Angiogenesis is a well known prerequisite for tumor growth and metastasis. It is believed that angiogenin initiates cell migration and aids cell proliferation. Based on this, the authors hypothesized that individuals who had increased plasma levels of angiogenin were at an elevated risk for carcinoma of the urinary bladder.
In this ongoing case–control study, the authors used an enzyme-linked immunosorbent assay to compare plasma levels of angiogenin in 209 patients with bladder carcinoma and in 208 healthy control participants who were matched according to age (± 5 years), gender, and ethnicity.
The mean plasma angiogenin concentration was significantly higher in patients compared with controls (343.2 ng/mL vs. 308.0 ng/mL, respectively; P < 0.01). High plasma angiogenin levels were associated with a two-fold increased risk for bladder carcinoma. Moreover, in patients who had superficial bladder carcinoma, plasma angiogenin levels were significantly higher among those who had recurrent disease than in those who were without recurrence (P < 0.01). Similarly, patients who had superficial bladder carcinoma with higher angiogenin levels had a shorter recurrence-free survival than patients who had lower angiogenin levels (P < 0.01). Finally, elevated angiogenin levels were associated with an increased recurrence risk, with hazard ratio of 2.85.
Angiogenesis, which is the development of new blood vessels from preexisting vasculature, is fundamental to tumor growth.1 It also is implicated in several other processes, including the initial progression from a premalignant tumor to carcinoma, the invasion of tumor cells into systemic circulation, and the growth of dormant micrometastases into frank metastatic lesions.2, 3 Such neovascularization may be stimulated by factors released from tumor cells, tumor-associated inflammatory cells, or the extracellular matrix.4 These factors include basic fibroblast growth factor (bFGF) family members, transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), platelet-derived endothelial cell growth factor (PDGF), E-cadherin, and angiogenin.5–9
Human angiogenin, a single-chain, 123-amino acid residue polypeptide with a molecular mass of 14.2 KDa, is a potent angiogenic factor that is believed to initiate cell migration and aid cell proliferation.10 Only a single copy of the human angiogenin gene exists and is localized to chromosome 14q11–q13.11 Angiogenin belongs to the family of ribonucleases called ribonucleases with special biologic actions (RIBASE).12 Unlike some family members that display cytotoxic activity, angiogenin shows RNase activity.13 However, angiogenin is not a tumor-specific product; rather, it is expressed in both normal and malignant cells and in plasma.14 In vitro, angiogenin binds to a cell-surface actin, and the ensuing complex then accelerates plasmin generation, which, in turn, directly degrades the laminin and fibronectin in the cellular membrane. This is a prerequisite for subsequent cell migration.15 It also has been found that angiogenin binds to components in the extracellular matrix.16 Angiogenin can affect vascular endothelium directly and can facilitate the role of other angiogenic growth factors, which are elevated significantly during disease progression in patients with carcinoma.17 Based on the available evidence, inhibiting the action of angiogenin may serve as an effective therapeutic approach for the treatment of malignant disease.18
Elevated serum/plasma angiogenin levels have been found in patients with cervical carcinoma, pancreatic carcinoma, colorectal carcinoma, melanoma, urothelial carcinoma, breast carcinoma, gastric carcinoma, and endometrial carcinoma.9, 17, 19–25 Moreover, the high serum/plasma angiogenin levels in those patients were related to either disease progression or poor survival. Two studies have suggested the important role of angiogenin in bladder carcinoma progression.22, 26 In one of those studies, the serum levels of angiogenin were significantly higher in invasive urothelial carcinoma than in superficial urothelial carcinoma, and patients who had invasive urothelial carcinoma and elevated angiogenin levels had a significantly lower survival rate compared with patients who had normal angiogenin levels.22 These observations suggest that angiogenin has clinical significance in bladder carcinoma. In the current study, we evaluated the plasma levels of angiogenin in patients with bladder carcinoma and in a healthy control group using an enzyme-linked immunosorbent assay (ELISA) method.
MATERIALS AND METHODS
The study population consisted of 209 patients with bladder carcinoma (cases) and 208 healthy control participants (controls). The cases were recruited from the Urology Departments of The University of Texas M. D. Anderson Cancer Center and the Baylor College of Medicine (Houston, TX). All cases were patients who had newly diagnosed, histologically confirmed bladder carcinoma; specifically, transitional cell carcinoma. The cases were eligible for the study only if they had not received chemotherapy or radiotherapy in the 6 months prior to recruitment. There were no age, gender, ethnic, or disease stage restrictions.
