Neuroprotective and Anti-Inflammatory Activities of Hybrid Small-Molecule SA-10 in Ischemia/Reperfusion-Induced Retinal Neuronal Injury Models.

Embolism, hyperglycemia, high intraocular pressure-induced increased reactive oxygen species (ROS) production, and microglial activation result in endothelial/retinal ganglion cell death. Here, we conducted in vitro and in vivo ischemia/reperfusion (I/R) efficacy studies of a hybrid antioxidant-nitric oxide donor small molecule, SA-10, to assess its therapeutic potential for ocular stroke. METHODS: To induce I/R injury and inflammation, we subjected R28 and primary microglial cells to oxygen glucose deprivation (OGD) for 6 h in vitro or treated these cells with a cocktail of TNF-α, IL-1ß and IFN-γ for 1 h, followed by the addition of SA-10 (10 µM). Inhibition of microglial activation, ROS scavenging, cytoprotective and anti-inflammatory activities were measured. In vivo I/R-injured mouse retinas were treated with either PBS or SA-10 (2%) intravitreally, and pattern electroretinogram (ERG), spectral-domain optical coherence tomography, flash ERG and retinal immunocytochemistry were performed. RESULTS: SA-10 significantly inhibited microglial activation and inflammation in vitro. Compared to the control, the compound SA-10 significantly attenuated cell death in both microglia (43% vs. 13%) and R28 cells (52% vs. 17%), decreased ROS (38% vs. 68%) production in retinal microglia cells, preserved neural retinal function and increased SOD1 in mouse eyes. CONCLUSION: SA-10 is protective to retinal neurons by decreasing oxidative stress and inflammatory cytokines.

UCHL1 Regulates Radiation Lung Injury via Sphingosine Kinase-1.

GADD45a is a gene we previously reported as a mediator of responses to acute lung injury. GADD45a-/- mice express decreased Akt and increased Akt ubiquitination due to the reduced expression of UCHL1 (ubiquitin c-terminal hydrolase L1), a deubiquitinating enzyme, while GADD45a-/- mice have increased their susceptibility to radiation-induced lung injury (RILI). Separately, we have reported a role for sphingolipids in RILI, evidenced by the increased RILI susceptibility of SphK1-/- (sphingosine kinase 1) mice. A mechanistic link between UCHL1 and sphingolipid signaling in RILI is suggested by the known polyubiquitination of SphK1. Thus, we hypothesized that the regulation of SphK1 ubiquitination by UCHL1 mediates RILI. Initially, human lung endothelial cells (EC) subjected to radiation demonstrated a significant upregulation of UCHL1 and SphK1. The ubiquitination of EC SphK1 after radiation was confirmed via the immunoprecipitation of SphK1 and Western blotting for ubiquitin. Further, EC transfected with siRNA specifically for UCHL1 or pretreated with LDN-5744, as a UCHL1 inhibitor, prior to radiation were noted to have decreased ubiquitinated SphK1 in both conditions. Further, the inhibition of UCHL1 attenuated sphingolipid-mediated EC barrier enhancement was measured by transendothelial electrical resistance. Finally, LDN pretreatment significantly augmented murine RILI severity. Our data support the fact that the regulation of SphK1 expression after radiation is mediated by UCHL1. The modulation of UCHL1 affecting sphingolipid signaling may represent a novel RILI therapeutic strategy.

MicroRNA-based engineering of mesenchymal stem cell extracellular vesicles for treatment of retinal ischemic disorders: Engineered extracellular vesiclesand retinal ischemia.

