Vn Expression Research Articles

High glucose/ox-LDL induced hepatic sinusoidal capillarization via αvβ5/FAK/ERK signaling pathway

ObjectiveThe main purpose of this study is to explore the role of integrin αvβ5 in hepatic sinusoidal capillarization under high glucose/ox-LDL conditions. MethodsEstablish rat model of diabetic fatty liver disease. LSECs were extracted from tissue obtained from rats of control group, cultured and treated with media containing glucose (25 mM, 24 h)/ox-LDL (100 μg/ml, 24 h) in different inhibitors. The expression of integrin αvβ5, FAK, ERK, VN in LSECs were detected by RT-PCR and Western blot. Hematoxylin-eosin staining and gomori methenaminutese silver stain was used to observe the basement membrane histopathological features of the liver tissue. Immunohistochemical to detected the protein expression of integrin αvβ5 and VN in liver tissue. Using scanning electron microscopy to visualise the fenestration frequency and fenestration diameter. Protein expression of VN was also testified by immunofluorescence assay. Results1.The expression of integrin αv, integrin β5, FAK, ERK, VN increased in a time- and concentration-dependent manner under high glucose or oxLDL. This effect is the most significant when they co-exist (P < 0.05).2.The expression of ERK, FAK and VN was down-regulated when LSECs were treated with.the integrin αvβ5 inhibitor RGDfv. DF228, inhibitor of FAK only suppressed expression of ERK and VN. Furthermore, VN expression was down-regulated by intervention of LSECs with the ERK inhibitor PD98059, while the expression of integrin αvβ5 and FAK was not significantly changed (P < 0.05).3.We observed that high glucose and oxLDL can lead to decreasing in the porosity, diameter.and number of fenestrae compared with the control group, which is more significant in the combination of high glucose and oxLDL (P < 0.05).4.Result of gomori methenaminutese silver stain showed that the T2DM + NAFLD group had.more obvious and darker brown coloration in basement membrane and sinus space. HE staining showed that in the T2DM + NAFLD group, the hepatocytes were loose in structure and disordered in arrangement, with a large number of lipid droplets in some cytoplasm, and most hepatocytes showed steatosis. In immunohistochemical staining, the positive areas of integrin αvβ5 and VN showed a increasing trend from the control group to T2DM group, NAFLD group and T2DM + NAFLD group (P < 0.05). ConclusionsIntegrin αvβ5 which induces LSECs dysfunction, promoting hepatic sinusoidal capillarization regulates VN expression via ERK/FAK pathway under high glucose/ox-LDL, may be a potential target for prevention and treatment of T2DM with fatty liver disease.

Evaluation of Mesenchymal stem cell adhesion to woven polymer meshes

Event Abstract Back to Event Evaluation of Mesenchymal stem cell adhesion to woven polymer meshes Jordon Gilmore1 and Karen J. Burg2 1 Clemson University, Bioengineering, United States 2 University of Georgia, Small Animal Medicine and Surgery, United States Introduction: Initial protein adhesion may be dominated by serum-based proteins such as Fibronectin (Fn) or Vitronectin (Vn). This layer may be quickly adsorbed or remodeled by the attaching cells. These cells may then express proteins such as Collagen (Col1) or Laminin (Lam2) that may attach to the biomaterial surface[1]. The goal of this work was to characterize the degree to which extracellular matrix (ECM) proteins adhered to woven poly-l-lactide (PL; Natureworks LLC, 2003d biopolymer, ~ 228,000 Da) meshes over 28 days. A change in the dominant surface protein over time was expected to correlate with the adhesion, proliferation, and differentiation of cells on the mesh. Materials and Methods: Murine bone marrow stromal D1 cells were cultured for 7-10 days in 24-well untreated, low-attachment polystyrene culture plates (Corning). Cells were seeded directly onto woven PL meshes. After day 10, medium was supplemented with an osteogenic cocktail (ascorbic acid (50 µg/mL), and beta-glycerophosphate (0.01 M)) for an additional 28 days. Adhesion of Fn, Vn, Col1, and Lam2 to the meshes over 28 days was evaluated through immuno-fluorescence (IF). Polyclonal primary antibodies rabbit-anti fibronectin, vitronectin, laminin alpha 2, and collagen type 1 were used (Bioss USA). A goat-anti-rabbit IGG antibody, conjugated with Alexa Fluor 488 was used as a secondary antibody. At 7 day intervals, samples were analyzed on a microplate reader (490 nm excitation, 525 nm emission). Results and Discussion: Results (Figure 1) show a significant change in Vn expression over the 28 days (p = 0.0031). Other proteins show a constant level of adhesion over time. These results suggest little displacement and reattachment by Fn, Col1, and Lam2 during the study. This constant expression was contrary to the expected increase in expression as osteoblastic differentiation was advanced. These results suggest that cell syntheses and deposition of Col1 and Lam2 into the ECM and onto meshes was not variable during the ECM maturation and mineralization phases of differentiation. Lam2 expression did not differ significantly from Vn or Col1 expression, suggesting that many of the attached cells may have been in the early stages of differentiation. Prior work has suggested that adhesion of MSCs differentiating toward osteoblasts is dominated by Laminins, followed by Vn, Col1, and lastly FN[2]. Lam2 is expressed equally with the two proteins cited to be strongly related to osteogenic differentiation, suggesting heterogeneity of differentiation level within the cells attached to the mesh. Perhaps early stage cells adhered primarily through Lam2, and mature cells through Col1 and Vn. Conclusion: This study demonstrated a heterogenic expression of ECM proteins during adhesion and differentiation. Further work will look to direct cell responses on meshes using ECM proteins.

