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1.  c-kit-Positive Cardiac Stem Cells Nested in Hypoxic Niches are Activated by Stem Cell Factor Reversing the Aging Myopathy 
Circulation research  2013;114(1):41-55.
Rationale
Hypoxia favors stem cell quiescence, while normoxia is required for their activation; but whether cardiac stem cell (CSC) function is regulated by the hypoxic/normoxic state of the cell is currently unknown.
Objective
A balance between hypoxic and normoxic CSCs may be present in the young heart, although this homeostatic control may be disrupted with aging. Defects in tissue oxygenation occur in the old myocardium, and this phenomenon may expand the pool of hypoxic CSCs, which are no longer involved in myocyte renewal.
Methods and Results
Here we show that the senescent heart is characterized by an increased number of quiescent CSCs with intact telomeres that cannot reenter the cell cycle and form a differentiated progeny. Conversely, myocyte replacement is controlled only by frequently dividing CSCs with shortened telomeres; these CSCs generate a myocyte population that is chronologically young but phenotypically old. Telomere dysfunction dictates their actual age and mechanical behavior. However, the residual subset of quiescent young CSCs can be stimulated in situ by stem cell factor reversing the aging myopathy.
Conclusions
Our findings support the notion that strategies targeting CSC activation and growth interfere with the manifestations of myocardial aging in an animal model. Although caution has to be exercised in the translation of animal studies to human beings, our data strongly suggests that a pool of functionally-competent CSCs persists in the senescent heart and this stem cell compartment can promote myocyte regeneration effectively, correcting partly the aging myopathy.
doi:10.1161/CIRCRESAHA.114.302500
PMCID: PMC3959163  PMID: 24170267
Cardiac stem cells; hypoxia; myocardial aging; stem cell factor
2.  Characterization of macroautophagic flux in vivo using a leupeptin-based assay 
Autophagy  2011;7(6):629-642.
Macroautophagy is a highly conserved catabolic process that is crucial for organ homeostasis in mammals. However, methods to directly measure macroautophagic activity (or flux) in vivo are limited. In this study we developed a quantitative macroautophagic flux assay based on measuring LC3b protein turnover in vivo after administering the protease inhibitor leupeptin. Using this assay we then characterized basal macroautophagic flux in different mouse organs. We found that the rate of LC3b accumulation after leupeptin treatment was greatest in the liver and lowest in spleen. Interestingly we found that LC3a, an ATG8/LC3b homologue and the LC3b-interacting protein p62 were degraded with similar kinetics to LC3b. However, the LC3b-related proteins GABARAP and GATE-16 were not rapidly turned over in mouse liver, implying that different LC3b homologues may contribute to macroautophagy via distinct mechanisms. Nutrient starvation augmented macroautophagic flux as measured by our assay, while refeeding the animals after a period of starvation significantly suppressed flux. We also confirmed that beclin 1 heterozygous mice had reduced basal macroautophagic flux compared to wild-type littermates. These results illustrate the usefulness of our leupeptin-based assay for studying the dynamics of macroautophagy in mice.
doi:10.4161/auto.7.6.15100
PMCID: PMC3127049  PMID: 21460622
macroautophagy; autophagy; flux; mice; in vivo; LC3; GABARAP; GATE-16; leupeptin; cycloheximide
3.  Autophagic Protein LC3B Confers Resistance against Hypoxia-induced Pulmonary Hypertension 
Rationale: Pulmonary hypertension (PH) is a progressive disease with unclear etiology. The significance of autophagy in PH remains unknown.
Objectives: To determine the mechanisms by which autophagic proteins regulate tissue responses during PH.
Methods: Lungs from patients with PH, lungs from mice exposed to chronic hypoxia, and human pulmonary vascular cells were examined for autophagy using electron microscopy and Western analysis. Mice deficient in microtubule-associated protein-1 light chain-3B (LC3B−/−), or early growth response-1 (Egr-1−/−), were evaluated for vascular morphology and hemodynamics.
Measurements and Main Results: Human PH lungs displayed elevated lipid-conjugated LC3B, and autophagosomes relative to normal lungs. These autophagic markers increased in hypoxic mice, and in human pulmonary vascular cells exposed to hypoxia. Egr-1, which regulates LC3B expression, was elevated in PH, and increased by hypoxia in vivo and in vitro. LC3B−/− or Egr-1−/−, but not Beclin 1+/−, mice displayed exaggerated PH during hypoxia. In vitro, LC3B knockdown increased reactive oxygen species production, hypoxia-inducible factor-1α stabilization, and hypoxic cell proliferation. LC3B and Egr-1 localized to caveolae, associated with caveolin-1, and trafficked to the cytosol during hypoxia.
