Brigham C. Willis

Induction of Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells by Transforming Growth Factor-β1: Potential Role in Idiopathic Pulmonary Fibrosis

The hallmark of idiopathic pulmonary fibrosis (IPF) is the myofibroblast, the cellular origin of which in the lung is unknown. We hypothesized that alveolar epithelial cells (AECs) may serve as a source of myofibroblasts through epithelial-mesenchymal transition (EMT). Effects of chronic exposure to transforming growth factor (TGF)-beta1 on the phenotype of isolated rat AECs in primary culture and a rat type II cell line (RLE-6TN) were evaluated. Additionally, tissue samples from patients with IPF were evaluated for cells co-expressing epithelial (thyroid transcription factor (TTF)-1 and pro-surfactant protein-B (pro-SP-B), and mesenchymal (alpha-smooth muscle actin (alpha-SMA)) markers. RLE-6TN cells exposed to TGF-beta1 for 6 days demonstrated increased expression of mesenchymal cell markers and a fibroblast-like morphology, an effect augmented by tumor necrosis factor-alpha (TNF-alpha). Exposure of rat AECs to TGF-beta1 (100 pmol/L) resulted in increased expression of alpha-SMA, type I collagen, vimentin, and desmin, with concurrent transition to a fibroblast-like morphology and decreased expression of TTF-1, aquaporin-5 (AQP5), zonula occludens-1 (ZO-1), and cytokeratins. Cells co-expressing epithelial markers and alpha-SMA were abundant in lung tissue from IPF patients. These results suggest that AECs undergo EMT when chronically exposed to TGF-beta1, raising the possibility that epithelial cells may serve as a novel source of myofibroblasts in IPF.

Role of Endoplasmic Reticulum Stress in Epithelial–Mesenchymal Transition of Alveolar Epithelial Cells: Effects of Misfolded Surfactant Protein

Endoplasmic reticulum (ER) stress has been implicated in alveolar epithelial type II (AT2) cell apoptosis in idiopathic pulmonary fibrosis. We hypothesized that ER stress (either chemically induced or due to accumulation of misfolded proteins) is also associated with epithelial-mesenchymal transition (EMT) in alveolar epithelial cells (AECs). ER stress inducers, thapsigargin (TG) or tunicamycin (TN), increased expression of ER chaperone, Grp78, and spliced X-box binding protein 1, decreased epithelial markers, E-cadherin and zonula occludens-1 (ZO-1), increased the myofibroblast marker, α-smooth muscle actin (α-SMA), and induced fibroblast-like morphology in both primary AECs and the AT2 cell line, RLE-6TN, consistent with EMT. Overexpression of the surfactant protein (SP)-C BRICHOS mutant SP-C(ΔExon4) in A549 cells increased Grp78 and α-SMA and disrupted ZO-1 distribution, and, in primary AECs, SP-C(ΔExon4) induced fibroblastic-like morphology, decreased ZO-1 and E-cadherin and increased α-SMA, mechanistically linking ER stress associated with mutant SP to fibrosis through EMT. Whereas EMT was evident at lower concentrations of TG or TN, higher concentrations caused apoptosis. The Src inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4]pyramidine) (PP2), abrogated EMT associated with TN or TG in primary AECs, whereas overexpression of SP-C(ΔExon4) increased Src phosphorylation, suggesting a common mechanism. Furthermore, increased Grp78 immunoreactivity was observed in AT2 cells of mice after bleomycin injury, supporting a role for ER stress in epithelial abnormalities in fibrosis in vivo. These results demonstrate that ER stress induces EMT in AECs, at least in part through Src-dependent pathways, suggesting a novel role for ER stress in fibroblast accumulation in pulmonary fibrosis.

