ResearchPad - adhesion-molecules https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Functional and structural consequences of epithelial cell invasion by <i>Bordetella pertussis</i> adenylate cyclase toxin]]> https://www.researchpad.co/article/elastic_article_7693 Bordetella pertussis, the causative agent of whopping cough, produces an adenylate cyclase toxin (CyaA) that plays a key role in the host colonization by targeting innate immune cells which express CD11b/CD18, the cellular receptor of CyaA. CyaA is also able to invade non-phagocytic cells, via a unique entry pathway consisting in a direct translocation of its catalytic domain across the cytoplasmic membrane of the cells. Within the cells, CyaA is activated by calmodulin to produce high levels of cyclic adenosine monophosphate (cAMP) and alter cellular physiology. In this study, we explored the effects of CyaA toxin on the cellular and molecular structure remodeling of A549 alveolar epithelial cells. Using classical imaging techniques, biochemical and functional tests, as well as advanced cell mechanics method, we quantify the structural and functional consequences of the massive increase of intracellular cyclic AMP induced by the toxin: cell shape rounding associated to adhesion weakening process, actin structure remodeling for the cortical and dense components, increase in cytoskeleton stiffness, and inhibition of migration and repair. We also show that, at low concentrations (0.5 nM), CyaA could significantly impair the migration and wound healing capacities of the intoxicated alveolar epithelial cells. As such concentrations might be reached locally during B. pertussis infection, our results suggest that the CyaA, beyond its major role in disabling innate immune cells, might also contribute to the local alteration of the epithelial barrier of the respiratory tract, a hallmark of pertussis.

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<![CDATA[Aspirin-triggered resolvin D1 attenuates PDGF-induced vascular smooth muscle cell migration via the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway]]> https://www.researchpad.co/article/5989db50ab0ee8fa60bdc0e7

Background and objectives

Resolvin D1 (RvD1) is a specialized pro-resolving lipid mediator that has been previously shown to attenuate vascular smooth muscle cell (VSMC) migration, a key process in the development of intimal hyperplasia. We sought to investigate the role of the cAMP/PKA pathway in mediating the effects of the aspirin-triggered epimer 17R-RvD1 (AT-RvD1) on VSMC migration.

Methods

VSMCs were harvested from human saphenous veins. VSMCs were analyzed for intracellular cAMP levels and PKA activity after exposure to AT-RvD1. Platelet-derived growth factor (PDGF)-induced migration and cytoskeletal changes in VSMCs were observed through scratch, Transwell, and cell shape assays in the presence or absence of a PKA inhibitor (Rp-8-Br-cAMP). Further investigation of the pathways involved in AT-RvD1 signaling was performed by measuring Rac1 activity, vasodilator stimulated phosphoprotein (VASP) phosphorylation and paxillin translocation. Finally, we examined the role of RvD1 receptors (GPR32 and ALX/FPR2) in AT-RvD1 induced effects on VSMC migration and PKA activity.

Results

Treatment with AT-RvD1 induced a significant increase in cAMP levels and PKA activity in VSMCs at 5 minutes and 30 minutes, respectively. AT-RvD1 attenuated PDGF-induced VSMC migration and cytoskeletal rearrangements. These effects were attenuated by the PKA inhibitor Rp-8-Br-cAMP, suggesting cAMP/PKA involvement. Treatment of VSMC with AT-RvD1 inhibited PDGF-stimulated Rac1 activity, increased VASP phosphorylation, and attenuated paxillin localization to focal adhesions; these effects were negated by the addition of Rp-8-Br-cAMP. The effects of AT-RvD1 on VSMC migration and PKA activity were attenuated by blocking ALX/FPR2, suggesting an important role of this G-protein coupled receptor.

Conclusions

Our results suggest that AT-RvD1 attenuates PDGF-induced VSMC migration via ALX/FPR2 and cAMP/PKA. Interference with Rac1, VASP and paxillin function appear to mediate the downstream effects of AT-RvD1 on VSMC migration.