Mesenchymal stem cell (MSCs)-derived extracellular vesicles (EVs) are emerging therapeutic tools. Hypoxic pre-conditioning (HPC) of MSCs altered the production of microRNAs (miRNAs) in EVs, and enhanced the cytoprotective, anti-inflammatory, and neuroprotective properties of their derivative EVs in retinal cells. EV miRNAs were identified as the primary contributors of these EV functions. Through miRNA seq analyses, miRNA-424 was identified as a candidate for the retina to overexpress in EVs for enhancing cytoprotection and anti-inflammatory effects. FEEs (functionally engineered EVs) overexpressing miR424 (FEE424) significantly enhanced neuroprotection and anti-inflammatory activities in vitro in retinal cells. FEE424 functioned by reducing inflammatory cytokine production in retinal microglia, and attenuating oxygen free radicals in retinal Muller cells and microvascular endothelial cells, providing a multi-pronged approach to enhancing recovery after retinal ischemic insult. In an in vivo model of retinal ischemia, native, HPC, and FEE424 MSC EVs robustly and similarly restored function to close to baseline, and prevented loss of retinal ganglion cells, but HPC EVs provided the most effective attenuation of apoptosis-related and inflammatory cytokine gene expression. These results indicate the potential for EV engineering to produce ameliorative effects for retinal diseases with a significant inflammatory component. STATEMENT OF SIGNIFICANCE: We show that functionally engineered extracellular vesicles (FEEs) from mesenchymal stem cells (MSCs) provide cytoprotection in rat retina subjected to ischemia. FEEs overexpressing microRNA 424 (FEE424) function by reducing inflammatory cytokine production in retinal microglia, and attenuating oxygen free radicals in Muller cells and microvascular endothelial cells, providing a multi-pronged approach to enhancing recovery. In an in vivo model of retinal ischemia in rats, native, hypoxic-preconditioned (HPC), and FEE424 MSC EVs robustly and similarly restored function, and prevented loss of retinal ganglion cells, but HPC EVs provided the most effective attenuation of apoptosis-related and inflammatory cytokine gene expression. The results indicate the potential for EV engineering to produce ameliorative effects for retinal diseases with a significant inflammatory component.

Emerging concepts in the treatment of optic neuritis: mesenchymal stem cell-derived extracellular vesicles.

BACKGROUND: Optic neuritis (ON) is frequently encountered in multiple sclerosis, neuromyelitis optica spectrum disorder, anti-myelin oligodendrocyte glycoprotein associated disease, and other systemic autoimmune disorders. The hallmarks are an abnormal optic nerve and inflammatory demyelination; episodes of optic neuritis tend to be recurrent, and particularly for neuromyelitis optica spectrum disorder, may result in permanent vision loss. MAIN BODY: Mesenchymal stem cell (MSC) therapy is a promising approach that results in remyelination, neuroprotection of axons, and has demonstrated success in clinical studies in other neuro-degenerative diseases and in animal models of ON. However, cell transplantation has significant disadvantages and complications. Cell-free approaches utilizing extracellular vesicles (EVs) produced by MSCs exhibit anti-inflammatory and neuroprotective effects in multiple animal models of neuro-degenerative diseases and in rodent models of multiple sclerosis (MS). EVs have potential to be an effective cell-free therapy in optic neuritis because of their anti-inflammatory and remyelination stimulating properties, ability to cross the blood brain barrier, and ability to be safely administered without immunosuppression. CONCLUSION: We review the potential application of MSC EVs as an emerging treatment strategy for optic neuritis by reviewing studies in multiple sclerosis and related disorders, and in neurodegeneration, and discuss the challenges and potential rewards of clinical translation of EVs including cell targeting, carrying of therapeutic microRNAs, and prolonging delivery for treatment of optic neuritis.

Uptake and Distribution of Administered Bone Marrow Mesenchymal Stem Cell Extracellular Vesicles in Retina.

Cell replacement therapy using mesenchymal (MSC) and other stem cells has been evaluated for diabetic retinopathy and glaucoma. This approach has significant limitations, including few cells integrated, aberrant growth, and surgical complications. Mesenchymal Stem Cell Exosomes/Extracellular Vesicles (MSC EVs), which include exosomes and microvesicles, are an emerging alternative, promoting immunomodulation, repair, and regeneration by mediating MSC's paracrine effects. For the clinical translation of EV therapy, it is important to determine the cellular destination and time course of EV uptake in the retina following administration. Here, we tested the cellular fate of EVs using in vivo rat retinas, ex vivo retinal explant, and primary retinal cells. Intravitreally administered fluorescent EVs were rapidly cleared from the vitreous. Retinal ganglion cells (RGCs) had maximal EV fluorescence at 14 days post administration, and microglia at 7 days. Both in vivo and in the explant model, most EVs were no deeper than the inner nuclear layer. Retinal astrocytes, microglia, and mixed neurons in vitro endocytosed EVs in a dose-dependent manner. Thus, our results indicate that intravitreal EVs are suited for the treatment of retinal diseases affecting the inner retina. Modification of the EV surface should be considered for maintaining EVs in the vitreous for prolonged delivery.

Autophagy and post-ischemic conditioning in retinal ischemia.

