Gene Expression Of Adhesion Molecules Research Articles

Evaluation of the Effects of STZ-Induced Diabetes onIn VitroFertilization and Early Embryogenesis Processes

The aim of this study was to investigate the effects of experimentally induced diabetes on (a) germ cells, (b) in vitro fertilization (IVF) success rate, and (c) gap junction and cell adhesion molecule gene and protein expressions during the early blastocyst period. Germ cells were obtained from healthy and diabetic rats, analyzed for number, motility, and morphology, and used for IVF. After reaching the early blastocyst stage, the expressions of genes encoding gap junction proteins and cell adhesion molecules were analyzed by quantitative RT-PCR. Histomorphologically and immunohistochemically analyses were also performed. Diabetes significantly affected sperm number and motility and the development of oocytes. Gene expressions of β-catenin and connexin family members and protein expressions of E-cadherin and connexin-43 significantly decreased in groups including germ cells isolated from diabetic rats. Connective tissue growth factor expression increased in groups that included sperm cells isolated from diabetic male rats, whereas mucin-1 expression increased in the group that included oocytes isolated from diabetic female rats paired with sperm cells isolated from healthy male rats. In summary, experimentally induced diabetes was found to influence gap junctions, cell adhesion molecules, and associated proteins which all have important roles in germ cell maturation, fertilization, and development.

Protective effects of branched-chain amino acids on hepatic ischemia-reperfusion-induced liver injury in rats: a direct attenuation of Kupffer cell activation

We determined whether there is a protective effect of branched-chain amino acid (BCAA) on hepatic ischemia-reperfusion (I/R)-induced acute liver injury. Wister rats were divided into the following four groups: simple laparotomy with vehicle; simple laparotomy with BCAA (1 g/kg body wt orally); I/R (30 min clamp) with vehicle; and I/R with BCAA. Serum liver function tests and the gene expression of adhesion molecules (intercellular adhesion molecule and vascular cell adhesion molecule) and vasoconstrictor-related genes (endothelin-1) in the liver were examined. In the in vivo study, portal venous pressure, leukocyte adhesion, and hepatic microcirculation were evaluated. Furthermore, Kupffer cells were isolated and cultured with various concentrations of BCAA in the presence or absence of lipopolysaccharide (LPS). Increased levels of liver function tests following I/R were significantly attenuated by BCAA treatment. The increased expression of adhesion molecules and endothelin-1 was also significantly attenuated by BCAA treatment. Moreover, increased portal venous pressure, enhanced leukocyte adhesion, and deteriorated hepatic microcirculation following I/R were all improved by BCAA treatment. In the experiment using isolated Kupffer cells, the expression of interleukin-6, interleukin-1β, and endothelin-1 in response to LPS stimulation was attenuated by BCAA in a dose-dependent fashion. These results indicate that perioperative oral administration of BCAA has excellent therapeutic potential to reduce I/R-induced liver injury. These beneficial effects may result from the direct attenuation of Kupffer cell activation under stressful conditions.

PS204. Mechanical (or “Gravitational”) Unloading Reduces Inflammatory and Cell Adhesion Molecule Gene Expression in Human Endothelial Cells

Mechanical forces including gravity affect mechanotransduction and cell function. The goal of this study was to investigate the impact of mechanical unloading (MU) and loading (ML) of endothelial cells (ECs) with microgravity and hypergravity respectively, with the hypothesis that MU alters expression of inflammatory and adhesion molecule gene expression and these changes are reversed by ML. Human umbilical vascular ECs grown to confluency were studied. A desktop random positioning machine and a gravitational cell-loading apparatus provided MU and ML. The experimental conditions included: 1) controls exposed to 1-gravity environment for 24 h (CL), 2) MU for 24 hours, 3) MU for 24 hours with three 30-minutes periods of ML of 12-gravity (MU/ML). Gene expression was studied with reverse transcription followed by real-time quantitative polymerase chain reaction (qRTPCR). MU led to a significant decrease in gene expression of the adhesion molecules ICAM-1, VCAM-1, E-Selectin, as well as TNF-α, IL-6 and VEGF. In contrast, NOS-3, Caveolin-1 and −2 were significantly increased with MU. The changes observed in gene expression with MU were reversed by gravitational mechanical loading (MU/ML). Gravitational MU decreases inflammatory and adhesion molecule gene expression and these changes are reversed by short periods of ML. This points towards the importance of gravitational loading in ECs function and cellular interactions.