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<![CDATA[The role of embryo contact and focal adhesions during maternal recognition of pregnancy]]> https://www.researchpad.co/article/5c8823f5d5eed0c484639429

Maternal recognition of pregnancy (MRP) in the mare is an unknown process. In a non-pregnant mare on day 14 post-ovulation (PO), prostaglandin F (PGF) is secreted by the endometrium causing regression of the corpus luteum. Prior to day 14, MRP must occur in order to attenuate secretion of PGF. The embryo is mobile throughout the uterus due to uterine contractions from day of entry to day 14. It is unknown what signaling is occurring. Literature stated that infusing oil or placing a glass marble into the equine uterus prolongs luteal lifespan and that in non-pregnant mares, serum exosomes contain miRNA that are targeting the focal adhesion (FA) pathway. The hypothesis of this study is embryo contact with endometrium causes a change in abundance of focal adhesion molecules (FA) in the endometrium leading to decrease in PGF secretion. Mares (n = 3/day) were utilized in a cross-over design with each mare serving as a pregnant and non-pregnant (non-mated) control on days 9 and 11 PO. Mares were randomly assigned to collection day and endometrial samples and embryos were collected on the specified day. Biopsy samples were divided into five pieces, four for culture for 24 hours and one immediately snap frozen. Endometrial biopsies for culture were placed in an incubator with one of four treatments: [1] an embryo in contact on the luminal side of the endometrium, [2] beads in contact on the luminal side of the endometrium, [3] peanut oil in contact on the luminal side of the endometrium or [4] the endometrium by itself. Biopsies and culture medium were frozen for further analysis. RNA and protein were isolated from biopsies for PCR and Western blot analysis for FA. PGF assays were performed on culture medium to determine concentration of PGF. Statistics were performed using SAS (P ≤ 0.05 indicated significance). The presence of beads on day 9 impacted samples from pregnant mares more than non-pregnant mares and had very little impact on day 11. Presence of oil decreased FA in samples from pregnant mares on day 9. On day 11, oil decreased FA abundance in samples from non-pregnant mares. Embryo contact caused multiple changes in RNA and protein abundance in endometrium from both pregnant and non-pregnant mares. The PGF secretion after 24 hours with each treatment was also determined. On day 9, there was no change in PGF secretion compared to any treatments. On day 11, presence of peanut oil increased PGF secretion in samples from non-pregnant mares. In samples from non-pregnant mares, presence of an embryo decreased PGF secretion compared to control samples from non-pregnant mares. Results revealed that while beads and peanut oil may impact abundance of FA RNA and protein in endometrial samples, it does not appear to impact PGF secretion. Conversely, embryo contact for 24 hours with endometrium from a non-pregnant mare causes a decrease in PGF secretion. These results suggest that it is not just contact of any substance/object causing attenuation of PGF secretion, but the embryo itself is necessary to decrease PGF secretion.

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<![CDATA[Equilin in conjugated equine estrogen increases monocyte-endothelial adhesion via NF-κB signaling]]> https://www.researchpad.co/article/5c5b5254d5eed0c4842bc66d

The adhesion of monocytes to endothelial cells, which is mediated by adhesion molecules, plays a crucial role in the onset of atherosclerosis. Conjugated equine estrogen, which is widely used for estrogen-replacement therapy, contains both estrone sulfate and various nonhuman estrogens, including equilin. To investigate the association between various estrogen types and atherosclerosis risk, we examined their effect on adhesion-molecule expression in human umbilical vein endothelial cells (HUVECs). In estrogen-treated HUVECs, the mRNA and protein expression levels of adhesion molecules were quantified by real-time polymerase chain reaction and enzyme immunoassay. Additionally, a flow-chamber system was used to assess the effects of estrogens on the adherence of U937 monocytoid cells to HUVECs. Equilin, but not 17β-estradiol (E2) or other types of estrogen, significantly increased the mRNA (P < 0.01) and protein (P < 0.05) expression of the adhesion molecules E-selectin and intercellular adhesion molecule-1 as compared with levels in controls. Equilin treatment increased the adherence of U937 monocytoid cells to HUVECs relative to the that in the control (P < 0.05), decreased estrogen receptor (ER)β expression, and increased the expression of proteins involved in nuclear factor kappa-B (NF-κB) activation relative to levels in controls. Furthermore, the accumulation of NF-κB subunit p65 in HUVEC nuclei was promoted by equilin treatment. By contrast, E2 treatment neither increased the number of adhered monocytoid cells to HUVECs nor altered the expression of ERβ or NF-κB-activating proteins. Our findings suggest that in terms of the adhesion of monocytes at the onset of atherosclerosis, E2 may be preferable for estrogen-replacement therapy. Further studies comparing equilin treatment with that of E2 are needed to investigate their differential impacts on atherosclerosis.

