Mahroo Mofarrahi

Mechanical Ventilation–induced Diaphragm Disuse in Humans Triggers Autophagy

RATIONALE Controlled mechanical ventilation (CMV) results in atrophy of the human diaphragm. The autophagy-lysosome pathway (ALP) contributes to skeletal muscle proteolysis, but its contribution to diaphragmatic protein degradation in mechanically ventilated patients is unknown. OBJECTIVES To evaluate the autophagy pathway responses to CMV in the diaphragm and limb muscles of humans and to identify the roles of FOXO transcription factors in these responses. METHODS Muscle biopsies were obtained from nine control subjects and nine brain-dead organ donors. Subjects were mechanically ventilated for 2 to 4 hours and 15 to 276 hours, respectively. Activation of the ubiquitin-proteasome system was detected by measuring mRNA expressions of Atrogin-1, MURF1, and protein expressions of UBC2, UBC4, and the α subunits of the 20S proteasome (MCP231). Activation of the ALP was detected by electron microscopy and by measuring the expressions of several autophagy-related genes. Total carbonyl content and HNE-protein adduct formation were measured to assess oxidative stress. Total AKT, phosphorylated and total FOXO1, and FOXO3A protein levels were also measured. MEASUREMENTS AND MAIN RESULTS Prolonged CMV triggered activation of the ALP as measured by the appearance of autophagosomes in the diaphragm and increased expressions of autophagy-related genes, as compared with controls. Induction of autophagy was associated with increased protein oxidation and enhanced expression of the FOXO1 gene, but not the FOXO3A gene. CMV also triggered the inhibition of both AKT expression and FOXO1 phosphorylation. CONCLUSIONS We propose that prolonged CMV causes diaphragm disuse, which, in turn, leads to activation of the ALP through oxidative stress and the induction of the FOXO1 transcription factor.

Skeletal muscle dysfunction in patients with chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a debilitating disease characterized by inflammation-induced airflow limitation and parenchymal destruction. In addition to pulmonary manifestations, patients with COPD develop systemic problems, including skeletal muscle and other organ-specific dysfunctions, nutritional abnormalities, weight loss, and adverse psychological responses. Patients with COPD often complain of dyspnea on exertion, reduced exercise capacity, and develop a progressive decline in lung function with increasing age. These symptoms have been attributed to increases in the work of breathing and in impairments in gas exchange that result from airflow limitation and dynamic hyperinflation. However, there is mounting evidence to suggest that skeletal muscle dysfunction, independent of lung function, contributes significantly to reduced exercise capacity and poor quality of life in these patients. Limb and ventilatory skeletal muscle dysfunction in COPD patients has been attributed to a myriad of factors, including the presence of low grade systemic inflammatory processes, nutritional depletion, corticosteroid medications, chronic inactivity, age, hypoxemia, smoking, oxidative and nitrosative stresses, protein degradation and changes in vascular density. This review briefly summarizes the contribution of these factors to overall skeletal muscle dysfunction in patients with COPD, with particular attention paid to the latest advances in the field.

Early Growth Response-1 Regulates Angiopoietin-1-Induced Endothelial Cell Proliferation, Migration, and Differentiation

Objective—Angiopoietin-1 (Ang-1) is an important regulator of angiogenesis in endothelial cells. It promotes migration, proliferation, and differentiation of cells, although the regulating factors involved in these processes remain unclear. In this study, we evaluated the contribution of the transcription factor early growth response-1 (Egr-1) to Ang-1–induced angiogenesis in human umbilical vein endothelial cells (HUVECs). Methods and Results—Expression of Egr-1 was evaluated with real-time PCR and immunoblotting, whereas Egr-1 DNA binding activity was monitored with electrophoretic mobility shift assays. Cell migration was measured with wound healing and Boyden chamber assays, whereas cell proliferation and differentiation of cells into capillary-like tube structures were monitored with cell counting, BrdU incorporation and Matrigels. To selectively inhibit Egr-1 expression, we used both siRNA oligonucleotides and specific DNAzymes. Egr-1 mRNA expression rose approximately 9-fold within 2 hours of Ang-1 exposure and declined thereafter. Upregulation of Egr-1 expression was accompanied by an increase in nuclear mobilization and augmented DNA binding. These processes were mediated through the Erk1/2, PI-3 kinase/AKT, and mTOR pathways. Knockdown of Egr-1 expression completely abrogated Ang-1–induced endothelial migration and significantly reduced proliferation and capillary-like tube formation of HUVECs that overexpress Ang-1. Conclusion—Ang-1 triggers significant and transient induction of Egr-1, and Egr-1 contributes to Ang-1–induced endothelial cell migration and proliferation.