The control participants without a history of cancer (except nonmelanoma skin cancer) were recruited from a large pool of potential volunteers from the Kelsey-Seybold clinics, Houston's largest multispecialty physician group. The identification and recruitment of the control group is detailed thoroughly in a report by Hudmon et al.27 Briefly, potential control participants first were surveyed using a short questionnaire to determine the demographic information necessary for matching and their willingness to participate in the epidemiological study. Controls were then selected randomly from this pool to match the cases by age (± 5 years), gender, and ethnicity. After a control participant was selected, the individual was be contacted by telephone to confirm their willingness to participate, and an appointment was scheduled at a Kelsey-Seybold clinic that was convenient to the participant. If the individual refused to participate or was ineligible, then another potential control was selected from the pool. It should be noted that cases and controls were recruited concurrently. Usually, a case was recruited first; then, a control was recruited within 1 month of case recruitment and was matched to the case by age, gender, and ethnicity. The majority of controls (> 95%) visited the clinic for annual health check-up.
Collection of Epidemiological Data
Eligible individuals who agreed to participate first were asked to sign an informed consent form and then were interviewed using a structured questionnaire administered by trained interviewers from The University of Texas M. D. Anderson Cancer Center. The questionnaire elicited information regarding demographics, smoking history, alcohol consumption, family history of cancer, medical history, dietary patterns, and occupational exposures.
After the interview, 40-mL blood specimens were obtained from each participant by venipuncture into heparinized tubes. The tubes first were coded with a unique identification number to ensure that laboratory personnel were blinded to the case–control status and then were transported immediately to the laboratory, where the specimens were processed. The plasma was collected after centrifugation of the blood at 1500 rpm for 10 minutes at room temperature and was stored immediately at − 80 °C. Information regarding the clinical stage of each tumor was collected from medical records. The staging was determined by the TNM classification system established by the American Joint Committee on Cancer. A tumor was considered superficial if it was classified as pathologic tumor stage a (pTa), pT in situ (pTis), or pT1.
Measurement of Angiogenin
An ELISA kit (R&D Systems, Minneapolis, MN) was used to measure the levels of angiogenin in the plasma. Briefly, first, each plasma sample was diluted 1:200 with buffer, then added to microtiter wells that were precoated with mouse monoclonal antihuman angiogenin antibody, and incubated for 1 hour. After incubation, polyclonal antihuman angiogenin antibody conjugated with horseradish peroxidase was added to the wells and incubated for 1 hour. Next, the wells were incubated with a tetramethylbenzidine/H2O2 mixture for 20 minutes to develop color. Color development was stopped by the addition of sulfuric acid. The absorbance of each well was determined using a Vmax Microplate Reader (Molecular Device, Sunnyvale, CA) with a 450-nm wavelength. The blank value was subtracted from the duplicate readings for each standard and sample. For all analyses, each sample was assayed twice, and the mean of the two values was used for data analysis.
The distributions of the demographic variables between the cases and controls were compared using the chi-square test for categorical variables (gender, ethnicity, and smoking status) and the two-sample Student t test for continuous variables (age). Because the distribution of angiogenin in the population was skewed positively, the levels of angiogenin were analyzed categorically on the basis of the quartile distribution in the control group. The median level of angiogenin in the control group was used as a cut-off point to calculate the overall risk for bladder carcinoma. To assess the strength of the association between bladder carcinoma risk and angiogenin levels, we calculated both crude and adjusted odds ratios (ORs) and their corresponding 95% confidence intervals (95%CIs) using univariate and multivariate unconditional logistic regression analysis, respectively.28 In the multivariate analysis, we controlled for gender, age, ethnicity (Caucasian, African American, or Hispanic), and cigarette smoking status (never, former, or current). Tests for linear trend were performed using quartile levels as continuous variables. Spearman correlation coefficients were used to examine the correlation between angiogenin levels and age, gender, ethnicity, and cigarette smoking status. The overall recurrence-free survival rates were calculated with the Kaplan–Meier method, and the difference was determined with the log-rank test. The prognostic significance was assessed with the Cox proportional hazards regression model. All P values were two-sided. Associations were considered statistically significant at P < 0.05.