Retinal ischemia is a major cause of vision loss and a common underlying mechanism associated with diseases, such as diabetic retinopathy and central retinal artery occlusion. We have previously demonstrated the robust neuroprotection in retina induced by post-conditioning (post-C), a brief period of ischemia, 24 h, following a prolonged and damaging initial ischemia. The mechanisms underlying post-C-mediated retinal protection are largely uncharacterized. We hypothesized that macroautophagy/autophagy is a mediator of post-C-induced neuroprotection. This study employed an in vitro model of oxygen glucose deprivation (OGD) in the retinal R28 neuronal cell line, and an in vivo rat model of retinal ischemic injury. In vivo, there were significant increases in autophagy proteins, MAP1LC3-II/LC3-II, and decreases in SQSTM1/p62 (sequestosome 1) in ischemia/post-C vs. ischemia/sham post-C. Blockade of Atg5 and Atg7 in vivo decreased LC3-II, increased SQSTM1, attenuated the functional protective effect of post-C, and increased histological damage and TUNEL compared to non-silencing siRNA. TUNEL after ischemia in vivo was found in retinal ganglion, amacrine, and photoreceptor cells. Blockade of Atg5 attenuated the post-C neuroprotection by a brief period of OGD in vitro. Moreover, in vitro, post-C attenuated cell death, loss of cellular proliferation, and defective autophagic flux from prolonged OGD. Stimulating autophagy using Tat-Beclin 1 rescued retinal neurons from cell death after OGD. As a whole, our results suggest that autophagy is required for the neuroprotective effect of retinal ischemic post-conditioning and augmentation of autophagy offers promise in the treatment of retinal ischemic injury.Abbreviations: BECN1: Beclin 1, autophagy related; DAPI: 4',6-diamidino-2-phenylindole; DR: diabetic retinopathy; EdU: 5-ethynyl-2'-deoxyuridine; ERG: Electroretinogram; FITC: Fluorescein isothiocyanate; GCL: Ganglion cell layer; GFAP: Glial fibrillary acidic protein; INL: Inner nuclear layer; IPL: Inner plexiform layer; MAP1LC3/LC3: Microtubule-associated protein 1 light chain 3; OGD: Oxygen-glucose deprivation; ONL: Outer nuclear layer; OP: Oscillatory potential; PFA: Paraformaldehyde; PL: Photoreceptor layer; post-C: post-conditioning; RFP: Red fluorescent protein; RGC: Retinal ganglion cell; RPE: Retinal pigment epithelium; RT-PCR: Real-time polymerase chain reaction; SEM: Standard error of the mean; siRNA: Small interfering RNA; SQSTM1: Sequestosome 1; STR: Scotopic threshold response; Tat: Trans-activator of transcription; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling.

Mesenchymal stem cell-derived extracellular vesicles and retinal ischemia-reperfusion.

Retinal ischemia is a major cause of vision loss and impairment and a common underlying mechanism associated with diseases such as glaucoma, diabetic retinopathy, and central retinal artery occlusion. The regenerative capacity of the diseased human retina is limited. Our previous studies have shown the neuroprotective effects of intravitreal injection of mesenchymal stem cells (MSC) and MSC-conditioned medium in retinal ischemia in rats. Based upon the hypothesis that the neuroprotective effects of MSCs and conditioned medium are largely mediated by extracellular vesicles (EVs), MSC derived EVs were tested in an in-vitro oxygen-glucose deprivation (OGD) model of retinal ischemia. Treatment of R28 retinal cells with MSC-derived EVs significantly reduced cell death and attenuated loss of cell proliferation. Mechanistic studies on the mode of EV endocytosis by retinal cells were performed in vitro. EV endocytosis was dose- and temperature-dependent, saturable, and occurred via cell surface heparin sulfate proteoglycans mediated by the caveolar endocytic pathway. The administration of MSC-EVs into the vitreous humor 24 h after retinal ischemia in a rat model significantly enhanced functional recovery, and decreased neuro-inflammation and apoptosis. EVs were taken up by retinal neurons, retinal ganglion cells, and microglia. They were present in the vitreous humor for four weeks after intravitreal administration, with saturable binding to vitreous humor components. Overall, this study highlights the potential of MSC-EV as biomaterials for neuroprotective and regenerative therapy in retinal disorders.

Attenuation of murine acute lung injury by PF-573,228, an inhibitor of focal adhesion kinase.

