Polyamine Pathway Inhibitor DENSPM Suppresses Lipid Metabolism in Pheochromocytoma Cell Line.

Pheochromocytomas (PCCs) are tumors arising from chromaffin cells in the adrenal medulla, and paragangliomas (PGLs) are tumors derived from extra-adrenal sympathetic or parasympathetic paraganglia; these tumors are collectively referred to as PPGL cancer. Treatment for PPGL primarily involves surgical removal of the tumor, and only limited options are available for treatment of the disease once it becomes metastatic. Human carriers of the heterozygous mutations in the succinate dehydrogenase subunit B (SDHB) gene are susceptible to the development of PPGL. A physiologically relevant PCC patient-derived cell line hPheo1 was developed, and SDHB_KD cells carrying a stable short hairpin knockdown of SDHB were derived from it. An untargeted metabolomic approach uncovered an overactive polyamine pathway in the SDHB_KD cells that was subsequently fully validated in a large set of human SDHB-mutant PPGL tumor samples. We previously reported that treatment with the polyamine metabolism inhibitor N1,N11-diethylnorspermine (DENSPM) drastically inhibited growth of these PCC-derived cells in culture as well as in xenograft mouse models. Here we explored the mechanisms underlying DENSPM action in hPheo1 and SDHB_KD cells. Specifically, by performing an RNAseq analysis, we have identified gene expression changes associated with DENSPM treatment that broadly interfere with all aspects of lipid metabolism, including fatty acid (FA) synthesis, desaturation, and import/uptake. Furthermore, by performing an untargeted lipidomic liquid chromatography-mass spectrometry (LC/MS)-based analysis we uncovered specific groups of lipids that are dramatically reduced as a result of DENSPM treatment. Specifically, the bulk of plasmanyl ether lipid species that have been recently reported as the major determinants of cancer cell fate are notably decreased. In summary, this work suggests an intersection between active polyamine and lipid pathways in PCC cells.

Genetic impairment of succinate metabolism disrupts bioenergetic sensing in adrenal neuroendocrine cancer.

Metabolic dysfunction mutations can impair energy sensing and cause cancer. Loss of function of the mitochondrial tricarboxylic acid (TCA) cycle enzyme subunit succinate dehydrogenase B (SDHB) results in various forms of cancer typified by pheochromocytoma (PC). Here we delineate a signaling cascade where the loss of SDHB induces the Warburg effect, triggers dysregulation of [Ca2+]i, and aberrantly activates calpain and protein kinase Cdk5, through conversion of its cofactor from p35 to p25. Consequently, aberrant Cdk5 initiates a phospho-signaling cascade where GSK3 inhibition inactivates energy sensing by AMP kinase through dephosphorylation of the AMP kinase γ subunit, PRKAG2. Overexpression of p25-GFP in mouse adrenal chromaffin cells also elicits this phosphorylation signaling and causes PC. A potent Cdk5 inhibitor, MRT3-007, reverses this phospho-cascade, invoking a senescence-like phenotype. This therapeutic approach halted tumor progression in vivo. Thus, we reveal an important mechanistic feature of metabolic sensing and demonstrate that its dysregulation underlies tumor progression in PC and likely other cancers.

Nicotine Exposure during Rodent Pregnancy Alters the Composition of Maternal Gut Microbiota and Abundance of Maternal and Amniotic Short Chain Fatty Acids.

Tobacco smoking is the leading cause of preventable death. Numerous reports link smoking in pregnancy with serious adverse outcomes, such as miscarriage, stillbirth, prematurity, low birth weight, perinatal morbidity, and infant mortality. Corollaries of consuming nicotine in pregnancy, separate from smoking, are less explored, and the mechanisms of nicotine action on maternal-fetal communication are poorly understood. This study examined alterations in the maternal gut microbiome in response to nicotine exposure during pregnancy. We report that changes in the maternal gut microbiota milieu are an important intermediary that may mediate the prenatal nicotine exposure effects, affect gene expression, and alter fetal exposure to circulating short-chain fatty acids (SCFAs) and leptin during in utero development.

Development of Human Adrenocortical Adenoma (HAA1) Cell Line from Zona Reticularis.

