The role of AMP-activated protein kinase α1-mediated endoplasmic reticulum stress in alleviating the toxic effect of uremic toxin indoxyl sulfate on vascular endothelial cells by Klotho.

Recently, the Klotho protein (Klotho) has received substantial attention as protective factor against cardiovascular complications of chronic kidney disease (CKD). However, the direct effect and mechanism of Klotho on endothelial cells injury are not well-known. In this study, we incubated human vein umbilical endothelial cells (HUVECs) with uremic toxin indoxyl sulfate (IS) to mimic CKD internal environment and investigated the direct effect of Klotho on the HUVECs injury induced by IS and to explore the mechanism in this process. We found IS inhibited cell viability, increased endoplasmic reticulum stress, and mediated apoptosis of HUVECs. Treatment with Klotho significantly attenuated IS-induced above effects. Furthermore, Klotho alleviated the IS toxic effect on HUVECs via promoting AMP-activated protein kinase (AMPK) α1 phosphorylation instead of directly upregulating AMPKα1, which could be partly blocked by AMPK pathway inhibitor-Compound C. In addition, Klotho also inhibited intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression induced by IS. Altogether, these results indicated that Klotho can protect HUVECs from IS-induced injury by alleviating AMPKα1-mediated endoplasmic reticulum stress.

Apixaban Downregulates Endothelial Inflammatory and Prothrombotic Phenotype in an In Vitro Model of Endothelial Dysfunction in Uremia.

PURPOSE: Chronic kidney disease (CKD) associates with inflammatory and prothrombotic phenotypes, resulting in higher cardiovascular risk. Factor Xa displays functions beyond coagulation, exhibiting proinflammatory effects. The aim of the present study was to investigate whether a direct FXa inhibitor protects from the endothelial dysfunction (ED) caused by uremia. METHODS: Macro (HUVEC) and microvascular (HMEC) endothelial cells (ECs) were exposed to serum from uremic patients or healthy donors, in absence and presence of apixaban (60 ng/ml). We evaluated changes in surface VCAM-1 and ICAM-1, intracellular eNOS, reactive oxygen species (ROS), and von Willebrand Factor (VWF) production by immunofluorescence, reactivity of the extracellular matrix (ECM) towards platelets, and intracellular signaling. RESULTS: ECs exposed to uremic serum triggered dysregulation of all the parameters. Presence of apixaban resulted in decreased expression of VCAM-1 (178 ± 14 to 89 ± 2% on HMEC and 324 ± 71 to 142 ± 25% on HUVEC) and ICAM-1 (388 ± 60 to 111 ± 10% on HMEC and 148 ± 9% to 90 ± 7% on HUVEC); increased eNOS (72 ± 8% to 95 ± 10% on HMEC); normalization of ROS levels (173 ± 21 to 114 ± 13% on HMEC and 165 ± 14 to 127 ± 7% on HUVEC); lower production of VWF (168 ± 14 to 92 ± 4% on HMEC and 151 ± 22 to 99 ± 11% on HUVEC); and decreased platelet adhesion onto ECM (134 ± 22 to 93 ± 23% on HMEC and 161 ± 14 to 117 ± 7% on HUVEC). Apixaban inhibited p38MAPK and p42/44 activation in HUVEC (139 ± 15 to 48 ± 15% and 411 ± 66 to 177 ± 57%, respectively) (p < 0.05 vs control for all parameters). CONCLUSION: Anti-FXa strategies, such as apixaban, prevented ED caused by the uremic milieu, exhibiting anti-inflammatory and antioxidant properties and modulating the reactivity of the ECM.

Effect of Tribulus Terrestris L. on Expression of ICAM-1, VCAM-1, E-Selectin and Proteome Profile of Human Endothelial Cells In-Vitro.

