Low-dose vanadium pentoxide perturbed lung metabolism associated with inflammation and fibrosis signaling in male animal and in vitro models.

Vanadium is available as a dietary supplement and also is known to be toxic if inhaled, yet little information is available concerning the effects of vanadium on mammalian metabolism when concentrations found in food and water. Vanadium pentoxide (V+5) is representative of the most common dietary and environmental exposures, and prior research shows that low-dose V+5 exposure causes oxidative stress measured by glutathione oxidation and protein S-glutathionylation. We examined the metabolic impact of V+5 at relevant dietary and environmental doses (0.01, 0.1, and 1 ppm for 24 h) in human lung fibroblasts (HLFs) and male C57BL/6J mice (0.02, 0.2, and 2 ppm in drinking water for 7 mo). Untargeted metabolomics using liquid chromatography-high-resolution mass spectrometry (LC-HRMS) showed that V+5 induced significant metabolic perturbations in both HLF cells and mouse lungs. We noted 30% of the significantly altered pathways in HLF cells, including pyrimidines and aminosugars, fatty acids, mitochondrial and redox pathways, showed similar dose-dependent patterns in mouse lung tissues. Alterations in lipid metabolism included leukotrienes and prostaglandins involved in inflammatory signaling, which have been associated with the pathogenesis of idiopathic pulmonary fibrosis (IPF) and other disease processes. Elevated hydroxyproline levels and excessive collagen deposition were also present in lungs from V+5-treated mice. Taken together, these results show that oxidative stress from environmental V+5, ingested at low levels, could alter metabolism to contribute to common human lung diseases.NEW & NOTEWORTHY We used relevant dietary and environmental doses of Vanadium pentoxide (V+5) to examine its metabolic impact in vitro and in vivo. Using liquid chromatography-high-resolution mass spectrometry (LC-HRMS), we found significant metabolic perturbations, with similar dose-dependent patterns observed in human lung fibroblasts and male mouse lungs. Alterations in lipid metabolism included inflammatory signaling, elevated hydroxyproline levels, and excessive collagen deposition were present in V+5-treated lungs. Our findings suggest that low levels of V+5 could trigger pulmonary fibrotic signaling.

Alcohol-Induced Interleukin-17 Expression Causes Murine Lung Fibroblast-to-Myofibroblast Transdifferentiation via Thy-1 Down-Regulation.

BACKGROUND: Alcohol exposure induces TGFß1 and renders the lung susceptible to injury and disrepair. We determined that TGFß1 regulates myofibroblast differentiation through the loss of Thy-1 expression and consequent induction of α-SMA. TGFß1 is important for T helper 17 (Th17) differentiation and IL-17 secretion, which in turn participates in tissue repair. We hypothesized that alcohol induces Th17 differentiation via TGFß1 and that IL-17 produced by these cells contributes to the development of profibrotic lung myofibroblasts. METHODS: Primary lung fibroblasts (PLFs) were treated with alcohol, TGFß1, and IL-17 and then analyzed for Thy-1 expression and cell morphology. Naïve and Th17-polarized CD4+ T cells were exposed to alcohol and assessed for IL-17 expression. CD4+ T cells from alcohol-fed mice were analyzed for Th17 and IL-17 expression. Lungs of control-fed, bleomycin-treated and alcohol-fed, bleomycin-treated mice were analyzed for IL-17 protein expression. RESULTS: Alcohol-treated PLFs expressed lower levels of Thy-1 than untreated cells. TGFß1 or IL-17 exposure suppressed PLF Thy-1 expression. When administered together, TGFß1 and IL-17 additively down-regulated Thy-1 expression. Exposure of naïve and Th17-polarized CD4+ T cells to alcohol induced the Th17 phenotype and augmented their production of IL-17. CD4+ Th17+ levels are elevated in the peripheral compartment but not in the lungs of alcohol-fed animals. Treatment of the PLFs with IL-17 and alcohol induced α-SMA expression. Induction of α-SMA and myofibroblast morphology by IL-17 occurred selectively in a Thy-1- fibroblast subpopulation. Chronic alcohol ingestion augmented lung-specific IL-17 expression following bleomycin-induced lung injury. CONCLUSIONS: Alcohol exposure skews T cells toward a Th17 immune response that in turn primes the lung for fibroproliferative disrepair through loss of Thy-1 expression and induction of myofibroblast differentiation. These effects suggest that IL-17 and TGFß1 contribute to fibroproliferative disrepair in the lung and targeting these proteins could limit morbidity and mortality following lung injury in alcoholic individuals.

