4) expressed VEGF in a significant higher level as high-grade bladder cells (J82 and T24). RT4 failed to express Ang-1, whereas UMUC3 cells do not express Ang-2.

Comparison of angiogenic factors between tumor patients and normal controls. We quantified the mRNA expression of Ang-1, Ang-2, Tie2, and VEGF in 5 cases of normal bladder epithelium and 113 cases of tumor samples. The VEGF expression was significantly 4-fold higher (P = 0.031), whereas the Ang-1 expression was 137-fold significantly lower (P < 0.001) in tumor than in normal-appearing tissue. Expression of Ang-2 and Tie2 in tumor did not differ significantly (P = 0.510 and 0.106) from the nonneoplastic mucosa; however, Tie2 was slightly higher in normal bladder (Fig. 1). No significant relationships were observed between Ang-1, Ang-2, Tie2, and VEGF mRNA abundance and patient's sex and smoking consumption. However, Ang-1 expression was higher (P = 0.01) in patients ages >65 years (Table 1).

Fig. 1.

Expression patterns of angiogenic factors by tumor stage and grade in bladder cancer. Boxes, 25th to 75th percentiles; horizontal lines, median values; circles, maximum expression. N, normal bladder epithelium.

Fig. 1.

Expression patterns of angiogenic factors by tumor stage and grade in bladder cancer. Boxes, 25th to 75th percentiles; horizontal lines, median values; circles, maximum expression. N, normal bladder epithelium.

Close modal

Immunohistochemical analysis. Also at the protein level, the immunostaining for VEGF was stronger in epithelium of Ta than in normal transitional epithelium (Fig. 2A and B). In the muscle invasive stages of bladder cancer, VEGF immunostaining was strongest in single tumor cells and tumor stromal cells (Supplementary Fig. S2C). The immunostaining for Tie2 was present in the superficial rows of normal transitional epithelium (Supplementary Fig. S2D), whereas, in tumor cells of Ta, no considerable Tie2 staining was seen (Supplementary Fig. S2E). In muscle invasive stages, single cells showed a strong staining for Tie2, whereas tumor cells themselves were almost negative (Supplementary Fig. S2F). Ang-1 immunostaining was found in the superficial cell rows of the normal transitional epithelium (Supplementary Fig. S2G), whereas tumor cells of Ta were almost negative for Ang-1 (Supplementary Fig. S2H). In muscle invasive stages of bladder cancer, a part of tumor stromal cells was strongly positive for Ang-1 (Supplementary Fig. S2I).

Fig. 2.

In tissue areas with normal transitional epithelium, a weak VEGF staining is visible only in the superficial cell row (arrows; A), whereas the majority of tumor cells of Ta show a strong VEGF staining (arrows; B). A strong Ang-1 immunostaining is found in superficial cell rows of normal transitional epithelium (arrows; C), whereas almost no Ang-1 staining is recognizable in tumor cells of Ta (D).

Fig. 2.

In tissue areas with normal transitional epithelium, a weak VEGF staining is visible only in the superficial cell row (arrows; A), whereas the majority of tumor cells of Ta show a strong VEGF staining (arrows; B). A strong Ang-1 immunostaining is found in superficial cell rows of normal transitional epithelium (arrows; C), whereas almost no Ang-1 staining is recognizable in tumor cells of Ta (D).

Close modal

Relationship between gene expression pattern and clinicopathologic variables. VEGF and Ang-2 expression was significantly decreased by increasing tumor stage (P < 0.001 and P = 0.001) and grade (P = 0.023 and 0.075), but VEGF level remained clearly above the level of normal bladder epithelium (Fig. 1; Table 1). In contrast, we found a modest increase of Ang-1 expression by tumor invasiveness and grade (both P < 0.001), but it remained far below the normal level. Along the increasing tumor stage and grade, the elevation of Tie2 expression was less obvious (P = 0.071 and 0.064).

Correlation analysis of VEGF, Ang-1, Ang-2, and Tie2 expression. A strong correlation was observed between VEGF and Ang-2 expression (P < 0.0001). Furthermore, Tie2 mRNA expression correlated significantly with all the three angiogenic factors we analyzed (VEGF, 0.005; Ang-1, <0.001; Ang-2, <0.001).