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<![CDATA[Fluctuations in cell density alter protein markers of multiple cellular compartments, confounding experimental outcomes]]> https://www.researchpad.co/article/5c61e8c6d5eed0c48496f14c

The life cycle of cultured proliferating cells is characterized by fluctuations in cell population density induced by periodic subculturing. This leads to corresponding changes in micro- and macroenvironment of the cells, accompanied by altered cellular metabolism, growth rate and locomotion. Studying cell density-dependent morphological, physiological and biochemical fluctuations is relevant for understanding basic cellular mechanisms and for uncovering the intrinsic variation of commonly used tissue culture experimental models. Using multiple cell lines, we found that expression levels of the autophagic markers p62 and LC3II, and lysosomal enzyme cathepsin D were altered in highly confluent cells as a consequence of nutrient depletion and cell crowding, which led to inactivation of the mTOR signaling pathway. Furthermore, both Lamp1 and active focal adhesion kinase (FAK) were reduced in high-density cells, while chemical inhibition or deletion of FAK led to alterations in lysosomal and autophagic proteins, as well as in the mTOR signaling. This was accompanied by alterations in the Hippo signaling pathway, while cell cycle checkpoint regulator p-cdc2 remained unaffected in at least one studied cell line. On the other hand, allometric scaling of cellular compartments in growing cell populations resulted in biochemically detectable changes in the plasma membrane proteins Na+K+-ATPase and cadherin, and nuclear proteins HDAC1 and Lamin B1. Finally, we demonstrate how treatment-induced changes in cell density and corresponding modulation of susceptible proteins may lead to ambiguous experimental outcomes, or erroneous interpretation of cell culture data. Together, our data emphasize the need to recognize cell density as an important experimental variable in order to improve scientific rigor of cell culture-based studies.

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<![CDATA[Insights into the Utility of the Focal Adhesion Scaffolding Proteins in the Anaerobic Fungus Orpinomyces sp. C1A]]> https://www.researchpad.co/article/5989dad3ab0ee8fa60bb7154

Focal adhesions (FAs) are large eukaryotic multiprotein complexes that are present in all metazoan cells and function as stable sites of tight adhesion between the extracellular matrix (ECM) and the cell’s cytoskeleton. FAs consist of anchor membrane protein (integrins), scaffolding proteins (e.g. α-actinin, talin, paxillin, and vinculin), signaling proteins of the IPP complex (e.g. integrin-linked kinase, α-parvin, and PINCH), and signaling kinases (e.g. focal adhesion kinase (FAK) and Src kinase). While genes encoding complete focal adhesion machineries are present in genomes of all multicellular Metazoa; incomplete machineries were identified in the genomes of multiple non-metazoan unicellular Holozoa, basal fungal lineages, and amoebozoan representatives. Since a complete FA machinery is required for functioning, the putative role, if any, of these incomplete FA machineries is currently unclear. We sought to examine the expression patterns of FA-associated genes in the anaerobic basal fungal isolate Orpinomyces sp. strain C1A under different growth conditions and at different developmental stages. Strain C1A lacks clear homologues of integrin, and the two signaling kinases FAK and Src, but encodes for all scaffolding proteins, and the IPP complex proteins. We developed a protocol for synchronizing growth of C1A cultures, allowing for the collection and mRNA extraction from flagellated spores, encysted germinating spores, active zoosporangia, and late inactive sporangia of strain C1A. We demonstrate that the genes encoding the FA scaffolding proteins α-actinin, talin, paxillin, and vinculin are indeed transcribed under all growth conditions, and at all developmental stages of growth. Further, analysis of the observed transcriptional patterns suggests the putative involvement of these components in alternative non-adhesion-specific functions, such as hyphal tip growth during germination and flagellar assembly during zoosporogenesis. Based on these results, we propose putative alternative functions for such proteins in the anaerobic gut fungi. Our results highlight the presumed diverse functionalities of FA scaffolding proteins in basal fungi.

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<![CDATA[Neural Crest Cell Survival Is Dependent on Rho Kinase and Is Required for Development of the Mid Face in Mouse Embryos]]> https://www.researchpad.co/article/5989da9eab0ee8fa60ba4ee7

Neural crest cells (NCC) give rise to much of the tissue that forms the vertebrate head and face, including cartilage and bone, cranial ganglia and teeth. In this study we show that conditional expression of a dominant-negative (DN) form of Rho kinase (Rock) in mouse NCC results in severe hypoplasia of the frontonasal processes and first pharyngeal arch, ultimately resulting in reduction of the maxilla and nasal bones and severe craniofacial clefting affecting the nose, palate and lip. These defects resemble frontonasal dysplasia in humans. Disruption of the actin cytoskeleton, which leads to abnormalities in cell-matrix attachment, is seen in the RockDN;Wnt1-cre mutant embryos. This leads to elevated cell death, resulting in NCC deficiency and hypoplastic NCC-derived craniofacial structures. Rock is thus essential for survival of NCC that form the craniofacial region. We propose that reduced NCC numbers in the frontonasal processes and first pharyngeal arch, resulting from exacerbated cell death, may be the common mechanism underlying frontonasal dysplasia.