BACKGROUND: Atherosclerosis is a chronic inflammation that interferes with blood arteries functions due to the accumulation of low density lipids and cholesterol. OBJECTIVE: To investigate the effect of aqueous extract and saponin fraction of Tribulus terrestris L. (TT) on the proteome and expression of intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin in the human umbilical vein endothelial cell (HUVEC) and human bone marrow endothelial cell (HBMEC) lines. METHODS: Two cell lines were cultured and induced with lipopolysaccharide (LPS). The primed cells were then treated with aqueous extract and saponin fraction of TT. The protein profile of the endothelial cells was assessed under normal and LPS-induced conditions using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 2D gel electrophoresis (2-DE). The levels of VCAM-1, ICAM-1, and E-selectin were estimated by use of western blotting. RESULTS: LPS-induced HUVECs and HBMECs were shown to significantly increase the expression of ICAM-1, VCAM-1, and E-selectin in comparison to control groups. Our findings revealed that TT extract resulted in significantly more reduced levels of proteome (80 spots) as well as all the three mentioned proteins compared with the effect of saponin fraction alone. CONCLUSION: TT extract and its saponin fraction exerted anti-inflammatory effects on HUVEC and HBMEC lines and reduced the expression of ICAM-1, VCAM-1, and E-selectin. However, the anti-inflammatory effect of aqueous extract was greater than that of saponin fraction. Therefore, TT could be considered as a potential candidate for the treatment or prevention of atherosclerosis.

Radial Glial Cell-Derived VCAM1 Regulates Cortical Angiogenesis Through Distinct Enrichments in the Proximal and Distal Radial Processes.

Angiogenesis in the developing cerebral cortex accompanies cortical neurogenesis. However, the precise mechanisms underlying cortical angiogenesis at the embryonic stage remain largely unknown. Here, we show that radial glia-derived vascular cell adhesion molecule 1 (VCAM1) coordinates cortical vascularization through different enrichments in the proximal and distal radial glial processes. We found that VCAM1 was highly enriched around the blood vessels in the inner ventricular zone (VZ), preventing the ingrowth of blood vessels into the mitotic cell layer along the ventricular surface. Disrupting the enrichment of VCAM1 surrounding the blood vessels by a tetraspanin-blocking peptide or conditional deletion of Vcam1 gene in neural progenitor cells increased angiogenesis in the inner VZ. Conversely, VCAM1 expressed in the basal endfeet of radial glial processes promoted angiogenic sprouting from the perineural vascular plexus (PNVP). In utero, overexpression of VCAM1 increased the vessel density in the cortical plate, while knockdown of Vcam1 accomplished the opposite. In vitro, we observed that VCAM1 bidirectionally affected endothelial cell proliferation in a concentration-dependent manner. Taken together, our findings identify that distinct concentrations of VCAM1 around VZ blood vessels and the PNVP differently organize cortical angiogenesis during late embryogenesis.

Effects of probiotic yogurt on glycemic indexes and endothelial dysfunction markers in patients with metabolic syndrome.

OBJECTIVES: The relationship between gut microflora and metabolic syndrome components such as obesity, low-grade chronic systemic inflammation, dyslipidemia, and altered glucose metabolism is now acknowledged. The aim of this study was to assess the effects of probiotic yogurt on glycemic indexes and endothelial dysfunction markers in patients with metabolic syndrome. METHODS: This was a randomized, double-blind, placebo-controlled clinical trial of 44 patients with metabolic syndrome (22 men and 22 women), who were 20 to 65 y of age. The patients were assigned to either a treatment or control group and consumed 300g/d of probiotic yogurt containing Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 or a regular yogurt for 2 mo, respectively. Each group contained 22 participants. Fasting blood glucose and serum insulin was performed to derive homeostasis model assessment of insulin resistance (HOMA-IR), insulin sensitivity (Quicki), and HOMA of ß-cell function (HOMA- ß). In addition, markers of vascular cell adhesion molecule cell (VCAM)-1, intercellular adhesion molecule cell (ICAM)-1, and plasminogen activator inhibitor (PAI)-1 were measured to evaluate endothelial function at the beginning and at the end of the study. RESULTS: Consumption of probiotic yogurt resulted in a significant reduction in the level of blood glucose and VCAM-1. Significant changes in PAI-1, VCAM-1, insulin, HOMA-IR, and Quicki were observed in the probiotic yogurt group after intervention compared with baseline. CONCLUSION: Consumption of probiotic yogurt improved fasting blood glucose and partly modified serum endothelial function markers. These results suggest that regular intake of probiotic yogurt may exert positive effects on the treatment of metabolic syndrome.

Long-Term Effects of Environmental Lead Exposure on Blood Pressure and Plasma Soluble Cell Adhesion Molecules in Young Adults: A Follow-Up Study of a Prospective Cohort in Kosovo.

