Endothelial NF-κB Research Articles

Abstract 5484: Activation of Endothelial NF- κ B Signaling Promotes Coagulation by Inhibiting Protein C Anticoagulant Mechanism in Endotoxemic Mice

Although the role of systemic activation of NF- κ B pathway in septic coagulation has been well documented, little is known about the mechanistic contribution of endothelial intrinsic NF- κ B signaling in this pathological process. This study defined the causative role of endothelial NF- κ B signaling in septic coagulation. Wild type (WT) and transgenic mice (TG) that conditionally overexpress a mutant I- κ B β , an inhibitor of NF- κ B, selectively on endothelium, were injected with saline (1 ml/kg, i.p.) or E Coli LPS (5 mg/kg, i.p.). Tissue fibrin deposition, plasma markers of coagulation, plasma levels of protein C and plasminogen activator inhibitor-1 (PAI-1) were measured at 6 hours after LPS injection. Immunohistochemical staining of frozen tissue sections of heart, liver and kidney from WT-LPS mice showed a widespread tissue fibrin deposition. Fibrin deposition was not evident in tissue sections of the three organs from TG-LPS mice. Compared with WT-Con and TG-Con mice, WT-LPS mice showed a 79% reduction in plasma fibrinogen level, which was restored to control level in TG-LPS mice. WT-LPS displayed 13.7- and 3.2-fold increase in plasma levels of thrombin-antithrombin and D-dimer, which were reduced by approximately 69% and 37% in TG-LPS mice. Plasma levels of total and activated protein C (mO.D. units) for WT-Con, TG-Con, WT-LPS and TG-LPS groups were: 73.4±2.4, 72.0±3.2, 26.0±6.2 and 58.5±4.7 (p < 0.01, WT-LPS vs other 3 groups), and 45.6±1.6, 43.0±1.2, 2.8±1.8 and 29.9±4.1 (p < 0.01, WT-LPS vs other 3 groups). Plasma level of PAI-1 (ng/ml) for WT-Con, TG-Con, WT-LPS and TG-LPS groups was 0.06±0.05, 0.0±0.0, 31.3±4.8 and 18.1±3.3 (p < 0.05, WT-LPS vs other 3 groups). These results indicate that endothelial intrinsic NF- κ B signaling plays a pivotal role in the activation of coagulation in endotoxemic mice, and suggest that activation of endothelial NF- κ B pathway promotes coagulation by impairing the protein C anticoagulant mechanism and by inhibiting the fibrinolytic system. (Supported by NIH R01GM063907).

Activation of endothelial intrinsic NF-κB pathway impairs protein C anticoagulation mechanism and promotes coagulation in endotoxemic mice

AbstractAlthough the role of systemic activation of the nuclear factor κB (NF-κB) pathway in septic coagulation has been well documented, little is known about the contribution of endothelial-specific NF-κB signaling in this pathologic process. Here, we used transgenic mice that conditionally overexpress a mutant I-κBα, an inhibitor of NF-κB, selectively on endothelium, and their wild-type littermates to define the role of endothelial-specific NF-κB in septic coagulation. In wild-type mice, lipopolysaccharide (LPS) challenge (5 mg/kg intraperitoneally) caused markedly increased plasma markers of coagulation, decreased plasma fibrinogen level, and widespread tissue fibrin deposition, which were abrogated by endothelial NF-κB blockade in transgenic mice. Endothelial NF-κB blockade inhibited tissue factor expression in endothelial cells, but not in leukocytes. Endothelial NF-κB blockade did not inhibit LPS-induced tissue factor expression in heart, kidney, and liver. Endothelial NF-κB blockade prevented LPS down-regulation of endothelial protein C receptor (EPCR) and thrombomodulin protein expressions, inhibited tissue tumor necrosis factor-α converting enzyme activity, reduced EPCR shedding, and restored plasma protein C level. Our data demonstrate that endothelial intrinsic NF-κB signaling plays a pivotal role in septic coagulation and suggests a link between endothelial-specific NF-κB activation and the impairment of the thrombomodulin-protein C-EPCR anticoagulation pathway.

