Mouse Vascular Endothelial Cells Research Articles

Peroxiredoxin 1 Stimulates Endothelial Cell Expression of VEGF via TLR4 Dependent Activation of HIF-1α

Chronic inflammation leads to the formation of a pro-tumorigenic microenvironment that can promote tumor development, growth and differentiation through augmentation of tumor angiogenesis. Prostate cancer (CaP) risk and prognosis are adversely correlated with a number of inflammatory and angiogenic mediators, including Toll-like receptors (TLRs), NF-κB and vascular endothelial growth factor (VEGF). Peroxiredoxin 1 (Prx1) was recently identified as an endogenous ligand for TLR4 that is secreted from CaP cells and promotes inflammation. Inhibition of Prx1 by CaP cells resulted in reduced expression of VEGF, diminished tumor vasculature and retarded tumor growth. The mechanism by which Prx1 regulates VEGF expression in normoxic conditions was investigated in the current study. Our results show that incubation of mouse vascular endothelial cells with recombinant Prx1 caused increases in VEGF expression that was dependent upon TLR4 and required hypoxia inducible factor-1 (HIF-1) interaction with the VEGF promoter. The induction of VEGF was also dependent upon NF-κB; however, NF-κB interaction with the VEGF promoter was not required for Prx1 induction of VEGF suggesting that NF-κB was acting indirectly to induce VEGF expression. The results presented here show that Prx1 stimulation increased NF-κB interaction with the HIF-1α promoter, leading to enhanced promoter activity and increases in HIF-1α mRNA levels, as well as augmented HIF-1 activity that resulted in VEGF expression. Prx1 induced HIF-1 also promoted NF-κB activity, suggesting the presence of a positive feedback loop that has the potential to perpetuate Prx1 induction of angiogenesis. Strikingly, inhibition of Prx1 expression in CaP was accompanied with reduced expression of HIF-1α. The combined findings of the current study and our previous study suggest that Prx1 interaction with TLR4 promotes CaP growth potentially through chronic activation of tumor angiogenesis.

Flavopiridol inhibits interferon-γ-induced nitric oxide production in mouse vascular endothelial cells

Flavopiridol (FP), a synthetic flavone, is a cyclin-dependent kinase inhibitor and possesses an anti-cancer activity. The effect of FP on interferon (IFN)-γ-induced nitric oxide (NO) production in mouse vascular endothelial cell line END-D was examined. FP significantly inhibited IFN-γ-induced NO production in END-D cells via reduced expression of an inducible NO synthase. FP inhibited the activation of STAT1, and subsequently IRF1 as a downstream molecule of STAT1, which is essential for IFN-γ-induced NO production. FP did not affect the cell surface expression of IFN-γ receptor. Taken together, FP was suggested to inhibit IFN-γ-induced NO production in vascular endothelial cells via preventing intracellular IFN-γ signaling. FP might be useful as an immunomodulatory drug as well as an anti-cancer drug.

Anti-Tumor Activity of a Novel HS-Mimetic-Vascular Endothelial Growth Factor Binding Small Molecule

The angiogenic process is controlled by variety of factors of which the vascular endothelial growth factor (VEGF) pathway plays a major role. A series of heparan sulfate mimetic small molecules targeting VEGF/VEGFR pathway has been synthesized. Among them, compound 8 (2-butyl-5-chloro-3-(4-nitro-benzyl)-3H-imidazole-4-carbaldehyde) was identified as a significant binding molecule for the heparin-binding domain of VEGF, determined by high-throughput-surface plasmon resonance assay. The data predicted strong binding of compound 8 with VEGF which may prevent the binding of VEGF to its receptor. We compared the structure of compound 8 with heparan sulfate (HS), which have in common the functional ionic groups such as sulfate, nitro and carbaldehyde that can be located in similar positions of the disaccharide structure of HS. Molecular docking studies predicted that compound 8 binds at the heparin binding domain of VEGF through strong hydrogen bonding with Lys-30 and Gln-20 amino acid residues, and consistent with the prediction, compound 8 inhibited binding of VEGF to immobilized heparin. In vitro studies showed that compound 8 inhibits the VEGF-induced proliferation migration and tube formation of mouse vascular endothelial cells, and finally the invasion of a murine osteosarcoma cell line (LM8G7) which secrets high levels of VEGF. In vivo, these effects produce significant decrease of tumor burden in an experimental model of liver metastasis. Collectively, these data indicate that compound 8 may prevent tumor growth through a direct effect on tumor cell proliferation and by inhibition of endothelial cell migration and angiogenesis mediated by VEGF. In conclusion, compound 8 may normalize the tumor vasculature and microenvironment in tumors probably by inhibiting the binding of VEGF to its receptor.