Conclusions: The results demonstrate elevated LC3B in the lungs of humans with PH, and of mice with hypoxic PH. The increased susceptibility of LC3B−/− and Egr-1−/− mice to hypoxia-induced PH and increased hypoxic proliferation of LC3B knockdown cells suggest adaptive functions of these proteins during hypoxic vascular remodeling. The results suggest that autophagic protein LC3B exerts a protective function during the pathogenesis of PH, through the regulation of hypoxic cell proliferation.
doi:10.1164/rccm.201005-0746OC
PMCID: PMC3081281  PMID: 20889906
autophagy; hypoxia; hypertension, pulmonary
4.  Carbon monoxide prevents ventilator induced lung injury via caveolin-1 
Critical care medicine  2009;37(5):1708-1715.
Objectives
Carbon monoxide (CO) can confer anti-inflammatory protection in rodent models of ventilator-induced lung injury (VILI). Caveolin-1 exerts a critical role in cellular responses to mechanical stress, and has been shown to mediate cytoprotective effects of CO in vitro. We sought to determine the role of caveolin-1 in lung susceptibility to VILI in mice. Furthermore, we assessed the role of caveolin-1 in the tissue protective effects of CO in the VILI model.
Design
Prospective experimental study
Setting
University laboratory
Subjects
Wild type (wt) and caveolin-1 deficient (cav-−/−) mice
Interventions
Mice were subjected to tracheostomy and arterial cannulation. Wt and cav-1−/− mice were ventilated with a tidal volume of 12 ml/kg body weight and a frequency of 80/min for 5 min as control, or for 8h with air in the absence or presence of CO (250 parts per million). Bronchoalveolar lavage (BAL) and histology were used to determine lung injury. Lung sections or homogenates were analyzed for caveolin-1 expression by immunohistochemical staining or Western Blotting, respectively.
Measurements and Main Results
Ventilation led to an increase in BAL protein concentration, cell count, neutrophil recruitment, and edema formation that was prevented in the presence of CO. While ventilation alone slightly induced caveolin-1 expression in epithelial cells, the application of CO during the ventilation significantly increased the expression of caveolin-1. In comparison to wt mice, mechanical ventilation of cav-1−/− mice led to a significantly higher degree of lung injury as compared to wt mice. In contrast to its effectiveness in wt mice, CO-administration failed to reduce lung injury markers in cav-1−/− mice.
Conclusions
Caveolin-1 null mice are more susceptible to VILI. Carbon monoxide executes lung protective effects during mechanical ventilation that are dependent in part, on caveolin-1 expression.
doi:10.1097/CCM.0b013e31819efa31
PMCID: PMC3086639  PMID: 19325477
ventilator induced lung injury; mechanical ventilation; carbon monoxide; caveolin-1; mechanotransduction; acute lung injury
5.  Deletion of Caveolin-1 Protects against Oxidative Lung Injury via Up-Regulation of Heme Oxygenase-1 
Acute lung injury (ALI) is a major cause of morbidity and mortality in critically ill patients. Hyperoxia causes lung injury in animals and humans, and is an established model of ALI. Caveolin-1, a major constituent of caveolae, regulates numerous biological processes, including cell death and proliferation. Here we demonstrate that caveolin-1–null mice (cav-1−/−) were resistant to hyperoxia-induced death and lung injury. Cav-1−/− mice sustained reduced lung injury after hyperoxia as determined by protein levels in bronchoalveolar lavage fluid and histologic analysis. Furthermore, cav-1−/− fibroblasts and endothelial cells and cav-1 knockdown epithelial cells resisted hyperoxia-induced cell death in vitro. Basal and inducible expression of the stress protein heme oxygenase-1 (HO-1) were markedly elevated in lung tissue or fibroblasts from cav-1−/− mice. Hyperoxia induced the physical interaction between cav-1 and HO-1 in fibroblasts assessed by co-immunoprecipitation studies, which resulted in attenuation of HO activity. Inhibition of HO activity with tin protoporphyrin-IX abolished the survival benefits of cav-1−/− cells and cav-1−/− mice exposed to hyperoxia. The cav-1−/− mice displayed elevated phospho-p38 mitogen-activated protein kinase (MAPK) and p38β expression in lung tissue/cells under basal conditions and during hyperoxia. Treatment with SB202190, an inhibitor of p38 MAPK, decreased hyperoxia-inducible HO-1 expression in wild-type and cav-1−/− fibroblasts. Taken together, our data demonstrated that cav-1 deletion protects against hyperoxia-induced lung injury, involving in part the modulation of the HO-1–cav-1 interaction, and the enhanced induction of HO-1 through a p38 MAPK–mediated pathway. These studies identify caveolin-1 as a novel component involved in hyperoxia-induced lung injury.
