S. K. Smith

Angiogenesis is important in the pathophysiology of endometriosis, a condition characterized by implantation of ectopic endometrium in the peritoneal cavity. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor involved in physiological and pathological angiogenesis, and elevated levels of VEGF are found in peritoneal fluid of patients with endometriosis. Our aim was to investigate the site of expression and regulation of VEGF in endometriosis. VEGF immunoreactivity was found in tissue macrophages present in ectopic endometrium and in activated peritoneal fluid macrophages. Macrophage activation was highest in women with endometriosis, and media conditioned by peritoneal fluid macrophages from these women caused a VEGF-dependent increase in endothelial cell proliferation above that seen from normal women. Peritoneal fluid macrophages secreted VEGF in response to ovarian steroids, and this secretion was enhanced after activation with lipopolysaccharide. Peritoneal fluid macrophages expressed receptors for steroid hormones. VEGF receptors flt and KDR (kinase domain receptor) were also detected, suggesting autocrine regulation. During the menstrual cycle, expression of flt was constant but that of KDR was increased in the luteal phase, at which time the cells migrated in response to VEGF. KDR expression and the migratory response were significantly higher in patients with endometriosis. This study demonstrates that activated macrophages are a major source of VEGF in endometriosis and that this expression is regulated directly by ovarian steroids.

BACKGROUND: Two thirds of patients with ovarian carcinoma have advanced disease at diagnosis and have poor prognoses because of the presence of highly invasive carcinoma cells and rapidly accumulating ascitic fluid. Vascular endothelial growth factor (VEGF), a potent mitogen of endothelial cells, is produced in elevated amounts by many tumors, including ovarian carcinomas. The known human receptors for VEGF, flt and KDR, are both cell surface tyrosine kinases and are expressed predominantly on endothelial cells. Acting through these receptors, VEGF may stimulate angiogenesis and promote tumor progression. PURPOSE: We aimed to clarify the function of VEGF in tumor development by identifying the cells in ovarian carcinoma tissue that express VEGF and its receptors. METHODS: VEGF, flt, and KDR expression was localized by in situ hybridization and immunohistochemistry in frozen sections of primary tumors from five patients with ovarian carcinoma and from metastases of ovarian carcinoma from three different patients. Reverse transcription followed by polymerase chain reaction (RT-PCR) and an enzyme-linked immunosorbent assay were used to analyze VEGF, flt, and KDR expression in six epithelial cell lines derived from ovarian carcinoma ascites from five additional patients. RESULTS: Messenger RNAs (mRNAs) encoding VEGF, flt, and KDR were detected in primary ascitic cells and in three of four ovarian carcinoma cell lines examined by RT-PCR. Two novel complementary DNAs that may encode truncated, soluble forms of flt were cloned from one primary source. VEGF levels of 20-120 pM were found in culture media conditioned by the cell lines. Elevated expression of VEGF mRNA was found in all primary tumors and metastases, especially at the margins of tumor acini. VEGF immunoreactivity was concentrated in clusters of tumor cells and patches of stromal matrix. flt immunoreactivity was confined to tumor blood vessels, but flt mRNA was not detected by in situ hybridization. In contrast, KDR mRNA was detected not only in vascular endothelial cells but also in tumor cells at primary malignant sites. CONCLUSIONS: VEGF is expressed by tumor cells in primary and metastatic ovarian carcinoma and accumulates in the stromal matrix. Its receptors, flt and KDR, are expressed by some tumor cells that coexpress VEGF. This is the first localization of KDR expression in nonendothelial cells. IMPLICATIONS: Coexpression of VEGF and KDR by tumor cells in ovarian carcinoma raises the possibility of autocrine stimulation and of therapeutic strategies targeting this receptor-ligand interaction.

Repair of human endometrium after menstruation and preparation of the endometrium for implantation involves profound angiogenic changes. Vascular endothelial cell growth factor (VEGF) is a recently identified growth factor with significant angiogenic properties. Four species of mRNA encoding VEGFs were identified in human endometrium and myometrium. All species were present throughout the menstrual cycle. Two species, VEGF165 and VEGF121, were present in peripheral leukocytes, indicating tissue-specific splicing of the two other VEGF transcripts. In situ hybridization of mRNA encoding VEGF was not restricted to vascular smooth muscle but was present in epithelial and stromal cells of endometrium throughout the cycle, and the distribution changed during the course of the cycle. All four species of VEGF were expressed by the endometrial carcinoma cell lines Ishikawa, HEC 1-A, and HEC 1-B. Estradiol increased steady-state levels of mRNA encoding VEGF in a dose- and time-dependent manner in HEC 1-A cells. Conditioned medium from these cells possessed angiogenic activity that was depleted by passage through a heparin affinity column. None of the cell lines demonstrated mRNA for acidic or basic fibroblast growth factor (FGF), despite previous reports of the identification of immunoreactive basic FGF in HEC 1-A and HEC 1-B cells. These findings show that VEGFs, not FGFs, are the principal angiogenic growth factors secreted by these cells and that human endometrium expresses a secreted angiogenic growth factor whose site of expression changes during the menstrual cycle.

Vascular endothelial growth factor (VEGF) is a potent secreted factor that promotes angiogenesis and maintains the integrity of the endothelium. Levels of VEGF are increased in many tumors and are elevated in women with pre-eclampsia, a serious disease of pregnancy. Here we show by in situ hybridization that the trophoblast contains the mRNA encoding a soluble version of the VEGF receptor known as Flt-1 (sFlt-1: initially described by Kendall and Thomas, PNAS 90:10705-10709). Binding assays and Western blotting of villus-conditioned media confirmed the production of sFlt-1. Serum from pregnant women was found to contain a VEGF-binding protein that was not present in serum from men or nonpregnant women. As determined by heparin affinity, column fractionation, and cross-linking, this protein was identical to sFlt-1. Taken together, these results show that the placenta secretes sFlt-1, which would be expected to be a VEGF antagonist. This is the first report of production of the sFlt-1 receptor in vivo, and it reveals a new mechanism for naturally regulating this potent angiogenic agent. The presence of such an antagonist suggests that regulation of VEGF action is essential to successful pregnancy. This has important implications for the activity of VEGF locally and systemically in other conditions.