Expression, Regulation, and Function of Atypical Chemerin Receptor CCRL2 on Endothelial Cells

Chemokine (CC motif) receptor-like 2 (CCRL2) binds leukocyte chemoattractant chemerin and can regulate local levels of the attractant, but does not itself support cell migration. In this study, we show that CCRL2 and VCAM-1 are upregulated on cultured human and mouse vascular endothelial cells (EC) and cell lines by proinflammatory stimuli. CCRL2 induction is dependent on NF-κB and JAK/STAT signaling pathways, and activated endothelial cells specifically bind chemerin. In vivo, CCRL2 is constitutively expressed at high levels by lung endothelial cells and at lower levels by liver endothelium; and liver but not lung EC respond to systemic LPS injection by further upregulation of the receptor. Plasma levels of total chemerin are elevated in CCRL2(-/-) mice and are significantly enhanced after systemic LPS treatment in CCRL2(-/-) mice compared with wild-type mice. Following acute LPS-induced pulmonary inflammation in vivo, chemokine-like receptor 1 (CMKLR1)(+) NK cell recruitment to the airways is significantly impaired in CCRL2(-/-) mice compared with wild-type mice. In vitro, chemerin binding to CCRL2 on endothelial cells triggers robust adhesion of CMKLR1(+) lymphoid cells through an α(4)β(1) integrin/VCAM-1-dependent mechanism. In conclusion, CCRL2 is expressed by EC in a tissue- and activation-dependent fashion, regulates circulating chemerin levels and its bioactivity, and enhances chemerin- and CMKLR1-dependent lymphocyte/EC adhesion in vitro and recruitment to inflamed airways in vivo. Its expression and/or induction on EC by proinflammatory stimuli provide a novel and specific mechanism for the local enrichment of chemerin at inflammatory sites, regulating the recruitment of CMKLR1(+) cells.

The PI3 Kinase δ Inhibitor, CAL-101 (GS-1101), Inhibits Chronic Lymphocytic Leukemia (CLL) Cell Survival in Endothelial and Marrow Stromal Cell Co-Cultures.