MiR-21-Mediated Suppression of Smad7 Induces TGFß1 and Can Be Inhibited by Activation of Nrf2 in Alcohol-Treated Lung Fibroblasts.

BACKGROUND: We previously demonstrated that chronic alcohol ingestion augments TGFß1 expression in the lung fibroblast and increases the risk of fibroproliferative disrepair in a mouse model of acute lung injury. The effect of alcohol on TGFß1 is mitigated by treatment with sulforaphane (SFP), which can activate nuclear factor (erythroid-derived 2)-like 2 (Nrf2). However, the mechanisms by which alcohol amplifies, or SFP attenuates, TGFß1 expression in the fibroblast are not known. MicroRNA (miR)-21 has been shown to inhibit Smad7, a TGFß1 signaling inhibitor. In this study, we hypothesized that alcohol augments TGFß1 expression through up-regulation of miR-21, which subsequently inhibits Smad7. METHODS: Primary mouse lung fibroblasts were cultured ± alcohol ± SFP and assessed for gene expression of miR-21, and gene and/or protein expression of Nrf2, Nrf2-regulated antioxidant enzymes, Smad7, STAT3, and TGFß1. NIH 3T3 fibroblasts were transfected with a miR-21 inhibitor and cultured ± alcohol. α-SMA, Smad7, and TGFß1 protein expression were then assessed. In parallel, NIH 3T3 lung fibroblasts were transfected with Nrf2 silencing RNA (siRNA) and cultured ± alcohol ± SFP. Gene expression of miR-21, Nrf2, Smad7, and TGFß1 was assessed. RESULTS: MiR-21 gene expression was increased by 12-fold at 48 hours, and Smad7 gene expression and protein expression were reduced by ~30% in alcohol-treated fibroblasts. In parallel, inhibition of miR-21 attenuated alcohol-mediated decrease in Smad7 and increase in TGFß1 and α-SMA protein expression. Treatment with SFP mitigated the effect of alcohol on miR-21, Smad7 and total and phosphorylated STAT3, and restored Nrf2-regulated antioxidant gene expression. Silencing of Nrf2 prevented the effect of SFP on miR-21, Smad7, and TGFß1 gene expression in alcohol-treated NIH 3T3 fibroblasts. CONCLUSIONS: Alcohol treatment increases TGFß1 in fibroblasts, at least in part, through augmentation of miR-21, which then inhibits Smad7 expression. These effects can be attenuated by activation of Nrf2 with SFP.

Nuclear Thioredoxin-1 Overexpression Attenuates Alcohol-Mediated Nrf2 Signaling and Lung Fibrosis.

BACKGROUND: Alcohol abuse, which impairs antioxidant defenses and promotes acute lung injury, increases Nrf2 nuclear translocation but nevertheless inhibits its activation of the antioxidant response element (ARE). Thioredoxin-1 (Trx1) is required for optimal Nrf2 binding and activation of the ARE, and we hypothesized that its inhibition contributes to impaired Nrf2-ARE signaling in the alcoholic lung. METHODS: Lung tissue and primary lung fibroblasts (PLFs) were isolated from C57/BL6 wild-type (WT) and transgenic mice overexpressing the human Trx1 gene with a nuclear localizing sequence (NLS-Tg); some mice consumed alcohol in water prior to lung tissue and PLF isolation; in some mice, acute lung injury was induced with intratracheal bleomycin. In other experiments, PLFs were isolated from WT and NLS-Tg mice and then exposed to alcohol. Finally, PLF isolated from WT mice were transfected with Trx1 expression vector containing either a cytosolic localized sequence (NES) or a nuclear localized sequence (NLS) prior to alcohol exposure. RESULTS: Alcohol treatment in vivo or in vitro decreased Trx1 expression, and bleomycin-treated alcohol-fed mice had fibrotic disrepair in their lungs. In parallel, whereas alcohol exposure in vitro increased TGFß1 expression and decreased Nrf2-ARE activity in PLF from WT mice, these effects were not observed in PLF from NLS-Tg mice. Finally, selective overexpression of Trx1 in the nucleus but not in the cytosol preserved Nrf2-ARE activity during alcohol exposure. CONCLUSIONS: Although alcohol-induced redox stress actually promotes Nrf2 nuclear translocation, the coincident suppression of Trx1 impairs Nrf2-ARE activity within the nuclear compartment. Nuclear overexpression of Trx1 restored Nrf2-ARE activity and attenuated alcohol-induced TGFß1 expression and alcohol-induced exaggerate response to bleomycin-induced acute lung injury.

