Low dose cadmium exposure regulates miR-381-ANO1 interaction in airway epithelial cells.

Chronic obstructive pulmonary disease (COPD) is the 3rd leading cause of death worldwide. Cigarette smoke which has approximately 2-3 µg of Cadmium (Cd) per cigarette contributes to the environmental exposure and development and severity of COPD. With the lack of a cadmium elimination mechanism in humans, the contribution of cadmium induced stress to lung epithelial cells remains unclear. Studies on cadmium responsive miRNAs suggest regulation of target genes with an emphasis on the critical role of miRNA-mRNA interaction for cellular homeostasis. Mir-381, the target miRNA in this study is negatively regulated by cadmium in airway epithelial cells. miR-381 is reported to also regulate ANO1 (Anoctamin 1) expression negatively and in this study low dose cadmium exposure to airway epithelial cells was observed to upregulate ANO1 mRNA expression via mir-381 inhibition. ANO1 which is a Ca2+-activated chloride channel has multiple effects on cellular functions such as proliferation, mucus hypersecretion and fibroblast differentiation in inflamed airways in chronic respiratory diseases. In vitro studies with cadmium at a high concentration range of 100-500 µM is reported to activate chloride channel, ANO1. The secretory epithelial cells are regulated by chloride channels like CFTR, ANO1 and SLC26A9. We examined "ever" smokers with COPD (n = 13) lung tissue sections compared to "never" smoker without COPD (n = 9). We found that "ever" smokers with COPD had higher ANO1 expression. Using mir-381 mimic to inhibit ANO1, we demonstrate here that ANO1 expression is significantly (p < 0.001) downregulated in COPD derived airway epithelial cells exposed to cadmium. Exposure to environmental cadmium contributes significantly to ANO1 expression.

Nicotinamide N-methyltransferase mediates lipofibroblast-myofibroblast transition and apoptosis resistance.

Metabolism controls cellular phenotype and fate. In this report, we demonstrate that nicotinamide N-methyltransferase (NNMT), a metabolic enzyme that regulates developmental stem cell transitions and tumor progression, is highly expressed in human idiopathic pulmonary fibrosis (IPF) lungs, and is induced by the pro-fibrotic cytokine, transforming growth factor-ß1 (TGF-ß1) in lung fibroblasts. NNMT silencing reduces the expression of extracellular matrix proteins, both constitutively and in response to TGF-ß1. Furthermore, NNMT controls the phenotypic transition from homeostatic, pro-regenerative lipofibroblasts to pro-fibrotic myofibroblasts. This effect of NNMT is mediated, in part, by the downregulation of lipogenic transcription factors, TCF21 and PPARγ, and the induction of a less proliferative but more differentiated myofibroblast phenotype. NNMT confers an apoptosis-resistant phenotype to myofibroblasts that is associated with the downregulation of pro-apoptotic members of the Bcl-2 family, including Bim and PUMA. Together, these studies indicate a critical role for NNMT in the metabolic reprogramming of fibroblasts to a pro-fibrotic and apoptosis-resistant phenotype and support the concept that targeting this enzyme may promote regenerative responses in chronic fibrotic disorders such as IPF.

Use of a pulmosphere model to evaluate drug antifibrotic responses in interstitial lung diseases.

