Nels Olson

Interactions between nitric oxide and hypoxia-inducible factor signaling pathways in inflammatory disease.

Induction and activation of nitric oxide (NO) synthases (NOS) and excessive production of NO are common features of almost all diseases associated with infection and acute or chronic inflammation, although the contribution of NO to the pathophysiology of these diseases is highly multifactorial and often still a matter of controversy. Because of its direct impact on tissue oxygenation and cellular oxygen (O(2)) consumption and re-distribution, the ability of NO to regulate various aspects of hypoxia-induced signaling has received widespread attention. Conditions of tissue hypoxia and the activation of hypoxia-inducible factors (HIF) have been implicated in hypoxia or in cancer biology, but are also being increasingly recognized as important features of acute and chronic inflammation. Thus, the activation of HIF transcription factors has been increasingly implicated in inflammatory diseases, and recent studies have indicated its critical importance in regulating phagocyte function, inflammatory mediator production, and regulation of epithelial integrity and repair processes. Finally, HIF also appears to contribute to important features of tissue fibrosis and epithelial-to-mesenchymal transition, processes that are associated with tissue remodeling in various non-malignant chronic inflammatory disorders. In this review, we briefly summarize the current state of knowledge with respect to the general mechanisms involved in HIF regulation and the impact of NO on HIF activation. Secondly, we will summarize the major recent findings demonstrating a role for HIF signaling in infection, inflammation, and tissue repair and remodeling, and will address the involvement of NO. The growing interest in hypoxia-induced signaling and its relation with NO biology is expected to lead to further insights into the complex roles of NO in acute or chronic inflammatory diseases and may point to the importance of HIF signaling as key feature of NO-mediated events during these disorders.

Circulating levels of TNF-α are associated with impaired glucose tolerance, increased insulin resistance, and ethnicity: the Insulin Resistance Atherosclerosis Study.

OBJECTIVE Although several epidemiological studies have investigated associations between TNF-α and insulin resistance, results have been inconsistent. We studied the relationship between TNF-α and glucose tolerance status as part of the Insulin Resistance Atherosclerosis Study. RESEARCH DESIGN AND METHODS Serum concentrations of TNF-α were measured in 1558 individuals in a triethnic population across a spectrum of glucose tolerance. Insulin sensitivity and insulin secretion were assessed by a frequently sampled iv glucose tolerance test (FSIGT). RESULTS Compared with those with normal glucose tolerance, circulating levels of TNF-α were elevated in individuals with impaired glucose tolerance (IGT) and type 2 diabetes mellitus (T2D) after adjusting for age, gender, ethnicity, clinic site, and body mass index (3.3, 3.5, and 3.7 pg/ml in subjects with normal glucose tolerance, IGT, and T2D, respectively; P<0.05). Age-, sex-, and body mass index-adjusted levels of TNF-α differed by ethnicity, with Hispanics having the highest levels and African-Americans having the lowest (4.1, 3.6, and 3.0 pg/ml in Hispanics, non-Hispanic whites, and African-Americans, respectively; P<0.05). TNF-α was correlated with waist circumference, high-density lipoprotein, triglycerides, plasminogen activator inhibitor-1 and insulin sensitivity index (SI) (r=0.22, -0.30, 0.35, 0.31, and -0.25; P<0.0001); however, correlations varied by ethnicity. After adjusting for demographics and adiposity, individuals characterized by increased insulin resistance (lower SI), had higher levels of TNF-α than subjects characterized by high insulin sensitivity (3.8 and 3.3 pg/ml in subjects with an SI below/above the median at baseline; P<0.0001). No differences were found for acute insulin response. CONCLUSIONS We confirm that TNF-α is associated with IGT and T2D in a large, multiethnic population, independent of measures of adiposity. Adjusted values of TNF-α, as well as relationships between TNF-α and variables related to T2D, varied by ethnicity. Increased TNF-α levels were predominantly associated with insulin resistance but not with primary defects in β-cell function.

