American journal of physiology. Lung cellular and molecular physiology最新文献

In pulmonary hypertension (PHTN), a metabolic shift to aerobic glycolysis promotes a hyperproliferative, apoptosis-resistant phenotype in pulmonary arterial smooth muscle cells (PASMCs). Enhanced glycolysis induces extracellular acidosis, which can activate proton-sensing membrane receptors and ion channels. We previously reported that activation of the proton-gated cation channel acid-sensing ion channel 1a (ASIC1a) contributes to the development of hypoxic PHTN. Therefore, we hypothesize that enhanced glycolysis and subsequent acidification of the PASMC extracellular microenvironment activate ASIC1a in hypoxic PHTN. We observed decreased oxygen consumption rate and increased extracellular acidification rate in PASMCs from chronic hypoxia (CH)-induced PHTN rats, indicating a shift to aerobic glycolysis. In addition, we found that intracellular alkalization and extracellular acidification occur in PASMCs following CH and in vitro hypoxia, which were prevented by the inhibition of glycolysis with 2-deoxy-d-glucose (2-DG). Inhibiting H⁺ transport/secretion through carbonic anhydrases, Na⁺/H⁺ exchanger 1, or vacuolar-type H⁺-ATPase did not prevent this pH shift following hypoxia. Although the putative monocarboxylate transporter 1 (MCT1) and -4 (MCT4) inhibitor syrosingopine prevented the pH shift, the specific MCT1 inhibitor AZD3965 and/or the MCT4 inhibitor VB124 were without effect, suggesting that syrosingopine targets the glycolytic pathway independent of H⁺ export. Furthermore, 2-DG and syrosingopine prevented enhanced ASIC1a-mediated store-operated Ca²⁺ entry in PASMCs from CH rats. These data suggest that multiple H⁺ transport mechanisms contribute to extracellular acidosis and that inhibiting glycolysis-rather than specific H⁺ transporters-more effectively prevents extracellular acidification and ASIC1a activation. Together, these data reveal a novel pathological relationship between glycolysis and ASIC1a activation in hypoxic PHTN.NEW & NOTEWORTHY In pulmonary hypertension, a metabolic shift to aerobic glycolysis drives a hyperproliferative, apoptosis-resistant phenotype in pulmonary arterial smooth muscle cells. We demonstrate that this enhanced glycolysis induces extracellular acidosis and activates the proton-gated ion channel, acid-sensing ion channel 1a (ASIC1a). Although multiple H⁺ transport/secretion mechanisms are upregulated in PHTN and likely contribute to extracellular acidosis, inhibiting glycolysis with 2-deoxy-d-glucose or syrosingopine effectively prevents extracellular acidification and ASIC1a activation, revealing a promising therapeutic avenue.

Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible respiratory disease with limited therapeutic options. A hallmark of IPF is excessive fibroblast activation and extracellular matrix (ECM) deposition. The resulting increase in tissue stiffness amplifies fibroblast activation and drives disease progression. Dampening stiffness-dependent activation of fibroblasts could slow disease progression. We performed an unbiased, next-generation sequencing (NGS) screen to identify signaling pathways involved in stiffness-dependent lung fibroblast activation. Adipocytokine signaling was downregulated in primary lung fibroblasts (PFs) cultured on stiff matrices. Re-activating adipocytokine signaling with adiponectin suppressed stiffness-dependent activation of human PFs. Adiponectin signaling depended on CDH13 expression and p38 mitogen-activated protein kinase gamma (p38MAPKγ) activation. CDH13 expression and p38MAPKγ activation were strongly reduced in lungs from IPF donors. Our data suggest that adiponectin-signaling via CDH13 and p38MAPKγ activation suppresses profibrotic activation of fibroblasts in the lung. Targeting of the adiponectin signaling cascade may provide therapeutic benefits in IPF.NEW & NOTEWORTHY A hallmark of idiopathic pulmonary fibrosis (IPF) is excessive fibroblast activation and extracellular matrix (ECM) deposition. The resulting increase in tissue stiffness amplifies fibroblast activation and drives disease progression. Dampening stiffness-dependent activation of fibroblasts could slow disease progression. We found that activation of the adipocytokine signaling pathway halts and reverses stiffness-induced, profibrotic fibroblast activation. Specific targeting of this signaling cascade may therefore provide therapeutic benefits in IPF.