TGF-ß1 epigenetically modifies Thy-1 expression in primary lung fibroblasts.

Idiopathic pulmonary fibrosis is a progressive lung disease that increases in incidence with age. We identified a profibrotic lung phenotype in aging mice characterized by an increase in the number of fibroblasts lacking the expression of thymocyte differentiation antigen 1 (Thy-1) and an increase in transforming growth factor (TGF)-ß1 expression. It has been shown that Thy-1 expression can be epigenetically modified. Lung fibroblasts (PLFs) were treated with TGF-ß1 ± DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-AZA) and analyzed for Thy-1 gene and protein expression, DNMT protein expression, and activity. α-Smooth muscle actin (α-SMA) and collagen type 1 (Col1A1) gene and protein expression was assessed. PLFs were transfected with DNMT1 silencing RNA ± TGF-ß1. TGF-ß1 inhibited Thy-1 gene and protein expression in PLFs, and cotreatment with 5-AZA ameliorated this effect and appeared to inhibit DNMT1 activation. TGF-ß1 induced Thy-1 promoter methylation as assessed by quantitative methyl PCR. Treatment with 5-AZA attenuated TGF-ß1-induced Col1A1 gene and protein expression and α-SMA gene expression (but not α-SMA protein expression). Inhibiting DNMT1 with silencing RNA attenuated TGF-ß1-induced DNMT activity and its downstream suppression of Thy-1 mRNA and protein expression as well as inhibited α-SMA mRNA and Col1A1 mRNA and protein expression, and showed a decreased trend in Thy-1 promoter methylation. Immunofluorescence for α-SMA suggested that 5-AZA inhibited stress fiber formation. These findings suggest that TGF-ß1 epigenetically regulates lung fibroblast phenotype through methylation of the Thy-1 promoter. Targeted inhibition of DNMT in the right clinical context might prevent fibroblast to myofibroblast transdifferentiation and collagen deposition, which in turn could prevent fibrogenesis in the lung and other organs.

The Use of TKM-100802 and Convalescent Plasma in 2 Patients With Ebola Virus Disease in the United States.

BACKGROUND: The current West Africa Ebola virus disease (EVD) outbreak has resulted in multiple individuals being medically evacuated to other countries for clinical management. METHODS: We report two patients who were transported from West Africa to the United States for treatment of EVD. Both patients received aggressive supportive care measures, as well as an investigational therapeutic (TKM-100802) and convalescent plasma. RESULTS: While one patient experienced critical illness with multi-organ failure requiring mechanical ventilation and renal replacement therapy, both patients recovered without serious long-term sequelae to date. CONCLUSIONS: It is unclear what role the experimental drug and convalescent plasma had in the recovery of these patients. Prospective clinical trials are needed to delineate the role of investigational therapies in the care of patients with EVD.

Critical Care for Multiple Organ Failure Secondary to Ebola Virus Disease in the United States.