Abstract 1769CLL cells are characterized by their ability to resist apoptosis in vivo, but in vitro they undergo spontaneous apoptosis. This suggests that interactions between CLL cells and accessory cells in the tissue microenvironments, such as mesenchymal stromal cells (MSC), nurselike cells (NLC), T-cells, and endothelial cells are critical for maintaining CLL cell survival. CLL cells display constitutive PI3K pathway activation, presumable due to CLL interactions with the microenvironment. CAL-101 is a potent and selective inhibitor of the p110d PI3K isoform and has shown promising clinical activity in chronic lymphocytic leukemia (CLL) in early stage clinical trials. Here, we investigated the ability of CAL-101 to disrupt interactions between CLL and endothelial cells (EC) or bone marrow stromal cells (BMSC). We tested two EC lines human umbilical vein endothelial cells (HUVEC) and UV-2 mouse vascular endothelial cells, and two BMSC lines, stroma-NKtert derived from human bone marrow, and KUSA-H1, a murine BMSC line. CLL cells were cultured for 72h in presence or absence of EC or BMSC. Fig A displays mean (±SEM) CLL cell viabilities of cells from 7 different patients. We found that both, EC and BMSC rescue CLL cells from spontaneous apoptosis with significantly higher CLL cell viabilities in the presence of EC and BMSC (*P< 0.05; **P< 0.01). For example, after 48h significantly higher CLL cell viabilities were noticed with HUVEC (53.2%±4.3%, p<0.05,), UV2 (61.8%±5.3%, p<0.01), stroma-NKtert (96.7%±5.3%, p<0.01) and KUSA-H1 (93.7%±0.95%, p<0.01), when compared to CLL cultured in medium alone (37.5%±4.1%).To test the effects of CAL-101 on EC- and BMSC-mediated CLL cell protection, CLL cells were cultured on ECs or BMSCs in presence or absence of CAL-101 (0.5μM and 5μM), and CLL cell viabilities were assessed at 24h, 48h and 72h. Viabilities of CAL-101 treated samples were normalized to the viabilities of control samples at the respective timepoints (100%). Fig B depicts the mean relative viabilities of CLL cells co-culture with ECs or BMCSs in presence of 5μM CAL-101, compared to CLL cells in the absence of CAL-101. We found a significant reduction of the viability of CLL cells in co-culture with EC and BMSC with both concentrations of CAL-101 (*P< 0.05; **P< 0.01; n=7).These data demonstrate that marrow stromal and endothelial cells both support the viability and protect CLL cells from apoptosis. When comparing BMSC with EC, we noticed that BMSC were more effective than EC in protecting CLL cells, which may explain why the marrow is a preferred site for residual disease and relapses in patients with CLL. CAL-101 can overcome both, BMSC- and EC-mediated CLL cell protection, indicating that CAL-101 inhibits BMSC- and EC-derived pro-survival signals. Ongoing experiments investigate the role of adhesion molecules on BMSC- and EC-derived survival signals and CLL cell adhesion to BMSC versus EC, and how adhesion molecule function is affected by CAL-101. These studies will give us better insight into the mechanism of action of this interesting new drug. [Display omitted] Disclosures:O'Brien:Gilead: Consultancy, Research Support. Lannutti:Gilead Sciences: Employment.

Abstract 4883: Peptide-based PET imaging agents using novel bifunctional chelates and 64Cu

Abstract Introduction: Developing peptide-based PET imaging agents would be highly advantageous due to their high affinity and specificity. Two novel bifunctional chelates (BFC), PCTA and Oxo-DO3A, have previously been used to label antibodies with 64Cu. Complexation of 64Cu with PCTA and Oxo-DO3A was more efficient than with DOTA; the PCTA and OXO-DO3A complexes were also more stable and had more favourable pharmacokinetics[1]. Similarly, PCTA was also found to complex 68Ga more efficiently than DOTA[2]. The utility of PCTA and Oxo-DO3A for radiolabeling peptides specific for tumour angiogenesis processes were further investigated. Methods: The receptor number and affinity of RGD peptides were determined in mouse vascular endothelial cells. Mouse vascular endothelial cells were grown and varying amounts of I-125 echistatin were added to 1 million cells until receptors were saturated. A competitive binding assay, where a set amount of I-125 echistatin was added to the cells in the presence of unlabeled peptides, confirmed the specificity of the peptides for the target. In vivo studies were carried out in HT29 human colorectal tumors grown subcutaneously in RAG2M mice. 64Cu radiolabeled bioconjugates of PCTA, Oxo-DO3A and DOTA were injected with and without excess cyclo-(RGDyK) peptide. The amount of activity in the blood, tumor, liver, kidney and muscle was measured and compared to each of the BFC. Results: The binding assay revealed the saturation of mouse vascular endothelial cells was achieved when 0.8µci of I-125 echistatin was added. The competitive binding assay indicated an IC50 at a concentration of 9pM of DOTA-RGD. In vivo biodistribution studies showed good tumor to blood ratios for all of the 64Cu radiolabeled bioconjugates and better tumor to muscle ratios for the 64Cu radiolabeled bioconjugates of PCTA and Oxo-DO3A. Conclusion: The bifunctional chelates PCTA and Oxo-DO3A were found to be effective for radiolabeling peptides with 64Cu; the resulting complexes appear to be more stable than those of DOTA. The data show that PCTA and Oxo-DO3A radiolabel peptides more efficiently than DOTA, and that the resulting complexes are more stable. Angiogenesis in HT-29 tumours was also imaged successfully using the peptides labeled with PCTA and Oxo-DO3A.

