Breast cancer is the most common malignancy among women in the USA (Jemal et al., 2006). The incidence of breast cancer is increasing worldwide, and this seems to be related to an increase in lifestyle risk factors, including physical inactivity and overweight/obesity (Huang et al., 1997; Edwards et al., 2005; Bardia et al., 2006). Physically active individuals have lower rates of many cancers including breast cancer. The probable protective effect of physical activity on breast cancer is associated with a reduction in risk ranging from 20 to 30% (Monninkhof et al., 2007; Friedenreich and Cust, 2008; Eliassen et al., 2010; Friedenreich et al., 2010). A prospective study of 64 777 premenopausal women indicates that leisure-time activity is associated with a reduced risk for premenopausal breast cancer (Maruti et al., 2008). Obese postmenopausal women have a 50% higher risk of breast cancer than nonobese women (Trentham-Dietz et al., 2000). Current physical activity guidelines developed by the American College of Sports Medicine recommend that healthy adults and cancer survivors perform a minimum of 30 min of moderate-intensity exercise 5 days/week to promote health (Schmitz et al., 2010). Although numerous observational studies have reported an association between physical activity, adiposity, and cancer, the evidence linking biomarkers with exercise, cancer etiology, and progression has not been clearly defined.
The growth of a tumor and its biological behavior are mainly dependent on angiogenesis, whereas avascular tumors cannot grow over a microscopic size and can be eliminated by the immune system. Angiogenesis is regulated by the net balance between the positive (angiogenic or proangiogenic) and the negative (angiostatic or antiangiogenic) regulators of blood vessel growth (O’Reilly et al., 1994; Hanahan and Folkman, 1996). Vascular endothelial growth factor (VEGF) is a key angiogenic factor that stimulates the growth of tumors including breast cancer (Ferrara et al., 2003; Young et al., 2010), in which VEGF exerts paracrine (especially angiogenesis) and autocrine (proliferation and migration) effects to promote progression of breast cancer. Many VEGF receptor tyrosine kinase inhibitors have been used in clinical trials as antitumor angiogenesis agents for breast cancer (Gasparini et al., 2005). Administration of angiostatic factors such as endostatin or soluble vascular endothelial growth factor receptor 1 [soluble fms-like tyrosine kinase-1 (sFlt-1)] inhibits the progression of cancers including breast cancer (Mahasreshti et al., 2001; Folkman, 2002; Cho et al., 2005). We have reported that exercise induces a circulating angiostatic phenotype, characterized by decreased free VEGF and increased endostatin and sFlt-1 plasma levels in men (Gu et al., 2004; Bailey et al., 2006) and in a rodent model (Gu et al., 2006). However, there are no current data on whether exercise can induce a circulating angiostatic phenotype in young adult women. We hypothesize that in women, exercise induces a circulating angiostatic phenotype characterized by increased endostatin and sFlt-1, and decreased free VEGF.
Epidemiological studies suggest that overweight/obesity is associated with increased adipokines, such as VEGF and leptin (Vona-Davis and Rose, 2009), and has been causally associated with breast cancer in postmenopausal populations and a poor prognosis irrespective of menopausal status. Several studies have reported increased plasma levels of VEGF in human obesity (Miyazawa-Hoshimoto et al., 2003; Seida et al., 2003; Gomez-Ambrosi et al., 2010), although others have failed to obtain the same results (Rehman et al., 2003). Another study has reported a higher plasma level of VEGF only in postmenopausal obese women (Silha et al., 2005). There are no data on whether overweight/obese young adult women have higher plasma levels of unbound VEGF than lean young adult women.
The present study aimed to determine the following: (a) whether moderate exercise induces a circulating angiostatic phenotype characterized by increased sFlt-1 and endostatin, and decreased unbound VEGF in young adult female volunteers; (b) whether overweight/obese young adult women have a higher plasma level of unbound VEGF than lean young adult women; and (c) whether there is a positive correlation between the basal levels of plasma sFlt-1 and age.
