Incidence of ovarian maldescent in women with mullerian duct anomalies: evaluation by MRI.

OBJECTIVE: The objective of our study was to evaluate the incidence of ovarian mal-descent in patients with and in those without müllerian duct anomalies. MATERIALS AND METHODS: Multiplanar MRI examinations of patients with (n = 65) and those without (n = 64) congenital uterine anomalies were evaluated for ovarian size, position, follicle count, and associated renal anomalies. Patients who were pregnant, had known prior pelvic surgery, or had large uterine leiomyomas were excluded. Two criteria were used to determine ovarian malposition: Was the upper pole of the ovary above the pelvic brim, as defined by the pubic symphysis-sacral promontory line, or was the upper pole of the ovary at or above the iliac artery bifurcation? RESULTS: The müllerian duct anomalies identified in the study group included hypoplasia, unicornuate, didelphys, bicornuate, and septate uterus. Ovarian maldescent was identified in 12 of 65 women with uterine anomalies (17%) as compared with two of 64 women with normal uterine anatomy (3%) using the criterion of the ovarian pole being above the iliac bifurcation. Among the women with müllerian duct anomalies, only three of 29 with septate uterus (10%) had ovarian maldescent compared with the remaining nine of 36 women with other anomalies (25%). Ovarian size did not vary significantly between the two groups. Follicle count was increased in women with müllerian duct anomalies. Renal anomalies were present in 16 of 65 patients, five of whom had concomitant ovarian maldescent. CONCLUSION: The incidence of ovarian maldescent is increased in patients with müllerian duct anomalies, with the highest association seen in those with didelphys, unicornuate, or bicornuate uterus.

In vitro liver tissue model established from transgenic mice: role of HIF-1alpha on hypoxic gene expression.

The instability of the hepatocyte phenotype in vitro has limited the ability to quantitatively investigate regulation of stress responses of the liver. Here, we adopt a tissue-engineering approach to form stable liver tissue in vitro by forming collagen "sandwich" cultures of transgenic murine hepatocytes harboring a regulatory gene of interest flanked by loxP sites. The floxed gene is excised in a subset of cultures by transfection with adenovirus carrying the gene for Cre-recombinase, thereby generating wild-type and null liver tissues from a single animal. In this study, we specifically investigated the role of hypoxia inducible factor 1 alpha (HIF-1alpha) in the hepatocellular response to hypoxia. Using high-density oligonucleotide arrays, we examined genome-wide gene expression after 8 h of hypoxia in wild-type and HIF- 1alpha null hepatocyte cultures. We identified more than 130 genes differentially expressed under hypoxia involved in metabolic adaptation, angiogenic signaling, immediate early response, and cell cycle regulation. Real-time polymerase chain reaction analysis verified that known hypoxia-responsive genes such as glucose transporter-1 and vascular endothelial growth factor were induced in a HIF-1alpha-dependent manner under hypoxia. Our results demonstrate the potential to integrate in vitro tissue models with transgenic and microarray technologies for the study of physiologic stress responses.

In vitro zonation and toxicity in a hepatocyte bioreactor.

The complex architecture of the liver is intertwined with its response to xenobiotic compounds. In particular, hepatocyte subpopulations are distributed along the sinusoid in zones 1 to 3, leading to prototypical "periportal" and "centrilobular" patterns of cell death in response to a toxic insult. In vitro models that more closely represent these zones of sub-specialization may therefore be valuable for the investigation of hepatic physiology and pathophysiology. We have established a perfused hepatocyte bioreactor that imposes physiologic oxygen gradients on co-cultures of rat hepatocytes and non-parenchymal cells, thereby producing an in vitro model of zonation. In order to predict and control oxygen gradients, oxygen transport in a parallel-plate bioreactor containing co-cultures was first mathematically modeled and experimentally validated. Co-cultures exposed to these physiologic oxygen gradients demonstrated regionally heterogeneity of CYP2B and CYP3A protein that mimics the distribution seen in the zonated liver. The distribution of CYP expression in the bioreactor was shown to vary with exposure to different chemical inducers and growth factors, providing a potential platform to study physiologic zonal responses. In order to explore zonal hepatotoxicity, bioreactors were perfused with APAP (acetominophen) for 24 h, resulting in maximal cell death at the low-oxygen outlet region similar to centrilobular necrotic patterns observed in vivo. This hepatocyte bioreactor system enables further in vitro investigation into zonation-dependent phenomena involving drug metabolism and toxicity.

