Trophoblast-Specific Expression of Hif-1α Results in Preeclampsia-Like Symptoms and Fetal Growth Restriction.

The placenta is an essential organ that is formed during pregnancy and its proper development is critical for embryonic survival. While several animal models have been shown to exhibit some of the pathological effects present in human preeclampsia, these models often do not represent the physiological aspects that have been identified. Hypoxia-inducible factor 1 alpha (Hif-1α) is a necessary component of the cellular oxygen-sensing machinery and has been implicated as a major regulator of trophoblast differentiation. Elevated levels of Hif-1α in the human placenta have been linked to the development of pregnancy-associated disorders, such as preeclampsia and fetal growth restriction. As oxygen regulation is a critical determinant for placentogenesis, we determined the effects of constitutively active Hif-1α, specifically in trophoblasts, on mouse placental development in vivo. Our research indicates that prolonged expression of trophoblast-specific Hif-1α leads to a significant decrease in fetal birth weight. In addition, we noted significant physiological alterations in placental differentiation that included reduced branching morphogenesis, alterations in maternal and fetal blood spaces, and failure to remodel the maternal spiral arteries. These placental alterations resulted in subsequent maternal hypertension with parturitional resolution and maternal kidney glomeruloendotheliosis with accompanying proteinuria, classic hallmarks of preeclampsia. Our findings identify Hif-1α as a critical molecular mediator of placental development and indicate that prolonged expression of Hif-1α, explicitly in placental trophoblasts causes maternal pathology and establishes a mouse model that significantly recapitulates the physiological and pathophysiological characteristics of preeclampsia with fetal growth restriction.

Important aspects of placental-specific gene transfer.

The placenta is a unique and highly complex organ that develops only during pregnancy and is essential for growth and survival of the developing fetus. The placenta provides the vital exchange of gases and wastes, the necessary nutrients for fetal development, acts as immune barrier that protects against maternal rejection, and produces numerous hormones and growth factors that promote fetal maturity to regulate pregnancy until parturition. Abnormal placental development is a major underlying cause of pregnancy-associated disorders that often result in preterm birth. Defects in placental stem cell propagation, growth, and differentiation are the major factors that affect embryonic and fetal well-being and dramatically increase the risk of pregnancy complications. Understanding the processes that regulate placentation is important in determining the underlying factors behind abnormal placental development. The ability to manipulate genes in a placenta-specific manner provides a unique tool to analyze development and eliminates potentially confounding results that can occur with traditional gene knockouts. Trophoblast stem cells and mouse embryos are not overly amenable to traditional gene transfer techniques. Most viral vectors, however, have a low infection rate and often lead to mosaic transgenesis. Although the traditional method of embryo transfer is intrauterine surgical implantation, the methodology reported here, combining lentiviral blastocyst infection and nonsurgical embryo transfer, leads to highly efficient and placental-specific gene transfer. Numerous advantages of our optimized procedures include increased investigator safety, a reduction in animal stress, rapid and noninvasive embryo transfer, and higher a rate of pregnancy and live birth.

Ovarian tumor initiating cell populations persist following paclitaxel and carboplatin chemotherapy treatment in vivo.

Development of recurrent platinum resistant disease following chemotherapy presents a challenge in managing ovarian cancer. Using tumors derived from genetically defined mouse ovarian cancer cells, we investigated the stem cell properties of residual cells post-chemotherapy. Utilizing CD133 and Sca-1 as markers of candidate tumor initiating cells (TIC), we determined that the relative levels of CD133+ and Sca-1+ cells were unaltered following chemotherapy. CD133+ and Sca-1+ cells exhibited increased stem cell-related gene expression, were enriched in G0/G1-early S phase and exhibited increased tumor initiating capacity, giving rise to heterogeneous tumors. Our findings suggest that residual TICs may contribute to recurrent disease.

Hypoxia activates constitutive luciferase reporter constructs.

Hypoxia has been identified as a contributing factor in the pathophysiology of several diseases and oxygen regulation is important during stem cell development, particularly in early embryogenesis. One aspect that has emerged is the role of hypoxia-inducible factors, or HIFs in regulating the effect of hypoxia. Studies in our laboratory sought to examine the hypoxic regulation of HIF activity in placental trophoblast cells, through the use of dual-reporter luciferase assays. Our study demonstrates that hypoxic conditions cause a significant increase in the level of constitutive luciferase reporter activity. We also show that this induction is not a cell type or species-specific phenomenon and provides an alternative method for normalizing transfection efficiency in luciferase assays under hypoxic conditions. Our results suggest that in studies dealing with hypoxic conditions, caution should be used when interpreting measurements of transcriptional activity by traditional dual-reporter assays.