Univariate analysis. Results of univariate analysis of angiogenic factors and prognostic endpoints (disease-specific, metastasis-free, and recurrence-free survival) are listed in Table 2. Figure 3 shows the Kaplan-Meier curves. The patients were subdivided into low or high groups for each gene expression using the median value as the cutoff. The cutoff values were as follows: 8.08 for VEGF, 0.20 for Ang-1, 396.21 for Ang-2, and 1.20 for Tie2 (Table 2).

Table 2.

Cox univariate analysis

VariablesDisease-specific survival
Metastasis-free survival
Recurrence-free survival
HR (95% CI)PHR (95% CI)PHR (95% CI)P
Age, 71.6 (36-96), y  0.850*  0.177*  0.779* 
    ≤65 (n = 51) Reference  Reference  Reference  
    >65 (n = 62) 1.056 (0.601-1.855) 0.850 0.576 (0.256-1.297) 0.183 1.097 (0.574-2.093) 0.780 
Sex  0.318*  0.953*  0.609* 
    Male (n = 88) Reference  Reference  Reference  
    Female (n = 25) 0.728 (0.391-1.356) 0.316 0.971 (0.362-2.603) 0.953 0.816 (0.816-0.373) 0.611 
    Noninvasive (n = 48) Reference <0.001* Reference <0.001* Reference 0.632* 
    Invasive (n = 65) 6.695 (3.000-14.942) <0.001 9.115 (2.706-30.705) <0.001 0.817 (0.356-1.875) 0.633 
Grade  <0.001*  0.005*  0.618* 
    Low-grade (n = 57) Reference  Reference  Reference  
    High-grade (n = 56) 3.8 (2.040-7.081) <0.001 3.19 (1.539-7.483) 0.008 0.8 (0.332-1.928) 0.620 
Prior recurrence  0.233*  0.621*  0.913* 
    Primer (n = 63) Reference  Reference  Reference  
    Recurrent (n = 50) 1.414 (0.798-2.506) 0.235 0.812 (0.354-1.859) 0.622 0.965 (0.511-1.822) 0.913 
Smoking  0.408*  0.534*  0.891* 
    Yes (n = 49) Reference  Reference  Reference  
    No (n = 54) 0.788 (0.447-1.389) 0.410 1.298 (0.569-2.964) 0.535 0.951 (0.462-1.957) 0.891 
    Unknown (n = 10)       
VEGF gene expression (n = 107)  0.269*  0.858*  0.265* 
    Not informative (n = 6)       
    Low (n = 53) Reference  Reference  Reference  
    High (n = 54) 0.718 (0.398-1.295) 0.271 0.928 (0.409-2.105) 0.858 1.461 (0.746-2.862) 0.269 
    Noninvasive cases  0.362*  0.827*  0.059* 
    Low (n = 15) Reference  Reference  Reference  
    High (n = 32) 0.496 (0.107-2.300) 0.371 1.309 (0.116-14.719) 0.827 2.22 (0.947-5.200) 0.066 
    Invasive cases  0.232*  0.072*  0.247* 
    Low (n = 38) Reference  Reference  Reference  
    High (n = 22) 1.474 (0.776-2.789) 0.236 2.216 (0.910-5.392) 0.08 0.311 (0.037-2.604) 0.282 
Ang-1 gene expression (n = 100)  0.041*  0.524*  0.992* 
    Not informative (n = 13)       
    Low (n = 50) Reference  Reference  Reference  
    High (n = 50) 1.841 (1.017-3.332) 0.044 1.321 (0.560-3.119) 0.525 0.996 (0.472-2.102) 0.992 
    Noninvasive cases  0.466*  0.494*  0.706* 