FGF coordinates air sac development by activation of the EGF ligand Vein through the transcription factor PntP2.

How several signaling pathways are coordinated to generate complex organs through regulation of tissue growth and patterning is a fundamental question in developmental biology. The larval trachea of Drosophila is composed of differentiated functional cells and groups of imaginal tracheoblasts that build the adult trachea during metamorphosis. Air sac primordium cells (ASP) are tracheal imaginal cells that form the dorsal air sacs that supply oxygen to the flight muscles of the Drosophila adult. The ASP emerges from the tracheal branch that connects to the wing disc by the activation of both Bnl-FGF/Btl and EGFR signaling pathways. Together, these pathways promote cell migration and proliferation. In this study we demonstrate that Vein (vn) is the EGF ligand responsible for the activation of the EGFR pathway in the ASP. We also find that the Bnl-FGF/Btl pathway regulates the expression of vn through the transcription factor PointedP2 (PntP2). Furthermore, we show that the FGF target gene escargot (esg) attenuates EGFR signaling at the tip cells of the developing ASP, reducing their mitotic rate to allow proper migration. Altogether, our results reveal a link between Bnl-FGF/Btl and EGFR signaling and provide novel insight into how the crosstalk of these pathways regulates migration and growth.

Abstract 1985: Vitronectin expression in primary lung cancers

Abstract Rationale Vitronectin (Vn) plays a role in extracellular matrix (ECM) remodeling during tumorigenesis. Vn is present in human bronchial submucosal glands and it is secreted by A549 lung adenocarcinoma cell line. However, whether Vn is differentially expressed in tissues of primary lung cancer has not been explored. Methods Lung tissue from 22 primary lung carcinoma and 36 non-lung cancer subjects were obtained from fibrobronchoscopy. Sections were analyzed by histo- immunohistochemical methods. The total area occupied by ECM, surface and gland epithelium, as well as the percentage area occupied by Vn at each of these localizations were measured. Chi-square, t-student, U-Mann Whitney and ANOVA tests were used in group comparisons. Statistical significance was tested at P&amp;lt;0.05. Results Vn expression was observed in bronchial glandular and surface epithelium, as well as in ECM . In total, the average area of ECM, surface or glandular epithelium was 0.289mm2 (± 0.032), 0.043mm2 (±0.009), 0.084mm2 (±0.031), respectively. The percentage area occupied by Vn in ECM, surface or glandular epithelium, was 4.899 (±1.186), 7.279 (±1.623), 3.285 (±1.038), respectively; with not significant statistical differences between lung cancer and non-cancer subjects or within different types of primary lung cancer (Table 1). Conclusion There were no statistically significant differences in Vn expression between lung cancer and non-cancer subjects, or within primary lung carcinomas. Demographic characteristics and measurement comparisons of subjects studiedConditionNon-cancer(n=36)Cancer(n=22)PAge (Years)59.810(+/- 13.556)65.790(+/- 10.648)0.074Sex*0.752Male18 (50.0%)11 (50.0%)Female18 (50.0%)11 (50.0%)ECM area (mm2)0.311(+/- 0.204)0.424(+/- 0.246)0.059Area occupied by Vn in ECM (percentage)2.894(+/- 2.717)3.584(+/- 3.220)0.384Surface epithelium area (mm2)0.221(+/- 0.678)0.052(+/- 0.023)0.398Area occupied by Vn in surface epithelium (percentage)5.583(+/- 3.939)6.478(+/- 7.757)0.662Gland epithelium area (mm2)0.142(+/- 0.129)0.086(+/- 0.079)0.389Area occupied by Vn in glandular epithelium (percentage)4.151(+/- 2.810)1.424(+/- 0.505)0.200 Note: This abstract was not presented at the meeting. Citation Format: Lina M. Salazar-Pelaez, Ivan Martinez-Forero. Vitronectin expression in primary lung cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1985. doi:10.1158/1538-7445.AM2014-1985

