Apoptosis Of Pulmonary Cells Research Articles

Abstract 15621: Checkpoint Kinases Important Players in Lung Fibrogenesis and Pulmonary Vascular Remodeling: A Translational Study

Introduction: Pulmonary fibrosis (PF) is an important commonality in pathogenesis of interstitial lung diseases, and pulmonary hypertension (PH) is a well-recognized severe complication of PF. Both significantly impact patients’ survival and functional status, but without validated treatments. In the lung, parenchymal and vascular remodeling share pathomechanisms, mainly including excessive proliferation and resistance to apoptosis of fibroblasts (LFs) and pulmonary arterial smooth muscle cells (PASMCs). In cancer, sustained cell proliferation is ensured, in part, by a fine tuning of cell cycle and DNA repair machinery, of which checkpoint kinases (CHK1 and CHK2) are critical regulators. They are upregulated in cancer and their inhibition are currently tested in clinical trials. However, these pathways have never been explored in PF-PH. We thus hypothesized that CHK1/2 are upregulated in PF and contribute to both fibrotic and vascular lesions in PF-PH patients. Methods and Results: Increased DNA damage (γH2Ax and p(S4/S8)-RPA32) as well as augmented expression and activity of CHK1/2 was observed by WB in lung and isolated LFs from PF patients compared to controls (p<0.05, n=8~15 per group) with marked expression in fibroblastic foci. Similarly, increased levels of CHK1/2 and DNA damage were detected by WB and IF in remodeled pulmonary arteries (PA) of PF patients with or without PH. Similar findings were noted in mice exposed to bleomycin. In isolated PF-LFs and PASMCs, dual inhibition of CHK1/2 using MK-8776 and LY2606368 significantly reduced expression of TCTP and RAD51 (two factors required for efficient DNA repair), leading to exacerbation of DNA damage and resulted in reduced cell proliferation (Ki67 labeling, WB PCNA) and resistance to apoptosis (Annexin V assay, WB Survivin). Furthermore, inhibition of CHK1/2 mitigated the hyper-activated state of PF-LFs, as illustrated by reduced expression of FN, CTGF, and pSTAT3 (WB). Similar results were observed in control LFs exposed to TGF-β1; all p<0.05. Conclusions: Our data provide compelling evidence that CHK1/2 are involved in lung fibrogenesis and PA remodeling in PF. Current experiments aim to determine whether CHK1/2 inhibitors elicit beneficial effects in animal models of PF-PH.

ADAM17 Deficiency Protects against Pulmonary Emphysema.

Pulmonary emphysema is the major debilitating component of chronic obstructive pulmonary disease (COPD), which is a leading cause of morbidity and mortality worldwide. The ADAM17 (A disintegrin and metalloproteinase 17) protease mediates inflammation via ectodomain shedding of numerous proinflammatory cytokines, cytokine receptors, and adhesion molecules; however, its role in the pathogenesis of emphysema and COPD is poorly understood. This study aims to define the role of the protease ADAM17 in the pathogenesis of pulmonary emphysema. ADAM17 protein expression and activation was investigated in lung biopsies from patients with emphysema, as well as lungs of the emphysematous gp130F/F mouse model and an acute (4 d) cigarette smoke (CS)-induced lung pathology model. The Adam17ex/ex mice, which display significantly reduced global ADAM17 expression, were coupled with emphysema-prone gp130F/F mice to produce gp130F/F:Adam17ex/ex. Both Adam17ex/ex and wild-type mice were subjected to acute CS exposure. Histological, immunohistochemical, immunofluorescence, and molecular analyses as well as lung function tests were performed to assess pulmonary emphysema, inflammation, and alveolar cell apoptosis. ADAM17 was hyperphosphorylated in the lungs of patients with emphysema and also in emphysematous gp130F/F and CS-exposed mice. ADAM17 deficiency ameliorated the development of pulmonary emphysema in gp130F/F mice by suppressing elevated alveolar cell apoptosis. In addition, genetic blockade of ADAM17 protected mice from CS-induced pulmonary inflammation and alveolar cell apoptosis. Our study places the protease ADAM17 as a central molecular switch implicated in the development of pulmonary emphysema, which paves the way for using ADAM17 inhibitors as potential therapeutic agents to treat COPD and emphysema.