    Low (n = 31) Reference  Reference  Reference  
    High (n = 10) 1.864 (0.341-10.199) 0.473 1.101 (0.230-1.160) 0.669 1.183 (0.494-2.836) 0.706 
    Invasive cases  0.469*  0.135*  0.556* 
    Low (n = 19) Reference  Reference  Reference  
    High (n = 40) 0.786 (0.409-1.512) 0.471 0.506 (0.203-1.260) 0.143 0.634 (0.134-3.004) 0.566 
Ang-2 gene expression (n = 108)  0.051*  0.038*  0.048* 
    Not informative (n = 5)       
    Low (n = 53) Reference  Reference  Reference  
    High (n = 55) 0.563 (0.314-1.011) 0.054 0.4 (0.163-0.983) 0.046 1.985 (0.992-3.973) 0.053 
    Noninvasive cases  0.974*  0.808*  0.009* 
    Low (n = 16) Reference  Reference  Reference  
    High (n = 31) 0.975 (0.211-4.500) 0.974 1.346 (0.121-14.987) 0.809 3.178 (1.273-7.939) 0.013 
    Invasive cases  0.598*  0.270*  0.420* 
    Low (n = 37) Reference  Reference  Reference  
    High (n = 24) 0.84 (0.439-1.608) 0.599 0.567 (0.204-1.587) 0.277 0.432 (0.051-3.633) 0.440 
Tie2 gene expression (n = 103)  0.890*  0.585*  0.681* 
    Not informative (n = 10)       
    Low (n = 52) Reference  Reference  Reference  
    High (n = 51) 0.96 (0.538-1.712) 0.890 0.795 (0.348-1.815) 0.586 1.154 (0.581-2.289) 0.682 
    Noninvasive cases  0.997*  0.977*  0.886* 
    Low (n = 28) Reference  Reference  Reference  
    High (n = 15) 1.004 (0.193-5.210) 0.997 1.037 (0.094-11.453) 0.977 1.057 (0.497-2.246) 0.886 
    Invasive cases  0.035*  0.034*  0.793* 
    Low (n = 24) Reference  Reference  Reference  
    High (n = 36) 0.511 (0.271-0.965) 0.039 0.395 (0.163-0.961) 0.041 1.259 (0.218-7.261) 0.796 
VariablesDisease-specific survival
Metastasis-free survival
Recurrence-free survival
HR (95% CI)PHR (95% CI)PHR (95% CI)P
Age, 71.6 (36-96), y  0.850*  0.177*  0.779* 
    ≤65 (n = 51) Reference  Reference  Reference  
    >65 (n = 62) 1.056 (0.601-1.855) 0.850 0.576 (0.256-1.297) 0.183 1.097 (0.574-2.093) 0.780 
Sex  0.318*  0.953*  0.609* 
    Male (n = 88) Reference  Reference  Reference  
    Female (n = 25) 0.728 (0.391-1.356) 0.316 0.971 (0.362-2.603) 0.953 0.816 (0.816-0.373) 0.611 
    Noninvasive (n = 48) Reference <0.001* Reference <0.001* Reference 0.632* 
    Invasive (n = 65) 6.695 (3.000-14.942) <0.001 9.115 (2.706-30.705) <0.001 0.817 (0.356-1.875) 0.633 
Grade  <0.001*  0.005*  0.618* 
    Low-grade (n = 57) Reference  Reference  Reference  
    High-grade (n = 56) 3.8 (2.040-7.081) <0.001 3.19 (1.539-7.483) 0.008 0.8 (0.332-1.928) 0.620 
Prior recurrence  0.233*  0.621*  0.913* 
    Primer (n = 63) Reference  Reference  Reference  
    Recurrent (n = 50) 1.414 (0.798-2.506) 0.235 0.812 (0.354-1.859) 0.622 0.965 (0.511-1.822) 0.913 
Smoking  0.408*  0.534*  0.891* 