Background and Aims: Epidemiologic studies examining the relationship between environmental lead (Pb) exposure and blood pressure (BP) generally report small associations between blood lead concentration (BPb) and BP. However, these studies are predominantly cross-sectional. In addition, no epidemiologic studies evaluate associations between either current or past Pb exposure and serum levels of markers of systemic inflammation and endothelial dysfunction, including soluble vascular adhesion molecule (sVCAM-1) and soluble intercellular cell adhesion molecule (sICAM-1). We prospectively investigate these associations later in life: Methods. From our original prospective birth cohort study in Mitrovica (a mining town) and Prishtina (a control town), Kosovo, from 1985 to 1998, we located and assessed BPb and BP in 101 participants (mean age of 24.9 years old) in 2011. Results: We found highly statistically significant association between concurrent BPb and sVCAM-1 in men and a marginally significant association between concurrent PBb and sICAM.-1 in women. We did not find evidence of mediation. Conclusion: Current study results, along with previously reported findings on this cohort, provide evidence for the hypothesis that exposure to Pb leads to small increases in sBP and perhaps to increased circulating levels of sVCAM-1 and sICAM-1 later in life.

Streptococcus pyogenes Phospholipase A2 Induces the Expression of Adhesion Molecules on Human Umbilical Vein Endothelial Cells and Aorta of Mice.

The Streptococcus pyogenes phospholipase A2 (SlaA) gene is highly conserved in the M3 serotype of group A S. pyogenes, which often involves hypervirulent clones. However, the role of SlaA in S. pyogenes pathogenesis is unclear. Herein, we report that SlaA induces the expression of intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) via the arachidonic acid signaling cascade. Notably, recombinant SlaA induced ICAM1 and VCAM1 expression in human umbilical vein endothelial cells (HUVECs), resulting in enhanced adhesion of human monocytic leukemia (THP-1) cells. However, C134A, a variant enzyme with no enzymatic activity, did not induce such events. In addition, culture supernatants from S. pyogenes SSI-1 enhanced the adhesion of THP-1 cells to HUVECs, but culture supernatants from the ΔslaA isogenic mutant strain had limited effects. Aspirin, a cyclooxygenase 2 inhibitor, prevented the adhesion of THP-1 cells to HUVECs and did not induce ICAM1 and VCAM1 expression in HUVECs treated with SlaA. However, zileuton, a 5-lipoxygenase inhibitor, did not exhibit such effects. Furthermore, pre-administration of aspirin in mice intravenously injected with SlaA attenuated the transcriptional abundance of ICAM1 and VCAM1 in the aorta. These results suggested that SlaA from S. pyogenes stimulates the expression of adhesion molecules in vascular endothelial cells. Thus, SlaA contributes to the inflammation of vascular endothelial cells upon S. pyogenes infection.

Bilirubin Prevents Atherosclerotic Lesion Formation in Low-Density Lipoprotein Receptor-Deficient Mice by Inhibiting Endothelial VCAM-1 and ICAM-1 Signaling.

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 (Ldlr-/- ) 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 Ldlr-/- 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 Ldlr-/- 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.

The α4ß1 Homing Pathway Is Essential for Ileal Homing of Crohn's Disease Effector T Cells In Vivo.

BACKGROUND: The precise mechanisms controlling homing of T effector (Teff) cells to the inflamed gut in Crohn's disease (CD) are still unclear, and clinical outcome data from patients with inflammatory bowel disease treated with the anti-α4ß7 integrin antibody vedolizumab suggest differences between ulcerative colitis and CD. METHODS: Expression of homing molecules was studied with flow cytometry and immunohistochemistry. Their functional role was investigated in in vitro adhesion assays and in a humanized mouse model of T cell homing to the inflamed gut in vivo. RESULTS: Despite in vitro blockade of CD Teff adhesion to mucosal vascular addressin cell adhesion molecule-1 (MadCAM-1) and in contrast to previous observations in ulcerative colitis, anti-α4ß7 treatment did not result in reduced Teff cell homing to the colon in vivo. However, the integrin α4ß1 was expressed in higher levels on Teffs from patients with CD compared with controls, while its expression in the peripheral blood declined, and its expression in the intestine increased during the course of clinical vedolizumab treatment. Consistently, adhesion of CD Teffs to vascular cell adhesion molecule-1 (VCAM-1) was blocked by inhibition of α4 and α4ß1 in vitro. Moreover, in vivo homing of CD Teffs to the ileum was reduced by inhibition of α4 and α4ß1 integrins, but not α4ß7 integrins. CONCLUSIONS: Our findings suggest that Teff cell homing to the ileum through the axis α4ß1-VCAM-1 is an essential and nonredundant pathway in CD in vivo, possibly affecting efficacy of clinical treatment with antiadhesion compounds.