Selection of participants
A total of 72 African American and White adult women volunteers ranging in age from 18 to 44 years were enrolled into the exercise study. Men, menopausal or pregnant women, or those who had any medical condition that would inhibit them from walking on a treadmill were excluded. Informed consent was obtained from all participants. The study was approved by the University of Mississippi’s Institutional Review Board.
Exercise study protocol
All the participants performed an exercise test by walking on a treadmill for 30 min at a moderate intensity. During exercise, submaximal oxygen consumption (VO2) was determined utilizing a ParvoMedics metabolic cart (Sandy, Utah, USA) that analyzed pulmonary and metabolic variables from the participants (Hodges et al., 2005). The participants walked at a speed and grade corresponding to 55–59% of their heart rate reserve while VO2 was measured (Steele et al., 2008). This test provided VO2 data on an actual 30-min bout of moderate-intensity endurance exercise. A volume of 6 ml of blood was collected into a venipuncture tube with an EDTA solution immediately before and after 30 min of exercise. The blood samples were immediately centrifuged at 700g in a microcentrifuge for 5 min at 4°C and the plasma was stored at −80°C. We had access to the blood samples from 63 of the participants. The exercise test was conducted between the first and the seventh day of the participant’s menstrual cycle.
Measurement of plasma levels of sFlt-1, unbound VEGF, and endostatin
Plasma levels of sFlt-1 and endostatin were measured using sFlt-1 and Endostatin ELISA kits (R&D Systems, Minneapolis, Minnesota, USA) according to the manufacturer’s protocol. Plasma levels of unbound VEGF were determined using a human ELISA kit with monoclonal anti-VEGF antibodies (R&D Systems) because Sandwich ELISA kits with monoclonal antibodies fail to detect the antigen if the epitopes are masked by soluble receptors, as is the case with VEGF bound to sFlt-1.
Where indicated, data are presented as mean±SE. Statistical significance was defined as a two-tailed Student’s t-test value of P less than 0.05. Paired Student’s t-test was used to compare the variables between two groups (before and after exercise, lean and overweight/obese, African American and White). The comparisons of the differences by obesity status and race were performed using analysis of variance and Dunnett’s t-test. Linear regression was performed for the correlation analysis between two continuous variables. All statistical calculations were performed using SPSS software (SPSS Inc., Chicago, Illinois, USA).
General characteristics and response to exercise
Of the 72 volunteers, 35 were African American and 37 were White. Average age, BMI, and VO2max determined during 30 min of exercise are summarized in Table 1. BMI less than 25 kg/m2 was considered lean. Overweight was defined as a BMI over 25 kg/m2 and obese as over 30 kg/m2. We found that in our population of women, 65% were overweight/obese (BMI: 29.1±0.82 kg/m2). The average BMI and the average age in years for the African American group and the White group were 28.5±1.21 kg/m2, 26.1±1.25 and 29.7±1.11 kg/m2, 22.1±0.84, respectively. There was no significant difference in the average BMI between the two groups (P<0.05). Submaximal VO2 (ml/kg/min) during 30 min of exercise averaged 31.4±1.20 ml/min/kg for the African American group and 27.1±1.18 ml/min/kg for the White group.
Effect of exercise on plasma levels of sFlt-1
Figure 1a shows significantly increased plasma sFlt-1 in adult women after a 30-min bout of moderate-intensity exercise walking on a treadmill. Plasma levels of sFlt-1 were 67.8±3.7 pg/ml immediately after 30 min of exercise, significantly higher than the basal levels, 54.5±3.3 pg/ml, before exercise (P<0.01; n=63). On the basis of the percent change of sFlt-1 in each individual before and after exercise, we calculated the average percent change to be 49%. There was no significant difference in the percent increase in sFlt-1 levels after exercise between African American and White women (P=0.563) or between lean and overweight/obese women (P=0.841).