BACKGROUND: Interstitial lung diseases (ILD) encompass a heterogenous group of diffuse parenchymal lung disorders characterized by variable degrees of inflammation and fibrosis. Pretherapeutic clinical testing models for such diseases can serve as a platform to test and develop effective therapeutic strategies. In this study, we developed patient derived 3D organoid model to recapitulate the disease process of ILDs. We characterized the inherent property of invasiveness in this model and tested for antifibrotic responses with an aim to develop a potential platform for personalized medicine in ILDs. METHODS: In this prospective study, 23 patients with ILD were recruited and underwent lung biopsy. 3D organoid-based models (pulmospheres) were developed from the lung biopsy tissues. Pulmonary functioning testing and other relevant clinical parameters were collected at the time of enrollment and follow up visits. The patient derived pulmospheres were compared to normal control pulmospheres obtained from 9 explant lung donor samples. These pulmospheres were characterized by their invasive capabilities and responsiveness to the antifibrotic drugs, pirfenidone and nintedanib. RESULTS: Invasiveness of the pulmospheres was measured by the zone of invasiveness percentage (ZOI%). The ILD pulmospheres (n = 23) had a higher ZOI% as compared to control pulmospheres (n = 9) (516.2 ± 115.6 versus 54.63 ± 19.6 respectively. ILD pulmospheres were responsive to pirfenidone in 12 of the 23 patients (52%) and responsive to nintedanib in all 23 patients (100%). Pirfenidone was noted to be selectively responsive in patients with connective tissue disease related ILD (CTD-ILD) at low doses. There was no correlation between the basal pulmosphere invasiveness, response to antifibrotics, and FVC change (Δ FVC). CONCLUSIONS: The 3D pulmosphere model demonstrates invasiveness which is unique to each individual subject and is greater in ILD pulmospheres as compared to controls. This property can be utilized to test responses to drugs such as antifibrotics. The 3D pulmosphere model could serve as a platform for the development of personalized approaches to therapeutics and drug development in ILDs and potentially other chronic lung diseases.

Fibrinogen mediates cadmium-induced macrophage activation and serves as a predictor of cadmium exposure in chronic obstructive pulmonary disease.

The etiologies of chronic obstructive pulmonary disease (COPD) remain unclear. Cadmium (Cd) causes both pulmonary fibrosis and emphysema; however, the predictors for Cd exposure and the mechanisms by which Cd causes COPD remain unknown. We demonstrated that Cd burden was increased in lung tissue from subjects with COPD and this was associated with cigarette smoking. Fibrinogen levels increased markedly in lung tissue of patients with smoked COPD compared with never-smokers and control subjects. Fibrinogen concentration also correlated positively with lung Cd load, but inversely with the predicted % of FEV1 and FEV1/FVC. Cd enhanced the secretion of fibrinogen in a cdc2-dependent manner, whereas fibrinogen further mediated Cd-induced peptidylarginine deiminase 2 (PAD2)-dependent macrophage activation. Using lung fibroblasts from CdCl2-treated Toll-like receptor 4 (TLR4) wild-type and mutant mice, we demonstrated that fibrinogen enhanced Cd-induced TLR4-dependent collagen synthesis and cytokine/chemokine production. We further showed that fibrinogen complexed with connective tissue growth factor (CTGF), which in turn promoted the synthesis of plasminogen activator inhibitor-2 (PAI-2) and fibrinogen and inhibited fibrinolysis in Cd-treated mice. The amounts of fibrinogen were increased in the bronchoalveolar lavage fluid (BALF) of Cd-exposed mice. Positive correlations were observed between fibrinogen with hydroxyproline. Our data suggest that fibrinogen is involved in Cd-induced macrophage activation and increases in fibrinogen in patients with COPD may be used as a marker of Cd exposure and predict disease progression.

Correlates of survival after autoantibody reduction therapy for acute IPF exacerbations.