TIE-ing off atherosclerosis

A Vanderbilt team has found that knocking out the tyrosine kinase Tie1 on vascular endothelial cells in mice decreases the formation of atherosclerotic plaques. The findings could represent a new indication for sanofi-aventis, which has licensed at least two therapies targeting TIE proteins in cancer and wound healing.

Thalidomide inhibits interferon-γ-mediated nitric oxide production in mouse vascular endothelial cells

Thalidomide is known as an anti-angiogenic, anti-tumor, and anti-proliferative agent, widely used in the treatment of some immunological disorders and cancers. The effect of thalidomide on interferon (IFN)-γ induced nitric oxide (NO) production in mouse vascular endothelial cells was examined in order to elucidate the anti-angiogenic or anti-inflammatory action. Thalidomide inhibited IFN-γ-induced NO production in mouse END-D cells via reduced expression of an inducible type of NO synthase (iNOS) protein and mRNA. Since thalidomide did not alter the cell surface expression of IFN-γ receptor, the NO inhibition was suggested to be due to the impairment of IFN-γ-induced intracellular event by thalidomide. Thalidomide inhibited the phosphorylation of IRF1, which was required for the iNOS expression. Moreover, it inhibited the phosphorylation of STAT1, an upstream molecule of IRF1, in IFN-γ signaling. Thalidomide did not inhibit the JAK activation in response to IFN-γ. A phosphatase inhibitor, sodium orthovanadate, abolished the inhibitory action of thalidomide. Therefore, thalidomide was suggested to inhibit IFN-γ-induced NO production via impaired STAT1 phosphorylation.

Cytosolic Phospholipase A2 and Lysophospholipids in Tumor Angiogenesis

BackgroundLung cancer and glioblastoma multiforme are highly angiogenic and, despite advances in treatment, remain resistant to therapy. Cytosolic phospholipase A2 (cPLA2) activation contributes to treatment resistance through transduction of prosurvival signals. We investigated cPLA2 as a novel molecular target for antiangiogenesis therapy.MethodsGlioblastoma (GL261) and Lewis lung carcinoma (LLC) heterotopic tumor models were used to study the effects of cPLA2 expression on tumor growth and vascularity in C57/BL6 mice wild type for (cPLA2α+/+) or deficient in (cPLA2α−/−) cPLA2α, the predominant isoform in endothelium (n = 6–7 mice per group). The effect of inhibiting cPLA2 activity on GL261 and LLC tumor growth was studied in mice treated with the chemical cPLA2 inhibitor 4-[2-[5-chloro-1-(diphenylmethyl)-2-methyl-1H-indol-3-yl]-ethoxy]benzoic acid (CDIBA). Endothelial cell proliferation and function were evaluated by Ki-67 immunofluorescence and migration assays in primary cultures of murine pulmonary microvascular endothelial cells (MPMEC) isolated from cPLA2α+/+ and cPLA2α−/− mice. Proliferation, invasive migration, and tubule formation were assayed in mouse vascular endothelial 3B-11 cells treated with CDIBA. Effects of lysophosphatidylcholine, arachidonic acid, and lysophosphatidic acid (lipid mediators of tumorigenesis and angiogenesis) on proliferation and migration were examined in 3B-11 cells and cPLA2α−/− MPMEC. All statistical tests were two-sided.ResultsGL261 tumor progression proceeded normally in cPLA2α+/+ mice, whereas no GL261 tumors formed in cPLA2α−/− mice. In the LLC tumor model, spontaneous tumor regression was observed in 50% of cPLA2α−/− mice. Immunohistochemical examination of the remaining tumors from cPLA2α−/− mice revealed attenuated vascularity (P ≤ .001) compared with tumors from cPLA2α+/+ mice. Inhibition of cPLA2 activity by CDIBA resulted in a delay in tumor growth (eg, LLC model: average number of days to reach tumor volume of 700 mm3, CDIBA vs vehicle: 16.8 vs 11.8, difference = 5, 95% confidence interval = 3.6 to 6.4, P = .04) and a decrease in tumor size (eg, GL261 model: mean volume on day 21, CDIBA vs vehicle: 40.1 vs 247.4 mm3, difference = 207.3 mm3, 95% confidence interval = 20.9 to 293.7 mm3, P = .021). cPLA2 deficiency statistically significantly reduced MPMEC proliferation and invasive migration (P = .002 and P = .004, respectively). Compared with untreated cells, cPLA2α−/− MPMEC treated with lysophosphatidylcholine and lysophosphatidic acid displayed increased cell proliferation (P = .011) and invasive migration (P < .001).ConclusionsIn these mouse models of brain and lung cancer, cPLA2 and lysophospholipids have key regulatory roles in tumor angiogenesis. cPLA2 inhibition may be a novel effective antiangiogenic therapy.