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<![CDATA[Differential Adhesion Molecule Expression during Murine Embryonic Stem Cell Commitment to the Hematopoietic and Endothelial Lineages]]> https://www.researchpad.co/article/5989da29ab0ee8fa60b81d71

Mouse embryonic stem cells (ESC) make cell fate decisions based on intrinsic and extrinsic factors. The decision of ESC to differentiate to multiple lineages in vitro occurs during the formation of embryoid bodies (EB) and is influenced by cell-environment interactions. However, molecular mechanisms underlying cell-environmental modulation of ESC fate decisions are incompletely understood. Since adhesion molecules (AM) influence proliferation and differentiation in developing and adult tissues, we hypothesized that specific AM interactions influence ESC commitment toward hematopoietic and endothelial lineages. Expression of AM in the adherens, tight and gap junction pathways in ESC subpopulations were quantified. E-cadherin (E-cad), Claudin-4 (Cldn4), Connexin-43 (Cx43), Zona Occludens-1 (ZO-1) and Zona Occludens-2 (ZO-2) transcript levels were differentially expressed during early stages of hematopoietic/endothelial commitment. Stable ESC lines were generated with reduced expression of E-cad, Cldn4, Cx43, ZO-1 and ZO-2 using shRNA technology. Functional and phenotypic consequences of modulating AM expression were assessed using hematopoietic colony forming assays, endothelial sprouting assays and surface protein expression. A decrease in E-cad, Cldn4, Cx43 and ZO-1 expression was associated with less commitment to the hematopoietic lineage and increased endothelial differentiation as evidenced by functional and phenotypic analysis. A reduction in ZO-2 expression did not influence endothelial differentiation, but decreased hematopoietic commitment two-fold. These data indicate that a subset of AM influence ESC decisions to commit to endothelial and hematopoietic lineages. Furthermore, differentially expressed AM may provide novel markers to delineate early stages of ESC commitment to hematopoietic/endothelial lineages.

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<![CDATA[Insulin Resistance in PCOS Patients Enhances Oxidative Stress and Leukocyte Adhesion: Role of Myeloperoxidase]]> https://www.researchpad.co/article/5989db41ab0ee8fa60bd6bdc

Cardiovascular diseases and oxidative stress are related to polycystic ovary syndrome (PCOS) and insulin resistance (IR). We have evaluated the relationship between myeloperoxidase (MPO) and leukocyte activation in PCOS patients according to homeostatic model assessment of IR (HOMA-IR), and have explored a possible correlation between these factors and endocrine and inflammatory parameters. This was a prospective controlled study conducted in an academic medical center. The study population consisted of 101 PCOS subjects and 105 control subjects. We divided PCOS subjects into PCOS non-IR (HOMA-IR<2.5) and PCOS IR (HOMA-IR>2.5). Metabolic and anthropometric parameters, total and mitochondrial reactive oxygen species (ROS) production, MPO levels, interactions between human umbilical vein endothelial cells and leukocytes, adhesion molecules (E-selectin, ICAM-1 and VCAM-1) and proinflammatory cytokines (IL-6 and TNF-α) were evaluated. Oxidative stress was observed in PCOS patients, in whom there was an increase in total and mitochondrial ROS production and MPO levels. Enhanced rolling flux and adhesion, and a decrease in polymorphonuclear cell rolling velocity were also detected in PCOS subjects. Increases in IL-6 and TNF-α and adhesion molecules (E-selectin, ICAM-1 and VCAM-1) were also observed, particularly in the PCOS IR group, providing evidence that inflammation and oxidative stress are related in PCOS patients. HOMA-IR was positively correlated with hsCRP (p<0.001, r = 0.304), ROS production (p<0.01, r = 0.593), leukocyte rolling flux (p<0.05, r = 0.446), E-selectin (p<0.01, r = 0.436) and IL-6 (p<0.001, r = 0.443). The results show an increase in the rate of ROS and MPO levels in PCOS patients in general, and particularly in those with IR. Inflammation in PCOS induces leukocyte-endothelium interactions and a simultaneous increase in IL-6, TNF-α, E-selectin, ICAM-1 and VCAM-1. These conditions are aggravated by the presence of IR.

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<![CDATA[Hypoxic Conditions Induce a Cancer-Like Phenotype in Human Breast Epithelial Cells]]> https://www.researchpad.co/article/5989daeeab0ee8fa60bc0305

Introduction

Solid tumors are less oxygenated than their tissue of origin. Low intra-tumor oxygen levels are associated with worse outcome, increased metastatic potential and immature phenotype in breast cancer. We have reported that tumor hypoxia correlates to low differentiation status in breast cancer. Less is known about effects of hypoxia on non-malignant cells. Here we address whether hypoxia influences the differentiation stage of non-malignant breast epithelial cells and potentially have bearing on early stages of tumorigenesis.