MUTYH Deficiency Is Associated with Attenuated Pulmonary Fibrosis in a Bleomycin-Induced Model

Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible lung disease of unknown etiology with limited survival. IPF incidence and prevalence increase significantly with aging, which is associated with an age-related accumulation of oxidative DNA damage. The Mutyh gene is involved in the base excision repair (BER) system, which is critical for repairing the misincorporated adenine that is opposite to the oxidized guanine base, 8-oxoguanine, and maintaining the fidelity of DNA replication. We used Mutyh knockout mice and a bleomycin-induced pulmonary fibrosis model to test the effect of MUTYH deficiency on lesion progression. Unexpectedly, a much less severe lesion of pulmonary fibrosis was observed in Mutyh−/− than in Mutyh+/+mice, which was supported by assay on protein levels of TGF-β1 and both fibrotic markers, α-SMA and Vimentin, in pulmonary tissues of the model animals. Mechanically, MUTYH deficiency prevented the genomic DNA of pulmonary tissue cells from the buildup of single-strand breaks (SSBs) of DNA and maintained the integrity of mtDNA. Furthermore, increased mitochondrial dynamic regulation and mitophagy were detected in pulmonary tissues of the bleomycin-induced Mutyh−/− model mice, which could reduce the pulmonary epithelial cell apoptosis. Our results suggested that MUTYH deficiency could even induce protective responses of pulmonary tissue under severe oxidative stress.

Knockdown of lncRNA MALAT1 Alleviates LPS-Induced Acute Lung Injury via Inhibiting Apoptosis Through the miR-194-5p/FOXP2 Axis.

PurposeWe aimed to identify and verify the key genes and lncRNAs associated with acute lung injury (ALI) and explore the pathogenesis of ALI. Research showed that lower expression of the lncRNA metastasis-associated lung carcinoma transcript 1 (MALAT1) alleviates lung injury induced by lipopolysaccharide (LPS). Nevertheless, the mechanisms of MALAT1 on cellular apoptosis remain unclear in LPS-stimulated ALI. We investigated the mechanism of MALAT1 in modulating the apoptosis of LPS-induced human pulmonary alveolar epithelial cells (HPAEpiC).MethodsDifferentially expressed lncRNAs between the ALI samples and normal controls were identified using gene expression profiles. ALI-related genes were determined by the overlap of differentially expressed genes (DEGs), genes correlated with lung, genes correlated with key lncRNAs, and genes sharing significantly high proportions of microRNA targets with MALAT1. Quantitative real-time PCR (qPCR) was applied to detect the expression of MALAT1, microRNA (miR)-194-5p, and forkhead box P2 (FOXP2) mRNA in 1 μg/ml LPS-treated HPAEpiC. MALAT1 knockdown vectors, miR-194-5p inhibitors, and ov-FOXP2 were constructed and used to transfect HPAEpiC. The influence of MALAT1 knockdown on LPS-induced HPAEpiC proliferation and apoptosis via the miR-194-5p/FOXP2 axis was determined using Cell counting kit-8 (CCK-8) assay, flow cytometry, and Western blotting analysis, respectively. The interactions between MALAT1, miR-194-5p, and FOXP2 were verified using dual-luciferase reporter gene assay.ResultsWe identified a key lncRNA (MALAT1) and three key genes (EYA1, WNT5A, and FOXP2) that are closely correlated with the pathogenesis of ALI. LPS stimulation promoted MALAT1 expression and apoptosis and also inhibited HPAEpiC viability. MALAT1 knockdown significantly improved viability and suppressed the apoptosis of LPS-stimulated HPAEpiC. Moreover, MALAT1 directly targeted miR-194-5p, a downregulated miRNA in LPS-stimulated HPAEpiC, when FOXP2 was overexpressed. MALAT1 knockdown led to the overexpression of miR-194-5p and restrained FOXP2 expression. Furthermore, inhibition of miR-194-5p exerted a rescue effect on MALAT1 knockdown of FOXP2, whereas the overexpression of FOXP2 reversed the effect of MALAT1 knockdown on viability and apoptosis of LPS-stimulated HPAEpiC.ConclusionOur results demonstrated that MALAT1 knockdown alleviated HPAEpiC apoptosis by competitively binding to miR-194-5p and then elevating the inhibitory effect on its target FOXP2. These data provide a novel insight into the role of MALAT1 in the progression of ALI and potential diagnostic and therapeutic strategies for ALI patients.

Oxidized DNA Precursors Cleanup by NUDT1 Contributes to Vascular Remodeling in Pulmonary Arterial Hypertension.

Rationale: Pulmonary arterial hypertension (PAH) is a life-threatening condition characterized by abnormally elevated pulmonary pressures and right ventricular failure. Excessive proliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs) is one of the most important drivers of vascular remodeling in PAH, for which available treatments have limited effectiveness.Objectives: To gain insights into the mechanisms leading to the development of the disease and identify new actionable targets.Methods: Protein expression profiling was conducted by two-dimensional liquid chromatography coupled to tandem mass spectrometry in isolated PASMCs from controls and patients with PAH. Multiple molecular, biochemical, and pharmacologic approaches were used to decipher the role of NUDT1 (nudrix hyrolase 1) in PAH.Measurements and Main Results: Increased expression of the detoxifying DNA enzyme NUDT1 was detected in cells and tissues from patients with PAH and animal models. In vitro, molecular or pharmacological inhibition of NUDT1 in PAH-PASMCs induced accumulation of oxidized nucleotides in the DNA, irresolvable DNA damage (comet assay), disruption of cellular bioenergetics (Seahorse), and cell death (terminal deoxynucleotidyl transferase dUTP nick end labeling assay). In two animal models with established PAH (i.e., monocrotaline and Sugen/hypoxia-treated rats), pharmacological inhibition of NUDT1 using (S)-Crizotinib significantly decreased pulmonary vascular remodeling and improved hemodynamics and cardiac function.Conclusions: Our results indicate that, by overexpressing NUDT1, PAH-PASMCs hijack persistent oxidative stress in preventing incorporation of oxidized nucleotides into DNA, thus allowing the cell to escape apoptosis and proliferate. Given that NUDT1 inhibitors are under clinical investigation for cancer, they may represent a new therapeutic option for PAH.

