IMPACT OF EXPERIMENTAL OBESITY ON DIAPHRAGM STRUCTURE, FUNCTION, AND BIOENERGETICS

Obesity is associated with bioenergetic dysfunction of peripheral muscles; however, little is known regarding the impact of obesity on the diaphragm. We hypothesized that obesity would be associated with diaphragm dysfunction attributable to mitochondrial oxygen consumption and structural and ultrastructural changes. Wistar rat litters were culled to 3 pups to induce early postnatal overfeeding and consequent obesity. Control animals were obtained from unculled litters. From postnatal day 150, diaphragm ultrasonography, computed tomography, high-resolution respirometry, immunohistochemical, biomolecular and ultrastructural histological analyses were performed. The diaphragms of obese animals, compared to those of controls, exhibited presented changes in morphology as increased thickening fraction, diaphragm excursion and diaphragm dome height, as well as increased mitochondrial respiratory capacity coupled to ATP synthesis and maximal respiratory capacity. Fatty acid synthase expression was also higher in obese animals, suggesting a source of energy for the respiratory chain. Myosin heavy chain-IIA was increased, indicating shift from glycolytic toward oxidative muscle fiber profile. Diaphragm tissue also exhibited ultrastructural changes, such as compact, round, and swollen mitochondria with fainter cristae and more lysosomal bodies. Dynamin-1 expression in the diaphragm was reduced in obese rats, suggesting decreased mitochondrial fission. Furthermore, gene expressions of peroxisome gamma proliferator-activated receptor co-activator-1\u3b1 and superoxide dismutase-2 were lower in obese animals than in controls, which may indicate a predisposition to oxidative injury. In conclusion, in the obesity model used herein, muscle fiber phenotype was altered in a manner likely associated with increased mitochondrial respiratory capability, suggesting respiratory adaptation to increased metabolic demand

Moderate Aerobic Training Improves Cardiorespiratory Parameters in Elastase-Induced Emphysema

Aim: We investigated the therapeutic effects of aerobic training on lung mechanics, inflammation, morphometry and biological markers associated with inflammation and endothelial cell damage, as well as cardiac function in a model of elastase-induced emphysema. Methods: Eighty-four BALB/c mice were randomly allocated to receive saline (control, C) or 0.1 IU porcine pancreatic elastase (emphysema, ELA) intratracheally once weekly for 4 weeks. After the end of administration period, once cardiorespiratory impairment associated with emphysema was confirmed, each group was further randomized into sedentary (S) and trained (T) subgroups. Trained mice ran on a motorized treadmill, at moderate intensity, 30 min/day, 3 times/week for 4 weeks. Results: Four weeks after the first instillation, ELA animals, compared to C, showed: 1) reduced static lung elastance (Est,L) and levels of vascular endothelial growth factor (VEGF) in lung tissue, 2) increased elastic and collagen fiber content, dynamic elastance (E, in vitro), alveolar hyperinflation, and levels of interleukin-1β and tumor necrosis factor (TNF)-α, and 3) increased right ventricular diastolic area (RVA). Four weeks after aerobic training, ELA-T group, compared to ELA-S, was associated with reduced lung hyperinflation, elastic and collagen fiber content, TNF-α levels, and RVA, as well as increased Est,L, E, and levels of VEGF. Conclusion: Four weeks of regular and moderate intensity aerobic training modulated lung inflammation and remodeling, thus improving pulmonary function, and reduced RVA and pulmonary arterial hypertension in this animal model of elastase-induced emphysema

Extracellular Vesicles from Different Sources of Mesenchymal Stromal Cells Have Distinct Effects on Lung and Distal Organs in Experimental Sepsis

The effects of the administration of mesenchymal stromal cells (MSC) may vary according to the source. We hypothesized that MSC-derived extracellular vesicles (EVs) obtained from bone marrow (BM), adipose (AD), or lung (L) tissues may also lead to different effects in sepsis. We profiled the proteome from EVs as a first step toward understanding their mechanisms of action. Polymicrobial sepsis was induced in C57BL/6 mice by cecal ligation and puncture (SEPSIS) and SHAM (control) animals only underwent laparotomy. Twenty-four hours after surgery, animals in the SEPSIS group were randomized to receive saline or 3 × 106 MSC-derived EVs from BM, AD, or L. The diffuse alveolar damage was decreased with EVs from all three sources. In kidneys, BM-, AD-, and L-EVs reduced edema and expression of interleukin-18. Kidney injury molecule-1 expression decreased only in BM- and L-EVs groups. In the liver, only BM-EVs reduced congestion and cell infiltration. The size and number of EVs from different sources were not different, but the proteome of the EVs differed. BM-EVs were enriched for anti-inflammatory proteins compared with AD-EVs and L-EVs. In conclusion, BM-EVs were associated with less organ damage compared with the other sources of EVs, which may be related to differences detected in their proteome

Antigen-induced pleural eosinophilia is suppressed in diabetic rats: role of corticosteroid hormones