In hypoxic and pseudohypoxic rodent models of pulmonary hypertension (PH), hypoxia-inducible factor (HIF) inhibition attenuates disease initiation. However, HIF activation alone, due to genetic alterations or use of inhibitors of prolyl hydroxylase domain (PHD) enzymes, has not been definitively shown to cause PH in humans, indicating the involvement of other mechanisms. Given the association between endothelial cell dysfunction and PH, the effects of pseudohypoxia and its underlying pathways were investigated in primary human lung endothelial cells. PHD2 silencing or inhibition, while activating HIF2α, induced apoptosis-resistance and IFN/STAT activation in endothelial cells, independent of HIF signaling. Mechanistically, PHD2 deficiency activated AKT and ERK, inhibited JNK, and reduced AIP1 (ASK1-interacting protein 1), all independent of HIF2α. Like PHD2, AIP1 silencing affected these same kinase pathways and produced a similar dysfunctional endothelial cell phenotype, which was partially reversed by AKT inhibition. Consistent with these in vitro findings, AIP1 protein levels in lung endothelial cells were decreased in Tie2-Cre/Phd2 knockout mice compared with wild-type controls. Lung vascular endothelial cells from patients with pulmonary arterial hypertension (PAH) showed IFN/STAT activation. Lung tissue from both SU5416/hypoxia PAH rats and patients with PAH all showed AKT activation and dysregulated AIP1 expression. In conclusion, PHD2 deficiency in lung vascular endothelial cells drives an apoptosis-resistant and inflammatory phenotype, mediated by AKT activation and AIP1 loss independent of HIF signaling. Targeting these pathways, including PHD2, AKT, and AIP1, holds the potential for developing new treatments for endothelial dysfunction in PH.NEW & NOTEWORTHY HIF activation alone does not conclusively lead to human PH, suggesting that HIF-independent signaling may also contribute to hypoxia-induced PH. This study demonstrated that PHD2 silencing-induced pseudohypoxia in human lung endothelial cells suppresses apoptosis and activates STAT, effects that persist despite HIF2α inhibition or knockdown and are attributed to AKT and ERK activation, JNK inhibition, and AIP1 loss. These findings align with observations in lung endothelial cells and tissues from PAH rodent models and patients.

Basal cells are adult stem cells in the airway epithelium and regenerate differentiated cell populations, including the mucosecretory and ciliated cells that enact mucociliary clearance. Human basal cells can proliferate and produce differentiated epithelium in vitro. However, studies of airway epithelial differentiation mostly rely on immunohistochemical or immunofluorescence-based staining approaches, meaning that a dynamic approach is lacking, and quantitative data are limited. Here, we use a lentiviral reporter gene approach to transduce primary human basal cells with bioluminescence reporter constructs to monitor airway epithelial differentiation longitudinally. We generated three constructs driven by promoter sequences from the TP63, MUC5AC, and FOXJ1 genes to quantitatively assess basal cell, mucosecretory cell, and ciliated cell abundance, respectively. We validated these constructs by tracking differentiation of basal cells in air-liquid interface and organoid ("bronchosphere") cultures. Transduced cells also responded appropriately to stimulation with interleukin 13 (IL-13; to increase mucosecretory differentiation and mucus production) and IL-6 (to increase ciliated cell differentiation). These constructs represent a new tool for monitoring airway epithelial cell differentiation in primary epithelial and/or induced pluripotent stem cell (iPSC)-derived cell cultures.NEW & NOTEWORTHY Orr et al. generated and validated new lentiviral vectors to monitor the differentiation of airway basal cells, goblet cells, or multiciliated cells using bioluminescence.

Hemolysis is associated with pulmonary hypertension (PH), but the direct contribution of circulating free heme to the PH pathogenesis remains unclear. Here, we show that the elevated levels of circulating free heme are sufficient to induce PH and inflammatory response in mice and confirm the critical role of mitogen-activated protein kinase kinase-3 (MKK3)-mediated pathway in free heme signaling. Following the continuous infusion of heme for 2 wk, wild-type (WT) but not MKK3 knockout (KO) mice develop PH, as evidenced by a significantly elevated right ventricular (RV) systolic pressure, RV hypertrophy, and pulmonary vascular remodeling. The MKK3/p38 axis, markedly activated by heme infusion in WTs, results in upregulated proliferative/cytokine signaling targets Akt, ERK1/2, and STAT3, which were abrogated in MKK3 KO mice. Moreover, the MKK3 KOs were protected against heme-mediated endothelial barrier dysfunction by restoring the tight junction protein zonula occludens-1 expression and diminishing the inflammatory cell infiltration in the lungs. Plasma cytokine multiplex analysis revealed a severe cytokine storm already 24 h after initiation of heme infusion, with a significant increase of 19 cytokines, including IL-1b, IL-2, IL-6, IL-9, and TNF-a, in WT animals and complete attenuation of cytokine production in MKK3 KO mice. Together, these findings reveal a causative role of circulating free heme in PH through activating inflammatory and proliferative responses. The central role of MKK3 in orchestrating the heme-mediated pathogenic response supports MKK3 as an attractive therapeutic target for PH and other lung inflammatory diseases linked to hemolytic anemia.NEW & NOTEWORTHY This study demonstrates that elevated levels of circulating free heme can induce pulmonary hypertension (PH) and inflammation in mice. Continuous heme infusion activated the MKK3/p38 pathway, leading to increased right ventricular pressure, right ventricular hypertrophy, and vascular remodeling. This activation upregulated signaling cascades such as Akt, ERK1/2, and STAT3, whereas MKK3 knockout mice were protected against these changes and had reduced inflammatory responses, highlighting MKK3's potential as a therapeutic target for PH.

