NDRG1 Signaling Is Essential for Endothelial Inflammation and Vascular Remodeling.

BACKGROUND: NDRG-1 (N-myc downstream-regulated gene 1) is a member of NDRG family that plays essential roles in cell differentiation, proliferation, and stress responses. Although the expression of NDRG1 is regulated by fluid shear stress, its roles in vascular biology remain poorly understood. The purpose of the study is to determine the functional significance of NDRG1 in vascular inflammation and remodeling. METHODS AND RESULTS: By using quantitative polymerase chain reaction, western blot, and immunohistochemistry, we demonstrate that the expression of NDRG1 is markedly increased in cytokine-stimulated endothelial cells and in human and mouse atherosclerotic lesions. To determine the role of NDRG1 in endothelial activation, we performed loss-of-function studies using NDRG1 short hairpin RNA. Our results demonstrate that NDRG1 knockdown by lentivirus bearing NDRG1 short hairpin RNA substantially attenuates both IL-1ß (interleukin-1ß) and TNF-α (tumor necrosis factor-α)-induced expression of cytokines/chemokines and adhesion molecules. Intriguingly, inhibition of NDRG1 also significantly attenuates the expression of procoagulant molecules, such as PAI-1 (plasminogen activator inhibitor type 1) and TF (tissue factor), and increases the expression of TM (thrombomodulin) and t-PA (tissue-type plasminogen activator), thus exerting potent antithrombotic effects in endothelial cells. Mechanistically, we showed that NDRG1 interacts with orphan Nur77 (nuclear receptor) and functionally inhibits the transcriptional activity of Nur77 and NF-κB (nuclear factor Kappa B) in endothelial cells. Moreover, in NDRG1 knockdown cells, both cytokine-induced mitogen-activated protein kinase activation, c-Jun phosphorylation, and AP-1 (activator protein 1) transcriptional activity are substantially inhibited. Neointima and atherosclerosis formation induced by carotid artery ligation and arterial thrombosis were markedly attenuated in endothelial cell-specific NDRG1 knockout mice compared with their wild-type littermates. CONCLUSIONS: Our results for the first time identify NDRG1 as a critical mediator implicated in regulating endothelial inflammation, thrombotic responses, and vascular remodeling, and suggest that inhibition of NDRG1 may represent a novel therapeutic strategy for inflammatory vascular diseases, such as atherothrombosis and restenosis.

α-Asarone attenuates chronic sciatica by inhibiting peripheral sensitization and promoting neural repair.

This study explored the therapeutic effect of α-asarone on chronic sciatica. Thirty-two Sprague-Dawley (SD) rats were divided into four groups: the sham group, chronic constriction injury (CCI) group, pregabalin group, and α-asarone group. Hot hyperalgesia was induced after the CCI operation, and α-asarone was found to relieve chronic neuralgia. Furthermore, α-asarone reduced IL1ß, IL6, TNF-α, CRP, and LPS levels and increased IL10 levels in serum. α-Asarone decreased the protein levels of TRPA1, TRPM8, and TRPV1-4 and the mRNA levels of TRPA1, TRPM8, TRPV1-4, IL1ß, and TNF-α in dorsal root ganglion neurons. In the sciatic nerve, α-asarone treatment reduced the number of inflammatory cells and promoted the proliferation of Schwann cells, favouring recovery of the nerve structure. In cellular experiments, LPS induced Schwann cell apoptosis via TLR4/p38MAPK signalling; α-asarone attenuated LPS-induced Schwann cell apoptosis by decreasing TLR4, p-p38MAPK, cleaved-caspase3, and cleaved-caspase7 levels and increasing Bcl-2 and Bcl-xl expression. Overall, these findings suggest that α-asarone relieves chronic sciatica by decreasing the levels of inflammatory factors, inhibiting peripheral sensitization, and favouring the repair of damaged nerves.

Macrophage NFATc3 prevents foam cell formation and atherosclerosis: evidence and mechanisms.

