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Objective: Integrins exert regulatory functions in placentogenesis. Null mutation of certain integrin α subunits leads to placental defects with subsequent fetal growth restriction or embryonic lethality in mice. So far, the placental role of α8 integrin remains to be determined. Methods: Localization of α8 integrin and its ligands, fibronectin (FN) and osteopontin (OPN), was studied by immunohistochemistry in human, rat, and mouse placenta. The vascularization of the placental labyrinth layer of α8 integrin-deficient mice was determined by CD31 staining. In humans, α8 integrin expression was assessed via real-time polymerase chain reaction in healthy placentas, in the placental pathologies such as intrauterine growth restriction (IUGR), preeclampsia, and HELLP-syndrome (hemolysis, elevated liver enzymes, low platelet count), as well as in primary extravillous trophoblasts (EVT) and villous trophoblasts. Results: In humans, α8 integrin was detected in first and third trimester syncytiotrophoblast and EVT. Although OPN showed the same localization, FN was observed in EVT only. No expressional changes in α8 integrin were detected in the placental pathologies studied. Rodent placenta showed α8 integrin expression in giant cells and in the labyrinth layer. The localization of OPN and FN, however, showed species-specific differences. Knockout of α8 integrin in mice did not cause IUGR, despite some reduction in labyrinth layer vascularization. Conclusion: α8 Integrin is expressed in functional placental compartments among its ligands, OPN and/or FN, across species. Although this may point to a regulatory role in trophoblast function, our data from α8 integrin-deficient mice indicated only mild placental pathology. Thus, the lack of placental α8 integrin seems to be largely compensated for.

Integrins play an important role in vascular biology. The α8 integrin chain attenuates smooth muscle cell migration but its functional role in the development of atherosclerosis is unclear. Therefore, we studied the contribution of α8 integrin to atherosclerosis and vascular remodelling. We hypothesized that α8 integrin expression is reduced in atherosclerotic lesions, and that its under-expression leads to a more severe course of atherosclerosis. α8 Integrin was detected by immunohistochemistry and qPCR and α8 integrin-deficient mice were used to induce two models of atherosclerotic lesions. First, ligation of the carotid artery led to medial thickening and neointima formation, which was quantified in carotid cross-sections. Second, after crossing into ApoE-deficient mice, the formation of advanced vascular lesions with atherosclerotic plaques was quantified in aortic en face preparations stained with Sudan IV. Parameters of renal physiology and histopathology were assessed: α8 integrin was detected in the media of human and murine vascular tissue and was down-regulated in arteries with advanced atherosclerotic lesions. In α8 integrin-deficient mice (α8(-/-) ) as well as α8(+/-) and α8(+/+) littermates, carotid artery ligation increased media:lumen ratios in all genotypes, with higher values in ligated α8(-/-) and α8(+/-) compared to ligated α8(+/+) animals. Carotid artery ligation increased smooth muscle cell number in the media of α8(+/+) mice and, more prominently, of α8(-/-) or α8(+/-) mice. On an ApoE(-/-) background, α8(+/-) and α8(-/-) mice developed more atherosclerotic plaques than α8(+/+) mice. α8 Integrin expression was reduced in α8(+/-) animals. Renal damage with increased serum creatinine and glomerulosclerosis was detected in α8(-/-) mice only. Thus, under-expression of α8 integrin aggravates vascular lesions, while a complete loss of α8 integrin results in reduced renal mass and additional renal disease in the presence of generalized atherosclerosis. Our data support the hypothesis that integrin α8β1 has a protective role in arterial remodelling and atherosclerosis.