    Yes (n = 49) Reference  Reference  Reference  
    No (n = 54) 0.788 (0.447-1.389) 0.410 1.298 (0.569-2.964) 0.535 0.951 (0.462-1.957) 0.891 
    Unknown (n = 10)       
VEGF gene expression (n = 107)  0.269*  0.858*  0.265* 
    Not informative (n = 6)       
    Low (n = 53) Reference  Reference  Reference  
    High (n = 54) 0.718 (0.398-1.295) 0.271 0.928 (0.409-2.105) 0.858 1.461 (0.746-2.862) 0.269 
    Noninvasive cases  0.362*  0.827*  0.059* 
    Low (n = 15) Reference  Reference  Reference  
    High (n = 32) 0.496 (0.107-2.300) 0.371 1.309 (0.116-14.719) 0.827 2.22 (0.947-5.200) 0.066 
    Invasive cases  0.232*  0.072*  0.247* 
    Low (n = 38) Reference  Reference  Reference  
    High (n = 22) 1.474 (0.776-2.789) 0.236 2.216 (0.910-5.392) 0.08 0.311 (0.037-2.604) 0.282 
Ang-1 gene expression (n = 100)  0.041*  0.524*  0.992* 
    Not informative (n = 13)       
    Low (n = 50) Reference  Reference  Reference  
    High (n = 50) 1.841 (1.017-3.332) 0.044 1.321 (0.560-3.119) 0.525 0.996 (0.472-2.102) 0.992 
    Noninvasive cases  0.466*  0.494*  0.706* 
    Low (n = 31) Reference  Reference  Reference  
    High (n = 10) 1.864 (0.341-10.199) 0.473 1.101 (0.230-1.160) 0.669 1.183 (0.494-2.836) 0.706 
    Invasive cases  0.469*  0.135*  0.556* 
    Low (n = 19) Reference  Reference  Reference  
    High (n = 40) 0.786 (0.409-1.512) 0.471 0.506 (0.203-1.260) 0.143 0.634 (0.134-3.004) 0.566 
Ang-2 gene expression (n = 108)  0.051*  0.038*  0.048* 
    Not informative (n = 5)       
    Low (n = 53) Reference  Reference  Reference  
    High (n = 55) 0.563 (0.314-1.011) 0.054 0.4 (0.163-0.983) 0.046 1.985 (0.992-3.973) 0.053 
    Noninvasive cases  0.974*  0.808*  0.009* 
    Low (n = 16) Reference  Reference  Reference  
    High (n = 31) 0.975 (0.211-4.500) 0.974 1.346 (0.121-14.987) 0.809 3.178 (1.273-7.939) 0.013 
    Invasive cases  0.598*  0.270*  0.420* 
    Low (n = 37) Reference  Reference  Reference  
    High (n = 24) 0.84 (0.439-1.608) 0.599 0.567 (0.204-1.587) 0.277 0.432 (0.051-3.633) 0.440 
Tie2 gene expression (n = 103)  0.890*  0.585*  0.681* 
    Not informative (n = 10)       
    Low (n = 52) Reference  Reference  Reference  
    High (n = 51) 0.96 (0.538-1.712) 0.890 0.795 (0.348-1.815) 0.586 1.154 (0.581-2.289) 0.682 
    Noninvasive cases  0.997*  0.977*  0.886* 
    Low (n = 28) Reference  Reference  Reference  
    High (n = 15) 1.004 (0.193-5.210) 0.997 1.037 (0.094-11.453) 0.977 1.057 (0.497-2.246) 0.886 
    Invasive cases  0.035*  0.034*  0.793* 
    Low (n = 24) Reference  Reference  Reference  
    High (n = 36) 0.511 (0.271-0.965) 0.039 0.395 (0.163-0.961) 0.041 1.259 (0.218-7.261) 0.796 