Effects of H2-B1 gene transfection on the immunogenicity of mouse vascular endothelial cells

Objective The pEGFP-N1 -H2B1 plasmid vector was transfected to mouse vascular endothelial cells (VECs) , the immunogenicity effect of VECs was investigated, and the possible mechanism of immune tolerance after heart transplantation mirroring matemal-fetal immune tolerance was speculated. Methods The mouse VECs were transfected with pEGFP-Nl-H2Bl plasmid vector. The efficiency of transfection was tested by flow cytometry, the expression of H2-B1 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) , and the cytolysis of peripheral blood mononuclear cells (PBMC)was investigated at 24, 48 and 72 h, respectively. Results The expression levels of H2-B1 in 0. 5 mg/L and 1.0 mg/L pEGFP-N1-H2B1 plasmid vector groups were higher than in control group (P <0.05, P <0. 01, respectively). After 48 h, the cytolysis of PBMC to VECs in H2-B1-transfected groups was significantly attenuated as compared with control group ( P <0.05). Conclusion The H2-B1 gene may have a possible immunoregulative effect. Transfection of pEGFP-N1-H2B1 plasmid vector to mouse VECs can reduce the immunogenicity of VECs, attenuate the cytotoxicity of PBMC, and induce immune tolerance. Key words: H2-B1 gene; Immunogenicity; Vascular endothelial cell

MURINE rVEGF164b, AN INHIBITORY VEGF REDUCES VEGF-A DEPENDENT ENDOTHELIAL PROLIFERATION AND BARRIER DYSFUNCTION

To investigate the effects of the murine inhibitory vascular endothelial growth factor (VEGF, rVEGF164b), we generated an adenoviral vector encoding rVEGF164b, and examined its effects on endothelial barrier, growth, and structure. Mouse vascular endothelial cells (MVEC) proliferation was determined by an MTT assay. Barrier of MVEC monolayers was measured by trans-endothelial electrical resistance (TEER). Reorganization of actin and zonula occludens-1 (ZO-1) were determined by fluorescent microscopy. Mouse venous endothelial cells treated with murine VEGF-A (VEGF-A) (50 ng/mL) increased proliferation (60.7 ± 0.1%) within 24 hours (p < 0.05) and rVEGF164b inhibited VEGF-A-induced proliferation. TEER was significantly decreased by VEGF-A (81.7 ± 6.2% of control). Treatment with rVEGF164b at 50 ng/mL transiently reduced MVEC barrier (p < 0.05) at 30 minutes post-treatment (87.9 ± 1.7% of control TEER), and returned to control levels by 40 minutes post-treatment. Treatment with rVEGF164b prevented barrier changes by subsequent exposure to VEGF-A. Treatment of MVECS with VEGF-A reorganized F-actin and ZO-1, which was attenuated by rVEGF164b. VEGF-A may dysregulate endothelial barrier through junctional cytoskeleton processes, which can be attenuated by rVEGF164b. The VEGF-A stimulated MVEC proliferation, barrier dysregulation, and cytoskeletal rearrangement. However, rVEGF164b blocks these effects, therefore it may be useful for regulation studies of VEGF-A/VEGF-R signaling in many different models.