N-acetylcysteine inhibits alveolar epithelial-mesenchymal transition

The ability of transforming growth factor-beta1 (TGF-beta1) to induce epithelial-mesenchymal transition (EMT) in alveolar epithelial cells (AEC) in vitro and in vivo, together with the demonstration of EMT in biopsies of idiopathic pulmonary fibrosis (IPF) patients, suggests a role for TGF-beta1-induced EMT in disease pathogenesis. We investigated the effects of N-acetylcysteine (NAC) on TGF-beta1-induced EMT in a rat epithelial cell line (RLE-6TN) and in primary rat alveolar epithelial cells (AEC). RLE-6TN cells exposed to TGF-beta1 for 5 days underwent EMT as evidenced by acquisition of a fibroblast-like morphology, downregulation of the epithelial-specific protein zonula occludens-1, and induction of the mesenchymal-specific proteins alpha-smooth muscle actin (alpha-SMA) and vimentin. These changes were inhibited by NAC, which also prevented Smad3 phosphorylation. Similarly, primary alveolar epithelial type II cells exposed to TGF-beta1 also underwent EMT that was prevented by NAC. TGF-beta1 decreased cellular GSH levels by 50-80%, whereas NAC restored them to approximately 150% of those found in TGF-beta1-treated cells. Treatment with glutathione monoethyl ester similarly prevented an increase in mesenchymal marker expression. Consistent with its role as an antioxidant and cellular redox stabilizer, NAC dramatically reduced intracellular reactive oxygen species production in the presence of TGF-beta1. Finally, inhibition of intracellular ROS generation during TGF-beta1 treatment prevented alveolar EMT, but treatment with H2O2 alone did not induce EMT. We conclude that NAC prevents EMT in AEC in vitro, at least in part through replenishment of intracellular GSH stores and limitation of TGF-beta1-induced intracellular ROS generation. We speculate that beneficial effects of NAC on pulmonary function in IPF may be mediated by inhibitory effects on alveolar EMT.

Prolonged use of dexmedetomidine in the paediatric cardiothoracic intensive care unit.

BACKGROUND Dexmedetomidine is an alpha2-adrenergic agonist that causes sleep-like sedation and mild analgesia without narcosis or respiratory depression, and has relative cardiovascular stability. Due to these properties, it may be an effective agent for prolonged use in the sedation of patients in the paediatric cardiothoracic intensive care unit. We reviewed our experience with the drug to detail its safety and efficacy. METHODS We conducted a retrospective chart review of all patients who received dexmedetomidine over a six month period in a dedicated paediatric cardiothoracic intensive care unit. Patients were identified from pharmacy records showing administration of drugs. We collected demographic data, information relating to doses of dexmedetomidine, physiologic parameters, and clinical outcomes. RESULTS We identified 54 patients who received the drug. The median age of recipients was 6 months, with a range from 1 day to 16 years. The mean duration of administration was 37.3 hours, with a range from 2 to 177 hours. The mean duration of continuation of administration after extubation was 16.7 hours, with a range from zero to 112.5 hours. Physiologically, there were no clinically significant changes in mean arterial pressure, heart rate, respiratory rate, or saturations of oxygen before, during, or after utilization of the drug. Use of dexmedetomidine significantly reduced the need to administer narcotics, and scores using the COMFORT system were not different between patients who received dexmedetomidine and those who did not. CONCLUSIONS In this limited and retrospective review, dexmedetomidine was found to be safe and efficacious. Its use as a sedative agent for extended periods of time in critically-ill children deserves investigation in a prospective and controlled manner.

Immunosuppression-induced bronchial epithelial–mesenchymal transition: A potential contributor to obliterative bronchiolitis