A Pivotal Role of Endothelial-Specific NF-κB Signaling in the Pathogenesis of Septic Shock and Septic Vascular Dysfunction

Although the role of NF-kappaB in the pathogenesis of sepsis and septic shock has been extensively studied, little is known about the causative contribution of endothelial-intrinsic NF-kappaB to these pathological processes. In this study, we used transgenic (TG) mice (on FVB genetic background) that conditionally overexpress the NF-kappaB inhibitor, mutant I-kappaBalpha, selectively on endothelium and their transgene-negative littermates (wild type (WT)) to define the causative role of endothelial-specific NF-kappaB signaling in septic shock and septic vascular dysfunction. In WT mice, LPS challenge caused systemic hypotension, a significantly blunted vasoconstrictor response to norepinephrine, and an impaired endothelium-dependent vasodilator response to acetylcholine, concomitant with a markedly increased aortic inducible NO synthase expression, significantly elevated plasma and aortic levels of nitrite/nitrate, increased aortic TNF-alpha expression, and decreased aortic endothelial NO synthase (eNOS) expression. In TG mice whose endothelial NF-kappaB was selectively blocked, LPS caused significantly less hypotension and no impairments in vasoconstrictor and endothelium-dependent vasodilator responses, associated with significantly reduced aortic inducible NO synthase expression, decreased plasma and aortic levels of nitrite/nitrate, reduced aortic TNF-alpha expression, and increased aortic eNOS expression. TNF-alpha knockout mice prevented LPS-induced eNOS down-regulation. WT mice subjected to cecal ligation and puncture showed significant systemic hypotension, which was prevented in TG mice. Our data show that selective blockade of endothelial-intrinsic NF-kappaB pathway is sufficient to abrogate the cascades of molecular events that lead to septic shock and septic vascular dysfunction, demonstrating a pivotal role of endothelial-specific NF-kappaB signaling in the pathogenesis of septic shock and septic vascular dysfunction.

Focal adhesion kinase phosphorylation in flow-activation of endothelial NF-κB. Focus on “Focal adhesion kinase modulates activation of NF-κB by flow in endothelial cells”

in this issue of American Journal of Physiology-Cell Physiol ogy, Petzold et al. (corresponding author Martin A. Schwartz) report their results from a very interesting study, “Focal adhesion kinase modulates activation of NF-κB by flow in endothelial cells” ([10][1]). Comparing endothelial

Role of NFκB in age-related vascular endothelial dysfunction in humans

Role of NFκB in age-related vascular endothelial dysfunction in humans

Immunology in Hypertension, Preeclampsia, and Target-Organ Damage

Hypertension generally brings to mind hemodynamic mechanisms, increased peripheral vascular resistance, the laws of Ohm or Hagen-Poisseule, and perhaps target-organ damage mediated through acceleration of atherosclerosis, vasculitis, or other concomitant processes. However, innate and acquired adaptive cellular or even antibody-mediated immunity plays an active role in the pathogenesis of hypertension (Figure 1). In primarily angiotensin (Ang) II-dependent animal models for end-organ damage, an intense inflammatory component, namely, inflammation, that was principally steered by the innate-immunity regulator nuclear factor κB (NF-κB) was observed. Immunosuppression with dexamethasone, the tumor necrosis factor (TNF)-α soluble receptor, etanercept, and mycophenolate decreased albuminuria, NF-κB activation, and infiltration of all of the immunocompetent cells in the animals but had little or no effect on blood pressure.1 Using mechanisms to block NF-κB either pharmacologically or subsequently locally in endothelial cells with Cre/lox transgenic mice harboring an endothelial cell-restricted NF-κB superrepressor Iκ-B-α-ΔN (Tie-1-ΔN mice), it became evident that innate immune mechanisms were certainly important in target-organ damage.2 Since then, numerous observations have been made regarding immune mechanisms that influence hypertension development and its resultant target-organ damage.3–6 These findings may offer important new insights into our understanding of mechanisms and could influence treatment. Figure 1. Immunity schematic: all of the various components can be activated by Ang II. Some components also influence the blood pressure-raising responses of Ang II. Guzik et al7 showed recently that mice lacking T and B cells (RAG-1−/− mice) have blunted hypertension and do not develop target-organ damage after Ang II or desoxycorticosterone acetate-salt treatment. Adoptive transfer of T but not B cells restored these abnormalities. Adoptive transfer of T cells lacking the G protein-coupled receptor Ang II type 1 (AT1) blunted Ang II-dependent hypertension. Ang II increased T-cell markers of activation and tissue homing in wild-type mice but not in …