Effect of exercise on plasma levels of unbound VEGF and endostatin
We also determined the effect of exercise on the plasma levels of free VEGF and endostatin. There was no change in plasma unbound VEGF (Fig. 1b) or endostatin (Fig. 1c) in young adult women after a 30-min bout of moderate-intensity exercise walking on a treadmill. The basal plasma levels of unbound VEGF (35.28±5.47 pg/ml) were similar to the plasma levels of unbound VEGF immediately after exercise (35.23±4.96 pg/ml; P=0.995). There was no significant difference in the plasma levels of endostatin before (111.12±5.48 ng/ml) and immediately after (115.45±7.15 ng/ml; P=0.632) exercise.
Comparison of basal plasma levels of unbound VEGF, sFlt-1, and endostatin in lean and overweight/obese young adult women
VEGF has been recognized as an adipokine (Gu et al., 2011). It is necessary to analyze whether increased plasma levels of VEGF are associated with increased BMI. Figure 2a shows that in our population of women aged 18–44 years, the average lean BMI was 21.95±0.47 kg/m2 and the average overweight/obese BMI was 32.83±0.79 kg/m2 (P<0.001). Figure 2b shows that overweight/obese women had a higher plasma level of unbound VEGF than lean women. Basal plasma levels of unbound VEGF in overweight/obese women were 52.26±9.6 pg/ml, significantly higher than the basal levels of unbound VEGF in lean women, 27.34±4.99 pg/ml (P<0.05).
We did not observe any significant changes in the basal plasma levels of sFlt-1 or endostatin between lean and overweight/obese young adult women. The basal plasma levels of sFlt-1 in lean women (48.37±4.66 pg/ml) compared with overweight/obese women (52.06±3.75 pg/ml) are shown in Fig. 2c (P=0.582). As shown in Fig. 2d, there was no significant difference in the basal plasma levels of endostatin in lean young adult women (120.81±9.66 ng/ml) compared with overweight/obese young adult women (109.77±9.04 ng/ml; P=0.471).
Correlation between basal levels of plasma sFlt-1, unbound VEGF, and endostatin versus age
Figure 3a shows a significant positive linear correlation between the basal levels of plasma sFlt-1 and age (R2=0.5326; P<0.001). We found no significant correlation between the basal levels of plasma unbound VEGF (R2=0.335; P<0.071) or endostatin (R2=0.025; P<0.898) with age, shown in Fig. 3b and c, respectively.
The present study has clearly shown that moderate exercise significantly increases the plasma sFlt-1 levels in young adult women. On the basis of the percent change of sFlt-1 in each individual before and after exercise, the percent increase in the sFlt-1 levels was 49% (n=63; P<0.01). These data support our hypothesis that exercise induces a circulating angiostatic factor, sFlt-1, in young adult women.
The VEGF family (including its ligands and receptors) are the prime molecular targets of proangiogenic and antiangiogenic therapies. sFlt-1 is an extracellular Ig-like domain of the VEGF receptor 1 that is released into the extracellular space and circulation, where it inhibits the activities of VEGF (Wu et al., 2010). The natural occurrence of sFlt-1 is predominantly from alternative splicing (Kendall and Thomas, 1993), but also from proteolytic cleavage of full-length VEGFR1 (Cai et al., 2006). There is growing interest in biomedical research to explore sFlt-1 as a disease marker (Bando et al., 2005; Jaroszewicz et al., 2008; Shapiro et al., 2008; Woolcok et al., 2008) and a therapeutic vector for inhibition of angiogenesis (Gehlbach et al., 2003; Mahendra et al., 2005; Kommareddy and Amiji, 2007). Some animal studies have shown that sFlt-1 gene therapy suppresses the growth of human ovarian carcinoma (Mahasreshti et al., 2001) and human breast adenocarcinoma (Kommareddy and Amiji, 2007). Evidence exists for a probable protective effect of physical activity on breast cancer, with a risk reduction ranging from 20 to 30% (Monninkhof et al., 2007; Friedenreich and Cust, 2008; Eliassen et al., 2010; Friedenreich et al., 2010). On the basis of the information presented above, we assume that increased plasma sFlt-1 is an important biomarker linking the protective effect of exercise on breast cancer. Currently, a clinical study in breast cancer patients to test this hypothesis is ongoing.
