Browsing by Author "Ayala, Pedro"

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    Acute lung injury by gastric fluid instillation: activation of myofibroblast apoptosis during injury resolution
    (2018) Ayala, Pedro; Torres, Jorge; Vivar, Raúl; Meneses, Manuel; Olmos Coelho, Pablo Roberto; San Martín, Tamara; Borzone, Gisella
    Abstract Background Gastric contents aspiration in humans has variable consequences depending on the volume of aspirate, ranging from subclinical pneumonitis to respiratory failure with up to 70% mortality. Several experimental approaches have been used to study this condition. In a model of single orotracheal instillation of gastric fluid we have shown that severe acute lung injury evolves from a pattern of diffuse alveolar damage to one of organizing pneumonia (OP), that later resolves leaving normal lung architecture. Little is known about mechanisms of injury resolution after a single aspiration that could be dysregulated with repetitive aspirations. We hypothesized that, in a similar way to cutaneous wound healing, apoptosis may participate in lung injury resolution by reducing the number of myofibroblasts and by affecting the balance between proteases and antiproteases. Our aim was to study activation of apoptosis as well as MMP-2/TIMP-2 balance in the sub-acute phase (4–14 days) of gastric fluid-induced lung injury. Methods Anesthesized Sprague-Dawley rats received a single orotracheal instillation of gastric fluid and were euthanized 4, 7 and 14 days later (n = 6/group). In lung tissue we studied caspase-3 activation and its location by double immunofluorescence for cleaved caspase-3 or TUNEL and alpha-SMA. MMP-2/TIMP-2 balance was studied by zymography and Western blot. BALF levels of TGF-β1 were measured by ELISA. Results An OP pattern with Masson bodies and granulomas was seen at days 4 and 7 that was no longer present at day 14. Cleaved caspase-3 increased at day 7 and was detected by immunofluorescence in Masson body-alpha-SMA-positive and –negative cells. TUNEL-positive cells at days 4 and 7 were located mainly in Masson bodies. Distribution of cleaved caspase-3 and TUNEL-positive cells at day 14 was similar to that in controls. At the peak of apoptosis (day 7), an imbalance between MMP-2 activity and TIMP-2 expression was produced by reduction in TIMP-2 expression. Conclusions Apoptosis is activated in Masson body-alpha-SMA–positive and –negative cells during the sub-acute phase of gastric fluid-induced lung injury. This mechanism likely contributes to OP resolution, by reducing myofibroblast number and new collagen production. In addition, pre-formed collagen degradation is favored by an associated MMP-2/TIMP-2 imbalance.
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    Acute lung injury induced by whole gastric fluid: hepatic acute phase response contributes to increase lung antiprotease protection
    (2016) Ayala, Pedro; Meneses, Manuel; Olmos Coelho, Pablo Roberto; Montalva, Rebeca; Droguett Quezada, Karla Denise.; Rios Raggio, Mariana; Borzone, Gisella
    Abstract Background Gastric contents aspiration in humans is a risk factor for severe respiratory failure with elevated mortality. Although aspiration-induced local lung inflammation has been studied in animal models, little is known about extrapulmonary effects of aspiration. We investigated whether a single orotracheal instillation of whole gastric fluid elicits a liver acute phase response and if this response contributes to enrich the alveolar spaces with proteins having antiprotease activity. Methods In anesthetized Sprague-Dawley rats receiving whole gastric fluid, we studied at different times after instillation (4 h −7 days): changes in blood cytokines and acute phase proteins (fibrinogen and the antiproteases alpha1-antitrypsin and alpha2-macroglobulin) as well as liver mRNA expression of the two antiproteases. The impact of the systemic changes on lung antiprotease defense was evaluated by measuring levels and bioactivity of antiproteases in broncho-alveolar lavage fluid (BALF). Markers of alveolar-capillary barrier derangement were also studied. Non-parametric ANOVA (Kruskall-Wallis) and linear regression analysis were used. Results Severe peribronchiolar injury involving edema, intra-alveolar proteinaceous debris, hemorrhage and PMNn cell infiltration was seen in the first 24 h and later resolved. Despite a large increase in several lung cytokines, only IL-6 was found elevated in blood, preceding increased liver expression and blood concentration of both antiproteases. These changes, with an acute phase response profile, were significantly larger for alpha2-macroglobulin (40-fold increment in expression with 12-fold elevation in blood protein concentration) than for alpha1-antitrypsin (2–3 fold increment in expression with 0.5-fold elevation in blood protein concentration). Both the increment in capillary-alveolar antiprotease concentration gradient due to increased antiprotease liver synthesis and a timely-associated derangement of the alveolar-capillary barrier induced by aspiration, contributed a 58-fold and a 190-fold increase in BALF alpha1-antitrypsin and alpha2-macroglobulin levels respectively (p < 0.001). Conclusions Gastric contents-induced acute lung injury elicits a liver acute phase response characterized by increased mRNA expression of antiproteases and elevation of blood antiprotease concentrations. Hepatic changes act in concert with derangement of the alveolar capillary barrier to enrich alveolar spaces with antiproteases. These findings may have significant implications decreasing protease burden, limiting injury in this and other models of acute lung injury and likely, in recurrent aspiration.