doi:10.1165/rcmb.2007-0323OC
PMCID: PMC2542454  PMID: 18323531
acute lung injury; acute respiratory distress syndrome; caveolin-1; heme oxygenase-1
6.  Carbon Monoxide Protects against Ventilator-induced Lung Injury via PPAR-γ and Inhibition of Egr-1 
Rationale: Ventilator-induced lung injury (VILI) leads to an unacceptably high mortality. In this regard, the antiinflammatory properties of inhaled carbon monoxide (CO) may provide a therapeutic option.
Objectives: This study explores the mechanisms of CO-dependent protection in a mouse model of VILI.
Methods: Mice were ventilated (12 ml/kg, 1–8 h) with air in the absence or presence of CO (250 ppm). Airway pressures, blood pressure, and blood gases were monitored. Lung tissue was analyzed for inflammation, injury, and gene expression. Bronchoalveolar lavage fluid was analyzed for protein, cell and neutrophil counts, and cytokines.
Measurements and Main Results: Mechanical ventilation caused significant lung injury reflected by increases in protein concentration, total cell and neutrophil counts in the bronchoalveolar lavage fluid, as well as the induction of heme oxygenase-1 and heat shock protein-70 in lung tissue. In contrast, CO application prevented lung injury during ventilation, inhibited stress-gene up-regulation, and decreased lung neutrophil infiltration. These effects were preceded by the inhibition of ventilation-induced cytokine and chemokine production. Furthermore, CO prevented the early ventilation-dependent up-regulation of early growth response-1 (Egr-1). Egr-1–deficient mice did not sustain lung injury after ventilation, relative to wild-type mice, suggesting that Egr-1 acts as a key proinflammatory regulator in VILI. Moreover, inhibition of peroxysome proliferator-activated receptor (PPAR)-γ, an antiinflammatory nuclear regulator, by GW9662 abolished the protective effects of CO.
Conclusions: Mechanical ventilation causes profound lung injury and inflammatory responses. CO treatment conferred protection in this model dependent on PPAR-γ and inhibition of Egr-1.
doi:10.1164/rccm.200708-1265OC
PMCID: PMC2408440  PMID: 18356564
carbon monoxide; early growth response-1; inflammation; peroxysome proliferator-activated receptor-γ; ventilator-induced lung injury
7.  Mitogen-Activated Protein Kinases Regulate Susceptibility to Ventilator-Induced Lung Injury 
PLoS ONE  2008;3(2):e1601.
Background
Mechanical ventilation causes ventilator-induced lung injury in animals and humans. Mitogen-activated protein kinases have been implicated in ventilator-induced lung injury though their functional significance remains incomplete. We characterize the role of p38 mitogen-activated protein kinase/mitogen activated protein kinase kinase-3 and c-Jun-NH2-terminal kinase-1 in ventilator-induced lung injury and investigate novel independent mechanisms contributing to lung injury during mechanical ventilation.
Methodology and Principle Findings
C57/BL6 wild-type mice and mice genetically deleted for mitogen-activated protein kinase kinase-3 (mkk-3−/−) or c-Jun-NH2-terminal kinase-1 (jnk1−/−) were ventilated, and lung injury parameters were assessed. We demonstrate that mkk3−/− or jnk1−/− mice displayed significantly reduced inflammatory lung injury and apoptosis relative to wild-type mice. Since jnk1−/− mice were highly resistant to ventilator-induced lung injury, we performed comprehensive gene expression profiling of ventilated wild-type or jnk1−/− mice to identify novel candidate genes which may play critical roles in the pathogenesis of ventilator-induced lung injury. Microarray analysis revealed many novel genes differentially expressed by ventilation including matrix metalloproteinase-8 (MMP8) and GADD45α. Functional characterization of MMP8 revealed that mmp8−/− mice were sensitized to ventilator-induced lung injury with increased lung vascular permeability.
Conclusions
We demonstrate that mitogen-activated protein kinase pathways mediate inflammatory lung injury during ventilator-induced lung injury. C-Jun-NH2-terminal kinase was also involved in alveolo-capillary leakage and edema formation, whereas MMP8 inhibited alveolo-capillary protein leakage.
doi:10.1371/journal.pone.0001601
PMCID: PMC2223071  PMID: 18270588
8.  Caveolin-1: a critical regulator of lung fibrosis in idiopathic pulmonary fibrosis 
The Journal of experimental medicine  2006;203(13):2895-2906.