In the kidney, the α8 integrin chain (itga8) is expressed in mesenchymal cells and is upregulated in fibrotic disease. We hypothesized that itga8 mediates a profibrotic phenotype of renal cells by promoting extracellular matrix and cytokine expression. Genetic itga8 deficiency caused complex changes in matrix expression patterns in mesangial and smooth-muscle cells, with the only concordant effect in both cell types being a reduction of collagen III expression. Silencing of itga8 with siRNA led to a decline of matrix turnover with repression of matrix metalloproteinases and reduction of matrix production. In contrast, de novo expression of itga8 in tubular epithelial cells resulted in reduced collagen synthesis. Overexpression of itga8 in fibroblasts did not change the expression of matrix molecules or regulators of matrix turnover. Thus, the influence of itga8 on the expression of matrix components was not uniform and celltype dependent. Itga8 seems unlikely to exert overall profibrotic effects in renal cells.

Introduction Gastrokine-1 (GKN1) is a secreted auto-/paracrine protein, described to be expressed in the gastric mucosa. In gastric cancers GKN1 expression is commonly down-regulated. While current research focusses on the exploration of tumor-suppressive properties of GKN1 with regard to its potential clinical use in the treatment of gastroenterologic tumor disease, nothing is known about GKN1 expression and function in other organ systems. We investigated GKN1 expression in placental tissue and cells. Materials and methods GKN1 was localized using immunohistochemistry in first and third trimester placental tissue, hydatidiform moles and various gestational trophoblastic neoplasias. We determined the expression of GKN1 in immunomagnetic bead-separated term placental cells and in choriocarcinoma cell lines. The role of GKN1 for JEG-3 migration was studied using live cell imaging. E-cadherin, MMP-2 and -9, TIMP-1 and -2, as well as urokinase (uPA) expression levels were determined. Results GKN1 is expressed in healthy third trimester placentas. Its expression is specifically limited to the extravillous trophoblast (EVT). GKN1 expression is significantly reduced in choriocarcinoma cell lines and gestational trophoblastic neoplasias. GKN1 attenuates the migration of JEG-3 choriocarcinoma cells in vitro, possibly via AKT-mediated induction of E-cadherin. GKN1 treatment reduced MMP-9 expression in JEG-3. Discussion Besides its role in gastric physiology our results clearly indicate regulatory functions of GKN1 in the EVT at the feto-maternal interface during pregnancy. Based on our findings in the JEG-3 choriocarcinoma cell line, an auto-/paracrine role of GKN1 for EVT motility and villous anchorage at the basal plate is conceivable. Thus, the tumor suppressor GKN1 is expressed in placental EVT and might contribute to the regulation of EVT migration/invasion.

Development of diabetic nephropathy is accompanied by changes in integrin-mediated cell-matrix interactions. The α8-integrin chain is specifically expressed in mesangial cells of the glomerulus. During experimental hypertension, α8-integrin plays a protective role in the glomerulus. We hypothesized that α8-integrin is involved in maintaining the integrity of the glomerulus in diabetic nephropathy. Experimental streptozotocin (STZ) diabetes led to an increased expression and glomerular deposition of α8-integrin. To test the functional role of α8-integrin, STZ diabetes was induced in mice with a homozygous (α8−/−) or heterozygous (α8+/−) deletion of the α8-integrin gene and in wild-type litters (α8+/+). Blood glucose and mean arterial blood pressure were not different in α8−/− and α8+/+ mice after 6 wk of diabetes. However, diabetic α8−/− mice developed significantly higher albuminuria and more glomerulosclerosis than diabetic α8+/+ mice. Moreover, in diabetic α8−/− mice, the number of glomerular cells staining positive for the podocyte markers WT-1 and vimentin were reduced more prominently than in diabetic α8+/+. The filtration barrier protein nephrin was downregulated in diabetic glomeruli with the strongest reduction observed in α8−/− mice. Taken together, α8−/− mice developed more severe glomerular lesions and podocyte damage after onset of STZ diabetes than α8+/+ mice, indicating that α8-integrin is protective for the structure and function of the glomerulus and maintains podocyte integrity during the development of diabetic nephropathy.

