Disruption of paraoxonase 3 impairs proliferation and antioxidant defenses in human A549 cells and causes embryonic lethality in mice.

We had shown previously that paraoxonase 3 (PON3), a putative circulating antioxidant, was systemically upregulated in late-gestation rat, sheep, and human fetuses. Our overarching hypothesis is that preterm human infants are delivered with low levels of PON3 and that this contributes to a state of oxidative stress. We sought to determine whether absence of Pon3 was associated with reduced neonatal viability in mice and studied the offspring from crosses between Pon3(+/-) mice. The number of Pon3(-/-) animals at E10.5 and E17.5 was significantly lower than the expected 25% (9.3 and 7.9% respectively, P < 0.001). On the first day of postnatal life, this was reduced further (2.4%, significantly less than the proportion in fetal life, P = 0.04). Pon3(+/-) animals had lower body and placental weights than wild-type littermates at E17.5, an effect that was independent of the parent of origin of the mutant allele. We then studied the effect of PON3 knockdown in a human cell line, A549. Stable knockdown of PON3 using short-hairpin RNA reduced cell proliferation in 21% oxygen. We then studied the effect of transient knockdown of PON3 using short interfering RNA (siRNA) in the same cell line in low (2%) or ambient (21%) oxygen. Knockdown of PON3 using siRNA reduced total antioxidant capacity in 21% (P = 0.008) but not 2% oxygen. We conclude that the absence of Pon3 in mice resulted in increased rates of early fetal and neonatal death. Knockdown of PON3 in human cells reduced cell proliferation and total antioxidant capacity.

Activation of mutated K-ras in donor endometrial epithelium and stroma promotes lesion growth in an intact immunocompetent murine model of endometriosis.

Endometriosis is a common chronic gynaecological condition, affecting 5-10% of women of child-bearing age. Its study has been hampered by lack of genetically tractable models. We transplanted steroid-manipulated, menstrual-like endometrium from K-ras(G12V/+) /Ah-Cre(+/+) /ROSA26R-LacZ(+/+) mice into gonad-intact immunocompetent wild-type mice. This led to endometriosis-like lesion development. Long-term lesion survival depended on the presence of the activated K-ras in the small proportion of the cells in the mature lesion that had undergone Cre-mediated K-ras activation. LacZ activity demonstrated Cre-mediated recombination in both endometrial epithelial cells and stromal cells, and transgenic K-ras expression was confirmed by RT-PCR. The endometriosis lesions developed without exogenous oestradiol supplementation and anti-oestrogen (fulvestrant, ICI 182780) treatment greatly suppressed their growth. Immunohistochemistry confirmed that as in human endometriosis, there was invasion and activation of fibroblasts, endothelial cells, and macrophages, with marked collagen deposition in the lesions. This model provides an opportunity to investigate endometriosis lesion establishment, growth, and regression in genetically tractable, immunocompetent, and hormonally intact mice. Furthermore, for the first time it provides a suitable model to test clinically validated driver genes in a faithful mouse model of the predisposing endometriotic lesion, thus providing the correct cellular context and microenvironment for ovarian clear cell carcinogenesis.

Localization and control of expression of VEGF-A and the VEGFR-2 receptor in fetal sheep intestines.

We studied expression of vascular endothelial growth factor A (VEGF-A) and its main receptor, VEGFR-2, in the small intestine from five groups of fetal sheep (each n = 5): 1) preterm controls, 2) term controls, 3) preterm animals where the fetus was infused with cortisol, or 4) saline, and 5) term animals where adrenalectomy had been performed preterm. The main transcript expressed in fetal small intestine was VEGF-A165. Comparing term with preterm animals, there were significantly higher levels of expression of VEGF-A protein (p = 0.005). Levels of VEGF-A protein expression were lower in term adrenalectomized animals (p = 0.01) and were higher in preterm animals infused with cortisol (p = 0.01), compared with their respective control groups. Immunohistochemistry demonstrated strongest expression of VEGF-A protein in the epithelial cells and lamina propria of the villi. Intestinal expression of mRNA encoding the VEGFR-2 receptor did not significantly vary with gestational age. In situ hybridization localized VEGFR-2 to the lamina propria of the villous core and receptor autoradiography using 125I VEGF-A demonstrated binding in the same site. These data show that intestinal VEGF-A is up-regulated with advancing gestation in a glucocorticoid-dependent manner--novel findings consistent with a role for VEGF-A stimulated angiogenesis in preparing the fetal gut for birth.