Methods

Normal human primary breast epithelial cells and immortalized non-malignant mammary epithelial MCF-10A cells were grown in a three-dimensional overlay culture on laminin-rich extracellular matrix for up to 21 days at normoxic or hypoxic conditions. Acinar morphogenesis and expression of markers of epithelial differentiation and cell polarization were analyzed by immunofluorescence, immunohistochemistry, qPCR and immunoblot.

Results

In large ductal carcinoma in situ patient-specimens, we find that epithelial cells with high HIF-1α levels and multiple cell layers away from the vasculature are immature compared to well-oxygenated cells. We show that hypoxic conditions impaired acinar morphogenesis of primary and immortalized breast epithelial cells grown ex vivo on laminin-rich matrix. Normoxic cultures formed polarized acini-like spheres with the anticipated distribution of marker proteins associated with mammary epithelial polarization e.g. α6-integrin, laminin 5 and Human Milk Fat Globule/MUC1. At hypoxia, cells were not polarized and the sub-cellular distribution pattern of the marker proteins rather resembled that reported in vivo in breast cancer. The hypoxic cells remained in a mitotic state, whereas proliferation ceased with acinar morphogenesis at normoxia. We found induced expression of the differentiation repressor ID1 in the undifferentiated hypoxic MCF-10A cell structures. Acinar morphogenesis was associated with global histone deacetylation whereas the hypoxic breast epithelial cells showed sustained global histone acetylation, which is generally associated with active transcription and an undifferentiated proliferative state.

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<![CDATA[Effects of Plectin Depletion on Keratin Network Dynamics and Organization]]> https://www.researchpad.co/article/5989dadcab0ee8fa60bba058

The keratin intermediate filament cytoskeleton protects epithelial cells against various types of stress and is involved in fundamental cellular processes such as signaling, differentiation and organelle trafficking. These functions rely on the cell type-specific arrangement and plasticity of the keratin system. It has been suggested that these properties are regulated by a complex cycle of assembly and disassembly. The exact mechanisms responsible for the underlying molecular processes, however, have not been clarified. Accumulating evidence implicates the cytolinker plectin in various aspects of the keratin cycle, i.e., by acting as a stabilizing anchor at hemidesmosomal adhesion sites and the nucleus, by affecting keratin bundling and branching and by linkage of keratins to actin filament and microtubule dynamics. In the present study we tested these hypotheses. To this end, plectin was downregulated by shRNA in vulvar carcinoma-derived A431 cells. As expected, integrin β4- and BPAG-1-positive hemidesmosomal structures were strongly reduced and cytosolic actin stress fibers were increased. In addition, integrins α3 and β1 were reduced. The experiments furthermore showed that loss of plectin led to a reduction in keratin filament branch length but did not alter overall mechanical properties as assessed by indentation analyses using atomic force microscopy and by displacement analyses of cytoplasmic superparamagnetic beads using magnetic tweezers. An increase in keratin movement was observed in plectin-depleted cells as was the case in control cells lacking hemidesmosome-like structures. Yet, keratin turnover was not significantly affected. We conclude that plectin alone is not needed for keratin assembly and disassembly and that other mechanisms exist to guarantee proper keratin cycling under steady state conditions in cultured single cells.

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<![CDATA[Advanced Glycation End Products in Infant Formulas Do Not Contribute to Insulin Resistance Associated with Their Consumption]]> https://www.researchpad.co/article/5989dabaab0ee8fa60bae494

Introduction

Infant formula-feeding is associated with reduced insulin sensitivity. In rodents and healthy humans, advanced glycation end product (AGE)-rich diets exert diabetogenic effects. In comparison with human breast-milk, infant formulas contain high amounts of AGEs. We assessed the role of AGEs in infant-formula-consumption-associated insulin resistance.

Methods

Total plasma levels of Nε-(carboxymethyl)lysine (CML), AGEs-associated fluorescence (λex = 370 nm/λem = 445 nm), soluble adhesion molecules, markers of micro- binflammation (hsCRP), oxidative stress (malondialdehyde, 8-isoprostanes) and leptinemia were determined, and correlated with insulin sensitivity in a cross-sectional study in 166 healthy term infants aged 3-to-14 months, subdivided according to feeding regimen (breast-milk- vs. infant formula-fed) and age (3-to-6-month-olds, 7-to-10-month-olds, and 11-to-14-month-old infants). Effects of the consumption of low- vs. high-CML-containing formulas were assessed. 36 infants aged 5.8±0.3 months were followed-up 7.5±0.3 months later.