Habitual aerobic exercise is associated with reduced vascular endothelial cell oxidative stress, NADPH oxidase and NFκB in middle‐aged and older humans

Middle‐aged and older (MA/O) adults who perform habitual aerobic exercise demonstrate enhanced vascular endothelial function compared with their sedentary peers, but the molecular mechanisms involved are unknown. Brachial artery flow‐mediated dilation, a measure of endothelium‐dependent dilation, was 34% higher (6.8 ± 0.9 vs. 5.1 ± 0.4 %δ, P<0.05) in regularly exercising (EX, n=12, 62 ± 2 yrs, 8M/4F) vs. sedentary (SED; n=25, age 62 ± 1 yrs, 12M/13F) healthy MA/O adults, whereas endothelium‐independent dilation to sublingual nitroglycerin was not different (29.3 ± 1.8 vs. 26.7 ± 1.4 %δ, P=0.29). In endothelial cells obtained from the brachial artery, expression of nitrotyrosine (NT), a cellular marker of oxidative stress (0.48 ± 0.07 vs. 0.74 ± 0.10 immunofluorescent intensity/HUVEC intensity), NADPH oxidase p47phox, a subunit of the oxidant enzyme NADPH oxidase (0.41 ± 0.04 vs. 0.76 ± 0.09), and nuclear factor κB (NFκB), a redox‐sensitive proinflammatory transcription factor (0.39 ± 0.04 vs. 0.64 ± 0.06), were 35‐50% lower in EX, whereas manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant enzyme, was 74% higher (0.48 ± 0.07 vs. 0.28 ± 0.04) (all P<0.05 vs. SED). NT was positively related to NADPH oxidase p47phox (r=0.64, P<0.01) and NFκB (r=0.29, P=0.05), but not to MnSOD. Compared with their sedentary peers, habitually exercising MA/O adults have reduced vascular endothelial cell oxidative stress related to decreased NADPH oxidase and NFκB. Supported by NIH AG000279, AG013038, AG022241, AG006537, and RR00051.

Functional characterization of bovine TIRAP and MyD88 in mediating bacterial lipopolysaccharide-induced endothelial NF-κB activation and apoptosis

Mastitis is a prevalent disease in dairy cows. Gram-negative bacteria, which express the pro-inflammatory molecule lipopolysaccharide (LPS), are responsible for the majority of acute clinical cases of mastitis. Previous studies have identified differential susceptibility of human and bovine endothelial cells (EC) to the pro-inflammatory and injury-inducing effects of LPS. The Toll-like receptor (TLR)-4 signaling pathway, which is activated by LPS, has been well studied in humans, but not in ruminants. Human myeloid differentiation-factor 88 (MyD88) and TIR-domain containing adaptor protein (TIRAP) are critical proteins in the LPS-induced NF-kappaB and apoptotic signaling pathways. To assess the role of the bovine orthologs of these proteins in bovine TLR-4 signaling, dominant-negative constructs were expressed in bovine EC, and LPS-induced NF-kappaB activation and apoptosis evaluated. The results from this study indicate that bovine MyD88 and TIRAP play functional roles in transducing LPS signaling from TLR-4 to downstream effector molecules involved in NF-kappaB activation, and that TIRAP promotes apoptotic signaling.