Acute lung injury (ALI) is characterized by endothelial barrier disruption resulting in increased vascular permeability. As focal adhesion kinase (FAK), a non-receptor protein tyrosine kinase, is involved in endothelial cell (EC) barrier regulation, we hypothesized that FAK inhibition could attenuate agonist-induced EC barrier disruption relevant to ALI. Human lung EC were pretreated with one of three pharmacologic FAK inhibitors, PF-573,228 (PF-228, 10 µM), PF-562,271 (PF-271, 5 µM) or NVP-TAE226 (TAE226, 5 µM) for 30 min prior to treatment with thrombin (1 U/ml, 30 min). Western blotting confirmed attenuated thrombin-induced FAK phosphorylation associated with all three inhibitors. Subsequently, EC were pretreated with either PF-228 (10 µM), TAE226 (5 µM) or PF-271 (5 µM) for 30 min prior to thrombin stimulation (1 U/ml) followed by measurements of barrier integrity by transendothelial electrical resistance (TER). Separately, EC grown in transwell inserts prior to thrombin (1 U/ml) with measurements of FITC-dextran flux after 30 min confirmed a significant attenuation of thrombin-induced EC barrier disruption by PF-228 alone. Finally, in a murine ALI model induced by LPS (1.25 mg/ml, IT), rescue treatment with PF-228 was associated with significantly reduced lung injury. Our findings PF-228, currently being studied in clinical trials, may serve as a novel and effective therapeutic agent for ALI.

Gene expression in retinal ischemic post-conditioning.

PURPOSE: The pathophysiology of retinal ischemia involves mechanisms including inflammation and apoptosis. Ischemic post-conditioning (Post-C), a brief non-lethal ischemia, induces a long-term ischemic tolerance, but the mechanisms of ischemic post-conditioning in the retina have only been described on a limited basis. Accordingly, we conducted this study to determine the molecular events in retinal ischemic post-conditioning and to identify targets for therapeutic strategies for retinal ischemia. METHODS: To determine global molecular events in ischemic post-conditioning, a comprehensive study of the transcriptome of whole retina was performed. We utilized RNA sequencing (RNA-Seq), a recently developed, deep sequencing technique enabling quantitative gene expression, with low background noise, dynamic detection range, and discovery of novel genes. Rat retina was subjected to ischemia in vivo by elevation of intraocular pressure above systolic blood pressure. At 24 h after ischemia, Post-C or sham Post-C was performed by another, briefer period of ischemia, and 24 h later, retinas were collected and RNA processed. RESULTS: There were 71 significantly affected pathways in post-conditioned/ischemic vs. normals and 43 in sham post conditioned/ischemic vs. normals. Of these, 28 were unique to Post-C and ischemia. Seven biological pathways relevant to ischemic injury, in Post-C as opposed to sham Post-C, were examined in detail. Apoptosis, p53, cell cycle, JAK-STAT, HIF-1, MAPK and PI3K-Akt pathways significantly differed in the number as well as degree of fold change in genes between conditions. CONCLUSION: Post-C is a complex molecular signaling process with a multitude of altered molecular pathways. We identified potential gene candidates in Post-C. Studying the impact of altering expression of these factors may yield insight into new methods for treating or preventing damage from retinal ischemic disorders.

Simvastatin-induced sphingosine 1-phosphate receptor 1 expression is KLF2-dependent in human lung endothelial cells.

We have demonstrated that simvastatin and sphingosine 1-phosphate (S1P) both attenuate increased vascular permeability in preclinical models of acute respiratory distress syndrome. However, the underlying mechanisms remain unclear. As Krüppel-like factor 2 (KLF2) serves as a critical regulator for cellular stress response in endothelial cells (EC), we hypothesized that simvastatin enhances endothelial barrier function via increasing expression of the barrier-promoting S1P receptor, S1PR1, via a KLF2-dependent mechanism. S1PR1 luciferase reporter promoter activity in human lung artery EC (HPAEC) was tested after simvastatin (5 µM), and S1PR1 and KLF2 protein expression detected by immunoblotting. In vivo, transcription and expression of S1PR1 and KLF2 in mice lungs were detected by microarray profiling and immunoblotting after exposure to simvastatin (10 mg/kg). Endothelial barrier function was measured by trans-endothelial electrical resistance with the S1PR1 agonist FTY720-(S)-phosphonate. Both S1PR1 and KLF2 gene expression (mRNA, protein) were significantly increased by simvastatin in vitro and in vivo. S1PR1 promoter activity was significantly increased by simvastatin (P < 0.05), which was significantly attenuated by KLF2 silencing (siRNA). Simvastatin induced KLF2 recruitment to the S1PR1 promoter, and consequently, significantly augmented the effects of the S1PR1 agonist on EC barrier enhancement (P < 0.05), which was significantly attenuated by KLF2 silencing (P < 0.05). These results suggest that simvastatin upregulates S1PR1 transcription and expression via the transcription factor KLF2, and consequently augments the effects of S1PR1 agonists on preserving vascular barrier integrity. These results may lead to novel combinatorial therapeutic strategies for lung inflammatory syndromes.