Trachea-Derived Dpp Controls Adult Midgut Homeostasis in Drosophila

Homeostasis in adult tissues is maintained by resident stem cells and their progeny. Little is known about the regulation of tissue homeostasis by organ-organ interaction. Here we demonstrate that trachea-derived Decapentaplegic (Dpp), the main bone morphogenetic protein ligand in Drosophila, is essential for adult midgut homeostasis. We show that Dpp signaling is primarily activated in enterocytes (ECs). Depletion of Dpp signaling in ECs results in excess amounts of intestinal stem-cell-like cells and their progeny. Importantly, we find that Dpp is expressed specifically in tracheal cells that reach the intestinal cells through the visceral muscles. Depletion of dpp expression in tracheal cells phenocopies the Dpp loss-of-function defects in ECs. Our data demonstrate that the Drosophila trachea not only exchanges air for bodily needs but also produces a Dpp morphogen essential for neighboring tissue homeostasis. This work will provide important insights into the mechanisms of tissue homeostasis control by interorgan communication.

Developmental expression of prion protein and its ligands stress‐inducible protein 1 and vitronectin

Prion protein (PrP(C)) is the normal isoform of PrP(Sc), a protein involved in neurodegenerative disorders. PrP(C) participates in neuritogenesis, neuroprotection, and memory consolidation through its interaction with the secreted protein stress-inducible protein 1 (STI1) and the extracellular matrix protein vitronectin (Vn). Although PrP(C) mRNA expression has been documented during embryogenesis, its protein expression patterns have not been evaluated. Furthermore, little is known about either Vn or STI protein expression. In this study, PrP(C), STI1, and Vn protein expression was explored throughout mouse embryonic life. We found that the distributions of the three proteins were spatiotemporally related. STI1 and Vn expression became evident at E8, earlier than PrP(C), in the nervous system and heart. At E10, we observed, in the spinal cord, a gradient of expression of the three proteins, more abundant in the notochord and floor plate, suggesting that they can have a role in axonal growth. As development proceeded, the three proteins were detected in other organs, suggesting that they may play a role in the development of nonneural tissues as well. Finally, although STI1 and Vn are PrP(C) ligands, their expression was not altered in PrP(C)-null mice.

Control of growth and patterning of the Drosophila wing imaginal disc by EGFR-mediated signaling.

The subdivision of the Drosophila wing imaginal disc into dorsoventral (DV) compartments and limb-body wall (wing-notum) primordia depends on Epidermal Growth Factor Receptor (EGFR) signaling, which heritably activates apterous (ap) in D compartment cells and maintains Iroquois Complex (Iro-C) gene expression in prospective notum cells. We examine the source, identity and mode of action of the EGFR ligand(s) that specify these subdivisions. Of the three known ligands for the Drosophila EGFR, only Vein (Vn), but not Spitz or Gurken, is required for wing disc development. We show that Vn activity is required specifically in the dorsoproximal region of the wing disc for ap and Iro-C gene expression. However, ectopic expression of Vn in other locations does not reorganize ap or Iro-C gene expression. Hence, Vn appears to play a permissive rather than an instructive role in organizing the DV and wing-notum segregations, implying the existance of other localized factors that control where Vn-EGFR signaling is effective. After ap is heritably activated, the level of EGFR activity declines in D compartment cells as they proliferate and move ventrally, away from the source of the instructive ligand. We present evidence that this reduction is necessary for D and V compartment cells to interact along the compartment boundary to induce signals, like Wingless (Wg), which organize the subsequent growth and differentiation of the wing primordium.