Endothelial Cell PECAM-1 Promotes Atherosclerotic Lesions in Areas of Disturbed Flow in ApoE-Deficient Mice

Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) has recently been shown to form an essential element of a mechanosensory complex that mediates endothelial responses to fluid shear stress. The aim of this study was to determine the in vivo role of PECAM-1 in atherosclerosis. We crossed C57BL/6 Pecam1(-/-) mice with apolipoprotein E-deficient (Apoe(-/-)) mice. On a Western diet, Pecam1(-/-)Apoe(-/-) mice showed reduced atherosclerotic lesion size compared to Apoe(-/-) mice. Striking differences were observed in the lesser curvature of the aortic arch, an area of disturbed flow, but not in the descending thoracic or abdominal aorta. Vascular cell adhesion molecule-1 (VCAM-1) expression, macrophage infiltration, and endothelial nuclear NF-kappaB were all reduced in Pecam1(-/-)Apoe(-/-) mice. Bone marrow transplantation suggested that endothelial PECAM-1 is the main determinant of atherosclerosis in the aortic arch, but that hematopoietic PECAM-1 promotes lesions in the abdominal aorta. In vitro data show that siRNA-based knockdown of PECAM-1 attenuates endothelial NF-kappaB activity and VCAM-1 expression under conditions of atheroprone flow. These results indicate that endothelial PECAM-1 contributes to atherosclerotic lesion formation in regions of disturbed flow by regulating NF-kappaB-mediated gene expression.

Endothelial dysfunction and vascular inflammation in Type 2 diabetes: interaction of AGE/RAGE and TNF-α signaling

the receptor for advanced glycation end products (RAGE) interacts with a diverse range of endogenous ligands termed advanced glycation end products (AGEs), which are formed by the Maillard reaction, a nonenzymatic process linking reducing sugar groups to proteins, lipids, and nucleic acids. AGEs

Divergent roles of endothelial NF-κB in multiple organ injury and bacterial clearance in mouse models of sepsis

Divergent roles of endothelial NF-κB in multiple organ injury and bacterial clearance in mouse models of sepsis

Divergent roles of endothelial NF-κB in multiple organ injury and bacterial clearance in mouse models of sepsis

To define the roles of endothelial-intrinsic nuclear factor κB (NF-κB) activity in host defense and multiple organ injury in response to sepsis, we generated double transgenic (TG) mice (EC-rtTA/I-κBαmt) that conditionally overexpress a degradation-resistant form of the NF-κB inhibitor I-κBα (I-κBαmt) selectively on vascular endothelium. The EC-rtTA/I-κBαmt mice had no basal, but a relatively high level of doxycycline-inducible, I-κBαmt expression. I-κBαmt expression was detected in endothelial cells, but not in fibroblasts, macrophages, and whole blood cells, confirming that transgene expression was restricted to the endothelium. When subjected to endotoxemia, EC-rtTA/I-κBαmt mice showed endothelial-selective blockade of NF-κB activation, repressed expression of multiple endothelial adhesion molecules, reduced neutrophil infiltration into multiple organs, decreased endothelial permeability, ameliorated multiple organ injury, reduced systemic hypotension, and abrogated intravascular coagulation. When subjected to cecal ligation and puncture–induced sepsis, the TG mice had less severe multiple organ injury and improved survival compared with wild-type (WT) mice. WT and EC-rtTA/I-κBαmt mice had comparable capacity to clear three different pathogenic bacteria. Our data demonstrate that endothelial NF-κB activity is an essential mediator of septic multiple organ inflammation and injury but plays little role in the host defense response to eradicate invading pathogenic bacteria.

Alterations in the Notch4 pathway in cerebral endothelial cells by the HIV aspartyl protease inhibitor, nelfinavir