Vascular development is disrupted by endothelial cell-specific expression of the anti-apoptotic protein Bcl-2.

Endothelial cell (EC) apoptosis has been detected in remodelling blood vessels in vivo, and inhibition of EC apoptosis appears to alter vascular morphogenesis in vitro, suggesting that EC apoptosis may play a role in blood vessel remodelling. However, apoptotic EC are difficult to quantify in vivo, and studies of the incidence of EC apoptosis and the sites at which it occurs in vivo have produced contradictory results. Therefore, the specific biological roles played by EC apoptosis remain unclear. Here, we have used a transgenic approach to determine the biological function of EC apoptosis in vivo. Anti-apoptotic Bcl-2 transgenes were expressed in mice under control of the EC-specific tie2 promoter. These transgenic mice died during the second half of gestation. While the development and remodelling of large vessels including aortic arch arteries and great veins proceeded normally, abnormally dense and disorganised networks of small vessels were present in the skin and internal organs. In addition, vessel organisation and lumen formation were disrupted in the placental labyrinth. This study provides direct experimental evidence that endothelial cell apoptosis plays an essential role during embryogenesis. Our results suggest that EC apoptosis plays an important role in determining the structure of the microcirculation but may be dispensable for large vessel development.

Localization of the VEGF and angiopoietin genes in uterine carcinosarcoma.

OBJECTIVE: Carcinosarcoma of the uterus is a highly aggressive tumor. However, the angiogenesis in this tumor remains unclear. This is the first study to examine the characteristics of angiogenesis in this tumor at the molecular level while also comparing the findings with those of high-grade endometrial carcinoma. METHODS: The expression of vascular endothelial growth factors (VEGF) and angiopoietins (Ang) genes were examined in 35 primary uterine carcinosarcomas as well as in 12 high-grade endometrial carcinomas by in situ hybridization. RESULTS: A strong expression of VEGF-A mRNA was significantly seen in carcinosarcomas compared to high-grade endometrial carcinomas. Interestingly, in uterine carcinosarcoma, VEGF-A mRNA was more strongly expressed in the carcinoma cells than in the sarcoma cells. In addition, a decrease in the VEGF-A mRNA expression was found in the transitional areas between carcinomatous and sarcomatous elements in most carcinosarcomas evaluated. Moreover, the Ang-2 mRNA expression was significantly seen in the vasculature adjacent to the periphery of the carcinoma cells in most carcinosarcomas, in comparison to that of endometrial carcinomas. CONCLUSIONS: A high angiogenic activity in uterine carcinosarcoma was shown, in comparison to that of endometrial carcinoma. Tumor angiogenesis in uterine carcinosarcoma might be chiefly influenced by VEGF-A in the carcinoma cells, in cooperation with Ang-2 in the surrounding microvessels, however, this is not fully usually the case in sarcoma cells.

Expression of vascular endothelial growth factor (VEGF)-D and its receptor, VEGF receptor 3, as a prognostic factor in endometrial carcinoma.