Tissue-Specific Regulation of Vein/EGF Receptor Signaling in Drosophila

Signaling by the Drosophila EGF receptor (DER) is modulated by four known EGF-like proteins: the agonists Vein (Vn), Spitz (Spi), and Gurken (Grk) and the antagonist Argos (Aos). DER is broadly expressed and thus tissue-specific regulation of ligand expression and activity is an important mechanism for controlling signaling. Here we investigate the tissue-specific regulation of Vn signaling by examining vn transcriptional control and Vn target gene activation in the embryo and the wing. The results show a complex temporal and spatial regulation of vn transcription involving multiple signaling pathways and tissue-specific activation of Vn target genes. In the embryo, vn is a target of Spi/DER signaling mediated by the ETS transcription factor PointedP1 (PntP1). This establishes a positive feedback loop in addition to the negative feedback loop involving Aos. The simultaneous production of Vn provides a mechanism for dampening Aos inhibition and thus fine-tunes signaling. In the larval wing pouch, vn is not a target of Spi/DER signaling but is expressed along the anterior–posterior boundary in response to Hedgehog (Hh) signaling. Repression by Wingless (Wg) signaling further refines the vn expression pattern by causing a discontinuity at the dorsal–ventral boundary. The potential for vn to activate DER target genes correlates with its roles in development: vn has a minor role in embryogenesis and does not induce DER target genes such as aos and pntP1 in the embryo. Conversely, vn has a major role in wing development and Vn/DER signaling is a potent inducer of DER target genes in the wing disc. Spi also has the potential to induce DER target genes in the wing disc. However, the ligands appear to evoke specific responses that result in different patterns of target gene expression. Finally, we show that other factors modulate the potential of Vn so that induction of Vn/DER target genes in the wing pouch is cell specific.

Hedgehog activates the EGF receptor pathway during Drosophila head development.

The Hedgehog (Hh) and Epidermal growth factor receptor (EGFR) signaling pathways play critical roles in pattern formation and cell proliferation in invertebrates and vertebrates. In this study, we demonstrate a direct link between these two pathways in Drosophila melanogaster. Hh and EGFR signaling are each required for the formation of a specific region of the head of the adult fruitfly. We show that hh and vein (vn), which encodes a ligand of the Drosophila EGFR (Schnepp, B., Grumbling, G., Donaldson, T. and Simcox, A. (1996) Genes Dev. 10, 2302-13), are expressed in adjacent domains within the imaginal primordium of this region. Using loss- and gain-of-function approaches, we demonstrate that Hh activates vn expression. We also show that Hh activation of vn is mediated through the gene cubitus interruptus (ci) and that this activation requires the C-terminal region of the Ci protein. Finally, we demonstrate that wingless (wg) represses vn expression, thereby limiting the domain of EGFR signaling.

Tumor necrosis factor-α stimulates attachment of small cell lung carcinoma to endothelial cells