OBJECTIVE: This report describes three patients with Ebola virus disease who were treated in the United States and developed for severe critical illness and multiple organ failure secondary to Ebola virus infection. The patients received mechanical ventilation, renal replacement therapy, invasive monitoring, vasopressor support, and investigational therapies for Ebola virus disease. DATA SOURCES: Patient medical records from three tertiary care centers (Emory University Hospital, University of Nebraska Medical Center, and Texas Health Presbyterian Dallas Hospital). STUDY SELECTION: Not applicable. DATA EXTRACTION: Not applicable. DATA SYNTHESIS: Not applicable. CONCLUSION: In the severe form, patients with Ebola virus disease may require life-sustaining therapy, including mechanical ventilation and renal replacement therapy. In conjunction with other reported cases, this series suggests that respiratory and renal failure may occur in severe Ebola virus disease, especially in patients burdened with high viral loads. Ebola virus disease complicated by multiple organ failure can be survivable with the application of advanced life support measures. This collective, multicenter experience is presented with the hope that it may inform future treatment of patients with Ebola virus disease requiring critical care treatment.

Endogenous distal airway progenitor cells, lung mechanics, and disproportionate lobar growth following long-term postpneumonectomy in mice.

Using a model of postpneumonectomy (PNY) compensatory lung growth in mice, we previously observed an increase in numbers of a putative endogenous distal airway progenitor cell population (CCSP(pos) /pro-SPC(pos) cells located at bronchoalveolar duct junctions [BADJs]), at 3, 7, and 14 days after pneumonectomy, returning to baseline at 28 days post-PNY. As the origin of these cells is poorly understood, we evaluated whether bone marrow cells contributed to the pool of these or other cells during prolonged post-PNY lung regrowth. Naïve and sex-mismatched chimeric mice underwent left PNY and were evaluated at 1, 2, and 3 months for numbers of BADJ CCSP(pos) /pro-SPC(pos) cells and presence of donor-derived marrow cells engrafted as airway or alveolar epithelium. Nonchimeric mice were also examined at 12 months after PNY for numbers of BADJ CCSP(pos) /pro-SPC(pos) cells. Notably, the right accessory lobe (RAL) continued to grow disproportionately over 12 months, a novel finding not previously described. Assessment of lung mechanics demonstrated an increase in lung stiffness following PNY, which significantly diminished over 1 year, but remained elevated relative to 1-year-old naïve controls. However, the number of CCSP(pos) /pro-SPC(pos) BADJ cells ≥1-month following PNY was equivalent to that found in naïve controls even after 12 months of continued RAL growth. Notably, no donor bone marrow-derived cells engrafted as airway or alveolar epithelial cells, including those at the BADJ, up to 3 months after PNY. These studies suggest that lung epithelial cells, including CCSP(pos) /pro-SPC(pos) cells, are not replenished from marrow-derived cells during post-PNY lung growth in mice.

TGFß1 mediates alcohol-induced Nrf2 suppression in lung fibroblasts.

BACKGROUND: Chronic alcohol ingestion induces the expression of transforming growth factor beta-1(TGFß1), inhibits nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated activation of the antioxidant response element (ARE), depletes alveolar glutathione pools, and potentiates acute lung injury. In this study, we examined the mechanistic relationship between TGFß1 and Nrf2-ARE signaling in the experimental alcoholic lung. METHODS: Wild-type mice were treated ± alcohol in drinking water for 8 weeks and their lungs were assessed for Nrf2 expression. In parallel, mouse lung fibroblasts were cultured ± alcohol and treated ± sulforaphane (SFP; an activator of Nrf2), ±TGFß1, ±TGFß1 neutralizing antibody, and/or ±activin receptor-like kinase 5 inhibitors (to block TGß1 receptor signaling) and then analyzed for the expression of Nrf2, Kelch-like ECH-associated protein 1 (Keap1) and TGFß1, Nrf2-ARE activity, and the expression of the Nrf2-ARE-dependent antioxidants glutathione s-transferase theta 2 (GSTT2) and glutamate-cysteine ligase catalytic subunit (GCLC). Finally, silencing RNA (siRNA) of Nrf2 was then performed prior to alcohol exposure and subsequent analysis of TGFß1 expression. RESULTS: Alcohol treatment in vivo or in vitro decreased Nrf2 expression in murine whole lung and lung fibroblasts, respectively. In parallel, alcohol exposure in vitro decreased Keap1 gene and protein expression in lung fibroblasts. Furthermore, alcohol exposure increased TGFß1 expression but decreased Nrf2-ARE activity and expression of the ARE-dependent genes for GSTT2 and GCLC. These effects of alcohol were prevented by treatment with SFP; in contrast, Nrf2 SiRNA expression exacerbated alcohol-induced TGFß1 expression. Finally, TGFß1 treatment directly suppressed Nrf2-ARE activity whereas blocking TGFß1 signaling attenuated alcohol-induced suppression of Nrf2-ARE activity. CONCLUSIONS: Alcohol-induced oxidative stress is mediated by TGFß1, which suppresses Nrf2-ARE-dependent expression of antioxidant defenses and creates a vicious cycle that feeds back to further increase TGFß1 expression. These effects of alcohol can be mitigated by activation of Nrf2, suggesting a potential therapy in individuals at risk for lung injury due to alcohol abuse.