The human adrenal cortex is composed of distinct zones that are the main source of steroid hormone production. The mechanism of adrenocortical cell differentiation into several functionally organized populations with distinctive identities remains poorly understood. Human adrenal disease has been difficult to study, in part due to the absence of cultured cell lines that faithfully represent adrenal cell precursors in the early stages of transformation. Here, Human Adrenocortical Adenoma (HAA1) cell line derived from a patient's macronodular adrenocortical hyperplasia and was treated with histone deacetylase inhibitors (HDACis) and gene expression was examined. We describe a patient-derived HAA1 cell line derived from the zona reticularis, the innermost zone of the adrenal cortex. The HAA1 cell line is unique in its ability to exit a latent state and respond with steroidogenic gene expression upon treatment with histone deacetylase inhibitors. The gene expression pattern of differentiated HAA1 cells partially recreates the roster of genes in the adrenal layer that they have been derived from. Gene ontology analysis of whole genome RNA-seq corroborated increased expression of steroidogenic genes upon HDAC inhibition. Surprisingly, HDACi treatment induced broad activation of the Tumor Necrosis Factor (TNF) alpha pathway. This novel cell line we developed will hopefully be instrumental in understanding the molecular and biochemical mechanisms controlling adrenocortical differentiation and steroidogenesis.

Targeting pheochromocytoma/paraganglioma with polyamine inhibitors.

BACKGROUND: Pheochromocytomas (PCCs) and paragangliomas (PGLs) are neuroendocrine tumors that are mostly benign. Metastatic disease does occur in about 10% of cases of PCC and up to 25% of PGL, and for these patients no effective therapies are available. Patients with mutations in the succinate dehydrogenase subunit B (SDHB) gene tend to have metastatic disease. We hypothesized that a down-regulation in the active succinate dehydrogenase B subunit should result in notable changes in cellular metabolic profile and could present a vulnerability point for successful pharmacological targeting. METHODS: Metabolomic analysis was performed on human hPheo1 cells and shRNA SDHB knockdown hPheo1 (hPheo1 SDHB KD) cells. Additional analysis of 115 human fresh frozen samples was conducted. In vitro studies using N1,N11-diethylnorspermine (DENSPM) and N1,N12- diethylspermine (DESPM) treatments were carried out. DENSPM efficacy was assessed in human cell line derived mouse xenografts. RESULTS: Components of the polyamine pathway were elevated in hPheo1 SDHB KD cells compared to wild-type cells. A similar observation was noted in SDHx PCC/PGLs tissues compared to their non-mutated counterparts. Specifically, spermidine, and spermine were significantly elevated in SDHx-mutated PCC/PGLs, with a similar trend in hPheo1 SDHB KD cells. Polyamine pathway inhibitors DENSPM and DESPM effectively inhibited growth of hPheo1 cells in vitro as well in mouse xenografts. CONCLUSIONS: This study demonstrates overactive polyamine pathway in PCC/PGL with SDHB mutations. Treatment with polyamine pathway inhibitors significantly inhibited hPheo1 cell growth and led to growth suppression in xenograft mice treated with DENSPM. These studies strongly implicate the polyamine pathway in PCC/PGL pathophysiology and provide new foundation for exploring the role for polyamine analogue inhibitors in treating metastatic PCC/PGL. PRéCIS: Cell line metabolomics on hPheo1 cells and PCC/PGL tumor tissue indicate that the polyamine pathway is activated. Polyamine inhibitors in vitro and in vivo demonstrate that polyamine inhibitors are promising for malignant PCC/PGL treatment. However, further research is warranted.

Adrenal Development in Mice Requires GATA4 and GATA6 Transcription Factors.