Bone-marrow mesenchymal stem-cell administration significantly improves outcome after retinal ischemia in rats.

PURPOSE: Ischemia-associated retinal degeneration is one of the leading causes of vision loss, and to date, there are no effective treatment options. We hypothesized that delayed injection of bone-marrow stem cells (BMSCs) 24 h after the onset of ischemia could effectively rescue ischemic retina from its consequences, including apoptosis, inflammation, and increased vascular permeability, thereby preventing retinal cell loss. METHODS: Retinal ischemia was induced in adult Wistar rats by increasing intraocular pressure (IOP) to 130-135 mmHg for 55 min. BMSCs harvested from rat femur were injected into the vitreous 24 h post-ischemia. Functional recovery was assessed 7 days later using electroretinography (ERG) measurements of the a-wave, b-wave, P2, scotopic threshold response (STR), and oscillatory potentials (OP). The retinal injury and anti-ischemic effects of BMSCs were quantitated by measuring apoptosis, autophagy, inflammatory markers, and retinal-blood barrier permeability. The distribution and fate of BMSC were qualitatively examined using real-time fundus imaging, and retinal flat mounts. RESULTS: Intravitreal delivery of BMSCs significantly improved recovery of the ERG a- and b-waves, OP, negative STR, and P2, and attenuated apoptosis as evidenced by decreased TUNEL and caspase-3 protein levels. BMSCs significantly increased autophagy, decreased inflammatory mediators (TNF-α, IL-1ß, IL-6), and diminished retinal vascular permeability. BMSCs persisted in the vitreous and were also found within ischemic retina. CONCLUSIONS: Taken together, our results indicate that intravitreal injection of BMSCs rescued the retina from ischemic damage in a rat model. The mechanisms include suppression of apoptosis, attenuation of inflammation and vascular permeability, and preservation of autophagy.

Hypoxic-Preconditioned Bone Marrow Stem Cell Medium Significantly Improves Outcome After Retinal Ischemia in Rats.

PURPOSE: We have previously demonstrated the protective effect of bone marrow stem cell (BMSC)-conditioned medium in retinal ischemic injury. We hypothesized here that hypoxic preconditioning of stem cells significantly enhances the neuroprotective effect of the conditioned medium and thereby augments the protective effect in ischemic retina. METHODS: Rats were subjected to retinal ischemia by increasing intraocular pressure to 130 to 135 mm Hg for 55 minutes. Hypoxic-preconditioned, hypoxic unconditioned, or normoxic medium was injected into the vitreous 24 hours after ischemia ended. Recovery was assessed 7 days after injections by comparing electroretinography measurements, histologic examination, and apoptosis (TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay). To compare proteins secreted into the medium in the groups and the effect of hypoxic exposure, we used rat cytokine arrays. RESULTS: Eyes injected with hypoxic BMSC-conditioned medium 24 hours after ischemia demonstrated significantly enhanced return of retinal function, decreased retinal ganglion cell layer loss, and attenuated apoptosis compared to those administered normoxic or hypoxic unconditioned medium. Hypoxic-preconditioned medium had 21 significantly increased protein levels compared to normoxic medium. CONCLUSIONS: The medium from hypoxic-preconditioned BMSCs robustly restored retinal function and prevented cell loss after ischemia when injected 24 hours after ischemia. The protective effect was even more pronounced than in our previous studies of normoxic conditioned medium. Prosurvival signals triggered by the secretome may play a role in this neuroprotective effect.

Nonmuscle myosin light chain kinase activity modulates radiation-induced lung injury.