AIMS: Our previous study demonstrated that Ca2+ influx through the Orai1 store-operated Ca2+ channel in macrophages contributes to foam cell formation and atherosclerosis via the calcineurin-ASK1 pathway, not the classical calcineurin-nuclear factor of activated T-cell (NFAT) pathway. Moreover, up-regulation of NFATc3 in macrophages inhibits foam cell formation, suggesting that macrophage NFATc3 is a negative regulator of atherogenesis. Hence, this study investigated the precise role of macrophage NFATc3 in atherogenesis. METHODS AND RESULTS: Macrophage-specific NFATc3 knockout mice were generated to determine the effect of NFATc3 on atherosclerosis in a mouse model of adeno-associated virus-mutant PCSK9-induced atherosclerosis. NFATc3 expression was decreased in macrophages within human and mouse atherosclerotic lesions. Moreover, NFATc3 levels in peripheral blood mononuclear cells from atherosclerotic patients were negatively associated with plaque instability. Furthermore, macrophage-specific ablation of NFATc3 in mice led to the atherosclerotic plaque formation, whereas macrophage-specific NFATc3 transgenic mice exhibited the opposite phenotype. NFATc3 deficiency in macrophages promoted foam cell formation by potentiating SR-A- and CD36-meditated lipid uptake. NFATc3 directly targeted and transcriptionally up-regulated miR-204 levels. Mature miR-204-5p suppressed SR-A expression via canonical regulation. Unexpectedly, miR-204-3p localized in the nucleus and inhibited CD36 transcription. Restoration of miR-204 abolished the proatherogenic phenotype observed in the macrophage-specific NFATc3 knockout mice, and blockade of miR-204 function reversed the beneficial effects of NFATc3 in macrophages. CONCLUSION: Macrophage NFATc3 up-regulates miR-204 to reduce SR-A and CD36 levels, thereby preventing foam cell formation and atherosclerosis, indicating that the NFATc3/miR-204 axis may be a potential therapeutic target against atherosclerosis.

Nur77 Attenuates Inflammasome Activation by Inhibiting Caspase-1 Expression in Pulmonary Vascular Endothelial Cells.

Inflammasomes are intracellular multiprotein complexes that help trigger and maintain the inflammatory response as part of the innate immune system. Recently, it has been increasingly recognized that aberrant inflammasome activation is critically involved in endothelial dysfunction in a variety of human diseases, such as atherosclerosis, acute lung injury (ALI), and type 2 diabetes. The molecular mechanisms underlying endothelial inflammasome activation, however, have not been completely elucidated. In the present study, we identified orphan nuclear receptor Nur77 as a novel regulator in controlling inflammasome activation in vascular endothelial cells (ECs). We demonstrated that LPS-induced inflammasome activation was significantly inhibited by ectopic overexpression of Nur77, predominantly through transcriptional suppression of caspase-1 expression in vascular ECs. Consistent with this observation, we found that LPS-induced inflammasome activation was significantly augmented in lung ECs isolated from Nur77-knockout mice. Mechanistically, we showed that Nur77-induced inhibition of caspase-1 expression was due to an inhibition of IRF1 (IFN regulatory factor 1) expression and its subsequent binding to the caspase-1 promoter. Importantly, in a mouse model of LPS-induced ALI, Nur77 knockout led to a marked activation of caspase-1 in the lung, increased alveolar and circulating IL-1ß levels, and exacerbated ALI, all of which were substantially inhibited by administration of caspase-1 inhibitor. Together, our results support the presence of an important role for Nur77 in controlling inflammasome activation in vascular ECs and suggest that Nur77 could be a novel therapeutic target for the treatment of human diseases associated with aberrant inflammasome activation, such as ALI and atherosclerosis.

Clinical characteristics of patients with chronic cough in Guangdong, China: a multicenter descriptive study.

BACKGROUND: The clinical characteristics of patients with chronic cough are reported only in single-center survey in China, being significantly different from that in western countries. Here, we performed a multicenter study to describe the clinical characteristics of chronic cough patients. METHODS: A cross-sectional observational survey was conducted in thirteen tertiary hospitals of Guangdong, South China. Relevant data were recorded using a standardized questionnaire and analyzed, including demographics, educational attainment, cough features, and concomitant symptoms. RESULTS: Of 933 patients in this study, the median age was 40.0 (IQR 31.0-52.0) years with a peaked age of 30-39 years. The proportion of females (487, 52.2 %) was comparable to that of males (446, 47.8 %). Up to 81.9 % of the patients were non-smokers. More than two-thirds of the subjects with chronic cough had a low educational level. The median cough duration was 6.0 (IQR 3.0-24.0) months, and 73.0 % of chronic cough patients presented with dry cough. Laryngeal paresthesia was the most common concomitant symptom (704, 75.5 %), followed by rhinitis/sinusitis-related (350, 37.5 %) and respiratory symptoms (322, 34.5 %). Rhinitis/sinusitis-related symptoms more frequently occurred in patients with productive cough than in those with dry cough (49.0 % vs. 33.0 %, P < 0.001). Moreover, female patients displayed an older age and a higher prevalence of nocturnal cough compared to male patients (both P < 0.05). CONCLUSIONS: Our results show an equal gender, young profile and laryngeal paresthesia in patients with chronic cough, and different clinical features between females and males.