Bevacizumab suppression of establishment of micrometastases in experimental ocular melanoma.

This study was undertaken to determine whether anti-vascular endothelial growth factor (VEGF) therapy inhibits growth of primary uveal melanoma and spread of its hepatic micrometastases. The human uveal melanoma cell lines Mel290 and Mel 270, HUVECs, mouse B16LS9 melanoma cells, and mouse vascular endothelial cells were separately cultured or co-cultured and incubated with bevacizumab or IgG1. The level of VEGF protein in the culture medium was measured by ELISA. In vitro angiogenesis and invasion assays were performed under bevacizumab or IgG1 treatment. Mel290 or B16LS9 cells were inoculated into NU/NU or C57Bl/6 mouse eyes which were enucleated after 7 days. The sizes of the intraocular tumors were determined. Time and dosage experiments were performed by using 50 or 250 microg bevacizumab starting at day 1 or 4 after inoculation. Hepatic micrometastases were enumerated. Proliferation, apoptosis, and angiogenesis markers were detected in the ocular tumor by immunofluorescence staining. Bevacizumab significantly reduced the level of VEGF in the culture media from human uveal melanoma cells, mouse melanoma cells, and co-cultured cells. It also inhibited cell tube formation and decreased in vitro invasion of tumor cells. In the mouse model, bevacizumab suppressed primary ocular melanoma growth and the formation of hepatic micrometastases in a dose-dependent manner. Furthermore, immunohistochemical staining showed decreased Ki67 and unchanged caspase 3 expression after treatment with bevacizumab. Treatment with bevacizumab suppressed in vitro growth and in vivo hepatic micrometastasis of ocular melanoma cells. Bevacizumab is a potential therapeutic agent for the treatment of uveal melanoma micrometastases.

Involvement of a disintegrin and metalloproteinase 10 and 17 in shedding of tumor necrosis factor-α

Tumor necrosis factor-alpha (TNF-alpha) is initially synthesized as a membrane-bound protein and converted into a soluble form by proteolytic cleavage. Although a disintegrin and metalloproteinase 17 (ADAM17) is considered to be the primary sheddase for TNF-alpha, it is not known whether ADAM17 is solely responsible for that process in any type of cells. To identify the TNF-alpha sheddase(s) in varieties of cells, we performed experiments using a unique screening system and observed that ADAM9, ADAM10, ADAM17, and ADAM19 were capable of cleaving TNF-alpha. We then performed RNA interference experiments and confirmed that ADAM10 and ADAM17 were in fact involved in TNF-alpha shedding in 293A cells. In mouse macrophages, ADAM17 was confirmed to be the primary sheddase, but the involvement of ADAM10 was also demonstrated. In NIH3T3 cells, ADAM10 could be more important in the shedding than ADAM17. In mouse vascular endothelial cell line UVfemale2, ADAM10 and ADAM17 were equally involved in TNF-alpha shedding, whereas ADAM17 was a major sheddase in human osteoarthritic chondrocytes. From these observations and others, we concluded that both ADAM10 and ADAM17 can be a TNF-alpha sheddase and that their significance could be determined by their expression levels and the abundance of tissue inhibitor of metalloproteinases.