Surfactant replacement therapy is crucial in managing neonatal respiratory distress syndrome (RDS). Currently licensed clinical surfactants in the United States and Europe, including Survanta, Infasurf, Curosurf, and Alveofact, are all derived from bovine or porcine sources. We conducted a comprehensive examination of the biophysical properties of these four clinical surfactant preparations under physiologically relevant conditions, using constrained drop surfactometry (CDS). The assessed biophysical properties included the adsorption rate, quasi-static and dynamic surface activity, resistance to surfactant inhibition by meconium, and the morphology of the adsorbed surfactant films. This comparative study unveiled distinct in vitro biophysical properties of these clinical surfactants and revealed correlations between their chemical composition, lateral film structure, and biophysical functionality. Notably, at 1 mg/mL, Survanta exhibited a significantly lower adsorption rate compared with the other preparations at the same surfactant concentration. At 10 mg/mL, Infasurf, Curosurf, and Survanta all demonstrated excellent dynamic surface activity, whereas Alveofact exhibited the poorest quasi-static and dynamic surface activity. The suboptimal surface activity of Alveofact is found to be correlated with its unique monolayer-predominant morphology, in contrast to other surfactants forming multilayers. Curosurf, in particular, showcased superior resistance to biophysical inhibition by meconium compared with other preparations. Understanding the diverse biophysical behaviors of clinical surfactants provides crucial insights for precision and personalized design in treating RDS and other respiratory conditions. The findings from this study contribute valuable perspectives for the development of more efficacious and fully synthetic surfactant preparations.NEW & NOTEWORTHY A thorough investigation into the biophysical properties of four animal-derived clinical surfactant preparations was conducted through constrained drop surfactometry under physiologically relevant conditions. This comparative study unveiled unique in vitro biophysical characteristics among these clinical surfactants, establishing correlations between their chemical composition, lateral film structure, and biophysical functionality. The acquired knowledge offers essential insights for the precise and personalized design of clinical surfactant for the treatment of respiratory distress syndrome and other respiratory conditions.

Integrative multiomics can help elucidate the pathophysiology of pulmonary fibrosis (PF)-associated pulmonary hypertension (PH) (PF-PH). Weighted gene coexpression network analysis (WGCNA) was performed on a transcriptomic dataset of explanted lung tissue from 116 patients with PF. Patients were stratified by pulmonary vascular resistance (PVR), and differential gene expression analysis was conducted. Gene modules were correlated with hemodynamics at the time of transplantation and tested for enrichment in the lung transcriptomics signature of an independent pulmonary arterial hypertension (PAH) cohort. We found 1,250 differentially expressed genes between high and low PVR groups. WGCNA identified that black and yellowgreen modules negatively correlated with PVR, whereas the tan and darkgrey modules are positively correlated with PVR in PF-PH. In addition, the tan module showed the strongest enrichment for an independent PAH gene signature, suggesting shared gene expression patterns between PAH and PF-PH. Pharmacotranscriptomic analysis using the Connectivity Map implicated the tan and darkgrey modules as potentially pathogenic in PF-PH, given their combined module signature demonstrated a high negative connectivity score for treprostinil, a medication used in the treatment of PF-PH, and a high positive connectivity score for bone morphogenetic protein (BMP) loss of function. Pathway enrichment analysis revealed that inflammatory pathways and oxidative phosphorylation were downregulated, whereas epithelial-mesenchymal transition was upregulated in modules associated with increased PVR. Our integrative systems biology approach to the lung transcriptome of PF with and without PH identified several PH-associated coexpression modules and gene targets with shared molecular features with PAH warranting further investigation to uncover potential new therapies for PF-PH.NEW & NOTEWORTHY An integrative systems biology approach that included transcriptomic analysis of explanted lung tissue from patients with pulmonary fibrosis (PF) with and without pulmonary hypertension (PH) undergoing lung transplantation, combined with hemodynamic correlation and pharmacotranscriptomics, identified modules of genes associated with pulmonary vascular disease severity. Comparison with an independent pulmonary arterial hypertension (PAH) dataset identified shared gene expression patterns between PAH and PF-PH.