Hsa_circNFXL1_009 modulates apoptosis, proliferation, migration, and potassium channel activation in pulmonary hypertension

In this study, we explored the circular RNA (circRNA) profile in pulmonary arterial hypertension (PAH) patients caused by chronic obstructive pulmonary disease (COPD) and the effects of hsa_circNFXL1_009 on abnormal proliferation, apoptosis, and migration of human pulmonary arterial smooth muscle cells (hPASMCs) driven by hypoxia. Using microarrays, we screened the circRNA profile in whole-blood samples from three pairs of subjects and found 158 dysregulated circRNAs in patients with PAH-COPD. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis further validated that hsa_circNFXL1_009 was dramatically downregulated with the highest area under a receiver operating characteristic curve (ROC) in 21 pairs of subjects. Consistently, exposure to hypoxia markedly reduced the hsa_circNFXL1_009 level in cultured hPASMCs. Delivery of exogenous hsa_circNFXL1_009 attenuated hypoxia-induced proliferation, apoptotic resistance, and migration of hPASMCs, as evidenced by immunocytochemistry, 5-ethynyl-2′-deoxyuridine incorporation, wound healing, and a TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling) assay. A luciferase assay showed that hsa_circNFXL1_009 directly sponged hsa-miR-29b-2-5p (miR-29b) and positively regulated the expression of voltage-gated potassium (K+) channel subfamily B member 1 (KCNB1) at the mRNA level. Using patch-clamp electrophysiology, we proved that overexpression of hsa_circNFXL1_009 promoted a whole-cell K+ current in hPASMCs. Taken together, these studies identify hsa_circNFXL1_009 as a key regulator of PAH, and it may be used as a potential therapeutic target for the treatment of PAH.

CircATP2B4 promotes hypoxia-induced proliferation and migration of pulmonary arterial smooth muscle cells via the miR-223/ATR axis

BackgroundCircular RNAs (circRNAs) function as important modulators in the progression of pulmonary arterial hypertension (PAH). Here, we aimed to discover the role and working mechanism of circATP2B4 in hypoxia-induced proliferation and migration of PASMCs. MethodsThe proliferation, migration and apoptosis of pulmonary arterial smooth muscle cells (PASMCs) were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), wound healing assay and flow cytometry. The expression of circATP2B4, ATPase plasma membrane Ca2+ transporting 4 (ATP2B4), microRNA-223 (miR-223) and ATR serine/threonine kinase (ATR) was quantified by quantitative real time polymerase chain reaction (qRT-PCR). Circular RNA Interactome and microT-CDS were used to search the targets of circATP2B4 and miR-223, respectively. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were used to validate the above predictions. Western blot assay was performed to detect the protein expression of ATR. ResultsHypoxia treatment promoted the proliferation and migration and impeded the apoptosis of PASMCs. A significant increase of circATP2B4 expression was observed in the serum of PAH patients and hypoxia-induced PASMCs compared with healthy volunteers and PASMCs under normoxia condition. MiR-223 is a target of circATP2B4, and the effects of circATP2B4 silencing on PASMCs were overturned by the transfection of anti-miR-223. ATR is a functional target of miR-223, and miR-223 inhibited the proliferation and migration while accelerated the apoptosis of PASMCs through targeting and down-regulating ATR. CircATP2B4 could up-regulate the level of ATR through sponging miR-223 in PSAMCs. ConclusionCircATP2B4 potentiated hypoxia-induced proliferation and migration of PASMCs through the miR-223/ATR axis. Restoration of the level of miR-223 might be an effective therapeutic method for the treatment of PAH.

Vital Roles of Gremlin-1 in Pulmonary Arterial Hypertension Induced by Systemic-to-Pulmonary Shunts.

