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Exaggerated exercise blood pressure (BP) is linked to cardiovascular disease (CVD). Although evening chronotypes have greater CVD risk than morning (Morn) types, it is unknown if exercise BP differs in intermediate (Int) types. Adults with obesity were classified as either Morn [ n = 23 (18 females), Morning-Eveningness Questionnaire (MEQ) = 63.96 ± 1.0, 54.74 ± 1.4 yr, 33.7 ± 0.6 kg/m 2 ] or Int [ n = 23 (19 females), MEQ = 51.36 ± 1.1, 55.96 ± 1.8 yr, 37.2 ± 1.2 kg/m 2 ] chronotype per MEQ. A graded, incremental treadmill test to maximal aerobic capacity (V̇o 2max ) was conducted. Systolic (SBP) and diastolic (DBP) blood pressure and mean arterial pressure (MAP), rate pressure product (RPP), heart rate (HR), and rate of perceived intensity (RPE) were determined at baseline, 4 min, 6 min, and maximal stages. HR recovery (HRR; maximum postexercise) was determined at 1 and 2 min postexercise. Preexercise fasted aortic waveforms (applanation tonometry), plasma leptin, nitrate/nitrite (nitric oxide bioavailability), and body composition (dual X-ray, DXA) were also collected. Int had lower V̇o 2max and plasma nitrate (both P ≤ 0.02) than Morn. No difference in preexercise BP, aortic waveforms, or body composition were noted between groups, although higher plasma leptin was seen in Int compared with Morn ( P = 0.04). Although Int had higher brachial DBP and MAP across exercise stages (both P ≤ 0.05) and higher HR, RPE, and RPP at 6 min of exercise (all P ≤ 0.05), covarying for V̇o 2max nullified the BP, but not HR or RPE, difference. HRR was greater in Morn independent of V̇o 2max ( P = 0.046). Fasted leptin correlated with HR at exercise stage 4 ( r = 0.421, P = 0.041) and 6 min ( r = 0.593, P = 0.002). This observational study suggests that Int has exaggerated BP and HR responses to exercise compared with Morn, although fitness abolished BP differences. NEW & NOTEWORTHY This study compares blood pressure and heart rate responses with graded, incremental exercise between morning and intermediate chronotype adults with obesity. Herein, blood pressure responses to exercise were elevated in intermediate compared with morning chronotype, although V̇o 2max abolished this observation. However, heart rate responses to exercise were higher in intermediate vs. morning chronotypes independent of fitness. Collectively, this exercise hemodynamic response among intermediate chronotype may be related to reduced aerobic fitness, altered nitric oxide metabolism, and/or elevated aortic waveforms.

E-cigarette consumption is under scrutiny by regulatory authorities due to concerns about product toxicity, lack of manufacturing standards, and increasing reports of e-cigarette- or vaping-associated acute lung injury. In vitro studies have demonstrated cytotoxicity, mitochondrial dysfunction, and oxidative stress induced by unflavored e-cigarette aerosols and flavoring additives. However, e-cigarette effects on the complex lung parenchyma remain unclear. Herein, the impact of e-cigarette condensates with or without menthol flavoring on functional, structural, and cellular responses was investigated using mouse precision cut lung slices (PCLS). PCLS were exposed to e-cigarette condensates prepared from aerosolized vehicle, nicotine, nicotine + menthol, and menthol e-fluids at doses from 50 to 500 mM. Doses were normalized to the glycerin content of vehicle. Video-microscopy of PCLS revealed impaired contractile responsiveness of airways to methacholine and dampened ciliary beating following exposure to menthol-containing condensates at concentrations greater than 300 mM. Following 500 mM menthol-containing condensate exposure, epithelial exfoliation in intrabronchial airways was identified in histological sections of PCLS. Measurement of lactate dehydrogenase release, mitochondrial water-soluble-tetrazolium salt-1 conversion, and glutathione content supported earlier findings of nicotine or nicotine + menthol e-cigarette-induced dose-dependent cytotoxicity and oxidative stress responses. Evaluation of PCLS metabolic activity revealed dose-related impairment of mitochondrial oxidative phosphorylation and glycolysis after exposure to menthol-containing condensates. Taken together, these data demonstrate prominent menthol-induced pulmonary toxicity and impairment of essential physiological functions in the lung, which warrants concerns about e-cigarette consumer safety and emphasizes the need for further investigations of molecular mechanisms of toxicity and menthol effects in an experimental model of disease.