The adrenal glands consist of an outer cortex and an inner medulla, and their primary purposes include hormone synthesis and secretion. The adrenal cortex produces a complex array of steroid hormones, whereas the medulla is part of the sympathetic nervous system and produces the catecholamines epinephrine and norepinephrine. In the mouse, GATA binding protein (GATA) 4 and GATA6 transcription factors are coexpressed in several embryonic tissues, including the adrenal cortex. To explore the roles of GATA4 and GATA6 in mouse adrenal development, we conditionally deleted these genes in adrenocortical cells using the Sf1Cre strain of animals. We report here that mice with Sf1Cre-mediated double deletion of Gata4 and Gata6 genes lack identifiable adrenal glands, steroidogenic factor 1-positive cortical cells and steroidogenic gene expression in the adrenal location. The inactivation of the Gata6 gene alone (Sf1Cre;Gata6(flox/flox)) drastically reduced the adrenal size and corticosterone production in the adult animals. Adrenocortical aplasia is expected to result in the demise of the animal within 2 weeks after birth unless glucocorticoids are provided. In accordance, Sf1Cre;Gata4(flox/flox)Gata6(flox/flox) females depend on steroid supplementation to survive after weaning. Surprisingly, Sf1Cre;Gata4(flox/flox)Gata6(flox/flox) males appear to live normal lifespans as vital steroidogenic synthesis shifts to their testes. Our results reveal a requirement for GATA factors in adrenal development and provide a novel tool to characterize the transcriptional network controlling adrenocortical cell fates.

Combined loss of the GATA4 and GATA6 transcription factors in male mice disrupts testicular development and confers adrenal-like function in the testes.

The roles of the GATA4 and GATA6 transcription factors in testis development were examined by simultaneously ablating Gata4 and Gata6 with Sf1Cre (Nr5a1Cre). The deletion of both genes resulted in a striking testicular phenotype. Embryonic Sf1Cre; Gata4(flox/flox) Gata6(flox/flox) (conditional double mutant) testes were smaller than control organs and contained irregular testis cords and fewer gonocytes. Gene expression analysis revealed significant down-regulation of Dmrt1 and Mvh. Surprisingly, Amh expression was strongly up-regulated and remained high beyond postnatal day 7, when it is normally extinguished. Neither DMRT1 nor GATA1 was detected in the Sertoli cells of the mutant postnatal testes. Furthermore, the expression of the steroidogenic genes Star, Cyp11a1, Hsd3b1, and Hsd17b3 was low throughout embryogenesis. Immunohistochemical analysis revealed a prominent reduction in cytochrome P450 side-chain cleavage enzyme (CYP11A1)- and 3ß-hydroxysteroid dehydrogenase-positive (3ßHSD) cells, with few 17α-hydroxylase/17,20 lyase-positive (CYP17A1) cells present. In contrast, in postnatal Sf1Cre; Gata4(flox/flox) Gata6(flox/flox) testes, the expression of the steroidogenic markers Star, Cyp11a1, and Hsd3b6 was increased, but a dramatic down-regulation of Hsd17b3, which is required for testosterone synthesis, was observed. The genes encoding adrenal enzymes Cyp21a1, Cyp11b1, Cyp11b2, and Mcr2 were strongly up-regulated, and clusters containing numerous CYP21A2-positive cells were localized in the interstitium. These data suggest a lack of testis functionality, with a loss of normal steroidogenic testis function, concomitant with an expansion of the adrenal-like cell population in postnatal conditional double mutant testes. Sf1Cre; Gata4(flox/flox) Gata6(flox/flox) animals of both sexes lack adrenal glands; however, despite this deficiency, males are viable in contrast to the females of the same genotype, which die shortly after birth.

Identification of a novel, non-tetrahydroquinoline variant of the cholesteryl ester transfer protein (CETP) inhibitor torcetrapib, with improved aqueous solubility.