COVID-19 commonly presents as pneumonia, with those most severely affected progressing to respiratory failure. Patient responses to SARS-CoV-2 infection are varied, with comorbidities acting as contributors to varied outcomes. Focusing on one such major comorbidity, we assessed whether pharmacological induction of Type I Diabetes Mellitus (T1DM) would increase the severity of lung injury in a murine model of COVID-19 pneumonia utilizing wild type mice infected with mouse-adapted SARS-CoV-2. Hyperglycemic mice exhibited increased weight loss and reduced blood oxygen saturation in comparison to their euglycemic counterparts, suggesting that these animals indeed experienced more severe lung injury. Transcriptomic analysis revealed a significant impairment of the adaptive immune response in the lungs of diabetic mice compared to those of control. In order to expand the options available for tissue analysis due to biosafety restrictions, we employed a new technique to digest highly fixed tissue into a single cell suspension, originally designed for scRNA-Seq, which we then adapted for flow cytometric analysis. Flow immunophenotyping and scRNA-Seq confirmed impaired recruitment of T cells into the lungs of T1DM animals. Additionally, scRNA-Seq revealed a distinct, highly inflammatory macrophage profile in the diabetic cohort that correlates with the more severe infection these mice experienced clinically, allowing insight into a possible mechanism for this phenomenon. Recognizing the near certainty that respiratory viruses will continue to present significant public health concerns for the foreseeable future, our study provides key insights into how T1DM results in a much more severe infection and identifies possible targets to ameliorate comorbidity-associated severe disease.

The intricate lung structure is crucial for gas exchange within the alveolar region. Despite extensive research, questions remain about the connection between capillaries and the vascular tree. We propose a computational approach combining three-dimensional morphological modeling with computational fluid dynamics simulations to explore alveolar capillary network connectivity based on blood flow dynamics.We developed three-dimensional sheet-flow models to accurately represent alveolar capillary morphology and conducted simulations to predict flow velocities and pressure distributions. Our approach leverages functional features to identify plausible system architectures. Given capillary flow velocities and arteriole-to-venule pressure drops, we deduced arteriole connectivity details. Preliminary analyses for non-human species indicate a single alveolus connects to at least two 20 µm arterioles or one 30 µm arteriole. Hence, our approach narrows down potential connectivity scenarios, but a unique solution may not always be expected.Integrating our blood flow model results into our previously published gas exchange application, Alvin, we linked these scenarios to gas exchange efficiency. We found that increased blood flow velocity correlates with higher gas exchange efficiency.Our study provides insights into pulmonary microvasculature structure by evaluating blood flow dynamics, offering a new strategy to explore the morphology-physiology relationship that is applicable to other tissues and organs. Future availability of experimental data will be crucial in validating and refining our computational models and hypotheses.

Phenotype distortion of lung resident mesenchymal stem cells (MSC) in preterm infants is a hallmark event in the pathogenesis of bronchopulmonary dysplasia. Here, we evaluated the impact of cyclic mechanical stretch (CMS) and hyperoxia (HOX). The negative action of HOX on proliferation and cell death was more pronounced at 80% than at 40%. While the impact of CMS alone was modest, CMS plus HOX displayed the strongest effect sizes. Exposure to CMS and/or HOX induced the downregulation of PDGFRα and cellular senescence preceded by p21 accumulation. p21 interference interfered with cellular senescence and resulted in aggravated cell death arguing for a pro-survival mechanism. HOX 40% and limited exposure to HOX 80% prevailed a reversible phenotype with reuptake of proliferation while prolonged exposure to HOX 80% resulted in definite MSC growth arrest. Our mechanistic data explain how HOX and CMS induce the effects on MSC phenotype disruption. The results are congruent with the clinical observation that preterm infants requiring supplemental oxygen plus mechanical ventilation are at particular risk for BPD. While inhibiting p21 is not a feasible approach, limiting the duration and magnitude of the exposures is promising.