BackgroundAspartyl protease inhibitors (PIs) used to treat HIV belong to an important group of drugs that influence significantly endothelial cell functioning and angiogenic capacity, although specific mechanisms are poorly understood. Recently, PIs, particularly Nelfinavir, were reported to disrupt Notch signaling in the HIV-related endothelial cell neoplasm, Kaposi's sarcoma. Given the importance of maintaining proper cerebral endothelial cell signaling at the blood brain barrier during HIV infection, we considered potential signaling pathways such as Notch, that may be vulnerable to dysregulation during exposure to PI-based anti-retroviral regimens. Notch processing by γ-secretase results in cleavage of the notch intracellular domain that travels to the nucleus to regulate expression of genes such as vascular endothelial cell growth factor and NFκB that are critical in endothelial cell functioning. Since, the effects of HIV PIs on γ-secretase substrate pathways in cerebral endothelial cell signaling have not been addressed, we sought to determine the effects of HIV PIs on Notch and amyloid precursor protein.ResultsExposure to reported physiological levels of Saquinavir, Indinavir, Nelfinavir and Ritonavir, significantly increased reactive oxygen species in cerebral endothelial cells, but had no effect on cell survival. Likewise, PIs decreased Notch 4-protein expression, but had no effect on Notch 1 or amyloid precursor protein expression. On the other hand, only Nelfinavir increased significantly Notch 4 processing, Notch4 intracellular domain nuclear localization and the expression of notch intracellular domain targets NFκB and matrix metalloproteinase 2. Pre-treatment with the antioxidant Vitamin E prevented PI-induced reactive oxygen species generation and partially prevented Nelfinavir-induced changes in both Notch 4 processing, and cellular localization patterns. Moreover, in support of increased expression of pro-angiogenic genes after Nelfinavir treatment, Nelfinavir did not inhibit angiogenic capacity.ConclusionNelfinavir affects Notch 4 processing that results in induction of expression of the pro-angiogenic genes NFκB and matrix metalloproteinase 2 in cerebral endothelial cells.

Abstract 2841: Endothelial Selective NF-κB Inhibition Improves Survival and Preserves Host Defense Capacity in Bacterial Model of Sepsis

NF-κB plays a predominant role in septic pathology and is a desired target for developing therapies for septic shock. However, NF-κB blockade impairs bacterial clearance capability (BCC), which is detrimental. We have demonstrated that endothelial-restricted NF-κB blockade effectively prevents endotoxemic multiple organ injury (Circulation, 114: II-257). In this study, we examined whether endothelial-selective NF-κB blockade preserves BCC and improves survival in bacterial model of sepsis. Wild type (WT) and transgenic mice (TG) that conditionally overexpress a degradation-resistant I-κBα (I-κBαmt) selectively on endothelial cells were intravenously injected with saline (1 ml/kg) or pathogenic bacteria. Bacterial colonies formed in blood and tissue homogenate cultures of lungs, liver, spleen and kidney were counted. TG mice were induced to express I-κBαmt by feeding with doxycycline in drinking water. No bacteria grew in blood and any tissue homogenate culture from WT and TG mice injected with saline. Bacterial colony counts in blood and the 4 tissue homogenate cultures were similar between TW and TG mice 12 hrs after 10 7 CFU S. pneumoniae or 10 8 CFU S. enterica injection or 24 hrs after 10 8 CFU L. monocytogenes injection (Table ). TG mice showed an improved survival in the cecal ligation and puncture model of sepsis. The survival rate was 33% and 67% for WT and TG mice. Our results demonstrate that endothelial-selective blockade of NF-κB activation improves survival and preserves bacterial clearance capacity in bacterial model of sepsis. Bacterial Colony Counts (CFU per ml blood or gram tissue)

Endothelial NF-κB As a Mediator of Kidney Damage

See related article, page 268–276 In the current issue of Circulation Research Henke and coworkers demonstrate a critical role for the transcription factor nuclear factor-kappaB (NF-κB) in the genesis of renal damage caused by hypertension.1 The authors created mice in which the NF-κB superrepressor IκBαΔN was induced in the endothelium using Cre/Lox technology. They then exposed these mice and appropriate controls to a rather complex model of hypertension involving angiotensin II infusion, high salt and inhibition of endogenous nitric oxide production. Although the NF-κB superrepressor did not prevent hypertension, it markedly diminished renal injury. Albuminuria in the NF-κB suppressed mice was reduced by half, perivascular and tubular fibrosis was decreased, TNF-α levels were diminished and the renal infiltration of inflammatory cells reduced. The study illustrates a previously unappreciated role of the endothelium and specifically endothelial NF-κB in coordinating numerous aspects of hypertensive renal damage. Prevention of hypertension and its attendant end organ damage is one of the major therapeutic aims in cardiovascular medicine. A vast amount of research has been devoted to understanding the mechanisms and pathogenesis of hypertension, but for the past two decades this knowledge has not led to new treatments. Although current drugs are effective in many patients, there are still numerous patients in whom even combination therapies are not successful.2 Moreover while we focus almost solely on bringing the blood pressure levels back to normal, the mechanisms of end-organ damage are in part independent of blood pressure and are influenced by factors such as race, socioeconomic status, diabetes, dyslipidemia and other unknown factors. This is particularly true of renal failure, which occurs much more commonly in blacks and is exacerbated by diabetes. Hypertensive end organ damage is predominantly caused by inflammatory cells such as macrophages and lymphocytes infiltrating target organs. Numerous aspects of …