432. Silencing α1,3GT Expression by Lentivirus-Mediated shRNA in Mouse Vascular Endothelial Cells

432. Silencing α1,3GT Expression by Lentivirus-Mediated shRNA in Mouse Vascular Endothelial Cells

Ciliary Polycystin-2 Is a Mechanosensitive Calcium Channel Involved in Nitric Oxide Signaling Cascades

Cardiovascular complications such as hypertension are a continuous concern in patients with autosomal dominant polycystic kidney disease (ADPKD). The PKD2 encoding for polycystin-2 is mutated in approximately 15% of ADPKD patients. Here, we show that polycystin-2 is localized to the cilia of mouse and human vascular endothelial cells. We demonstrate that the normal expression level and localization of polycystin-2 to cilia is required for the endothelial cilia to sense fluid shear stress through a complex biochemical cascade, involving calcium, calmodulin, Akt/PKB, and protein kinase C. In response to fluid shear stress, mouse endothelial cells with knockdown or knockout of Pkd2 lose the ability to generate nitric oxide (NO). Consistent with mouse data, endothelial cells generated from ADPKD patients do not show polycystin-2 in the cilia and are unable to sense fluid flow. In the isolated artery, we further show that ciliary polycystin-2 responds specifically to shear stress and not to mechanical stretch, a pressurized biomechanical force that involves purinergic receptor activation. We propose a new role for polycystin-2 in transmitting extracellular shear stress to intracellular NO biosynthesis. Thus, aberrant expression or localization of polycystin-2 to cilia could promote high blood pressure because of inability to synthesize NO in response to an increase in shear stress (blood flow).

S0201-2-2 糖鎖が静置並びに流れ負荷下の血管内皮細胞の挙動に及ぼす影響(マイクロ・ナノバイオメカニクス:細胞生物学への接近(2)刺激と応答)

It has been reported that glycans play pivotal roles in biomechanical responses of cells. To reveal the roles of glycans in mechanical response of endothelial cells, we observed morphological changes of cultured mouse vascular endothelial cells treated with deoxy-D-glucose (DG), which modifies protein glycosylation. When confluent cell monolayer in static culture was treated with DG, it formed mesh-like structure in 48 hours. This change was accompanied by decrease in the total area of focal adhesions that indicates the weakening of cell-substrate adhesion. Such weakening may cause loss of ability to repair gaps between cells that may form in cell monolayer temporally. When exposed to fluid shear stress, normal cells aligned in the direction of flow while treated cells did not. These results indicate that glycans have significant effects on mechanical response of vascular endothelial cells.

AIP1 Is Critical in Transducing IRE1-mediated Endoplasmic Reticulum Stress Response

We have previously shown that ASK1-interacting protein 1 (AIP1) transduces tumor necrosis factor-induced ASK1-JNK signaling. Because endoplasmic reticulum (ER) stress activates ASK1-JNK signaling cascade, we investigated the role of AIP1 in ER stress-induced signaling. We created AIP1-deficient mice (AIP1-KO) from which mouse embryonic fibroblasts and vascular endothelial cells were isolated. AIP1-KO cells show dramatic reductions in ER stress-induced, but not oxidative stress-induced, ASK1-JNK activation and cell apoptosis. The ER stress-induced IRE1-JNK/XBP-1 axis, but not the PERK-CHOP1 axis, is blunted in AIP1-KO cells. ER stress induced formation of an AIP1-IRE1 complex, and the PH domain of AIP1 is critical for the IRE1 interaction. Furthermore, reconstitution of AIP1-KO cells with AIP1 wild type, not an AIP1 mutant with a deletion of the PH domain (AIP1-DeltaPH), restores ER stress-induced IRE1-JNK/XBP-1 signaling. AIP1-IRE1 association facilitates IRE1 dimerization, a critical step for activation of IRE1 signaling. More importantly, AIP1-KO mice show impaired ER stress-induced IRE1-dependent signaling in vivo. We conclude that AIP1 is essential for transducing the IRE1-mediated ER stress response.