Results

Cross-sectional study: 3-to-6-month-olds and 7-to-10-month-old formula-fed infants presented higher total plasma CML levels and AGEs-associated fluorescence (p<0.01, both), while only the 3-to-6-month-olds displayed lower insulin sensitivity (p<0.01) than their breast-milk-fed counterparts. 3-to-6-month-olds fed low-CML-containing formulas presented lower total plasma CML levels (p<0.01), but similar insulin sensitivity compared to those on high-CML-containing formulas. Markers of oxidative stress and inflammation, levels of leptin and adhesion molecules did not differ significantly between the groups. Follow-up study: at initial investigation, the breast-milk-consuming infants displayed lower total plasma CML levels (p<0.01) and AGEs-associated fluorescence (p<0.05), but higher insulin sensitivity (p<0.05) than the formulas-consuming infants. At follow-up, the groups did not differ significantly in either determined parameter.

Conclusions

In healthy term infants, high dietary load with CML does not play a pathophysiological role in the induction of infant formula-associated insulin resistance. Whether a high load of AGEs in early childhood affects postnatal programming remains to be elucidated.

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<![CDATA[A Systematic Screen for Micro-RNAs Regulating the Canonical Wnt Pathway]]> https://www.researchpad.co/article/5989dac7ab0ee8fa60bb2c07

MicroRNAs (miRs) and the canonical Wnt pathway are known to be dysregulated in human cancers and play key roles during cancer initiation and progression. To identify miRs that can modulate the activity of the Wnt pathway we performed a cell-based overexpression screen of 470 miRs in human HEK293 cells. We identified 38 candidate miRs that either activate or repress the canonical Wnt pathway. A literature survey of all verified candidate miRs revealed that the Wnt-repressing miRs tend to be anti-oncomiRs and down-regulated in cancers while Wnt-activating miRs tend to be oncomiRs and upregulated during tumorigenesis. Epistasis-based functional validation of three candidate miRs, miR-1, miR-25 and miR-613, confirmed their inhibitory role in repressing the Wnt pathway and suggest that while miR-25 may function at the level of â-catenin (β-cat), miR-1 and miR-613 act upstream of β-cat. Both miR-25 and miR-1 inhibit cell proliferation and viability during selection of human colon cancer cell lines that exhibit dysregulated Wnt signaling. Finally, transduction of miR-1 expressing lentiviruses into primary mammary organoids derived from Conductin-lacZ mice significantly reduced the expression of the Wnt-sensitive β-gal reporter. In summary, these findings suggest the potential use of Wnt-modulating miRs as diagnostic and therapeutic tools in Wnt-dependent diseases, such as cancer.

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<![CDATA[Computational Tension Mapping of Adherent Cells Based on Actin Imaging]]> https://www.researchpad.co/article/5989daedab0ee8fa60bbfc10

Forces transiting through the cytoskeleton are known to play a role in adherent cell activity. Up to now few approaches haves been able to determine theses intracellular forces. We thus developed a computational mechanical model based on a reconstruction of the cytoskeleton of an adherent cell from fluorescence staining of the actin network and focal adhesions (FA). Our custom made algorithm converted the 2D image of an actin network into a map of contractile interactions inside a 2D node grid, each node representing a group of pixels. We assumed that actin filaments observed under fluorescence microscopy, appear brighter when thicker, we thus presumed that nodes corresponding to pixels with higher actin density were linked by stiffer interactions. This enabled us to create a system of heterogeneous interactions which represent the spatial organization of the contractile actin network. The contractility of this interaction system was then adapted to match the level of force the cell truly exerted on focal adhesions; forces on focal adhesions were estimated from their vinculin expressed size. This enabled the model to compute consistent mechanical forces transiting throughout the cell. After computation, we applied a graphical approach on the original actin image, which enabled us to calculate tension forces throughout the cell, or in a particular region or even in single stress fibers. It also enabled us to study different scenarios which may indicate the mechanical role of other cytoskeletal components such as microtubules. For instance, our results stated that the ratio between intra and extra cellular compression is inversely proportional to intracellular tension.