Gastrokines (GKNs) were originally described as stomach-specific tumor suppressor genes. Recently, we identified GKN1 in extravillous trophoblasts (EVT) of human placenta. GKN1 treatment reduced the migration of the trophoblast cell line JEG-3. GKN2 is known to inhibit the proliferation, migration and invasion of gastric cancer cells and may interact with GKN1. Recently, GKN2 was detected in the placental yolk sac of mice. We therefore aimed to further characterize placental GKN2 expression. By immunohistochemistry, healthy first-trimester placenta showed ubiquitous staining for GKN2 at its early gestational stage. At later gestational stages, a more differentiated expression pattern in EVT and villous cytotrophoblasts became evident. In healthy third-trimester placenta, only EVT retained strong GKN2 immunoreactivity. In contrast, HELLP placentas showed a tendency of increased levels of GKN2 expression with a more prominent GKN2 staining in their syncytiotrophoblast. Choriocarcinoma cell lines did not express GKN2. Besides its trophoblastic expression, we found human GKN2 in fibrotic villi, in amniotic membrane and umbilical cord. GKN2 co-localized with smooth muscle actin in villous myofibroblasts and with HLA-G and GKN1 in EVT. In the rodent placenta, GKN2 was specifically located in the spongiotrophoblast layer. Thus, the gestational age-dependent and compartment-specific expression pattern of GKN2 points to a role for placental development. The syncytial expression of GKN2 in HELLP placentas might represent a reduced state of functional differentiation of the syncytiotrophoblast. Moreover, the specific GKN2 expression in the rodent spongiotrophoblast layer (equivalent to human EVT) might suggest an important role in EVT physiology.

Fibrillin-1 is a microfibrillar extracellular matrix protein that was described to be a ligand for α8 integrin. α8 integrin is a matrix receptor specifically expressed in mesangial and smooth muscle cells of the kidney. In previous studies we detected glomerular expression of fibrillin-1. Moreover, fibrillin-1 promoted adhesion, migration, and proliferation of mesangial cells. We hypothesized that fibrillin-1 and α8 integrin might interact in the glomerulus, and thus, regulate mesangial cell properties. Our studies showed that fibrillin-1 and α8 integrin colocalize in the glomerular mesangium. Induction of experimental glomerulonephritis led to an increase of both fibrillin-1 and α8 integrin expression. In vitro studies revealed that mesangial cells deficient for α8 integrin adhere weaker to fibrillin-1 and migrate more easily on fibrillin-1 than wild-type mesangial cells. Baseline proliferation on fibrillin-1 is higher in α8 integrin-deficient mesangial cells, but the induction of proliferation is not different in α8 integrin-deficient and wild-type mesangial cells. We conclude that fibrillin-1 and α8 integrin interact, and thus, regulate mesangial cell adhesion and migration. The concomitant induction of both fibrillin-1 and α8 integrin in a self-limited model of glomerular injury points to a protective role of the interaction of fibrillin-1 with α8 integrin in the glomerulus resulting in reduced damage of the glomerular tuft as a consequence of firm adhesion of mesangial cells.

Integrins play an important role in vascular biology. The α8 integrin chain attenuates smooth muscle cell migration but its functional role in the development of atherosclerosis is unclear. Therefore, we studied the contribution of α8 integrin to atherosclerosis and vascular remodelling. We hypothesized that α8 integrin expression is reduced in atherosclerotic lesions, and that its under-expression leads to a more severe course of atherosclerosis. α8 Integrin was detected by immunohistochemistry and qPCR and α8 integrin-deficient mice were used to induce two models of atherosclerotic lesions. First, ligation of the carotid artery led to medial thickening and neointima formation, which was quantified in carotid cross-sections. Second, after crossing into ApoE-deficient mice, the formation of advanced vascular lesions with atherosclerotic plaques was quantified in aortic en face preparations stained with Sudan IV. Parameters of renal physiology and histopathology were assessed: α8 integrin was detected in the media of human and murine vascular tissue and was down-regulated in arteries with advanced atherosclerotic lesions. In α8 integrin-deficient mice (α8(-/-) ) as well as α8(+/-) and α8(+/+) littermates, carotid artery ligation increased media:lumen ratios in all genotypes, with higher values in ligated α8(-/-) and α8(+/-) compared to ligated α8(+/+) animals. Carotid artery ligation increased smooth muscle cell number in the media of α8(+/+) mice and, more prominently, of α8(-/-) or α8(+/-) mice. On an ApoE(-/-) background, α8(+/-) and α8(-/-) mice developed more atherosclerotic plaques than α8(+/+) mice. α8 Integrin expression was reduced in α8(+/-) animals. Renal damage with increased serum creatinine and glomerulosclerosis was detected in α8(-/-) mice only. Thus, under-expression of α8 integrin aggravates vascular lesions, while a complete loss of α8 integrin results in reduced renal mass and additional renal disease in the presence of generalized atherosclerosis. Our data support the hypothesis that integrin α8β1 has a protective role in arterial remodelling and atherosclerosis.

