Does Resveratrol Inhibit Vascularization in Endometriosis?
Does Resveratrol Inhibit Vascularization in Endometriosis?
Resveratrol represents one of the most frequently analyzed phytochemical compounds in life sciences during the last decades. Because resveratrol exerts a broad spectrum of beneficial effects under various pathological conditions, it has been suggested as a promising therapeutic agent for the treatment of cancer as well as several inflammatory, metabolic and cardiovascular diseases (Beaudeux et al., 2010; Petrovski et al., 2011; Aluyen et al., 2012). Recently, Bruner-Tran et al. (2011) reported for the first time that resveratrol may also be suitable for the prevention and treatment of endometriosis by inhibiting the establishment and growth of endometriotic lesions. Herein, we now provide the novel finding that the anti-angiogenic activity of resveratrol crucially contributes to this observation.
Animals were orally treated with 40 mg/kg resveratrol over 4 weeks, because this dose and route of application has previously been shown to effectively suppress the formation of new blood vessels in tumors (Tseng et al., 2004; Harikumar et al., 2010). In this context, it should be noted that resveratrol acts as a typical pleiotropic agent, which targets several key steps of the angiogenic process (Chen and Tseng, 2007). In fact, resveratrol has been shown to inhibit hypoxia-mediated activation of Erk1/2 and Akt, resulting in decreased expression of hypoxia-inducible factor-1α and vascular endothelial growth factor (Cao et al., 2004; Tseng et al., 2004; Zhang et al., 2005). Moreover, resveratrol reduces the activity of matrix metalloproteinase-2 and -9 (Ganapathy et al., 2010; Kaneko et al., 2011), which are both involved in extracellular matrix disruption in the early angiogenic phase of vascular bud and sprout formation (Carmeliet, 2000). In addition, resveratrol directly inhibits the proliferation and migration of endothelial cells and vascular smooth muscle cells (Hu et al., 2007; Lee et al., 2009). Accordingly, we found in the present study that resveratrol-treated endometriotic lesions exhibited a reduced microvessel density when compared with vehicle-treated controls, independently of their localization at the peritoneal wall or in the intestinal mesentery. More detailed immunohistochemical analyses further revealed that this was caused by a decreased proliferating activity of endothelial cells in the newly developing microvasculature of the lesions.
For the non-invasive evaluation of peritoneal endometriotic lesions, we used the technique of high-resolution ultrasound imaging. Using this approach, it is possible to distinguish between the proliferation of stromal tissue and the formation of endometrial cysts as possible causes for increasing lesion volumes over time (Laschke et al., 2010). We could demonstrate that resveratrol treatment reduced the secretory activity of endometrial cysts, resulting in decreased cyst volumes when compared with controls. Furthermore, resveratrol treatment induced a regression of the stromal tissue, which was confirmed by additional histomorphometric analyses. The latter observation may be attributed to an up-regulation of apoptotic cell death, as previously reported by Bruner-Tran et al. (2011), studying the growth of ectopic human endometrial tissue in nude mice. However, unexpectedly we could not detect any caspase-3-positive apoptotic cells inside our endometriotic lesions. These discrepant results may be due to the different tissue types that were transplanted in these studies for the induction of endometriotic lesions. In fact, in the present study endometriotic lesions were surgically induced by transplantation of uterine tissue from donor mice into the peritoneal cavity of recipient animals without the use of endometrium or pathological endometriotic tissue of human nature. Because there may be marked differences between human and mouse tissue, the results obtained in our mouse model may thus not fully correlate to human patients with endometriosis. On the other hand, it is well known that there is a biphasic dose-dependent effect of resveratrol on apoptosis (Mukherjee et al., 2010). Because we treated mice with a resveratrol dose, which was ~8-fold lower than that used by Bruner-Tran et al. (2011), it may thus be speculated that the resveratrol concentrations achieved in our study were not sufficient to induce programmed cell death.
In addition, we analyzed the proliferative activity of stromal and glandular cells. By this, we found that resveratrol treatment suppressed the proliferation of both cell types in peritoneal and mesenteric lesions. However, our data from the analyses of PCNA- and Ki67-stained tissue sections did not correlate well. Numbers of Ki67-positive cells were in general much lower in endometriotic lesions than those of PCNA-positive cells. Moreover, marked differences in proliferating activity of PCNA-stained lesions were found in stromal cells, whereas Ki67 expression mainly differed in the glandular cells. This may be explained by a prolonged half-life of PCNA or by the finding that growth factors are capable of stabilizing PCNA mRNA, resulting in the accumulation of the protein also in non-proliferating cells (Hall et al., 1990; Kordek et al., 1996). Besides, PCNA has also been shown to be expressed in cells with increased DNA repair activity (Gramantieri et al., 2003). Nonetheless, despite these discrepancies both markers indicated an inhibitory effect of resveratrol on the ectopic endometrial tissue.
In summary, we could demonstrate that resveratrol suppresses the development of new microvessels in endometriotic lesions by inhibiting endothelial cell proliferation. This is associated with a decreased lesion size, further supporting the concept that the establishment and progression of endometriosis is crucially dependent on angiogenesis (Feng et al., 2012). Importantly, we used in our study a resveratrol dose that is in the dose range of clinical trials (Scott et al., 2012; Smoliga et al., 2011). Accordingly, we are optimistic that the observed beneficial effects of resveratrol may be also reproducible under clinical conditions. Further studies have to clarify now whether this succeeds without exerting severe side effects in endometriosis patients.