Biomimetic Cascade Polymer Nanoreactors for Starvation and Photodynamic Cancer Therapy.

The selective disruption of nutritional supplements and the metabolic routes of cancer cells offer a promising opportunity for more efficient cancer therapeutics. Herein, a biomimetic cascade polymer nanoreactor (GOx/CAT-NC) was fabricated by encapsulating glucose oxidase (GOx) and catalase (CAT) in a porphyrin polymer nanocapsule for combined starvation and photodynamic anticancer therapy. Internalized by cancer cells, the GOx/CAT-NCs facilitate microenvironmental oxidation by catalyzing endogenous H2O2 to form O2, thereby accelerating intracellular glucose catabolism and enhancing cytotoxic singlet oxygen (1O2) production with infrared irradiation. The GOx/CAT-NCs have demonstrated synergistic advantages in long-term starvation therapy and powerful photodynamic therapy (PDT) in cancer treatment, which inhibits tumor cells at more than twice the rate of starvation therapy alone. The biomimetic polymer nanoreactor will further contribute to the advancement of complementary modes of spatiotemporal control of cancer therapy.

The histone deacetylase inhibitor tubacin mitigates endothelial dysfunction by up-regulating the expression of endothelial nitric oxide synthase.

Endothelial nitric oxide (NO) synthase (eNOS) plays a critical role in the maintenance of blood vessel homeostasis. Recent findings suggest that cytoskeletal dynamics play an essential role in regulating eNOS expression and activation. Here, we sought to test whether modulation of cytoskeletal dynamics through pharmacological regulation of histone deacetylase 6 (HDAC6)-mediated tubulin deacetylation affects eNOS expression and endothelial function in vitro and in vivo We found that tubulin acetylation inducer (tubacin), a compound that appears to selectively inhibit HDAC6 activity, dramatically increased eNOS expression in several different endothelial cell lines, as determined by both immunoblotting and NO production assays. Mechanistically, we found that these effects were not mediated by tubacin's inhibitory effect on HDAC6 activity, but rather were due to its ability to stabilize eNOS mRNA transcripts. Consistent with these findings, tubacin also inhibited proinflammatory cytokine-induced degradation of eNOS transcripts and impairment of endothelium-dependent relaxation in the mouse aorta. Furthermore, we found that tubacin-induced up-regulation in eNOS expression in vivo is associated with improved endothelial function in diabetic db/db mice and with a marked attenuation of ischemic brain injury in a murine stroke model. Our findings indicate that tubacin exhibits potent eNOS-inducing effects and suggest that this compound might be useful for the prevention or management of endothelial dysfunction-associated cardiovascular diseases.

A new simple score of chronic cough: cough evaluation test.

BACKGROUND: Chronic cough has an important impact on physical, social and psychological aspects. A simple and effective method to assess different aspects of chronic cough severity is required. We aimed to develop a simple, self-completed test, Cough Evaluation Test (CET), to evaluate cough severity and its impact on health. METHOD: The items of preliminary CET were generated based on literature review and clinical practice. Items reduction was conducted by modified Delphi method. Patients with chronic cough were recruited to complete CET, Cough Visual Analog Scales (VAS), Mandarin Chinese version of the Leicester Cough Questionnaire (LCQ-MC), and Cough Symptom Score (CSS). Reassessments were performed at 1 week apart before treatment, and after more than 2 weeks treatments. Concurrent validation, internal consistency, repeatability, responsiveness and the minimal important difference (MID) were determined. RESULTS: CET consists of five items with a 5-point Likert scale (1-5 scaling of items, 5-25 score range). The Cronbach's alpha values for CET was 0.80. CET showed a stronger correlation with LCQ-MC (r = - 0.74) compared to that between LCQ-MC with VAS (r = - 0.61). CET also showed a stronger correlation with VAS (r = 0.70) compared to that between VAS with other measures. Intraclass correlation coefficients for CET was 0.84. In patients undergoing treatment, CET scores significantly changed (p < 0.0001). The MID of CET was 2. CONCLUSION: Cough Evaluate Test is a reliable, valid and responsive tool to simply evaluate impact of cough on physical, social and psychological aspects.