Idiopathic pulmonary fibrosis (IPF) is a progressive chronic disorder characterized by activation of fibroblasts and overproduction of extracellular matrix (ECM). Caveolin-1 (cav-1), a principal component of caveolae, has been implicated in the regulation of numerous signaling pathways and biological processes. We observed marked reduction of cav-1 expression in lung tissues and in primary pulmonary fibroblasts from IPF patients compared with controls. We also demonstrated that cav-1 markedly ameliorated bleomycin (BLM)-induced pulmonary fibrosis, as indicated by histological analysis, hydroxyproline content, and immunoblot analysis. Additionally, transforming growth factor β1 (TGF-β1), the well-known profibrotic cytokine, decreased cav-1 expression in human pulmonary fibroblasts. cav-1 was able to suppress TGF-β1–induced ECM production in cultured fibroblasts through the regulation of the c-Jun N-terminal kinase (JNK) pathway. Interestingly, highly activated JNK was detected in IPF- and BLM-instilled lung tissue samples, which was dramatically suppressed by ad–cav-1 infection. Moreover, JNK1-null fibroblasts showed reduced smad signaling cascades, mimicking the effects of cav-1. This study indicates a pivotal role for cav-1 in ECM regulation and suggests a novel therapeutic target for patients with pulmonary fibrosis.
doi:10.1084/jem.20061536
PMCID: PMC1850940  PMID: 17178917
9.  Caveolin-1: a critical regulator of lung fibrosis in idiopathic pulmonary fibrosis 
The Journal of Experimental Medicine  2006;203(13):2895-2906.
Idiopathic pulmonary fibrosis (IPF) is a progressive chronic disorder characterized by activation of fibroblasts and overproduction of extracellular matrix (ECM). Caveolin-1 (cav-1), a principal component of caveolae, has been implicated in the regulation of numerous signaling pathways and biological processes. We observed marked reduction of cav-1 expression in lung tissues and in primary pulmonary fibroblasts from IPF patients compared with controls. We also demonstrated that cav-1 markedly ameliorated bleomycin (BLM)-induced pulmonary fibrosis, as indicated by histological analysis, hydroxyproline content, and immunoblot analysis. Additionally, transforming growth factor β1 (TGF-β1), the well-known profibrotic cytokine, decreased cav-1 expression in human pulmonary fibroblasts. cav-1 was able to suppress TGF-β1–induced ECM production in cultured fibroblasts through the regulation of the c-Jun N-terminal kinase (JNK) pathway. Interestingly, highly activated JNK was detected in IPF- and BLM-instilled lung tissue samples, which was dramatically suppressed by ad–cav-1 infection. Moreover, JNK1-null fibroblasts showed reduced smad signaling cascades, mimicking the effects of cav-1. This study indicates a pivotal role for cav-1 in ECM regulation and suggests a novel therapeutic target for patients with pulmonary fibrosis.
doi:10.1084/jem.20061536
PMCID: PMC1850940  PMID: 17178917
10.  Carbon monoxide reverses established pulmonary hypertension 
The Journal of Experimental Medicine  2006;203(9):2109-2119.
Pulmonary arterial hypertension (PAH) is an incurable disease characterized by a progressive increase in pulmonary vascular resistance leading to right heart failure. Carbon monoxide (CO) has emerged as a potently protective, homeostatic molecule that prevents the development of vascular disorders when administered prophylactically. The data presented in this paper demonstrate that CO can also act as a therapeutic (i.e., where exposure to CO is initiated after pathology is established). In three rodent models of PAH, a 1 hour/day exposure to CO reverses established PAH and right ventricular hypertrophy, restoring right ventricular and pulmonary arterial pressures, as well as the pulmonary vascular architecture, to near normal. The ability of CO to reverse PAH requires functional endothelial nitric oxide synthase (eNOS/NOS3) and NO generation, as indicated by the inability of CO to reverse chronic hypoxia-induced PAH in eNOS-deficient (nos3−/−) mice versus wild-type mice. The restorative function of CO was associated with a simultaneous increase in apoptosis and decrease in cellular proliferation of vascular smooth muscle cells, which was regulated in part by the endothelial cells in the hypertrophied vessels. In conclusion, these data demonstrate that CO reverses established PAH dependent on NO generation supporting the use of CO clinically to treat pulmonary hypertension.
doi:10.1084/jem.20052267
PMCID: PMC2118401  PMID: 16908624

Results 1-10 (10)