Vascular Endothelial Cell–Specific NF-κB Suppression Attenuates Hypertension-Induced Renal Damage

Nuclear factor kappa B (NF-kappaB) participates in hypertension-induced vascular and target-organ damage. We tested whether or not endothelial cell-specific NF-kappaB suppression would be ameliorative. We generated Cre/lox transgenic mice with endothelial cell-restricted NF-kappaB super-repressor IkappaBalphaDeltaN (Tie-1-DeltaN mice) overexpression. We confirmed cell-specific IkappaBalphaDeltaN expression and reduced NF-kappaB activity after TNF-alpha stimulation in primary endothelial cell culture. To induce hypertension with target-organ damage, we fed mice a high-salt diet and N(omega)-nitro-l-arginine-methyl-ester (L-NAME) and infused angiotensin (Ang) II. This treatment caused a 40-mm Hg blood pressure increase in both Tie-1-DeltaN and control mice. In contrast to control mice, Tie-1-DeltaN mice developed a milder renal injury, reduced inflammation, and less albuminuria. RT-PCR showed significantly reduced expression of the NF-kappaB targets VCAM-1 and ICAM-1, compared with control mice. Thus, the data demonstrate a causal link between endothelial NF-kappaB activation and hypertension-induced renal damage. We conclude that in vivo NF-kappaB suppression in endothelial cells stops a signaling cascade leading to reduced hypertension-induced renal damage despite high blood pressure.

Increased mitochondrial H2O2 production promotes endothelial NF-κB activation in aged rat arteries

Previous studies have shown that the aging vascular system undergoes pro-atherogenic phenotypic changes, including increased oxidative stress and a pro-inflammatory shift in endothelial gene expression profile. To elucidate the link between increased oxidative stress and vascular inflammation in aging, we compared the carotid arteries and aortas of young and aged (24 mo old) Fisher 344 rats. In aged vessels there was an increased NF-kappaB activity (assessed by luciferase reporter gene assay and NF-kappaB binding assay), which was attenuated by scavenging H(2)O(2). Aging did not alter the vascular mRNA and protein expression of p65 and p50 subunits of NF-kappaB. In endothelial cells of aged vessels there was an increased production of H(2)O(2) (assessed by 5,6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate-acetyl ester fluorescence), which was attenuated by the mitochondrial uncoupler FCCP. In young arteries and cultured endothelial cells, antimycin A plus succinate significantly increased FCCP-sensitive mitochondrial H(2)O(2) generation, which was associated with activation of NF-kappaB. In aged vessels inhibition of NF-kappaB (by pyrrolidenedithiocarbamate, resveratrol) significantly attenuated inflammatory gene expression and inhibited monocyte adhesiveness. Thus increased mitochondrial oxidative stress contributes to endothelial NF-kappaB activation, which contributes to the pro-inflammatory phenotypic alterations in the aged vaculature. Our model predicts that by reducing mitochondrial H(2)O(2) production and/or directly inhibiting NF-kappaB novel anti-aging pharmacological treatments (e.g., calorie restriction mimetics) will exert significant anti-inflammatory and vasoprotective effects.

Absence of TRAM Restricts Toll-Like Receptor 4 Signaling in Vascular Endothelial Cells to the MyD88 Pathway