1. Elaborate studies of cholesteryl ester transfer protein (CETP) polymorphisms and genetic deficiency in humans suggest direct links between CETP, high-density lipoprotein cholesterol (HDL-c) levels and coronary heart diseases. The hypothesis that CETP inhibition by small molecule inhibitors raises HDL-c has been validated clinically with structurally-diverse CETP inhibitors such as torcetrapib, anacetrapib, dalcetrapib and evacetrapib. 2. Despite promising phase 2 results with respect to HDL-c elevation, torcetrapib was discontinued in phase 3 trials due to increased mortality rates in the cardiovascular outcomes study. Emerging evidence for the adverse effects hints at off-target chemotype-specific cardiovascular toxicity, possibly related to the pressor effects of torcetrapib, since structurally diverse CETP inhibitors such as anacetrapib, evacetrapib and dalcetrapib are not associated with blood pressure increases in humans. Nonclinical follow-up studies showed that torcetrapib induces aldosterone biosynthesis and secretion in vivo and in vitro, an effect which is not observed with other CETP inhibitors in clinical development. 3. As part of ongoing efforts to identify novel CETP inhibitors devoid of pressor effects, strategies were implemented towards the design of compounds, which lack the 1,2,3,4-tetrahydroquinoline (THQ) scaffold present in torcetrapib. In this article, we disclose results of structure-activity relationship studies for a series of novel non-THQ CETP inhibitors, which resulted in the identification of a novel isonipecotic acid derivative 10 (also referred to as PF-04445597) with vastly improved oral pharmacokinetic properties mainly as a result of improved aqueous solubility. This feature is attractive in that, it bypasses significant investments needed to develop compatible solubilizing formulation(s) for oral drug delivery of highly lipophilic and poorly soluble compounds; attributes, which are usually associated with small molecule CETP inhibitors. PF-04445597 was also devoid of aldosterone secretion in human H295R adrenal carcinoma cells.

Role of transporters in the disposition of the selective phosphodiesterase-4 inhibitor (+)-2-[4-({[2-(benzo[1,3]dioxol-5-yloxy)-pyridine-3-carbonyl]-amino}-methyl)-3-fluoro-phenoxy]-propionic acid in rat and human.

The role of transporters in the disposition of (+)-2-[4-({[2-(benzo[1,3]dioxol-5-yloxy)-pyridine-3-carbonyl]-amino}-methyl)-3-fluoro-phenoxy]-propionic acid (CP-671,305), an orally active inhibitor of phosphodiesterase-4, was examined. In bile duct-exteriorized rats, a 7.4-fold decrease in the half-life of CP-671,305 was observed, implicating enterohepatic recirculation. Statistically significant differences in CP-671,305 pharmacokinetics (clearance and area under the curve) were discernible in cyclosporin A- or rifampicin-pretreated rats. Considering that cyclosporin A and rifampicin inhibit multiple uptake/efflux transporters, the interactions of CP-671,305 with major human hepatic drug transporters, multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 2 (MRP2), breast cancer resistant protein (BCRP), and organic anion-transporting polypeptide (OATPs) were evaluated in vitro. CP-671,305 was identified as a substrate of MRP2 and BCRP, but not MDR1. CP-671,305 was a substrate of human OATP2B1 with a high affinity (Km = 4 microM) but not a substrate for human OATP1B1 or OATP1B3. Consistent with these results, examination of hepatobiliary transport of CP-671,305 in hepatocytes indicated active uptake followed by efflux into bile canaliculi. Upon examination as a substrate for major rat hepatic Oatps, CP-671,305 displayed high affinity (Km = 12 microM) for Oatp1a4. The role of rat Mrp2 in the biliary excretion was also examined in Mrp2-deficient rats. The observations that CP-671,305 pharmacokinetics were largely unaltered suggested that compromised biliary clearance of CP-671,305 was compensated by increased urinary clearance. Overall, these studies suggest that hepatic transporters play an important role in the disposition and clearance of CP-671,305 in rat and human, and as such, these studies should aid in the design of clinical drug-drug interaction studies.

Preclinical pharmacokinetics and metabolism of 6-(4-(2,5-difluorophenyl)oxazol-5-yl)-3-isopropyl-[1,2,4]-triazolo[4,3-a]pyridine, a novel and selective p38alpha inhibitor: identification of an active metabolite in preclinical species and human liver microsomes.