Abstract 353: Mechanical (or “Gravitational”) Unloading Reduces Inflammatory and Cell Adhesion Molecule Gene Expression in Human Endothelial Cells

Objectives: Mechanical forces including gravity affect mechanotransduction and subsequent cell function. The goal of this study was to investigate the impact of mechanical unloading (MU) and loading (ML) of endothelial cells (ECs) with microgravity and hypergravity respectively, with the hypothesis that MU alters expression of inflammatory and adhesion molecule gene expression and these changes are reversed by ML. Methods: Human umbilical vascular endothelial cells (HUVECs) grown to confluency were studied. A desktop random positioning machine and a gravitational cell-loading apparatus provided MU and ML conditions, respectively. The experimental conditions included: 1) controls exposed to 1-gravity environment for 24 h (CL), 2) MU for 24 hours, 3) MU for 24 hours with three 30-minutes periods of ML of 12-gravity (MU/ML). Gene expression was studied with reverse transcription followed by real-time quantitative polymerase chain reaction (qRTPCR). Results: MU led to a significant decrease in gene expression of the adhesion molecules ICAM-1, VCAM-1, E-Selectin, as well as TNF-α, IL-6 and VEGF. In contrast, NOS-3, Caveolin-1 and -2 were significantly increased with MU. The changes observed in gene expression with MU were reversed by gravitational mechanical loading (MU/ML). Conclusions: Gravitational MU decreases inflammatory and adhesion molecule gene expression and these changes are reversed by short periods of ML. This points towards the importance of gravitational loading in ECs function and cellular interactions.

Mechanisms of the Anti‐inflammatory Action of Pulsatile Laminar Flow: Role of AMPK in Chromatin Remodeling

Pulsatile laminar flow (PS) mediates many anti‐inflammatory functions in endothelial cells (ECs). However, the mechanisms involved in the repression of inflammatory responses by mechanical stimuli have not been elucidated. Transcriptional repression can occur through epigenetic modifications of gene promoter regions. We performed experiments to test the hypothesis that the PS‐dependent protective mechanism occurs through signaling pathways that downregulate the expression of inflammatory and adhesion molecule genes and that this mechanotransduction process is mediated through an AMP‐activated protein kinase (AMPK) pathway and subsequent chromatin modifications. We found that 24‐hr PS downregulated EC gene expression of monocyte chemoattractant protein‐1 (MCP1) and vascular cell adhesion molecule‐1 (VCAM) and inhibited monocyte attachment to ECs via an AMPK‐dependent pathway. PS also induced the AMPK‐dependent recruitment of the corepressor enzymes histone deacetylase‐3 and ‐5 to the MCP1 and VCAM proximal promoters as well as concomitant histone hypoacetylation. Our findings indicate that PS suppresses inflammatory processes through chromatin modifications and downregulation of AMPK‐dependent inflammatory genes and monocyte adhesion, thus providing new insights into the role of chromatin remodeling in mechanotransduction. 3R01 HL106579 (S.C.) and 3R01 HL106579‐01S1 (L.F.)

Human Leukotactin‐1/CCL15 up‐regulates expression of cell adhesion molecules and stimulates monocyte adhesion to endothelial cells via JAK2/STAT3 signaling pathway

Leukotactin‐1 (Lkn‐1)/CCL15 is a potent chemo‐attractant for leukocytes and endothelial cell and is implicated in inflammatory diseases. Lkn‐1/CCL‐15 enhances cell migration and activation of neutrophils and endothelial cells mainly through CCR1 signal pathway. We found that Lkn‐1/CCL15 significantly increased mRNA and protein expression of ICAM‐1 and VCAM‐1 compared with those in non‐stimulated endothelial cells. We showed that Lkn‐1/CCL15 increased cell adhesion of human U937 monocytoid cells to the endothelial cell. Furthermore, we provide evidence that Janus kinase (JAK) and STAT pathway plays a role in Lkn‐ 1/CCL15‐induced gene expression of cell adhesion molecules in endothelial cells. We observed that phosphorylation of JAK and STAT3 was stimulated after Lkn‐1/CCL15 treatment of HUVECs. Pretreatment of these cells with inhibitors for JAK, PI3K, and AKT prevented the Lkn‐1/CCL15‐induced expression of ICAM‐1, suggesting the involvement of JAK pathway in ICAM‐1 gene activation by Lkn‐1/CCL15. The results suggest that Lkn‐1/CCL15 and its signaling pathway may be a potential target for treating inflammatory diseases involving up‐regulation of cell adhesion molecules. .