Abbreviation: 95% CI, 95% confidence interval.

*

P values recorded are the results from log-rank tests.

Fig. 3.

Kaplan-Meier curves of cancer-specific and metastasis-free survival stratified by Tie2 in muscle invasive (T>1) bladder tumors and recurrence-free survival in noninvasive (Ta, T1) bladder tumors stratified by Ang-2 expression.

Fig. 3.

Kaplan-Meier curves of cancer-specific and metastasis-free survival stratified by Tie2 in muscle invasive (T>1) bladder tumors and recurrence-free survival in noninvasive (Ta, T1) bladder tumors stratified by Ang-2 expression.

Close modal

Association of VEGF, Ang-1, Ang-2, and Tie2 with disease-specific survival. We could not detect a prognostic effect on cancer-related survival for VEGF gene expression (P = 0.269). In contrast, Ang-1 and Ang-2 significantly correlated with disease-specific survival (P = 0.041 and 0.051). This, however, was not independent from tumor stage and grade. Muscle invasive tumors with high Tie2 expression were found to have a more favorable prognosis (P = 0.035; Table 2; Fig. 3).

Association of VEGF, Ang-1, Ang-2, and Tie2 with metastasis-free survival. Because metastasis of superficial bladder cancer is extremely rare, we preferred to seek correlations with metastasis-free survival mainly in invasive cases. Regarding Ang-1 and Ang-2 expression levels, there was no significant difference in metastasis-free survival (P = 0.135 and 0.270). Higher VEGF mRNA tended to indicate an unfavorable prognosis, although this correlation failed to reach significance (P = 0.072). High Tie2 expression significantly correlated with favorable metastasis-free survival (P = 0.034; Table 2; Fig. 3).

Association of VEGF, Ang-1, Ang-2, and Tie2 with recurrence-free survival. The majority of invasive bladder tumors (55 of 65) were treated with radical cystectomy. In these cases, the probability to observe a local recurrence is limited. Therefore, we discuss the recurrence-free survival only in noninvasive cases. We did not find any correlation between recurrence-free survival and the expression level of Ang-1 and Tie2 (P = 0.706 and 0.886). In contrast, high Ang-2 expression found to be a strong predictor of recurrence (P = 0.009). High VEGF level showed an unfavorable borderline correlation with recurrence-free survival (P = 0.059; Table 2; Fig. 3).

Multivariate analysis. Multivariate analysis indicated that Tie2 high expression is an independent favorable prognostic factor of disease-specific survival (P = 0.003) and metastasis-free survival (P = 0.029). VEGF had a tendency to be an independent unfavorable prognostic factor of metastasis-free survival. However, this correlation did not reach prognostic significance (P = 0.070). Furthermore, high Ang-2 emerged as an independent predictor of recurrence (P = 0.001). Multivariate Cox models are listed in Table 3.

Table 3.

Multivariate Cox regression analyses of histopathologic variables, risk factors, and gene expressions on disease-specific, metastasis-free, and recurrence-free survival in bladder cancer patients