Tumor cell attachment to endothelial cells (ECs) is an important step in the metastasis of small cell lung carcinoma (SCLC). Tumor necrosis factor-α (TNF-α) stimulation of ECs increases the attachment of some malignant cell types to ECs by affecting the expression of cell adhesion molecules (CAMs). Similarly, the inhibition of EC protein kinase C (PKC) and tyrosine kinase (TK) pathways modulates TNF-α-mediated effects on CAM expression. We hypothesized that TNF-α would increase SCLC attachment to ECs by affecting CAM expression through activation of PKC and TK pathways. To test this hypothesis, human umbilical vein endothelial cells (HUVECs) were stimulated with TNF-α (0 to 500 U/mL) for variable time periods (1 to 24 hours), and the attachment of H82 cells (an SCLC cell line) to the HUVECs was quantified. TNF-α stimulation of the HUVECs increased H82 attachment from 28.1% ± 1.6% to 48.8% ± 1.7% (P < .05). Preincubation of HUVECs with the PKC inhibitors bis-indolylmaleimide (BIN) or calphostin C or the TK inhibitors genistein or herbimycin A (HMA) blocked the TNF-α-induced increase in H82 cell attachment. The addition of antibodies to vitronectin (Vn) or β1-integrin to TNF-α-activated HUVECs before the addition of the H82 cells also significantly decreased H82 attachment, whereas the addition of antibodies to E-selectin, P-selectin, vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM), neural cell adhesion molecule (NCAM), sialyl-Lewisx, fibronectin (Fn), αv-integrin, α3-integrin, α4-integrin, or α5-integrin had no effect on SCLC attachment. In summary, the TNF-α-mediated increase in SCLC attachment to ECs appears to be mediated by the activation of EC PKC and TK pathways as well as through effects on the function or expression of EC Vn and β1 integrin.

Human Ovarian Adenocarcinoma Cells Synthesize Vitronectin and Use It to Organize Their Adhesion

Extracellular matrix components and integrin receptors are frequently altered in cancer, including ovarian adenocarcinoma. Vitronectin (Vn) is a matrix protein mainly synthesized by liver cells; it is present in normal ovarian surface epithelium and differentiated ovarian adenocarcinoma, but is frequently undetectable in undifferentiated carcinoma (F. Carreiraset al.,1996, Gynecol Oncol 62:260–267). Wondering about the cellular origin of Vn in ovarian carcinoma, we searched for evidence of Vn synthesis by these tumors. We demonstrated that three human ovarian adenocarcinoma cell lines were able to synthesize Vn, as revealed by the presence of Vn mRNA and the protein. The Vn matrix promotes adhesion of ovarian tumor cells through αv integrins. Moreover, duringin vitrogrowth, Vn is progressively organized into a particular pattern in combination with the recruitment of αv into focal contacts. Our results suggest that Vn synthesis may participate in ovarian adenocarcinoma cell biology and raise the possibility that altered expression of Vn in some ovarian carcinomas could result from a defect in Vn synthesis.

Vein is a novel component in the Drosophila epidermal growth factor receptor pathway with similarity to the neuregulins.

The activation signal from tyrosine kinase receptors, such as the epidermal growth factor receptor (EGFR), is relayed via a highly conserved intracellular pathway involving Ras, Raf, and MAPK. In Drosophila, the EGFR and components of the intracellular pathway are broadly expressed, yet receptor activation evokes tissue-specific cell responses. Extracellular events that lead to receptor activation are one mechanism by which signaling is modulated. Here we show molecular and genetic evidence that Drosophila vein (vn) encodes a candidate EGFR ligand and that vn expression is spatially restricted. Consequently, vn may promote tissue-specific receptor activation. Unlike two other ligands, Gurken (Grk) and Spitz (Spi), which are transforming growth factor alpha-like proteins, Vn has both an immunoglobulin-like and an EGF-like domain. This combination of domains mirrors those in the vertebrate neuregulins that bind EGFR relatives.

Expression and Localization of αv Integrins and Their Ligand Vitronectin in Normal Ovarian Epithelium and in Ovarian Carcinoma

In an extension of a previousin vitrostudy [Carreiraset al., Int. J. Cancer63, 530–536 (1995)] and in an effort to understand the adhesive interactions mediated by integrins within epithelial ovarian tumors, the presence of the αv and β3 subunits and that of vitronectin (Vn) in ovarian carcinomas at various stages of differentiation and in normal ovarian epithelium were comparatively investigated. The study was performed on material from 34 patients. By immunofluorescence, cryostat sections were analyzed for their expression of αv (34 cases), β3 (19 cases), and Vn (29 cases). αv was expressed in normal epithelium and in highly differentiated tumors as well as in a majority of moderately and poorly differentiated carcinomas with identical staining pattern. β3 subunit and Vn were also expressed in normal cases and highly differentiated carcinomas. However, they were lacking in most of the less differentiated tumors. The analysis of cases which were simultaneously tested for the presence of αv, β3, and Vn revealed that a large proportion of normal ovarian epithelium and highly differentiated tumors simultaneously expressed αv, β3, and Vn; in contrast, in all moderately and poorly differentiated carcinomas either β3 or Vn was absent. The potential role of the αvβ3/Vn system in ovarian epithelium functions is discussed. It is also speculated that modifications of this system in ovarian carcinomas might contribute to tumor progression.