Regulation of angiopoietin expression by bacterial lipopolysaccharide

Angiopoietins are ligands for Tie-2 receptors and play important roles in angiogenesis and inflammation. While angiopoietin-1 (Ang-1) inhibits inflammatory responses, angiopoietin-2 (Ang-2) promotes cytokine production and vascular leakage. In this study, we evaluated in vivo and in vitro effects of Escherichia coli lipopolysaccharides (LPS) on angiopoietin expression. Wild-type C57/BL6 mice were injected with saline (control) or E. coli LPS (20 mg/ml ip) and killed 6, 12, and 24 h later. The diaphragm, lung, and liver were excised and assayed for mRNA and protein expression of Ang-1, Ang-2, and Tie-2 protein and tyrosine phosphorylation. LPS injection elicited a severalfold rise in Ang-2 mRNA and protein levels in the three organs. By comparison, both Ang-1 and Tie-2 levels in the diaphragm, liver, and lung were significantly attenuated by LPS administration. In addition, Tie-2 tyrosine phosphorylation in the lung was significantly reduced in response to LPS injection. In vitro exposure to E. coli LPS elicited cell-specific changes in Ang-1 expression, with significant induction in Ang-1 expression being observed in cultured human epithelial cells, whereas significant attenuation of Ang-1 expression was observed in response to E. coli LPS exposure in primary human skeletal myoblasts. In both cell types, E. coli LPS elicited substantial induction of Ang-2 mRNA, a response that was mediated in part through NF-kappaB. We conclude that in vivo endotoxemia triggers functional inhibition of the Ang-1/Tie-2 receptor pathway by reducing Ang-1 and Tie-2 expression and inducing Ang-2 levels and that this response may contribute to enhanced vascular leakage in sepsis.

Profiling of mRNA Expression in Quadriceps of Patients with COPD and Muscle Wasting

Peripheral muscle wasting is a feature of chronic obstructive pulmonary disease (COPD). Potent therapeutic strategies are needed to improve peripheral muscle mass in these patients. We hypothesized that the evaluation of the mRNA expression profile of quadriceps muscle could be useful in identifying key biochemical pathways involved in the wasting process. We monitored mRNA expression profile of quadriceps muscle in four patients with COPD with muscle atrophy (age: 71.3 ± 2.1 years, mean SD; FEV1 28.3 ± 10.8 % predicted) and four control subjects (age: 66.5 ± 1.3 years) using HuU95v2 gene chips. Fifty-seven mRNAs transcripts (0.5%) were found to be differentially expressed in muscles of COPD patients (i.e., p < 0.01). Among them, forkhead box O -1 and -3 and insulin-like growth factor-1 expressions being significantly elevated in COPD subjects. Concomitantly, a significant reduction in mRNA expression of two myofilament proteins was observed. Energy production appears to be impaired as indicated by the significant rise in nicotinamide N-methyltransferase mRNA expression. This study provides for the first time evidence that genes are selectively expressed in limb muscles of COPD patients and further research need to focus on their functional roles in the pathogenesis of muscle dysfunction.

Expression and functional significance of nicotinamide N-methyl transferase in skeletal muscles of patients with chronic obstructive pulmonary disease.

RATIONALE Nicotinamide N-methyl transferase (NNMT) is highly expressed in quadriceps muscles of patients with chronic obstructive pulmonary disease (COPD). However, its expression in the diaphragm of these patients has not been assessed. The functional significance of NNMT induction in skeletal muscles of patients with COPD is also unknown. OBJECTIVES (1) To compare NNMT expressions in the diaphragm and quadriceps muscles of patients with COPD. (2) To identify the influence of proinflammatory cytokines on NNMT expression. (3) To assess the influence of NNMT on indices of myogenesis (satellite cell migration and proliferation) and the defense against oxidative stress. METHODS Costal diaphragm muscle biopsies were acquired from 13 patients with moderate and severe COPD and 8 control subjects. Quadriceps muscle biopsies were obtained from 12 patients with COPD and 14 control subjects. MEASUREMENTS AND MAIN RESULTS NNMT expressions were significantly elevated in the diaphragm and quadriceps muscles of patients with COPD; however, the relative induction of NNMT expression was greater in the quadriceps muscle (10-fold) than it was in the diaphragm (2-fold). NNMT expressions correlated negatively with the severity of COPD and limb muscle wasting. In skeletal myoblasts, NNMT expression was significantly induced by IL-6, transforming growth factor beta, and tumor necrosis factor-alpha. Overexpression of NNMT in myoblasts triggered a significant increase in proliferation and migration, but had no influence on cell death. Carbonyl formation, induced by exposing myoblasts to H(2)O(2), was significantly attenuated when NNMT was overexpressed. CONCLUSIONS Up-regulation of NNMT expression in the skeletal muscles of patients with COPD may represent an adaptive response designed to improve myogenesis and defend against oxidative stress.