BackgroundHeterozygous mutation in BMP (bone morphogenetic protein) receptor 2 is rare, but BMP cascade suppression is common in congenital heart disease–associated pulmonary arterial hypertension (CHD‐PAH); however, the underling mechanism of BMP cascade suppression independent of BMP receptor 2 mutation is unknown.Methods and ResultsPulmonary hypertensive status observed in CHD‐PAH was surgically reproduced in rats. Gremlin‐1 expression was increased, but BMP cascade was suppressed, in lungs from CHD‐PAH patients and shunted rats, whereas shunt correction retarded these trends in rats. Immunostaining demonstrated increased gremlin‐1 was mainly in the endothelium and media of remodeled pulmonary arteries. However, mechanical stretch time‐ and amplitude‐dependently stimulated gremlin‐1 secretion and suppressed BMP cascade in distal pulmonary arterial smooth muscle cells from healthy rats. Under static condition, gremlin‐1 significantly promoted the proliferation and inhibited the apoptosis of distal pulmonary arterial smooth muscle cells from healthy rats via BMP cascade. Furthermore, plasma gremlin‐1 closely correlated with hemodynamic parameters in CHD‐PAH patients and shunted rats.ConclusionsServing as an endogenous antagonist of BMP cascade, the increase of gremlin‐1 in CHD‐PAH may present a reasonable mechanism explanation for BMP cascade suppression independent of BMP receptor 2 mutation.

Long-Chain Non-Coding RNA Metastasis-Related Lung Adenocarcinoma Transcript 1 (MALAT1) Promotes the Proliferation and Migration of Human Pulmonary Artery Smooth Muscle Cells (hPASMCs) by Regulating the MicroRNA-503 (miR-503)/Toll-Like Receptor 4 (TLR4) Signal Axis.

BackgroundTo study the role of the long-chain noncoding RNA (lncRNA) metastasis-related lung adenocarcinoma transcript 1 (MALAT1), microRNA-503 (miR-503), Toll-like receptor 4 (TLR4) signal axis in the pathogenesis of pulmonary arterial hypertension (PAH).Material/MethodsTotal RNA was extracted from the plasma of 45 PAH patients and 45 healthy subjects, and the expression of lncRNA MALAT1 and miR-503 was measured by quantitative real-time polymerase chain reaction (qRT-PCR). The effects of lncRNA MALAT1 and miR-503 on Toll-like receptor 4 (TLR4) and the proliferation, migration, and apoptosis of human pulmonary artery smooth muscle cells (hPASMCs) were tested following in vitro transfection of hPASMCs.ResultslncRNA MALAT1 was highly expressed in the plasma of PAH patients and in hypoxia-induced hPASMCs. Silencing lncRNA MALAT1 inhibited the proliferation and migration of hPASMC cells while promoting their apoptosis. MiR-503 is underexpressed in plasma and hPASMCs of patients with PAH. TLR4 was a target gene of miR-503 and was highly expressed in peripheral blood mononuclear cells (PBMCs) of PAH patients. lncRNA MALAT1 was a “molecular sponge” of miR-503, regulating the expression of TLR4 and the proliferation, migration, and apoptosis of hPASMCs through miR-503.ConclusionslncRNA MALAT1 promotes the proliferation and migration of hPASMCs and inhibits their apoptosis by inhibiting the miR-503/TLR4 signal axis.

Dexmedetomidine alleviates LPS‑induced acute lung injury via regulation of the p38/HO‑1 pathway.

Acute lung injury (ALI) is a common critical illness in clinical anesthesia and the intensive care unit that can cause acute hypoxic respiratory insufficiency. Despite various therapeutic regimes having been investigated, there is currently no effective pharmacotherapy available to treat ALI. Previous studies have reported that the NOD-like receptor protein 3 (NLRP3) signaling pathway plays an important role in the inflammatory response and is involved in the pathogenesis of ALI. Moreover, dexmedetomidine (Dex), an α2-adrenergic receptor activating agent, has been routinely used as an adjuvant therapy in treating inflammatory diseases, including ALI. However, the precise pathological mechanisms of Dex in ALI remain to be elucidated. Thus, the present study aimed to investigate the effects of the p38/heme oxygenase 1 (HO-1) signaling pathways in the pathological mechanisms of Dex in ALI. Newborn male Sprague-Dawley rats (n=48) were randomly divided into four groups (n=12 each), and an intravenous injection of lipopolysaccharide (LPS) was used to successfully induce the ALI model, with increased pulmonary damage, cell apoptosis, interleukin-1β (IL-1β) secretion and edema fluid in lungs. Moreover, the mRNA and protein expression levels of NLRP3 were significantly upregulated, while that of HO-1 were downregulated by LPS treatment. Furthermore, the levels of phosphorylated p38 were also upregulated in ALI rats. It was demonstrated that Dex administration significantly alleviated LPS-induced ALI, downregulated the secretion of IL-1β, decreased the expression of NLRP3, inhibited the phospho-activation of p38 and increased HO-1 expression. In addition, pharmacological inhibition of p38 using the inhibitor SB20380 further enhanced the effect of Dex. Collectively, these preliminarily results identified the effects of Dex intervention on the pathogenesis of ALI via the regulation of p38/HO-1 signaling pathways, which impacted the inflammatory effects, thus providing a theoretical basis and novel evidence for the development of new targets for clinical treatment of ALI.

Ma xing shi gan decoction eliminates PM2.5-induced lung injury by reducing pulmonary cell apoptosis through Akt/mTOR/p70S6K pathway in rats.