Radiotherapy as a primary treatment for thoracic malignancies induces deleterious effects, such as acute or subacute radiation-induced lung injury (RILI). Although the molecular etiology of RILI is controversial and likely multifactorial, a potentially important cellular target is the lung endothelial cytoskeleton that regulates paracellular gap formation and the influx of macromolecules and fluid to the alveolar space. Here we investigate the central role of a key endothelial cytoskeletal regulatory protein, the nonmuscle isoform of myosin light chain kinase (nmMLCK), in an established murine RILI model. Our results indicate that thoracic irradiation significantly augmented nmMLCK protein expression and enzymatic activity in murine lungs. Furthermore, genetically engineered mice harboring a deletion of the nmMLCK gene (nmMLCK(-/-) mice) exhibited protection from RILI, as assessed by attenuated vascular leakage and leukocyte infiltration. In addition, irradiated wild-type mice treated with two distinct MLCK enzymatic inhibitors, ML-7 and PIK (peptide inhibitor of kinase), also demonstrated attenuated RILI. Taken together, these data suggests a key role for nmMLCK in vascular barrier regulation in RILI and warrants further examination of RILI strategies that target nmMLCK.

Role of GADD45a in murine models of radiation- and bleomycin-induced lung injury.

We previously reported protective effects of GADD45a (growth arrest and DNA damage-inducible gene 45 alpha) in murine ventilator-induced lung injury (VILI) via effects on Akt-mediated endothelial cell signaling. In the present study we investigated the role of GADD45a in separate murine models of radiation- and bleomycin-induced lung injury. Initial studies of wild-type mice subjected to single-dose thoracic radiation (10 Gy) confirmed a significant increase in lung GADD45a expression within 24 h and persistent at 6 wk. Mice deficient in GADD45a (GADD45a(-/-)) demonstrated increased susceptibility to radiation-induced lung injury (RILI, 10 Gy) evidenced by increased bronchoalveolar lavage (BAL) fluid total cell counts, protein and albumin levels, and levels of inflammatory cytokines compared with RILI-challenged wild-type animals at 2 and 4 wk. Furthermore, GADD45a(-/-) mice had decreased total and phosphorylated lung Akt levels both at baseline and 6 wk after RILI challenge relative to wild-type mice while increased RILI susceptibility was observed in both Akt(+/-) mice and mice treated with an Akt inhibitor beginning 1 wk prior to irradiation. Additionally, overexpression of a constitutively active Akt1 transgene reversed RILI-susceptibility in GADD45a(-/-) mice. In separate studies, lung fibrotic changes 2 wk after treatment with bleomycin (0.25 U/kg IT) was significantly increased in GADD45a(-/-) mice compared with wild-type mice assessed by lung collagen content and histology. These data implicate GADD45a as an important modulator of lung inflammatory responses across different injury models and highlight GADD45a-mediated signaling as a novel target in inflammatory lung injury clinically.

Sphingosine-1-phosphate lyase is an endogenous suppressor of pulmonary fibrosis: role of S1P signalling and autophagy.

INTRODUCTION: Idiopathic pulmonary fibrosis (IPF) is characterised by accumulation of fibroblasts and myofibroblasts and deposition of extracellular matrix proteins. Sphingosine-1-phosphate (S1P) signalling plays a critical role in pulmonary fibrosis. METHODS: S1P lyase (S1PL) expression in peripheral blood mononuclear cells (PBMCs) was correlated with pulmonary functions and overall survival; used a murine model to check the role of S1PL on the fibrogenesis and a cell culture system to study the effect of S1PL expression on transforming growth factor (TGF)-ß- and S1P-induced fibroblast differentiation. RESULTS: S1PL expression was upregulated in fibrotic lung tissues and primary lung fibroblasts isolated from patients with IPF and bleomycin-challenged mice. TGF-ß increased the expression of S1PL in human lung fibroblasts via activation and binding of Smad3 transcription factor to Sgpl1 promoter. Overexpression of S1PL attenuated TGF-ß-induced and S1P-induced differentiation of human lung fibroblasts through regulation of the expression of LC3 and beclin 1. Knockdown of S1PL (Sgpl1(+/-)) in mice augmented bleomycin-induced pulmonary fibrosis, and patients with IPF reduced Sgpl1 mRNA expression in PBMCs exhibited higher severity of fibrosis and lower survival rate. CONCLUSION: These studies suggest that S1PL is a novel endogenous suppressor of pulmonary fibrosis in human IPF and animal models.

The mitochondrial cardiolipin remodeling enzyme lysocardiolipin acyltransferase is a novel target in pulmonary fibrosis.