Lipopolysaccharide and interferon-γ enhance Fas-mediated cell death in mouse vascular endothelial cells via augmentation of Fas expression

The effect of interferon (IFN)-gamma and/or lipopolysaccharide (LPS) on Fas-mediated cell death with anti-Fas agonistic antibody in vascular endothelial cells was examined using a mouse END-D cell line. Anti-Fas agonistic antibody exhibited cytotoxic actions on END-D cells. Fas-mediated cell death was enhanced by LPS or IFN-gamma. The combination of IFN-gamma and LPS significantly enhanced cell death compared to IFN-gamma or LPS alone. IFN-gamma and LPS augmented cell surface expression of Fas, but not tumour necrosis factor (TNF) receptor 1. Inhibitors of p38 mitogen-activated protein kinase (MAPK) prevented augmentation of Fas expression in IFN-gamma and LPS-treated END-D cells. IFN-gamma and LPS-treated END-D cells did not become susceptible to TNF-alpha or nitric oxide-mediated cytotoxicity. IFN-gamma and LPS thus appear to augment selectively Fas expression via activation of p38 MAPK and enhance Fas-mediated cell death in END-D cells. Furthermore, administration of IFN-gamma and LPS into mice induced in vivo expression of Fas on vascular endothelial cells and Fas ligand (FasL) on peripheral blood leucocytes. The relationship between enhancement of Fas-mediated cell death by IFN-gamma and LPS and the development of vascular endothelial injury is discussed.

Amelioration of lacrimal gland inflammation by oral administration of K-13182 in Sjögren's syndrome model mice

Regulation of the adhesion of mononuclear cells to endothelial cells is considered to be a critical step for the treatment of inflammatory diseases, including autoimmune diseases. K-13182 was identified as a novel inhibitor for these adhesions. K-13182 inhibited the expression of vascular cell adhesion molecule-1 (VCAM-1, CD106) on human umbilical vein endothelial cells (HUVECs) and on mouse vascular endothelial cell line (MAECs) induced by tumour necrosis factor (TNF)-alpha. K-13182 also inhibited the adhesion of mononuclear cells to these HUVECs and MAECs, indicating that K-13182 suppressed these adhesions mediated by cellular adhesion molecules including VCAM-1. To evaluate the therapeutic effect in autoimmune disease model mice, K-13182 was orally administered to non-obese diabetic (NOD) mice as Sjögren's syndrome (SS) model mice. Severe destructive inflammatory lesions were observed in the lacrimal glands of vehicle-treated control mice; however, 8-week administration of K-13182 inhibited the mononuclear cell infiltration into the inflammatory lesions of the lacrimal glands. In K-13182-treated mice, the decrease in tear secretion was also prevented compared to the control mice. In addition, the apoptosis and the expression of FasL (CD178), perforin, and granzyme A was suppressed in the lacrimal glands of K-13182-treated mice. Therefore, K-13182 demonstrated the possibility of therapeutic efficacy for the inflammatory region of autoimmune disease model mice. These data reveal that VCAM-1 is a promising target molecule for the treatment of autoimmune diseases as a therapeutic strategy and that K-13182 has the potential as a new anti-inflammatory drug for SS.

Redundant roles of Sox17 and Sox18 in early cardiovascular development of mouse embryos

Sox7, -17 and -18 constitute the Sox subgroup F ( SoxF) of HMG box transcription factor genes, which all are co-expressed in developing vascular endothelial cells in mice. Here we characterized cardiovascular phenotypes of Sox17/ Sox18-double and Sox17-single null embryos during early-somite stages. Whole-mount PECAM staining demonstrated the aberrant heart looping, enlarged cardinal vein and mild defects in anterior dorsal aorta formation in Sox17 single-null embryos. The Sox17/ Sox18 double-null embryos showed more severe defects in formation of anterior dorsal aorta and head/cervical microvasculature, and in some cases, aberrant differentiation of endocardial cells and defective fusion of the endocardial tube. However, the posterior dorsal aorta and allantoic microvasculature was properly formed in all of the Sox17/ Sox18 double-null embryos. The anomalies in both anterior dorsal aorta and head/cervical vasculature corresponded with the weak Sox7 expression sites. This suggests the region-specific redundant activities of three SoxF members along the anteroposterior axis of embryonic vascular network.