Diana Álvarez

mTORC1 activation decreases autophagy in aging and idiopathic pulmonary fibrosis and contributes to apoptosis resistance in IPF fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually lethal disease associated with aging. However, the molecular mechanisms of the aging process that contribute to the pathogenesis of IPF have not been elucidated. IPF is characterized by abundant foci of highly active fibroblasts and myofibroblasts resistant to apoptosis. Remarkably, the role of aging in the autophagy activity of lung fibroblasts and its relationship with apoptosis, as adaptive responses, has not been evaluated previously in this disease. In the present study, we analyzed the dynamics of autophagy in primary lung fibroblasts from IPF compared to young and age‐matched normal lung fibroblasts. Our results showed that aging contributes for a lower induction of autophagy on basal conditions and under starvation which is mediated by mTOR pathway activation. Treatment with rapamycin and PP242, that target the PI3K/AKT/mTOR signaling pathway, modified starvation‐induced autophagy and apoptosis in IPF fibroblasts. Interestingly, we found a persistent activation of this pathway under starvation that contributes to the apoptosis resistance in IPF fibroblasts. These findings indicate that aging affects adaptive responses to stress decreasing autophagy through activation of mTORC1 in lung fibroblasts. The activation of this pathway also contributes to the resistance to cell death in IPF lung fibroblasts.

miR-155 in the progression of lung fibrosis in systemic sclerosis

BackgroundMicroRNA (miRNA) control key elements of mRNA stability and likely contribute to the dysregulated lung gene expression observed in systemic sclerosis associated interstitial lung disease (SSc-ILD). We analyzed the miRNA gene expression of tissue and cells from patients with SSc-ILD. A chronic lung fibrotic murine model was used.MethodsRNA was isolated from lung tissue of 12 patients with SSc-ILD and 5 controls. High-resolution computed tomography (HRCT) was performed at baseline and 2–3 years after treatment. Lung fibroblasts and peripheral blood mononuclear cells (PBMC) were isolated from healthy controls and patients with SSc-ILD. miRNA and mRNA were analyzed by microarray, quantitative polymerase chain reaction, and/or Nanostring; pathway analysis was performed by DNA Intelligent Analysis (DIANA)-miRPath v2.0 software. Wild-type and miR-155 deficient (miR-155ko) mice were exposed to bleomycin.ResultsLung miRNA microarray data distinguished patients with SSc-ILD from healthy controls with 185 miRNA differentially expressed (q < 0.25). DIANA-miRPath revealed 57 Kyoto Encyclopedia of Genes and Genomes pathways related to the most dysregulated miRNA. miR-155 and miR-143 were strongly correlated with progression of the HRCT score. Lung fibroblasts only mildly expressed miR-155/miR-21 after several stimuli. miR-155 PBMC expression strongly correlated with lung function tests in SSc-ILD. miR-155ko mice developed milder lung fibrosis, survived longer, and weaker lung induction of several genes after bleomycin exposure compared to wild-type mice.ConclusionsmiRNA are dysregulated in the lungs and PBMC of patients with SSc-ILD. Based on mRNA-miRNA interaction analysis and pathway tools, miRNA may play a role in the progression of the disease. Our findings suggest that targeting miR-155 might provide a novel therapeutic strategy for SSc-ILD.

IPF lung fibroblasts have a senescent phenotype

The mechanisms of aging that are involved in the development of idiopathic pulmonary fibrosis (IPF) are still unclear. Although it has been hypothesized that the proliferation and activation of human lung fibroblasts (hLFs) are essential in IPF, no studies have assessed how this process works in an aging lung. Our goal was to elucidate if there were age-related changes on primary hLFs isolated from IPF lungs compared with age-matched controls. We investigated several hallmarks of aging in hLFs from IPF patients and age-matched controls. IPF hLFs have increased cellular senescence with higher expression of β-galactosidase, p21, p16, p53, and cytokines related to the senescence-associated secretory phenotype (SASP) as well as decreased proliferation/apoptosis compared with age-matched controls. Additionally, we observed shorter telomeres, mitochondrial dysfunction, and upon transforming growth factor-β stimulation, increased markers of endoplasmic reticulum stress. Our data suggest that IPF hLFs develop senescence resulting in a decreased apoptosis and that the development of SASP may be an important contributor to the fibrotic process observed in IPF. These results might change the existing paradigm, which describes fibroblasts as aberrantly activated cells, to a cell with a senescence phenotype.

