Endothelial Adhesion Molecule Expression Research Articles

Alpha-melanocyte stimulating hormone inhibits monocytes adhesion to vascular endothelium.

Inflammation and its subsequent endothelial dysfunction have been reported to play a pivotal role in the initiation and progression of chronic vascular diseases. Inhibiting the attachment of monocytes to endothelium is a potential therapeutic strategy for vascular diseases treatment. α-Melanocyte stimulating hormone is generated from a precursor hormone called proopiomelanocortin by post-translational processing. However, whether α-melanocyte stimulating hormone plays a role in regulating endothelial inflammation is still unknown. In this study, the effects of α-melanocyte stimulating hormone on endothelial inflammation in human umbilical vein endothelial cell lines were investigated. And the result indicated that α-melanocyte stimulating hormone inhibits the expression of endothelial adhesion molecules, including vascular adhesion molecule-1 and E-selectin, thereby attenuating the adhesion of THP-1 cells to the surface of endothelial cells. Mechanistically, α-melanocyte stimulating hormone was found to inhibit NF-κB transcriptional activity. Finally, we found that the effect of α-melanocyte stimulating hormone on endothelial inflammation is dependent on its receptor melanocortin receptor 1.

Cerebrovascular and blood-brain barrier morphology in spontaneously hypertensive rats: effect of treatment with choline alphoscerate.

Cholinergic precursors increasing choline availability and acetylcholine synthesis/release may represent a therapeutic approach for countering cognitive impairment occurring in adult-onset dementia disorders. Choline alphoscerate (alpha-gliceryl-phosphoryl-choline, GPC) is among cholinergic precursors the most effective in enhancing acetylcholine biosynthesis and release in animal models. This study was designed to assess if a long-term treatment with GPC modify cerebrovascular components [perivascular astrocytes, blood-brain barrier (BBB) and microvessels] and endothelial inflammatory markers expression in spontaneously hypertensive rats (SHR) used as a model of brain vascular injury. Male SHR aged 32 weeks and age-matched normotensive Wistar-Kyoto rats were treated for 4 weeks with GPC (150 mg/kg/day) or a vehicle. Intracerebral arteries of different brain areas, perivascular astrocytes, BBB and endothelial inflammatory markers were assessed by quantitative morphological and immunohistochemical techniques. No significant changes in the size of perivascular astrocytes were observed in SHR versus normotensive Wistar-Kyoto rats, whereas the expression of the BBB marker aquaporin-4 increased in SHR. This phenomenon was countered by GPC treatment. On the contrary, GPC has no vasodilator effect on brain micro-vessels. Endothelial markers and vascular adhesion molecules expression were not homogeneously affected by hypertension and GPC treatment in intracerebral vessels. The observation that treatment with GPC reversed BBB changes and countered to some extent micro-vessels changes occurring in SHR could explain data of clinical trials reporting an improvement of cognitive function in subjects suffering from cerebrovascular disorders and treated with GPC. These preclinical data suggest that the compound could have a cerebrovascular protective effect deserving a further characterization.

Mo1689 Nicotine Significantly Affects the Expression of Vascular Endothelial Adhesion Molecules and Ameliorates DSS Induced Colitis

Introduction Anti-adhesion molecule therapy is useful for inflammatory bowel diseases. Smoking has been reported to have a harmful effect on Crohn's disease, but it is still controversial in ulcerative colitis. Although effect of nicotine on adhesion molecules of cardiovascular system has been well recognized, it has not been studied in the intestine. We investigated effect of nicotine on murine colitis induced by dextran sodium sulfate (DSS) and compared expression of adhesion molecules in vitro in several conditions including preconditioning exposure. Involvement of nicotinic acetylcholine receptor (nAChR) was also examined. Method In in vivo study C57BL/6J mice were treated with 3% DSS and 0.1mg/ ml nicotine in drinking water for 7 days. Disease activity of colitis was assessed by DAI score. mRNA expressions of ICAM-1, VCAM-1 and MAdCAM-1 in colonic tissues were measured by qPCR. Microvascular endothelial cell line, bEnd3, was used for in vitro study. The cultured cells (2x105/ml) were treated with 5ng/ml TNF-α and 200μg/ml nicotine for 12 hours. mRNA expressions of ICAM-1, VCAM-1 and MAdCAM-1 in cells were measured by qPCR. Various concentrations of nicotine (0.01-1000μM) were tested. In pre-treatment protocol, cells were treated with nicotine for 3 days, and after 12 hours interval without nicotine, exposed to TNF-α and nicotine for 12 hours as treatment. In some experiments cells were cultured with TNF-α and nicotine under the presence of 0.01μM mecamylamine (Meca, nonselective nAChR antagonist) or 0.01μM methyllycaconitine (MLA, selective α7nAChR antagonist). Result In in vivo study, nicotine treatment decreased DAI score induced by DSS. Although nicotine alone did not affect expression of ICAM-1, VCAM-1 and MAdCAM-1 in control mice, addition of nicotine to DSS mice significantly attenuated the increased expression of VCAM-1 and MAdCAM-1 induced by DSS, and tended to inhibit ICAM-1. In in vitro study, expression of ICAM-1, VCAM-1 and MAdCAM-1 was increased by TNF-α. Increased expression of MAdCAM-1 was significantly attenuated by simultaneous administration of nicotine. In dose-response study, degree of ICAM-1 inhibition by nicotine was only observed at high doses, whereas that of MAdCAM-1 was observed in a low dose already. Interestingly pretreatment with nicotine before TNF-α decreased inhibitory effect of nicotine. These inhibitory effects tended to be attenuated by Meca or MLA, but not