BACKGROUND: No medical treatment has proven efficacy for acute exacerbations of idiopathic pulmonary fibrosis (AE-IPF), and this syndrome has a very high mortality. Based on data indicating humoral autoimmune processes are involved in IPF pathogenesis, we treated AE-IPF patients with an autoantibody reduction regimen of therapeutic plasma exchange, rituximab, and intravenous immunoglobulin. This study aimed to identify clinical and autoantibody determinants associated with survival after autoantibody reduction in AE-IPF. METHODS: Twenty-four(24) AE-IPF patients received the autoantibody reduction regimen. Plasma anti-epithelial autoantibody titers were determined by HEp-2 indirect immunofluorescence assays in 22 patients. RESULTS: Mean age of the patients was 70 + 7 years old, and 70% were male. Beneficial clinical responses that occurred early during therapy were a favorable prognostic indicator: supplemental O2 flows needed to maintain resting SaO2>92% significantly decreased and/or walk distances increased among all 10 patients who survived for at least one year. Plasma anti-HEp-2 autoantibody titers were ~-three-fold greater in survivors compared to non-survivors (p<0.02). Anti-HEp-2 titers >1:160 were present in 75% of the evaluable one-year survivors, compared to 29% of non-survivors, and 10 of 12 patients (83%) with anti-HEP-2 titers <1:160 died during the observation period (Hazard Ratio = 3.3, 95% Confidence Interval = 1.02-10.6, p = 0.047). CONCLUSIONS: Autoantibody reduction therapy is associated with rapid reduction of supplemental oxygen requirements and/or improved ability to ambulate in many AE-IPF patients. Facile anti-epithelial autoantibody assays may help identify those most likely to benefit from these treatments.

Mesenchymal stromal cell aging impairs the self-organizing capacity of lung alveolar epithelial stem cells.

Multicellular organisms maintain structure and function of tissues/organs through emergent, self-organizing behavior. In this report, we demonstrate a critical role for lung mesenchymal stromal cell (L-MSC) aging in determining the capacity to form three-dimensional organoids or 'alveolospheres' with type 2 alveolar epithelial cells (AEC2s). In contrast to L-MSCs from aged mice, young L-MSCs support the efficient formation of alveolospheres when co-cultured with young or aged AEC2s. Aged L-MSCs demonstrated features of cellular senescence, altered bioenergetics, and a senescence-associated secretory profile (SASP). The reactive oxygen species generating enzyme, NADPH oxidase 4 (Nox4), was highly activated in aged L-MSCs and Nox4 downregulation was sufficient to, at least partially, reverse this age-related energy deficit, while restoring the self-organizing capacity of alveolospheres. Together, these data indicate a critical role for cellular bioenergetics and redox homeostasis in an organoid model of self-organization and support the concept of thermodynamic entropy in aging biology.

Many tissues in the body are capable of regenerating by replacing defective or worn-out cells with new ones. This process relies heavily on stem cells, which are precursor cells that lack a set role in the body and can develop into different types of cells under the right conditions. Tissues often have their own pool of stem cells that they use to replenish damaged cells. But as we age, this regeneration process becomes less effective. Many of our organs, such as the lungs, are lined with epithelial cells. These cells form a protective barrier, controlling what substances get in and out of the tissue. Alveoli are parts of the lungs that allow oxygen and carbon dioxide to move between the blood and the air in the lungs. And alveoli rely on an effective epithelial cell lining to work properly. To replenish these epithelial cells, alveoli have pockets, in which a type of epithelial cell, known as AEC2, lives. These cells can serve as stem cells, developing into a different type of cell under the right conditions. To work properly, AEC2 cells require close interactions with another type of cell called L-MSC, which supports the maintenance of other cells and also has the ability to differentiate into several other cell types. Both cell types can be found close together in these stem cell pockets. So far, it has been unclear how aging affects how these cells work together to replenish the epithelial lining of the alveoli. To investigate, Chanda et al. probed AEC2s and L-MSCs in the alveoli of young and old mice. The researchers collected both cell types from young (2-3 months) and aged (22-24 months) mice. Various combinations of these cells were grown to form 3D structures, mimicking how the cells grow in the lungs. Young L-MSCs formed normal 3D structures with both young and aged AEC2 cells. But aged L-MSCs developed abnormal, loose structures with AEC2 cells (both young and old cells). Aged L-MSCs were found to have higher levels of an enzyme (called Nox4) that produces oxidants and other 'pro-aging' factors, compared to young L-MSCs. However, reducing Nox4 levels in aged L-MSCs allowed these cells to form normal 3D structures with young AEC2 cells, but not aged AEC2 cells. These findings highlight the varying effects specific stem cells have, and how their behaviour is affected by pro-aging factors. Moreover, the pro-aging enzyme Nox4 shows potential as a therapeutic target ­ downregulating its activity may reverse critical effects of aging in cells.