OBJECTIVE Obliterative bronchiolitis is the predominant histopathologic finding in patients with chronic rejection after lung transplant. This fibroproliferative transformation within small airways of lung allograft is poorly understood; however, studies suggest epithelial-mesenchymal transition plays a role. Transplant immunosuppressive therapy has been shown to cause epithelial-mesenchymal transition in renal tubular epithelial cells, with subsequent fibrosis. This study explored whether immunosuppressive therapy contributes to epithelial-mesenchymal transition in airway epithelial cells. METHODS Bronchial epithelial cell line RL-65 was treated 3 to 5 days with several immunosuppressive agents, including cyclosporine (INN ciclosporin), tacrolimus, azathioprine, mycophenolic acid, sirolimus, prednisone, and transforming growth factor β1 as control. We then analyzed cells for presence of mesenchymal morphology and protein markers. RESULTS Treatment with cyclosporine, azathioprine, mycophenolate, and sirolimus resulted in elongated and irregular cell shape, and all but azathioprine showed loss of cell-cell adhesions relative to vehicle-treated cells. Expressions of extracellular matrix proteins, fibronectin and collagen, along with mesenchymal marker, vimentin, were significantly upregulated. Immunofluorescence showed loss of E-cadherin at cell membranes and cytoskeletal rearrangements typical of epithelial-mesenchymal transition. These immunosuppressive agents also increased transforming growth factor produced by cells; however, tacrolimus- and prednisone-treated cells maintained epithelial morphology, baseline levels of matrix protein expression, and transforming growth factor production levels. CONCLUSIONS Overall, we found that certain immunosuppressive agents may contribute to partial epithelial-mesenchymal transition in bronchial epithelial cells, specifically increasing production of excessive extracellular matrix proteins. This may provide novel insights into the pathogenesis of obliterative bronchiolitis after lung transplant.

Respiratory inductance plethysmography used to diagnose bilateral diaphragmatic paralysis: a case report.

Objective: To report the use of respiratory inductance plethysmography in the diagnosis and management for a case of bilateral diaphragmatic paralysis after repeated sternotomies in a 23-month-old child. Design: Case report. Setting: A 15-bed pediatric cardiothoracic intensive care unit in an academic childrens hospital. Interventions: The patient could not be weaned from the ventilator after a repeat sternotomy for pulmonary artery reconstruction. Pulmonary function test results were within normal limits, and plain film radiography, ultrasonography, and fluoroscopy were unable to establish a definitive diagnosis. Evaluation of thoracoabdominal synchrony was undertaken using respiratory inductance plethysmography (RespiTrace). The work of breathing was assessed using esophageal manometry to obtain the pressure-rate product. Results: During spontaneous breathing, complete thoracoabdominal asynchrony was noted, with clockwise Konno–Mead loops and associated phase angles of nearly 180 degrees. The pressure-rate product was 120 cm H2O/min, indicating elevated work of breathing. The pressure-rate product decreased dramatically, as indicated by measurement and observation, in response to increased levels of continuous positive airway pressure. Conclusions: The diagnosis of bilateral diaphragmatic paralysis can be confirmed by measurement of thoracoabdominal synchrony. Therapeutic and diagnostic application of continuous positive airway pressure may predict response to diaphragmatic plication. Controlled trials comparing measurement of thoracoabdominal synchrony with standard methods for the early diagnosis of diaphragmatic paralysis are needed.

Epithelial-mesenchymal transition: potential role in obliterative bronchiolitis?

Lung transplantation remains the only viable option for many patients suffering from a variety of progressive or intractable end-stage lung diseases. Despite significant advances in the prevention of early graft rejection, ischaemia-reperfusion injury and acute management of lung transplant recipients, significant challenges remain in the chronic management of patients after lung transplantation.1 In this issue of Thorax , Borthwick and colleagues2 provide intriguing new evidence that implicates the airway epithelium directly in the pathogenesis of bronchiolitis obliterans syndrome (BOS), the most significant factor in determining long-term lung graft survival ( see page 770 ). As discussed in the study, the pathological lesion of BOS is obliterative bronchiolitis (OB), which recently has been postulated to be at least partially a disease of aberrant epithelial repair processes.3 Borthwick and colleagues provide evidence that epithelial to mesenchymal transition (EMT), a process whereby epithelial cells undergo a complete lineage transition to become fibroblasts and/or myofibroblasts, may underlie the dysfunctional airway repair processes that lead to OB. This study, and others like it, attempt to redefine traditional paradigms regarding normal airway epithelial biology and disease pathogenesis, and have the potential to lead to entirely new therapeutic avenues for previously untreatable disease processes such as BOS. OB is characterised by initial inflammation of the small airways followed by airway remodelling, aberrant epithelial regeneration and repair, proliferation of fibroblasts and myofibroblasts, deposition of extracellular matrix (ECM) and eventual airway obstruction.4 The initial inflammatory response is the result of an allogeneic immune response initiated against donor antigens in the graft endothelial and airway epithelial cells. This response characteristically generates antigen-specific graft-infiltrating destructive lymphocytes. The lymphocytes facilitate activation of macrophages and a variety of other inflammatory cells, with resultant …