Ethanol extract of Pinus eldarica needle reduces atherosclerosis in ApoE‐deficient mice

Atherosclerosis is a chronic inflammatory disease initiated by vascular inflammation and monocyte recruitment in part. Pinus eldarica needle have been widely used in traditional medicine for the treatment of various diseases. However, the effect of ethanolic extract of Pine eldarica needle on inflammatory diseases like atherosclerosis is not known yet. Herein we evaluated whether ethanolic extract of Pine eldarica needle has a protective effect against atherosclerosis in experimental mice. 6 male ApoE –/– mice and 6 male control (C57BL/6J) mice were fed with 30% high‐cholesterol diet for 21 weeks. 6 of the ApoE –/– mice were orally administered pine needle extract(10 mg/kg and 20 mg/kg body weight, every two days for 21 weeks). Pine needle extractshowed the suppressive effects on the expression of endothelial intercellular adhesion molecule 1 (ICAM‐1) and vascular cell adhesion molecule 1 (VCAM‐1) and the serum levels of CCL2, CCL5, IL‐6 and TNF‐α in ApoE –/– mice. In addition,administration of pine needle extractattenuated the serum levels of oxLDL, HDL, LDH, triglyceride and total cholesterol. Furthermore, the serum levels of SREBP‐2, LOX‐1 and LXRs were inhibited by the pine needle extract. Collectively, these results suggest that of ethanolic extract of Pine eldarica needle might play a protective role against atherosclerosis.

Multiple anti-inflammatory and anti-atherosclerotic properties of red wine polyphenolic extracts: differential role of hydroxycinnamic acids, flavonols and stilbenes on endothelial inflammatory gene expression.

The aim of the study was to evaluate the vascular anti-inflammatory effects of polyphenolic extracts from two typical South Italy red wines, the specific contribution of individual polyphenols and the underlying mechanisms of action. Human endothelial cells were incubated with increasing concentrations (1-50 μg/mL) of Primitivo and Negroamaro polyphenolic extracts (PWPE and NWPE, respectively) or pure polyphenols (1-25 μmol/L), including hydroxycinnamic acids (p-coumaric, caffeic and caftaric acids), flavonols (kaempferol, quercetin, myricetin) or stilbenes (trans-resveratrol, trans-piceid) before stimulation with lipopolysaccharide. Through multiple assays, we analyzed the endothelial-monocyte adhesion, the endothelial expression of adhesion molecules (ICAM-1, VCAM-1 and E-Selectin), monocyte chemoattractant protein-1 (MCP-1) and macrophage colony-stimulating factor (M-CSF), as well as ROS intracellular levels and the activation of NF-κB and AP-1. Both PWPE and NWPE, already at 1 μg/mL, inhibited monocyte adhesion to stimulated endothelial cells, a key event in triggering vascular inflammation. They down-regulated the expression of adhesion molecules, ICAM-1, VCAM-1, E-Selectin, as well as MCP-1 and M-CSF, at mRNA and protein levels. All polyphenols reduced intracellular ROS, and everything, except caftaric acid, inhibited the endothelial expression of adhesion molecules and MCP-1, although with different potency. Flavonols and resveratrol significantly reduced also the endothelial expression and release of M-CSF. The decrease in endothelial inflammatory gene expression was related to the inhibition of NF-κB and AP-1 activation but not to intracellular oxidative stress. This study showed multiple anti-inflammatory and anti-atherosclerotic properties of red wine polyphenolic extracts and indentified specific bioactive polyphenols which could counteract inflammatory diseases including atherosclerosis.