Hypoxic inhibition of 3-methylcholanthrene-induced CYP1A1 expression is independent of HIF-1alpha.

Hypoxia-inducible factor-1alpha (HIF-1alpha) and aryl hydrocarbon receptor (AhR) both require dimerization with AhR nuclear translocator (ARNT) to initiate transcription of their respective target genes. It has been proposed that competition for ARNT results in decreased targeting of AhR to cytochrome P450 1A1 (CYP1A1) under hypoxia. We established primary cultures of HIF-1alpha null hepatocytes to examine the interaction between HIF-1alpha and AhR signaling. Gene expression of known HIF targets phosphoglycerate kinase (PGK), vascular endothelial growth factor (VEGF) and glucose transporter-1 (GLUT-1) increased under hypoxia, but was reduced in the HIF null cultures. Concomitant treatment of cultures with hypoxia (1% O2) and 3-methylcholanthrene (an AhR ligand) did not significantly alter HIF target gene expression. Furthermore, enzymatic activity and transcription of CYP1A1 was inhibited by hypoxia in HIF-1alpha null cultures, indicating that HIF-1alpha is not directly involved in negative regulation of AhR signaling.

Formation of steady-state oxygen gradients in vitro: application to liver zonation.

We have developed a perfusion bioreactor system that allows the formation of steady state oxygen gradients in cell culture. In this study, gradients were formed in cultures of rat hepatocytes to study the role of oxygen in modulating cellular functions. A model of oxygen transport in our flat-plate reactor was developed to estimate oxygen distribution at the cell surface. Experimental measurements of outlet oxygen concentration from various flow conditions were used to validate model predictions. We showed that cell viability was maintained over a 24-h period when operating with a physiologic oxygen gradient at the cell surface from 76 to 5 mmHg O(2) at the outlet. Oxygen gradients have been implicated in the maintenance of regional compartmentalized metabolic and detoxification functions in the liver, termed zonation. In this system, physiologic oxygen gradients in reactor cultures contributed to a heterogeneous distribution of phosphoenolpyruvate carboxykinase (predominantly localized upstream) and cytochrome p450 2B (predominantly localized downstream) that correlates with the distribution of these enzymes in vivo. The oxygen gradient chamber provides a means of probing the oxygen effects in vitro over a continuous range of O(2) tensions. In addition, this system serves as an in vitro model of zonation that could be further extended to study the role of gradients in ischemia-reperfusion injury, toxicity, and bioartificial liver design.

Engineering liver therapies for the future.

Treatment of liver disease has been greatly improved by the advent and evolution of liver transplantation. However, as demand for donor organs continues to increase beyond their availability, the need for alternative liver therapies is clear. Several approaches including extracorporeal devices, cell transplantation, and tissue-engineered constructs have been proposed as potential adjuncts or even replacements for transplantation. Simultaneously, experience from the liver biology community have provided valuable insight into tissue morphogenesis and in vitro stabilization of the hepatocyte phenotype. The next generation of cellular therapies must therefore consider incorporating cell sources and cellular microenvironments that provide both a large population of cells and strategies to maintain liver-specific functions over extended time frames. As cell-based therapies evolve, their success will require contribution from many diverse disciplines including regenerative medicine, developmental biology, and transplant medicine.

Improving the next generation of bioartificial liver devices.

Several extracorporeal bioartificial liver (BAL) devices are currently being evaluated as an alternative or adjunct therapy for liver disease. While these hybrid systems show promise, in order to become a clinical reality, BAL devices must clearly demonstrate efficacy in improving patient outcomes. Here, we present aspects of BAL devices that could benefit from fundamental advances in cell and developmental biology. In particular, we examine the development of human hepatocyte cell lines, strategies to stabilize the hepatocyte phenotype in vitro, and emphasize the importance of the cellular microenvironment in bioreactor design. Consideration of these key components of BAL systems will greatly improve next generation devices.