A Review of Indigo Naturalis as an Alternative Treatment for Nail Psoriasis.

INTRODUCTION: Nail psoriasis is challenging to treat. The few currently available therapies are limited in efficacy, and often produce unfavorable side effects. A plant extract widely used in Traditional Chinese Medicine, indigo naturalis (Qing Dai), is presented in this review as an alternative topical treatment for skin and nail psoriasis. The purpose of this article is to present information on a viable alternative treatment with a favorable side effect profile for a difficult disease to treat. METHODS: A PubMed search for the term "indigo naturalis" was performed, and literature from 2006 to the present relevant to indigo naturalis and treatment of psoriasis and nail psoriasis was reviewed. RESULTS: Indigo naturalis shares several therapeutic mechanisms with current psoriasis treatments, such as regulation of keratinocyte proliferation and differentiation, restoration of epidermal barrier function, and reduction of inflammatory processes. Clinically, it is well tolerated. CONCLUSION: Recent research of indigo naturalis suggests that it is a safe, inexpensive, and effective alternative topical treatment for skin and nail psoriasis.

Rutin improves endotoxin-induced acute lung injury via inhibition of iNOS and VCAM-1 expression.

Endotoxins exist anywhere including in water pools, dust, humidifier systems, and machining fluids. The major causal factor is endotoxins in many serious diseases, such as fever, sepsis, multi-organ failure, meningococcemia, and severe morbidities like neurologic disability, or hearing loss. Endotoxins are also called lipopolysaccharide (LPS) and are important pathogens of acute lung injury (ALI). Rutin has potential beneficial effects including anti-inflammation, antioxidation, anti-hyperlipidemia, and anti-platelet aggregation. Pre-treatment with rutin inhibited LPS-induced neutrophil infiltration in the lungs. LPS-induced expression of vascular cell adhesion molecule (VCAM)-1 and inducible nitric oxide synthase (iNOS) was suppressed by rutin, but there was no influence on expression of intercellular adhesion molecule-1 and cyclooxygenase-2. In addition, activation of the nuclear factor (NF)κB was reduced by rutin. Furthermore, we found that the inhibitory concentration of rutin on expression of VCAM-1 and iNOS was similar to NFκB activation. In conclusion, rutin is a potential protective agent for ALI via inhibition of neutrophil infiltration, expression of VCAM-1 and iNOS, and NFκB activation.

17ß estradiol regulates adhesion molecule expression in mesangial cells during glomerulonephritis.

We showed previously that 17ß estradiol (E2) led to improved survival in nephrotoxic serum induced nephritis (NTN) in male mice. In this study we determined whether E2 regulates vascular cell adhesion molecule (VCAM)-1, an adhesion molecule that is upregulated in kidney during autoimmune nephritis, in mesangial cells (MC). We show that E2 inhibited VCAM-1 up-regulation in kidneys in vivo during NTN, and in MCs upon TNFα stimulation. VCAM-1 up-regulation in MCs was controlled by the transcription factor NFκB. E2 inhibited RNA polymerase II recruitment to the VCAM-1 promoter, but not p65 recruitment. Interestingly E2 inhibited TNFα stimulated interaction between poly (ADP-ribose) polymerase-1 (PARP-1) and p65. As PARP-1 is required for VCAM-1 upregulation in MCs, our data suggest that E2 may inhibit pre-initiation complex formation at VCAM-1 promoter by inhibiting PARP-1 recruitment to p65. We propose that E2 plays an important role in regulating renal inflammation locally.

Nucleotide-binding oligomerization domain 1 regulates Porphyromonas gingivalis-induced vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 expression in endothelial cells through NF-κB pathway.

BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis has been shown to actively invade endothelial cells and induce vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) overexpression. Nucleotide-binding oligomerization domain 1 (NOD1) is an intracellular pattern recognition reporter, and its involvement in this process was unknown. This study focused on endothelial cells infected with P. gingivalis, the detection of NOD1 expression and the role that NOD1 plays in the upregulation of VCAM-1 and ICAM-1. MATERIAL AND METHODS: The human umbilical vein endothelial cell line (ECV-304) was intruded by P. gingivalis W83, and cells without any treatment were the control group. Expression levels of NOD1, VCAM-1, ICAM-1, phosphorylated P65 between cells with and without treatment on both mRNA and protein levels were compared. Then we examined whether mesodiaminopimelic acid (NOD1 agonist) could increase VCAM-1 and ICAM-1 expression, meanwhile, NOD1 gene silence by RNA interference could reduce VCAM-1, ICAM-1 and phosphorylated P65 release. At last, we examined whether inhibition of NF-κB by Bay117082 could reduce VCAM-1 and ICAM- 1 expression. The mRNA levels were measured by real-time polymerase chain reaction, and protein levels by western blot or electrophoretic mobility shift assays (for phosphorylated P65). RESULTS: P. gingivalis invasion showed significant upregulation of NOD1, VCAM-1 and ICAM-1. NOD1 activation by meso-diaminopimelic acid increased VCAM-1 and ICAM-1 expression, and NOD1 gene silence reduced VCAM-1 and ICAM-1 release markedly. The NF-κB signaling pathway was activated by P. gingivalis, while NOD1 gene silence decreased the activation of NF-κB. Moreover, inhibition of NF-κB reduced VCAM-1 and ICAM-1 expression induced by P. gingivalis in endothelial cells. CONCLUSION: The results revealed that P. gingivalis induced NOD1 overexpression in endothelial cells and that NOD1 played an important role in the process of VCAM-1 and ICAM-1 expression in endothelial cells infected with P. gingivalis through the NF-κB signaling pathway.

The effects of iodixanol and iopamidol on adhesion molecule serum levels in patients with angina pectoris undergoing coronary angiography: a randomized study.

OBJECTIVE: To compare intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) serum levels between patients with stable (SAP) and unstable angina pectoris (USAP) undergoing coronary angiography (CAG), investigate effects of CAG on ICAM-1, VCAM-1 levels in SAP, USAP patients; probable different effects of non-ionic radiocontrast media (RCM), iso-osmotic iodixanol and low osmolar iopamidol, on these adhesion molecules (AM). METHODS: In this randomized, prospective study, 2 groups consisting of patients with SAP (n=22) and USAP (n=22) undergoing CAG were included. For halves of each group iopamidol, for the other halves iodixanol were used as RCM, in turn for randomization. The patients were divided into 4 subgroups according to clinical presentations and used RCM(SAP-iodixanol, SAP-iopamidol USAP-iodixanol, USAP-iopamidol). ICAM-1, VCAM-1 levels were measured just before and 12 hours after CAG. Repeated measurements were compared with two-way ANOVA test. RESULTS: Baseline VCAM-1 concentration was higher in USAP group than SAP group (p=0.001). ICAM-1, VCAM-1 concentrations increased significantly following CAG in SAP, USAP groups. ICAM-1, VCAM-1 concentration increments; didn't reach statistical significance in SAP-iodixanol subgroup, reached a borderline significance in SAP-iopamidol subgroup (p=0.06). In USAP-iodixanol subgroup; only VCAM-1 (p<0.001), in USAP-iopamidol subgroup; ICAM-1 (p=0.009), VCAM-1 (p=0.006) levels increased significantly following CAG. No complication was observed. CONCLUSION: To our knowledge, this is the first study indicating ICAM-1, VCAM-1 inducing effect of CAG in patients with SAP, USAP and differential effects of iodixanol and iopamidol on ICAM-1, VCAM-1 serum levels. Further studies are needed to clarify the effects of CAG and different RCM on vascular inflammation, vessel injury, serum AM levels and their clinical significance. This study should be taken as a pilot, hypothesis-generating study.

Mycophenolic acid attenuates the tumour necrosis factor-α-mediated proinflammatory response in endothelial cells by blocking the MAPK/NF-κB and ROS pathways.

