Introduction of short interfering RNA to silence endogenous E-selectin in vascular endothelium leads to successful inhibition of leukocyte adhesion

Nishiwaki, Yasunobu; Yokota, Takanori; Hiraoka, M; Miyagishi, Makoto; Taira, Kazunari; Isobe, Mitsuaki; Mizusawa, Hidehiro; Yoshida, Masayuki

doi:10.1016/j.bbrc.2003.09.125

Cited by 16 publications

(18 citation statements)

References 11 publications

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“…1B). Multiple E-selectin protein bands with molecular masses of ϳ115 kDa suggest the posttranslational modification of E-selectin, as has previously been reported (40,56). The HOXA9 protein (ϳ32 kDa) was constitutively expressed in EC, and a 3-h TNF-␣ treatment had little, if any, effect on the protein level.…”

Section: Vol 27 2007 Hoxa9 In E-selectin Expression 4209supporting

confidence: 66%

HOXA9 Participates in the Transcriptional Activation of E-Selectin in Endothelial Cells

Bandyopadhyay

Ashraf

Daher

et al. 2007

Molecular and Cellular Biology

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The homeobox gene HOXA9 has recently been shown to be an important regulator of endothelial cell (EC) differentiation and activation in addition to its role in embryonic development and hematopoiesis. In this report, we have determined that the EC-leukocyte adhesion molecule E-selectin is a key target for HOXA9. The depletion of HOXA9 protein in ECs resulted in a significant and specific decrease in tumor necrosis factor alpha (TNF-␣)-induced E-selectin gene expression. In addition, HOXA9 specifically activated the E-selectin gene promoter in ECs. Progressive deletional analyses together with site-specific mutagenesis of the E-selectin promoter indicated that the Abd-B-like HOX DNA-binding motif, CAATTTTATTAA, located in the proximal region spanning bp ؊210 to ؊221 upstream of the transcription start site was crucial for the promoter induction by HOXA9. Both HOXA9 in EC nuclear extract and recombinant HOXA9 protein bound to this sequence in vitro. Moreover, we showed that HOXA9 binds temporally, in a TNF-␣-dependent manner, to the region containing this Abd-B-like element in vivo. We have thus identified a novel and functionally critical cis-regulatory element for TNF-␣-mediated transient expression of the E-selectin gene. Further, we provide evidence that HOXA9 acts as an obligate proinflammatory factor by mediating cytokine induction of E-selectin.

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Section: Vol 27 2007 Hoxa9 In E-selectin Expression 4209supporting

confidence: 66%

HOXA9 Participates in the Transcriptional Activation of E-Selectin in Endothelial Cells

Bandyopadhyay

Ashraf

Daher

et al. 2007

Molecular and Cellular Biology

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“…Recombinant human TNF-␣ was obtained from R&D Systems (Minneapolis, MN). A monoclonal antibody against E-selectin (clone 7A9) was obtained from American Type Culture Collection (Manassas, VA) (13). Antibodies against ICAM-1, VCAM-1, the NF-B p65 subunit, and the phospho-NF-B p65 subunit and a monoclonal blocking antibody against mouse E-selectin (clone UZ4) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).…”

Section: Methodsmentioning

confidence: 99%

Indoxyl Sulfate Induces Leukocyte-Endothelial Interactions through Up-regulation of E-selectin

Ito

Osaka

Higuchi

et al. 2010

Journal of Biological Chemistry

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146

133

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Cardiovascular disease is a major cause of death in chronic kidney disease (CKD) 2 (1). Atherosclerosis is highly prevalent in patients with severe renal failure and advances more rapidly in individuals with renal dysfunction compared with the general population (2). Reduced kidney function is associated with the risk of cardiovascular events, even when the dysfunction is mild (3).Leukocyte-endothelial interactions play an important role in the development of atherosclerosis (4). Cell adhesion molecules belonging to the immunoglobulin superfamily, such as ICAM-1 (intercellular cell adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1), together with members of the selectin family, including E-selectin, are upregulated to mediate monocyte/macrophage infiltration into atherosclerotic lesions (4, 5).Indoxyl sulfate is a uremic toxin synthesized in the liver from indole, a metabolite of tryptophan produced by the intestinal flora (6). In CKD patients, the serum levels of indoxyl sulfate are increased significantly compared with those in healthy individuals (7), and a number of studies have indicated that indoxyl sulfate accelerates glomerular sclerosis, whereas its accumulation promotes renal failure (8 -10). Other studies also showed that indoxyl sulfate induces endothelial dysfunction by releasing endothelial microparticles (11) and producing reactive oxygen species (ROS) (12). However, its effect on endothelial inflammatory processes such as leukocyte recruitment to vascular endothelium has not been reported.We report for the first time that indoxyl sulfate enhances monocyte adhesion to vascular endothelium through up-regulation of E-selectin and augmentation of oxidative stress in both in vitro and in vivo models. The underlying mechanisms seem to involve activation of JNK and NF-B. Our findings reveal a previously unrecognized molecular link between uremic toxins and cardiovascular diseases. EXPERIMENTAL PROCEDURESReagents-Indoxyl sulfate, N-acetylcysteine, probenecid, RPMI 1640 medium, and Dulbecco's PBS were obtained from Sigma. The JNK phosphorylation inhibitor SP600125, the p38 MAPK phosphorylation inhibitor SB203580, the ERK1/2 inhibitor U0126, and the IB phosphorylation inhibitor BAY11-7082 were purchased from Calbiochem. Recombinant human TNF-␣ was obtained from R&D Systems (Minneapolis, MN). A monoclonal antibody against E-selectin (clone 7A9) was obtained from American Type Culture Collection (Manassas, VA) (13). Antibodies against ICAM-1, VCAM-1, the NF-B p65 subunit, and the phospho-NF-B p65 subunit and a monoclonal blocking antibody against mouse E-selectin (clone UZ4) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-ERK, anti-phospho-ERK, anti-p38 MAPK, anti-phospho-p38 MAPK, anti-JNK, and anti-phos-* This work was supported in part by Ministry of Education, Science, and Technology Grant-in-aid for Scientific Research 10178102 and special coordination funds, a grant-in-aid from the Ministry of Culture of Japan, a grant from the Ministry of Health, Labor, and We...