PURPOSE: To evaluate the prognostic value of vascular endothelial growth factor (VEGF)-D and VEGF receptor (VEGFR)-3 in endometrial carcinoma. EXPERIMENTAL DESIGN: We assessed the levels of immunoreactivity for VEGF-D and VEGFR-3 in 71 endometrial carcinomas, 14 complex atypical endometrial hyperplasias, and 16 normal endometria by immunohistochemistry. RESULTS: VEGF-D was stained in both tumor cells and adjacent stromal cells. VEGFR-3 was stained in both tumor cells and adjacent endothelial cells. Immunoreactivity for VEGF-D in tumor cells and adjacent stromal cells became significantly stronger as lesions progressed from normal endometrium to advanced carcinoma. Similarly, immunoreactivity for VEGFR-3 in tumor cells and adjacent endothelial cells was significantly greater as lesions progressed from normal endometrium to advanced carcinoma. A strong correlation was found between high levels of VEGF-D immunoreactivity in carcinoma cells and VEGFR-3 in both carcinoma cells and adjacent endothelial cells. Similarly, high levels of VEGF-D immunoreactivity in stromal cells were significantly correlated with those of VEGFR-3 in both carcinoma cells and endothelial cells. High levels of VEGF-D in carcinoma cells and stromal cells, as well as those of VEGFR-3 in carcinoma cells and endothelial cells, were significantly related to myometrial invasion and lymph node metastasis. A strong correlation was found between poor survival and high levels of VEGF-D in both carcinoma cells and stromal cells and between poor survival and high levels of VEGFR-3 in carcinoma cells. Moreover, the high levels of VEGF-D in stromal cells and VEGFR-3 in carcinoma cells were independent prognostic factors in endometrial carcinoma. CONCLUSIONS: The presence of VEGF-D and VEGFR-3 in endometrial carcinoma may predict myometrial invasion and lymph node metastasis and may prospectively identify patients who are at increased risk for poor outcome. In addition, VEGF-D and VEGFR-3 may be promising targets for new therapeutic strategies in endometrial carcinoma.

Soluble vascular endothelial growth factor receptor 1 inhibits edema and epithelial proliferation induced by 17beta-estradiol in the mouse uterus.

The uterine response to 17beta-estradiol (E2) includes increased water retention, enhanced vascular permeability, DNA and RNA synthesis, and increased cellular mitosis. We have used the natural antagonist of vascular endothelial growth factor A (VEGF-A), sflt-1 (soluble form of flt-1), to determine whether the edematous and proliferative effects of E2 in the uterus are mediated by VEGF-A. Female BALB/c mice were ovariectomized and treated with E2 (10 micro g/kg) in the absence or presence of sflt-1 (0.8 and 4.0 mg/kg) for 24 h. E2 induced increases in uterine mass from 25.3 to 36.8 mg, in total cross-sectional uterine area from 771 to 1133 micro m(2), in cross-sectional endometrial area from 268 to 569 micro m(2), and in the mitotic index of lumenal epithelial cells from 0% to 53%. Antagonism with sflt-1 reduced the E2-induced increases in total uterine area to 779 micro m(2), endometrial area to 398 micro m(2) and the mitotic index of lumenal epithelial cells to 25%, but the E2-induced increase in uterine mass was not significantly reduced. From these data we conclude that the edematous response and proliferation of lumenal epithelial cells in the murine uterus are mediated in part through VEGF-A. These data suggest that sflt-1 could be a useful anti-VEGF-A agent and may be effective in modifying uterine biology.

VavCre transgenic mice: a tool for mutagenesis in hematopoietic and endothelial lineages.

Cre transgenic mice can be used to delete gene sequences flanked by loxP sites in specific somatic tissues. We have generated vavCre transgenic mice, which can be used to inactivate genes specifically in adult hematopoietic and endothelial cells. In these animals, a Cre transgene is expressed under control of murine vav gene regulatory elements. To assess their usefulness, vavCre transgenic mice were bred with R26R mice, which express a lacZ reporter gene only in cells where Cre-mediated recombination has occurred. VavCre/R26R double-heterozygous offspring were analyzed by beta-galactosidase histochemistry and flow cytometry. VavCre-mediated recombination occurred in most hematopoietic cells of all hematopoietic organs, including the hematopoietic progenitor-rich bone marrow. Recombination also occurred in most endothelial and germ cells, but only rarely in other cell types. The recombination in both hematopoietic and endothelial lineages may partly reflect their putative shared ontogeny and provides a unique tool for simultaneous pan-hematopoietic and endothelial mutagenesis.