Effect of ovarian steroids on gene expression related to synapse assembly in serotonin neurons of macaques

Dendritic spines are the elementary structural units of neural plasticity. In a model of hormone replacement therapy (HT), we sought to determine the effect of estradiol (E) and progesterone (P) on gene expression related to synapse assembly in a laser-captured preparation enriched for serotonin neurons from rhesus macaques. Microarray analysis was conducted (n = 2 animals/treatment), and the results were confirmed for pivotal genes with qRT-PCR on additional laser-captured material (n = 3 animals/treatment). Ovariectomized rhesus macaques were treated with placebo, E, or E + P via Silastic implants for 1 month. The midbrain was obtained, sectioned, and immunostained for tryptophan hydroxylase (TPH). TPH-positive neurons were laser captured using an arcturus laser dissection microscope (Pixel II). RNA from laser-captured serotonin neurons was hybridized to Rhesus Affymetrix GeneChips for screening purposes. There was a twofold or greater change in the expression of 63 probe sets in the cell adhesion molecule (CAM) category, and 31 probe sets in the synapse assembly category were similarly altered in E- and E + P-treated animals. qRT-PCR assays showed that E treatment induced a significant increase in ephrin receptor A4 (EPHA4) and in integrin A8 (ITGA8) but not in ephrin receptor B4 (EPHB4) or integrin B8 (ITGB8) expression. E also increased expression of cadherin 11 (CDH11), neuroligin 3 (NLGN3), neurexin 3 (NRXN3), syndecan 2 (SCD2), and neural cell adhesion molecule (NCAM) compared with placebo. Supplemental P treatment suppressed E-induced gene expression. In summary, ovarian steroids target gene expression of adhesion molecules in serotonin neurons that are important for synapse assembly.

The anti-TNF-α antibody infliximab indirectly regulates PECAM-1 gene expression in two models of in vitro blood cell activation

Chronic inflammatory bowel diseases can be successfully treated with antibodies against the acute phase mediator TNF-α. The process of activation and of extravasation of inflammatory cells from the blood into the ‘stressed' tissue site is controlled by cytokines and chemokines, which attract leukocytes and by adhesion molecules, which mediate their attachment and transmigration toward the affected cell(s). The changes in the gene expression of adhesion molecules taking place in those cells before attachment have been less investigated. Changes of PECAM-1, ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1) gene expression were studied in phytohaemagglutinin (PHA)- and lipolysaccharide (LPS)-treated human peripheral blood leukocytes (PBLs), granulocytes and the human monocyte cell line U-937. Cells were treated either with PHA or with LPS in the presence or absence of infliximab and incubated with TNF-α, IFN-γ and/or transforming growth factor beta (TGF-β) and treated as above. Activation of PBLs by PHA or LPS treatment triggered a sharp upregulation of ICAM-1, VCAM-1 gene expression and a time-dependent downregulation of PECAM-1 gene expression reaching a minimum 4 h from start of the experiment. The anti-TNF-α antibody infliximab, by neutralizing TNF-α and IFN-γ production, completely reversed PECAM-1 mRNA downregulation and ICAM-1 and VCAM-1 upregulation. Immunostaining of PBLs cytospins with antibodies against PECAM-1 and ICAM-1 confirmed RT-PCR and western blot results. PBLs IFN-γ or TNF-α treatment downregulated PECAM-1 in parallel with the upregulation of ICAM-1 and VCAM-1 gene expression, whereas TGF-β upregulated PECAM-1- and downregulated ICAM-1 and VCAM-1 gene expression counteracting the effect of TNF-α or IFN-γ. Similar results were obtained in human U937 cells and in granulocyte cultures by TNF-α or IFN-γ treatment. Taken together, these results suggest that infliximab, blocking TNF-α and IFN-γ production, exerts its anti-inflammatory effect through inhibiting downregulation of PECAM-1 gene expression and upregulation of ICAM-1 and VCAM-1 expression in leukocytes of the peripheral blood. These results also suggest that TGF-β may thus be of therapeutic importance as an anti-inflammatory agent.