Transcriptional up-regulation of relaxin-3 by Nur77 attenuates ß-adrenergic agonist-induced apoptosis in cardiomyocytes.

The relaxin family peptides have been shown to exert several beneficial effects on the heart, including anti-apoptosis, anti-fibrosis, and anti-hypertrophy activity. Understanding their regulation might provide new opportunities for therapeutic interventions, but the molecular mechanism(s) coordinating relaxin expression in the heart remain largely obscured. Previous work demonstrated a role for the orphan nuclear receptor Nur77 in regulating cardiomyocyte apoptosis. We therefore investigated Nur77 in the hopes of identifying novel relaxin regulators. Quantitative real-time PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) data indicated that ectopic expression of orphan nuclear receptor Nur77 markedly increased the expression of latexin-3 (RLN3), but not relaxin-1 (RLN1), in neonatal rat ventricular cardiomyocytes (NRVMs). Furthermore, we found that the ß-adrenergic agonist isoproterenol (ISO) markedly stimulated RLN3 expression, and this stimulation was significantly attenuated in Nur77 knockdown cardiomyocytes and Nur77 knockout hearts. We showed that Nur77 significantly increased RLN3 promoter activity via specific binding to the RLN3 promoter, as demonstrated by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays. Furthermore, we found that Nur77 overexpression potently inhibited ISO-induced cardiomyocyte apoptosis, whereas this protective effect was significantly attenuated in RLN3 knockdown cardiomyocytes, suggesting that Nur77-induced RLN3 expression is an important mediator for the suppression of cardiomyocyte apoptosis. These findings show that Nur77 regulates RLN3 expression, therefore suppressing apoptosis in the heart, and suggest that activation of Nur77 may represent a useful therapeutic strategy for inhibition of cardiac fibrosis and heart failure.

Nur77 limits endothelial barrier disruption to LPS in the mouse lung.

Nur77 is an orphan nuclear receptor implicated in the regulation of a wide range of biological processes, including the maintenance of systemic blood vessel homeostasis. Although Nur77 is known to be expressed in the lung, its role in regulating pulmonary vascular functions remains entirely unknown. In this study, we found that Nur77 is expressed at high levels in the lung, and its expression is markedly upregulated in response to LPS administration. While the pulmonary vasculature of mice that lacked Nur77 appeared to function normally under homeostatic conditions, we observed a dramatic decrease in its barrier functions after exposure to LPS, as demonstrated by an increase in serum proteins in the bronchoalveolar lavage fluid and a reduction in the expression of endothelial junctional proteins, such as vascular endothelial cadherin (VE-cadherin) and ß-catenin. Similarly, we found that siRNA knockdown of Nur77 in lung microvascular endothelial cells also reduced VE-cadherin and ß-catenin expression and increased the quantity of fluorescein isothiocyanate-labeled dextran transporting across LPS-injured endothelial monolayers. Consistent with Nur77 playing a vascular protective role, we found that adenoviral-mediated overexpression of Nur77 both enhanced expression of VE-cadherin and ß-catenin and augmented endothelial barrier protection to LPS in cultured cells. Mechanistically, Nur77 appeared to mediate its protective effects, at least in part, by binding to ß-catenin and preventing its degradation. Our findings demonstrate a key role for Nur77 in the maintenance of lung endothelial barrier protection to LPS and suggest that therapeutic strategies aimed at augmenting Nur77 levels might be effective in treating a wide variety of inflammatory vascular diseases of the lung.

MicroRNA-638 inhibits human airway smooth muscle cell proliferation and migration through targeting cyclin D1 and NOR1.