Fragments of characteristic size retaining the ability of sequence-specific DNA binding were generated by partial proteolysis of transcription factor Stat3 with trypsin, chymotrypsin, or Staphylococcus V8 proteinase. The molecular masses of the smallest DNA-binding fragments were 75, 48, and 32 kDa after digestion with V8 proteinase, chymotrypsin, and trypsin, respectively. The fragments contained major parts of the domain controlling the sequence specificity of DNA binding (amino acids 406-514), the SH3 and SH2 domains, and the phosphorylated tyrosine residue Tyr-705, but not the C-terminal 20 amino acids. The N terminus of the 32-kDa tryptic fragment (ANCDASLIV) matched the sequence of amino acids 424-432 deduced from cDNA. The fragments were observed after proteolytic treatment of preformed complexes between DNA and native factors eluted from rat liver nuclei or recombinant, tyrosine-phosphorylated rat Stat3 from insect cells. It was possible to elute all three minimal fragments from their complexes with DNA and to obtain specific re-binding. The minimal fragments eluted from complexes with DNA still contained the phosphorylated Tyr-705 and the SH2 domain suggesting that they were probably bound to DNA as dimers. The DNA-binding domain of Stat3 identified by these experiments overlapped the domain previously identified by genetic experiments as the domain controlling the sequence specificity of DNA binding. The DNA-binding domain defined here by partial proteolysis probably represents an autonomously folding portion of Stat3.

Background Extracellular matrix receptors of the integrin family are known to regulate cell adhesion, shape and functions. The α8 integrin chain is expressed in glomerular mesangial cells and in vascular smooth muscle cells. Mice deficient for α8 integrin have structural alterations in glomeruli but not in renal arteries. For this reason we hypothesized that mesangial cells and vascular smooth muscle cells differ in their respective capacity to compensate for the lack of α8 integrin. Results Wild type and α8 integrin-deficient mesangial cells varied markedly in cell morphology and expression or localization of cytoskeletal molecules. In α8 integrin-deficient mesangial cells α-smooth muscle actin and CTGF were downregulated. In contrast, there were no comparable differences between α8 integrin-deficient and wild type vascular smooth muscle cells. Expression patterns of integrins were altered in α8 integrin-deficient mesangial cells compared to wild type mesangial cells, displaying a prominent overexpression of α2 and α6 integrins, while expression patterns of the these integrins were not different between wild type and α8 integrin-deficient vascular smooth muscle cells, respectively. Cell proliferation was augmented in α8 integrin-deficient mesangial cells, but not in vascular smooth muscle cells, compared to wild type cells. Conclusions Our findings suggest that α8 integrin deficiency has differential effects in mesangial cells and vascular smooth muscle cells. While the phenotype of vascular smooth muscle cells lacking α8 integrin is not altered, mesangial cells lacking α8 integrin differ considerably from wild type mesangial cells which might be a consequence of compensatory changes in the expression patterns of other integrins. This could result in glomerular changes in α8 integrin-deficient mice, while the vasculature is not affected in these mice.

