Formation Of Tubular Structures Research Articles

Advanced tube formation assay using human endothelial colony forming cells for in vitro evaluation of angiogenesis.

The tube formation assay is a widely used in vitro experiment model to evaluate angiogenic properties by measuring the formation of tubular structures from vascular endothelial cells (ECs). in vitro experimental results are crucial when considered the advisability of moving forward to in vivo studies. Thus, the additional attentions to the in vitro assay is necessary to improve the quality of the pre-clinical data, leading to better decision-making for successful drug discovery. In this study, we improved the tube formation assay system in three aspects. First, we used human endothelial colony forming cells (ECFCs), which are endothelial precursors that have a robust proliferative capacity and more defined angiogenic characteristics compared to mature ECs. Second, we utilized a real-time cell recorder to track the progression of tube formation for 48 hours. Third, to minimize analysis error due to the limited observation area, we used image-stitching software to increase the microscope field of view to a 2×2 stitched area from the 4× object lens. Our advanced tube formation assay system successfully demonstrated the time-dependent dynamic progression of tube formation in the presence and absence of VEGF and FGF-2. Vatalanib, VEGF inhibitor, was tested by our assay system. Of note, IC50 values of vatalanib was different at each observation time point. Collectively, these results indicate that our advanced tube formation assay system replicates the dynamic progression of tube formation in response to angiogenic modulators. Therefore, this new system provides a sensitive and versatile assay model for evaluating pro- or anti-angiogenic drugs.

β-Cryptoxanthin Inhibits Angiogenesis in Human Umbilical Vein Endothelial Cells Through Retinoic Acid Receptor.

β-Cryptoxanthin is an abundant carotenoid in fruits and vegetables that can be quantified in human blood serum. Yet, contrary to other carotenoids, its effects on endothelial cells and angiogenesis remain unknown. Human umbilical vein endothelial cells (HUVEC) are treated with 0.01, 0.1, or 1 μm of β-cryptoxanthin. Antioxidant activity is determined by its free radical scavenging and oxygen-radical absorbance capacity. The effect on migration and formation of tubular structures is studied. Additionally, effect on angiogenesis is also analyzed using an in vivo model. β-Cryptoxanthin exhibits scavenging ability, having an antioxidant effect on HUVEC. Interestingly, β-cryptoxanthin reduces their migration and angiogenesis, even in the presence of vascular endothelial growth factor (VEGF). Additionally, such carotenoid inhibits in vivo angiogenesis induced by VEGF. In addition, treatment of HUVEC with LE540 (retinoic acid receptor [RAR] panantagonist) inhibits β-cryptoxanthin antiangiogenic effect on HUVEC. β-Cryptoxanthin inhibits angiogenesis through RAR. Thus, this carotenoid and food containing it may be useful for the prevention and treatment of angiogenic pathologies. That includes tumoral growth and wet macular degeneration associated with aging. To the best of our knowledge, this is the first report of the antioxidant effect and antiangiogenic activity of this carotenoid on HUVEC, both in vitro and in vivo.

Staphylococcus aureus Alpha-Toxin Induces the Formation of Dynamic Tubules Labeled with LC3 within Host Cells in a Rab7 and Rab1b-Dependent Manner.

Staphylococcus aureus is a pathogen that causes severe infectious diseases that eventually lead to septic and toxic shock. S. aureus infection is characterized by the production of virulence factors, including enzymes and toxins. After internalization S. aureus resides in a phagosome labeled with Rab7 protein. Here, we show that S. aureus generates tubular structures marked with the small GTPases Rab1b and Rab7 and by the autophagic protein LC3 at early times post-infection. As shown by live cell imaging these tubular structures are highly dynamic, extend, branch and grow in length. We have named them S. aureus induced filaments (Saf). Furthermore, we demonstrate that the formation of these filaments depends on the integrity of microtubules and the activity of the motor protein Kinesin-1 (Kif5B) and the Rab-interacting lysosomal protein (RILP). Our group has previously reported that α-hemolysin, a secreted toxin of S. aureus, is responsible of the activation of the autophagic pathway induced by the bacteria. In the present report, we demonstrate that the autophagic protein LC3 is recruited to the membrane of S. aureus induced filaments and that α-hemolysin is the toxin that induces Saf formation. Interestingly, increasing the levels of intracellular cAMP significantly inhibited Saf biogenesis. Remarkably in this report we show the formation of tubular structures that emerge from the S. aureus-containing phagosome and that these tubules generation seems to be required for efficient bacteria replication.