Abnormal airway smooth muscle cell (ASMC) proliferation and migration contribute significantly to increased ASM mass associated with asthma. MicroRNA (miR)-638 is a primate-specific miRNA that plays important roles in development, DNA damage repair, hematopoiesis, and tumorigenesis. Although it is highly expressed in ASMCs, its function in ASM remodeling remains unknown. In the current study, we found that in response to various mitogenic stimuli, including platelet-derived growth factor-two B chains (PDGF-BB), transforming growth factor ß1, and fetal bovine serum, the expression of miR-638, as determined by quantitative real-time polymerase chain reaction (qRT-PCR), was significantly downregulated in the proliferative human ASMCs. Both gain- and loss-of-function studies were performed to study the role of miR-638 in ASMC proliferation and migration. We found that adenovirus-mediated miR-638 overexpression markedly inhibits ASMC proliferation and migration, while ablation of miR-638 by anti-miR-638 markedly increases cell proliferation and migration, as determined by WST-8 proliferation and scratch wound assays. Dual-luciferase reporter assay, qRT-PCR, and immunoblot analysis were used to investigate the effects of miR-638 on the expression of the downstream target genes in ASMCs. Our results demonstrated that miR-638 overexpression significantly reduced the expression of downstream target cyclin D1 and NOR1, both of which have been shown to be essential for cell proliferation and migration. Together, our study provides the first in vitro evidence highlighting the antiproliferative and antimigratory roles of miR-638 in human ASMC remodeling and suggests that targeted overexpression of miR-638 in ASMCs may provide a novel therapeutic strategy for preventing ASM hyperplasia associated with asthma.

Pim-1 Kinase Phosphorylates Cardiac Troponin I and Regulates Cardiac Myofilament Function.

BACKGROUND/AIMS: Pim-1 is a serine/threonine kinase that is highly expressed in the heart, and exerts potent cardiac protective effects through enhancing survival, proliferation, and regeneration of cardiomyocytes. Its myocardial specific substrates, however, remain unknown. In the present study, we aim to investigate whether Pim-1 modulates myofilament activity through phosphorylation of cardiac troponin I (cTnI), a key component in regulating myofilament function in the heart. METHODS: Coimmunoprecipitation and immunofluorescent assays were employed to investigate the interaction of Pim-1 with cTnI in cardiomyocytes. Biochemical, site directed mutagenesis, and mass spectrometric analyses were utilized to identify the phosphorylation sites of Pim1 in cTnI. Myofilament functional assay using skinned cardiac fiber was used to assess the effect of Pim1-mediated phosphorylation on cardiac myofilament activity. Lastly, the functional significance of Pim1-mediated cTnI in heart disease was determined in diabetic mice. RESULTS: We found that Pim-1 specifically interacts with cTnI in cardiomyocytes and this interaction leads to Pim1-mediated cTnI phosphorylation, predominantly at Ser23/24 and Ser150. Furthermore, our functional assay demonstrated that Pim-1 induces a robust phosphorylation of cTnI within the troponin complex, thus leading to a decreased Ca2+ sensitivity. Insulin-like growth factor 1 (IGF-1), a peptide growth factor that has been shown to stimulate myocardial contractility, markedly induces cTnI phosphorylation at Ser23/24 and Ser150 through increasing Pim-1 expression in cardiomyocytes. In a high-fat diabetic mice model, the expression of Pim1 in the heart is significantly decreased, which is accompanied by a decreased phosphorylation of cTnI at Ser23/24 and Ser150, further implicating the pathological significance of the Pim1/cTnI axis in the development of diabetic cardiomyopathy. CONCLUSION: Our results demonstrate that Pim-1 is a novel kinase that phosphorylates cTnI primarily at Ser23/24 and Ser150 in cardiomyocytes, which in turn may modulate myofilament function under a variety of physiological and pathophysiological conditions.

Correlation of simulation/finite element analysis to the separation of intrinsically magnetic spores and red blood cells using a microfluidic magnetic deposition system.

Magnetic separation of cells has been, and continues to be, widely used in a variety of applications, ranging from healthcare diagnostics to detection of food contamination. Typically, these technologies require cells labeled with antibody magnetic particle conjugate and a high magnetic energy gradient created in the flow containing the labeled cells (i.e., a column packed with magnetically inducible material), or dense packing of magnetic particles next to the flow cell. Such designs, while creating high magnetic energy gradients, are not amenable to easy, highly detailed, mathematic characterization. Our laboratories have been characterizing and developing analysis and separation technology that can be used on intrinsically magnetic cells or spores which are typically orders of magnitude weaker than typically immunomagnetically labeled cells. One such separation system is magnetic deposition microscopy (MDM) which not only separates cells, but deposits them in specific locations on slides for further microscopic analysis. In this study, the MDM system has been further characterized, using finite element and computational fluid mechanics software, and separation performance predicted, using a model which combines: 1) the distribution of the intrinsic magnetophoretic mobility of the cells (spores); 2) the fluid flow within the separation device; and 3) accurate maps of the values of the magnetic field (max 2.27 T), and magnetic energy gradient (max of 4.41 T2 /mm) within the system. Guided by this model, experimental studies indicated that greater than 95% of the intrinsically magnetic Bacillus spores can be separated with the MDM system. Further, this model allows analysis of cell trajectories which can assist in the design of higher throughput systems.