Previous studies have evidenced for the existence of interactive regulatory mechanisms between insulin and steroid hormones in different systems. In this study, we have investigated whether endogenous corticosteroids could be implicated in the hyporeactivity to antigen challenge observed in sensitized diabetic rats. Alloxinated rats showed a long-lasting increase in the blood glucose levels and a reduction in the number of pleural mast cells at 48 and 72 hr, but not at 24 hr after alloxan administration. In parallel, they also showed a significant elevation in the plasma levels of corticosterone together with an increase in the adrenal/body weight ratio. Antigen-evoked eosinophil accumulation appeared significantly reduced in rats pretreated with dexamethasone as well as in those rendered diabetic 72 hr after alloxan. In the same way, naive animals treated with dexamethasone also responded with a significant decrease in the number of pleural mast cells. Interestingly, when sensitized diabetic rats were pretreated with the steroid antagonist RU 38486 a reversion of the reduction in the allergen-induced eosinophil accumulation was noted. We conclude that the down-regulation of the allergic inflammatory response in diabetic rats is close-related to reduction in mast cell numbers and over expression of endogenous corticosteroids

Proteomic Approaches Identify Members of Cofilin Pathway Involved in Oral Tumorigenesis

<div><p>The prediction of tumor behavior for patients with oral carcinomas remains a challenge for clinicians. The presence of lymph node metastasis is the most important prognostic factor but it is limited in predicting local relapse or survival. This highlights the need for identifying biomarkers that may effectively contribute to prediction of recurrence and tumor spread. In this study, we used one- and two-dimensional gel electrophoresis, mass spectrometry and immunodetection methods to analyze protein expression in oral squamous cell carcinomas. Using a refinement for classifying oral carcinomas in regard to prognosis, we analyzed small but lymph node metastasis-positive versus large, lymph node metastasis-negative tumors in order to contribute to the molecular characterization of subgroups with risk of dissemination. Specific protein patterns favoring metastasis were observed in the “more-aggressive” group defined by the present study. This group displayed upregulation of proteins involved in migration, adhesion, angiogenesis, cell cycle regulation, anti-apoptosis and epithelial to mesenchymal transition, whereas the “less-aggressive” group was engaged in keratinocyte differentiation, epidermis development, inflammation and immune response. Besides the identification of several proteins not yet described as deregulated in oral carcinomas, the present study demonstrated for the first time the role of cofilin-1 in modulating cell invasion in oral carcinomas.</p> </div

Immunodetection of keratin 4 expression in OSCC samples. Immunohistochemistry analysis:

<p>pattern of keratin 4 immunostaining in (A) superficial layers of epithelium in margin showing intense positivity in stratum corneum (A, insert); (B) absence of keratin 4 immunostaining in nests of well differentiated and (C) poorly differentiated areas of OSCC. Scale bar indicates 100 µm. <b>Western blot</b>: (D) tumor samples (lanes 1, 3, 5, 7) and matched margins (lanes 2, 4, 6, 8) from patients with T1N0, T4N2, T4N1 and T4N1 carcinomas, respectively; (E) Surgical margin (lane 1) and tumor samples (lanes 2, 3, 4, 5) from patients with T4N2, T4N2, T4N2, T1N0 and T2N2, respectively. β-actin was used as an internal control. MW, PageRuler™ Prestained Protein Ladder.</p

Cofilin pathway.

<p>Microenvironmental stimuli signal through Rho-GTPases and their regulating kinases (ROCK1 and Pak-1), stimulating LIMK to phosphorylate and inactivate cofilin-1. Otherwise, SSH phosphatases dephosphorylate cofilin. Rap proteins may increase the enzymatic activity of SSHs, possibly by promoting their release from 14-3-3 proteins. Cofilin is sequestered by PIP2 and released after hydrolysis of PIP2 by phosphorylated PLC to IP3 and DAG. The active cofilin severs “old” actin filaments to generate free actin barbed ends. ATP-actin assembles into these barbed ends and ADP-actin subunits are, in turn, dissociated from the pointed end. Free actin monomers exchange ADP to ATP, frequently with the help of profilin and CAP proteins. ARP2/3 complex binds to F-actin and nucleates the growth of daughter filaments, generating a dendritic network at the leading edge of migratory cells. Other members of this pathway include Hsp90, which promotes stability of LIMK, and CAPZ, which interacts with barbed ends and inhibits filament assembly. ARP = actin-related protein 2/3 complex; CAP = adenylyl cyclase-associated protein 1; CAPZ = F-actin-capping protein subunit alpha-1; CFL = cofilin-1; F-actin = filamentous actin; DAG =  diacylglycerol; G-actin = globular actin; GF = growth factor; HSP90 = heat shock protein HSP 90-alpha; IP3 = inositoltrisphosphate; LIMK = LIM kinases; Pak-1 = serine/threonine-protein kinase PAK 1; PIP2 = phosphatidylinositol-4-5-biphosphate; PLC = phospholipase C; RAP = Ras-related protein; ROCK-1 = Rho-associated protein kinase 1; SSH = slingshot phosphatase.</p

Immunodetection <i>of</i> cofilin-1 and p-cofilin in OSCC samples.

<p>Immunostaining for total cofilin-1 and p-cofilin in FFPE sections of (A and B, respectively) surgical margins and (C and D, respectively) OSCC samples. Note the low positivity of total cofilin (A) and the nuclear staining for p-cofilin (B) in the more basal layers of epithelium in margins, and (C and D, inserts) the more intense staining of tumor cell nuclei for p-cofilin than for total cofilin. Figures and inserts = 100X and 400X magnification, respectively.</p