The present study was designed to investigate the anti-apoptosis effect of Ma xing shi gan decoction (MXD) on PM2.5-induced lung injury via protein kinase B (Akt)/mTOR/p70S6K pathway. A UPLC-MS/MS system was introduced for component analysis of MXD. Rats were instilled with PM2.5 solution suspension intratracheally to induce acute lung injury. The rats were then orally administered with MXD (16, 8, and 4 g/kg) once a day for 7 consecutive days. The therapeutic effects of MXD were evaluated by Hematoxylin and Eosin (HE) staining. The apoptotic cell death was analyzed by terminal-deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assay. The alterations in cytochrome c (Cytc) and cleaved-caspase-3 (C-caspase-3) were measured by immunohistochemistry (IHC). The expressions of Bax, B-cell lymphoma 2 (Bcl-2), p-Akt, p-mTOR and p-p70S6K were detected by Western blot. In vitro, PM2.5 exposure model was introduced in A549 cell, followed by incubation with MXD-medicated serum. Hoechst staining was used to determine apoptotic rate. The levels of Bax, Bcl-2, p-Akt, p-mTOR and p-p70S6K were detected by Western blot. Our results in vivo indicated that treatment with MXD decreased histopathological changes score, TUNEL-positive cells rate, expressions of Cytc and C-caspase-3. The in vitro results revealed that incubation with MXD-mediated serum decreased apoptotic rate. Both results in vivo and in vitro demonstrated that MXD inhibited pro-apoptotic protein Bax and promoted anti-apoptotic protein Bcl-2 expression. Likewise, MXD activated Akt/mTOR/p70S6K signal pathway, which was also confirmed by Western immunoblotting. In conclusion, MXD attenuates lung injury and the underlying mechanisms may relate to regulating the apoptosis via Akt/mTOR/p70S6K signaling pathway activation.

MiR-143-3p impacts on pulmonary inflammatory factors and cell apoptosis in mice with mycoplasmal pneumonia by regulating TLR4/MyD88/NF-κB pathway.

miR-143-3p is correlated with inflammatory pain responses, such as hsa-miR-143-3p expression reduction in fibromyalgia. The present study aimed to explore the effects of miR-143-3p and Toll-like receptor (TLR) 4/myeloid differentiation factor 88 (MyD88)/NF-κB signaling pathway on pulmonary inflammatory factors levels and alveolar epithelial cell apoptosis in mycoplasmal pneumonia mice. Twenty mice were selected as normal group. The 120 successfully modeled Mycoplasma pneumoniae (MP) infection mice were randomly divided into model group (without any treatment), negative control (NC) group (injected with NC mimic), miR-143-3p mimic group (injected with miR-143-3p mimic), miR-143-3p inhibitor group (injected with miR-143-3p inhibitor), TAK-242 group (treatment with TAK-242), and miR-143-3p inhibitor + TAK-242 group (treatment with miR-143-3p inhibitor + TAK-242). Compared with model group, model mice had up-regulated miR-143-3p expression and decreased MyD88 and p-NF-κB p50 protein expressions (all P<0.05); Model mice treated with miR-143-3p mimic and TAK-242 had reduced interleukin (IL)-2 and tumor necrosis factor (TNF)-α contents and protein expressions of MyD88, p-NF-κB p50, increased IL-10 content, fewer alveolar epithelial cell apoptosis, lower Bax expression and higher Bcl-2 expression (all P<0.05); however, mice with miR-143-3p inhibitor treatment showed opposite trends in terms of above indicators. The exacerbation of mycoplasmal pneumonia caused by miR-143-3p inhibitor was partly improved by miR-143-3p inhibitor + TAK-242 combination treatment (all P<0.05). Therefore, up-regulation of miR-143-3p expression may ameliorate pulmonary inflammatory factors levels and reduce alveolar epithelial cell apoptosis in mycoplasmal pneumonia mice by inhibiting TLR4/MyD88/NF-κB signaling pathway.

Diet-Induced Obesity Mice Execute Pulmonary Cell Apoptosis via Death Receptor and ER-Stress Pathways after E. coli Infection.

Obesity has developed into a considerable health problem in the whole world. Escherichia coli (E. coli) can cause nosocomial pneumonia and induce cell apoptosis during injury and infection. Normal (lean) and diet-induced obesity mice (DIO, fed with high-fat diet) were chosen to perform nasal instillation with E. coli to establish a nonfatal acute pneumonia model. At 0 h, 12 h, 24 h, and 72 h postinfection, lung tissues were obtained to measure cell apoptosis. As shown in this study, both lean and DIO mice exhibited histopathological lesions of acute pneumonia and increased cell apoptosis in the lung infected with E. coli. Interestingly, the relative mRNA and protein expressions associated with either endoplasmic reticulum stress or death receptor apoptotic pathway were all dramatically increased in the DIO mice after infection, while only significant upregulation of death receptor apoptotic pathway in the lean mice at 72 h. These results indicated that the DIO mice executed excess cell apoptosis in the nonfatal acute pneumonia induced by E. coli infection through endoplasmic reticulum stress and death receptor apoptotic pathway.