Oligomer procyanidins (F2) repress HIF-1α expression in human U87 glioma cells by inhibiting the EGFR/ AKT/mTOR and MAPK/ERK1/2 signaling pathways in vitro and in vivo.

Hypoxia-inducible factor-1 (HIF-1) is over-expressed in gliomas and has become one of the most compelling tumor targets. In this study, we found that oligomer procyanidins (F2) can suppress the expressions of HIF-1α and its target genes in U87 cells, and also down-regulate the EGFR/PI3K/AKT/mTOR and MAPK/ERK1/2 pathways in vitro and in vivo. Furthermore, hypoxia-induced formation of tubular structures by human umbilical vascular endothelial cells and the migration and invasion of U87 cells could be inhibited by F2 in a HIF-1 dependent manner. Moreover, in a U87 xenograft tumor model, F2 significantly reduced intra-tumor vessel density and cell proliferation and finally retarded tumor growth, indicating that F2 may be a potential HIF-1 inhibitor and serve as one of candidates for glioma therapy.

ADENOMATOID ODONTOGENIC TUMOR: A CASE REPORT

The adenomatoid odontogenic tumor is a rare benign neoplasm. This is a case of an adenomatoid odontogenic tumor in the maxilla. A female patient, 25 years old, presented to the specialized service with volumetric increase of the right maxilla. The intraoral physical examination revealed bulging buccal maxilla of same side. Imaging tests revealed radiolucent lesion, well defined, and delimited with radiopaque points and associated with the upper lateral incisor included. After careful clinical and imaging evaluation, excisional biopsy was chosen to perform complete enucleation of the lesion. Histopathology showed odontogenic neoplasm composed of massive fusiform epithelial cells forming nests and rosettes. Formation of tubular structures and extensive areas of mineralized matrix were also observed. The diagnosis was conclusive for adenomatoid odontogenic tumor. The patient is being monitored and has no signs of recurrence.

BLOC-1 deficiency causes alterations in amino acid profile and in phospholipid and adenosine metabolism in the postnatal mouse hippocampus

Biogenesis of lysosome-related organelles complex-1 (BLOC-1) is a protein complex involved in the formation of endosomal tubular structures that mediates the sorting of protein cargoes to specialised compartments. In this study, we present insights into the metabolic consequences caused by BLOC-1 deficiency in pallid mice, which carry a null mutation in the Bloc1s6 gene encoding an essential component of this complex. The metabolome of the hippocampus of pallid mice was analysed using an untargeted, liquid chromatography-coupled mass spectrometric approach. After data pre-treatment, statistical analysis and pathway enrichment, we have identified 28 metabolites that showed statistically significant changes between pallid and wild-type control. These metabolites included amino acids, nucleobase-containing compounds and lysophospholipids. Interestingly, pallid mice displayed increased hippocampal levels of the neurotransmitters glutamate and N-acetyl-aspartyl-glutamic acid (NAAG) and their precursor glutamine. Expression of the sodium-coupled neutral amino acid transporter 1 (SNAT1), which transports glutamine into neurons, was also upregulated. Conversely, levels of the neurotransmitter precursors phenylalanine and tryptophan were decreased. Interestingly, many of these changes could be mapped to overlapping metabolic pathways. The observed metabolic alterations are likely to affect neurotransmission and neuronal homeostasis and in turn could mediate the memory and behavioural impairments observed in BLOC-1-deficient mice.

