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Acute Respiratory Distress Syndrome (ARDS) is a severe lung condition with a high mortality rate for which there are no effective therapeutics. The failure of the alveolar–capillary barrier, composed of lung endothelial (EC) and alveolar epithelial (AEC) cells, is a critical factor leading to excessive inflammation and edema characteristic of acute lung injury (ALI) pathophysiology. Phosphodiesterases (PDE) are enzymes well-recognized for their roles in regulating endothelial permeability and inflammation. Although PDE inhibitors are used as therapeutics for inflammatory diseases like COPD (chronic obstructive pulmonary disease), their efficacy in treating ARDS has not yet been established. In this study, we investigated the effects of ensifentrine, an FDA-approved novel dual PDE 3/4 inhibitor, on lung endothelial and epithelial dysfunction caused by methicillin-resistant S. aureus (MRSA), a pathogen involved in bacterial ARDS. Human primary lung endothelial cells and alveolar epithelial cell lines (A549 and immortalized AEC) were treated with heat-killed MRSA, and their responses were assessed in the presence or absence of ensifentrine. Ensifentrine given either pre- or post-exposure attenuated MRSA-induced increased lung endothelial permeability. VE-cadherin junctions, which serve to stabilize the EC barrier, were disrupted by MRSA; however, ensifentrine effectively prevented this disruption. Pre-treatment with ensifentrine protected against MRSA-induced EC pro-inflammatory signaling by inhibiting the expression of VCAM-1, ICAM-1, and by reducing the IL-6 and IL-8 release. In AEC, MRSA caused the upregulation of ICAM-1, the activation of NF-kB, and the production of IL-8, all of which were inhibited by ensifentrine. These results indicate that the dual inhibition of phosphodiesterases 3 and 4 by ensifentrine is barrier protective and attenuates MRSA-induced inflammation in both lung endothelial and epithelial cells. The PDE3/4 inhibitor ensifentrine may represent a promising novel strategy for the treatment of MRSA-induced ARDS. [ABSTRACT FROM AUTHOR]

Background: Analysis of existing mutations of Angiotensin-I-Converting Enzyme (ACE) led us to hypothesize that the carriers of damaging ACE mutations (accompanied by low ACE levels) could be at risk for the development of late-onset Alzheimer's disease (AD). Methodology/Principal findings: We quantified blood ACE levels in EDTA-containing plasma from 15 patients with 11 different heterozygous ACE mutations and estimated the effects of these mutations on ACE phenotypes, using a set of mAbs to ACE and two ACE substrates. We confirmed prior observations that the relatively frequent Y215C mutation in the N domain of ACE (present in ~1% of the population) is associated with both Alzheimer's disease (AD) and reduced plasma levels of ACE (~50% of controls), indicating that it likely results in a transport-deficient protein. In addition, we identified another 4 mutations in both ACE domains (M118T, C734Y, V992M and V997M) which are also associated with decreased ACE levels in the blood, and, thus, could be putative risk factors for late-onset AD. One of these mutations, C734Y, is likely transport-deficient, while the other mutations appear to influence ACE catalytic properties. The precipitation of mutant M118T by mAb 2D1 and ACE mutant C734Y by mAb 3F10 increased 2-3-fold compared to native ACE, and therefore, these mAbs could be markers of these mutations. Also, we identified a mutation I989T, which is associated with increased ACE levels in the blood. Conclusions/Significance: Conducting a systematic analysis of blood ACE levels in patients with ACE mutations holds promise for identifying individuals with low blood ACE levels. Such individuals may be at increased risk for late-onset AD. The patients with transport-deficient ACE mutations may benefit from therapeutic treatment with a combination of chemical and pharmacological chaperones and proteasome inhibitors, as was demonstrated previously using a cell model of the transport-deficient ACE mutation, Q1069R [Danilov et al, PLoS One, 2010]. [ABSTRACT FROM AUTHOR]