Obesity-Induced Endoplasmic Reticulum Stress Causes Lung Endothelial Dysfunction and Promotes Acute Lung Injury.

RETRACTED: Obesity is a significant risk factor for acute respiratory distress syndrome. The mechanisms underlying this association are unknown. We recently showed that diet-induced obese mice exhibit pulmonary vascular endothelial dysfunction, which is associated with enhanced susceptibility to LPS-induced acute lung injury. Here, we demonstrate that lung endothelial dysfunction in diet-induced obese mice coincides with increased endoplasmic reticulum (ER) stress. Specifically, we observed enhanced expression of the major sensors of misfolded proteins, including protein kinase R-like ER kinase, inositol-requiring enzyme α, and activating transcription factor 6, in whole lung and in primary lung endothelial cells isolated from diet-induced obese mice. Furthermore, we found that primary lung endothelial cells exposed to serum from obese mice, or to saturated fatty acids that mimic obese serum, resulted in enhanced expression of markers of ER stress and the induction of other biological responses that typify the lung endothelium of diet-induced obese mice, including an increase in expression of endothelial adhesion molecules and a decrease in expression of endothelial cell-cell junctional proteins. Similar changes were observed in lung endothelial cells and in whole-lung tissue after exposure to tunicamycin, a compound that causes ER stress by blocking N-linked glycosylation, indicating that ER stress causes endothelial dysfunction in the lung. Treatment with 4-phenylbutyric acid, a chemical protein chaperone that reduces ER stress, restored vascular endothelial cell expression of adhesion molecules and protected against LPS-induced acute lung injury in diet-induced obese mice. Our work indicates that fatty acids in obese serum induce ER stress in the pulmonary endothelium, leading to pulmonary endothelial cell dysfunction. Our work suggests that reducing protein load in the ER of pulmonary endothelial cells might protect against acute respiratory distress syndrome in obese individuals.

Increased Krüppel-like factor 12 in recurrent implantation failure impairs endometrial decidualization by repressing Nur77 expression.

BACKGROUND: Decidualization is a prerequisite for successful implantation and the establishment of pregnancy. A critical role of impaired decidualization in subfertility has been established. In human endometrial stromal cells (hESCs), Krüppel-like factor 12 (KLF12) and Nur77 are novel regulators of decidualization. We investigated whether KLF12 impaired the decidualization of hESCs in recurrent implantation failure (RIF) patients. METHODS: Endometrial tissues and hESCs were collected from RIF patients (n = 34) and fertile controls (n = 30) for in vitro analysis. Primary hESCs isolated from RIF endometrial tissues were used to evaluate the biological functions of KLF12 and Nur77. In addition, their molecular mechanisms were investigated by adenovirus-mediated overexpression. Gene expression regulation was examined by real-time-quantitative PCR (qRT-PCR), immunostaining and luciferase reporter assay. Further, blastocyst-like spheroid (BLS) and blastocyst implantation models were performed to examine the roles of KLF12 and Nur77 during embryo expansion on hESCs. RESULTS: hESCs from the RIF patients showed a poor decidual response, mainly characterized by decreased decidual prolactin (dPRL) secretion, impaired transformation and limited BLS expansion. In addition, KLF12 expression was increased in endometrial tissues from the RIF patients compared with those from the fertile controls, especially in stromal compartments. The opposite results were observed for Nur77 expression in these tissues. KLF12 repressed hESC decidualization by decreasing Nur77 expression. Mechanistically, KLF12 bound to a conserved site in the Nur77 promoter region. Nur77 overexpression significantly reversed the KLF12-mediated repression of dPRL expression, decidual transformation and BLS/blastocyst expansion. CONCLUSIONS: KLF12 impairs endometrial decidualization by transcriptionally repressing Nur77, and Nur77 overexpression reverses the poor decidual response of hESCs in RIF patients.

Antithrombotic Effects of Nur77 and Nor1 Are Mediated Through Upregulating Thrombomodulin Expression in Endothelial Cells.