Discussion
Resveratrol represents one of the most frequently analyzed phytochemical compounds in life sciences during the last decades. Because resveratrol exerts a broad spectrum of beneficial effects under various pathological conditions, it has been suggested as a promising therapeutic agent for the treatment of cancer as well as several inflammatory, metabolic and cardiovascular diseases (Beaudeux et al., 2010; Petrovski et al., 2011; Aluyen et al., 2012). Recently, Bruner-Tran et al. (2011) reported for the first time that resveratrol may also be suitable for the prevention and treatment of endometriosis by inhibiting the establishment and growth of endometriotic lesions. Herein, we now provide the novel finding that the anti-angiogenic activity of resveratrol crucially contributes to this observation.
Animals were orally treated with 40 mg/kg resveratrol over 4 weeks, because this dose and route of application has previously been shown to effectively suppress the formation of new blood vessels in tumors (Tseng et al., 2004; Harikumar et al., 2010). In this context, it should be noted that resveratrol acts as a typical pleiotropic agent, which targets several key steps of the angiogenic process (Chen and Tseng, 2007). In fact, resveratrol has been shown to inhibit hypoxia-mediated activation of Erk1/2 and Akt, resulting in decreased expression of hypoxia-inducible factor-1α and vascular endothelial growth factor (Cao et al., 2004; Tseng et al., 2004; Zhang et al., 2005). Moreover, resveratrol reduces the activity of matrix metalloproteinase-2 and -9 (Ganapathy et al., 2010; Kaneko et al., 2011), which are both involved in extracellular matrix disruption in the early angiogenic phase of vascular bud and sprout formation (Carmeliet, 2000). In addition, resveratrol directly inhibits the proliferation and migration of endothelial cells and vascular smooth muscle cells (Hu et al., 2007; Lee et al., 2009). Accordingly, we found in the present study that resveratrol-treated endometriotic lesions exhibited a reduced microvessel density when compared with vehicle-treated controls, independently of their localization at the peritoneal wall or in the intestinal mesentery. More detailed immunohistochemical analyses further revealed that this was caused by a decreased proliferating activity of endothelial cells in the newly developing microvasculature of the lesions.
For the non-invasive evaluation of peritoneal endometriotic lesions, we used the technique of high-resolution ultrasound imaging. Using this approach, it is possible to distinguish between the proliferation of stromal tissue and the formation of endometrial cysts as possible causes for increasing lesion volumes over time (Laschke et al., 2010). We could demonstrate that resveratrol treatment reduced the secretory activity of endometrial cysts, resulting in decreased cyst volumes when compared with controls. Furthermore, resveratrol treatment induced a regression of the stromal tissue, which was confirmed by additional histomorphometric analyses. The latter observation may be attributed to an up-regulation of apoptotic cell death, as previously reported by Bruner-Tran et al. (2011), studying the growth of ectopic human endometrial tissue in nude mice. However, unexpectedly we could not detect any caspase-3-positive apoptotic cells inside our endometriotic lesions. These discrepant results may be due to the different tissue types that were transplanted in these studies for the induction of endometriotic lesions. In fact, in the present study endometriotic lesions were surgically induced by transplantation of uterine tissue from donor mice into the peritoneal cavity of recipient animals without the use of endometrium or pathological endometriotic tissue of human nature. Because there may be marked differences between human and mouse tissue, the results obtained in our mouse model may thus not fully correlate to human patients with endometriosis. On the other hand, it is well known that there is a biphasic dose-dependent effect of resveratrol on apoptosis (Mukherjee et al., 2010). Because we treated mice with a resveratrol dose, which was ~8-fold lower than that used by Bruner-Tran et al. (2011), it may thus be speculated that the resveratrol concentrations achieved in our study were not sufficient to induce programmed cell death.
In addition, we analyzed the proliferative activity of stromal and glandular cells. By this, we found that resveratrol treatment suppressed the proliferation of both cell types in peritoneal and mesenteric lesions. However, our data from the analyses of PCNA- and Ki67-stained tissue sections did not correlate well. Numbers of Ki67-positive cells were in general much lower in endometriotic lesions than those of PCNA-positive cells. Moreover, marked differences in proliferating activity of PCNA-stained lesions were found in stromal cells, whereas Ki67 expression mainly differed in the glandular cells. This may be explained by a prolonged half-life of PCNA or by the finding that growth factors are capable of stabilizing PCNA mRNA, resulting in the accumulation of the protein also in non-proliferating cells (Hall et al., 1990; Kordek et al., 1996). Besides, PCNA has also been shown to be expressed in cells with increased DNA repair activity (Gramantieri et al., 2003). Nonetheless, despite these discrepancies both markers indicated an inhibitory effect of resveratrol on the ectopic endometrial tissue.
In summary, we could demonstrate that resveratrol suppresses the development of new microvessels in endometriotic lesions by inhibiting endothelial cell proliferation. This is associated with a decreased lesion size, further supporting the concept that the establishment and progression of endometriosis is crucially dependent on angiogenesis (Feng et al., 2012). Importantly, we used in our study a resveratrol dose that is in the dose range of clinical trials (Scott et al., 2012; Smoliga et al., 2011). Accordingly, we are optimistic that the observed beneficial effects of resveratrol may be also reproducible under clinical conditions. Further studies have to clarify now whether this succeeds without exerting severe side effects in endometriosis patients.
