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Mouse laser-induced choroidal neovascularization (mouse LCNV) recapitulates the "wet" form of human age-related macular degeneration (AMD). Vascular cell adhesion molecule-1 (VCAM-1) is a known inflammatory biomarker, and it increases in the choroidal neovascular tissues characteristic of this experimental model. We have designed and constructed gold nanoparticles (AuNPs) functionalized with hairpin-DNA that incorporates an antisense sequence complementary to VCAM-1 mRNA (AS-VCAM-1 hAuNPs) and tested them as optical imaging probes. The 3' end of the hairpin is coupled to a near-infrared fluorophore that is quenched by the AuNP surface via Förster resonance energy transfer (FRET). Hybridization of the antisense sequence to VCAM-1 mRNA displaces the fluorophore away from the AuNP surface, inducing fluorescent activity. In vitro testing showed that hAuNPs hybridize to an exogenous complementary oligonucleotide within a pH range of 4.5-7.4, and that they are stable at reduced pH. LCNV mice received tail-vein injections of AS-VCAM-1 hAuNPs. Hyperspectral imaging revealed the delivery of AS-VCAM-1 hAuNPs to excised choroidal tissues. Fluorescent images of CNV lesions were obtained, presumably in response to the hybridization of AS-hAuNPs to LCNV-induced VCAM-1 mRNA. This is the first demonstration of systemic delivery of hAuNPs to ocular tissues to facilitate mRNA imaging of any target.
Vascular cell adhesion molecule 1 (VCAM-1) is an important inflammatory biomarker correlating with retinal disease progression. Thus, detection of VCAM-1 mRNA expression levels at an early disease stage could be an important predictive biomarker to assess the risk of disease progression and monitoring treatment response. We have developed VCAM-1 targeted antisense hairpin DNA-functionalized gold nanoparticles (AS-VCAM-1 hAuNP) for the real time detection of VCAM-1 mRNA expression levels in retinal endothelial cells. The AS-VCAM-1 hAuNP fluorescence enhancement clearly visualized the TNF-α induced cellular VCAM-1 mRNA levels with high signal to noise ratios compared to normal serum treated cells. The scrambled hAuNP probes were minimally detectable under same image acquisition conditions. Intracellular hAuNPs were detected using transmission electron microscopy (TEM) analysis of the intact cells. In addition, the AS-VCAM-1 hAuNP probes exhibited no acute toxicity to the retinal microvascular endothelial cells as measured by live-dead assay.
Copyright © 2017 Elsevier Inc. All rights reserved.
RATIONALE - Improving the prospective identification of patients with systemic inflammatory response syndrome (SIRS) and sepsis at low risk for organ dysfunction and death is a major clinical challenge.
OBJECTIVES - To develop and validate a multibiomarker-based prediction model for 28-day mortality in critically ill patients with SIRS and sepsis.
METHODS - A derivation cohort (n = 888) and internal test cohort (n = 278) were taken from a prospective study of critically ill intensive care unit (ICU) patients meeting two of four SIRS criteria at an academic medical center for whom plasma was obtained within 24 hours. The validation cohort (n = 759) was taken from a prospective cohort enrolled at another academic medical center ICU for whom plasma was obtained within 48 hours. We measured concentrations of angiopoietin-1, angiopoietin-2, IL-6, IL-8, soluble tumor necrosis factor receptor-1, soluble vascular cell adhesion molecule-1, granulocyte colony-stimulating factor, and soluble Fas.
MEASUREMENTS AND MAIN RESULTS - We identified a two-biomarker model in the derivation cohort that predicted mortality (area under the receiver operator characteristic curve [AUC], 0.79; 95% confidence interval [CI], 0.74-0.83). It performed well in the internal test cohort (AUC, 0.75; 95% CI, 0.65-0.85) and the external validation cohort (AUC, 0.77; 95% CI, 0.72-0.83). We determined a model score threshold demonstrating high negative predictive value (0.95) for death. In addition to a low risk of death, patients below this threshold had shorter ICU length of stay, lower incidence of acute kidney injury, acute respiratory distress syndrome, and need for vasopressors.
CONCLUSIONS - We have developed a simple, robust biomarker-based model that identifies patients with SIRS/sepsis at low risk for death and organ dysfunction.
BACKGROUND - Numerous epidemiological studies support an inverse association between serum bilirubin levels and the incidence of cardiovascular disease; however, the mechanism(s) by which bilirubin may protect against atherosclerosis is undefined. The goals of the present investigations were to assess the ability of bilirubin to prevent atherosclerotic plaque formation in low-density lipoprotein receptor-deficient ( ) mice and elucidate the molecular processes underlying this effect.