The major limitation of the present study of exercise in women is that only one blood sample was taken immediately after exercise. In our previous exercise study in men, we found: (a) significantly increased plasma sFlt-1 after 30 min of exercise; (b) significantly increased plasma endostatin after 2 h of exercise; and (c) significantly decreased plasma unbound VEGF after 2 h of exercise (Gu et al., 2004; Bailey et al., 2006). However, the present study shows that a 30-min bout of moderate exercise significantly increases plasma sFlt-1 levels in adult women and there is no change in plasma unbound VEGF or endostatin in adult women. In the future exercise study in women, we will take multiple blood samples after exercise, for example at 30 min and 2 h, to measure sFlt-1, free VEGF, and endostatin. We hypothesize that similar to our findings in men, exercise in women can induce a circulating angiostatic phenotype characterized by increased endostatin and sFlt-1, and decreased free VEGF.
The sFtl-1 is produced in the microvascular and macrovascular endothelial cells (Kendall and Thomas, 1993; Karumanchi and Bdolah, 2004), vascular smooth muscle cells (Sela et al., 2008), and activated peripheral blood mononuclear monocytes (Karumanchi and Bdolah, 2004), which exist throughout skeletal muscle tissues. It is therefore plausible that the release of sFlt-1 from skeletal muscle into the circulation might be because of exercise-dependent reductions in oxygen tension in the skeletal muscle. In future studies, we will determine whether sFlt-1 can be released directly from the exercised muscle.
The present study has also shown that the basal plasma level of unbound VEGF in overweight/obese young adult women was 52.26±9.6 pg/ml, significantly higher than the basal level of unbound VEGF in lean young adult women, 27.34±4.99 pg/ml (P<0.05). These results indicate that obesity has a proangiogenic phenotype characterized by increased plasma levels of VEGF. These data are consistent with our previous reports on increased plasma VEGF in MC4R−/− mice (Brandon et al., 2009) and postmenopausal obese mice (Gu et al., 2011), and some clinical studies of increased plasma levels of VEGF in obese men (Miyazawa-Hoshimoto et al., 2003; Seida et al., 2003; Gomez-Ambrosi et al., 2010). It is a significant finding that overweight/obese young adult women have a higher plasma level of unbound VEGF than lean young adult women because adipose tissue-derived VEGF promoted breast cancer progression in animal models (Gu et al., 2011). Further studies are needed.
Aging is associated with a decreased capacity for angiogenesis because of impairments in the function of some molecules such as VEGF, hypoxia-inducible factor-1α, and endothelial nitric oxide synthase (Revard et al., 1999; Hoenig et al., 2008). Decreased metabolic activity and sex hormones probably contribute toward impaired angiogenesis in the elderly. On analysis of the correlation between the basal levels of plasma sFlt-1 and age in women ranging in age from 18 to 44 years, we found that there was a significant positive linear correlation (R2=0.5326; P<0.001). These data suggest that younger individuals show a more angiogenic phenotype. However, further studies are needed to determine whether individuals older than 50 years of age have higher plasma levels of sFlt-1.
The measurement of plasma sFlt-1 levels before and after exercise in 63 African American and White adult women volunteers ranging in age from 18 to 44 years shows that moderate exercise significantly increases plasma sFlt-1 levels. The data support the hypothesis that exercise-induced plasma levels of sFlt-1 could be an important clinical biomarker to explore the mechanisms of exercise training in reducing progression of breast cancer. The present study also shows that in overweight/obese women, the increased levels of plasma unbound VEGF are linked to the relationship between obesity and progression of breast cancer. However, the conclusions are based on 62 participants, which is a small sample size. Further studies with more participants and different age groups are necessary. Identification of the biomarkers associated with the VEGF pathway could be an important strategy for the treatment and prevention of breast cancer, especially in obesity.
This work was supported by the National Institute on Alcohol Abuse and Alcoholism Grant AA-013821 and the National Heart, Lung and Blood Institute Grant HL-51971.
Conflicts of interest
There are no conflicts of interest.
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