Backgrounds. Our recent analysis of 1200+ existing missense ACE mutations revealed that 400+ mutations are damaging and led us to hypothesize that carriers of heterozygous loss-of-function (LoF) ACE mutations (which result in low ACE levels) could be at risk for the development of late-onset Alzheimer's disease (AD). Methods. Here, we quantified blood ACE levels in EDTA plasma from 41 subjects with 10 different heterozygous ACE mutations, as well as 33 controls, and estimated the effect of these mutations on ACE phenotype using a set of mAbs to ACE and two ACE substrates. Results. We found that relatively frequent (~1%) AD-associated ACE mutations in the N domain of ACE, Y215C, and G325R are truly damaging and likely transport-deficient, with the ACE levels in plasma at only ~50% of controls. Another AD-associated ACE mutation, R1250Q, in the cytoplasmic tail, did not cause a decrease in ACE and likely did not affect surface ACE expression. We have also developed a method to identify patients with anti-catalytic mutations in the N domain. These mutations may result in reduced degradation of amyloid beta peptide Aβ42, an important component for amyloid deposition. Consequently, these could pose a risk factor for the development of AD. Conclusions. Therefore, a systematic analysis of blood ACE levels in patients with all ACE mutations has the potential to identify individuals at an increased risk of late-onset AD. These individuals may benefit from future preventive or therapeutic interventions involving a combination of chemical and pharmacological chaperones, as well as proteasome inhibitors, aiming to enhance ACE protein traffic. This approach has been previously demonstrated in our cell model of the transport-deficient ACE mutation Q1069R. [ABSTRACT FROM AUTHOR]

Lung endothelium resides at the interface between the circulation and the underlying tissue, where it senses biochemical and mechanical properties of both the blood as it flows through the vascular circuit and the vessel wall. The endothelium performs the bidirectional signaling between the blood and tissue compartments that is necessary to maintain homeostasis while physically separating both, facilitating a tightly regulated exchange of water, solutes, cells, and signals. Disruption in endothelial function contributes to vascular disease, which can manifest in discrete vascular locations along the artery-to-capillary-to-vein axis. Although our understanding of mechanisms that contribute to endothelial cell injury and repair in acute and chronic vascular disease have advanced, pathophysiological mechanisms that underlie site-specific vascular disease remain incompletely understood. In an effort to improve the translatability of mechanistic studies of the endothelium, the American Thoracic Society convened a workshop to optimize rigor, reproducibility, and translation of discovery to advance our understanding of endothelial cell function in health and disease. [ABSTRACT FROM AUTHOR]

Rationale: Cortactin, an actin-binding cytoskeletal protein, plays a crucial role in maintaining endothelial cell (EC) barrier integrity and regulating vascular permeability. The gene encoding cortactin, CTTN, is implicated in various lung inflammatory disorders. Despite this, the transcriptional regulation of CTTN by inflammatory stimuli and promoter SNPs remains unexplored. Methods: We transfected human lung ECs with a full-length CTTN promoters linked to a luciferase reporter to measure promoter activity. SNP-containing CTTN promoter was created via site-directed mutagenesis. Transfected ECs were exposed to LPS (PAMP), TNF-α (cytokine), cyclic stretch (CS), FG-4592 (HIF-inducer), NRF2 (anti-oxidant modulator), FTY-(S)-phosphate (endothelial barrier enhancer), and 5α-Aza (demethylation inducer). Immunohistochemistry was used to assess cortactin expression in mouse lungs exposed to LPS. Results: LPS, TNF-α, and 18%CS significantly increased CTTN promoter activities in a time-dependent manner (P<0.05). The variant rs34612166 (-212T/C) markedly enhanced LPS- and 18%CS- induced CTTN promoter activities (P<0.05). FG-4592 signifi- cantly boosted CTTN promoter activities (P<0.01), which were partially inhibited by HIF1α (KC7F2) and HIF2α (PT2385) inhibitors (P<0.05). NRF2 activator Bixin increased CTTN promoter activities, whereas NRF2 inhibitor Brusatol reduced them (P<0.05). 5α-Aza increased CTTN promoter activities by 2.9-fold (P<0.05). NF-κB response element mutations significantly reduced CTTN promoter activities response to LPS and TNFα. FTY-(S)-phosphate significantly increased CTTN promoter activities in 24 h. In vivo, cortactin levels were significantly elevated in inflammatory mouse lungs exposed to LPS for 18 h. Conclusion: CTTN transcriptional is significantly influenced by inflammatory factors and promoter variants. Cortactin, essential in mitigating inflammatory edema, presents a promising therapeutic target to alleviate severe inflammatory disorders. [ABSTRACT FROM AUTHOR]