VariablesDisease-specific survival
Metastasis-free survival
Recurrence-free survival
HR (95% CI)PHR (95% CI)PHR (95% CI)P
Stage 9.655 (2.666-34.960) 0.001 22.211 (3.357-146.945) 0.001 8.853 (2.220-35.299) 0.002 
    Noninvasive (n = 48)       
    Invasive (n = 65)       
Grade 0.97 (0.397-2.365) 0.946 0.653 (0.196-2.176) 0.487 0.347 (0.097-1.246) 0.105 
    Low grade (n = 57)       
    High grade (n = 56)       
Smoking 0.650 (0.324-1.305) 0.226 1.115 (0.397-3.131) 0.837 0.632 (0.227-1.755) 0.378 
    Yes (n = 49)       
    No (n = 54)       
    Unknown (n = 10)       
Age 0.993 (0.488-2.019) 0.985 0.685 (0.234-2.009) 0.491 1.011 (0.340-3.007) 0.984 
    ≤65 (n = 51)       
    >65 (n = 62)       
Recurrent 1.341 (0.672-2.676) 0.406 0.942 (0.342-2.593) 0.907 0.209 (0.072-0.605) 0.004 
    Yes (n = 63)       
    No (n = 50)       
VEGF expression 1.499 (0.735-3.059) 0.266 2.55 (0.928-7.011) 0.070 0.549 (0.190-1.583) 0.267 
    Low (n = 53)       
    High (n = 54)       
Ang-1 expression 1.655 (0.668-4.100) 0.277 — (—) — 1.072 (0.331-3.472) 0.908 
    Low (n = 50)       
    High (n = 50)       
Ang-2 expression 1.040 (0.430-2.516) 0.931 0.711 (0.216-2.342) 0.575 10.179 (2.691-38.498) 0.001 
    Low (n = 53)       
    High (n = 55)       
Tie2 expression 0.249 (0.100-0.619) 0.003 0.311 (0.109-0.889) 0.029 0.727 (0.241-2.204) 0.573 
    Low (n = 52)       
    High (n = 51)       
VariablesDisease-specific survival
Metastasis-free survival
Recurrence-free survival
HR (95% CI)PHR (95% CI)PHR (95% CI)P
Stage 9.655 (2.666-34.960) 0.001 22.211 (3.357-146.945) 0.001 8.853 (2.220-35.299) 0.002 
    Noninvasive (n = 48)       
    Invasive (n = 65)       
Grade 0.97 (0.397-2.365) 0.946 0.653 (0.196-2.176) 0.487 0.347 (0.097-1.246) 0.105 
    Low grade (n = 57)       
    High grade (n = 56)       
Smoking 0.650 (0.324-1.305) 0.226 1.115 (0.397-3.131) 0.837 0.632 (0.227-1.755) 0.378 
    Yes (n = 49)       
    No (n = 54)       
    Unknown (n = 10)       
Age 0.993 (0.488-2.019) 0.985 0.685 (0.234-2.009) 0.491 1.011 (0.340-3.007) 0.984 
    ≤65 (n = 51)       
    >65 (n = 62)       
Recurrent 1.341 (0.672-2.676) 0.406 0.942 (0.342-2.593) 0.907 0.209 (0.072-0.605) 0.004 
    Yes (n = 63)       
    No (n = 50)       
VEGF expression 1.499 (0.735-3.059) 0.266 2.55 (0.928-7.011) 0.070 0.549 (0.190-1.583) 0.267 
    Low (n = 53)       
    High (n = 54)       
Ang-1 expression 1.655 (0.668-4.100) 0.277 — (—) — 1.072 (0.331-3.472) 0.908 
    Low (n = 50)       
    High (n = 50)       
Ang-2 expression 1.040 (0.430-2.516) 0.931 0.711 (0.216-2.342) 0.575 10.179 (2.691-38.498) 0.001 
    Low (n = 53)       
    High (n = 55)       
Tie2 expression 0.249 (0.100-0.619) 0.003 0.311 (0.109-0.889) 0.029 0.727 (0.241-2.204) 0.573 
    Low (n = 52)       
    High (n = 51)       

The present results show a switch in the expression of factors involved in vascular destabilization and angiogenic activation at the superficial and noninvasive stage of bladder cancer. Briefly, we show here that (a) the Ang-1 expression is dramatically reduced in tumor stage Ta in comparison with normal bladder epithelium; (b) in contrast, the highest level of Ang-2 and VEGF expression is measured in this tumor stage; (c) high level of Ang-2 expression seems to be a strong independent predictor of tumor recurrence; and (d) enhanced expression of Tie2 is identified as an independent favorable prognostic factor of disease-specific survival. Particularly, the reduced expression of Ang-1 accompanied by enhanced expression of Ang-2 and VEGF in bladder cancer stage Ta in comparison with normal bladder epithelium suggests an establishment of a potent proangiogenic stimulus at the superficial bladder cancer stage Ta.

Angiopoietins represent a family of extracellular ligands of the tyrosine kinase receptor Tie2. Ang-1 induces tyrosine phosphorylation of Tie2, whereas Ang-2 blocks this effect of Ang-1. Ang-1 inhibits apoptosis of endothelial cells and promotes the stabilization and maturation of newly formed blood vessels via assembling of periendothelial cells such as pericytes or smooth muscle cells into the vascular wall (22). In contrast, Ang-2 antagonizes the stabilizing effect of Ang-1 and thereby sensitizes endothelial cells to VEGF action (2, 23). This destabilization effect of Ang-2 can also occur on preexisting vessels before vessel sprouting or regression (2). Already during the embryonic development of blood vessels, the Ang-1-Tie2 system is crucial for stabilization, maintenance, and remodeling of the nascent primitive vascular plexus (24). In contrast, a uniform model for the role of the angiopoietins in tumorigenesis is not yet established from the diverse data published to date. Tait and Jones reviewed the literature about the expression of angiopoietins in human tumors and concluded that both Ang-1 and Ang-2 are elevated in a wide variety of tumors (25). However, the change of Ang-1 expression was not as obvious as that of Ang-2. Recent studies on hepatocellular carcinoma, lung cancer, and bladder cancer reported conflicting results regarding Ang-1 expression (8, 26, 27). Regarding bladder cancer, there are only two publications dealing with expression of angiopoietins. Oka et al. immunohistochemically analyzed bladder cancer specimens and found no difference in the abundance of the two angiopoietins between normal and tumor specimens (15). They found correlation between Ang-2 but not Ang-1 expression and tumor stage and grade. They concluded that Ang-2 might provide prognostic information in bladder cancer. In contrast Quentin et al. found a 7-fold decreased level of Ang-1 expression and a slightly elevated level of Ang-2 expression in bladder tumors using real-time PCR (14). Furthermore, they analyzed VEGF expression and found a considerable elevation in tumors by immunohistochemistry and also by real-time PCR. Our data regarding the expression pattern of Ang-1, Ang-2, and VEGF in tumor versus normal bladder tissue are in line with that of Quentin et al. (14).