Surfactant protein-D (SP-D) is a regulator of pulmonary innate immunity whose oligomeric state can be altered through S -nitrosylation to regulate its signaling function in macrophages. Here, we examined how nitrosylation of SP-D alters the phenotypic response of macrophages to stimuli both in vivo and in vitro. Bronchoalveolar lavage (BAL) from C57BL6/J and SP-D-overexpressing (SP-D OE) mice was incubated with RAW264.7 cells ± LPS. LPS induces the expression of the inflammatory genes Il1b and Nos2 , which is reduced 10-fold by SP-D OE-BAL. S -nitrosylation of the SP-D OE-BAL (SNO-SP-D OE-BAL) abrogated this inhibition. SNO-SP-D OE-BAL alone induced Il1b and Nos2 expression. PCR array analysis of macrophages incubated with SP-D OE-BAL (±LPS) shows increased expression of repair genes, Ccl20 , Cxcl1 , and Vcam1 , that was accentuated by LPS. LPS increases inflammatory gene expression, Il1a , Nos2 , Tnf , and Ptgs2 , which was accentuated by SNO-SP-D OE-BAL but inhibited by SP-D OE-BAL. The transcription factor NF-κB was identified as a target for SNO-SP-D by IPA, which was confirmed by Trans-AM ELISA in vitro. In vivo, SP-D overexpression increases the burden of infection in a Pneumocystis model while increasing cellular recruitment. Expression of iNOS and the production of NO metabolites were significantly reduced in SP-D OE mice relative to C57BL6/J. Inflammatory gene expression was increased in infected C57BL6/J mice but decreased in SP-D OE. SP-D oligomeric structure was disrupted in C57BL6/J infected mice but unaltered within SP-D OE. Thus SP-D modulates macrophage phenotype and the balance of multimeric to trimeric SP-D is critical to this regulation.

Nitrogen mustard (NM) is a vesicant that causes lung injury and fibrosis, accompanied by a persistent macrophage inflammatory response. In these studies we analyzed the spleen as a source of these cells. Splenectomized (SPX) and sham control rats were treated intratracheally with NM (0.125 mg/kg) or PBS control. Macrophage responses were analyzed 1-7 days later. Splenectomy resulted in an increase in lung macrophages expressing CCR2, but a decrease in ATR-1α(+) cells, receptors important in bone marrow and spleen monocyte trafficking, respectively. Splenectomy was also associated with an increase in proinflammatory M1 (iNOS(+), CD11b(+)CD43(+)) macrophages in lungs of NM-treated rats, as well as greater upregulation of iNOS and COX-2 mRNA expression. Conversely, a decrease in CD11b(+)CD43(-) M2 macrophages was observed in SPX rats, with no changes in CD68(+), CD163(+), CD206(+), or YM-1(+) M2 macrophages, suggesting distinct origins of M2 subpopulations responding to NM. Macrophage expression of M2 genes including IL-10, ApoE, PTX-2, PTX-3, 5-HT2α, and 5-HT7 was also reduced in NM-treated SPX rats compared with shams, indicating impaired M2 activity. Changes in lung macrophages responding to NM as a consequence of splenectomy were correlated with exacerbated tissue injury and more rapid fibrogenesis. These data demonstrate that the spleen is a source of a subset of M2 macrophages with anti-inflammatory activity; moreover, in their absence, proinflammatory/cytotoxic M1 macrophages predominate in the lung, resulting in heightened pathology. Understanding the origin of macrophages and characterizing their phenotype after vesicant exposure may lead to more targeted therapeutics aimed at reducing toxicity and disease pathogenesis., (Copyright © 2015 the American Physiological Society.)