Adiponectin ameliorates lung injury induced by intermittent hypoxia through inhibition of ROS-associated pulmonary cell apoptosis.

Obstructive sleep apnea hypopnea syndrome has been reported to be associated with pulmonary hypertension (PH). Adiponectin (Ad) has many protective roles in the human body, including its function as an anti-inflammatory and an anti-oxidant, as well as its role in preventing insulin resistance and atherosclerosis. This study aimed to investigate the molecular mechanism of chronic intermittent hypoxia (CIH)-induced pulmonary injury and the protective role of Ad in experimental rats. Thirty male Sprague-Dawley rats were randomly divided into three groups with 10 rats in each group: normal control (NC) group, CIH group, and CIH + Ad group. Rats in the NC group were kept breathing room air for 12weeks. Rats in the CIH group were intermittently exposed to a hypoxic environment for 8h/day for 12weeks. Rats in the CIH + Ad group received 10μg Ad twice weekly via intravenous injection. After 12weeks of CIH exposure, we detected the pulmonary function, pulmonary artery pressure, lung histology, pulmonary cell apoptosis, pulmonary artery endothelial cell apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) level. We also analyzed expression proteins involved in the mitochondria-, endoplasmic reticulum (ER) stress-, and Fas receptor-associated pulmonary apoptosis pathways, as well as the SIRT3/SOD2 pathway. CIH exposure for 12weeks did not lead to abnormal pulmonary function, PH, or pulmonary artery endothelial cell apoptosis. However, we observed a significant increase in the rate of pulmonary cell apoptosis, the expression of proteins involved in mitochondria-, ER stress-, and Fas receptor-associated pulmonary apoptosis pathways, and the generation of ROS in the CIH group compared with the NC group. In contrast, the MMP and protein expressions of SIRT3/SOD2 pathway were significantly decreased in the CIH group compared with the NC group. Ad supplementation in the CIH + Ad group partially improved these changes induced by CIH. Even though CIH did not cause abnormal pulmonary function or PH, early lung injury was detected at the molecular level in rats exposed to CIH. Treatment with Ad ameliorated the pulmonary injury by activating the SIRT3/SOD2 pathway, reducing ROS generation, and inhibiting ROS-associated lung cell apoptosis.

Conditioned medium from M2b macrophages modulates the proliferation, migration, and apoptosis of pulmonary artery smooth muscle cells by deregulating the PI3K/Akt/FoxO3a pathway.

BackgroundImmunity and inflammation are considered to be central features of pulmonary artery hypertension (PAH), in which macrophages are one of the main components of inflammatory cell infiltration around the pulmonary artery. M2b macrophages, which are different from M1 and M2 macrophages, are believed to have immunomodulatory activities and produce little fibrosis. The purpose of this study was to explore the effect of M2b macrophages on pulmonary artery smooth muscle cells (PASMCs) derived from monocrotaline-induced PAH rats.MethodsPASMCs were cultured in serum-free medium, the supernatant of M0 macrophages, or the supernatant of M2b macrophages for 24 hours. Then cell proliferation was assessed by cell counting kit-8 and cell migration ability was detected by wound healing and transwell assays. The apoptosis rate of cells was determined by TUNEL staining and annexin V-PE/7-ADD staining. Western blot was used to detect the expression of Bcl-2 family proteins, cleaved caspase-9 and PI3K/Akt/FoxO3a pathway. LY294002 (a specific inhibitor of PI3K) was used to investigate its effect on PASMCs and its relationship with M2b macrophages.ResultsConditioned medium from M2b macrophages significantly inhibited the proliferation and migration of PASMCs compared with the control group and M0 macrophage group. Furthermore, conditioned medium from M2b macrophages promote PASMC apoptosis and increased the expression of pro-apoptotic proteins Bax and cleaved caspase-9, inhibited the expression of anti-apoptotic proteins Bcl-2 and Bcl-xl. Finally, conditioned medium from M2b macrophages inhibited the PI3K/Akt/FoxO3a pathway. Inhibition of PI3K/Akt/FoxO3a pathway also significantly inhibit the proliferation, migration, and apoptosis resistance of PASMCs.ConclusionConditioned medium from M2b macrophages can inhibit the proliferation, migration, and apoptosis resistance of PASMCs, which may be at least partially by deregulating the PI3K/Akt/FoxO3a pathway.