The disposition of 6-(4-(2,5-difluorophenyl)oxazol-5-yl)-3-isopropyl-[1,2,4]-triazolo[4,3-a]pyridine (1), a potent and selective inhibitor of mitogen activated protein (MAP) kinase p38alpha, was characterized in several animal species in support of its selection for preclinical safety studies and potential clinical development. 1 demonstrated generally favorable pharmacokinetic properties in all species examined. Following intravenous (i.v.) administration, 1 exhibited low volumes of distribution at steady state (Vd(ss)) ranging from 0.4-1.3 l/kg (2.4-26 l/m(2)) in the rat, dog and monkey. Systemic plasma clearance was low in cynomolgus monkeys (6.00 ml/min/kg, 72.0 ml/min/m(2)) and Sprague-Dawley rats (7.65+/-1.08 ml/min/kg, 45.9+/-6.48 ml/min/m(2) in male rats and 3.15+/-0.27 ml/min/kg, 18.9+/-1.62 ml/min/m(2) in female rats) and moderate in beagle dogs (12.3+/-5.1 ml/min/kg, 246+/-102 ml/min/m(2)) resulting in plasma half-lives ranging from 1 to 5 h in preclinical species. Moderate to high bioavailability of 1 was observed in rats (30-65%), dogs (87%) and monkeys (40%) after oral (p.o.) dosing consistent with the in vitro absorption profile of 1 in the Caco-2 permeability assay. In rats, the oral pharmacokinetics were dose dependent over the dose range studied (5, 50 and 100 mg/kg). The principal route of clearance of 1 in rat, dog, monkey and human liver microsomes and in vivo in preclinical species involved oxidative metabolism mediated by cytochrome P450 enzymes. The major metabolic fate of 1 in preclinical species and humans involved hydroxylation on the isopropyl group to yield the tertiary alcohol metabolite 2. In human liver microsomes, this transformation was catalysed by CYP3A4 as judged from reaction phenotyping analysis using isozyme-specific inhibitors and recombinant CYP enzymes. Metabolite 2 was also shown to possess inhibitory potency against p38alpha in a variety of in vitro assays. 1 as well as the active metabolite 2 were moderately to highly bound to plasma proteins (f(u) approximately 0.1-0.33) in rat, mouse, dog, monkey and human. 1 as well as the active metabolite 2 did not exhibit competitive inhibition of the five major cytochrome P450 enzymes namely CYP1A2, 2C9, 2C19, 2D6 and 3A4 (IC(50)>50 microM). Overall, these results indicate that the absorption, distribution, metabolism and excretion (ADME) profile of 1 is relatively consistent across preclinical species and predict potentially favorable pharmacokinetic properties in humans, supporting its selection for toxicity/safety assessment studies and possible investigations in humans as an anti-inflammatory agent.

Teratoma formation leads to failure of treatment for type I diabetes using embryonic stem cell-derived insulin-producing cells.

Embryonic stem (ES) cells have been proposed to be a powerful tool in the study of pancreatic disease, as well as a potential source for cell replacement therapy in the treatment of diabetes. However, data demonstrating the feasibility of using pancreatic islet-like cells differentiated from ES cells remain controversial. In this study we characterized ES cell-derived insulin-expressing cells and assessed their suitability for the treatment of type I diabetes. ES cell-derived insulin-stained cell clusters expressed insulin mRNA and transcription factors associated with pancreatic development. The majority of insulin-positive cells in the clusters also showed immunoreactivity for C-peptide. Insulin was stored in the cytoplasm and released into the culture medium in a glucose-dependent manner. When the cultured cells were transplanted into diabetic mice, they reversed the hyperglycemic state for approximately 3 weeks, but the rescue failed due to immature teratoma formation. Our studies demonstrate that reversal of hyperglycemia by transplantation of ES cell-derived insulin-producing cells is possible. However, the risk of teratoma formation would need to be eliminated before ES cell-based therapies for the treatment of diabetes are considered.

The impact of P-glycoprotein on the disposition of drugs targeted for indications of the central nervous system: evaluation using the MDR1A/1B knockout mouse model.