Abstract 789: Establishment of an in vitro model for the study of vasculogenic mimicry in ovarian and gastrointestinal cancer cells

Abstract Introduction: A key step in cancer progression is tumor irrigation. The process of angiogenesis can be complemented in sub-sets of highly aggressive cancers by the process of vasculogenic mimicry, which is the formation of tubular structures by tumor cells. Herein, we sought to establish an in vitro assay of vasculogenic mimicry and determine the percentage of ovarian and gastrointestinal primary cultures capable of undergoing this process. Materials & Methods: Gastric cancer cell lines, AGS and Hs746T, and the ovarian cancer cell lines SKOV-3 and HEY were used in conjunction with gastrointestinal and ovarian primary cultured cancer cells extracted from peritoneal fluid. Cells were seeded on matrigel and monitored for a week. Hollow channels formation was evaluated by fluorescent dye microinyection, periodic acid Schiff staining, confocal microscopy and X-Ray microtomography (Micro-CT). Results: SKOV-3, HEY and AGS cell lines underwent the process of vasculogenic mimicry. Both confocal microscopy and Micro-CT reconstruction were able to show the presence of a lumen of the structures in 3D. In 22 primary cancer cultures (Ovarian, Colon and Gastric) only 38% could underwent vasculogenic mimicry. Discussion: We have standardized an in vitro assay to assay and quantify vasculogenic mimicry. Regardless of origin, only a low percentage of cultures have the ability to undergo vasculogenic mimicry. An understanding of this process could shed light on new therapies for this sub-set of highly aggressive cancers. Funding: FONDECYT 1120292, 3150028 & 1140970. CORFO L2 13IDL2-18608, BMRC 13CTI 21526-P6, IMII P09/016-F, CONICYT-FONDAP #1513001 Citation Format: Andres Valdivia, Dusan Racordon, Raul Aravena, Gabriel Mingo, Alejandra Sandoval, Maria Loreto Bravo, Mauricio A. Cuello, Sumie Kato, Rafaela Erices, Carolina Ramirez, Pamela Gonzalez, Beatriz Sanchez, Alejandro H. Corvalan, Gareth I. Owen. Establishment of an in vitro model for the study of vasculogenic mimicry in ovarian and gastrointestinal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 789. doi:10.1158/1538-7445.AM2017-789

171 Amphiphysin ii (bin1) driven transverse tubule formation in cardiac muscle

Transverse (t)-tubules are vital for maintaining normal contractility of the heart through the tight regulation of excitation coupling. In cardiac disease, such as heart failure, t-tubule loss is closely associated...

A heparin conjugate, LHbisD4, inhibits lymphangiogenesis and attenuates lymph node metastasis by blocking VEGF-C signaling pathway.

Clinical studies have found that the incidence of cancer metastasis through the lymphatic vessels are 3-5 times higher than that through the blood vessels. These findings suggest the potency of anti-lymphangiogenic therapy in reducing the incidence of cancer metastasis. Previously, we reported LHbisD4, which is the conjugate of low molecular weight heparin (LMWH) and four bis-deoxycholates as a potent anti-angiogenic drug with less toxicity and orally active property. Here, we show that LHbisD4 could also suppress the formation of new lymphatic vessels and attenuate the incidence of metastasis by blocking VEGF-C signaling pathway. LHbisD4 significantly enhanced binding affinity with VEGF-C when compared with LMWH, which enables LHbisD4 to suppress the proliferation, migration and formation of tubular structures of human dermal lymphatic endothelial cells(HDLECs) in invitro condition even in the presence of excessive amounts of VEGF-C. Similarly, we found that the density of lymphatic vessels in the primary tumor tissue in breast cancer bearing mice was significantly diminished when LHbisD4 was administered compared with the control group. Also, the incidence of axillary lymph nodes and distant organ metastasis was significantly reduced in the LHbisD4 administered group, which demonstrates that LHbisD4 could successfully lower the incidence of metastasis through blocking VEGF-C induced lymphangiogenesis. Based on these results, we propose LHbisD4 as a potent anti-cancer drug that can reduce the incidence of metastasis by suppressing lymphangiogenesis through blocking VEGF-C signaling pathway.