s / Placenta 35 (2014) A1eA112 A52 alterations in maternal behaviour. Three cohorts of wild type dams carrying fetuses that possessed either a 50% reduction in the spongiotrophoblast (Phlda2 TG) or a 200% increase (Phlda2 KO) were generated using recipient embryo transfer (RET) and their behaviour was studied. Result: Distinct changes in pup retrieval and nest building behaviour were observed, alongside differences in anxiety, assessed using the elevated plus maze (EPM) assay. Specific brain regions and placental samples were subject to microarray analysis to examine gene expression. A number of genes, and one gene family, were significantly altered in relation to WT mothers with a particular genotype litter. Conclusions: This work provides the first experimental evidence that the fetal Phlda2 gene acts on the mother to stimulate maternal behaviours ensuring improved survival of the fetus when it is born. Translated into the human condition this work has relevance to postnatal depression, a disorder that affects 10-15\% of pregnancies. Future directions of this work could offer a new approach to understanding such debilitating maternal mood disorders, whichmay originate in placental abnormalities, providing the potential for generating new therapeutics. P1.134. CHARACTERISATION OF THE TUMOR-SUPPRESSOR GENE GASTROKINE 2 IN THE HUMAN PLACENTA Fabian Fahlbusch , Hannah Bartunik , Gudrun Volkert , Andrea Hartner , Hanna Huebner , Matthias Ruebner , Sven Kehl , Regine Schneider-Stock , Wolfgang Rascher a Department of Pediatrics and Adolescent Medicine, Friedrich-Alexander University Erlangen-Nurnberg, Erlangen, Germany; Department of Obstetrics and Gynaecology, FriedrichAlexander University Erlangen-Nurnberg, Erlangen, Germany; c Experimental Tumorpathology, Friedrich-Alexander University ErlangenNurnberg, Erlangen, Erlangen, Germany Objectives: Gastrokine-1 and -2 (GKNs) act as stomach-specific tumor suppressor genes. Beyond its role in the gastric mucosa, we have recently identified GKN1 in human placenta. Its expression was specific for extravillous trophoblasts (EVT) and treatment of the extravillous cell line JEG3withGKN1 inhibited cellmigration. Similarly, GKN2 is known to inhibit the proliferation, migration and invasion of gastric cancer cells. This study was designed to analyse expression and localisation of GKN2 in the human placenta. Methods: Placental localisation of GKN2 was determined by immunohistochemistry in first and third trimester placentas. We analysed the expression levels of GKN2 via sqPCR in healthy placental tissue, in placental pathologies (IUGR, PE and HELLP) and in various trophoblast-like cell lines (JEG-3, Jar, BeWo, Swan-71). Results: In the first trimester, GKN2 was located in all trophoblast compartments. In the third trimester, EVTs stained strongly for GKN2. While in healthy placental tissue the syncytiotrophoblast only showed minor positive staining for GKN2, we found a more pronounced GKN2 positivity in the syncytiotrophoblast of IUGR. Additionally, GKN2 was detected in the stroma of fibrotic villi of IUGR and in smooth muscle cells of the villous vessels. SqPCR indicated a significant induction of GKN2 expression in all placental pathologies investigated. No expression of GKN2 was found in trophoblast-like cell lines. Conclusion: Our study is the first to characterise GKN2 in placenta. Its expression was absent in invasive trophoblast-like cell lines, supporting its role as a tumor suppressor. The fact that GKN2 is expressed in all first trimester trophoblast compartments,whilemoredistinctly distributed in the third trimester, points to a role of GKN2 in placental development. Whether the increase of GKN2 in placental pathologies is of trophoblast origin or secondary to the accompanying villous fibrosis remains to be elucidated. P1.135. PLACENTAL HORMONE CONTRIBUTION TO FETAL BRAIN DAMAGE Anna Penn , Wendy Koss , Megha Agrawal , Suresh Volate , Danielle Leuenberger , Marianna Kiraly , Anca Pasca , Karen Chisholm Children's National Medical Center, Washington DC, USA; b Stanford University, Stanford CA, USA Objectives: The placenta produces a wide array of neuroactive hormones; this endocrine function can be disrupted