Thirty-two structurally diverse drugs used for the treatment of various conditions of the central nervous system (CNS), along with two active metabolites, and eight non-CNS drugs were measured in brain, plasma, and cerebrospinal fluid in the P-glycoprotein (P-gp) knockout mouse model after subcutaneous administration, and the data were compared with corresponding data obtained in wild-type mice. Total brain-to-plasma (B/P) ratios for the CNS agents ranged from 0.060 to 24. Of the 34 CNS-active agents, only 7 demonstrated B/P area under the plasma concentration curve ratios between P-gp knockout and wild-type mice that did not differ significantly from unity. Most of the remaining drugs demonstrated 1.1- to 2.6-fold greater B/P ratios in P-gp knockout mice versus wild-type mice. Three, risperidone, its active metabolite 9-hydroxyrisperidone, and metoclopramide, showed marked differences in B/P ratios between knockout and wild-type mice (6.6- to 17-fold). Differences in B/P ratios and cerebrospinal fluid/plasma ratios between wild-type and knockout animals were correlated. Through the use of this model, it appears that most CNS-active agents demonstrate at least some P-gp-mediated transport that can affect brain concentrations. However, the impact for the majority of agents is probably minor. The example of risperidone illustrates that even good P-gp substrates can still be clinically useful CNS-active agents. However, for such agents, unbound plasma concentrations may need to be greater than values projected using receptor affinity data to achieve adequate receptor occupancy for effect.

Adult bone marrow-derived cells trans-differentiating into insulin-producing cells for the treatment of type I diabetes.

Recent findings suggest that bone marrow (BM) cells have the capacity to differentiate into a variety of cell types including endocrine cells of the pancreas. We report that BM derived cells, when cultured under defined conditions, were induced to trans-differentiate into insulin-producing cells. Furthermore, these insulin-producing cells formed aggregates that, upon transplantation into mice, acquired architecture similar to islets of Langerhans. These aggregates showed endocrine gene expression for insulin (I and II), glucagon, somatostatin and pancreatic polypeptide. Immunohistochemistry also confirmed that these aggregates were positive for insulin, somatostatin, pancreatic polypeptide and C-peptide. Also, Western and ELISA analysis demonstrated expression of proinsulin and/or secretion of active insulin upon glucose challenge. Subcapsular renal transplantation of these aggregates into hyperglycemic mice lowered circulating blood glucose levels and maintained comparatively normal glucose levels for up to 90 days post-transplantation. Graft removal resulted in rapid relapse and death in experimental animals. In addition, electron microscopy revealed these aggregates had acquired ultrastructure typically associated with mature beta (beta) cells. These results demonstrate that adult BM cells are capable of trans-differentiating into a pancreatic lineage in vitro and may represent a pool of cells for the treatment of diabetes mellitus.

Mouse A6-positive hepatic oval cells also express several hematopoietic stem cell markers.

Hepatic oval cells (HOC) are thought to be a type of facultative stem cell that arises as a result of certain forms of hepatic injury. A new and more efficient model has been established to activate the oval cell compartment in mice by incorporating 3,5-diethoxycarbonyl-1,4-dihydro-collidine (DDC) in a standard chow at a concentration of 0.1%. At the present time, very few markers exist for the mouse oval cells. One accepted marker is A6, an uncharacterized epitope recognized by mouse hepatic oval cells and it is accepted to be an oval cell marker. Sca-1 is a cell surface marker used to identify hematopoietic stem cells in conjunction with Thy-1+, CD34+, and lineage-specific markers. Both the CD34 and Sca-1 antigens are not normally expressed in adult liver, but are expressed in fetal liver, presumably on the hematopoietic cells. We report herein that mouse oval cells express high levels of Sca-1 and CD34, as well as CD45 surface proteins. Immunohistochemistry revealed that the cells expressing Sca-1/CD34/CD45 were indeed oval cells because they co-expressed the oval cell-specific marker A6 (94.57% +/- 0.033%), as well as alpha-fetoprotein (AFP) (75.92% +/- 0.071%). By using Sca-1 antibody in conjunction with magnetic activated cell sorting (MACS), followed with a flow cytometric cell sorting (FACS) method for CD34 and CD45, we have developed a rapid oval cell isolation protocol with high yields of greater than 90%. In conclusion, we have an efficient murine model for the production and isolation of large numbers of highly purified oval cells. Our system works with most strains of mouse, which will facilitate both in vivo and in vitro studies of mouse hepatic oval cells.

Hepatic oval 'stem' cell in liver regeneration.