Chronic alcohol ingestion primes the lung for bleomycin-induced fibrosis in mice.

BACKGROUND: Alcohol abuse increases the risk for acute lung injury (ALI). In both experimental models and in clinical studies, chronic alcohol ingestion causes airway oxidative stress and glutathione depletion and increases the expression of transforming growth factor beta-1 (TGFß1), a potent inducer of fibrosis, in the lung. Therefore, we hypothesized that alcohol ingestion could promote aberrant fibrosis following experimental ALI and that treatment with the glutathione precursor s-adenosylmethionine (SAMe) could mitigate these effects. METHODS: Three-month-old C57BL/6 mice were fed standard chow ± alcohol (20% v/v) in their drinking water for 8 weeks and ±SAMe (4% w/v) during the last 4 weeks. ALI was induced by intratracheal instillation of bleomycin (2.5 units/kg), and lungs were assessed histologically at 7 and 14 days for fibrosis and at 14 days for the expression of extracellular matrix proteins and TGFß1. RESULTS: Alcohol ingestion had no apparent effect on lung inflammation at 7 days, but at 14 days after bleomycin treatment, it increased lung tissue collagen deposition, hydroxyproline content, and the release of activated TGFß1 into the airway. In contrast, SAMe supplementation completely mitigated alcohol-induced priming of these aberrant fibrotic changes through decreased TGFß1 expression in the lung. In parallel, SAMe decreased alcohol-induced TGFß1 and Smad3 mRNA expressions by lung fibroblasts in vitro. CONCLUSIONS: These new experimental findings demonstrate that chronic alcohol ingestion renders the experimental mouse lung susceptible to fibrosis following bleomycin-induced ALI, and that these effects are likely driven by alcohol-mediated oxidative stress and its induction and activation of TGFß1.

Ethanol-exposed lung fibroblasts cause airway epithelial barrier dysfunction.

BACKGROUND: Chronic alcohol ingestion predisposes to lung injury and disrepair during sepsis. Our previous studies outlined roles for transforming growth factor-beta 1 (TGFß1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in epithelial barrier homeostasis and how alcohol perturbs their expression and signaling. Here we hypothesize that ethanol-exposed lung fibroblasts (LF) are a source of dysregulated TGFß1 and GM-CSF and thereby alter airway epithelial barrier function. METHODS: Human or rat LF were cultured ± ethanol for 2 weeks and then co-cultured with human or rat airway epithelial cells (AEC) seeded on Transwell permeable supports. In selected groups, a TGFß1 receptor type 1 (TGFßR1) inhibitor (SB431542) or a TGFß1 neutralizing antibody was applied. Transepithelial electrical resistance (TER) was measured prior to co-culture and on day 5 of co-culture. AEC were then analyzed for the expression of selected tight junction and mesenchymal proteins, and transwell membranes were analyzed by immunofluorescence microscopy for ZO-1 expression and localization. TGFß1 and GM-CSF levels in conditioned media from the co-cultures were quantified by ELISA. RESULTS: AEC co-cultured with ethanol-exposed LF (ELF) showed a significant reduction in TER and corresponding decreases in ZO-1 expression, whereas collagen type 1A1 and α-smooth muscle actin protein expression were increased. In parallel, in conditioned media from the ELF + AEC co-cultures, activated TGFß1 levels increased and GM-CSF levels decreased. Notably, all the effects of ELF on the AEC were prevented by blocking TGFß1 activity. CONCLUSIONS: Prior ethanol exposure to LF induces barrier dysfunction in naive AEC in a paracrine fashion through activation of TGFß1 signaling and suppression of GM-CSF. These experimental findings provide a potential mechanism by which chronic alcohol ingestion impairs airway epithelial integrity and renders individuals susceptible to lung injury.