Dysregulated gene expression of extracellular matrix and adhesion molecules in saphenous vein conduits of hemodialysis patients

The incidence of cardiovascular disease was approximately 10 times higher in hemodialysis patients with end-stage renal disease than in the general population. The saphenous vein is the most commonly used conduit for coronary artery bypass grafting. However, the extracellular matrix and adhesion molecule characteristics of saphenous vein in hemodialysis patients remain unclear. The aim of the present study was to survey the extracellular matrix gene expression profile of the saphenous vein in hemodialysis patients undergoing coronary artery bypass grafting. A total of 34 patients undergoing elective coronary artery bypass grafting were enrolled. Of the 34 patients, 15 with end-stage renal disease required maintenance hemodialysis. The control group consisted of the other 19 patients without preoperative renal disease. Samples of the saphenous vein were obtained at coronary artery bypass grafting. The expression profile of the extracellular matrix genes was analyzed by microarray. The tissue matrix metallopeptidase/tissue inhibitor of metallopeptidase protein activities in the saphenous vein were evaluated by immunocytochemistry and Western blotting. Nineteen extracellular matrix and adhesion molecule-focused genes demonstrated at least a threefold difference in expression between the 2 groups. Upregulation was observed in 16 genes, and 3 genes appeared to be downregulated. Notable imbalanced matrix metallopeptidase/tissue inhibitor of metallopeptidase protein activities of saphenous vein exposed to end-stage renal disease conditions was found. The results from present study suggest that the native extracellular matrix gene expression profile of the saphenous vein conduits in hemodialysis patients show signs of the vein graft disease process before coronary surgery. Furthermore, some preoperative profiles of hemodialysis patients undergoing coronary artery bypass grafting might provide some useful clues regarding vein graft quality and prompt adjustment in surgical strategy.

Disorder gene expression of extracellular matrix and adhesion molecules in saphenous vein conduits of diabetic patients

Saphenous vein (SV) is the most commonly employed conduit in coronary surgery. However, the extracellular matrix (ECM) characteristics of SV in diabetic patients still remain unclear. This study was to survey the ECM gene expression profile of SV in diabetic patients. Thirty-five patients had type 2 diabetic mellitus; the non-diabetic (control) group comprised 49 patients. The expression profile of ECM genes was analysed by microarray. Tissue MMP/TIMP protein activities were evaluated by immunocytochemistry and western-blot. In this microarray, 25 genes demonstrated at least a 3-fold difference in expression. Upregulation was observed in 20 genes, while five genes appeared to be downregulated. SV exposed to DM conditions demonstrated a notable increase in MMP-2 and MMP-9 but a significant decrease in TIMP-2 and TIMP-3 in protein concentration compared with control group. This study suggests that native ECM gene expression profile of SV in diabetic patients has showed signs of the vein graft disease process before coronary surgery. Preoperative profiles of diabetic patients might provide some useful clues regarding vein graft quality and prompt adjustment in surgical strategy.

Bacterial DNA Induces Pulmonary Damage Via TLR-9 Through Cross-talk With Neutrophils

Bacterial DNA (bDNA) contains hypomethylated "CpG" repeats that can be recognized by Toll-like receptor 9 (TLR-9) as a pathogen-associated molecular pattern. The ability of bDNA to initiate lung injury via TLR-9 has been inferred on the basis of studies using artificial CpG DNA. But the role of authentic bDNA in lung injury is still unknown. Moreover, the mechanisms by which CpG DNA species can lead to pulmonary injury are unknown, although neutrophils (PMNs) are thought to play a key role in the genesis of septic acute lung injury. We evaluated the effects of bDNA on PMN-endothelial cell (EC) interactions thought critical for initiation of acute lung injury. Using a biocapacitance system to monitor real-time changes in endothelial permeability, we demonstrate here that bDNA causes EC permeability in a dose-dependent manner uniquely in the presence of PMNs. These permeability changes are inhibited by chloroquine, suggesting TLR-9 dependency. When PMNs were preincubated with bDNA and applied to ECs or when bDNA was applied to ECs without PMNs, no permeability changes were detected. To study the underlying mechanisms, we evaluated the effects of bDNA on PMN-EC adherence. Bacterial DNA significantly increased PMN adherence to ECs in association with upregulated adhesion molecules in both cell types. Taken together, our results strongly support the conclusion that bDNA can initiate lung injury by stimulating PMN-EC adhesive interactions predisposing to endothelial permeability. Bacterial DNA stimulation of TLR-9 appears to promote enhanced gene expression of adhesion molecules in both cell types. This leads to PMN-EC cross-talk, which is required for injury to occur.