BACKGROUND: Mycophenolate mofetil (MMF) has beneficial effects in cardiac transplant patients beyond the suppression of tissue rejection. Moreover, mycophenolic acid (MPA), its active metabolite, has been associated with positive effects on atherosclerosis in animal models. The attachment of leukocytes to the vascular endothelium and the subsequent migration of these cells into the vessel wall are early events in inflammation and atherosclerosis. The aim of this study was to investigate the effects of MPA on tumour necrosis-α (TNF-α)-induced, endothelial cell proinflammatory responses and the underlying mechanisms. METHODS AND RESULTS: Human aortic endothelial cells (HAECs) were treated with different concentrations (primarily 50 µM) of MPA before treatment with TNF-α. The surface protein and mRNA expressions of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were determined by flow cytometry and real-time RT-PCR, respectively. Adhesion of leukocytes to TNF-α-treated HAECs was evaluated by an adhesion assay. Activation of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) was evaluated by measuring the levels of their phosphorylation using flow cytometry. NF-κB p65 translocation was detected by Western blotting. The production of reactive oxygen species (ROS) was determined by reduction in fluorescent 2',7'-dichlorofluorescein diacetate (H2 DCFH-DA). MPA significantly inhibits TNF-α-induced ICAM-1, VCAM-1 surface protein and mRNA expression as well as adhesion of mononuclear leukocytes to HAEC. ICAM-1 and VCAM-1 expressions were also reduced by antioxidants such as pyrrolidine dithiocarbamate, diphenylene iodonium and apocynin. MPA inhibited TNF-α-stimulated ROS generation similarly to apocynin. TNF-α increased ICAM-1 and VCAM-1 expression via c-Jun NH2 -terminal kinase (JNK), extracellular signal-regulated kinase (ERK1/2) and p38 MAPK. MPA and apocynin inhibited TNF-α-induced phosphorylation of all three MAP kinases. Furthermore, TNF-α-induced NF-κB activation was attenuated by SP600125 (JNK inhibitor), PD98059 (ERK1/2 inhibitor, SB203580 (p38 MAPK inhibitor) and MPA. MPA also inhibited TNF-α-induced nuclear translocation of NF-κB p65. CONCLUSION: These results suggest that, in addition to the prevention of rejection, MPA may be a promising approach for the treatment of inflammatory vascular disease.

Shear stress modulates VCAM-1 expression in response to TNF-α and dietary lipids via interferon regulatory factor-1 in cultured endothelium.

Dyslipidemia is a primary risk factor for cardiovascular disease, but the specific mechanisms that determine the localization of atherosclerotic plaques in arteries are not well defined. Triglyceride-rich lipoproteins (TGRL) isolated from human plasma after a high-fat meal modulate TNF-α-induced VCAM-1 expression in cultured human aortic endothelial cells (HAECs) via an interferon regulatory factor (IRF)-1-dependent transcriptional mechanism. We examined whether fluid shear stress acts as a mediator of IRF-1-dependent VCAM-1 expression in response to cytokine and dietary lipids. IRF-1 and VCAM-1 were examined by immunofluorescence in TNF-α-stimulated HAEC monolayers exposed to TGRL and a linear gradient of shear stress ranging from 0 to 16 dyn/cm(2) in a microfluidic device. Shear stress alone modulated TNF-α-induced VCAM-1 expression, eliciting a 150% increase at low shear stress (2 dyn/cm(2)) and a 70% decrease at high shear stress (12 dyn/cm(2)) relative to static. These differences correlated with a 60% increase in IRF-1 expression under low shear stress and a 40% decrease under high shear stress. The addition of TGRL along with cytokine activated a fourfold increase in VCAM-1 expression and a twofold increase in IRF-1 expression. The combined effect of shear stress and TGRL on the upregulation of membrane VCAM-1 was abolished by transfection of HAECs with IRF-1-specific small interfering RNA. In a healthy swine model, elevated levels of endothelial IRF-1 were also observed within atherosusceptible regions of the aorta by Western blot analysis and immunohistochemistry, implicating arterial hemodynamics in the regulation of IRF-1 expression. These data demonstrate direct roles for fluid shear stress and postprandial TGRL from human serum in the regulation of IRF-1 expression and downstream inflammatory responses in HAECs.