Activation of hypoxia-inducible factor-1 protects airway epithelium against oxidant-induced barrier dysfunction

The respiratory epithelium forms an important barrier against inhaled pollutants and microorganisms, and its barrier function is often compromised during inflammatory airway diseases. Epithelial activation of hypoxia-inducible factor-1 (HIF-1) represents one feature of airway inflammation, but the functional importance of HIF-1 within the respiratory epithelium is largely unknown. Using primary mouse tracheal epithelial (MTE) cells or immortalized human bronchial epithelial cells (16HBE14o-), we evaluated the impact of HIF-1 activation on loss of epithelial barrier function during oxidative stress. Exposure of either 16HBE14o- or MTE cells to H(2)O(2) resulted in significant loss of transepithelial electrical resistance and increased permeability to fluorescein isothiocyanate-dextran (4 kDa), and this was attenuated significantly after prior activation of HIF-1 by preexposure to hypoxia (2% O(2); 6 h) or the hypoxia mimics CoCl(2) or dimethyloxalylglycine (DMOG). Oxidative barrier loss was associated with reduced levels of the tight junction protein occludin and with hyperoxidation of the antioxidant enzyme peroxiredoxin (Prx-SO(2)H), events that were also attenuated by prior activation of HIF-1. Involvement of HIF-1 in these protective effects was confirmed using the pharmacological inhibitor YC-1 and by short-hairpin RNA knockdown of HIF-1α. The protective effects of HIF-1 were associated with induction of sestrin-2, a hypoxia-inducible enzyme known to reduce oxidative stress and minimize Prx hyperoxidation. Together, our results suggest that loss of epithelial barrier integrity by oxidative stress is minimized by activation of HIF-1, in part by induction of sestrin-2.

Inflammatory Levels of Nitric Oxide Inhibit Airway Epithelial Cell Migration by Inhibition of the Kinase ERK1/2 and Activation of Hypoxia-inducible Factor-1α

Increased synthesis of NO during airway inflammation, caused by induction of nitric-oxide synthase 2 in several lung cell types, may contribute to epithelial injury and permeability. To investigate the consequence of elevated NO production on epithelial function, we exposed cultured monolayers of human bronchial epithelial cells to the NO donor diethylenetriaamine NONOate. At concentrations generating high nanomolar levels of NO, representative of inflammatory conditions, diethylenetriaamine NONOate markedly reduced wound closure in an in vitro scratch injury model, primarily by inhibiting epithelial cell migration. Analysis of signaling pathways and gene expression profiles indicated a rapid induction of the mitogen-activated protein kinase phosphatase (MPK)-1 and decrease in extracellular signal-regulated kinase (ERK)1/2 activation, as well as marked stabilization of hypoxia-inducible factor (HIF)-1α and activation of hypoxia-responsive genes, under these conditions. Inhibition of ERK1/2 signaling using U0126 enhanced HIF-1α stabilization, implicating ERK1/2 dephosphorylation as a contributing mechanism in NO-mediated HIF-1α activation. Activation of HIF-1α by the hypoxia mimic cobalt chloride, or cell transfection with a degradation-resistant HIF-1α mutant construct inhibited epithelial wound repair, implicating HIF-1α in NO-mediated inhibition of cell migration. Conversely, NO-mediated inhibition of epithelial wound closure was largely prevented after small interfering RNA suppression of HIF-1α. Finally, NO-mediated inhibition of cell migration was associated with HIF-1α-dependent induction of PAI-1 and activation of p53, both negative regulators of epithelial cell migration. Collectively, our results demonstrate that inflammatory levels of NO inhibit epithelial cell migration, because of suppression of ERK1/2 signaling, and activation of HIF-1α and p53, with potential consequences for epithelial repair and remodeling during airway inflammation.