Implementación de un manojo de medidas (bundle) de inserción para prevenir la infección del torrente sanguíneo asociada a dispositivo intravascular central en Cuidado Intensivo en Colombia

Las infecciones asociadas a la instalacion y manejo de cateteres venosos centrales (CVC) son eventos frecuentes en unidades de cuidados intensivos pero evitables. Objetivo: Evaluar la eficacia en disminuir la tasa de infeccion asociada a cateter (IACVC) obtenida con la implementacion de un manojo de medidas (bundle) durante la insercion del dispositivo. Material y Metodos: Se condujo un estudio que compara un periodo pre-intervencion con uno de intervencion, de 6 meses cada uno, consistente en la implementacion de un manojo de medidas para la insercion de cateter venoso central (CVC), en la Unidad de Cuidado Intensivo (UCI) del Hospital Universitario de Neiva, Colombia. En el periodo pre-intervencion (2010) se evaluo la tasa de IACVC y las caracteristicas de la poblacion. Durante la intervencion (2011) se implemento un manojo de medidas para la insercion de CVC que consistio en: higiene de manos, uso de clorhexidina 2%, empleo de maximas barreras esteriles y evitar el acceso femoral. Resultados: Se obtuvo reduccion de la tasa de IACVC de 5,56 a 3,26 X 1.000 dias CVC. Los dias de estancia en UCI y de exposicion al CVC se asociaron a mayor riesgo de desarrollar IACVC (p < 0,05); el cumplimiento del manojo de medidas fue un factor protector contra IACVC (OR 0,45; p = 0,615). La adherencia del personal al manojo de medidas fue mayor de 80%. Conclusion: La implementacion de un manojo de medidas para la insercion de CVC resulto ser una medida util para la prevencion de IACVC en nuestro hospital, lo que podria implementarse en otras instituciones hospitalarias de complejidad similar.

Regenerative medicine in the treatment of idiopathic pulmonary fibrosis: current position.

Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible disease of the lung that has no lasting option for therapy other than transplantation. It is characterized by replacement of the normal lung tissue by fibrotic scarring, honeycombing, and increased levels of myofibroblasts. The underlying causes of IPF are still largely unknown. The focus of the current review is the possible use of stem cell therapy, specifically mesenchymal stem cells (MSCs), a multipotent stromal cell population, which have demonstrated promising data in multiple animal models of pulmonary fibrosis (PF). The most studied source of MSCs is the bone marrow, although they can be found also in the adipose tissue and umbilical cord, as well as in the placenta. MSCs have immunomodulatory and tissue-protective properties that allow them to manipulate the local environment of the injured tissue, ameliorating the inflammation and promoting repair. Because IPF primarily affects older patients, the issue of aging is intrinsically linked to many aspects of the disease, including the age of the stem cells. Animal models have shown the success of MSC therapy in mitigating the fibrotic effects of bleomycin-induced PF. However, bleomycin, the most commonly used model for PF, is imperfect in mimicking IPF as it presents in humans, as the duration of the illness is not parallel or reversible, and honeycombing is not produced. Furthermore, the time of MSC dosage has proven to be critical in determining whether the cells will ultimately have a positive or negative effect on disease progression, since it has been demonstrated that the maximal beneficial effect of MSCs occurs during the early inflammatory phase of the disease and that there is no or negative effect during the late fibrotic phase. Therefore, all the current clinical trials of MSCs and IPF, though promising, should proceed with caution as we move toward true stem cell therapy for this disease.

Modified mesenchymal stem cells using miRNA transduction alter lung injury in a bleomycin model

Although different preclinical models have demonstrated a favorable role for bone marrow-derived mesenchymal stem cells (B-MSC) in preventing fibrosis, this protective effect is not observed with late administration of these cells, when fibrotic changes are consolidated. We sought to investigate whether the late administration of B-MSCs overexpressing microRNAs (miRNAs) let-7d (antifibrotic) or miR-154 (profibrotic) could alter lung fibrosis in a murine bleomycin model. Using lentiviral vectors, we transduced miRNAs (let-7d or miR-154) or a control sequence into human B-MSCs. Overexpression of let-7d or miR-154 was associated with changes in the mesenchymal properties of B-MSCs and in their cytokine expression. Modified B-MSCs were intravenously administered to mice at day 7 after bleomycin instillation, and the mice were euthanized at day 14 Bleomycin-injured animals that were treated with let-7d cells were found to recover quicker from the initial weight loss compared with the other treatment groups. Interestingly, animals treated with miR-154 cells had the lowest survival rate. Although a slight reduction in collagen mRNA levels was observed in lung tissue from let-7d mice, no significant differences were observed in Ashcroft score and OH-proline. However, the distinctive expression in cytokines and CD45-positive cells in the lung suggests that the differential effects observed in both miRNA mice groups were related to an effect on the immunomodulation function. Our results establish the use of miRNA-modified mesenchymal stem cells as a potential future research in lung fibrosis.