Altered expression of extracellular matrix molecules and their receptors in chronic pancreatitis and pancreatic adenocarcinoma in comparison with normal pancreas

The aim of this study was to elucidate the expression and distribution patterns of both integrins and extracellular matrix (ECM) molecules in chronic pancreatitis (CP) and pancreatic adenocarcinoma (PC) compared with normal pancreas (NP). Expression of nine alpha-subunits (alpha 2-alpha 6, alpha V, alpha L, alpha M, and alpha X), four beta-subunits (beta 1, beta 3-beta 5), and four ECM molecules (type IV collagen, laminin, fibronectin, and vitronectin) was investigated immunohistochemically. In CP, all integrins except alpha V showed nearly the same staining patterns compared with NP. Some acinar cells in CP expressed alpha V. Whereas alpha 2, alpha 3, and alpha 6 expression was stronger and diffuse, no alpha 5 expression was seen in PC. Basement membrane (BM) showed continuous staining in CP, whereas it showed discontinuous/absent staining in PC with antitype IV collagen, laminin, and vitronectin antibodies. Some carcinoma cells showed reverse correlation between alpha 2, alpha 3, and alpha 6 expression and type IV collagen and laminin expression. Fibronectin showed diffuse stromal expression in CP and PC. Some acinar cells or duct cells in CP carcinoma cells in PC showed intracellular VN expression. These results suggest that these integrins and ECM molecules are involved in inflammatory and malignant processes in pancreas.

IL-6 stimulates vitronectin gene expression in vivo.

We tested the hypothesis that vitronectin (Vn) is regulated as an acute phase reactant in response to inflammatory stimuli. In initial experiments, Vn levels were measured during the surgically induced acute phase response in humans. The plasma concentration of Vn increased approximately twofold following elective orthopedic surgery and remained elevated up to 5 days. To examine the mechanism(s) of increased Vn synthesis, hepatic Vn mRNA expression and serum levels were examined in three rat models of acute inflammation: LPS (i.v.), CFA (i.p.), or turpentine (s.c.) injection. The serum concentration of Vn increased approximately twofold 24 h following treatment with turpentine. The expression of Vn mRNA in the liver increased markedly as early as 3 h after treatment in these models and remained elevated up to 18 h. Northern blot analysis of RNA isolated from fractionated liver cells derived from rats treated with LPS indicated that Vn was mainly expressed in hepatocytes, but not in the endothelial or nonparenchymal cell fractions. To analyze the individual effects of raised corticosterone and IL-6 levels on the expression of hepatic Vn mRNA, rats were injected (i.p.) with either dexamethasone or purified recombinant rat IL-6. Vn mRNA expression was elevated within 1 h after IL-6 injection, whereas dexamethasone-injected rats showed unchanged Vn expression. Vn mRNA also was increased in rats chronically injected with IL-6. These results indicate that the Vn gene is up-regulated in acute and chronic inflammation, and this induction is primarily mediated by IL-6.

Expression of vitronectin and fibronectin binding by Candida albicans yeast cells

Expression of binding to vitronectin (Vn or S-protein) and fibronectin (Fn) was common among clinical isolates of Candida albicans. Growth at 37 degrees C enhanced expression of both Vn and Fn binding. Some strains expressed higher binding after growth in liquid media and others after growth on solid media. Most strains expressed higher cell surface hydrophobicity after growth on agar media. Vn binding was less influenced by expression of cell surface hydrophobicity than Fn binding. Vn binding to yeast cells was optimal around pH 4 and Fn binding around pH 6. Binding to soluble Vn was inhibited by unlabelled Vn and to a lesser extent by Fn. Fn binding to the same C. albicans strain was inhibited by unlabelled Fn, Vn, fibrinogen and to some extent collagens. C. albicans strain 3248 expressed specific high binding of Vn, and high binding of Fn. Binding of both proteins was sensitive to heat and protease treatment, but in different ways. Vn binding differed significantly from the earlier reported Fn binding and may represent a novel type of tissue adherence.