Hepatic oval cell activation, proliferation, and differentiation has been observed under certain physiological conditions, mainly when the proliferation of existing hepatocytes has been inhibited followed by severe hepatic injury. Hepatic oval cells display a distinct phenotype and have been shown to be a bipotential progenitor of two types of epithelial cells found in the liver, hepatocytes and bile ductular cells. Bone marrow stem cells have recently been shown to be a potential source of the hepatic oval cells and that reconstitution of an injured liver from a purified stem cell population is possible. The focus of this review is on the studies involving the activation, proliferation, and differentiation of these hepatic oval cells and the role that they play in regeneration of the damaged liver. In order to present the potentiality of the hepatic oval cell, an experimental model that involves the inhibition of normal hepatic growth and division as well as severe hepatic injury via chemical or surgical means has been employed. In this model, an as yet undetermined signal or perhaps the lack of regenerative capability in the hepatocytes activates the hepatic oval cell compartment. However, other than understanding a potential origin of these cells and some of the markers that characterize them, it still remains unclear as to how these cells migrate ('home') into the damaged areas and how they begin their differentiation into mature and functioning hepatic cells.

In vitro trans-differentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells.

Although organ-specific stem cells possess plasticity that permit differentiation along new lineages, production of endocrine pancreas and insulin-secreting beta cells from adult nonpancreatic stem cells has not been demonstrated. We present evidence that highly purified adult rat hepatic oval "stem" cells, which are capable of differentiation to hepatocytes and bile duct epithelium, can trans-differentiate into pancreatic endocrine hormone-producing cells when cultured in a high-glucose environment. These differentiated cells can self-assemble to form three-dimensional islet cell-like clusters that express pancreatic islet cell differentiation-related transcripts detectable by reverse transcription-PCR/nested PCR (e.g., PDX-1, PAX-4, PAX-6, Nkx2.2 and Nkx6.1, insulin I, insulin II, glucose transporter 2, and glucagon) and islet-specific hormones detectable by immunocytochemistry (e.g., insulin, glucagon, and pancreatic polypeptide). In addition, these cells concomitantly lose expression of the hepatocyte protein Hep-par. When stimulated with glucose, these cells synthesize and secrete insulin, a response enhanced by nicotinamide. In a pilot study, the oval cell-derived islet cell-like clusters displayed the ability to reverse hyperglycemia in a diabetic NOD-scid mouse. These results indicate that primary adult liver stem cells can differentiate in a nonlineage-restricted manner. Trans-differentiation into endocrine pancreas could have significant implications for future therapies of diabetes.

SDF-1alpha/CXCR4: a mechanism for hepatic oval cell activation and bone marrow stem cell recruitment to the injured liver of rats.

Stromal derived factor-1 alpha (SDF-1alpha) and its receptor CXCR4 have been shown to play a role in the systematic movement of hematopoietic stem cells (HSC) in the fetal and adult stages of hematopoiesis. Under certain physiological conditions liver oval cells can participate in the regeneration of the liver. We have shown that a percentage of oval cells are of hematopoietic origin. Others have shown that bone marrow derived stem cells can participate in liver regeneration as well. In this study we examined the role of SDF-1alpha and its receptor CXCR4 as a possible mechanism for oval cell activation in oval cell aided liver regeneration. In massive liver injury models where oval cell repair is involved hepatocytes up-regulate the expression of SDF-1alpha, a potent chemoattractant for hematopoietic cells. However, when moderate liver injury occurs, proliferation of resident hepatocytes repairs the injury. Under these conditions SDF-1alpha expression is not up-regulated and oval cells are not activated in the liver. In addition, we show that oval cells express CXCR4, the only known receptor for SDF-1alpha. Lastly, in vitro chemotaxis assays demonstrated that oval cells migrate along a SDF-1alpha gradient which suggests that the SDF-1alpha/CXCR4 interaction is a mechanism by which the oval cell compartment could be activated and possibly recruit a second wave of bone marrow stem cells to the injured liver. In conclusion, these experiments begin to shed light on a possible mechanism, which may someday lead to a better understanding of the hepatic and hematopoietic interaction in oval cell aided liver regeneration.