Loss of mouse P2Y4 nucleotide receptor protects against myocardial infarction through endothelin-1 downregulation.

Nucleotides are released in the heart under pathological conditions, but little is known about their contribution to cardiac inflammation. The present study defines the P2Y4 nucleotide receptor, expressed on cardiac microvascular endothelial cells and involved in postnatal heart development, as an important regulator of the inflammatory response to cardiac ischemia. P2Y4-null mice displayed smaller infarcts in the left descending artery ligation model, as well as reduced neutrophil infiltration and fibrosis. Gene profiling identified inter alia endothelin-1 (ET-1) as one of the target genes of P2Y4 in ischemic heart. The reduced level of ET-1 was correlated with reduction of microvascular hyperpermeability, neutrophil infiltration, and endothelial adhesion molecule expression, and it could be explained by the decreased number of endothelial cells in P2Y4-null mice. Expression analysis of metalloproteinases and their tissue inhibitors in ischemic heart revealed reduced expression of matrix metalloproteinase (MMP)-9, reported to be potentially regulated by ET-1, and MMP-8, considered as neutrophil collagenase, as well as reduction of tissue inhibitor of MMP-1 and tissue inhibitor of MMP-4 in P2Y4-null mice. Reduction of cardiac permeability and neutrophil infiltration was also observed in P2Y4-null mice in LPS-induced inflammation model. Protection against infarction resulting from loss of P2Y4 brings new therapeutic perspectives for cardiac ischemia and remodeling.

Carbon monoxide protects against hemorrhagic shock and resuscitation-induced microcirculatory injury and tissue injury.

Traumatic injury is a significant cause of morbidity and mortality worldwide. Microcirculatory activation and injury from hemorrhage contribute to organ injury. Many adaptive responses occur within the microcirculatory beds to limit injury including upregulation of heme oxygenase (HO) enzymes, the rate-limiting enzymes in the breakdown of heme to carbon monoxide (CO), iron, and biliverdin. Here we tested the hypothesis that CO abrogates trauma-induced injury and inflammation protecting the microcirculatory beds. C57Bl/6 mice underwent sham operation or hemorrhagic shock to a mean arterial pressure of 25 mmHg for 120 minutes. Mice were resuscitated with lactated Ringer's at 2× the volume of maximal shed blood. Mice were randomized to receive CO-releasing molecule or inactive CO-releasing molecule at resuscitation. A cohort of mice was pretreated with tin protoporphyrin-IX to inhibit endogenous CO generation by HOs. Primary mouse liver sinusoidal endothelial cells were cultured for in vitro experiments. Carbon monoxide-releasing molecule protected against hemorrhagic shock/resuscitation organ injury and systemic inflammation and reduced hepatic sinusoidal endothelial injury. Inhibition of HO activity with tin protoporphyrin-IX exacerbated liver hepatic sinusoidal injury. Hemorrhagic shock/resuscitation in vivo or cytokine stimulation in vitro resulted in increased endothelial expression of adhesion molecules that was associated with decreased leukocyte adhesion in vivo and in vitro. Hemorrhagic shock/resuscitation is associated with endothelial injury. Heme oxygenase enzymes and CO are involved in part in diminishing this injury and may prove useful as a therapeutic adjunct that can be harnessed to protect against endothelial activation and damage.

Estradiol Inhibits Cytokine-Induced Expression of VCAM-1 and ICAM-1 in Cultured Human Endothelial Cells Via AMPK/PPARα Activation.