Background: Endotracheal intubations (EIs) in the intensive care unit are high-risk procedures often performed by pulmonary and critical care medicine (PCCM) providers. The Accreditation Council for Graduate Medical Education mandates PCCM fellows' competency in this procedure; however, the learning experiences vary across programs. After conducting a needs assessment, we developed a curriculum unique to our institution to supplement our fellows' existing EI experiences in the operating room and the intensive care unit. Objective: To assess the curriculum's short-term objectives: knowledge acquisition, maintenance, and practical skills 1 year after participation. Methods: We administered a survey to the graduating PCCM fellows for two consecutive years. We designed the comprehensive airway curriculum to include didactic lectures and simulation-based education. The knowledge acquisition and maintenance were measured by administering a 26-question knowledge survey before and after curriculum participation and after 1 year. The fellows also received a practical examination 1 year after participation. To compare knowledge survey scores, we used paired t tests and permutation tests. Results: In the needs assessment, 56% of graduating fellows believed they were proficient in performing EI, whereas 33% were undecided and 11% believed they were unprepared. Most believed they would need more than two courses after graduation to be confident in independently performing EIs. Most will only occasionally have backup for EI from anesthesiology or emergency medicine in their future jobs. One identified barrier to learning EI was the lack of a formal curriculum. In the knowledge assessment, nine first-year fellows participated in the curriculum. The cohort's mean presurvey score was 13.0 (standard deviation [SD], 4.5) versus 18.6 (SD, 3.6) mean postsurvey score. One year after participation, the mean survey score was 17 (SD, 1.2). The postsurvey and 1-year postparticipation survey scores were significantly higher than the presurvey scores (P < 0.05). One year after participation, the practical examination showed most fellows retained skills in EI using ramped position, video and direct laryngoscopy, bag-mask ventilation, and oropharyngeal airway placement. Conclusion: The airway curriculum enhances fellows' knowledge acquisition and maintenance 1 year after participation. The practical examination 1 year after participation highlighted the skills retained and those still needing improvement. [ABSTRACT FROM AUTHOR]

Chronic cigarette smoke exposure decreases lung expression of WWOX which is known to protect the endothelial barrier during infectious models of acute respiratory distress syndrome (ARDS). Proteomic analysis of WWOX-silenced endothelial cells (ECs) was done using tandem mass tag mass spectrometry (TMT-MS). WWOX-silenced ECs as well as those isolated from endothelial cell Wwox knockout (EC Wwox KO) mice were subjected to cyclic stretch (18% elongation, 0.5 Hz, 4 h). Cellular lysates and media supernatant were harvested for assays of cellular signaling, protein expression, and cytokine release. These were repeated with dual silencing of WWOX and zyxin. Control and EC Wwox KO mice were subjected to high tidal volume ventilation. Bronchoalveolar lavage fluid and mouse lung tissue were harvested for cellular signaling, cytokine secretion, and histological assays. TMT-MS revealed upregulation of zyxin expression during WWOX knockdown which predicted a heightened inflammatory response to mechanical stretch. WWOX-silenced ECs and ECs isolated from EC Wwox mice displayed significantly increased cyclic stretch-mediated secretion of various cytokines (IL-6, KC/IL-8, IL-1β, and MCP-1) relative to controls. This was associated with increased ERK and JNK phosphorylation but decreased p38 mitogen-activated kinases (MAPK) phosphorylation. EC Wwox KO mice subjected to VILI sustained a greater degree of injury than corresponding controls. Silencing of zyxin during WWOX knockdown abrogated stretch-induced increases in IL-8 secretion but not in IL-6. Loss of WWOX function in ECs is associated with a heightened inflammatory response during mechanical stretch that is associated with increased MAPK phosphorylation and appears, in part, to be dependent on the upregulation of zyxin. NEW & NOTEWORTHY Prior tobacco smoke exposure is associated with an increased risk of acute respiratory distress syndrome (ARDS) during critical illness. Our laboratory is investigating one of the gene expression changes that occurs in the lung following smoke exposure: WWOX downregulation. Here we describe changes in protein expression associated with WWOX knockdown and its influence on ventilator-induced ARDS in a mouse model. [ABSTRACT FROM AUTHOR]