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<![CDATA[Forsythia suspensa Suppresses House Dust Mite Extract-Induced Atopic Dermatitis in NC/Nga Mice]]> https://www.researchpad.co/article/5989db32ab0ee8fa60bd21af

Forsythia suspensa (F. suspensa) is a traditional medicine for treatment of inflammation. In this study, we evaluated the therapeutic effects of an ethanol extract from F. suspensa fruits on atopic dermatitis both in vivo and in vitro. We investigated the inhibitory effects of F. suspensa extract on the development of atopic dermatitis-like skin lesions in an NC/Nga mouse model exposed to Dermatophagoides farinae crude extract. Topical application of F. suspensa extract to the mice attenuated the atopic dermatitis symptoms, including increased dermatitis severity score, ear thickness, infiltration of inflammatory cells in the skin lesions, serum levels of IgE, TNF-α, and histamine, and expression of chemokines, cytokines, and adhesion molecules in ear tissue. In addition, F. suspensa extract inhibited the production of chemokines in TNF-α/IFN-γ-activated human keratinocytes. High-performance liquid chromatography analysis of FSE revealed the presence of four chemical constituents (forsythiaside, phillyrin, pinoresinol, and phylligenin). These compounds inhibited the production of chemokines in TNF-α/IFN-γ-activated human keratinocytes. These results suggest that the F. suspensa might be a useful candidate for treating allergic skin inflammatory disorders.

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<![CDATA[Ve-ptp Modulates Vascular Integrity by Promoting Adherens Junction Maturation]]> https://www.researchpad.co/article/5989da36ab0ee8fa60b86346

Background

Endothelial cell junctions control blood vessel permeability. Altered permeability can be associated with vascular fragility that leads to vessel weakness and haemorrhage formation. In vivo studies on the function of genes involved in the maintenance of vascular integrity are essential to better understand the molecular basis of diseases linked to permeability defects. Ve-ptp (Vascular Endothelial-Protein Tyrosine Phosphatase) is a transmembrane protein present at endothelial adherens junctions (AJs).

Methodology/Principal Findings

We investigated the role of Ve-ptp in AJ maturation/stability and in the modulation of endothelial permeability using zebrafish (Danio rerio). Whole-mount in situ hybridizations revealed zve-ptp expression exclusively in the developing vascular system. Generation of altered zve-ptp transcripts, induced separately by two different splicing morpholinos, resulted in permeability defects closely linked to vascular wall fragility. The ultrastructural analysis revealed a statistically significant reduction of junction complexes and the presence of immature AJs in zve-ptp morphants but not in control embryos.

Conclusions/Significance

Here we show the first in vivo evidence of a potentially critical role played by Ve-ptp in AJ maturation, an important event for permeability modulation and for the development of a functional vascular system.

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<![CDATA[Plakophilin-3 Is Required for Late Embryonic Amphibian Development, Exhibiting Roles in Ectodermal and Neural Tissues]]> https://www.researchpad.co/article/5989da5cab0ee8fa60b90311

The p120-catenin family has undergone a significant expansion during the evolution of vertebrates, resulting in varied functions that have yet to be discerned or fully characterized. Likewise, members of the plakophilins, a related catenin subfamily, are found throughout the cell with little known about their functions outside the desmosomal plaque. While the plakophilin-3 (Pkp3) knockout mouse resulted in skin defects, we find larger, including lethal effects following its depletion in Xenopus. Pkp3, unlike some other characterized catenins in amphibians, does not have significant maternal deposits of mRNA. However, during embryogenesis, two Pkp3 protein products whose temporal expression is partially complimentary become expressed. Only the smaller of these products is found in adult Xenopus tissues, with an expression pattern exhibiting distinctions as well as overlaps with those observed in mammalian studies. We determined that Xenopus Pkp3 depletion causes a skin fragility phenotype in keeping with the mouse knockout, but more novel, Xenopus tailbud embryos are hyposensitive to touch even in embryos lacking outward discernable phenotypes, and we additionally resolved disruptions in certain peripheral neural structures, altered establishment and migration of neural crest, and defects in ectodermal multiciliated cells. The use of two distinct morpholinos, as well as rescue approaches, indicated the specificity of these effects. Our results point to the requirement of Pkp3 in amphibian embryogenesis, with functional roles in a number of tissue types.

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<![CDATA[Soluble Forms of Intercellular and Vascular Cell Adhesion Molecules Independently Predict Progression to Type 2 Diabetes in Mexican American Families]]> https://www.researchpad.co/article/5989dae4ab0ee8fa60bbcdc7

Objective

While the role of type 2 diabetes (T2D) in inducing endothelial dysfunction is fairly well-established the etiological role of endothelial dysfunction in the onset of T2D is still a matter of debate. In the light of conflicting evidence in this regard, we conducted a prospective study to determine the association of circulating levels of soluble intercellular adhesion molecule 1 (sICAM-1) and soluble vessel cell adhesion molecule 1 (sVCAM-1) with incident T2D.

