BACKGROUND AND AIMS:Chronic intermittent hypoxia (CIH) exposure causes atherosclerosis, although the underlying mechanisms are poorly understood. This study defines the role of endothelial intrinsic NF-κB signaling in the atherogenic response to CIH. METHODS:We created ApoE-ECI-κBmt mice that are deficient in the apolipoprotein E gene (ApoE-/-) and overexpress an I-κBα mutant (I-κBmt) selectively in endothelial cells. ApoE-/- and ApoE-ECI-κBmt mice were fed a normal chow diet (NCD) or high cholesterol diet (HCD) and exposed to sham or CIH, and atherosclerotic lesions were quantified. RESULTS:CIH exposure activated NF-κB in aortas, and induced the expression of endothelial-specific and NF-κB-dependent genes, E-selectin and vascular cell adhesion molecule (VCAM)-1, in the aortas and hearts. Endothelial I-κBmt overexpression in ApoE-ECI-κBmt mice significantly inhibited CIH-induced NF-κB activity, and suppressed E-selectin and VCAM-1 expressions, confirming endothelial NF-κB inhibition in ApoE-ECI-κBmt mice. ApoE-/- mice, on NCD, developed mild atherosclerotic lesions spontaneously, and developed advanced and larger areas of atherosclerotic plaques when exposed to CIH. ApoE-/- mice also developed advanced atherosclerotic lesions when fed an HCD alone. The HCD-induced atherosclerotic plaques became more advanced, and plaque area was doubled in mice exposed to HCD + CIH. Endothelial I-κBmt overexpression in ApoE-ECI-κBmt mice attenuated spontaneously developed atherosclerotic lesions, abrogated CIH-induced atherosclerosis and mitigated CIH-mediated facilitation of HCD-induced atherosclerosis. CONCLUSIONS:These results suggest that endothelial intrinsic NF-kB signaling may play a pivotal role in CIH-induced atherosclerosis.

Objective— Disruption of endothelial barrier integrity is a characteristic of many inflammatory conditions. However, the origin and function of endothelial cells (ECs) restoring endothelial barrier function remain unknown. This study defined the roles of resident ECs (RECs) and bone marrow–derived endothelial progenitor cells (BMDEPCs) in endothelial barrier restoration after endotoxemic lung injury. Approach and Results— We generated mice that enable to quantify proliferating RECs or BMDEPCs and also to study the causal link between REC or BMDEPC proliferation and endothelial barrier restoration. Using these mouse models, we showed that endothelial barrier restoration was associated with increased REC and BMDEPC proliferation. RECs and BMDEPCs participate in barrier repair. Immunofluorescence staining demonstrated that RECs proliferate in situ on endothelial layer and that BMDEPCs are engrafted into endothelial layer of lung microvessels at the active barrier repair phase. In lungs, 8 weeks after lipopolysaccharide-induced injury, the number of REC-derived ECs (CD45 − /CD31 + /BrdU + /rtTA + ) or BMDEPC-derived ECs (CD45 − /CD31 + /eNOS + /GFP + ) increased by 22- or 121-fold, respectively. The suppression of REC or BMDEPC proliferation by blocking REC or BMDEPC intrinsic nuclear factor-κB at the barrier repair phase was associated with an augmented endothelial permeability and impeded endothelial barrier recovery. RECs and BMDEPCs contributed differently to endothelial barrier repair. In lungs, 8 weeks after lipopolysaccharide-induced injury, REC-derived ECs constituted 22%, but BMDEPC-derived ECs constituted only 3.7% of the total new ECs. Conclusions— REC is a major and BMDEPC is a complementary source of new ECs in endothelial barrier restoration. RECs and BMDEPCs play important roles in endothelial barrier restoration after inflammatory lung injury.

Background Specificity protein (Sp) 1 mediates the transcription of a large number of constitutive genes encoding physiological mediators. NF-κB mediates the expression of hundreds of inducible genes encoding pathological mediators. Crosstalk between Sp1 and NF-κB pathways could be pathophysiologically significant, but has not been studied. This study examined the crosstalk between the two pathways and defined the role of NF-κB signaling in LPS-induced down-regulation of Sp1 activity. Methods and Main Findings Challenge of wild type mice with samonelia enteritidis LPS (10 mg/kg, i.p.) down-regulated Sp1 binding activity in lungs in a time-dependent manner, which was concomitantly associated with an increased NF-κB activity. LPS down-regulates Sp1 activity by inducing an LPS inducible Sp1-degrading enzyme (LISPDE) activity, which selectively degrades Sp1 protein, resulting in Sp1 down-regulation. Blockade of NF-κB activation in mice deficient in NF-κB p50 gene (NF-κB-KO) suppressed LISPDE activity, prevented Sp1 protein degradation, and reversed the down-regulation of Sp1 DNA binding activity and eNOS expression (an indicator of Sp1 transactivation activity). Inhibition of LISPDE activity using a selective LISPDE inhibitor mimicked the effects of NF-κB blockade. Pretreatment of LPS-challenged WT mice with a selective LISPDE inhibitor increased nuclear Sp1 protein content, restored Sp1 DNA binding activity and reversed eNOS protein down-regulation in lungs. Enhancing tissue level of Sp1 activity by inhibiting NF-κB-mediated Sp1 down-regulation increased tissue level of IL-10 and decreased tissue level of TNF- αin the lungs. Conclusions NF-κB signaling mediates LPS-induced down-regulation of Sp1 activity. Activation of NF-κB pathway suppresses Sp1 activity and Sp1-mediated anti-inflammatory signals. Conversely, Sp1 signaling counter-regulates NF-κB-mediated inflammatory response. Crosstalk between NF-κB and Sp1 pathways regulates the balance between pro- and anti-inflammatory cytokines.