Angiopoietin-1 enhances skeletal muscle regeneration in mice

Activation of muscle progenitor cell myogenesis and endothelial cell angiogenesis is critical for the recovery of skeletal muscle from injury. Angiopoietin-1 (Ang-1), a ligand of Tie-2 receptors, enhances angiogenesis and skeletal muscle satellite cell survival; however, its role in skeletal muscle regeneration after injury is unknown. We assessed the effects of Ang-1 on fiber regeneration, myogenesis, and angiogenesis in injured skeletal muscle (tibialis anterior, TA) in mice. We also assessed endogenous Ang-1 levels and localization in intact and injured TA muscles. TA fiber injury was triggered by cardiotoxin injection. Endogenous Ang-1 mRNA levels immediately decreased in response to cardiotoxin then increased during the 2 wk. Ang-1 protein was expressed in satellite cells, both in noninjured and recovering TA muscles. Positive Ang-1 staining was present in blood vessels but not in nerve fibers. Four days after the initiation of injury, injection of adenoviral Ang-1 into injured muscles resulted in significant increases in in situ TA muscle contractility, muscle fiber regeneration, and capillary density. In cultured human skeletal myoblasts, recombinant Ang-1 protein increased survival, proliferation, migration, and differentiation into myotubes. The latter effect was associated with significant upregulation of the expression of the myogenic regulatory factors MyoD and Myogenin and certain genes involved in cell cycle regulation. We conclude that Ang-1 strongly enhances skeletal muscle regeneration in response to fiber injury and that this effect is mediated through induction of the myogenesis program in muscle progenitor cells and the angiogenesis program in endothelial cells.

Angiopoietin-1 and Vascular Endothelial Growth Factor Regulation of Leukocyte Adhesion to Endothelial Cells: Role of Nuclear Receptor-77

Objective—Vascular endothelial growth factor (VEGF) promotes leukocyte adhesion to endothelial cells (ECs). Angiopoietin-1 (Ang-1) inhibits this response. Nuclear receptor-77 (Nur77) is a proangiogenic nuclear receptor. In the present study, we assessed the influence of Ang-1 and VEGF on Nur77 expression in ECs, and evaluated its role in Ang-1/VEGF-mediated leukocyte adhesion. Methods and Results—Expression of Nur77 was evaluated with real-time polymerase chain reaction and immunoblotting. Adhesion of leukocytes to ECs was monitored with inverted microscopy. Nur77 expression or activity was inhibited using adenoviruses expressing dominant-negative form of Nur77, retroviruses expressing Nur77 in the antisense direction, and small interfering RNA oligos. Both Ang-1 and VEGF induce Nur77 expression, by >5- and 30-fold, respectively. When combined, Ang-1 potentiates VEGF-induced Nur77 expression. Ang-1 induces Nur77 through the phosphoinositide 3-kinase and extracellular signal-regulated protein kinase 1/2 pathways. VEGF induces Nur77 expression through the protein kinase D/histone deacetylase 7/myocyte enhancer factor 2 and extracellular signal-regulated protein kinase 1/2 pathways. VEGF induces nuclear factor-kappaB transcription factor, vascular cell adhesion molecule-1, and E-selectin expressions, and promotes leukocyte adhesion to ECs. Ang-1 inhibits these responses. This inhibitory effect of Ang-1 disappears when Nur77 expression is disrupted, restoring the inductive effects of VEGF on adhesion molecule expression, and increased leukocyte adhesion to ECs. Conclusion—Nur77 promotes anti-inflammatory effects of Ang-1, and functions as a negative feedback inhibitor of VEGF-induced EC activation.

Prolonged controlled mechanical ventilation in humans triggers myofibrillar contractile dysfunction and myofilament protein loss in the diaphragm

Background Prolonged controlled mechanical ventilation (CMV) in humans and experimental animals results in diaphragm fibre atrophy and injury. In animals, prolonged CMV also triggers significant declines in diaphragm myofibril contractility. In humans, the impact of prolonged CMV on myofibril contractility remains unknown. The objective of this study was to evaluate the effects of prolonged CMV on active and passive human diaphragm myofibrillar force generation and myofilament protein levels. Methods and results Diaphragm biopsies were obtained from 13 subjects undergoing cardiac surgery (control group) and 12 brain-dead organ donors (CMV group). Subjects in each group had been mechanically ventilated for 2–4 and 12–74 h, respectively. Specific force generation of diaphragm myofibrils was measured with atomic force cantilevers. Rates of force development (Kact), force redevelopment after a shortening protocol (Ktr) and relaxation (Krel) in fully activated myofibrils (pCa2+=4.5) were calculated to assess myosin cross-bridge kinetics. Myofilament protein levels were measured with immunoblotting and specific antibodies. Prolonged CMV significantly decreased active and passive diaphragm myofibrillar force generation, Kact, Ktr and Krel. Myosin heavy chain (slow), troponin-C, troponin-I, troponin-T, tropomyosin and titin protein levels significantly decreased in response to prolonged CMV, but no effects on α-actin, α-actinin or nebulin levels were observed. Conclusions Prolonged CMV in humans triggers significant decreases in active and passive diaphragm myofibrillar force generation. This response is mediated, in part, by impaired myosin cross-bridge kinetics and decreased myofibrillar protein levels.