Intracellular Localization, Interactions and Functions of Capsicum Chlorosis Virus Proteins.

Tospoviruses are among the most devastating viruses of horticultural and field crops. Capsicum chlorosis virus (CaCV) has emerged as an important pathogen of capsicum and tomato in Australia and South-east Asia. Present knowledge about CaCV protein functions in host cells is lacking. We determined intracellular localization and interactions of CaCV proteins by live plant cell imaging to gain insight into the associations of viral proteins during infection. Proteins were transiently expressed as fusions to autofluorescent proteins in leaf epidermal cells of Nicotiana benthamiana and capsicum. All viral proteins localized at least partially in the cell periphery suggestive of cytoplasmic replication and assembly of CaCV. Nucleocapsid (N) and non-structural movement (NSm) proteins localized exclusively in the cell periphery, while non-structural suppressor of silencing (NSs) protein and Gc and Gn glycoproteins accumulated in both the cell periphery and the nucleus. Nuclear localization of CaCV Gn and NSs is unique among tospoviruses. We validated nuclear localization of NSs by immunofluorescence in protoplasts. Bimolecular fluorescence complementation showed self-interactions of CaCV N, NSs and NSm, and heterotypic interactions of N with NSs and Gn. All interactions occurred in the cytoplasm, except NSs self-interaction was exclusively nuclear. Interactions of a tospoviral NSs protein with itself and with N had not been reported previously. Functionally, CaCV NSs showed strong local and systemic RNA silencing suppressor activity and appears to delay short-distance spread of silencing signal. Cell-to-cell movement activity of NSm was demonstrated by trans-complementation of a movement-defective tobamovirus replicon. CaCV NSm localized at plasmodesmata and its transient expression led to the formation of tubular structures that protruded from protoplasts. The D155 residue in the 30K-like movement protein-specific LxD/N50-70G motif of NSm was critical for plasmodesmata localization and movement activity. Compared to other tospoviruses, CaCV proteins have both conserved and unique properties in terms of in planta localization, interactions and protein functions which will effect viral multiplication and movement in host plants.

Formation of ZnIn2S4 nanosheets and tubular structures in organic media

Hot-injection method for synthesis of ZnIn2S4 (ZIS) in organic media was studied. Nanosized ZnIn2S4 is successfully synthesized in oleic acid/oleylamine mixture in different ratios, which are both: complexing and/or capping agents. Nanosheets/nanoscrolls of about 50nm are obtained. The band gaps of synthesized nanostructures were calculated to be in the range from 2.6 to 3.0eV, values which are increased compared to bulk value (2.3–2.5eV). The increase of band gap can be assigned to nanodimensions of the particles. Potential of band edges is calculated using empirical equation and find to be in the range from −0.16 to −0.36V for conduction band, and from 2.24 to 2.69V for valence band. By using XRD and TEM techniques, crystal structure and morphology of obtained materials were investigated, and detailed formation and growth mechanism of nanosheets/nanoscroles is proposed accordingly.

Construction of a patterned hydrogel-fibrous mat bilayer structure to mimic choroid and Bruch's membrane layers of retina.

Deterioration of retina and death of the retinal cells due to age, diabetes, or occlusion can cause retinal degeneration which leads to loss of vision. In this study, it is aimed to design a bilayered matrix to mimic the choroid and the Bruch's membrane of the retinal tissue. As choroid, a microchanneled network resembling a fractal tree design was fabricated by photolithography over photo-cross-linkable methacrylated hyaluronic acid hydrogel. Gelatin or collagen was immobilized into the microchannels to enhance adherence of Human Umbilical Vein Endothelial Cells (HUVEC). At late culture periods (2 weeks), formation of tubular structures due to proliferation of the attached cells was observed. As Bruch's membrane, an electrospun fibroin nanofiber mat was produced to grow retinal pigment epithelium (RPE) cells on. Cellular interactions between RPE and HUVEC in the microchannels were investigated in a coculture model in a noncontact mode. It was deduced that by combining the RPE layer on the highly permeable Bruch's membrane with the choroid layer populated by HUVECs, a retinal substitute which might have a potential for use in the treatment of retinal diseases is possible. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2166-2177, 2016.