OBJECTIVE: Thrombomodulin is highly expressed on the lumenal surface of vascular endothelial cells (ECs) and possesses potent anticoagulant, antifibrinolytic, and anti-inflammatory activities in the vessel wall. However, the regulation of thrombomodulin expression in ECs remains largely unknown. APPROACHES AND RESULTS: In this study, we characterized nuclear receptor 4A family as a novel regulator of thrombomodulin expression in vascular ECs. We demonstrated that both nuclear receptors 4A, Nur77 and Nor1, robustly increase thrombomodulin mRNA and protein levels in human vascular ECs and in mouse liver tissues after adenovirus-mediated transduction of Nur77 and Nor1 cDNAs. Moreover, Nur77 deficiency and knockdown of Nur77 and Nor1 expression markedly attenuated the basal and vascular endothelial growth factor165-stimulated thrombomodulin expression. Mechanistically, we found that Nur77 and Nor1 increase thrombomodulin expression by acting through 2 different mechanisms. We showed that Nur77 barely affects thrombomodulin promoter activity, but significantly increases thrombomodulin mRNA stability, whereas Nor1 enhances thrombomodulin expression mainly through induction of Kruppel-like factors 2 and 4 in vascular ECs. Furthermore, we demonstrated that both Nur77 and Nor1 significantly increase protein C activity and inhibit tumor necrosis factor α-induced prothrombotic effects in human ECs. Deficiency of Nur77 increases susceptibility to arterial thrombosis, whereas enhanced expression of Nur77 and Nor1 protects mice from arterial thrombus formation. CONCLUSIONS: Our results identified nuclear receptors 4A as novel regulators of thrombomodulin expression and function in vascular ECs and provided a proof-of-concept demonstration that targeted increasing expression of Nur77 and Nor1 in the vascular endothelium might represent a novel therapeutic approach for the treatment of thrombotic disorders.

Mitochondrial Reactive Oxygen Species Mediate Lysophosphatidylcholine-Induced Endothelial Cell Activation.

OBJECTIVE: Hyperlipidemia-induced endothelial cell (EC) activation is considered as an initial event responsible for monocyte recruitment in atherogenesis. However, it remains poorly defined what is the mechanism underlying hyperlipidemia-induced EC activation. Here, we tested a novel hypothesis that mitochondrial reactive oxygen species (mtROS) serve as signaling mediators for EC activation in early atherosclerosis. APPROACH AND RESULTS: Metabolomics and transcriptomics analyses revealed that several lysophosphatidylcholine (LPC) species, such as 16:0, 18:0, and 18:1, and their processing enzymes, including Pla2g7 and Pla2g4c, were significantly induced in the aortas of apolipoprotein E knockout mice during early atherosclerosis. Using electron spin resonance and flow cytometry, we found that LPC 16:0, 18:0, and 18:1 induced mtROS in primary human aortic ECs, independently of the activities of nicotinamide adenine dinucleotide phosphate oxidase. Mechanistically, using confocal microscopy and Seahorse XF mitochondrial analyzer, we showed that LPC induced mtROS via unique calcium entry-mediated increase of proton leak and mitochondrial O2 reduction. In addition, we found that mtROS contributed to LPC-induced EC activation by regulating nuclear binding of activator protein-1 and inducing intercellular adhesion molecule-1 gene expression in vitro. Furthermore, we showed that mtROS inhibitor MitoTEMPO suppressed EC activation and aortic monocyte recruitment in apolipoprotein E knockout mice using intravital microscopy and flow cytometry methods. CONCLUSIONS: ATP synthesis-uncoupled, but proton leak-coupled, mtROS increase mediates LPC-induced EC activation during early atherosclerosis. These results indicate that mitochondrial antioxidants are promising therapies for vascular inflammation and cardiovascular diseases.

C1q Deficiency Promotes Pulmonary Vascular Inflammation and Enhances the Susceptibility of the Lung Endothelium to Injury.