Cellular adaptive response to glutathione depletion modulates endothelial dysfunction triggered by TNF-α

Several interrelated cellular signaling molecules are involved in modulating adaptive compensatory changes elicited by low exposures to toxins and other stressors. The most prominent example of signaling pathway typically involved in this adaptive stress response, is represented by the activation of a redox-sensitive gene regulatory network mediated by the NF-E2-related factor-2 (Nrf2) which is intimately involved in mediating the Antioxidant Responsive Element (ARE)-driven response to oxidative stress and xenobiotics.We investigated if Nrf2 pathway activation following intracellular glutathione depletion through buthionine sulfoximine (BSO) exposure, might be able to alter the response to TNF-α, a proinflammatory cytokine, in cultured human umbilical vein endothelial cells. Herein, we revealed that such a change in the cellular redox status is able to reduce TNF-α induced endothelial activation (as shown by a decreased gene expression of adhesion molecules) by activating an adaptive response mediated by an increased Nrf2 nuclear translocation and overexpression of the ARE genes HO-1 and NQO-1. Furthermore, we have demonstrated the involvement of ERK1/2 kinases in Nrf2 nuclear translocation activated by BSO-induced glutathione depletion. The coordinate induction of endogenous cytoprotective proteins through adaptive activation of Nrf2 pathway is a field of great interest for potential application in prevention and therapy of inflammatory diseases such as atherosclerosis.

Leukocyte rolling and adhesion via ICAM-1 signals to endothelial permeability. Focus on “Leukocyte rolling and adhesion both contribute to regulation of microvascular permeability to albumin via ligation of ICAM-1”

THE VASCULAR ENDOTHELIUM plays a vital role in the inflammatory response by induction and surface expression of adhesion molecules and chemokines. At sites of acute inflammation, blood flow is increased and postcapillary venules exhibit increased permeability (leakiness) and support the influx of blood leukocytes, primarily neutrophils. The relationship between neutrophil influx and increased vessel permeability has been a topic of investigation for decades. Many investigations have reported a direct neutrophil-dependent increase in vessel permeability during inflammation using in vivo and in vitro models (reviewed in Ref. 4). One aspect that has received increased attention is whether endothelial cell adhesion molecules contribute to the regulation of vessel permeability, in addition to their well-characterized role in neutrophil rolling and adhesion. Intercellular adhesion molecule-1 (ICAM-1, also known as CD54) is an immunoglobulin superfamily member that is abundantly expressed on the endothelial cell surface and is enriched at endothelial cell borders in vivo and in vitro (2, 12, 14, 17). ICAM-1 surface expression is upregulated in endothelial cells by proinflammatory cytokines like TNF-, IL-1, IFN-, or bacterial endotoxins, and ICAM-1 serves as a receptor for leukocyte 2-integrins (LFA-1 and Mac-1) (reviewed in Ref. 15). In an experiment of nature, patients with leukocyte adhesion deficiency-1 (LAD-1) have a severe primary immune deficiency in which blood neutrophils fail to localize to sites of inflammation or injury. This defect occurs because patients’ neutrophils either lack 2-integrins or contain mutations in these molecules. Studies using neutrophils from LAD-1 patients or 2-integrin-deficient mice clearly demonstrate that these cells have normal selectin-dependent rolling on activated endothelium but fail to stably arrest and transmigrate. Additional studies have demonstrated that endothelial-expressed ICAM-1 contributes to both kinetics of leukocyte rolling and arrest in vivo as determined by intravital microscopy of the cremaster muscle microcirculation in ICAM-1 / mice (16). In this issue of American Journal of Physiology-Cell Physiology, Sumagin and colleagues (18) use elegant in vivo studies to demonstrate that microvascular permeability in the cremaster muscle model is regulated by leukocyte engagement of ICAM-1. The authors monitored permeability (Ps) in cremaster microcirculation by efflux of luminal fluorescent-tagged albumin by fluorescence confocal intravital microscopy. Prior studies by Sarelius and colleagues (19) in the same model demonstrated that ICAM-1 engagement induced a localized increase in vessel permeability in unstimulated arterioles and in TNF-stimulated venules. In agreement with this finding ,4ho f TNF- stimulation of arterioles and venules in ICAM-1 knockout (KO) animals did not result in increased vessel permeability as compared with wild-type (WT) mice under identical conditions. Similar results were seen with 2-integrin KO mice. In the current study, the authors provide further evidence linking ICAM-1 expression to regulation of endothelial permeability. Comparisons between unstimulated venules and TNF--treated venules reveal that neutrophil rolling regulates venule permeability in unstimulated venules while leukocyte adhesion regulates venule permeability in TNF--stimulated venules. The 2-integrin-blocking antibodies, which inhibit neutrophil arrest on endothelium, reduced the vessel permeability, and this was further reduced by treatment with a combination of 2-integrin and P-selectin blocking antibodies that inhibits both rolling and arrest. Interestingly, the reduction in venule permeability observed by blockade of P-selectin and 2-integrin function were suggested to be due to insufficient ICAM-1 engagement due to the absence of rolling leukocytes. To examine the role that neutrophils play, circulating neutrophils were depleted by antineutrophil GR-1 antibody treatment. Notably, depletion of neutrophils caused a marked decrease in