Platelet lysate-based pro-angiogenic nanocoatings

Human platelet lysate (PL) is a cost-effective and human source of autologous multiple and potent pro-angiogenic factors, such as vascular endothelial growth factor A (VEGF A), fibroblast growth factor b (FGF b) and angiopoietin-1. Nanocoatings previously characterized were prepared by layer-by-layer assembling incorporating PL with marine-origin polysaccharides and were shown to activate human umbilical vein endothelial cells (HUVECs). Within 20h of incubation, the more sulfated coatings induced the HUVECS to the form tube-like structures accompanied by an increased expression of angiogenic-associated genes, such as angiopoietin-1 and VEGF A. This may be a cost-effective approach to modify 2D/3D constructs to instruct angiogenic cells towards the formation of neo-vascularization, driven by multiple and synergistic stimulations from the PL combined with sulfated polysaccharides. Statement of SignificanceThe presence, or fast induction, of a stable and mature vasculature inside 3D constructs is crucial for new tissue formation and its viability. This has been one of the major tissue engineering challenges, limiting the dimensions of efficient tissue constructs. Many approaches based on cells, growth factors, 3D bioprinting and channel incorporation have been proposed. Herein, we explored a versatile technique, layer-by-layer assembling in combination with platelet lysate (PL), that is a cost-effective source of many potent pro-angiogenic proteins and growth factors. Results suggest that the combination of PL with sulfated polyelectrolytes might be used to introduce interfaces onto 2D/3D constructs with potential to induce the formation of cell-based tubular structures.

Long noncoding RNA MALAT1 as a potential therapeutic target in osteosarcoma.

Recent studies have revealed that long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays an important role in the development of several solid tumors. However, the function of MALAT1 in the tumorigenesis of osteosarcoma remains unknown. In the present study, levels of MALAT1 in human osteosarcoma cell lines and tissues were detected by quantitative real-time polymerase chain reaction (RT-PCR). The roles of MALAT1 in osteosarcoma were investigated by using in vitro and in vivo assays. We observed that MALAT1 expression was up-regulated in human osteosarcoma cell lines and tissues. In vitro knockdown of MALAT1 by siRNA significantly inhibited cell proliferation and migration, and induced cell cycle arrest and apoptosis in osteosarcoma cells. In addition, MALAT1 knockdown markedly suppressed the formation of tubular network structures and caused breakage of stress fibers in osteosarcoma cell lines U2OS and MNNG/HOS. Furthermore, MALAT1 knockdown delayed tumor growth in an osteosarcoma xenograft model. Specifically, we found that administration of MALAT1 siRNA decreased the protein levels of RhoA and its downstream effectors Rho-associated coiled-coil containing protein kinases (ROCKs). Taken together, these findings suggest that MALAT1 plays an oncogenic role in osteosarcoma and may be a promising therapeutic target for the treatment of osteosarcoma patients. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:932-941, 2016.

Sequential Functions of CPEB1 and CPEB4 Regulate Pathologic Expression of Vascular Endothelial Growth Factor and Angiogenesis in Chronic Liver Disease