Pulmonary lymphangioleiomyomatosis (LAM), a rare progressive lung disease associated with mutations of the tuberous sclerosis complex 2 (Tsc2) tumor suppressor gene, manifests by neoplastic growth of LAM cells, induction of cystic lung destruction, and respiratory failure. LAM severity correlates with upregulation in serum of the prolymphangiogenic vascular endothelial growth factor D (VEGF-D) that distinguishes LAM from other cystic diseases. The goals of our study was to determine whether Tsc2 deficiency upregulates VEGF-D, and whether axitinib, the Food and Drug Administration-approved small-molecule inhibitor of VEGF receptor (VEGFR) signaling, will reduce Tsc2-null lung lesion growth in a mouse model of LAM. Our data demonstrate upregulation of VEGF-D in the serum and lung lining in mice with Tsc2-null lesions. Progressive growth of Tsc2-null lesions induces recruitment and activation of inflammatory cells and increased nitric oxide production. Recruited cells isolated from the lung lining of mice with Tsc2-null lesions demonstrate upregulated expression of provasculogenic Vegfa, prolymphangiogenic Figf, and proinflammatory Nos2, Il6, and Ccl2 genes. Importantly, axitinib is an effective inhibitor of Tsc2-null lesion growth and inflammatory cell recruitment, which correlates with reduced VEGF-D levels in serum and lung lining. Our data demonstrate that pharmacological inhibition of VEGFR signaling with axitinib inhibits Tsc2-null lesion growth, attenuates recruitment and activation of inflammatory cells, and reduces VEGF-D levels systemically and in the lung lining. Our study suggests a potential therapeutic benefit of inhibition of VEGFR signaling for treatment of LAM., (Copyright © 2015 the American Physiological Society.)

Surfactant protein D (SP-D) modulates the lung's immune system. Its absence leads to NOS2-independent alveolar lipoproteinosis and NOS2-dependent chronic inflammation, which is critical for early emphysematous remodeling. With aging, SP-D knockout mice develop an additional interstitial fibrotic component. We hypothesize that this age-related interstitial septal wall remodeling is mediated by NOS2. Using invasive pulmonary function testing such as the forced oscillation technique and quasistatic pressure-volume perturbation and design-based stereology, we compared 29-wk-old SP-D knockout (Sftpd(-/-)) mice, SP-D/NOS2 double-knockout (DiNOS) mice, and wild-type mice (WT). Structural changes, including alveolar epithelial surface area, distribution of septal wall thickness, and volumes of septal wall components (alveolar epithelium, interstitial tissue, and endothelium) were quantified. Twenty-nine-week-old Sftpd(-/-) mice had preserved lung mechanics at the organ level, whereas elastance was increased in DiNOS. Airspace enlargement and loss of surface area of alveolar epithelium coexist with increased septal wall thickness in Sftpd(-/-) mice. These changes were reduced in DiNOS, and compared with Sftpd(-/-) mice a decrease in volumes of interstitial tissue and alveolar epithelium was found. To understand the effects of lung pathology on measured lung mechanics, structural data were used to inform a computational model, simulating lung mechanics as a function of airspace derecruitment, septal wall destruction (loss of surface area), and septal wall thickening. In conclusion, NOS2 mediates remodeling of septal walls, resulting in deposition of interstitial tissue in Sftpd(-/-). Forward modeling linking structure and lung mechanics describes the complex mechanical properties by parenchymatous destruction (emphysema), interstitial remodeling (septal wall thickening), and altered recruitability of acinar airspaces., (Copyright © 2015 the American Physiological Society.)

In these studies we determined whether progressive pulmonary inflammation associated with aging in surfactant protein D (Sftpd)-/- mice leads to an exacerbated response to ozone. In Sftpd-/- mice, but not wild-type (WT) mice, age-related increases in numbers of enlarged vacuolated macrophages were observed in the lung, along with alveolar wall rupture, type 2 cell hyperplasia, and increased bronchoalveolar lavage protein and cell content. Numbers of heme oxygenase+ macrophages also increased with age in Sftpd-/- mice, together with classically (iNOS+) and alternatively (mannose receptor+, YM-1+, or galectin-3+) activated macrophages. In both WT and Sftpd-/- mice, increasing age from 8 to 27 wk was associated with reduced lung stiffness, as reflected by decreases in resistance and elastance spectra; however, this response was reversed in 80-wk-old Sftpd-/- mice. Ozone exposure (0.8 ppm, 3 h) caused increases in lung pathology, alveolar epithelial barrier dysfunction, and numbers of iNOS+ macrophages in 8- and 27-wk-old Sftpd-/-, but not WT mice at 72 h postexposure. Conversely, increases in alternatively activated macrophages were observed in 8-wk-old WT mice following ozone exposure, but not in Sftpd-/- mice. Ozone also caused alterations in both airway and tissue mechanics in Sftpd-/- mice at 8 and 27 wk, but not at 80 wk. These data demonstrate that mild to moderate pulmonary inflammation results in increased sensitivity to ozone; however, in senescent mice, these responses are overwhelmed by the larger effects of age-related increases in baseline inflammation and lung injury.