Expression and regulation of tumor necrosis factor-α-induced protein-8-like 2 is associated with acute lung injury induced by myocardial ischemia reperfusion in diabetic rats

AimsThe purpose of the present study was to investigate the possible role of TIPE2 on acute lung injury (ALI) induced by myocardial ischemia/reperfusion (MIR) in diabetic rats. MethodsSprague-Dawley (SD) rats were randomly separated into four groups: control+sham (C + sham); control+MIR (C + MIR); diabetes+sham (D + sham); diabetes+MIR (D + MIR). Diabetes was induced using streptozotocin. Eight weeks after diabetes induction, MIR was conducted. At 2 h after MIR, myocardial injury indices were assessed; arterial blood, bronchoalveolar lavage fluid (BALF) and lung tissues were collected for corresponding detection. ResultsRats subjected to MIR showed serious ALI (estimated via pathological changes, lung injury score and Wet/Dry weight ratio), lung inflammation and pulmonary cell apoptosis compared with sham groups, especially in D + MIR group. Evaluation of protein expression in lung tissues showed that p-JNK and nuclear NF-κB p65 protein levels were higher in D + MIR group as compared with C + MIR group. Besides, either hyperglycemia or MIR can significantly upregulate TIPE2 protein levels. ConclusionsIn conclusion, diabetic lungs are more susceptible to MIR. TIPE2 may involve in this pathological process, possibly through regulation of inflammation, oxidative stress and apoptosis.

DDAH1 Overexpression in Human Pulmonary Vascular Endothelial Cells Co‐Cultured with Human Pulmonary Artery Smooth Muscle Cells

Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease of preterm infants. Pulmonary hypertension (PH) develops in 25–40% of BPD patients as a result of pathologic vascular remodeling, and PH is an independent predictor of death in BPD patients. Nitric oxide (NO) is a potent vasodilator and apoptotic mediator made in the pulmonary endothelium by nitric oxide synthase (eNOS). Asymmetric dimethylarginine (ADMA) is an endogenous competitive NOS inhibitor. Dimethylarginine dimethylaminohydrolase (DDAH) metabolizes the majority of ADMA in vivo and there are two isoforms, DDAH1 and DDAH2. Translational studies from our group analyzed a cohort of BPD patients and identified a potential biomarker, DDAH1 single nucleotide polymorphism (rs480414), that decreased the risk of PH in BPD by 61%. In the present study, we hypothesized that overexpression of DDAH1 in human pulmonary microvascular endothelial cells (hPMVEC) would lead to greater apoptosis and lower proliferation of human pulmonary artery smooth muscle cells (hPASMC) via effects in hPMVEC. We utilized a co‐culture system where hPMVEC were transfected with AdDDAH1 or AdGFP control for 24h and then incubated in co‐culture with hPASMC for 24h. Analysis included 1) western blot analysis for cleaved and total caspase‐3, ‐8, ‐9, p21, PCNA, and β‐actin and 2) trypan blue exclusion for viable cell numbers. Transfection of hPMVEC with AdDDAH1 resulted in greater DDAH1 protein expression (n=6, p=0.003) as compared to control. After AdDDAH1 transfection of hPMVEC and co‐culture with hPASMC, we found greater cleaved caspase‐3 (n=6, p=0.01), greater cleaved caspase‐8 (n=6, p=0.03), similar cleaved caspase‐9 (n=6, p=0.94), similar p21 (n=6, p=0.7), and similar PCNA (n=3, p=0.82) protein expression as compared to control. After AdDDAH1 transfection of hPMVEC and co‐culture with hPASMC, we found lower hPASMC viable cell numbers (n=3, p=0.008) as compared to control. After AdDDAH1 transfection of hPMVEC and co‐culture with hPASMC treated with DETA NONOate 250μM or DETA NONOate 250μM + Hemoglobin 100μM, the hPASMC viable cell numbers were lower compared to control (n=3, p=0.001; n=3, p=0.003, respectively). There was no difference detected between DETA NONOate 250μM and DETA NONOate 250μM + Hemoglobin 100μM groups (n=3, p=0.429). In conclusion, DDAH1 overexpression in hPMVEC resulted in greater apoptosis and lower proliferation in hPASMC, consistent with our hypothesis. We propose that DDAH1 is a potential new therapy for BPD‐PH via relieving pulmonary vascular endothelial ADMA‐inhibition of eNOS, resulting in greater endogenous NO production, greater pulmonary vascular wall apoptosis, and potentially less pathologic vascular remodeling. Future studies will determine if DDAH1 endothelial knockout mice have worse lung function as compared to wild‐type, after exposure to BPD‐PH model of LPS and hyperoxia.Support or Funding InformationK08 HL129080 (PI: Trittmann)

Regulatory effects of Prohibitin 1 on proliferation and apoptosis of pulmonary arterial smooth muscle cells in monocrotaline-induced PAH rats