Vascular endothelial growth factor (VEGF) regulates angiogenesis, yet therapeutic strategies to disrupt VEGF signaling can interfere with physiologic angiogenesis. In a search for ways to inhibit pathologic production or activities of VEGF without affecting its normal productionor functions, we investigated the post-transcriptional regulation of VEGF by the cytoplasmic polyadenylation element-binding proteins CPEB1 and CPEB4 during development of portal hypertension and liver disease. We obtained transjugular liver biopsies from patients with hepatitis C virus-associated cirrhosis or liver tissues removed during transplantation; healthy human liver tissue was obtained from a commercial source (control). We also performed experiments with male Sprague-Dawley rats and CPEB-deficient mice (C57BL6 or mixed C57BL6/129 background) and their wild-type littermates. Secondary biliary cirrhosis was induced in rats by bile duct ligation, and portal hypertension was induced by partial portal vein ligation. Liver and mesenteric tissues were collected and analyzed in angiogenesis, reverse transcription polymerase chain reaction, polyA tail, 3' rapid amplification of complementary DNA ends, Southern blot, immunoblot, histologic, immunohistochemical, immunofluorescence, and confocal microscopy assays. CPEB was knocked down with small interfering RNAs in H5V endothelial cells, and translation of luciferase reporters constructs was assessed. Activation of CPEB1 promoted alternative nuclear processing within noncoding 3'-untranslated regions of VEGF and CPEB4 messenger RNAs in H5V cells, resulting in deletion of translation repressor elements. The subsequent overexpression of CPEB4 promoted cytoplasmic polyadenylation of VEGF messenger RNA, increasing its translation; the high levels of VEGF produced bythese cells led to their formation of tubular structures in Matrigel assays. We observed increased levels of CPEB1 and CPEB4 in cirrhotic liver tissues from patients, compared withcontrol tissue, as well as in livers and mesenteries of ratsand mice with cirrhosis or/and portal hypertension. Micewith knockdown of CPEB1 or CPEB4 did notoverexpress VEGF or have signs of mesenteric neovascularization, and developed less-severe forms of portal hypertension afterportal vein ligation. We identified a mechanism of VEGF overexpression in liver and mesentery that promotes pathologic, but not physiologic, angiogenesis, via sequential and nonredundant functions of CPEB1 and CPEB4. Regulation of CPEB4 by CPEB1 and the CPEB4 autoamplification loop induces pathologic angiogenesis. Strategies to block the activities of CPEBs might be developed to treat chronic liver and other angiogenesis-dependent diseases.

Incarvine C suppresses proliferation and vasculogenic mimicry of hepatocellular carcinoma cells via targeting ROCK inhibition.

BackgroundStudies have described vasculogenic mimicry (VM) as an alternative circulatory system to blood vessels in multiple malignant tumor types, including hepatocellular carcinoma (HCC). In the current study, we aimed to seek novel and more efficient treatment strategies by targeting VM and explore the underlying mechanisms in HCC cells.MethodsCell counting kit-8 (CCK-8) assay and colony survival assay were performed to explore the inhibitory effect of incarvine C (IVC) on human cancer cell proliferation. Flow cytometry was performed to analyze the cell cycle distribution after DNA staining and cell apoptosis by the Annexin V-PE and 7-AAD assay. The effect of IVC on Rho-associated, coiled-coil-containing protein kinase (ROCK) was determined by western blotting and stress fiber formation assay. The inhibitory role of IVC on MHCC97H cell VM formation was determined by formation of tubular network structures on Matrigel in vitro, real time-qPCR, confocal microscopy and western blotting techniques.ResultsWe explored an anti-metastatic HCC agent, IVC, derived from traditional Chinese medicinal herbs, and found that IVC dose-dependently inhibited the growth of MHCC97H cells. IVC induced MHCC97H cell cycle arrest at G1 transition, which was associated with cyclin-dependent kinase 2 (CDK-2)/cyclin-E1 degradation and p21/p53 up-regulation. In addition, IVC induced apoptotic death of MHCC97H cells. Furthermore, IVC strongly suppressed the phosphorylation of the ROCK substrate myosin phosphatase target subunit-1 (MYPT-1) and ROCK-mediated actin fiber formation. Finally, IVC inhibited cell-dominant tube formation in vitro, which was accompanied with the down-regulation of VM-key factors as detected by real time-qPCR and immunofluorescence.ConclusionsTaken together, the effective inhibitory effect of IVC on MHCC97H cell proliferation and neovascularization was associated with ROCK inhibition, suggesting that IVC may be a new potential drug candidate for the treatment of HCC.