Senescence of bone marrow-derived mesenchymal stem cells from patients with idiopathic pulmonary fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic lung disease for which age is the most important risk factor. Different mechanisms associated with aging, including stem cell dysfunction, have been described to participate in the pathophysiology of IPF. We observed an extrapulmonary effect associated with IPF: increase in cell senescence of bone marrow-derived mesenchymal stem cells (B-MSCs).MethodsB-MSCs were obtained from vertebral bodies procured from IPF patients and age-matched normal controls. Cell senescence was determined by cell proliferation and expression of markers of cell senescence p16INK4A, p21, and β-galactosidase activity. Mitochondrial function and DNA damage were measured. Paracrine induction of senescence and profibrotic responses were analyzed in vitro using human lung fibroblasts. The reparative capacity of B-MSCs was examined in vivo using the bleomycin-induced lung fibrosis model.ResultsIn our study, we demonstrate for the first time that B-MSCs from IPF patients are senescent with significant differences in mitochondrial function, with accumulation of DNA damage resulting in defects in critical cell functions when compared with age-matched controls. Senescent IPF B-MSCs have the capability of paracrine senescence by inducing senescence in normal-aged fibroblasts, suggesting a possible link between senescent B-MSCs and the late onset of the disease. IPF B-MSCs also showed a diminished capacity to migrate and were less effective in preventing fibrotic changes observed in mice after bleomycin-induced injury, increasing illness severity and proinflammatory responses.ConclusionsWe describe extrapulmonary alterations in B-MSCs from IPF patients. The consequences of having senescent B-MSCs are not completely understood, but the decrease in their ability to respond to normal activation and the risk of having a negative impact on the local niche by inducing inflammation and senescence in the neighboring cells suggests a new link between B-MSC and the onset of the disease.

Ex vivo lung perfusion as a human platform for preclinical small molecule testing

The acute respiratory distress syndrome (ARDS) causes an estimated 70,000 US deaths annually. Multiple pharmacologic interventions for ARDS have been tested and failed. An unmet need is a suitable laboratory human model to predictively assess emerging therapeutics on organ function in ARDS. We previously demonstrated that the small molecule BC1215 blocks actions of a proinflammatory E3 ligase-associated protein, FBXO3, to suppress NF-κB signaling in animal models of lung injury. Ex vivo lung perfusion (EVLP) is a clinical technique that maintains lung function for possible transplant after organ donation. We used human lungs unacceptable for transplant to model endotoxemic injury with EVLP for 6 hours. LPS infusion induced inflammatory injury with impaired oxygenation of pulmonary venous circulation. BC1215 treatment after LPS rescued oxygenation and decreased inflammatory cytokines in bronchoalveolar lavage. RNA sequencing transcriptomics from biopsies taken during EVLP revealed robust inflammatory gene induction by LPS with a strong signal for NF-κB-associated transcripts. BC1215 treatment reduced the LPS induction of genes associated with inflammatory and host defense gene responses by Gene Ontology (GOterm) and pathways analysis. BC1215 also significantly antagonized LPS-mediated NF-κB activity. EVLP may provide a unique human platform for preclinical study of chemical entities such as FBXO3 inhibitors on tissue physiology.

RevisiónInfecciones pulmonares en pacientes con VIH 20 años después de la terapia antirretroviral combinada. ¿Qué ha cambiado?Pulmonary infections in patients with HIV, 20 years after combined antiretroviral therapy. What has changed?

Background Prior to 1996, HIV was considered practically fatal. However, after highly active antiretroviral therapy was introduced, HIV became a chronic disease with a great increase in life expectancy. Despite this therapy, there are many complications to which an HIV infected person is exposed, with the lung the most commonly affected organ. Opportunistic infections such as pneumocystis pneumonia, histoplasmosis, tuberculosis, nontuberculous mycobacteria, among others, are still important threats to the HIV population.

Infecciones pulmonares en pacientes con VIH 20 años después de la terapia antirretroviral combinada. ¿Qué ha cambiado?

Background Prior to 1996, HIV was considered practically fatal. However, after highly active antiretroviral therapy was introduced, HIV became a chronic disease with a great increase in life expectancy. Despite this therapy, there are many complications to which an HIV infected person is exposed, with the lung the most commonly affected organ. Opportunistic infections such as pneumocystis pneumonia, histoplasmosis, tuberculosis, nontuberculous mycobacteria, among others, are still important threats to the HIV population.