Polyphenolics from Açaí (Euterpe oleracea Mart.) and Red Muscadine Grape (Vitis rotundifolia) Protect Human Umbilical Vascular Endothelial Cells (HUVEC) from Glucose- and Lipopolysaccharide (LPS)-Induced Inflammation and Target MicroRNA-126

Endothelial anti-inflammatory effects of açaí (Ac) and red muscadine grape (Gp) polyphenolics have not been extensively investigated. It was hypothesized that polyphenolics from Ac and Gp exert comparable protective effects in human vascular endothelial cells (HUVEC) upon inflammatory stress. Furthermore, this study investigated whether microRNAs relevant to endothelial function might be regulated by Ac and Gp. Results showed that Ac and Gp (5-20 mg gallic acid equivalent/L) protected HUVEC against glucose-induced oxidative stress and inflammation. Glucose-induced expression of interleukin-6 and -8 was down-regulated by Ac and Gp at mRNA and protein levels. Upon lipopolysaccharide (LPS; 1 μg/L)-induced inflammation, Ac and Gp inhibited gene expression of adhesion molecules and NF-κB activation to similar extents, although Gp was more effective in decreasing PECAM-1 and ICAM-1 protein. Of the screened microRNAs, only microRNA-126 expression was found to be modulated by Ac and Gp as the underlying mechanism to inhibit gene and protein expression of VCAM-1.

Beneficial effect of anti-platelet therapies on atherosclerotic lesion formation assessed by phase-contrast X-ray CT imaging

We have applied an imaging system of phase-contrast X-ray CT to the detection of atherosclerotic plaque components by means of the differences of tissue mass densities. In this study, we investigated the effect of the anti-platelet therapies, widely used for secondly prevention of cardiovascular events, on plaque stability and examined whether this novel technique could detect the changes of plaque components under the therapy. Apolipoprotein E-deficient mice were fed on high-cholesterol diet alone and either with 0.1% cilostazol or clopidogrel for 10 weeks. We assessed atherosclerotic lesion volumes and components at brachiocephalic artery by the phase-contrast X-ray CT imaging and histochemistry. The phase-contrast X-ray CT imaging could reveal that cilostazol and clopidogrel significantly decreased atherosclerotic lesion volumes at brachiocephalic artery (31.2% reduction in cilostazol group and 37.4% reduction in clopidogrel group), compared with control group. In addition, the mass densities calculated by this method revealed the anti-platelet treatment increased stable plaque areas including high collagen content, but decreased unstable plaque areas including lipid and macrophage content. These findings were confirmed by histological analyses. Real-time PCR analyses indicated that anti-platelets inhibited gene expressions of cytokines and adhesion molecules, such as IFNγ and ICAM-1. Anti-platelet therapies had a beneficial effect on plaque stability maybe due to anti-inflammatory actions. Phase-contrast X-ray CT imaging could quantify the plaque volume and qualify the plaque components affected by anti-platelet therapies. This novel phase-contrast X-ray CT imaging system could be a plausible method to detect the unstable plaque non-invasively in the future.