Expression of cell adhesion molecules by vascular endothelial cells (ECs) plays an important role in migration and adhesion of leukocytes into the surrounding tissues, which is a vital step in inflammation and atherogenesis. Estradiol (E2), a human sex hormone and steroid, has differential anti-inflammatory properties, increases arterial vasodilation, inhibits the cellular response to vascular injury, and prevents atherosclerosis. However, the related mechanisms are poorly understood. Here, we studied the effect of E2 on expression of endothelial leukocyte adhesion molecules by cytokine-stimulated human aortic endothelial cells (HAECs). Pre-treatment of HAEC with E2 inhibited tumor necrosis factor-α (TNFα)-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) in a time- and dose-dependent manner. E2 at 10nmol/L inhibited interleukin-1 (IL-1)-stimulated expression of VCAM-1 and ICAM-1. The effect of E2 on cytokine-stimulated VCAM-1 expression was more prominent than that of ICAM-1. E2 reduced TNF-induced mRNA expression of VCAM-1 and ICAM-1. Repressed expression of VCAM-1 and ICAM-1 was associated with decreased adherence of lymphocytes and monocytes to cytokine-stimulated HAEC. E2 suppressed TNF-induced activation of nuclear factor-κB (NF-κB) in HAEC. Importantly, E2 improved AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor α (PPARα) expression in HAEC. Pharmaceutical inhibition of AMPK and PPARα blocks the effect of E2 on NF-κB, VCAM-1, and ICAM-1. These results indicate that E2 may have anti-inflammatory properties in ECs. The anti-inflammatory and anti-atherogenic properties of E2 may partly be ascribed to an effect on activation of AMPK and PPARα and subsequent inhibition of TNF-induced NF-κB activation, leading to the decreased expression of VCAM-1 and ICAM-1. Our findings support further investigations on the therapeutic benefits of E2 in several pathological events involving endothelial dysfunction and atherosclerosis.

Nafamostat Mesilate Inhibits TNF-α-Induced Vascular Endothelial Cell Dysfunction by Inhibiting Reactive Oxygen Species Production.

Nafamostat mesilate (NM) is a serine protease inhibitor with anticoagulant and anti-inflammatory effects. NM has been used in Asia for anticoagulation during extracorporeal circulation in patients undergoing continuous renal replacement therapy and extra corporeal membrane oxygenation. Oxidative stress is an independent risk factor for atherosclerotic vascular disease and is associated with vascular endothelial function. We investigated whether NM could inhibit endothelial dysfunction induced by tumor necrosis factor-α (TNF-α). Human umbilical vein endothelial cells (HUVECs) were treated with TNF-α for 24 h. The effects of NM on monocyte adhesion, vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) protein expression, p38 mitogen-activated protein kinase (MAPK) activation, and intracellular superoxide production were then examined. NM (0.01~100 µg/mL) did not affect HUVEC viability; however, it inhibited the increases in reactive oxygen species (ROS) production and p66shc expression elicited by TNF-α (3 ng/mL), and it dose dependently prevented the TNF-α-induced upregulation of endothelial VCAM-1 and ICAM-1. In addition, it mitigated TNF-α-induced p38 MAPK phosphorylation and the adhesion of U937 monocytes. These data suggest that NM mitigates TNF-α-induced monocyte adhesion and the expression of endothelial cell adhesion molecules, and that the anti-adhesive effect of NM is mediated through the inhibition of p66shc, ROS production, and p38 MAPK activation.

The olive oil-based lipid clinoleic blocks leukocyte recruitment and improves survival during systemic inflammation: a comparative in vivo study of different parenteral lipid emulsions.

Although fish oil-based and olive oil-based lipid emulsions have been shown to exert anti-inflammatory functions, the immunomodulating properties of lipids are still controversial. Therefore, we investigated the anti-inflammatory effect of three different parenterally administered lipid emulsions in vivo: olive oil-based Clinoleic, fish oil-based Smoflipid, and soybean oil-based Lipofundin. We observed leukocyte recruitment in inflamed murine cremaster muscle using intravital microscopy and survival in a murine model of LPS-induced systemic inflammation and analyzed expression of leukocyte and endothelial adhesion molecules. Olive oil-based Clinoleic and fish oil-based Smoflipid profoundly inhibited leukocyte adhesion compared to Lipofundin during LPS-induced inflammation of the murine cremaster muscle. In the trauma model of cremaster muscle inflammation, Lipofundin was the only lipid emulsion that even augmented leukocyte adhesion. In contrast to Smoflipid and Lipofundin, Clinoleic effectively blocked leukocyte recruitment and increased survival during lethal endotoxemia. Flow chamber experiments and analysis of adhesion molecule expression suggest that both endothelial and leukocyte driven mechanisms might contribute to anti-inflammatory effects of Clinoleic. We conclude that the anti-inflammatory properties of Clinoleic are superior to those of Smoflipid and Lipofundin even during systemic inflammation. Thus, these results should stimulate further studies investigating parenteral lipids as an anti-inflammatory strategy in critically ill patients.

Milk-derived bioactive peptides inhibit human endothelial-monocyte interactions via PPAR-γ dependent regulation of NF-κB.