Predicting glycemic control status and high blood glucose levels through voice characteristic analysis in patients with cystic fibrosis-related diabetes (CFRD).

Cystic fibrosis-related diabetes (CFRD) is associated with reduced life expectancy in adults with cystic fibrosis (CF). Voice analysis may be a convenient method for diagnosing and monitoring CFRD. This study aims to determine the relationship between voice characteristics and markers of glucose and glycemic control and to identify if voice analysis can predict high blood glucose levels and glycemic control in adults with CFRD. We conducted a prospective cross-sectional study in adults with CF from March to December 2021. We recorded 3-second voice samples of a sustained /a/ vowel and analyzed voice characteristic using the Computerized Speech Lab with the Multi-Dimensional Voice Program. In female participants with CFRD, the noise-to-harmonic ratio was significantly lower in those with HbA1c ≥ 7. Furthermore, fundamental frequency variation was significantly lower in both male and female participants with CFRD who had a glucose level of 200 mg/dL or higher at the time of collection. This finding was also associated with a high level of point-of-care glucose. The human voice has potential as a non-invasive tool for measuring glucose levels and glycemic control status in CFRD patients in the future.

Bone marrow-derived mesenchymal stromal cells inhibit Th2-mediated allergic airways inflammation in mice.

Bone marrow-derived mesenchymal stromal cells (BMSCs) mitigate inflammation in mouse models of acute lung injury. However, specific mechanisms of BMSC actions on CD4 T lymphocyte-mediated inflammation in vivo remain poorly understood. Limited data suggests promotion of Th2 phenotype in models of Th1-mediated diseases. However, whether this might alleviate or worsen Th2-mediated diseases such as allergic asthma is unknown. To ascertain the effects of systemic administration of BMSCs in a mouse model of Th2-mediated allergic airways inflammation, ovalbumin (OVA)-induced allergic airways inflammation was induced in wild-type C57BL/6 and BALB/c mice as well as in interferon-γ (IFNγ) receptor null mice. Effects of systemic administration during antigen sensitization of either syngeneic or allogeneic BMSC on airways hyperreactivity, lung inflammation, antigen-specific CD4 T lymphocytes, and serum immunoglobulins were assessed. Both syngeneic and allogeneic BMSCs inhibited airways hyperreactivity and lung inflammation through a mechanism partly dependent on IFNγ. However, contrary to existing data, BMSCs did not affect antigen-specific CD4 T lymphocyte proliferation but rather promoted Th1 phenotype in vivo as assessed by both OVA-specific CD4 T lymphocyte cytokine production and OVA-specific circulating immunoglobulins. BMSCs treated to prevent release of soluble mediators and a control cell population of primary dermal skin fibroblasts only partly mimicked the BMSC effects and in some cases worsened inflammation. In conclusion, BMSCs inhibit Th2-mediated allergic airways inflammation by influencing antigen-specific CD4 T lymphocyte differentiation. Promotion of a Th1 phenotype in antigen-specific CD4 T lymphocytes by BMSCs is sufficient to inhibit Th2-mediated allergic airways inflammation through an IFNγ-dependent process.

Use of estrogen supplementation is associated with higher quality of life scores in women with cystic fibrosis.

