Citations

ABSTRACT Inflammatory response of blood–brain barrier (BBB) endothelial cells plays an important role in pathogenesis of many central nervous system inflammatory diseases, including multiple sclerosis; however, the molecular mechanism mediating BBB endothelial cell inflammatory response remains unclear. In this study, we first observed that knockdown of neuropilin-1 (NRP1), a co-receptor of several structurally diverse ligands, suppressed interferon-γ (IFNγ)-induced C-X-C motif chemokine 10 expression and activation of STAT1 in brain microvascular endothelial cells in a Rac1-dependent manner. Moreover, endothelial-specific NRP1-knockout mice, VECadherin-Cre-ERT2/NRP1flox/flox mice, showed attenuated disease progression during experimental autoimmune encephalomyelitis, a mouse neuroinflammatory disease model. Detailed analysis utilizing histological staining, quantitative PCR, flow cytometry and magnetic resonance imaging demonstrated that deletion of endothelial NRP1 suppressed neuron demyelination, altered lymphocyte infiltration, preserved BBB function and decreased activation of the STAT1–CXCL10 pathway. Furthermore, increased expression of NRP1 was observed in endothelial cells of acute multiple sclerosis lesions. Our data identify a new molecular mechanism of brain microvascular endothelial inflammatory response through NRP1–IFNγ crosstalk that could be a potential target for intervention of endothelial cell dysfunction in neuroinflammatory diseases.

AbstractA lipid‐conjugated, estrogenic derivative molecule, ESC8, compared with other estrogenic molecules, encourages cell death in both ER‐positive and ER‐negative breast cancer cells. A rapid and highly sensitive assay method has been developed and validated for the estimation of a ESC8 in rat plasma using liquid chromatography coupled with mass spectrometry under positive‐ion mode with electrospray ionization. The sample process includes using methanol for precipitation of ESC8 and dextromethorphan (internal standard, IS) from plasma. Chromatographic separation was achieved with methanol–water–formic acid (70:30:0.1% v/v/v) pumped at a flow rate of 0.3mL/min and a C18 column (50 × 2.1 mm i.d., 1.7 μm particle size) with a total run time of 5 min. The m/z ions monitored were 568.5 and 272.1 for ESC8 and IS, respectively. The lower limit of quantitation achieved was 1.08 ng/mL and linearity was observed from 5 to 500 ng/mL. The intra‐ and inter‐day precisions were <4%. The proposed method was successfully applied to a preliminary pharmacokinetic study of ESC8 liposomal formulation following an intraperitoneal administration of 3.67 mg/kg in rats. The concentrations of ESC8 in plasma were quantifiable up to 36 h. The peak concentration of ESC8 was found to be 110.72 ng/mL, the area under the concentration–time curve was 1625.23ng/mL h and the half‐life was 11.72 h.

Our recent study defined the chemokine-induced human monocyte signaling under normoglycemic condition. To explore the hyperglycemia-induced monocyte signaling, we performed adhesion, migration, and transmigration assays on human monocytes obtained from THP-1 cell line in the presence of normal (5 mM) and high (10 and 20 mM) glucose concentrations without chemokines. We observed augmented ( P < 0.01) monocyte adhesion to human umbilical vein endothelial cell monolayer at 10 than 5 mM glucose with no further increase at 20-mM glucose concentration ( P < 0.07 vs 10 mM; P < 0.01 vs 5 mM). But incremental increases in monocyte migration ( P < 0.01), transmigration ( P < 0.01), and stress fiber response ( P < 0.01) were observed at 10- and 20-mM glucose concentrations in comparison to 5-mM glucose concentrations. We found gradational increase ( P < 0.01) in phosphorylation of Akt S473 and glycogen synthase kinase (GSK3β S9 ) in hyperglycemia (10 and 20 mM) when compared with 5 mM glucose. Furthermore, hyperglycemia (both 10 and 20 mM)-treated monocyte showed up-regulated phosphorylation of p101 and p110γ subunits of PI-3 kinase in comparison to 5 mM glucose. Hyperglycemia-induced monocyte migration was restored to basal levels in the presence of PI-3 kinase inhibitor, LY. These observations imply that modest hyperglycemia per se, as is commonly observed in diabetic individuals, is a potent stimulator of monocyte activity even without chemokines.

Objective Inadequate vascular remodeling is contributory to increased cardiovascular events in people with type 2 diabetes mellitus (DM) and impaired fasting glucose (IFG). Vascular endothelial growth factor (VEGF) and its regulatory molecule soluble Flt-1(sFlt-1) play important roles in atherogenesis. Research Design We measured fasting plasma concentrations of VEGF and sFlt-1 in 11 nondiabetic (ND) (aged 46.1 ± 2.1 years; body mass index [BMI], 26.1 ± 0.9 kg/m 2 ; glucose, 5.0 ± 0.1 mM), 15 IFG (aged 52.9 ± 1.8 years; BMI, 32.7 ± 1.3 kg/m 2 ; glucose, 6.4 ± 0.1 mM), and 8 DM (aged 55.8 ± 3.2 years; BMI, 30.0 ± 1.0 kg/m 2 ; glucose, 9.3 ± 0.5 mM) subjects. Results Plasma VEGF (42.1 ± 4.0 vs 24.2 ± 0.9 vs 29.4 ± 3.8 pg/mL, respectively) and sFlt-1 (119.4 ± 4.9 vs 58.9 ± 3.2 vs 56.7 ± 1.2 pg/mL, respectively) concentrations were higher ( P < 0.04) in DM than IFG and ND subjects. Whereas VEGF concentrations were significantly lower ( P < 0.05) in IFG than in ND subjects, sFlt-1 concentrations did not differ between the IFG and ND subjects. Conclusions Although plasma VEGF concentrations were higher (35%) in DM than in ND subjects, VEGF action on vascular remodeling was likely attenuated by higher sFlt-1 concentrations in DM. In contrast, IFG subjects did not have major perturbations in either VEGF or sFlt-1 levels. Further studies defining the roles of these mediators in DM and IFG are necessary to extend these observations.