RATIONALE: Lysocardiolipin acyltransferase (LYCAT), a cardiolipin-remodeling enzyme regulating the 18:2 linoleic acid pattern of mammalian mitochondrial cardiolipin, is necessary for maintaining normal mitochondrial function and vascular development. We hypothesized that modulation of LYCAT expression in lung epithelium regulates development of pulmonary fibrosis. OBJECTIVES: To define a role for LYCAT in human and murine models of pulmonary fibrosis. METHODS: We analyzed the correlation of LYCAT expression in peripheral blood mononuclear cells (PBMCs) with the outcomes of pulmonary functions and overall survival, and used the murine models to establish the role of LYCAT in fibrogenesis. We studied the LYCAT action on cardiolipin remodeling, mitochondrial reactive oxygen species generation, and apoptosis of alveolar epithelial cells under bleomycin challenge. MEASUREMENTS AND MAIN RESULTS: LYCAT expression was significantly altered in PBMCs and lung tissues from patients with idiopathic pulmonary fibrosis (IPF), which was confirmed in two preclinical murine models of IPF, bleomycin- and radiation-induced pulmonary fibrosis. LYCAT mRNA expression in PBMCs directly and significantly correlated with carbon monoxide diffusion capacity, pulmonary function outcomes, and overall survival. In both bleomycin- and radiation-induced pulmonary fibrosis murine models, hLYCAT overexpression reduced several indices of lung fibrosis, whereas down-regulation of native LYCAT expression by siRNA accentuated fibrogenesis. In vitro studies demonstrated that LYCAT modulated bleomycin-induced cardiolipin remodeling, mitochondrial membrane potential, reactive oxygen species generation, and apoptosis of alveolar epithelial cells, potential mechanisms of LYCAT-mediated lung protection. CONCLUSIONS: This study is the first to identify modulation of LYCAT expression in fibrotic lungs and offers a novel therapeutic approach for ameliorating lung inflammation and pulmonary fibrosis.

Sphingosine-1-phosphate, FTY720, and sphingosine-1-phosphate receptors in the pathobiology of acute lung injury.

Acute lung injury (ALI) attributable to sepsis or mechanical ventilation and subacute lung injury because of ionizing radiation (RILI) share profound increases in vascular permeability as a key element and a common pathway driving increased morbidity and mortality. Unfortunately, despite advances in the understanding of lung pathophysiology, specific therapies do not yet exist for the treatment of ALI or RILI, or for the alleviation of unremitting pulmonary leakage, which serves as a defining feature of the illness. A critical need exists for new mechanistic insights that can lead to novel strategies, biomarkers, and therapies to reduce lung injury. Sphingosine 1-phosphate (S1P) is a naturally occurring bioactive sphingolipid that acts extracellularly via its G protein-coupled S1P1-5 as well as intracellularly on various targets. S1P-mediated cellular responses are regulated by the synthesis of S1P, catalyzed by sphingosine kinases 1 and 2, and by the degradation of S1P mediated by lipid phosphate phosphatases, S1P phosphatases, and S1P lyase. We and others have demonstrated that S1P is a potent angiogenic factor that enhances lung endothelial cell integrity and an inhibitor of vascular permeability and alveolar flooding in preclinical animal models of ALI. In addition to S1P, S1P analogues such as 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol (FTY720), FTY720 phosphate, and FTY720 phosphonates offer therapeutic potential in murine models of lung injury. This translational review summarizes the roles of S1P, S1P analogues, S1P-metabolizing enzymes, and S1P receptors in the pathophysiology of lung injury, with particular emphasis on the development of potential novel biomarkers and S1P-based therapies for ALI and RILI.

Role of migratory inhibition factor in age-related susceptibility to radiation lung injury via NF-E2-related factor-2 and antioxidant regulation.

