These results are the first to demonstrate that AT1-AA level is significantly elevated in EOC patients. Enhanced AT1-AA titer was associated with advanced stage and grade of the EOC and positively correlated with VEGF level in patients. Using cultured OVCAR3 cells and the CAM of chick embryo, we found that AT1-AA has direct effect on cell migration and angiogenesis through activating Ang II AT1 receptor.
AT1-AA, an autoantibody against angiotensin II type 1 receptor, which is characterized to activate the receptor via specifically interacting with the second extracellular loop of the Ang II AT1 receptor, has been documented to play a role in the pathogenesis of preeclampsia and hypertension
[10, 11, 13–15]. However, AT1-AA level and function has not been examined or identified in the ovarian cancer. In the current study, we found that serum titer and positive rate of AT1-AA were significantly increased in EOC patients. More importantly, this study revealed that the level of AT1-AA is significantly elevated with an advanced FGIO stage and grade in EOC patients, supporting the concept that AT1-AA may participate in ovarian cancer development and progression. As it has well been demonstrated, the FIGO stage and grade are poor prognostic factors for overall survival in EOC patients
. Therefore, monitoring serum AT1-AA level might be of great value as a single marker in detecting all stages of EOC patients for clinical screening test, diagnosis and prognosis after therapeutic intervention.
VEGF is a main angiogenic factor in development of ovarian cancer through promoting angiogenesis and significantly associated with tumor progression and poor prognosis
[16–18]. Recent studies have shown that targeting inhibition of tumor angiogenesis through VEGF and its various signaling pathways is an effective therapy to suppress tumor growth and progression
[8, 17]. Our results showed that higher AT1-AA titer is positively correlated with VEGF level in advanced stages of EOC patients, consistent with previous findings showing a role of Ang II in cancer development through VEGF gene expression and secretion
Stimulation of AT1 receptor by Ang II has been reported to be involved in tumor progression in a number of cancers including EOC
[12, 13]. The postulated role of AT1-AA in cell migration and tumor spread led us to test if AT1-AA has direct stimulating effect on ovarian cell migration. We selected either autoantibody-neutralizing AT1-AA peptide, AT1R-ECII as an inhibitor or selective AT1 receptor antagonist, losartan to test the direct effect of AT1-AA on cell migration and illustrate if this process is mediated by AT1 receptor
. We found that the migratory number of OVCAR3 cells was significantly increased in AT1-AA treated group, which was blocked either by AT1R-ECII or losartan. These data suggested that AT1-AA has direct effect on migration of ovarian cancer cells through activating AT1 receptor, consistent with a previous report showing that Ang II-induced tumor cell invasion, angiogenesis and peritoneal dissemination are blocked by Ang II AT1-receptor antagonist
. However, mechanistic studies are needed to further elucidate how AT1-AA activates the Ang II AT1 receptor. In line with our data, it has previously postulated that AT1-AA may alter the structural conformation of Ang II AT1 receptor so that the receptor’s ability binding to circulating Ang II is enhanced
The CAM of chick embryo has widely been selected to study the morphological aspects of tumor angiogenesis and metastasis
. We chose the CAM of chick embryo as a test model to demonstrate angiogenic substances in our study because of its extensive vascularization and easy accessibility to investigate mechanisms of action of proangiogenic and antiangiogenic molecules
. We found that addition of AT1-AA at the same dose that causes OVCAR3 cell migration is effective in stimulating angiogenesis in the CAM, which was parallel with data showing elevation of VEGF in EOC patients. This increased microvascular density elicited by AT1-AA was comparable to the level as that in the Ang II group. Furthermore, we showed that the use of AT1R-ECII or AT1 receptor blocker, losartan completely inhibits AT1-AA-induced angiogenesis of the CAM. These findings suggest that an enhancement of angiogenesis by AT1-AA involves activation of Ang II AT1 receptor, thus selective Ang II AT1 blockade therapy could efficiently inhibit the AT1-AA-elicited angiogenesis under conditions exposed to AT1-AA as it has previously been reported
There are several limitations to this study that need to be mentioned. First, although in vitro studies speculated the mechanisms responsible for the migration of cancer cells and angiogenesis through AT1 receptor, this study did not measure AT1 receptor expression to show whether such a change is associated with AT1-AA-mediated effects. Second, although a raised titer of AT1-AA was detected in EOC patients, the “cause-effect” relationship remains to be investigated. In this regard, it will be interesting to determine whether the AT1-AA titer falls in patients undergoing treatment. Third, the size of the study population was relatively small and limited only in the Asian patients. Therefore, future large-scale clinical trials will be necessary to further determine whether AT1-AA titer is also altered in EOC patients of different ethnicities.