Meconium Increases Type 1 Angiotensin II Receptor Expression and Alveolar Cell Death

The pulmonary renin-angiotensin system (RAS) contributes to inflammation and epithelial apoptosis in meconium aspiration. It is unclear if both angiotensin II receptors (ATR) contribute, where they are expressed and if meconium modifies subtype expression. We examined ATR subtypes in 2 wk rabbit pup lungs before and after meconium exposure and with and without captopril pretreatment or type 1 receptor (AT1R) inhibition with losartan, determining expression and cellular localization with immunoblots, RT-PCR and immunohistochemistry, respectively. Responses of cultured rat alveolar type II pneumocytes were also examined. Type 2 ATR were undetected in newborn lung before and after meconium instillation. AT1R were expressed in pulmonary vascular and bronchial smooth muscle and alveolar and bronchial epithelium. Meconium increased total lung AT1R protein approximately 3-fold (p = 0.006), mRNA 29% (p = 0.006) and immunostaining in bronchial and alveolar epithelium and smooth muscle, which were unaffected by captopril and losartan. Meconium also increased AT1R expression >3-fold in cultured type II pneumocytes and caused concentration-dependent cell death inhibited by losartan. Meconium increases AT1R expression in newborn rabbit lung and cultured type II pneumocytes and induces AT1R-mediated cell death. The pulmonary RAS contributes to the pathogenesis of meconium aspiration through increased receptor expression.

250: METHYLENE BLUE TREATMENT OF VASOPLEGIC SYNDROME IN PEDIATRIC PATIENTS

Critical Care Medicine • Volume 46 • Number 1 (Supplement) www.ccmjournal.org Learning Objectives: Vasoplegia is hypothesized to result from the disruption of blood vessel endothelial homeostasis through dysregulation of the nitric oxide (NO) pathway. In cases of NO upregulation, methylene blue appears to be effective at inhibiting the final cellular messengers of the NO pathway without the myocardial depression and other adverse side effects seen in nonspecific NO inhibition. The objective of this study is to investigate if there is an association between the use of methylene blue and an increase in mean arterial blood pressure subsequently twelve hours after treatment. Methods: A single center, retrospective cohort study was performed by reviewing the charts of patients who received methylene blue in the Cardiovascular Intensive Care Unit of Phoenix Children’s Hospital from February 1st, 2013 to June 30th, 2016. Trained investigators manually extracted demographic data, vital sign data and vasoactive inotrope scores during a designated collection period after the administration of methylene blue. Mixed effects models were used to estimate the change in mean arterial pressure over time after initiating treatment with methylene blue while controlling for standardized inotrope dose (vasoactive inotrope score or VIS) and other confounding variables. Results: Thirty-two patients were identified as receiving methylene blue during the study period. Four were excluded because methylene blue was used for diagnostic procedures and not for treatment of vasoplegia. Described uses of methylene blue included treatment of vasoplegia from cardiogenic shock (n = 7), post cardiopulmonary bypass vasoplegia (n = 16), ECMO decannulation hemodynamic instability (n = 2), and septic shock (n = 3). Analysis of mean arterial blood pressure after controlling for inotrope dose using vasoactive inotrope scores revealed a statistically significant correlation between methylene blue use and an increase in mean arterial blood pressure of approximately 0.9 mmHg per hour or 10.8 mmHg over a twelve hour period (p = 0.0003). Conclusions: Our study suggests that using methylene blue to treat vasoplegic syndrome may be associated with an increase in mean arterial blood pressure over time. Further studies are needed to investigate the efficacy of methylene blue use in pediatric patients.