**3.3 Angiogenesis**

The establishment of new blood vessels is essential in growth and survival of endometriosis. Increased angiogenic activity has been demonstrated in peritoneal fluid of women with endometriosis and strong expression of angiogenic factors has been shown in active lesions (Donnez *et al.*, 1998; Nisolle *et al.*, 1993). Moreover, inhibition of endometrial implants by anti-angiogenic agents or VEGF receptors (VEGFR) blocker was observed in animal studies (Dabrosin *et al.*, 2002; Nap *et al.*, 2004). Many anti-angiogenic compounds are studied extensively in animal models of endometriosis. Vlahos stated that pentoxifylline used in the treatment of peripheral vascular disease for many years may cause suppression of endometriotic tissue by inhibiting angiogenesis through VEGF-C and VEGFR-2 expression in rat model (Vlahos *et al.*). Besides, progestins already used in the treatment of endometriosis inhibit human ectopic endometrial lesions in a mouse model by regulating cysteine-rich angiogenic inducer (CYR61), basic fibroblast growth factor (bFGF) and VEGFA (Monckedieck *et al.*, 2009). Endostain, a potent endogenous inhibitor of blood vessel growth, suppress angiogenesis by inhibiting endothelial migration without effecting normal estrous cycles (Becker *et al.*, 2006). What's more, either selective cyclooxygenase-2 (COX-2) inhibitor or immunoconjugate molecule (Icon) suppress angiogenesis in animal models by microvessels density assessment (Krikun *et al.*). However, there is no study to investigate the early process of angiogenesis in endometriosis, which makes new anti-angiogenesis therapy possible for prevention of reoccurrence after surgical treatment.

Pathophysiological Changes in Early Endometriosis 467

In order to monitor the oxidative stress response in early development of endometriosis, an experimental endometriosis model in C57 mice was established by subcutaneous injection of mouse endometrium fragments. A chemiluminescent probe, L-012 (25mg/kg s.c.), was injected to the mice for the noninvasive *in vivo* oxidative stress imaging. L-012 is a new luminol derivative and sensitive chemiluminescence probe reacting with various types of ROS. ROS and reactive nitrogen species (RNS) production in the transplanted lesion can be monitored longitudinally by Xenogen IVIS 200 Imaging System. The results showed that *In vivo* imaging demonstrated significant increased bioluminescence signals for ROS/RNS from the transplanted lesions at the first hour interval. The signal reached a peak after 4 hours of transplantation. Then, the signal gradually decreased and maintained at minimal intensity in the rest of experiment. Immunohistochemistry showed positive lag correlation for the stained Hypoxia-inducible factors (HIF-1) in glandular epithelial cells and stromal tissue from the isolated lesions across the later time after transplantation. For angiogenesis, CD34, VEGF and Von Willebrand factor (vWF) signals were increased in parallel with HIF expression at 1 week thereafter. The non-invasive *in vivo* imaging method provides a valuable tool for monitoring oxidative stress in endometriosis and to understand its role in the early development and growth of endometriosis. The study indicated oxidative stress preceded HIF activation and VEGF angiogenesis in the pathogenesis of early endometriosis.

Both donor and recipient BALB/c mice at 7 weeks old were subjected to ovariectomy (OVX) and then were supplemented with 100ug/kg estradiol. Uterine horns from the donor mice were removed into F12 medium. Endometrium was punched into endometrial fragments after peeling off the serosa and myometrium under microscope. Fragments suspended in 0.3ml PBS were injected into peritoneal cavity of recipient mice. Peritoneal fluid was collected at experiment time intervals after transplantation. Cytokines profiles in peritoneal fluid were detected simultaneously. Differentially expressed cytokines were confirmed by

The results showed that the levels of CD30, CD36/SR-B3, Dickkopf-related protein (Dkk-1), epidermal growth factor (EGF), Eotaxin, IL-1 receptor antagonist (IL-1ra), IL-6 and Vascular cell adhesion protein 1 (VCAM-1) were significantly increased with the first hour of transplantation. This is the first report to analyze the peritoneal fluid cytokines profiles in experimental endometriosis in mice. The change pattern of cytokines could provide insights in understanding the early development of endometriosis. From the results, we can see that the oxidative stress and abnormal cytokine profiles might contribute to the early

Mice were randomly treated with epigallocatchin-3-gallate (EGCG) extracted from green tea, Vitamin E (antioxidant controls) or vehicle (negative controls) for *in vivo* and *in vitro*  microvessel imaging at the end of intervention. Microvascular networks in the endometriotic lesions *in vivo* were imaged by Cellvizio LAB LSU-488 with ProFlex Microprobe S1500. Microvessel length and area were measured using Cellvizio LAB Vessel Detection software and averaged from 4 perpendicular regions of the lesion in replicate.

**4.1 Oxidative stress in early endometriosis** 

**4.2 Cytokine profiling in early endometriosis** 

ELISA quantification.

development of endometriosis.

**4.3 Angiogenesis in early endometriosis** 