BackgroundMilk-derived bioactive peptides retain many biological properties and have therapeutic effects in cardiovascular disorders such as atherosclerosis. Under inflammatory conditions the expression of endothelial cells adhesion molecules is induced, increasing monocyte adhesion to human vessel wall, a critical step in the pathogenesis of atherosclerosis. In the present work we explored the effects of milk-derived bioactive peptides on the expression of the inflammatory phenotype of human endothelial cells and their effects on monocyte adherence to endothelial cells.ResultsTreatment of endothelial cells with milk-derived hydrolysate inhibited their production of inflammatory proteins MCP-1 and IL-8 and expression of VCAM-1, ICAM-1 and E-selectin. Milk derived hydrolysate also attenuated the adhesion of human monocytes to activated endothelial cells. The effect was similar to that obtained in endothelial cells treated with troglitazone, a ligand of peroxisome proliferators-activator receptor-gamma (PPAR-γ). PPAR-γ is a transcription factor which when activated antagonises the pro-inflammatory capability of nuclear factor κB (NF-κB). We further examined whether the effects of milk-derived hydrolysates on endothelial cells may be mediated through NF-κB activation via a PPAR-γ dependent mechanism. The specific PPAR-γ inhibitor, GW9662 blocked the effects of the hydrolysate on the NF-κB-mediated chemokines and adhesion molecules expression in endothelial cells.ConclusionsThese results suggest that milk-derived bioactive peptides work as anti-atherogenic agents through the inhibition of endothelial-dependent adhesive interactions with monocytes by inhibiting the NF-κB pathway through a PPAR-γ dependent mechanism.Electronic supplementary materialThe online version of this article (doi:10.1186/s12950-014-0044-1) contains supplementary material, which is available to authorized users.

Circulating Vascular Endothelial Growth Factor and Vascular Cell Adhesion Molecule in Laryngeal Squamous Cell Carcinomas.

Objective: To assess the relationship between the immunohistochemical expression of vascular endothelial growth factor (VEGF) and vascular cell adhesion molecule (VCAM) with the clinical feature of laryngeal squamous cell carcinoma (LSCC). Study Design and Methods: Twenty-nine patients with LSCC were examined as the LSCC group. Twenty-one healthy people who showed no signs of regional or systemic disorders were used as the control group. The staging system for LSCC was determined using American Joint Committee on Cancer (AJCC) 2010 criteria. A quantitative enzyme-linked immunosorbent assay (ELISA) was performed to measure the concentration of soluble serum VEGF and VCAM-1. Results: VEGF levels were higher in the LSCC group compared to the control group (p = 0.001). VCAM levels were not significantly different (p = 0.617). VEGF levels were not correlated to the T and N stage in the LSCC group (p = 0.402). VCAM levels were significantly correlated with the T and N stage (p < 0.0001). Conclusions: VCAM levels could not be used as a tumor marker for diagnosis but were correlated with the stage and, therefore, the aggressivity of the cancer disease. Keywords: Larynx, squamous cell carcinomas, vascular endothelial growth factor, vascular cell adhesion molecule, tumor marker, stages, lymph node metastasis, aggressivity. Read more →

Vascular Geometry and Flow Profile Mediate Pathological Cell-Cell Interactions in Sickle Cell Disease As Measured with “Do-It-Yourself” “Endothelial-Ized” Microfluidics