Vasculogenesis and angiogenesis are controlled by vascular permeability factor/vascular endothelial growth factor A (VEGF). Dysregulation of these physiological processes contributes to the pathologies of heart disease, cancer and stroke. Rho GTPase proteins play an integral role in VEGF-mediated formation and maintenance of blood vessels. The regulatory functions of RhoA and RhoB in vasculogenesis and angiogenesis are well defined, whereas the purpose of RhoC remains poorly understood. Here, we describe how RhoC promotes vascular homeostasis by modulating endothelial cell migration, proliferation, and permeability. RhoC stimulates proliferation of human umbilical vein endothelial cells (HUVEC) by stabilizing nuclear β-catenin, which promotes transcription of cyclin D1 and subsequently drives cell cycle progression. RhoC negatively regulates endothelial cell migration through MAPK and downstream MLC-2 signaling and decreases vascular permeability through downregulation of the phospholipase Cγ (PLCγ)/Ca2+/eNOS cascade in HUVEC. Using a VEGF-inducible zebrafish (Danio rerio) model, we observed significantly less vascular permeability in RhoC morpholino (MO)-injected zebrafish than control MO-injected zebrafish. Taken together, our findings suggest RhoC is a key regulator of vascular homeostasis in endothelial cells.

Background and objectives New-onset diabetes and concomitant weight loss occurring several months before the clinical presentation of pancreatic cancer (PC) appear to be paraneoplastic phenomena caused by tumour-secreted products. Our recent findings have shown exosomal adrenomedullin (AM) is important in development of diabetes in PC. Adipose tissue lipolysis might explain early onset weight loss in PC. We hypothesise that lipolysis-inducing cargo is carried in exosomes shed by PC and is responsible for the paraneoplastic effects. Therefore, in this study we investigate if exosomes secreted by PC induce lipolysis in adipocytes and explore the role of AM in PC-exosomes as the mediator of this lipolysis. Design Exosomes from patient-derived cell lines and from plasma of patients with PC and non-PC controls were isolated and characterised. Differentiated murine (3T3-L1) and human adipocytes were exposed to these exosomes to study lipolysis. Glycerol assay and western blotting were used to study lipolysis. Duolink Assay was used to study AM and adrenomedullin receptor (ADMR) interaction in adipocytes treated with exosomes. Results In murine and human adipocytes, we found that both AM and PC-exosomes promoted lipolysis, which was abrogated by ADMR blockade. AM interacted with its receptor on the adipocytes, activated p38 and extracellular signal-regulated (ERK1/2) mitogen-activated protein kinases and promoted lipolysis by phosphorylating hormone-sensitive lipase. PKH67-labelled PC-exosomes were readily internalised into adipocytes and involved both caveolin and macropinocytosis as possible mechanisms for endocytosis. Conclusions PC-secreted exosomes induce lipolysis in subcutaneous adipose tissue; exosomal AM is a candidate mediator of this effect.

Abstract Purpose: Pancreatic cancer frequently causes diabetes. We recently proposed adrenomedullin as a candidate mediator of pancreatic β-cell dysfunction in pancreatic cancer. How pancreatic cancer–derived adrenomedullin reaches β cells remote from the cancer to induce β-cell dysfunction is unknown. We tested a novel hypothesis that pancreatic cancer sheds adrenomedullin-containing exosomes into circulation, which are transported to β cells and impair insulin secretion. Experimental Methods: We characterized exosomes from conditioned media of pancreatic cancer cell lines (n = 5) and portal/peripheral venous blood of patients with pancreatic cancer (n = 20). Western blot analysis showed the presence of adrenomedullin in pancreatic cancer-exosomes. We determined the effect of adrenomedullin-containing pancreatic cancer exosomes on insulin secretion from INS-1 β cells and human islets, and demonstrated the mechanism of exosome internalization into β cells. We studied the interaction between β-cell adrenomedullin receptors and adrenomedullin present in pancreatic cancer-exosomes. In addition, the effect of adrenomedullin on endoplasmic reticulum (ER) stress response genes and reactive oxygen/nitrogen species generation in β cells was shown. Results: Exosomes were found to be the predominant extracellular vesicles secreted by pancreatic cancer into culture media and patient plasma. Pancreatic cancer-exosomes contained adrenomedullin and CA19-9, readily entered β cells through caveolin-mediated endocytosis or macropinocytosis, and inhibited insulin secretion. Adrenomedullin in pancreatic cancer exosomes interacted with its receptor on β cells. Adrenomedullin receptor blockade abrogated the inhibitory effect of exosomes on insulin secretion. β cells exposed to adrenomedullin or pancreatic cancer exosomes showed upregulation of ER stress genes and increased reactive oxygen/nitrogen species. Conclusions: Pancreatic cancer causes paraneoplastic β-cell dysfunction by shedding adrenomedullin+/CA19-9+ exosomes into circulation that inhibit insulin secretion, likely through adrenomedullin-induced ER stress and failure of the unfolded protein response. Clin Cancer Res; 21(7); 1722–33. ©2014 AACR. See related commentary by Korc, p. 1508