Effects of plant lectin and extracts on adhesion molecules of endothelial progenitors

Abstract Promise of cell therapy has advanced the use of adult stem cells towards the development of novel approaches to promote regeneration of injured endothelium. The aim of this study was to stimulate endothelial progenitor cells (EPCs) with lectin isolated from Solanum tuberosum (potato) shoot and Calendula officinalis (marigold) extracts, in order to increase EPCs proliferation and gene expression of molecules with roles in chemotaxis and adhesion for a better attachment to injured vascular tissue. EPCs were differentiated from umbilical cord blood-derived mononuclear cells and characterized by light microscopy, flow cytometry, and vascular tube-like structures formation on Matrigel. Cell proliferation was determined by MTS assay, and gene expression of molecules involved in EPCs adhesion (VCAM-1, VE-cadherin, ICAM-1, PECAM-1, P-selectin) and chemotaxis was determined (CXCR4, Tie-2) by RT-PCR. For the assessment of cell motility, wound-healing assay was employed. Both potato shoot lectin and marigold extracts stimulated EPCs proliferation in a concentration dependent manner and were able to increase expression of adhesion and chemotactic molecules. Marigold flower extract proved to be more efficient. This study demonstrates the usefulness of potato lectin and marigold extracts to increase EPCs proliferation and modulate gene expression of chemotactic and adhesion molecules, which may facilitate EPCs attachment to injured endothelium.

Mucosal Gene Expression of Cell Adhesion Molecules, Chemokines, and Chemokine Receptors in Patients With Inflammatory Bowel Disease Before and After Infliximab Treatment

Inflammatory bowel disease (IBD) is characterized by a continuous influx of leukocytes into the gut wall. This migration is regulated by cell adhesion molecules (CAMs), and selective antimigration therapies have been developed. This study investigated the effect of infliximab therapy on the mucosal gene expression of CAMs in IBD. Mucosal gene expression of 69 leukocyte/endothelial CAMs and E-cadherin was investigated in 61 IBD patients before and after first infliximab infusion and in 12 normal controls, using Affymetrix gene expression microarrays. Quantitative reverse transcriptase-PCR (qRT-PCR), immunohistochemistry, and western blotting were used to confirm the microarray data. When compared with control colons, the colonic mucosal gene expression of most leukocyte/endothelial adhesion molecules was upregulated and E-cadherin gene expression was downregulated in active colonic IBD (IBDc) before therapy, with no significant colonic gene expression differences between ulcerative colitis and colonic Crohn's disease. Infliximab therapy restored the upregulations of leukocyte CAMs in IBDc responders to infliximab that paralleled the disappearance of the inflammatory cells from the colonic lamina propria. Also, the colonic gene expression of endothelial CAMs and of most chemokines/chemokine receptors returned to normal after therapy in IBDc responders, and only CCL20 and CXCL1-2 expression remained increased after therapy in IBDc responders vs. control colons. When compared with control ileums, the ileal gene expression of MADCAM1, THY1, PECAM1, CCL28, CXCL1, -2, -5, -6, and -11, and IL8 was increased and CD58 expression was decreased in active ileal Crohn's disease (CDi) before therapy, and none of the genes remained dysregulated after therapy in CDi responders vs. control ileums. This microarray study identified a number of interesting targets for antiadhesion therapy including PECAM1, IL8, and CCL20, besides the currently studied α4β7 integrin-MADCAM1 axis. Our data demonstrate that many leukocyte/endothelial CAMs and chemokines/chemokine receptors are upregulated in inflamed IBD mucosa. Controlling the inflammation with infliximab restores most of these dysregulations in IBD. These results show that at least part of the mechanism of anti-tumor necrosis factor-α therapy goes through downregulation of certain adhesion molecules.