Background and Significance: Cell-cell interactions between blood cells and endothelial cells play an important role in sickle cell disease (SCD) pathophysiology. While in vivo transgenic animal models and in vitro systems have both contributed to our understanding of these pathologic cell-cell interactions in SCD, isolating the causes and effects of cellular interactions is exceedingly difficult in the former and recapitulating the complex vascular geometries found in vivo is not readily available with current systems in the latter. The vascular system comprises diverse geometries that range from normal (e.g. curves and bifurcations) to pathologic (e.g. aneurysms and stenoses) and as blood flows from one vascular geometry to another, the local shear stress profile acutely changes. Interestingly, changes in shear stress are known to alter endothelial pro-inflammatory signaling pathways and expression of cell adhesion molecules, especially vascular cell adhesion molecule-1(VCAM-1) (Tzima, Nature, 2005), which is implicated in SCD vasculopathy. Here we present a rapid and inexpensive method using only off-the-shelf materials to create “do-it-yourself” (DIY) microfluidic devices that incorporate endothelial cells and clinically relevant vascular geometries; this system effectively and bridges current in vitro and in vivo models to study SCD. Using this technique, we developed a vascularized bifurcation, and observed that shear stress changes can be extremely localized, affecting only several 10s of cells, and are associated with changes in VCAM1 expression. We used this in vitro vascularized bifurcation to test the hypothesis that SS RBC-endothelial cell adhesion occurs primarily at bifurcations, which are difficult to visualize in vivo (Nagel,Arterioscler Thromb Vasc Biol, 1999). We demonstrate that SCD RBCs do primarily aggregate at bifurcations, specifically in locations where the shear stress has decreased and VCAM-1 is upregulated.Methods: In order to bridge in vitro data with the complex vascular geometric environments found in vivo, we developed a “DIY” endothelialized microfluidic model (Figure 1A). A strand of 500um diameter polymethylmethacrylate (PMMA) optical fiber is laid flat on top of a layer of polydimethylsiloxane (PDMS) and covered with a second, thin layer of PDMS. After curing, the optical fiber is pulled out, exposing a hollow, circular, channel that can be used as a microchannel to seed endothelial cells. A wide variety of endothelial cells can be successfully seeded in these devices, such as human umbilical vein endothelial cells, human aortic endothelial cells, and human microvascular endothelial cells. Slight alterations to this fabrication method result in the creation of multiple vascular geometries, such as curved or bifurcated channels with or without aneurysms or stenoses.Results: Curved channels & bifurcations (Figure 1B-C) are seeded with endothelial cells (Figure 1E-F). Computational fluidic dynamics calculations show that the shear varies by 2.5 fold within the bifurcation. As shear affects endothelial expression, we tested if the extremely localized shear changes created in this system were sufficient to alter local endothelial expression of VCAM-1 Indeed, in our system, VCAM1 expression significantly correlated with shear variation (Figure1G), and was highest near the bifurcation point. Noting this localized variation in adhesion molecule expression, we tested whether the bifurcations are implicated in SCD RBC adhesion to the endothelium. With our vascularized bifurcation model and custom image analysis software that quantifies RBC aggregation, we observed that SCD RBC adhesion predominantly occurred at the point of bifurcation where the shear is lowest and VCAM1 expression is greatest, and minimal endothelial adhesion occurred with healthy control RBCs (Figure 2). This phenomenon persisted with tumor necrosis factor-stimulation of the endothelium.Conclusion: This DIY system represents an easily accessible technique that allows any researcher to bridge the gap between in vitro and in vivo models of pathological cell-cell interactions in SCD. We demonstrate that recapitulating the complex vascular geometries in vivo is vital to understanding blood cell-endothelial interactions and this system will not only be useful for studying SCD, but a myriad of hematologic and vascular diseases as well. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Focal disturbances in the blood–brain barrier are associated with formation of neuroinflammatory lesions

Early changes in the normal appearing white matter of multiple sclerosis (MS) patients precede the appearance of gadolinium-enhancing lesions. Although these findings suggest blood–brain barrier (BBB) breakdown as an important feature in MS pathogenesis, limited information is available on the BBB alterations during lesion genesis. Here, we perform a longitudinal characterization of the vascular, neuropathological and immunological changes before lesion formation in mice developing spontaneous relapsing–remitting experimental autoimmune encephalomyelitis (sRR-EAE). We found a significant upregulation of Th1 and Th17 cytokines in the periphery of sRR-EAE mice before any evident neuropathology. In the CNS, BBB and astroglial activations were the first pathological changes occurring after 45days of age and were followed by immune cell infiltration by day 50. These pathological alterations subsequently led to perivascular demyelination and disease onset. In MS, (p)reactive lesions mirrored the changes seen in early sRR-EAE by displaying considerable BBB disruption, perivascular astrogliosis, redistribution of junctional proteins and increased expression of endothelial cell adhesion molecules. Our findings suggest that BBB breach occurs before significant immune cell infiltration and demyelination. In addition, peripheral immune activation during sRR-EAE precedes CNS pathology, suggesting that outside in signaling mechanisms play a role in the development of neuroinflammatory lesions.

Endothelial Na+/H+ exchanger NHE1 participates in redox-sensitive leukocyte recruitment triggered by methylglyoxal.