The association of estrogen supplementation use and quality of life in women with cystic fibrosis (CF) is not well characterized. In this cross-sectional study, women with CF completed quality of life questionnaires during a routine CF clinic visit. The use of estrogen supplementation was associated with higher quality of life scores in all domains of the CF questionnaire-revised (CFQ-R) and was significant in the role limitations and respiratory domains. Most participants who were not currently using estrogen supplementation had previously used estrogen supplementation. Most participants had used estrogen to regulate menses, prevent pregnancy and control symptoms around menses. Use of estrogen supplementation was not associated with differences in life-space mobility or screening for sexual dysfunction. This is the largest study to date investigating the association of estrogen supplementation and quality of life in women with CF. Prospective randomized studies are needed to clarify the association of estrogen supplementation and quality of life in women with CF.

Cell therapy approaches for lung diseases: current status.

Recent findings suggest that embryonic stem cells and stem cells derived from adult tissues, including bone marrow and umbilical cord blood, could be utilized in repair and regeneration of injured or diseased lungs. This is an exciting and rapidly moving field that holds promise as a therapeutic approach for variety of lung diseases. Although initial emphasis was on engraftment of stem cells in lung, more recent studies demonstrate that mesenchymal stem cells (MSCs) can modulate local inflammatory and immune responses in mouse lung disease models including acute lung injury and pulmonary fibrosis. Further, on the basis of initial reports of safety and efficacy following allogeneic administration of MSCs to patients with Crohn's disease or with graft-versus-host disease, a recent trial has been initiated to study the effect of MSCs in patients with chronic obstructive pulmonary disease. Notably, several recent clinical trials have demonstrated potential benefit of autologous stem cell administration in patient with pulmonary hypertension. In this review, we will describe recent advances in cell therapy with the focus on MSCs and their potential roles in lung development and repair.

Alcohol induces TGFß1 via downregulation of miR-1946a in murine lung fibroblast.

Exaggerated transforming growth factor-beta 1 (TGFß1) expression worsens fibroproliferation following bleomycin-induced lung injury in alcohol-fed mice. MicroRNA (miR)-1946a is predicted to bind to the TGFß1 3' untranslated region (UTR), thereby inhibiting its transcription. We hypothesize that alcohol suppresses miR-1946a and induces TGFß1. Primary murine lung fibroblasts (PLFs) were cultured ± alcohol, miR-1946a mimic or inhibitor, and TGFß1 signaling inhibitors. miR-1946a was analyzed after alcohol treatment in vitro and in vivo. TGFß1 expression and TGFß1 3'UTR-luciferase activity was quantified. We showed that alcohol suppressed miR-1946a in the alcohol-fed mouse lungs and PLFs. MiR-1946a inhibitor increased TGFß1 expression in the fibroblast. MiR-1946a mimic treatment suppressed TGFß1 gene expression and TGFß1 3'UTR activity. Overexpression of miR1946a inhibited alcohol-induced TGFß1 gene and protein expression as well as alcohol-induced TGFß1 and α-smooth muscle actin (SMA) protein expression in PLFs. In conclusion, miR-1946a modulates TGFß1 expression through direct interaction with TGFß1 3'UTR. These findings identify a novel mechanism by which alcohol induces TGFß1 in the lung.

Slow on the Uptake, Progression to Heartbreak.

The prevalence of serotonin syndrome increases over the past several years as more serotonergic medications are being used in clinical practice. It is a potentially lethal condition caused by excessive serotonergic activity. Common causes of serotonin syndrome are the use of prescription medications, illicit drugs, or a combination of substances, leading to an increase in the activity of serotonin in the central and peripheral nervous system. The clinical symptoms range from mild to severe. We report a case of a 25-year-old woman with polysubstance abuse, including cocaine, who presented with confusion, rigidity, high-grade fever, and reduced biventricular function on echocardiogram. Based on the combination of substance used history, clinical presentation, and echocardiogram findings, she was diagnosed with serotonin syndrome complicated by takotsubo cardiomyopathy. She improved after being treated in the intensive care unit and was discharged from the hospital. This patient demonstrates the importance of recognizing and promptly initiating management of serotonin syndrome in order to improve morbidity and mortality.

Derivation of lung epithelium from human cord blood-derived mesenchymal stem cells.