P38. Strong inhibitory effect of preeclampsia serum on angiogenesis using in vitro angiogenesis test

Introduction The angiogenic response of preeclampsia patient serum samples were studied by using human in vitro angiogenesis test. The hypothesis was that preeclampsia patients’ serum may contain factors that disturb angiogenesis and that could explain formation of some of the adverse effects in patients. Objective To measure in vitro angiogenetic effects of serum samples from preeclamptic mothers and umbilical blood. Methods Serum samples were collected from ten primiparous preeclampsia patients and ten matched controls at gestational age of 28–38 weeks. After birth giving, also umbilical blood samples were taken. The test method consisted of a co-culture of human fibroblasts and endothelial cells in the culture medium with specific exogenous growth factors to induce formation of tubular structures and tubular networks. During the test, the co-culture was exposed to patient serum samples (dilution 1:15) and cultured for further 6 days (with one replenishment of the growth medium). After the exposure cytotoxicity was evaluated using mitochondrial viability (WST-1) assay. Tubular structures were evaluated microscopically after immunostaining with anti von Willebrand factor. Results There was no cytotoxicity shown with any of the samples, but the umbilical blood samples seemed to induce cell growth to some extent. There were no stimulatory effects found on tubule formation with any of the samples, but there was a strong inhibitory effect on tubule formation with serum from preeclampsia patients. Also umbilical blood from preeclamptic pregnancies showed inhibitory effect on tubule formation, but there was no clear correlation of serum from mothers compared to corresponding umbilical serum. No significant correlations to the severity of preeclampsia or any clinical features of the disease were noticed. Conclusion Both maternal and umbilical blood serum after established preeclampsia show inhibitory effects on angiogenesis, as tested by in vitro -model.

Abstract A11: Ets transcription factor Etv5 regulates ductal morphogenesis and differentiation in association with Sox9 in vitro and increases susceptibility and delays recovery from pancreatitis in vivo

Abstract Introduction: The exocrine pancreas comprises a branched network of ducts that are connected to acini and lined by a monolayered epithelium that derives from the endoderm and is surrounded by mesenchyme. The formation and maturation of pancreatic ductal cells during branching morphogenesis is controlled by a complex hierarchy of transcription factors, which is not fully understood. The Ets-transcription factor Etv5 has been reported to play an important role during the development of organs that undergo branching morphogenesis such as mammary and salivary glands. We therefore aimed to characterize the functional role of Etv5 in pancreatic ductal development and differentiation. Methods: Cells were isolated from wild-type mouse pancreata and lentiviral transduction was used to induce Etv5 overexpression. Cells were grown in 3D organotypic culture and analyzed with time lapsed microscopy for morphology and dynamics in the formation of duct-like cystic structures. Differentiation status of cells grown in 3D was analyzed by immunofluorescence-staining (IF) and qPCR. To asses the role of Etv5 in the normal mouse pancreas and its role in inflammation and regeneration in vivo, Pdx1cre;Etv5-/-; RosaYFP mice were generated and aged up to six months and subjected to a cerulein induced acute pancreatitis protocol. Results: Normal pancreatic ductal cells grown in 3D-organotypic culture form spheroid cysts that resemble pancreatic ductal structures. Etv5 overexpression leads to early and exuberant formation of spheroid cysts within 2 days after seeding. Time-lapse microscopy demonstrated a significantly increased movement of cellular structures along the cyst as well as fusion of cysts into larger tubular structures. mRNA analysis of cysts harvested at day 7 displayed a strong upregulation of Sox9 and Foxa2, important regulators of ductal differentiation. Concurrently, E-cadherin was upregulated significantly whereas N-cadherin was downregulated indicating the terminal differentiation of these cells. IF-staining revealed co-localization of Etv5 and Sox9 in spheroid cysts of Etv5-overexpressing cells. Knockdown of Sox9 in Etv5-overexpressing cells with siRNA partially abrogated the formation of tubular structures and disrupted cyst architecture. Etv5 knockout mice (Pdx1Cre;Etv5-/-) were generated and we performed a cerulein-induced acute pancreatitis model. We found significantly elevated levels of serum amylase in Etv5-/- mice on Days 1 and 3 of the protocol. In a semiquantitative, blinded review of the histology, there was significantly more intense edema, inflammation, vacuolization and necrosis in Etv5-/- mice in all time points, which was also confirmed by quantitative amylase area scoring. Conclusion: Our data suggest a novel role for Etv5 in pancreatic ductal morphogenesis and lumen formation that is at least in part mediated by Sox9. In addition, our data suggests that the loss of Etv5 expression increases susceptibility to pancreatitis and results in persistent pancreatitis with delayed regeneration. Citation Format: Koushik K. Das, Steffen Heeg, Maximilian Reichert, Shigetsugu Takano, Basil S. Bakir, Gregory P. Botta, Christopher Hahn, Andrew D. Rhim, Anil K. Rustgi. Ets transcription factor Etv5 regulates ductal morphogenesis and differentiation in association with Sox9 in vitro and increases susceptibility and delays recovery from pancreatitis in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A11.