BackgroundExcessive levels of methylglyoxal (MG) encountered in diabetes foster enhanced leukocyte-endothelial cell interactions, mechanisms of which are incompletely understood. MG genomically upregulates endothelial serum- and glucocorticoid-inducible kinase 1 (SGK1) which orchestrates leukocyte recruitment by regulating the activation and expression of transcription factors and adhesion molecules. SGK1 regulates a myriad of ion channels and carriers including the Na+/H+ exchanger NHE1. Here, we explored the effect of MG on SGK1-dependent NHE1 activation and the putative role of NHE1 activation in MG-induced leukocyte recruitment and microvascular hyperpermeability.MethodsUsing RT-PCR and immunoblotting, we analyzed NHE1 mRNA and protein levels in murine microvascular SVEC4-10EE2 endothelial cells (EE2 ECs). NHE1 phosphorylation was detected using a specific antibody against the 14-3-3 binding motif at phospho-Ser703. SGK in EE2 ECs was silenced using targeted siRNA. ROS production was determined using DCF-dependent fluorescence. Leukocyte recruitment and microvascular permeability in murine cremasteric microvasculature were measured using intravital microscopy. The expression of endothelial adhesion molecules was determined by immunoblotting and confocal imaging analysis.ResultsMG treatment significantly upregulated NHE1 mRNA and dose-dependently increased total- and phospho-NHE1. Treatment with SGK1 inhibitor GSK650394, antioxidant Tempol and silencing SGK all blunted MG-triggered phospho-NHE1 upregulation in EE2 ECs. NHE1 inhibitor cariporide attenuated MG-triggered ROS production, leukocyte adhesion and emigration and microvascular hyperpermeability, without affecting leukocyte rolling. Cariporide treatment did not alter MG-triggered upregulation of P- and E-selectins, but reduced endothelial ICAM-1 expression.ConclusionMG elicits SGK1-dependent activation of endothelial Na+/H+ exchanger NHE1 which participates in MG-induced ROS production, upregulation of endothelial ICAM-1, leukocyte recruitment and microvascular hyperpermeability. Pharmacological inhibition of NHE1 attenuates the proinflammatory effects of excessive MG and may, thus, be beneficial in diabetes-associated inflammation.

Leptin enhances endothelial cell differentiation and angiogenesis in murine embryonic stem cells

The metabolic regulation of leptin and its angiogenic effects have been well characterized in adult mammals. However, the role of leptin in the differentiation of embryonic stem cells (ESCs) to endothelial cells (ECs) has not been characterized. We hypothesized that leptin enhances the generation of ECs derived from ESCs and, in this way, promotes angiogenesis in embryonic vessels. To address this hypothesis, we utilized an in vitro model consisting of murine ESCs-derived embryoid bodies (EBs). Vascular density, EC and angiogenesis markers as well as phosphorylation levels of signal transducer and activator of transcription 3 (pSTAT3) were investigated in leptin-treated EBs and in untreated EBs as controls. ESC-derived ECs were isolated by magnetic sorting based on the expression of platelet endothelial cell adhesion molecule (PECAM-1/CD31). Significant upregulation of EC and angiogenic markers as well as higher vessel density were found in leptin-treated EBs compared to controls. CD31 positive enriched cells derived from leptin-treated EBs had improved proliferation and survival rate and showed higher levels of pSTAT3. These results suggested that leptin promotes EC differentiation and angiogenesis in mouse EBs and that janus tyrosine kinase (JAK)/STAT pathway can play a role in this biological process. Leptin-mediated EC differentiation and angiogenesis in ESCs can be a useful application towards regenerative medicine and tissue engineering.

Effects of pitavastatin on the expression of VCAM-1 and its target gene miR-126 in cultured human umbilical vein endothelial cells.

Reducing the expression of endothelial cell adhesion molecules is conducive to the decrease of inflammation-induced vascular complications. In this study, we observed pitavastatin on expression of vascular cell adhesion molecule-1 (VCAM-1) and its influence on VCAM-1's target gene miR-126 in endothelial cells. The purpose of this study is to explore the mechanism of pitavastatin in prevention and treatment of atherosclerosis. HUVEC were cultured in M1640 and passages 2-5 were used in experiments. The cells were randomly divided into three groups, control, TNF-α and pitavastatin group. Cells of TNF-α group were co-incubated with different concentrations (10, 20, 30μg/L) of TNF-α for 24h. Cells of pitavastatin group were firstly coincubated with (0.01, 0.1, 1μmol/L) pitavastatin, respectively, for 1h, then coincubated with 30μg/L TNF-α for 24h. VCAM-1 and miR-126 mRNA were detected by RT-PCR, and Western blotting was used to detect protein expression of VCAM-1. Both detection methods have showed that TNF-α stimulation significantly increased the mRNA and protein expression of VCAM-1 in a dose-dependent manner, and miR-126 mRNA expression exhibited a decreasing trend. The increase of VCAM-1 mRNA and protein expression induced by TNF-α was inhibited by pitavastatin in a dose-dependent manner, too. However, there were no differences of the expression of miR-126 among three groups. These effects may explain the ability of pitavastatin to reduce the progression of atherosclerosis. The findings further suggest that inhibitory effect of pitavastatin on VCAM-1 is not related to miR-126 but depends on other ways.