Methods

Data from this study came from 1,269 Mexican Americans of whom 821 initially T2D-free individuals were longitudinally followed up in the San Antonio Family Heart Study. These individuals were followed for 9752.95 person-years for development of T2D. Prospective association of sICAM-1 and sVCAM-1 with incident T2D was studied using Kaplan-Meier survival plots and mixed effects Cox proportional hazards modeling to account for relatedness among study participants. Incremental value of adhesion molecule biomarkers was studied using integrated discrimination improvement (IDI) and net reclassification improvement (NRI) indexes.

Results

Decreasing median values for serum concentrations of sICAM-1 and sVCAM-1 were observed in the following groups in this order: individuals with T2D at baseline, individuals who developed T2D during follow-up, individuals with prediabetes at baseline and normal glucose tolerant (NGT) individuals who remained T2D-free during follow-up. Top quartiles for sICAM-1 and sVCAM-1 were strongly and significantly associated with homeostatic model of assessment—insulin resistance (HOMA-IR). Mixed effects Cox proportional hazards modeling revealed that after correcting for important clinical confounders, high sICAM-1 and sVCAM-1 concentrations were associated with 2.52 and 1.99 times faster progression to T2D as compared to low concentrations, respectively. Individuals with high concentrations for both sICAM-1 and sVCAM-1 progressed to T2D 3.42 times faster than those with low values for both sICAM-1 and sVCAM-1. The results were similar in women in reproductive age group and the remainder of the cohort. Inclusion of sICAM-1 and sVCAM-1 in predictive models significantly improved reclassification and discrimination. The majority of these results were seen even when the analyses were restricted to NGT individuals.

Conclusion

Serum concentrations of sICAM-1 and sVCAM-1 independently and additively predict future T2D and represent important candidate biomarkers of T2D.

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<![CDATA[Serum after Autologous Transplantation Stimulates Proliferation and Expansion of Human Hematopoietic Progenitor Cells]]> https://www.researchpad.co/article/5989dabaab0ee8fa60bae64b

Regeneration after hematopoietic stem cell transplantation (HSCT) depends on enormous activation of the stem cell pool. So far, it is hardly understood how these cells are recruited into proliferation and self-renewal. In this study, we have addressed the question if systemically released factors are involved in activation of hematopoietic stem and progenitor cells (HPC) after autologous HSCT. Serum was taken from patients before chemotherapy, during neutropenia and after hematopoietic recovery. Subsequently, it was used as supplement for in vitro culture of CD34+ cord blood HPC. Serum taken under hematopoietic stress (4 to 11 days after HSCT) significantly enhanced proliferation, maintained primitive immunophenotype (CD34+, CD133+, CD45) for more cell divisions and increased colony forming units (CFU) as well as the number of cobblestone area-forming cells (CAFC). The stimulatory effect decays to normal levels after hematopoietic recovery (more than 2 weeks after HSCT). Chemokine profiling revealed a decline of several growth-factors during neutropenia, including platelet-derived growth factors PDGF-AA, PDGF-AB and PDGF-BB, whereas expression of monocyte chemotactic protein-1 (MCP-1) increased. These results demonstrate that systemically released factors play an important role for stimulation of hematopoietic regeneration after autologous HSCT. This feedback mechanism opens new perspectives for in vivo stimulation of the stem cell pool.

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<![CDATA[Phospholipid binding to the FAK catalytic domain impacts function]]> https://www.researchpad.co/article/5989db4fab0ee8fa60bdba02

Focal adhesion kinase is an essential nonreceptor tyrosine kinase that plays an important role in development, in homeostasis and in the progression of human disease. Multiple stimuli activate FAK, which requires a change in structure from an autoinhibited to activated conformation. In the autoinhibited conformation the FERM domain associates with the catalytic domain of FAK and PI(4,5)P2 binding to the FERM domain plays a role in the release of autoinhibition, activating the enzyme. An in silico model of FAK/PI(4,5)P2 interaction suggests that residues on the catalytic domain interact with PI(4,5)P2, in addition to the known FERM domain PI(4,5)P2 binding site. This study was undertaken to test the significance of this in silico observation. Mutations designed to disrupt the putative PI(4,5)P2 binding site were engineered into FAK. These mutants exhibited defects in phosphorylation and failed to completely rescue the phenotype associated with fak -/- phenotype fibroblasts demonstrating the importance of these residues in FAK function. The catalytic domain of FAK exhibited PI(4,5)P2 binding in vitro and binding activity was lost upon mutation of putative PI(4,5)P2 binding site basic residues. However, binding was not selective for PI(4,5)P2, and the catalytic domain bound to several phosphatidylinositol phosphorylation variants. The mutant exhibiting the most severe biological defect was defective for phosphatidylinositol phosphate binding, supporting the model that catalytic domain phospholipid binding is important for biochemical and biological function.

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