Restoration of SIRT3 gene expression by airway delivery resolves age-associated persistent lung fibrosis in mice.

Aging is a risk factor for progressive fibrotic disorders involving diverse organ systems, including the lung. Idiopathic pulmonary fibrosis, an age-associated degenerative lung disorder, is characterized by persistence of apoptosis-resistant myofibroblasts. In this report, we demonstrate that sirtuin-3 (SIRT3), a mitochondrial deacetylase, is downregulated in lungs of IPF human subjects and in mice subjected to lung injury. Over-expression of the SIRT3 cDNA via airway delivery restored capacity for fibrosis resolution in aged mice, in association with activation of the forkhead box transcription factor, FoxO3a, in fibroblasts, upregulation of pro-apoptotic members of the Bcl-2 family, and recovery of apoptosis susceptibility. While transforming growth factor-ß1 reduced levels of SIRT3 and FoxO3a in lung fibroblasts, cell non-autonomous effects involving macrophage secreted products were necessary for SIRT3-mediated activation of FoxO3a. Together, these findings reveal a novel role of SIRT3 in pro-resolution macrophage functions that restore susceptibility to apoptosis in fibroblasts via a FoxO3a-dependent mechanism.

Vimentin intermediate filament assembly regulates fibroblast invasion in fibrogenic lung injury.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease, with a median survival of 3-5 years following diagnosis. Lung remodeling by invasive fibroblasts is a hallmark of IPF. In this study, we demonstrate that inhibition of vimentin intermediate filaments (VimIFs) decreases the invasiveness of IPF fibroblasts and confers protection against fibrosis in a murine model of experimental lung injury. Increased expression and organization of VimIFs contribute to the invasive property of IPF fibroblasts in connection with deficient cellular autophagy. Blocking VimIF assembly by pharmacologic and genetic means also increases autophagic clearance of collagen type I. Furthermore, inhibition of expression of collagen type I by siRNA decreased invasiveness of fibroblasts. In a bleomycin injury model, enhancing autophagy in fibroblasts by an inhibitor of VimIF assembly, withaferin A (WFA), protected from fibrotic lung injury. Additionally, in 3D lung organoids, or pulmospheres, from patients with IPF, WFA reduced the invasiveness of lung fibroblasts in the majority of subjects tested. These studies provide insights into the functional role of vimentin, which regulates autophagy and restricts the invasiveness of lung fibroblasts.

"Preleukemic or smoldering" chronic myelogenous leukemia (CML):BCR-ABL1 positive: A brief case report.

Chronic myelogenous leukemia (CML), in the Chronic Phase (CP), is often suspected as a result of a complete blood count (CBC), which shows increased granulocytes, mostly mature including a peak in myelocytes, increased basophils, and rarely blasts and/or promyelocytes. Morphologic dysplasia is not present. CML is confirmed by detecting the characteristic Philadelphia chromosome (Ph)[t(9;22)(q34;q11.2)] by routine cytogenetics or fluorescent in situ hybridization (FISH) or molecular studies (RT-PCR) for the bcr-abl fusion gene. The most common feature of CML is an elevated WBC count, usually above 25×10(3)/µL, and frequently above 100×10(3)/µL. We report a case of confirmed Ph+CML with a normal CBC detected because of the presence of rare myelocytes and 2% basophils [Fig. 1]. Previous leukocyte counts for the preceding eight years were normal with the exception of one done four months prior to his presentation that showed an abnormal differential with 1% basophils, 2% metamyelocytes and 2% myelocytes.