American Heart Association's Life's Simple 7 and Risk of Venous Thromboembolism: The Reasons for Geographic and Racial Differences in Stroke (REGARDS) Study

Background The American Heart Associations Lifes Simple 7 metric is being used to track the populations cardiovascular health (CVH) toward a 2020 goal for improvement. The metric includes body mass index (BMI), blood pressure, cholesterol, glucose, physical activity (PA), cigarette smoking, and diet. We hypothesized a lower risk of venous thromboembolism (VTE) with favorable Lifes Simple 7 scores. Methods and Results REGARDS recruited 30 239 black and white participants ≥45 years of age across the United States in 2003–2007. A 14‐point summary score for Lifes Simple 7 classified participants into inadequate (0 to 4 points), average (5 to 9 points), and optimal (10 to 14 points) categories. Hazard ratios (HRs) of incident VTE were calculated for these categories, adjusting for age, sex, race, income, education, and region of residence. For comparison, HRs of VTE were calculated using the Framingham 10‐year coronary risk score. There were 263 incident VTE cases over 5.0 years of follow‐up; incidence rates per 1000 person‐years declined from 2.9 (95% confidence interval [CI], 2.3 to 3.7) among those in the inadequate category to 1.8 (95% CI, 1.4 to 2.4) in the optimal category. Compared to the inadequate category, participants in the average category had a 38% lower VTE risk (95% CI, 11 to 57) and participants in the optimal category had a 44% lower risk (95% CI, 18 to 62). The individual score components related to lower VTE risk were ideal PA and BMI. There was no association of Framingham Score with VTE. Conclusions Lifes Simple 7, a CVH metric, was associated with reduced VTE risk. Findings suggest that efforts to improve the populations CVH may reduce VTE incidence.

Decreased Naive and Increased Memory CD4+ T Cells Are Associated with Subclinical Atherosclerosis: The Multi-Ethnic Study of Atherosclerosis

Background Adaptive immunity has been implicated in atherosclerosis in animal models and small clinical studies. Whether chronic immune activation is associated with atherosclerosis in otherwise healthy individuals remains underexplored. We hypothesized that activation of adaptive immune responses, as reflected by higher proportions of circulating CD4+ memory cells and lower proportions of naive cells, would be associated with subclinical atherosclerosis. Methods and Findings We examined cross-sectional relationships of circulating CD4+ naive and memory T cells with biomarkers of inflammation, serologies, and subclinical atherosclerosis in 912 participants of the Multi-Ethnic Study of Atherosclerosis (MESA). Circulating CD4+ naive cells were higher in women than men and decreased with age (all p-values <0.0001). European-Americans had higher levels of naive cells and lower levels of memory cells compared with African-Americans and Hispanic-Americans (all p-values ≤0.0005). Lower naive/higher memory cells were associated with interleukin-6 levels. In multivariate models, cytomegalovirus (CMV) and H. Pylori titers were strongly associated with higher memory and lower naive cells (all p-values <0.05). Higher memory cells were associated with coronary artery calcification (CAC) level in the overall population [β-Coefficient (95% confidence interval (CI))  = 0.20 (0.03, 0.37)]. Memory and naive (inversely) cells were associated with common carotid artery intimal media thickness (CC IMT) in European-Americans [memory: β =  0.02 (0.006, 0.04); naive: β = −0.02 (−0.004, −0.03)]. Conclusions These results demonstrate that the degree of chronic adaptive immune activation is associated with both CAC and CC IMT in otherwise healthy individuals, consistent with the known role of CD4+ T cells, and with innate immunity (inflammation), in atherosclerosis. These data are also consistent with the hypothesis that immunosenescence accelerates chronic diseases by putting a greater burden on the innate immune system, and suggest the importance of prospective studies and research into strategies to modulate adaptive immune activation in chronic disease states such as atherosclerosis.

Inflammation markers and incident venous thromboembolism: the REasons for Geographic And Racial Differences in Stroke (REGARDS) cohort

Inflammation biomarkers are associated with the venous thromboembolism (VTE) risk factors obesity and age; however, the relationships of inflammation with VTE risk remain controversial.