Table 1 shows the distribution of demographic variables in the study population. Because the controls were selected to match the patients by gender, age, and ethnicity, no statistically significant differences were observed between the two groups with respect to these variables. The difference in cigarette smoking status between the cases and controls, as expected, was statistically significant (P < 0.01). The cases included more current smokers than the controls (26.8% vs. 8.2%). The mean age of the cases and controls was approximately 62 years. Both groups consisted mainly of Caucasian males. The mean and quartile values of angiogenin in the study participants also are shown in Table 1. The mean levels of angiogenin were significantly higher in cases than in controls (343.2 ng/mL vs. 308.0 ng/mL; P < 0.01).
Table 1. Association of Bladder Carcinoma with Angiogenin Levels and Other Variables
SD: standard deviation; 95% CI: 95% confidence interval.
All P values are 2-sided, and associations are considered statistically significant at P < 0.05.
Mean ± SD
62.1 ± 12.2
61.5 ± 11.9
No. of patients (%)
Angiogenin levels (ng/mL)
The risk estimates from the univariate and multivariate analyses, both of which are shown in Table 2, were similar. In the multivariate analysis, when age, gender, ethnicity, and cigarette smoking status were included in the regression model, the OR was 2.07 (95%CI, 1.36–3.15) for the overall risk of bladder carcinoma. When the study participants were categorized by the quartile values in the control group, there were significant differences between cases and controls in the quartile distributions for angiogenin, with cases showing higher angiogenin levels than controls. Compared with the controls, the distribution of the cases was skewed toward the higher quartiles (P < 0.01), with close to 70% of cases in the third and fourth quartiles. An apparent dose-response relation for angiogenin was observed in the quartile analysis. Individuals in the third and forth (highest) quartiles of angiogenin displayed ORs of 1.76 (95%CI, 1.00–3.14) and 1.85 (95%CI, 1.09–3.13), respectively, compared with individuals in the lowest quartile. The trend test for the quartiles was statistically significant in both the univariate model (P for trend < 0.01) and the multivariate model (P for trend < 0.01). This was more evident in older individuals (age ≥ 62.5 years, the median age of the control population), but not in younger individuals (age < 62.5 years) (data not shown).
Table 2. Levels of Angiogenin in Patients with Bladder Carcinoma and in Healthy Control Participants
In addition, we evaluated angiogenin levels in patients with bladder carcinoma according to clinical variables, including disease stage (superficial vs. invasive) and recurrence status. A tumor was considered superficial (Ta, Tis, or T1) if there was no bladder muscle involvement and invasive (T2, T2, or T4) if the tumor had invaded the bladder muscle. Patients in the cases group who had superficial or invasive bladder carcinoma had significantly higher levels of angiogenin compared with the control group (Table 3). Patients (cases) who had invasive bladder tumors had slightly higher angiogenin levels compared with patients (cases) who had superficial bladder tumors; however, the difference was not significant (P = 0.63). Further analysis in the patients with superficial bladder carcinoma showed that the plasma levels of angiogenin were significantly higher in patients who had recurrent disease compared with the levels in patients who were without recurrence (383.8 ng/mL vs. 278.0 ng/mL; P < 0.001). Higher angiogenin levels were associated with shorter recurrence-free survival compared with lower angiogenien levels in Kaplan–Meier analysis (P = 0.001) (Fig. 1). In the Cox regression analysis, after adjusting for age, gender, ethnicity, and smoking status, we found that higher levels of angiogenin were associated with increased risk of bladder carcinoma recurrence (hazard ratio, 2.85; 95%CI, 1.46–5.59) (Table 4).
Table 3. Levels of Angiogenin in Patients with Bladder Carcinoma by Tumor Classificationa
Adjusted for age, gender, ethnicity, and smoking status.