RATIONALE: Recent studies have suggested that both embryonic stem cells and adult bone marrow stem cells can participate in the regeneration and repair of diseased adult organs, including the lungs. However, the extent of airway epithelial remodeling with adult marrow stem cells is low, and there are no available in vivo data with embryonic stem cells. Human umbilical cord blood contains both hematopoietic and nonhematopoietic stem cells, which have been used clinically as an alternative to bone marrow transplantation for hematologic malignancies and other diseases. OBJECTIVES: We hypothesized that human umbilical cord blood stem cells might be an effective alternative to adult bone marrow and embryonic stem cells for regeneration and repair of injured airway epithelium. METHODS: Human cord blood was obtained from normal deliveries at the University of Vermont. Cultured plastic adherent cells were characterized as mesenchymal stem cells (MSCs) by flow cytometry and differentiation assays. Cord blood-derived MSCs (CB-MSCs) were cultured in specialized airway growth media or with specific growth factors, including keratinocyte growth factor and retinoic acid. mRNA and protein expression were analyzed with PCR and immunofluorescent staining. CB-MSCs were systematically administered to immunotolerant, nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice. Lungs were analyzed for presence of human cells. MEASUREMENTS AND MAIN RESULTS: When cultured in specialized airway growth media or with specific growth factors, CB-MSCs differentially expressed Clara cell secretory protein (CCSP), cystic fibrosis transmembrane conductance regulator (CFTR), surfactant protein C, and thyroid transcription factor-1 mRNA, and CCSP and CFTR protein. Furthermore, CB-MSCs were easily transduced with recombinant lentiviral vectors to express human CFTR. After systemic administration to immunotolerant, NOD-SCID, mice, rare cells were found in the airway epithelium that had acquired cytokeratin and human CFTR expression. CONCLUSIONS: CB-MSCs appear to be comparable to MSCs obtained from adult bone marrow in ability to express phenotypic markers of airway epithelium and to participate in airway remodeling in vivo.

HIV-1 Protein gp120 Induces Mouse Lung Fibroblast-to-Myofibroblast Transdifferentiation via CXCR4 Activation.

BACKGROUND: Individuals with HIV have ∼2-fold increased risk of developing pulmonary fibrosis. The mechanism(s) by which this occurs has yet to be determined. HIV-1 protein gp120 activates CXCR4 in the lymphocyte, promoting a variety of intracellular signaling pathways including those common to TGFß1 associated with lung fibroblast-to-myofibroblast transdifferentiation. We hypothesized that gp120 promotes pulmonary fibrotic changes via activation of CXCR4 in the lung fibroblast. METHODS: Mouse primary lung fibroblasts (PLFs) were cultured ± gp120, then analyzed for α-SMA expression and stress fiber formation. In parallel, PLFs were cultured ± gp120 ± AMD3100 (a CXCR4 antagonist), and α-SMA, pan and phospho-Akt, and total and phospho-MAPK (or ERK1/2) protein expression was quantified. Finally, lungs and PLFs from wild-type and HIV-1 transgenic mice were analyzed for hydroxyproline and α-SMA content. RESULTS: gp120 treatment increased α-SMA expression and myofibroblast differentiation in PLFs. gp120 treatment activated phosphorylation of ERK1/2, but not PI3K-Akt. Pretreatment with AMD3100 inhibited gp120-induced ERK1/2 phosphorylation and gp120-induced α-SMA expression. In parallel, there was a significant increase in hydroxyproline content in lungs from older HIV-1 transgenic mice and a >3-fold increase in α-SMA expression in PLFs isolated from HIV-1 transgenic mice. CONCLUSIONS: gp120 induces α-SMA expression and fibroblast-to-myofibroblast transdifferentiation by activating the CXCR4-ERK1/2 signaling pathway in mouse PLFs. Lungs of older HIV-1 transgenic mice contain higher hydroxyproline content and their PLFs have a striking increase in α-SMA expression. These results suggest a mechanism by which individuals with HIV are at increased risk of developing pulmonary fibrotic changes as they age.