METHODS AND RESULTS - Bilirubin, at physiological concentrations (≤20 μmol/L), dose-dependently inhibits THP-1 monocyte migration across tumor necrosis factor α-activated human umbilical vein endothelial cell monolayers without altering leukocyte binding or cytokine production. A potent antioxidant, bilirubin effectively blocks the generation of cellular reactive oxygen species induced by the cross-linking of endothelial vascular cell adhesion molecule 1 (VCAM-1) or intercellular adhesion molecule 1 (ICAM-1). These findings were validated by treating cells with blocking antibodies or with specific inhibitors of VCAM-1 and ICAM-1 signaling. When administered to mice on a Western diet, bilirubin (30 mg/kg intraperitoneally) prevents atherosclerotic plaque formation, but does not alter circulating cholesterol or chemokine levels. Aortic roots from bilirubin-treated animals exhibit reduced lipid and collagen deposition, decreased infiltration of monocytes and lymphocytes, fewer smooth muscle cells, and diminished levels of chlorotyrosine and nitrotyrosine, without changes in VCAM-1 or ICAM-1 expression.
CONCLUSIONS - Bilirubin suppresses atherosclerotic plaque formation in mice by disrupting endothelial VCAM-1- and ICAM-1-mediated leukocyte migration through the scavenging of reactive oxygen species signaling intermediaries. These findings suggest a potential mechanism for the apparent cardioprotective effects of bilirubin.
© 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.
The objective of the present study was to assess the effect of elevating epoxygenated fatty acids on retinal vascular inflammation. To stimulate inflammation we utilized TNFα, a potent pro-inflammatory mediator that is elevated in the serum and vitreous of diabetic patients. In TNFα-stimulated primary human retinal microvascular endothelial cells, total levels of epoxyeicosatrienoic acids (EETs), but not epoxydocosapentaenoic acids (EDPs), were significantly decreased. Exogenous addition of 11,12-EET or 19,20-EDP when combined with 12-(3-adamantane-1-yl-ureido)-dodecanoic acid (AUDA), an inhibitor of epoxide hydrolysis, inhibited VCAM-1 and ICAM-1 expression and protein levels; conversely the diol product of 19,20-EDP hydrolysis, 19,20-DHDP, induced VCAM1 and ICAM1 expression. 11,12-EET and 19,20-EDP also inhibited leukocyte adherence to human retinal microvascular endothelial cell monolayers and leukostasis in an acute mouse model of retinal inflammation. Our results indicate that this inhibition may be mediated through an indirect effect on NFκB activation. This is the first study demonstrating a direct comparison of EET and EDP on vascular inflammatory endpoints, and we have confirmed a comparable efficacy from each isomer, suggesting a similar mechanism of action. Taken together, these data establish that epoxygenated fatty acid elevation will inhibit early pathology related to TNFα-induced inflammation in retinal vascular diseases.
Leucine Rich Repeat Containing 8A (LRRC8A) is a required component of volume-regulated anion channels (VRACs). In vascular smooth muscle cells, tumor necrosis factor-α (TNFα) activates VRAC via type 1 TNFα receptors (TNFR1), and this requires superoxide (O) production by NADPH oxidase 1 (Nox1). VRAC inhibitors suppress the inflammatory response to TNFα by an unknown mechanism. We hypothesized that LRRC8A directly supports Nox1 activity, providing a link between VRAC current and inflammatory signaling. VRAC inhibition by 4-(2-butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl) oxobutyric acid (DCPIB) impaired NF-κB activation by TNFα. LRRC8A siRNA reduced the magnitude of VRAC and inhibited TNFα-induced NF-κB activation, iNOS and VCAM expression, and proliferation of VSMCs. Signaling steps disrupted by both siLRRC8A and DCPIB included; extracellular O production by Nox1, c-Jun N-terminal kinase (JNK) phosphorylation and endocytosis of TNFR1. Extracellular superoxide dismutase, but not catalase, selectively inhibited TNFR1 endocytosis and JNK phosphorylation. Thus, O is the critical extracellular oxidant for TNFR signal transduction. Reducing JNK expression (siJNK) increased extracellular O suggesting that JNK provides important negative feedback regulation to Nox1 at the plasma membrane. LRRC8A co-localized by immunostaining, and co-immunoprecipitated with, both Nox1 and its p22phox subunit. LRRC8A is a component of the Nox1 signaling complex. It is required for extracellular O production, which is in turn essential for TNFR1 endocytosis. These data are the first to provide a molecular mechanism for the potent anti-proliferative and anti-inflammatory effects of VRAC inhibition.
Copyright © 2016 Elsevier Inc. All rights reserved.
The objective of this study was to determine the role of individual NFAT isoforms in TNFα-induced retinal leukostasis. To this end, human retinal microvascular endothelial cells (HRMEC) transfected with siRNA targeting individual NFAT isoforms were treated with TNFα, and qRT-PCR was used to examine the contribution of each isoform to the TNFα-induced upregulation of leukocyte adhesion proteins. This showed that NFATc1 siRNA increased ICAM1 expression, NFATc2 siRNA reduced CX3CL1, VCAM1, SELE, and ICAM1 expression, NFATc3 siRNA increased CX3CL1 and SELE expression, and NFATc4 siRNA reduced SELE expression. Transfected HRMEC monolayers were also treated with TNFα and assayed using a parallel plate flow chamber, and both NFATc2 and NFATc4 knockdown reduced TNFα-induced cell adhesion. The effect of isoform-specific knockdown on TNFα-induced cytokine production was also measured using protein ELISAs and conditioned cell culture medium, and showed that NFATc4 siRNA reduced CXCL10, CXCL11, and MCP-1 protein levels. Lastly, the CN/NFAT-signaling inhibitor INCA-6 was shown to reduce TNFα-induced retinal leukostasis in vivo. Together, these studies show a clear role for NFAT-signaling in TNFα-induced retinal leukostasis, and identify NFATc2 and NFATc4 as potentially valuable therapeutic targets for treating retinopathies in which TNFα plays a pathogenic role.
