Tackling Brain and Muscle Dysfunction in Acute Respiratory Distress Syndrome Survivors: NHLBI Workshop Report.

Acute respiratory distress syndrome (ARDS) is associated with long-term impairments in brain and muscle function that significantly impact the quality of life of those who survive the acute illness. The mechanisms underlying these impairments are not yet well understood, and evidence-based interventions to minimize the burden on patients remain unproved. The NHLBI of the NIH assembled a workshop in April 2023 to review the state of the science regarding ARDS-associated brain and muscle dysfunction, to identify gaps in current knowledge, and to determine priorities for future investigation. The workshop included presentations by scientific leaders across the translational science spectrum and was open to the public as well as the scientific community. This report describes the themes discussed at the workshop as well as recommendations to advance the field toward the goal of improving the health and well-being of ARDS survivors.

Biphasic regulation of miR-17∼92 transcription during hypoxia: roles of HIF1 and p53 hyperphosphorylation at ser15.

We have reported previously that during hypoxia exposure, the expression of mature miR-17∼92 was first upregulated and then downregulated in pulmonary artery smooth muscle cells (PASMC) and in mouse lungs in vitro and in vivo. Here, we investigated the mechanisms regulating this biphasic expression of miR-17∼92 in PASMC in hypoxia. We measured the level of primary miR-17∼92 in PASMC during hypoxia exposure and found that short-term hypoxia exposure (3% O2, 6 h) induced the level of primary miR-17∼92, whereas long-term hypoxia exposure (3% O2, 24 h) decreased its level, suggesting a biphasic regulation of miR-17∼92 expression at the transcriptional level. We found that short-term hypoxia-induced upregulation of miR-17∼92 was hypoxia-inducible factor 1α (HIF1α) and E2F1 dependent. Two HIF1α binding sites on miR-17∼92 promoter were identified. We also found that long-term hypoxia-induced suppression of miR-17∼92 expression could be restored by silencing of p53. Mutation of the p53-binding sites in the miR-17∼92 promoter increased miR-17∼92 promoter activity in both normoxia and hypoxia. Our findings suggest that the biphasic transcriptional regulation of miR-17∼92 during hypoxia is controlled by HIF1/E2F1 and p53 in PASMC: during short-term hypoxia exposure, stabilization of HIF1 and induction of E2F1 induce the transcription of miR-17∼92, whereas during long-term hypoxia exposure, hyperphosphorylation of p53 suppresses the expression of miR-17∼92.NEW & NOTEWORTHY We showed that the biphasic transcriptional regulation of miR-17∼92 during hypoxia is controlled by two distinct mechanisms: during short-term hypoxia exposure, induction of HIF1 and E2F1 upregulates miR-17∼92. Longer hypoxia exposure induces hyperphosphorylation of p53 at ser15, which leads to its binding to miR-17∼92 promoter and inhibition of its expression. Our findings provide novel insights into the spatiotemporal regulation of miR-17∼92 that may play a role in the development of human lung diseases including pulmonary hypertension (PH).

HIF and HOIL-1L-mediated PKCζ degradation stabilizes plasma membrane Na,K-ATPase to protect against hypoxia-induced lung injury.