Inhibition of Hedgehog signaling antagonizes serous ovarian cancer growth in a primary xenograft model.

BACKGROUND: Recent evidence links aberrant activation of Hedgehog (Hh) signaling with the pathogenesis of several cancers including medulloblastoma, basal cell, small cell lung, pancreatic, prostate and ovarian. This investigation was designed to determine if inhibition of this pathway could inhibit serous ovarian cancer growth. METHODOLOGY: We utilized an in vivo pre-clinical model of serous ovarian cancer to characterize the anti-tumor activity of Hh pathway inhibitors cyclopamine and a clinically applicable derivative, IPI-926. Primary human serous ovarian tumor tissue was used to generate tumor xenografts in mice that were subsequently treated with cyclopamine or IPI-926. PRINCIPAL FINDINGS: Both compounds demonstrated significant anti-tumor activity as single agents. When IPI-926 was used in combination with paclitaxel and carboplatinum (T/C), no synergistic effect was observed, though sustained treatment with IPI-926 after cessation of T/C continued to suppress tumor growth. Hh pathway activity was analyzed by RT-PCR to assess changes in Gli1 transcript levels. A single dose of IPI-926 inhibited mouse stromal Gli1 transcript levels at 24 hours with unchanged human intra-tumor Gli1 levels. Chronic IPI-926 therapy for 21 days, however, inhibited Hh signaling in both mouse stromal and human tumor cells. Expression data from the micro-dissected stroma in human serous ovarian tumors confirmed the presence of Gli1 transcript and a significant association between elevated Gli1 transcript levels and worsened survival. CONCLUSIONS/SIGNIFICANCE: IPI-926 treatment inhibits serous tumor growth suggesting the Hh signaling pathway contributes to the pathogenesis of ovarian cancer and may hold promise as a novel therapeutic target, especially in the maintenance setting.

BITC Sensitizes Pancreatic Adenocarcinomas to TRAIL-induced Apoptosis.

Pancreatic adenocarcinoma is an aggressive cancer with a greater than 95% mortality rate and short survival after diagnosis. Chemotherapeutic resistance hinders successful treatment. This resistance is often associated with mutations in codon 12 of the K-Ras gene (K-Ras 12), which is present in over 90% of all pancreatic adenocarcinomas. Codon 12 mutations maintain Ras in a constitutively active state leading to continuous cellular proliferation. Our study determined if TRAIL resistance in pancreatic adenocarcinomas with K-Ras 12 mutations could be overcome by first sensitizing the cells with Benzyl isothiocyanate (BITC). BITC is a component of cruciferous vegetables and a cell cycle inhibitor. BxPC3, MiaPaCa2 and Panc-1 human pancreatic adenocarcinoma cell lines were examined for TRAIL resistance. Our studies show BITC induced TRAIL sensitization by dual activation of both the extrinsic and intrinsic apoptotic pathways.

Hypoxia-inducible factor 1alpha (HIF1A) mediates distinct steps of rat trophoblast differentiation in gradient oxygen.

Defective differentiation of invasive, placental trophoblast cells has been associated with several pregnancy-related disorders. This study examines the molecular, functional, and morphological differentiation of lineage-specific, trophoblast giant cells under a gradient of oxygen concentrations. Low oxygen (3%) inhibited differentiation, but this inhibition was relieved in a stepwise fashion with increasing levels of oxygen. The oxygen-sensitive hypoxia-inducible factor 1alpha (HIF1A) is a major transcriptional regulator of the cellular response to low oxygen, and increased HIF1A protein levels and activity corresponded with the maintenance of the stem cell-like state and inhibition of trophoblast differentiation in low oxygen. Furthermore, constitutive expression of an oxygen-insensitive, active form of HIF1A protein mimicked the effects of low oxygen, inhibiting the differentiation of trophoblast giant cells. This study is the first to delineate the stepwise effects of oxygen on the activation of the trophoblast giant cell differentiation process and establishes a new paradigm from which to investigate trophoblast differentiation. In addition, this is the first reported study to demonstrate that constitutive HIF1A activity mediates oxygen's inhibition of differentiation. These results suggest that a dysregulation of HIF1A could contribute to impaired placental development.