We detected a considerable shift in the balance of angiogenic factors between normal bladder epithelium and superficial carcinoma. In contrast to some published findings by other groups, we found elevated levels of Ang-2 only in Ta tumors (8). Furthermore, we show here a 137-fold decrease of Ang-1 expression together with the accompanying significant increase of VEGF and considerable elevation of Ang-2 expression at the mRNA level in tumor specimens with Ta stage. Because these findings were confirmed at the protein level for VEGF and Ang-1 by immunohistochemistry, this switch of the angiogenic balance represents a crucial process in early bladder tumor development leading to vascular destabilization and subsequent initiation of angiogenesis (28). Although the immunohistochemically shown distribution pattern of VEGF and Ang-1 proteins does not necessarily represent their exact cellular production place because of diffusion and receptor binding processes, these data together with mRNA expression pattern suggest the strongest angiogenic shift at the bladder cancer stage Ta.

The high correlation coefficient (P < 0.0001) of VEGF and Ang-2 underlines the strong cooperative effect of both factors. Recently, it was shown that the endothelial up-regulation of the proangiogenic cell adhesion molecule CEACAM1 in Ta stage of bladder cancer leads to increased angiogenesis (29). Furthermore, it has also been shown that down-regulation of Ang-1 together with accompanied up-regulation of VEGF and Ang-2 in high-grade prostate intraepithelial neoplasia, a noninvasive precancerous stage of prostate tumor, results in vascular destabilization including endothelial fenestrations and gaps, detachment of pericytes from endothelial cells, and degradation of vascular basement membrane (30). Although we did not performed ultrastructural analyses in Ta stage of bladder cancer because the tissue specimens were used for expression analyses, the results presented here assume that the copresence of VEGF and Ang-2 at high level together with the presence of Ang-1 at a low level is one of the main driving forces of angiogenic activation in bladder cancer. Remarkably, this switch is most prominent in Ta when we compare the known pathologic defined stages of bladder cancer with each other. Thus, the present findings suggest that the strongest proangiogenic stimulus is already generated in superficial noninvasive bladder cancer stages. This may be related to the fact that the superficial (Ta) tumors are not vascularized and need the generation of such a strong angiogenic stimulus to activate new vessel sprouting from the existing neighboring blood vessels. The nonvascularized growth of tumor cells in Ta might cause hypoxia, an essential driving force of angiogenesis, whereas the vascularized state of invasive bladder cancer stages might reduce hypoxia. This might be responsible for the relatively low level of angiogenic stimuli in late bladder cancer stages.

In in vitro studies on bladder cancer cell lines, we could only detect a considerable difference between invasive and noninvasive cell lines in case of VEGF expression in a similar pattern as observed in tissue studies. Notable, VEGF expression was significantly higher in superficial than in invasive bladder cancer cell lines. Regarding the expression of other factors studied here, no such a correlation was found, indicating that cell culture experiments do not reflect the complex relationship between tumor cells and matrix.