Endothelial barrier disruption is a hallmark of multiple organ injury (MOI). However, mechanisms governing the restoration of endothelial barrier function are poorly understood. Here, we uncovered an NF-κB-to-AP-1 switch that regulates the transition from barrier injury to repair following endotoxemic MOI. Endothelial NF-κB mediates barrier repair by inhibiting endothelial cell (EC) apoptosis. Blockade of endothelial NF-κB pathway activated the activator protein (AP)-1 pathway (NF-κB-to-AP-1 switch), which compensated for the anti-apoptotic and barrier-repair functions of NF-κB. The NF-κB-to-AP-1 switch occurred at 24 hours (injury to repair transition phase), but not at 48 hours (repair phase) post-LPS and required an inflammatory signal within the endothelium. In the absence of an inflammatory signal, the NF-κB-to-AP-1 switch failed, resulting in enhanced EC apoptosis, augmented endothelial permeability and impeded transition from barrier injury to recovery. The NF-κB-to-AP-1 switch is a protective mechanism to ensure timely transition from endothelial barrier injury to repair, accelerating barrier restoration following MOI.

Background Recruitment of bone marrow derived endothelial progenitor cells (BMDEPCs) alleviates multiple organ injury (MOI) and improves outcomes. However, mechanisms mediating BMDEPC recruitment following septic MOI remain largely unknown. This study characterized the kinetics of BMDEPC recruitment and proliferation and defined the role of NF-κB in regulating BMDEPC recruitment and proliferation. Methods and Main Findings Chimeric mice with an intact or disrupted NF-κB p50 gene and BMDEPC-restricted expression of green fluorescent protein were created and injected with LPS (2 mg/kg, i.p.). BMDEPC recruitment and proliferation in multiple organs were quantified. BMDEPC recruitment and proliferation are highly organ-dependent. Lungs had the highest number of BMDEPC recruitment, whereas heart, liver and kidney had only a small fraction of the number of BMDEPCs in lungs. Number of proliferating BMDEPCs was several-fold higher in lungs than in other 3 organs. Kinetically, BMDEPC recruitment into different organs showed different time course profiles. NF-κB plays obligatory roles in mediating BMDEPC recruitment and proliferation. Universal deletion of NF-κB p50 gene inhibited LPS-induced BMDEPC recruitment and proliferation by 95% and 69% in heart. However, the contribution of NF-κB to these regulations varies significantly between organs. In liver, universal p50 gene deletion reduced LPS-induced BMDEPC recruitment and proliferation only by 49% and 35%. NF-κB activities in different tissue compartments play distinct roles. Selective p50 gene deletion either in stromal/parenchymal cells or in BM/blood cells inhibited BMDEPC recruitment by a similar extent. However, selective p50 gene deletion in BM/blood cells inhibited, but in stromal/parenchymal cells augmented BMDEPC proliferation. Conclusions BMDEPC recruitment and proliferation display different kinetics in different organs following endotoxemic MOI. NF-κB plays obligatory and organ-dependent roles in regulating BMDEPC recruitment and proliferation. NF-κB activities in different tissue compartments play distinct roles in regulating BMDEPC proliferation.

Objectives: Patients with obstructive sleep apnea have an impaired endothelium-dependent vasodilator response. The mechanisms underlying this impairment remain unclear. We tested the hypothesis that chronic intermittent hypoxia (CIH) impairs endothelium-dependent vasodilatation by NF-kappa B-mediated down-regulation of endothelial nitric oxide synthase (eNOS) expression.Methods: Wild type (WT) mice and mice deficient in NF-kappa B p50 or TNF-alpha gene were exposed to sham or CIH. Aortic NF-kappa B activity and aortic expression of TNF-alpha were determined. Aortic and mesenteric artery levels of eNOS expression were examined and their correlation to endothelium-dependent vasodilator response in vitro and vasodepressor response in vivo were analyzed.Results: WT mice exposed to CIH for five to eight weeks showed significantly reduced eNOS protein expression in aortas and mesenteric arteries, associated with significantly blunted vasodilator and vasodepressor responses to acetylcholine, but not to sodium nitroprusside. CIH activated NF-kappa B, which preceded TNF-alpha up-regulation and eNOS down-regulation. NF-kappa B p50 gene deletion blocked NF-kappa B activation, inhibited TNF-alpha expression, prevented eNOS down-regulation and reversed the impaired endothelium-dependent vasodepressor response induced by CIH. TNF-alpha knockout prevented CIH-induced eNOS down-regulation and restored the endothelium-dependent vasodepressor response.Conclusions: CIH exposure impairs endothelium-dependent vasodilator mechanism by stimulating NF-kappa B-mediated TNF-alpha generation, which in turn, down-regulates eNOS expression, resulting in an impaired endothelium-dependent vasodilatation. (C) 2012 Elsevier B. V. All rights reserved.