Aquaporin 11 (AQP11) is a newly described member of the protein family of transport channels. AQP11 associates with the endoplasmic reticulum (ER) and is highly expressed in proximal tubular epithelial cells in the kidney. Previously, we identified and characterized a recessive mutation of the highly conserved Cys227 to Ser227 in mouse AQP11 that caused proximal tubule (PT) injury and kidney failure in mutant mice. The current study revealed induction of ER stress, unfolded protein response, and apoptosis as molecular mechanisms of this PT injury. Cys227Ser mutation interfered with maintenance of AQP11 oligomeric structure. AQP11 is abundantly expressed in the S1 PT segment, a site of major renal glucose flux, and Aqp11 mutant mice developed PT-specific mitochondrial injury. Glucose increased AQP11 protein expression in wild-type kidney and upregulation of AQP11 expression by glucose in vitro was prevented by phlorizin, an inhibitor of sodium-dependent glucose transport across PT. Total AQP11 levels in heterozygotes were higher than in wild-type mice but were not further increased in response to glucose. In Aqp11 insufficient PT cells, glucose potentiated increases in reactive oxygen species (ROS) production. ROS production was also elevated in Aqp11 mutation carriers. Phenotypically normal mice heterozygous for the Aqp11 mutation repeatedly treated with glucose showed increased blood urea nitrogen levels that were prevented by the antioxidant sulforaphane or by phlorizin. Our results indicate an important role for AQP11 to prevent glucose-induced oxidative stress in proximal tubules.

Surfactant protein D (SP-D), a member of the collectin superfamily, modulates pulmonary inflammatory responses and innate immunity. Disruption of the SP-D gene in mice induces peribronchiolar inflammation, accumulation of large, foamy macrophages, increased bronchoalveolar lavage (BAL) phospholipid, and pulmonary emphysema. We hypothesized that absence of SP-D aggravates hyperoxia-induced injury. To test this, SP-D-deficient (SP-D-/-) and wild-type (SP-D+/+) mice were exposed to 80% or 21% oxygen. Paradoxically, during 14 days of hyperoxia, SP-D-/- mice had 100% survival vs. 30% in SP-D+/+. The survival advantage in SP-D-/- mice was accompanied by lower histopathological injury scores at days 5 and 14, although total BAL cells (8.2 +/- 1.4 x 10(5) in SP-D-/- vs. 4.04 +/- 0.25 x 10(5) in SP-D+/+ mice) and neutrophils (1.2 +/- 0.4 x 10(5) vs. 0.03 +/- 0.02 x 10(5) in SP-D-/- and SP-D+/+, respectively) were increased. In addition, BAL protein and lung-to-body weight ratios were similarly elevated in both groups after 3, 5, and 14 days of continuous exposure. Biochemically, in contrast to SP-D+/+, SP-D-/- mice had higher levels of surfactant phospholipid and SP-B at baseline and 5 days after hyperoxia accompanied by a preservation of surfactant biophysical activity. From a multiplex assay of nine cytokines, we found elevated levels of IL-13 in BAL fluid of normoxic SP-D-/- mice compared with SP-D+/+. We conclude that the resistance of SP-D-deficient mice to hyperoxia reflects homeostatic changes in the SP-D-/- phenotype involving both phospholipid and SP-B-mediated induced resistance of surfactant to inactivation as well as changes in the immunomodulatory BAL cytokine profile.

Nitric oxide (NO), despite an apparently simple diatomic structure, has a wide variety of functions in both physiology and pathology and within every major organ system. It has become an increasingly important scientific challenge to decipher how this wide range of activity is achieved. To this end a number of investigators have begun to explore how NO-mediated posttranslational modifications of proteins may represent mechanisms of cellular signaling. These modifications include: 1). binding to metal centers; 2). nitrosylation of thiol and amine groups; 3). nitration of tyrosine, tryptophan, amine, carboxylic acid, and phenylalanine groups; and 4). oxidation of thiols (both cysteine and methionine residues) and tyrosine. However, two particular modifications have recently received much attention, nitrosylation of thiols to produce S-nitrosothiol and nitration of tyrosine residues to produce nitrotyrosine. It is the purpose of this review to examine the possibility that these modifications may play a role in NO-mediated signaling.