Organisms have evolved adaptive mechanisms in response to stress for cellular survival. During acute hypoxic stress, cells down-regulate energy-consuming enzymes such as Na,K-ATPase. Within minutes of alveolar epithelial cell (AEC) exposure to hypoxia, protein kinase C zeta (PKCζ) phosphorylates the α1-Na,K-ATPase subunit and triggers it for endocytosis, independently of the hypoxia-inducible factor (HIF). However, the Na,K-ATPase activity is essential for cell homeostasis. HIF induces the heme-oxidized IRP2 ubiquitin ligase 1L (HOIL-1L), which leads to PKCζ degradation. Here we report a mechanism of prosurvival adaptation of AECs to prolonged hypoxia where PKCζ degradation allows plasma membrane Na,K-ATPase stabilization at ∼50% of normoxic levels, preventing its excessive down-regulation and cell death. Mice lacking HOIL-1L in lung epithelial cells (CreSPC/HOIL-1Lfl/fl ) were sensitized to hypoxia because they express higher levels of PKCζ and, consequently, lower plasma membrane Na,K-ATPase levels, which increased cell death and worsened lung injury. In AECs, expression of an α1-Na,K-ATPase construct bearing an S18A (α1-S18A) mutation, which precludes PKCζ phosphorylation, stabilized the Na,K-ATPase at the plasma membrane and prevented hypoxia-induced cell death even in the absence of HOIL-1L. Adenoviral overexpression of the α1-S18A mutant Na,K-ATPase in vivo rescued the enhanced sensitivity of CreSPC/HOIL-1Lfl/fl mice to hypoxic lung injury. These data suggest that stabilization of Na,K-ATPase during severe hypoxia is a HIF-dependent process involving PKCζ degradation. Accordingly, we provide evidence of an important adaptive mechanism to severe hypoxia, whereby halting the exaggerated down-regulation of plasma membrane Na,K-ATPase prevents cell death and lung injury.

Sphingosine Kinase 1/S1P Signaling Contributes to Pulmonary Fibrosis by Activating Hippo/YAP Pathway and Mitochondrial Reactive Oxygen Species in Lung Fibroblasts.

The sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) signaling axis is emerging as a key player in the development of idiopathic pulmonary fibrosis (IPF) and bleomycin (BLM)-induced lung fibrosis in mice. Recent evidence implicates the involvement of the Hippo/Yes-associated protein (YAP) 1 pathway in lung diseases, including IPF, but its plausible link to the SPHK1/S1P signaling pathway is unclear. Herein, we demonstrate the increased co-localization of YAP1 with the fibroblast marker FSP1 in the lung fibroblasts of BLM-challenged mice, and the genetic deletion of Sphk1 in mouse lung fibroblasts (MLFs) reduced YAP1 localization in fibrotic foci. The PF543 inhibition of SPHK1 activity in mice attenuated YAP1 co-localization with FSP1 in lung fibroblasts. In vitro, TGF-ß stimulated YAP1 translocation to the nucleus in primary MLFs, and the deletion of Sphk1 or inhibition with PF543 attenuated TGF-ß-mediated YAP1 nuclear localization. Moreover, the PF543 inhibition of SPHK1, or the verteporfin inhibition of YAP1, decreased the TGF-ß- or BLM-induced mitochondrial reactive oxygen species (mtROS) in human lung fibroblasts (HLFs) and the expression of fibronectin (FN) and alpha-smooth muscle actin (α-SMA). Furthermore, scavenging mtROS with MitoTEMPO attenuated the TGF-ß-induced expression of FN and α-SMA. The addition of the S1P antibody to HLFs reduced TGF-ß- or S1P-mediated YAP1 activation, mtROS, and the expression of FN and α-SMA. These results suggest a role for SPHK1/S1P signaling in TGF-ß-induced YAP1 activation and mtROS generation, resulting in fibroblast activation, a critical driver of pulmonary fibrosis.

PAI-1 is a novel component of the miR-17~92 signaling that regulates pulmonary artery smooth muscle cell phenotypes.