In this study we employ atomic force microscopy, supported by finite element analysis and fluorescence microscopy, to characterize the elastic properties accompanying cytoskeletal structural rearrangements of lung microvascular endothelial cells in response to barrier altering stimuli. Statistical analysis of elasticity data obtained from multiple cells demonstrates a heterogeneous cellular elastic response to barrier-enhancing and barrier-disrupting agents; sphingosine 1-phosphate (S1P) and thrombin, respectively. A small but detectable (10%) increase in the average elastic modulus of all cells is observed for S1P, which is accompanied by a corresponding significant decrease in cell thickness. Variable effects of thrombin on these parameters were observed. To account for possible substrate effects in our elasticity analysis, we analyzed only the low-force sections of the force-displacement curves and utilized a finite-thickness correction to the Hertzian model. Our finite element analysis results substantiate this approach. The heterogeneous elastic behavior correlates with differential cytoskeletal rearrangements observed with fluorescence microscopy.From the clinical editorThis team of investigators employed atomic force microscopy coupled with finite element analysis and fluorescence microscopy to characterize the elastic properties accompanying cytoskeletal structural rearrangements of lung microvascular endothelial cells in response to barrier altering stimuli, demonstrating the validity of their approach.

The article focuses on addressing the substantial heterogeneity in acute respiratory distress syndrome (ARDS) by examining the genotypic association of the MICBG406A polymorphism with clinical parameters in post-lung transplant primary graft dysfunction (PGD) and sepsis-induced ARDS. Topics include the potential of precision medicine approaches to improve therapeutic selection based on characterizing clinical and biological sources of heterogeneity.

The pathogenesis of pulmonary fibrosis involves complex interplay between cell types and signaling pathways. Recurrent alveolar epithelial injury can occur during pulmonary inflammation, causing dysregulation of epithelial repair. Dysregulated repair interacts with mesenchymal, inflammatory, and endothelial cells to trigger fibroblast‐to‐myofibroblast activation. CD26/dipeptidyl peptidase‐4 (DPP4) is a type II membrane protein mediating pleiotropic effect. However, the mechanistic role of CD26/DPP4 in pulmonary fibrosis remains unclear. In this study, we aimed to characterize Dpp4 deficiency in a mouse bleomycin (BLM)‐induced pulmonary fibrosis model and in cell culture systems of human lung fibroblasts (HLFs). Dpp4 knockout (Dpp4 KO) mouse lungs exhibited lower Ashcroft scale indices, collagen content, and numbers of fibroblasts and myofibroblasts compared with those in C57BL/6 wild‐type (WT) mice. Upregulation of Tgfb1 and Tgfb2 mRNA levels in the lungs after BLM treatment was lower in Dpp4 KO mice compared with those in WT mice. Although TGF‐β‐driven endothelial‐to‐mesenchymal transition (EndMT) has been implicated as one of the mechanisms of pulmonary fibrosis, a number of partial EndMT cells in lungs did not differ between Dpp4 KO mice and WT mice. The proliferation capacity and mRNA levels of COL1A1, a collagen deposition‐related gene, in cultured HLFs were suppressed in DPP4 small interfering RNA‐treated cells. This study indicates that the genetic deficiency of DPP4 has protective effects against BLM‐induced pulmonary fibrosis, partly through the reduction in TGF‐β expression and inhibition of fibroblast activation in the lung. Our study suggests that CD26/DPP4 inhibition is a potential therapeutic strategy for pulmonary fibrosis. [ABSTRACT FROM AUTHOR]