Cox multivariate analysis identified Ang-2 as an independent, unfavorable prognostic factor for recurrence in noninvasive cases. Patients with high Ang-2 had 10 times the risk of recurrence compared with those with low Ang-2 expression [hazard ratio (HR), 10.18]. This is in accordance with former findings indicating that high Ang-2 expression is associated with adverse prognosis in several tumors (11, 31, 32). The prognostic significance of Ang-2 was not so obvious in muscle invasive bladder cancer. Regarding VEGF, a key regulator of tumor angiogenesis (33), our results tend to support the previous findings, suggesting VEGF as a predictor for disease recurrence in bladder cancer (34), but the borderline significance in univariate analysis was not proven to be independent from other variables included in multivariate Cox model (Table 3). Furthermore, VEGF emerged as a borderline significant independent risk factor for metastasis-free survival.

Conflicting results have been reported concerning changes in Tie2 expressions in various tumors. Elevated Tie2 levels were detected in breast, gastric, and endometrial cancers (3537). In contrast, decreased Tie2 level was found in ovarian carcinoma and lung cancer (10, 38). Some authors found correlation between Tie2 level and malignancy (e.g., in gastric cancer), whereas others in ovarian cancer did not (11, 39). Despite its major role in angiogenesis, little is known about the prognostic effect of Tie2 in cancer. Immunohistologic examination of breast cancer identified Tie2 as an independent unfavorable prognostic indicator. However, this study evaluated the localization (tumor surface positive versus tumor surface negative) but not the intensity of Tie2 expression (40). In contrast, Harris et al. reported positive correlation between high soluble Tie2 levels and improved survival and positive response for antiangiogenic therapy in renal cancer (41).

To our knowledge, this is the first investigation analyzing the expression of Tie2 in bladder cancer at the mRNA and protein levels. The Tie2 immunostaining in umbrella cells of normal transitional epithelium disappeared in superficial tumor stage Ta, which might be related to dysplastic transformation of normal transitional epithelium. In the muscle invasive stages of Ta, tumor-associated single cells, probably macrophages, were positive for Tie2. This finding is in line with our result showing a slightly increased expression of Tie2 at the mRNA in muscle invasive tumor stages in comparison with Ta. We observed a considerable correlation between Tie2 expression and disease-specific as well as metastasis-free survival. Our multivariate Cox analysis revealed that Tie2 expression is an independent favorable prognostic factor in bladder cancer. This is in accordance with the notion that the Ang-1-Tie2 interaction has an inhibitory effect on tumor growth through improved vessel stabilization (42). It has been shown that vascular stabilization per se results in extensive necrosis of tumor tissue (43, 44). The decrease of Ang-1 expression in Ta compared with normal bladder epithelium as we show here also supports this concept and is probably an important step in bladder carcinogenesis. Additionally, it has also been postulated that vascular stabilization or “vascular normalization” would open a therapeutic window, increasing the efficiency of chemotherapy or radiation therapy of tumors (45). Furthermore, in a rat cutaneous tumor model, the use of soluble Tie2 systematically blocked tumor vessel proliferation and inhibited the growth of primary tumors and metastases (46). This could be an alternative explanation how enhanced Tie2 expression could improve prognosis. However, further research is needed to clarify the link between Tie2 expression, soluble Tie2 level, and disease prognosis.

Conclusions. Here, we show a characteristic “angiogenic switch” in gene expression pattern with strong down-regulation of Ang-1 and concurrent up-regulation of Ang-2 and VEGF expression in bladder tumor stage Ta, a superficial noninvasive tumor stage. This shift is probably a main driving force of vascular destabilization and initiation of angiogenesis in bladder cancer. Remarkably, this switch is less pronounced in later stages of bladder cancer. A further interesting finding of the present study is the strong prognostic significance of Tie2 and Ang-2 expression in bladder cancer. Further studies are needed to assess the clinical relevance of these factors in a greater collective of bladder cancer and to better characterize their prognostic and therapeutic role in bladder cancer.

No potential conflicts of interest were disclosed.

Grant support: National Federal Ministry of Education and Research grant 0313659B.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

We thank Sabine Hertel for help in statistical analysis, Jacqueline Wittschier for excellent technical assistance, and Dr. Derya Tilki for critical reading of this article.

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Supplementary data