BackgroundPulmonary arterial hypertension (PAH) is a severe pulmonary vascular disease characterized by unbalanced proliferation and apoptosis of pulmonary arterial smooth muscle cells (PASMCs). Prohibitin 1 (PHB1) is known for its significant anti-proliferative activity. However, the role of PHB1 in PASMCs and PAH have not been elucidated. MethodsMonocrotaline (MCT 60 mg/kg) was used to build a PAH model in SD rats. Right ventricular systolic pressure (RVSP) and right ventricle (RV) hypertrophy were measured. Morphology of pulmonary vessels was observed by Hematoxylin-Eosin (HE) staining. Expression of PHB1 in pulmonary arteries and PASMCs was determinated by immunoblot and immunofluorescence. Cell proliferation was detected by CCK8 and EDU when PASMCs were stimulated by PDGF-BB (20 ng/mL). Furthermore, siRNA for PHB1 and Akt inhibitor were conducted to investigate the mechanism behind the role of PHB1 and AKT signaling pathway in PASMCs proliferation and apoptosis. ResultsThe protein expression of PHB1 in PAH rats lung tissue was significantly up-regulated accompanied by elevated RVSP and enhanced RV hypertrophy. Immunohistochemistry showed that PHB1 was mainly localized in the pulmonary vascular smooth muscle layer. PDGF-BB significantly up-regulated the expression of PHB1 in rat primary PASMCs in a time- and dose-dependent manner. After PHB1 knock down, PASMCs proliferation was significantly suppressed while apoptosis was significantly recovered. Meanwhile the level of proliferating cell nuclear antigen (PCNA) and P-Akt were significantly down-regulated. Perifosine (Akt inhibitor) also significantly inhibit proliferation of PASMCs. ConclusionPHB1 contributes to pulmonary vascular remodeling by accelerating proliferation of PASMCs which involves AKT phosphorylation.

LJ-2698, an Adenosine A3 Receptor Antagonist, Alleviates Elastase-Induced Pulmonary Emphysema in Mice.

Emphysema, a major component of chronic obstructive pulmonary disease (COPD), is a leading cause of human death worldwide. The progressive deterioration of lung function that occurs in the disease is caused by chronic inflammation of the airway and destruction of the lung parenchyma. Despite the main impact of inflammation on the pathogenesis of emphysema, current therapeutic regimens mainly offer symptomatic relief and preservation of lung function with little therapeutic impact. In the present study, we aimed to discover novel therapeutics that suppress the pathogenesis of emphysema. Here, we show that LJ-2698, a novel and highly selective antagonist of the adenosine A3 receptor, a G protein-coupled receptor involved in various inflammatory diseases, significantly reversed the elastase-induced destructive changes in murine lungs. We found that LJ-2698 significantly prevented elastase-induced airspace enlargement, resulting in restoration of pulmonary function without causing any obvious changes in body weight in mice. LJ-2698 was found to inhibit matrix metalloproteinase activity and pulmonary cell apoptosis in the murine lung. LJ-2698 treatment induced increases in anti-inflammatory cytokines in macrophages at doses that displayed no significant cytotoxicity in normal cell lines derived from various organs. Treatment with LJ-2698 significantly increased the number of anti-inflammatory M2 macrophages in the lungs. These results implicate the adenosine A3 receptor in the pathogenesis of emphysema. Our findings also demonstrate the potential of LJ-2698 as a novel therapeutic/preventive agent in suppressing disease development with limited toxicity.

Protective Effect of Dexmedetomidine on Acute Lung Injury via the Upregulation of Tumour Necrosis Factor-α-Induced Protein-8-like 2 in Septic Mice.

The aim of the present study was to investigate whether TIPE2 participates in the protective actions of dexmedetomidine (DEX) in a mouse model of sepsis-induced acute lung injury (ALI). We administered TIPE2 adeno-associated virus (AAV-TIPE2) intratracheally into the lungs of mice. Control mice were infected with an adeno-associated virus expressing no transgene. Three weeks later, an animal model of caecal ligation-perforation (CLP)-induced sepsis was established. DEX was administered intravenously 30 min after CLP. Twenty-four hours after sepsis, lung injury was assayed by lung histology, the ratio of polymorphonuclear leukocytes (PMNs) to total cells in the bronchoalveolar lavage fluid (BALF), myeloperoxidase (MPO) activity, BALF protein content and the lung wet-to-dry (W/D) weight ratio. Proinflammatory factor levels in the BALF of mice were measured. The protein expression levels in lung tissues were analysed by Western blotting. The results showed that DEX treatment markedly mitigated sepsis-induced lung injury, which was characterized by the deterioration of histopathology, histologic scores, the W/D weight ratio and total protein levels in the BALF. Moreover, DEX markedly attenuated sepsis-induced lung inflammation, as evidenced by the decrease in the number of PMNs in the BALF, lung MPO activity and proinflammatory cytokines in the BALF. In addition, DEX dramatically prevented sepsis-induced pulmonary cell apoptosis in mice, as reflected by decreases in the number of TUNEL-positive cells, the protein expression of cleaved caspase-9 and cleaved caspase 3 and the Bax/Bcl-2 ratio. In addition, evaluation of protein expression showed that DEX blocked sepsis-activated JNK phosphorylation and NF-κB p65 nuclear translocation. Similar results were also observed in the TIPE2 overexpression group. Our study demonstrated that DEX inhibits acute inflammation and apoptosis in a murine model of sepsis-stimulated ALI via the upregulation of TIPE2 and the suppression of the activation of the NF-κB and JNK signalling pathways.