Bilirubin is thought to exert anti-inflammatory effects by inhibiting vascular cell adhesion molecule-1 (VCAM-1)-dependent leukocyte migration and by suppressing the expression of inducible nitric oxide synthase (iNOS). As VCAM-1 and iNOS are important mediators of tissue injury in the dextran sodium sulfate (DSS) murine model of inflammatory colitis, we examined whether bilirubin prevents colonic injury in DSS-treated mice. Male C57BL/6 mice were administered 2.5% DSS in the drinking water for 7 days, while simultaneously receiving intraperitoneal injections of bilirubin (30 mg/kg) or potassium phosphate vehicle. Disease activity was monitored, peripheral blood counts and serum nitrate levels were determined, and intestinal specimens were analyzed for histological injury, leukocyte infiltration, and iNOS expression. The effect of bilirubin on IL-5 production by HSB-2 cells and on Jurkat cell transendothelial migration also was determined. DSS-treated mice that simultaneously received bilirubin lost less body weight, had lower serum nitrate levels, and exhibited reduced disease severity than vehicle-treated animals. Concordantly, histopathological analyses revealed that bilirubin-treated mice manifested significantly less colonic injury, including reduced infiltration of eosinophils, lymphocytes, and monocytes, and diminished iNOS expression. Bilirubin administration also was associated with decreased eosinophil and monocyte infiltration into the small intestine, with a corresponding increase in peripheral blood eosinophilia. Bilirubin prevented Jurkat migration but did not alter IL-5 production. In conclusion, bilirubin prevents DSS-induced colitis by inhibiting the migration of leukocytes across the vascular endothelium and by suppressing iNOS expression.
Copyright © 2015 the American Physiological Society.
RATIONALE - Human and murine Vcam1 promoters contain 2 adjacent nuclear factor-κB (NF-κB)-binding elements. Both are essential for cytokine-induced transcription of transiently transfected promoter-reporter constructs. However, the relevance of these insights to regulation of the endogenous Vcam1 gene and to pathophysiological processes in vivo remained unknown.
OBJECTIVE - Determine the role of the 5' NF-κB-binding element in expression of the endogenous Vcam1 gene.
METHODS AND RESULTS - Homologous recombination in embryonic stem cells was used to inactivate the 5' NF-κB element in the Vcam1 promoter and alter 3 nucleotides in the 5' untranslated region to allow direct comparison of wild-type versus mutant allele RNA expression and chromatin configuration in heterozygous mice. Systemic treatment with inflammatory cytokines or endotoxin (lipopolysaccharide) induced lower expression of the mutant allele relative to wild-type by endothelial cells in the aorta, heart, and lungs. The mutant allele also showed lower endothelial expression in 2-week atherosclerotic lesions in Vcam1 heterozygous/low-density lipoprotein receptor-deficient mice fed a cholesterol-rich diet. In vivo chromatin immunoprecipitation assays of heart showed diminished lipopolysaccharide-induced association of RNA polymerase 2 and NF-κB p65 with the mutant promoter. In contrast, expression of mutant and wild-type alleles was comparable in intimal cells of wire-injured carotid artery and 4- to 12-week atherosclerotic lesions.
CONCLUSIONS - This study highlights differences between in vivo and in vitro promoter analyses, and reveals a differential role for a NF-κB transcriptional response element in endothelial vascular cell adhesion molecule-1 expression induced by inflammatory cytokines or a cholesterol-rich diet versus intimal cell expression in atherosclerotic lesions and injured arteries.
© 2015 American Heart Association, Inc.
Leukocyte residence in lymphoid organs is controlled by a balance between retention and egress-promoting chemoattractants sensed by pertussis toxin (PTX)-sensitive Gαi protein-coupled receptors (GPCRs). Here, we use two-photon intravital microscopy to show that immature B cell retention within bone marrow (BM) was strictly dependent on amoeboid motility mediated by CXCR4 and CXCL12 and by α4β1 integrin-mediated adhesion to VCAM-1. However, B lineage cell egress from BM is independent of PTX-sensitive GPCR signaling. B lineage cells expressing PTX rapidly exited BM even though their motility within BM parenchyma was significantly reduced. Our experiments reveal that when immature B cells are near BM sinusoids their motility is reduced, their morphology is predominantly rounded, and cells reverse transmigrate across sinusoidal endothelium in a largely nonamoeboid manner. Immature B cell egress from BM was dependent on a twofold CXCR4 down-regulation that was antagonized by antigen-induced BCR signaling. This passive mode of cell egress from BM also contributes significantly to the export of other hematopoietic cells, including granulocytes, monocytes, and NK cells, and is reminiscent of erythrocyte egress.
© 2014 Beck et al.