Histone deacetylase 3 expression correlates with vasculogenic mimicry through the phosphoinositide3-kinase/ERK-MMP-laminin5γ2 signaling pathway.

Vasculogenic mimicry (VM) refers to the process by which highly aggressive tumor cells mimic endothelial cells to form vessel-like structures that aid in supplying enough nutrients to rapidly growing tumors. Histone deacetylases (HDACs) regulate the expression and activity of numerous molecules involved in cancer initiation and progression. Notably, HDAC3 is overexpressed in the majority of carcinomas. However, thus far, no data are available to support the role of HDAC3 in VM. In this study, we subjected glioma specimens to immunohistochemical and histochemical double-staining methods and found that VM and HDAC3 expression were related to the pathological grade of gliomas. The presence of VM correlated with HDAC3 expression in glioma tissues. The formation of tubular structures, as determined by the tube formation assay to evaluate VM, was impaired in U87MG cells when transfected by siRNA or treated with an HDAC3 inhibitor. Importantly, the expression of VM-related molecules such as MMP-2/14 and laminin5γ2 was also affected when HDAC3 expression was altered. Furthermore, U87MG cells were treated with a phosphoinositide 3-kinase (PI3K) inhibitor or/and ERK inhibitor and found that the PI3K and ERK signaling pathways play key roles in VM; whereas, in VM, the two signaling pathways did not act upstream or downstream from each other. Taken together, our findings showed that HDAC3 contributed to VM in gliomas, possibly through the PI3K/ERK–MMPs–laminin5γ2 signaling pathway, which could potentially be a novel therapeutic target for gliomas.

Mitochondrial Genome Maintenance 1 (Mgm1) Protein Alters Membrane Topology and Promotes Local Membrane Bending

Large GTPases of the dynamin superfamily promote membrane fusion and division, processes that are crucial for intracellular trafficking and organellar dynamics. To promote membrane scission, dynamin proteins polymerize, wrap around, and constrict the membrane; however, the mechanism underlying their role in membrane fusion remains unclear. We previously reported that the mitochondrial dynamin-related protein mitochondrial genome maintenance 1 (Mgm1) mediates fusion by first tethering opposing membranes and then undergoing a nucleotide-dependent structural transition. However, it is still unclear how Mgm1 directly affects the membrane to drive fusion of tethered membranes. Here, we show that Mgm1 association with the membrane alters the topography of the membrane, promoting local membrane bending. We also demonstrate that Mgm1 creates membrane ruffles resulting in the formation of tubular structures on both supported lipid bilayers and liposomes. These data suggest that Mgm1 membrane interactions impose a mechanical force on the membrane to overcome the hydrophilic repulsion of the phospholipid head groups and initiate the fusion reaction. The work reported here provides new insights into a possible mechanism of Mgm1-driven mitochondrial membrane fusion and sheds light into how members of the dynamin superfamily function as fusion molecules.