Ultra-sensitive molecular MRI of vascular cell adhesion molecule-1 reveals a dynamic inflammatory penumbra after strokes.

BACKGROUND AND PURPOSE: Our aim was to assess the spatiotemporal evolution of the cerebrovascular inflammation occurring after ischemic and hemorrhagic strokes using a recently developed, fast, and ultra-sensitive molecular MRI method. METHODS: We first assessed longitudinally the cerebrovascular inflammation triggered by collagenase-induced hemorrhage and by permanent/transient middle cerebral artery occlusion in mice, using MRI after injection of microparticles of iron oxide targeted to vascular cell adhesion molecule-1 (MPIOs-αVCAM-1). Thereafter, we used this method to study the anti-inflammatory effects of celecoxib, atorvastatin, and dipyridamole after stroke. RESULTS: Using multiparametric MRI, we demonstrated that the level and the kinetics of cerebrovascular VCAM-1 expression depend on several parameters, including stroke pathogenesis, the natural history of the disease, and the administration of inflammation-modulating drugs. Interestingly, in transient middle cerebral artery occlusion and intracranial hemorrhage models, VCAM-1 expression was maximal at 24 hours and almost returned to baseline 5 days after stroke onset. In contrast, after permanent middle cerebral artery occlusion, VCAM-1 overexpression was sustained between 24 hours and 5 days, and was particularly significant in the peri-infarct areas. Our results suggest that these perilesional areas expressing VCAM-1 constitute an inflammatory penumbra that is recruited by the ischemic core during the subacute phase. Using MPIOs-αVCAM-1-enhanced imaging, we also provided evidence that celecoxib and atorvastatin (but not dipyridamole) alleviate VCAM-1 overexpression after stroke and prevent formation of the inflammatory penumbra. CONCLUSIONS: MPIOs-αVCAM-1-enhanced imaging seems to be promising in the detection of individuals presenting with severe cerebrovascular responses after stroke, which could therefore benefit from anti-inflammatory treatments.

Effects of Aggregatibacter actinomycetemcomitans leukotoxin on endothelial cells.

Aggregatibacter actinomycetemcomitans is a human pathogen that produces leukotoxin (LtxA) as a major virulence factor. In this study the effect of LtxA on microvascular endothelial cell viability and phenotype was studied. High doses of single LtxA treatment (500 ng/ml to 5 µg/ml) significantly and irreversibly decreased cell proliferation and induced apoptosis, as assessed by tetrazolium salt and annexin V assay, respectively. Apoptosis was partially inhibited by the pan-caspase inhibitor, z-VAD-fmk. LtxA caused a cell cycle arrest in the G2/M phase after 72 h. Between 500 ng/ml and 5 µg/ml, after long- or short-term stimulation LtxA increased the expression of ICAM-1 and VCAM-1, as well as the percentages of endothelial cells expressing these adhesion molecules. Thus, A. actinomycetemcomitans LtxA has substantial pro-inflammatory effects on human brain endothelial cells by upregulation of ICAM-1 and VCAM-1. Furthermore, LtxA in higher concentration was found to decrease proliferation and induces apoptosis in microvascular endothelial cells.

Anti-inflammatory functions of apolipoprotein A-I and high-density lipoprotein are preserved in trimeric apolipoprotein A-I.

Raising high-density lipoprotein (HDL) levels is proposed as an attractive target to treat cardiovascular disease. However, a number of clinical studies examining the effect of HDL-raising therapies have been prematurely halted due to futility. Therefore there is a need for alternative therapies. Infusion of reconstituted HDL (rHDL) particles is still considered as a viable approach to increasing HDL levels. In this study we have profiled the anti-inflammatory effects of a trimeric-HDL particle. We show that trimeric apoA-I and rHDL particles promote cholesterol efflux to a similar rate as native apoA-I particles in both ABCA1-dependent and -independent pathways. Trimeric particles inhibited ICAM-1 and VCAM-1 expression and the ability of the endothelium to capture monocytes under shear flow. Monocyte activation, CD11b-dependent adhesion, and monocyte recruitment under shear flow conditions were perturbed by the trimeric particles. Our data suggest that trimeric rHDL particles can be constructed without any loss of function, preserving the anti-inflammatory effects of HDL that are key to its in vivo actions.