MIP-1α enhances Jurkat cell transendothelial migration by up-regulating endothelial adhesion molecules VCAM-1 and ICAM-1

The aim of this study is to evaluate the expression of macrophage inflammatory protein-1α (MIP-1α) in Jurkat cells and its effect on transendothelial migration. In the present study, human acute lymphoblastic leukemia Jurkat cells (Jurkat cells) were used as a model of T cells in human T-cell acute lymphoblastic leukemia (T-ALL), which demonstrated significantly higher MIP-1α expression compared with that in normal T-cell controls. The ability of Jurkat cells to cross a human brain microvascular endothelial cell (HBMEC) monolayer was almost completely abrogated by MIP-1α siRNA. In addition, the overexpression of MIP-1α resulted in the up-regulated expression of endothelial adhesion molecules, which enhanced the migration of Jurkat cells through a monolayer of HBMEC. MIP-1α levels in Jurkat cells appeared to be an important factor for its transendothelial migration, which may provide the theoretical basis to understand the mechanisms of brain metastases of T-ALL at cellular and molecular levels.

Regulation of methylglyoxal-elicited leukocyte recruitment by endothelial SGK1/GSK3 signaling

Excessive levels of the glycolysis metabolite methylglyoxal (MG) elicit enhanced expression of adhesion molecules which foster leukocyte–endothelial cell interactions. The signaling mechanisms involved remain elusive. To address this, we investigated the signal transduction of leukocyte- and endothelial-expressed phosphoinositide 3-kinase (PI3K) effector kinases glycogen synthase kinase 3 (GSK3) and serum- and glucocorticoid-inducible kinase 1 (SGK1) in the regulation of MG-elicited leukocyte recruitment. Using intravital microscopy of mouse cremasteric microvasculature, we demonstrate that GSK3 inhibitors lithium and SB216763 mitigate MG-elicited leukocyte recruitment and microvascular hyperpermeability. In SVEC4-10EE2 endothelial cells, but not in neutrophils, MG transiently activates GSK3 by reducing inhibitory phospho-GSK3α/β (Ser21/9) which parallels decrease of phospho-Akt at early time points (<30min). At later time points (≥1h), MG induces GSK3 deactivation which is dissipated by siRNA silencing of SGK. MG treatment potentiates endothelial SGK1 mRNA, total SGK1, phospho-SGK1 and phospho-NDRG1. The SGK1 inhibitor GSK650394 attenuates MG-elicited leukocyte recruitment. Pharmacological inhibition or silencing endothelial GSK3 or SGK attenuates MG-triggered nuclear factor (NF)-κB activity. Furthermore, silencing SGK blunts MG-triggered redox-sensitive phosphorylation of endothelial transcription factor CREB. Inhibition of SGK1 or GSK3 mitigates the expression of endothelial adhesion molecules P- and E-selectins and ICAM-1. Moreover, SGK1-dependent CREB activation participates in MG-elicited ICAM-1 upregulation. We conclude that temporal activation of endothelial SGK1 and GSK3 is decisive in MG-elicited upregulation of transcription factors, adhesion molecule expression, and leukocyte-vascular endothelium interactions. This novel signaling pathway may link excessive MG levels in vivo to inflammation, thus, unraveling potential therapeutic targets.

Mechanoresponsive networks controlling vascular inflammation.

Atherosclerosis is a chronic inflammatory disease of arteries that develops preferentially at branches and bends that are exposed to disturbed blood flow. Vascular function is modified by flow, in part, via the generation of mechanical forces that alter multiple physiological processes in endothelial cells. Shear stress has profound effects on vascular inflammation; high uniform shear stress prevents leukocyte recruitment to the vascular wall by reducing endothelial expression of adhesion molecules and other inflammatory proteins, whereas low oscillatory shear stress has the opposite effects. Here, we review the molecular mechanisms that underpin the effects of shear stress on endothelial inflammatory responses. They include shear stress regulation of inflammatory mitogen-activated protein kinase and nuclear factor-κB signaling. High shear suppresses these pathways through the induction of several negative regulators of inflammation, whereas low shear promotes inflammatory signaling. Furthermore, we summarize recent studies indicating that inflammatory signaling is highly sensitive to pulse wave frequencies, magnitude, and direction of flow. Finally, the importance of systems biology approaches (including omics studies and functional screening) to identify novel mechanosensitive pathways is discussed.