The collectin proteins are innate immune molecules found in high concentrations on the epithelial and endothelial surfaces of the lung. While these proteins are known to have important anti-inflammatory actions in the airways of the lung little is known of their functional importance in the pulmonary circulation. We recently demonstrated that the circulating collectin protein adiponectin has potent anti-inflammatory effects on the lung endothelium, leading us to reason that other structurally related proteins might have similar effects. To test this hypothesis, we investigated the anti-inflammatory actions of C1q in lung endothelial homeostasis and the pulmonary vascular response to LPS or HCl injury. We show that lung endothelium from C1q-deficient (C1q(-/-)) mice expresses higher baseline levels of the vascular adhesion markers ICAM-1, VCAM-1, and E-selectin when compared with wild-type mice. Further, we demonstrate that these changes are associated with enhanced susceptibility of the lung to injury as evident by increased expression of adhesion markers, enhanced production of pro-inflammatory cytokines, and augmented neutrophil recruitment. Additionally, we found that C1q(-/-) mice also exhibited enhanced endothelial barrier dysfunction after injury as manifested by decreased expression of junctional adherens proteins and enhanced vascular leakage. Mechanistically, C1q appears to mediate its effects by inhibiting phosphorylation of p38 mitogen-activated protein kinase (MAPK) and blocking nuclear translocation of the P65 subunit of nuclear factor (NF)-κB. In summary, our findings indicate a previously unrecognized role for C1q in pulmonary vascular homeostasis and provide added support for the hypothesis that circulating collectin proteins have protective effects on the lung endothelium.

Hypoxia-elicited impairment of cell wall integrity, glycosylation precursor synthesis, and growth in scaled-up high-cell density fed-batch cultures of Saccharomyces cerevisiae.

BACKGROUND: In this study we examine the integrity of the cell wall during scale up of a yeast fermentation process from laboratory scale (10 L) to industrial scale (10,000 L). In a previous study we observed a clear difference in the volume fraction occupied by yeast cells as revealed by wet cell weight (WCW) measurements between these scales. That study also included metabolite analysis which suggested hypoxia during scale up. Here we hypothesize that hypoxia weakens the yeast cell wall during the scale up, leading to changes in cell permeability, and/or cell mechanical resistance, which in turn may lead to the observed difference in WCW. We tested the cell wall integrity by probing the cell wall sensitivity to Zymolyase. Also exometabolomics data showed changes in supply of precursors for the glycosylation pathway. RESULTS: The results show a more sensitive cell wall later in the production process at industrial scale, while the sensitivity at early time points was similar at both scales. We also report exometabolomics data, in particular a link with the protein glycosylation pathway. Significantly lower levels of Man6P and progressively higher GDP-mannose indicated partially impaired incorporation of this sugar nucleotide during co- or post-translational protein glycosylation pathways at the 10,000 L compared to the 10 L scale. This impairment in glycosylation would be expected to affect cell wall integrity. Although cell viability from samples obtained at both scales were similar, cells harvested from 10 L bioreactors were able to re-initiate growth faster in fresh shake flask media than those harvested from the industrial scale. CONCLUSIONS: The results obtained help explain the WCW differences observed at both scales by hypoxia-triggered weakening of the yeast cell wall during the scale up.

Post-Transcriptional Regulation of Endothelial Nitric Oxide Synthase Expression by Polypyrimidine Tract-Binding Protein 1.

OBJECTIVE: Endothelial nitric oxide synthase (eNOS) is an important regulator of vascular function and its expression is regulated at post-transcriptional levels through a yet unknown mechanism. The purpose of this study is to elucidate the post-transcriptional factors regulating eNOS expression and function in endothelium. APPROACHES AND RESULTS: To elucidate the molecular basis of tumor necrosis factor (TNF)-α-mediated eNOS mRNA instability, biotinylated eNOS 3'-untranslational region (UTR) was used to purify its associated proteins by RNA affinity chromatography from cytosolic fractions of TNF-α-stimulated human umbilical vein endothelial cells (HUVECs). We identified 2 cytosolic proteins, with molecular weight of 52 and 57 kDa, which specifically bind to eNOS 3'-UTR in response to TNF-α stimulation. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis identified the 57-kDa protein as polypyrimidine tract-binding protein 1 (PTB1). RNA gel mobility shift and UV cross-linking assays demonstrated that PTB1 binds to a UCUU-rich sequence in eNOS 3'-UTR, and the C-terminal half of PTB1 is critical to this interaction. Importantly, PTB1 overexpression leads to decreased activity of luciferase gene fused with eNOS 3'-UTR as well as reduced eNOS expression and activity in human ECs. In HUVECs, we show that TNF-α markedly increased PTB1 expression, whereas adenovirus-mediated PTB1 overexpression decreased eNOS mRNA stability and reduced protein expression and endothelium-dependent relaxation. Furthermore, knockdown of PTB1 substantially attenuated TNF-α-induced destabilization of eNOS transcript and downregulation of eNOS expression. CONCLUSIONS: These results indicate that PTB1 is essential for regulating eNOS expression at post-transcriptional levels and suggest a novel therapeutic target for treatment of vascular diseases associated with inflammatory endothelial dysfunction.