inmanywaysdby abnormal gene expression, infection, prematuritydresulting in long-term damage. Compromised placental function has been linked to diverse developmental disorders including cerebral palsy, autism, and schizophrenia. We investigate changes in placental hormones that shape fetal brain development or modify susceptibility to neurological damage. Preterm birth, affecting one tenth of all deliveries, provides the most extreme case of hormone loss, but we hypothesize that it is just one of many cases inwhich placental dysfunction leads to developmental brain damage. We have developed novel animal models in which individual hormones are specifically removed from the placenta to test this hypothesis directly. Methods: We have targeted placental production of allopregnanolone (ALLO or 3a, 5a tetrohydroprogestrone) to investigate its contribution to embryonic cortical neurogenesis. To achieve placenta-specific suppression, mouse blastocysts were infected with lentivirus coding for shRNA targeting 3a hydroxysteroid dehydrogenase (3aHSD), the enzyme responsible for the second step of conversion from progesterone to ALLO, allowing ALLO production to be suppressed specifically in extra-embryonic tissues following blastocyst transfer into pseudopregnant dams. Comparison was made to scrambled shRNA control virus infection. Specificity of infection was monitored by qRT-PCR of 3aHSD and a fluorophore expressed by the virus for each placenta/embryo pair produced. Results: Suppression of placental 3aHSD resulted in abnormal cortical development when assessed in near-term (embryonic day 17) mice. Significantly fewer proliferating cortical neuron precursors were seen, with more cells quitting the cell cycle compared to control. However, the proliferative zone itself (the cortical subventricular zone) enlarged, suggesting a shift in the kinetics of neurogensis produced by loss of placental ALLO. Concurrently, we assessed neural circuit formation in the neonatal cortex and hippocampus following fetal ALLOmanipulation and found that ALLO suppression may delay circuit maturation. In human placental samples, measurements of steroid-converting enzymes show changes in expression across gestation, as well as differential changes with specific pathologies, suggesting relevance to human placental and neuropathology. Preclinical investigation of neurosteroid replacement as a therapeutic treatment for preterm brain injury is underway. Conclusion: Taken together, our experiments provide a direct demonstration that hormone production by the placenta (not from the maternal or fetal circulation) is needed for normal anatomical and functional development of the fetal brain. These findings open the door for new therapeutic approaches to developmental brain disorders through replacement of one or more hormones to which the fetus or neonate is normally exposed to prevent or ameliorate developmental brain injury. P1.136-N. ANTIRETROVIRAL-THERAPY (ART) INDUCED ADVERSE PREGNANCY OUTCOMES: THE POTENTIAL ROLE OF PROGESTERONE Eszter Papp, Hakimeh Mohammadi, Lena Serghides University Health Network, Toronto, ON, Canada Objectives: Each year, millions of HIV-positive pregnant women receive antiretroviral therapy (ART), which effectively prevents mother-to-child transmission of the virus. On the other hand, an increasing body of evidence suggests an association between ART exposure and adverse birth outcomes, including an increased rate of miscarriage, preterm birth, and small birth weight. Studies have also reported significant interaction between contraceptive steroid hormones and HIV drugs. We investigated if alterations in steroid hormone balance could play a role in ART-related adverse pregnancy outcomes. Methods: 1.Human choriocarcinoma cells (BeWo) were exposed to various antiretroviral drugs singly or in clinically relevant combinations. After 48 hours progesterone production was quantified. 2. Pregnant C57Bl/6 mice were exposed to human-equivalent doses of combination ART (cART) or waterdaily fromgestational day0.5.Damswere sacrificedongestationalday 14.5. Another set of micewere exposed to cARTor water as described above and supplemented with 0.5 mg progesterone in 4 day intervals throughout