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    Changes in the pattern of fibrosis in the rat lung with repetitive orotracheal instillations of gastric contents: evidence of persistent collagen accumulation
    (2018) Ayala, Pedro; Torres, Jorge; Vivar, Raul; Olmos Coelho, Pablo Roberto; Meneses, Manuel; Borzone, Gisella
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    Elastin degradation products in acute lung injury induced by gastric contents aspiration
    (2018) Ayala, Pedro; Vivar, Raúl; Montalva, Rebeca; Olmos Coelho, Pablo Roberto; Borzone, Gisella; Meneses, Manuel
    Abstract Background Gastric contents aspiration is a high-risk condition for acute lung injury (ALI). Consequences range from subclinical pneumonitis to respiratory failure, depending on the volume of aspirate. A large increment in inflammatory cells, an important source of elastase, potentially capable of damaging lung tissue, has been described in experimental models of aspiration. We hypothesized that in early stages of aspiration-induced ALI, there is proteolytic degradation of elastin, preceding collagen deposition. Our aim was to evaluate whether after a single orotracheal instillation of gastric fluid, there is evidence of elastin degradation. Methods Anesthesized Sprague-Dawley rats received a single orotracheal instillation of gastric fluid and were euthanized 4, 12 and 24 h and at day 4 after instillation (n = 6/group). We used immunodetection of soluble elastin in lung tissue and BALF and correlated BALF levels of elastin degradation products with markers of ALI. We investigated possible factors involved in elastin degradation and evaluated whether a similar pattern of elastin degradation can be found in BALF samples of patients with interstitial lung diseases known to have aspirated. Non-parametric ANOVA (Kruskall-Wallis) and linear regression analysis were used. Results We found evidence of early proteolytic degradation of lung elastin. Elastin degradation products are detected both in lung tissue and BALF in the first 24 h and are significantly reduced at day 4. They correlate significantly with ALI markers, particularly PMN cell count, are independent of acidity and have a similar molecular weight as those obtained using pancreatic elastase. Evaluation of BALF from patients revealed the presence of elastin degradation products not present in controls that are similar to those found in BALF of rats treated with gastric fluid. Conclusions A single instillation of gastric fluid into the lungs induces early proteolytic degradation of elastin, in relation to the magnitude of alveolar-capillary barrier derangement. PMN-derived proteases released during ALI are mostly responsible for this damage. BALF from patients showed elastin degradation products similar to those found in rats treated with gastric fluid. Long-lasting effects on lung elastic properties could be expected under conditions of repeated instillations of gastric fluid in experimental animals or repeated aspiration events in humans.
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    EPAC expression and function in cardiac fibroblasts and myofibroblasts
    (2013) Olmedo, Ivonne; Muñoz, Claudia; Guzmán, Nancy; Catalán, Mabel; Vivar, Raúl; Ayala, Pedro; Humeres, Claudio; Aránguiz, Pablo; García, Lorena; Velarde, Victoria; Díaz Araya, Guillermo
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    Lipopolysaccharide Activates Toll-Like Receptor 4 and Prevents Cardiac Fibroblast-to-Myofibroblast Differentiation
    (HUMANA PRESS INC, 2017) Bolivar, Samir; Santana, Roxana; Ayala, Pedro; Landaeta, Rodolfo; Boza, Pia; Humeres, Claudio; Vivar, Raul; Munoz, Claudia; Pardo, Viviana; Fernandez, Samuel; Anfossi, Renatto; Diaz Araya, Guillermo
    Bacterial lipopolysaccharide (LPS) is a known ligand of Toll-like receptor 4 (TLR4) which is expressed in cardiac fibroblasts (CF). Differentiation of CF to cardiac myofibroblasts (CMF) is induced by transforming growth factor-beta 1 (TGF-beta 1), increasing alpha-smooth muscle actin (alpha-SMA) expression. In endothelial cells, an antagonist effect between LPS-induced signaling and canonical TGF-beta 1 signaling was described; however, it has not been studied whether in CF and CMF the expression of alpha-SMA induced by TGF-beta 1 is antagonized by LPS and the mechanism involved. In adult rat CF and CMF, alpha-SMA, ERK1/2, Akt, NF-kappa beta, Smad3, and Smad7 protein levels were determined by western blot, TGF-beta isoforms by ELISA, and alpha-SMA stress fibers by immunocytochemistry. CF and CMF secrete the three TGF-beta isoforms, and the secretion levels of TGF-beta 2 was affected by LPS treatment. In CF, LPS treatment decreased the protein levels of alpha-SMA, and this effect was prevented by TAK-242 (TLR4 inhibitor) and LY294002 (Akt inhibitor), but not by BAY 11-7082 (NF-kappa beta inhibitor) and PD98059 (ERK1/2 inhibitor). TGF-beta 1 increased alpha-SMA protein levels in CF, and LPS prevented partially this effect. In addition, in CMF alpha-SMA protein levels were decreased by LPS treatment, which was abolished by TAK-242. Finally, in CF LPS decreased the p-Smad3 phosphorylation and increased the Smad7 protein levels. LPS treatment prevents the CF-to-CMF differentiation and reverses the CMF phenotype induced by TGF-beta 1, through decreasing p-Smad3 and increasing Smad7 protein levels.
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    Near-apneic ventilation decreases lung injury and fibroproliferation in an acute respiratory distress syndrome model with extracorporeal membrane oxygenation
    (2019) Araos, J.; Alegría Aguirre, Luz Katiushka; Garcia, P.; Cruces, P.; Soto, D.; Erranz, B.; Amthauer, M.; Ayala, Pedro; Borzone, Gisella; Damiani Rebolledo, L. Felipe
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    Resolution of Lung Injury after a Single Event of Aspiration. A Model of Bilateral Instillation of Whole Gastric Fluid
    (2015) Araos, Joaquín D.; Ayala, Pedro; Meneses, Manuel; Contreras, Rafael; Cutiño, Andrea; Montalva, Rebeca M.; Tazelaar, Henry D.; Borzone, Gisella