We also investigated the correlations between angiogenin levels and gender, age, ethnicity, and smoking status in the control group. Angiogenin levels did not differ by gender (301.25 ng/mL vs. 289.21 ng/mL; P = 0.17) and did not correlate with age (r = − 0.06 and P = 0.41). African Americans had slightly higher levels of angiogenin than Caucasians and Hispanics, although the difference was not statistically significant (African Americans vs. Caucasians: 336.76 ng/mL vs. 323.66 ng/mL; P = 0.54; African Americans vs. Hispanics: 336.76 ng/mL vs. 316.75 ng/mL; P = 0.63). Current smokers had higher levels of aniogenin than never smokers and former smokers, but the difference was not statistically significant (current smokers vs. never smokers: 331.46 ng/mL vs. 307.76 ng/mL; P = 0.32; current smokers vs. former smokers: 331.46 ng/mL vs. 321.01 ng/mL; P = 0.86).
It is believed that angiogenin plays a role in the growth of malignant tumors and metastasis. The serum/plasma levels of angiogenin reportedly are higher in patients with certain malignancies than in healthy control individuals.9, 17, 19–25 However, no study to date has investigated the detailed expression profiles of angiogenin levels in the population. Evaluating the plasma angiogenin level may provide important prognostic information: For example, it could be used as a potential biomarker in bladder carcinogenesis. Therefore, in the current study, we compared the plasma levels of angiogenin between patients with bladder carcinoma and a group well matched, healthy control participants, and we assessed the risk of bladder carcinoma associated with angiogenin levels.
Although the current understanding of the molecular function of angiogenin in tumor development is limited, the evidence clearly suggests that angiogenin plays a role in early tumor growth. Experiments have been carried out to block the effects of human angiogenin using monoclonal antibodies.18, 29 These antibodies stopped the growth of human large intestine tumors HT-29, pulmonary adenocarcinoma, and fibrosarcoma transplanted to athymic mice. In the future, angiogenin inhibition may develop into an effective treatment for tumor growth. The results of our study provide further evidence of the role of angiogenin in tumor development. We found that plasma angiogenin levels were significantly higher in patients with bladder carcinoma compared with the levels in healthy controls. This result is in accordance with other studies that found elevated serum/plasma angiogenin levels in patients with several types of carcinoma.9, 17, 19–25
We also found that older individuals (age ≥ 62.5 years) with elevated levels of angiogenin had a greater risk for developing bladder carcinoma compared with younger individuals (age < 62.5 yrs). Several studies have shown that angiogenesis slows with aging.30, 31 Because older individuals already have accumulated more carcinogenic exposure burdens than younger individuals, it is hypothesized higher angiogenin levels will pose greater risk of malignant disease among older individuals compared with younger individuals. Thus, in this scenario, it may be reasonable to assume that high levels of angiogenin will have a more striking effect in older individuals than in younger individuals. Our findings support this hypothesis.
Angiogenin is involved not only in tumor initiation but also in tumor progression. Evidence from other studies has shown that elevated angiogenin levels predict advanced tumor stage, increased recurrence, and poor survival.19–25 For example, Miyake et al. found that patients with invasive urothelial carcinoma had significantly higher angiogenin levels (514.6 ng/mL) than patients with superficial carcinoma (381.7 ng/mL). In the same study, the authors also concluded that elevated angiogenin levels were associated with poor recurrence-free survival in patients with invasive carcinoma.22 In contrast, we did not detect any significant difference in angiogenin levels between patients with invasive bladder tumors and patients with superficial bladder tumors. However, similar to the findings of Miyake et al., we observed that elevated angiogenin levels were associated with poor recurrence-free survival in patients with superficial carcinoma. Although angiogenin may have had slightly different clinical implications in these two studies, the findings are in agreement that measuring the preoperative concentration of serum/plasma angiogenin may provide additional useful information about disease progression in patients with bladder carcinoma.
The current findings clearly demonstrate that a higher level of angiogenin in plasma is associated strongly with an increased risk of bladder carcinoma. Combined with the finding that angiogenin is related to superficial bladder carcinoma recurrence, this also suggests that angiogenin may serve as a novel predictor for the risk of bladder carcinoma. In addition to angiogenin, several other genes involved in angiogenesis, such as bFGF, VEGF, E-cadherin, and TGF-β, are related to the risk of carcinoma.32 In conclusion, the identification of plasma angiogenin level as a biomarker in bladder carcinoma is important because, by integrating this marker with a panel of epidemiological and clinical markers, our objective is to build a comprehensive risk-assessment model for bladder carcinoma in the future.