T-Helper Type 1 Bias in Healthy People Is Associated With Cytomegalovirus Serology and Atherosclerosis: The Multi-Ethnic Study of Atherosclerosis

Background Although T‐helper type 1 (Th1) cells are considered important in atherosclerosis, the relationships between Th1 and Th2 cells and atherosclerosis have not been examined in population‐based studies. Methods and Results We measured Th cells as a percentage of lymphocytes by flow cytometry using CD4 staining (%CD4) in 917 participants of the Multi‐Ethnic Study of Atherosclerosis. We also measured interferon gamma–positive and interleukin‐4‐positive CD4+ cells, representing Th1 and Th2 subpopulations (%Th1 and %Th2), respectively. We found that %CD4 was 1.5% lower per 10 years of age (P<0.0001). Whites had higher %CD4 and lower %Th1 and %Th2 values than other race/ethnic groups. Body mass index (BMI) and blood pressure were associated with %CD4, but no traditional cardiovascular disease (CVD) risk factors were associated with %Th1 or %Th2. In multivariable models, the major independent variable associated with %Th1 was cytomegalovirus (CMV) antibody titer, with minor contributions from age, sex, seasonality, and interleukin‐6. In models with coronary artery calcification level as the outcome, significant independent variables included age, sex, smoking status, and %Th1 (β=0.25; P≤0.01). Both %Th1 and %Th2 were associated with common carotid intimal media thickness (β=0.02 and −0.02, respectively; both P<0.05), as were age, sex, race/ethnicity, blood pressure, and BMI. Conclusions Th1 bias is associated with subclinical atherosclerosis in a multiethnic population. The main Th1 correlate was CMV infectious burden. These findings are consistent with a role of Th1 cells in atherosclerosis and suggest the importance of prospective studies of T‐helper cell biasing in CVD.

T Helper Cell Polarization in Healthy People: Implications for Cardiovascular Disease

Atherosclerosis is a chronic inflammatory disease characterized by T lymphocyte infiltration into the atherosclerotic plaque. Assessments of T cell subtypes have demonstrated a predominance of CD4+ T helper (Th) cells, implicated Th1 and Th17 immunity in both human and mouse atherogenesis, and provided some evidence suggesting protective roles of Th2 and T regulatory cells. Observations that certain inbred mouse strains have an inherent T helper bias suggest a genetic predisposition toward developing a particular T helper phenotype. This review summarizes our current understanding of mechanisms of antigen processing for major histocompatibility complex molecules, describes the different T helper cell subsets and their roles in atherosclerosis, and discusses mechanisms of genetic predisposition toward Th1/Th2 bias in mice. We also present data from our laboratory demonstrating inherent Th1/Th2 phenotypes in apparently healthy human volunteers that are stable over time and discuss the potential implications for cardiovascular disease.

Nitric Oxide and Airway Epithelial Barrier Function: Regulation of Tight Junction Proteins and Epithelial Permeability

Acute airway inflammation is associated with enhanced production of nitric oxide (NO(.)) and altered airway epithelial barrier function, suggesting a role of NO(.) or its metabolites in epithelial permeability. While high concentrations of S-nitrosothiols disrupted transepithelial resistance (TER) and increased permeability in 16HBE14o- cells, no significant barrier disruption was observed by NONOates, in spite of altered distribution and expression of some TJ proteins. Barrier disruption of mouse tracheal epithelial (MTE) cell monolayers in response to inflammatory cytokines was independent of NOS2, based on similar effects in MTE cells from NOS2-/- mice and a lack of effect of the NOS2-inhibitor 1400W. Cell pre-incubation with LPS protected MTE cells from TER loss and increased permeability by H2O2, which was independent of NOS2. However, NOS2 was found to contribute to epithelial wound repair and TER recovery after mechanical injury. Overall, our results demonstrate that epithelial NOS2 is not responsible for epithelial barrier dysfunction during inflammation, but may contribute to restoration of epithelial integrity.