Mammalian cells respond to bacterial lipopolysaccharide (LPS) through a cognate receptor: Toll-like receptor 4 (TLR4). The signaling pathways, which link TLR4 to the proinflammatory transcription factor nuclear factor kappaB (NF-kappaB), occur through the intracellular docking proteins MyD88 and Trif. We hypothesize that unlike antigen-presenting cells, vascular endothelial cells (ECs) lack the Trif protein TRAM and are therefore incapable of eliciting Trif-dependent immune responses to LPS. Stimulation of wild-type mice with LPS leads to the activation of NF-kappaB in ECs and macrophages in vitro and in vivo. In contrast to macrophages, LPS did not activate endothelial NF-kappaB or NF-kappaB-dependent genes in MyD88(-/-) mice, suggesting the absence of a functional Trif pathway in vascular ECs. Indeed, the Trif-dependent gene cxcl10 was not expressed in ECs after LPS stimulation. This correlated with diminished expression of the Trif accessory TIR protein TRAM in ECs. Overexpression of TRAM cDNA in ECs reconstituted LPS-induced Trif-dependent NF-kappaB activation and cxcl10 promoter activity. The functional absence of TRAM in vascular ECs restricts TLR4 signaling to MyD88-dependent pathway. This is in contrast to macrophages, which respond to LPS via both Trif- and MyD88-dependent pathways. These findings indicate that vascular ECs do not express the Trif-dependent gene subset. This implies that these genes may be dispensable for the endothelial response to bacterial infection and play no role in the endothelial contribution to the development of atherosclerosis.

Age‐related impaired endothelium‐dependent dilation is associated with increased vascular endothelial cell protein expression of NF‐kB in humans

Chronic vascular inflammation is involved in all stages atherosclerosis including endothelial dysfunction. Age, a major atherogenic risk factor, is considered a state of systemic chronic inflammation and associated with impaired endothelium dependent-dilation (EDD), the central feature of endothelial dysfunction. The main aims of this study were to determine if: 1) vascular (venous) endothelial cell protein expression (ECPE) of NF-κB (quantitative immunofluorescence), a major redox-sensitive transcription factor that mediates expression of inflammatory mediators, is increased with aging; and 2) the age-related decline in EDD is associated with increased endothelial NF-κB. Endothelial cell protein expression of NF-κB was 52 % greater in older (n=11, 64±2 years, MEAN±SE) compared with young (n=8, 24±1 years) subjects (0.21±0.02 vs. 0.44±0.07 ECPE/HUVEC; P<0.001). Brachial artery flow-mediated dilation (FMD), a measure of EDD, was 60 % lower in older than young subjects (3.1±0.6 vs. 7.6±0.8 %, P<0.001), whereas endothelium independent dilation (EID) was similar in both groups. Brachial artery FMD was inversely related to NF-κB in the pool sample (r=−0.70, P=0.008), but not to EID. These preliminary results indicate that endothelial NF-κB protein expression in increased with age and related to impaired EDD in healthy humans. Supported by NIH AG006537, AG013038, and AG022241.

ERK is an anti-inflammatory signal that suppresses expression of NF-κB-dependent inflammatory genes by inhibiting IKK activity in endothelial cells

Unveiling of endothelial nuclear factor-κB (NF-κB) activation is pivotal for understanding the inflammatory reaction and the pathogenesis of inflammatory vascular diseases. We here report the novel function of extracellular signal-related kinase (ERK) in controlling endothelial NF-κB activation and inflammatory responses. In human endothelial cells, vascular endothelial growth factor (VEGF) induced NF-κB-dependent transcription of cell adhesion molecules (CAMs) and monocyte adhesion. These effects were prominently enhanced by either pretreatment with the MEK inhibitors, PD98059 and U0126 or overexpression of a dominant negative form of MEK, but blocked by a wild type ERK. Consistently, inhibition of ERK significantly increased IκB kinase (IKK) activity, IκBα phosphorylation, and nuclear translocation of NF-κB induced by VEGF, whereas overexpression of ERK resulted in the loss of these responses to VEGF. Using two PKC inhibitors has demonstrated that VEGF concomitantly stimulates IKK and its negative regulatory signal ERK through PKC that lies downstream of KDR/Flk-1. Strikingly, elevation of ERK in endothelial cells markedly inhibited CAM expression and NF-κB activation as well as monocyte adhesion induced by IL-1β and TNF-α. The data collectively suggest that ERK serves as an anti-inflammatory signal that suppresses expression of NF-κB-dependent inflammatory genes by inhibiting IKK activity in endothelial cells. Measuring the existence of ERK activity in vascular endothelial cells may be useful for predicting the feasibility and potency of inflammatory reactions in the vasculature.