Microvascular injury and increased vascular leakage are prominent features of radiation-induced lung injury (RILI), and often follow cancer-associated thoracic irradiation. Our previous studies demonstrated that polymorphisms in the gene (MIF) encoding macrophage migratory inhibition factor (MIF), a multifunctional pleiotropic cytokine, confer susceptibility to acute inflammatory lung injury and increased vascular permeability, particularly in senescent mice. In this study, we exposed wild-type and genetically engineered mif(-/-) mice to 20 Gy single-fraction thoracic radiation to investigate the age-related role of MIF in murine RILI (mice were aged 8 wk, 8 mo, or 16 mo). Relative to 8-week-old mice, decreased MIF was observed in bronchoalveolar lavage fluid and lung tissue of 8- to 16-month-old wild-type mice. In addition, radiated 8- to 16-month-old mif(-/-) mice exhibited significantly decreased bronchoalveolar lavage fluid total antioxidant concentrations with progressive age-related decreases in the nuclear expression of NF-E2-related factor-2 (Nrf2), a transcription factor involved in antioxidant gene up-regulation in response to reactive oxygen species. This was accompanied by decreases in both protein concentrations (NQO1, GCLC, and heme oxygenase-1) and mRNA concentrations (Gpx1, Prdx1, and Txn1) of Nrf2-influenced antioxidant gene targets. In addition, MIF-silenced (short, interfering RNA) human lung endothelial cells failed to express Nrf2 after oxidative (H2O2) challenge, an effect reversed by recombinant MIF administration. However, treatment with an antioxidant (glutathione reduced ester), but not an Nrf2 substrate (N-acetyl cysteine), protected aged mif(-/-) mice from RILI. These findings implicate an important role for MIF in radiation-induced changes in lung-cell antioxidant concentrations via Nrf2, and suggest that MIF may contribute to age-related susceptibility to thoracic radiation.

Targeting sphingosine kinase 1 attenuates bleomycin-induced pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease, wherein transforming growth factor ß (TGF-ß) and sphingosine-1-phosphate (S1P) contribute to the pathogenesis of fibrosis. However, the in vivo contribution of sphingosine kinase (SphK) in fibrotic processes has not been documented. Microarray analysis of blood mononuclear cells from patients with IPF and SphK1- or SphK2-knockdown mice and SphK inhibitor were used to assess the role of SphKs in fibrogenesis. The expression of SphK1/2 negatively correlated with lung function and survival in patients with IPF. Also, the expression of SphK1 was increased in lung tissues from patients with IPF and bleomycin-challenged mice. Knockdown of SphK1, but not SphK2, increased survival and resistance to pulmonary fibrosis in bleomycin-challenged mice. Administration of SphK inhibitor reduced bleomycin-induced mortality and pulmonary fibrosis in mice. Knockdown of SphK1 or treatment with SphK inhibitor attenuated S1P generation and TGF-ß secretion in a bleomycin-induced lung fibrosis mouse model that was accompanied by reduced phosphorylation of Smad2 and MAPKs in lung tissue. In vitro, bleomycin-induced expression of SphK1 in lung fibroblast was found to be TGF-ß dependent. Taken together, these data indicate that SphK1 plays a critical role in the pathology of lung fibrosis and is a novel therapeutic target.

Particulate matter air pollution disrupts endothelial cell barrier via calpain-mediated tight junction protein degradation.

BACKGROUND: Exposure to particulate matter (PM) is a significant risk factor for increased cardiopulmonary morbidity and mortality. The mechanism of PM-mediated pathophysiology remains unknown. However, PM is proinflammatory to the endothelium and increases vascular permeability in vitro and in vivo via ROS generation. OBJECTIVES: We explored the role of tight junction proteins as targets for PM-induced loss of lung endothelial cell (EC) barrier integrity and enhanced cardiopulmonary dysfunction. METHODS: Changes in human lung EC monolayer permeability were assessed by Transendothelial Electrical Resistance (TER) in response to PM challenge (collected from Ft. McHenry Tunnel, Baltimore, MD, particle size >0.1 µm). Biochemical assessment of ROS generation and Ca2+ mobilization were also measured. RESULTS: PM exposure induced tight junction protein Zona occludens-1 (ZO-1) relocation from the cell periphery, which was accompanied by significant reductions in ZO-1 protein levels but not in adherens junction proteins (VE-cadherin and ß-catenin). N-acetyl-cysteine (NAC, 5 mM) reduced PM-induced ROS generation in ECs, which further prevented TER decreases and atteneuated ZO-1 degradation. PM also mediated intracellular calcium mobilization via the transient receptor potential cation channel M2 (TRPM2), in a ROS-dependent manner with subsequent activation of the Ca2+-dependent protease calpain. PM-activated calpain is responsible for ZO-1 degradation and EC barrier disruption. Overexpression of ZO-1 attenuated PM-induced endothelial barrier disruption and vascular hyperpermeability in vivo and in vitro. CONCLUSIONS: These results demonstrate that PM induces marked increases in vascular permeability via ROS-mediated calcium leakage via activated TRPM2, and via ZO-1 degradation by activated calpain. These findings support a novel mechanism for PM-induced lung damage and adverse cardiovascular outcomes.