To better understand the mechanism(s) underlying nitric oxide (. NO)-mediated toxicity, in the presence and absence of concomitant oxidant exposure, postmitotic terminally differentiated NT2N cells, which are incapable of producing. NO, were exposed to PAPA-NONOate (PAPA/NO) and 3-morpholinosydnonimine (SIN-1). Exposure to SIN-1, which generated peroxynitrite in the range of 25-750 nM/min, produced a concentration- and time-dependent delayed cell death. In contrast, a critical threshold concentration (>440 nM/min) was required for. NO to produce significant cell injury. Examination of cells by electron microscopy shows a largely necrotic injury after peroxynitrite exposure but mainly apoptotic-like morphology after. NO exposure. Cellular levels of reduced thiols correlated with cell death, and pretreatment with N-acetylcysteine (NAC) fully protected from cell death in either PAPA/NO or SIN-1 exposure. NAC given within the first 3 h posttreatment further delayed cell death and increased the intracellular thiol level in SIN-1 but not. NO-exposed cells. Cell injury from. NO was independent of cGMP, caspases, and superoxide or peroxynitrite formation. Overall, exposure of non-. NO-producing cells to. NO or peroxynitrite results in delayed cell death, which, although occurring by different mechanisms, appears to be mediated by the loss of intracellular redox balance.

Extracellular and intracellular reactive oxygen species attack different targets and may, therefore, result in different forms of oxidative stress. To specifically study an oxidative stress induced by a regulated intracellular flux of a defined reactive oxygen species in endothelium, we used immunotargeting of the H(2)O(2)-generating enzyme glucose oxidase (GOX) conjugated with an antibody to platelet-endothelial cell adhesion molecule (PECAM)-1, an endothelial surface antigen. Anti-PECAM-(125)I-GOX conjugates specifically bind to both endothelial and PECAM-transfected cells. Approximately 70% of cell-bound anti-PECAM-(125)I-GOX was internalized. The cell-bound conjugate was enzymatically active and generated H(2)O(2) from glucose. Use of the fluorescent dye dihydrorhodamine 123 revealed that 70% of H(2)O(2) was generated intracellularly, whereas 30% of H(2)O(2) was detected in the cell medium. Catalase added to the cells eliminated H(2)O(2) in the medium but had little effect on the intracellular generation of H(2)O(2) by anti-PECAM-GOX. Both H(2)O(2) added exogenously to the cell medium (extracellular H(2)O(2)) and that generated by anti-PECAM-GOX caused oxidative stress manifested by time- and dose-dependent irreversible plasma membrane damage. Inactivation of cellular catalase by aminotriazole treatment augmented damage caused by either extracellular H(2)O(2) or anti-PECAM-GOX. Catalase added to the medium protected either normal or aminotriazole-treated cells against extracellular H(2)O(2), yet failed to protect cells against injury induced by anti-PECAM-GOX. Therefore, treatment of PECAM-positive cells with anti-PECAM-GOX leads to conjugate internalization, predominantly intracellular H(2)O(2) generation and intracellular oxidative stress. These results indicate that anti-PECAM-GOX 1) provides cell-specific intracellular delivery of an active enzyme and 2) causes intracellular oxidative stress in PECAM-positive cells.

Nitric oxide (.NO) can be produced within the lung, and recently inhaled nitric oxide has been used as a therapeutic agent. Peroxynitrite1 (ONOO-), the product of the nearly diffusion-limited reaction between .NO and superoxide, may represent the proximal reactive species mediating .NO injury to pulmonary cells. To investigate the physiological and pathological reactivities of .NO and ONOO- at the molecular and cellular levels, bovine pulmonary artery endothelial cells (BPAEC) and rat type II epithelial cells were exposed to .NO (0.01-2.5 microM/min for 2 h) generated by spermine-NONOate and papa-NONOate and to the same fluxes of ONOO- generated by 1,3-morpholinosydnonimine (SIN-1). Exposure to SIN-1 resulted in cellular injury and death in both cell types. Epithelial cells displayed a concentration-dependent loss of cellular viability within 8 h of exposure. In contrast, BPAEC loss of cellular viability was evident after 18 h postexposure. Events preceding cell death in BPAEC include depolarization of the mitochondrial membrane, evident as early as 6 h postexposure, loss of cellular redox activity at 16 h, and DNA fragmentation detected by in situ staining at 18 h after exposure. Exposure of BPAEC to .NO did not affect the cellular viability, but type II cells were injured in a manner similar to ONOO- exposure. .NO-mediated cellular injury within type II cells was reduced by preincubation with N-acetylcysteine. The data imply that the pathological and physiological effects of .NO may be regulated by its reactions with superoxide and reduced thiols.