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“…47 Gene silencing by RNAi-based technology overcomes some of the previous difficulties at achieving gene knockout in primary human endothelial cells, which can only undergo a limited number of cell divisions in culture without phenotypic change, loss of telomerase activity, or evidence of senescence. 37,[72][73][74] Unlike other gene-silencing approaches that require clonal selection and multiple passaging, RNAi is shown here to represent a powerful tool for angiogenesis research. Application of this method has uncovered a previously unrecognized function of NRP-1 as a critical regulator of endothelial cell attachment to extracellular matrix.…”

Section: Discussionmentioning

confidence: 99%

Neuropilin-1 regulates attachment in human endothelial cells independently of vascular endothelial growth factor receptor-2

Murga

Fernández-Capetillo

Tosato

2005

Blood

112

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IntroductionVascular endothelial growth factor (VEGF, also named vascular permeability factor, VPF) is a key regulator of physiologic and pathologic angiogenesis, and a molecular target for effective therapeutic intervention. [1][2][3][4][5][6] The biologic activities of VEGF are mediated, for the most part, by 2 receptor tyrosine kinases, Flt-1 (VEGFR-1) and KDR (VEGFR-2). 7,8 VEGFR-1 and VEGFR-2 are essential for fetal angiogenesis, and mouse embryos null for either receptor die in utero between days 8.5 and 9.5. 9,10 Compelling evidence indicates that VEGFR-2, and not VEGFR-1, is the major mediator of VEGF-induced proliferation and migration of endothelial cells, despite both receptors having high affinity binding to VEGF. [11][12][13][14] Chimeric fusion receptors containing the intracellular domain of VEGFR-1 or VEGFR-2 and the extracellular domain of other receptors provided evidence that VEGFR-1 can serve as a competitive inhibitor of VEGFR-2 mitogenic signals. 13,14 Gene targeting studies have shown that whereas hematopoietic and vascular development are severely impaired in VEGFR-2 null mice, 10 overgrowth of endothelial cells and disorganized blood vessels are the cause of death in VEGFR-1 null embryos, 9 providing additional evidence for VEGFR-1's role as a negative regulator of VEGF function. Besides these canonical VEGF receptors, the angiogenic roles of VEGF are modulated by the co-receptor molecules neuropilin NRP-1 and NRP-2. 15,16 NRP-1 and NRP-2, originally identified as receptors for class-3 semaphorins mediating neuronal guidance, 17,18 were subsequently found to bind several isoforms of VEGF and to form complexes with VEGFR-1 and VEGFR-2. [19][20][21][22] When coexpressed with VEGFR-2, NRP-1 enhanced the binding of VEGF to VEGFR-2 and enhanced VEGF-mediated chemotaxis. 16 When complexed with VEGFR-1, NRP-1 prevents the binding of VEGF to NRP-1, providing evidence for a common surface for interaction of NRP-1 with either VEGFR-1 or VEGF. 23 Consistent with NRP-1 playing an important role in regulating VEGF availability and function, NRP-1-deficient mice die at the embryo stage with defects in the heart, vasculature, and nervous system. 24,25 Transgenic mice overexpressing NRP-1 also exhibited abnormalities in the cardiovascular and nervous systems, contributing to embryonic lethality. 26,27 NRP-1 knockin mice expressing NRP-1 mutants that bind either VEGF or class 3 semaphorins demonstrated that VEGF/NRP-1 signaling is required for vascular development, whereas semaphorin-3A/NRP-1 signaling is required for neuronal, but not vascular, development. 28 In addition, endothelial cell-specific NRP-1 null mice displayed marked vascular abnormalities, confirming that NRP-1 expression in endothelial cells is essential for normal vascular development. 28 NRP-1 is a single spanning transmembrane protein with only a short intracytoplasmic tail of 40 amino acids. Deletion studies indicated that semaphorin function in neuronal cells is not dependent upon the intracellular domain of NRP-1 but requires plexi...

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Introduction of short interfering RNA to silence endogenous E-selectin in vascular endothelium leads to successful inhibition of leukocyte adhesion

Cited by 16 publications

References 11 publications

HOXA9 Participates in the Transcriptional Activation of E-Selectin in Endothelial Cells

HOXA9 Participates in the Transcriptional Activation of E-Selectin in Endothelial Cells

Indoxyl Sulfate Induces Leukocyte-Endothelial Interactions through Up-regulation of E-selectin

Neuropilin-1 regulates attachment in human endothelial cells independently of vascular endothelial growth factor receptor-2

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