We have previously reported that miR-17~92 is critically involved in the pathogenesis of pulmonary hypertension (PH). We also identified two novel mR-17/20a direct targets, PDZ and LIM domain protein 5 (PDLIM5) and prolyl hydroxylase 2 (PHD2), and elucidated the signaling pathways by which PDLIM5 and PHD2 regulate functions of pulmonary artery smooth muscle cells (PASMCs). In addition, we have shown that plasminogen activator inhibitor-1 (PAI-1) is also downregulated in PASMCs that overexpress miR-17~92. However, it is unclear whether PAI-1 is a direct target of miR-17~92 and whether it plays a role in regulating the PASMC phenotype. In this study, we have identified PAI-1 as a novel target of miR-19a/b, two members of the miR-17~92 cluster. We found that the 3'-untranslated region (UTR) of PAI-1 contains a miR-19a/b binding site and that miR-19a/b can target this site to suppress PAI-1 protein expression. MiR-17/20a, two other members of miR-17~92, may also indirectly suppress PAI-1 expression through PDLIM5. PAI-1 is a negative regulator of miR-17~92-mediated PASMC proliferation. Silencing of PAI-1 induces Smad2/calponin signaling in PASMCs, suggesting that PAI-1 is a negative regulator of the PASMC contractile phenotype. We also found that PAI-1 is essential for the metabolic gene expression in PASMCs. Furthermore, although there is no significant change in PAI-1 levels in PASMCs isolated from idiopathic pulmonary arterial hypertension and associated pulmonary arterial hypertension patients, PAI-1 is downregulated in hypoxia/Sugen-induced hypertensive rat lungs. These results suggest that miR-17~92 regulates the PASMC contractile phenotype and proliferation coordinately and synergistically by direct and indirect targeting of PAI-1.

IL-11 facilitates a novel connection between RA joint fibroblasts and endothelial cells.

IL-11 has been detected in inflamed joints; however, its role in the pathogenesis of arthritis is not yet clear. Studies were conducted to characterize the expression and functional significance of IL-11 and IL-11Rα in rheumatoid arthritis (RA). IL-11 levels were elevated in RA synovial fluid (SF) compared to osteoarthritis (OA) SF and plasma from RA, OA and normal individuals (NLs). Morphologic studies established that IL-11 was detected in lining fibroblasts and macrophages in addition to sublining endothelial cells and macrophages at higher levels in RA compared to NL synovial tissues. Since IL-11Rα was exclusively expressed in RA fibroblasts and endothelial cells, macrophages were not involved in IL-11 effector function. Ligation of IL-11 to IL-11Rα strongly provoked fibroblast infiltration into RA joint, while cell proliferation was unaffected by this process. Secretion of IL-8 and VEGF from IL-11 activated RA fibroblasts was responsible for the indirect effect of IL-11 on endothelial cell transmigration and tube formation. Moreover, IL-11 blockade impaired RA SF capacity to elicit endothelial cell transmigration and tube formation. We conclude that IL-11 binding to endothelial IL-11Rα can directly induce RA angiogenesis. In addition, secretion of proangiogenic factors from migrating fibroblasts potentiated by IL-11 can indirectly contribute to RA neovascularization.

Suppression of von Hippel-Lindau Protein in Fibroblasts Protects against Bleomycin-Induced Pulmonary Fibrosis.

We have reported that von Hippel-Lindau protein (pVHL) expression is elevated in human and mouse fibrotic lungs and that overexpression of pVHL stimulates fibroblast proliferation. We sought to determine whether loss of pVHL in fibroblasts prevents injury and fibrosis in mice that are treated with bleomycin. We generated heterozygous fibroblast-specific pVHL (Fsp-VHL) knockdown mice (Fsp-VHL(+/-)) and homozygous Fsp-VHL knockout mice (Fsp-VHL(-/-)) by crossbreeding vhlh 2-lox mice (VHL(fl/fl)) with Fsp-Cre recombinase mice. Our data show that Fsp-VHL(-/-) mice, but not Fsp-VHL(+/-) mice, have elevated red blood cell counts, hematocrit, hemoglobin content, and expression of hypoxia-inducible factor (HIF) targets, indicating HIF activation. To examine the role of pVHL in bleomycin-induced lung injury and fibrosis in vivo, we administered PBS or bleomycin to age-, sex-, and strain-matched 8-week-old VHL(fl/fl), Fsp-VHL(+/-), and Fsp-VHL(-/-) mice. In Fsp-VHL(+/-) and Fsp-VHL(-/-) mice, bleomycin-induced collagen accumulation, fibroblast proliferation, differentiation, and matrix protein dysregulation were markedly attenuated. Suppression of pVHL also decreased bleomycin-induced Wnt signaling and prostaglandin E2 signaling but did not affect bleomycin-induced initial acute lung injury and lung inflammation. These results indicate that pVHL has a pivotal role in bleomycin-induced pulmonary fibrosis, possibly via an HIF-independent pathway. Paradoxically, pVHL does not affect bleomycin-induced lung injury and inflammation, indicating a separation of the mechanisms involved in injury/inflammation from those involved in pulmonary fibrosis.