Lactosylceramide Mediates the Expression of Adhesion Molecules in TNF-α and IFNγ-stimulated Primary Cultured Astrocytes

Here we have investigated how lactosylceramide (LacCer) modulates gene expression of adhesion molecules in TNF-α and IFNγ (CM)-stimulated astrocytes. We have observed that stimulation of astrocytes with CM increased the gene expression of ICAM-1 and VCAM-1. D-Threo-1-phenyl- 2-decanoylamino-3-morpholino-1-propanol (PDMP) and N-butyldeoxynojirimycin (NBDNJ), inhibitors of glucosylceramide synthase (GLS) and LacCer synthase (galactosyltransferase, GalT-2), inhibited the gene expression of ICAM-1 and VCAM-1 and activation of their gene promoter induced by CM, which were reversed by exogenously supplied LacCer. Silencing of GalT-2 gene using its antisense oligonucleotides also attenuated CM-induced ICAM-1 and VCAM-1 expression, which were reversed by LacCer. PDMP treatment and silencing of GalT-2 gene significantly reduced CM-induced luciferase activities in NF-KB, AP-1, GAS, and STAT-3 luciferase vectors-transfected cells. In addition, LacCer reversed the inhibition of NF-KB and STAT-1 luciferase activities by PDMP. Taken together, our results suggest that LacCer may play a crucial role in the expression of ICAM-1 and VCAM-1 via modulating transcription factors, such as NF-KB, AP-1, STAT-1, and STAT-3 in CM-stimulated astrocytes.

Dupuytren's: a systems biology disease.

Dupuytren's disease (DD) is an ill-defined fibroproliferative disorder of the palm of the hands leading to digital contracture. DD commonly occurs in individuals of northern European extraction. Cellular components and processes associated with DD pathogenesis include altered gene and protein expression of cytokines, growth factors, adhesion molecules, and extracellular matrix components. Histology has shown increased but varying levels of particular types of collagen, myofibroblasts and myoglobin proteins in DD tissue. Free radicals and localised ischaemia have been suggested to trigger the proliferation of DD tissue. Although the existing available biological information on DD may contain potentially valuable (though largely uninterpreted) information, the precise aetiology of DD remains unknown. Systems biology combines mechanistic modelling with quantitative experimentation in studies of networks and better understanding of the interaction of multiple components in disease processes. Adopting systems biology may be the ideal approach for future research in order to improve understanding of complex diseases of multifactorial origin. In this review, we propose that DD is a disease of several networks rather than of a single gene, and show that this accounts for the experimental observations obtained to date from a variety of sources. We outline how DD may be investigated more effectively by employing a systems biology approach that considers the disease network as a whole rather than focusing on any specific single molecule.

Endothelial Argininosuccinate Synthetase 1 Regulates Nitric Oxide Production and Monocyte Adhesion under Static and Laminar Shear Stress Conditions

Laminar shear stress (LSS) is known to increase endothelial nitric oxide (NO) production, which is essential for vascular health, through expression and activation of nitric oxide synthase 3 (NOS3). Recent studies demonstrated that LSS also increases the expression of argininosuccinate synthetase 1 (ASS1) that regulates the provision of L-arginine, the substrate of NOS3. It was thus hypothesized that ASS1 might contribute to vascular health by enhancing NO production in response to LSS. This hypothesis was pursued in the present study by modulating NOS3 and ASS1 levels in cultured endothelial cells. Exogenous expression of either NOS3 or ASS1 in human umbilical vein endothelial cells increased NO production and decreased monocyte adhesion stimulated by tumor necrosis factor-α (TNF-α). The latter effect of overexpressed ASS1 was reduced when human umbilical vein endothelial cells were co-treated with small interfering RNAs (siRNAs) for ASS1 or NOS3. SiRNAs of NOS3 and ASS1 attenuated the increase of NO production in human aortic endothelial cells stimulated by LSS (12 dynes·cm(-2)) for 24 h. LSS inhibited monocyte adhesion to human aortic endothelial cells stimulated by TNF-α, but this effect of LSS was abrogated by siRNAs of NOS3 and ASS1 that recovered the expression of vascular cell adhesion molecule-1. The current study suggests that the expression of ASS1 harmonized with that of NOS3 may be important for the optimized endothelial NO production and the prevention of the inflammatory monocyte adhesion to endothelial cells.