Loss of microRNA-17∼92 in smooth muscle cells attenuates experimental pulmonary hypertension via induction of PDZ and LIM domain 5.

RATIONALE: Recent studies suggest that microRNAs (miRNAs) play important roles in regulation of pulmonary artery smooth muscle cell (PASMC) phenotype and are implicated in pulmonary arterial hypertension (PAH). However, the underlying molecular mechanisms remain elusive. OBJECTIVES: This study aims to understand the mechanisms regulating PASMC proliferation and differentiation by microRNA-17∼92 (miR-17∼92) and to elucidate its implication in PAH. METHODS: We generated smooth muscle cell (SMC)-specific miR-17∼92 and PDZ and LIM domain 5 (PDLIM5) knockout mice and overexpressed miR-17∼92 and PDLIM5 by injection of miR-17∼92 mimics or PDLIM5-V5-His plasmids and measured their responses to hypoxia. We used miR-17∼92 mimics, inhibitors, overexpression vectors, small interfering RNAs against PDLIM5, Smad, and transforming growth factor (TGF)-ß to determine the role of miR-17∼92 and its downstream targets in PASMC proliferation and differentiation. MEASUREMENTS AND MAIN RESULTS: We found that human PASMC (HPASMC) from patients with PAH expressed decreased levels of the miR-17∼92 cluster, TGF-ß, and SMC markers. Overexpression of miR-17∼92 increased and restored the expression of TGF-ß3, Smad3, and SMC markers in HPASMC of normal subjects and patients with idiopathic PAH, respectively. Knockdown of Smad3 but not Smad2 prevented miR-17∼92-induced expression of SMC markers. SMC-specific knockout of miR-17∼92 attenuated hypoxia-induced pulmonary hypertension (PH) in mice, whereas reconstitution of miR-17∼92 restored hypoxia-induced PH in these mice. We also found that PDLIM5 is a direct target of miR-17/20a, and hypertensive HPASMC and mouse PASMC expressed elevated PDLIM5 levels. Suppression of PDLIM5 increased expression of SMC markers and enhanced TGF-ß/Smad2/3 activity in vitro and enhanced hypoxia-induced PH in vivo, whereas overexpression of PDLIM5 attenuated hypoxia-induced PH. CONCLUSIONS: We provided the first evidence that miR-17∼92 inhibits PDLIM5 to induce the TGF-ß3/SMAD3 pathway, contributing to the pathogenesis of PAH.

MicroRNAs in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a devastating disease without effective treatment. Despite decades of research and the development of novel treatments, PAH remains a fatal disease, suggesting an urgent need for better understanding of the pathogenesis of PAH. Recent studies suggest that microRNAs (miRNAs) are dysregulated in patients with PAH and in experimental pulmonary hypertension. Furthermore, normalization of a few miRNAs is reported to inhibit experimental pulmonary hypertension. We have reviewed the current knowledge about miRNA biogenesis, miRNA expression pattern, and their roles in regulation of pulmonary artery smooth muscle cells, endothelial cells, and fibroblasts. We have also identified emerging trends in our understanding of the role of miRNAs in the pathogenesis of PAH and propose future studies that might lead to novel therapeutic strategies for the treatment of PAH.