The Protective Effects of Κ-Opioid Receptor Stimulation in Hypoxic Pulmonary Hypertension Involve Inhibition of Autophagy Through the AMPK-MTOR Pathway

Background/Aims: In a previous study, we showed that κ-opioid receptor stimulation with the selective agonist U50,488H ameliorated hypoxic pulmonary hypertension (HPH). However, the roles that pulmonary arterial smooth muscle cell (PASMC) proliferation, apoptosis, and autophagy play in κ-opioid receptor-mediated protection against HPH are still unknown. The goal of the present study was to investigate the role of autophagy in U50,488H-induced HPH protection and the underlying mechanisms. Methods: Rats were exposed to 10% oxygen for three weeks to induce HPH. After hypoxia, the mean pulmonary arterial pressure (mPAP) and the right ventricular pressure (RVP) were measured. Cell viability was monitored using the Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis was detected by flow cytometry and Western blot. Autophagy was assessed by means of the mRFP-GFP-LC3 adenovirus transfection assay and by Western blot. Results: Inhibition of autophagy by the administration of chloroquine prevented the development of HPH in the rat model, as evidenced by significantly reduced mPAP and RVP, as well as decreased autophagy. U50,488H mimicked the effects of chloroquine, and the effects of U50,488H were blocked by nor-BNI, a selective κ-opioid receptor antagonist. In vitro experiments showed that the inhibition of autophagy by chloroquine was associated with decreased proliferation and increased apoptosis of PASMCs. Under hypoxia, U50,488H also significantly inhibited autophagy, reduced proliferation and increased apoptosis of PASMCs. These effects of U50,488H were blocked by nor-BNI. Moreover, exposure to hypoxic conditions significantly increased AMPK phosphorylation and reduced mTOR phosphorylation, and these effects were abrogated by U50,488H. The effects of U50,488H on PASMC autophagy were inhibited by AICAR, a selective AMPK agonist, or by rapamycin, a selective mTOR inhibitor. Conclusion: Our data provide evidence for the first time that κ-opioid receptor stimulation protects against HPH by inhibiting PASMCs autophagy via the AMPK-mTOR pathway.

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Hydrogen Sulfide Metabolism and Pulmonary Hypertension

Cells ◽

10.3390/cells10061477 ◽

2021 ◽

Vol 10 (6) ◽

pp. 1477

Author(s):

Lukas Roubenne ◽

Roger Marthan ◽

Bruno Le Le Grand ◽

Christelle Guibert

Keyword(s):

Pulmonary Hypertension ◽

Hydrogen Sulfide ◽

Pulmonary Circulation ◽

Pulmonary Arteries ◽

K Channel ◽

Protective Effects ◽

Pulmonary Arterial ◽

Regulatory Functions

Pulmonary hypertension (PH) is a severe and multifactorial disease characterized by a progressive elevation of pulmonary arterial resistance and pressure due to remodeling, inflammation, oxidative stress, and vasoreactive alterations of pulmonary arteries (PAs). Currently, the etiology of these pathological features is not clearly understood and, therefore, no curative treatment is available. Since the 1990s, hydrogen sulfide (H2S) has been described as the third gasotransmitter with plethoric regulatory functions in cardiovascular tissues, especially in pulmonary circulation. Alteration in H2S biogenesis has been associated with the hallmarks of PH. H2S is also involved in pulmonary vascular cell homeostasis via the regulation of hypoxia response and mitochondrial bioenergetics, which are critical phenomena affected during the development of PH. In addition, H2S modulates ATP-sensitive K+ channel (KATP) activity, and is associated with PA relaxation. In vitro or in vivo H2S supplementation exerts antioxidative and anti-inflammatory properties, and reduces PA remodeling. Altogether, current findings suggest that H2S promotes protective effects against PH, and could be a relevant target for a new therapeutic strategy, using attractive H2S-releasing molecules. Thus, the present review discusses the involvement and dysregulation of H2S metabolism in pulmonary circulation pathophysiology.

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Depletion of Alveolar Macrophages Attenuates Hypoxic Pulmonary Hypertension but not Hypoxia-Induced Increase in Serum Concentration of MCP-1

Physiological Research ◽

10.33549/physiolres.933187 ◽

2016 ◽

pp. 763-768 ◽

Cited By ~ 13

Author(s):

M. ŽALOUDÍKOVÁ ◽

R. VYTÁŠEK ◽

O. VAJNEROVÁ ◽

O. HNILIČKOVÁ ◽

M. VÍZEK ◽

...

Keyword(s):

Pulmonary Hypertension ◽

Alveolar Macrophages ◽

Pulmonary Arterial Pressure ◽

Hypoxic Pulmonary Hypertension ◽

Monocyte Chemoattractant Protein 1 ◽

Monocyte Chemoattractant ◽

Mean Pulmonary Arterial Pressure ◽

Selective Elimination ◽

Pulmonary Arterial

Exposure to hypoxia, leading to hypoxic pulmonary hypertension (HPH), is associated with activation of alveolar macrophages (AM). However, it remains unclear how AM participate in this process. There are studies which imply that the AM product monocyte chemoattractant protein-1 (MCP-1) plays an important role. Thus we tested: 1. if the selective elimination of AM attenuates HPH in rats, 2. the correlation of MCP-1 plasmatic concentrations with the presence and absence of AM during exposure to hypoxia, 3. the direct influence of hypoxia on MCP-1 production in isolated AM. We found that experimental depletion of AM attenuated the chronic hypoxia-induced increase in mean pulmonary arterial pressure, but did not affect the serum MCP-1 concentrations. Furthermore, the MCP-1 production by AM in vitro was unaffected by hypoxia. Thus we conclude that AM play a significant role in the mechanism of HPH, but MCP-1 release from these cells is most likely not involved in this process. The increase of MCP-1 accompanying the development of HPH probably originates from other sources than AM.

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κ-Opioid Receptor Stimulation Improves Endothelial Function in Hypoxic Pulmonary Hypertension

PLoS ONE ◽

10.1371/journal.pone.0060850 ◽

2013 ◽

Vol 8 (5) ◽

pp. e60850 ◽

Cited By ~ 17

Author(s):

Qi Wu ◽

Hai-Yan Wang ◽

Juan Li ◽

Peng Zhou ◽

Qiu-Lin Wang ◽

...

Keyword(s):

Pulmonary Hypertension ◽

Endothelial Function ◽

Opioid Receptor ◽

Receptor Stimulation ◽

Hypoxic Pulmonary Hypertension

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CGRP and somatostatin modulate chronic hypoxic pulmonary hypertension

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1992.263.3.h681 ◽

1992 ◽

Vol 263 (3) ◽

pp. H681-H690 ◽

Cited By ~ 14

Author(s):

S. Tjen-A-Looi ◽

R. Ekman ◽

H. Lippton ◽

J. Cary ◽

I. Keith

Keyword(s):

Pulmonary Hypertension ◽

Main Pulmonary Artery ◽

Pulmonary Vasculature ◽

Hypoxic Pulmonary Hypertension ◽

Calcitonin Gene ◽

Pulmonary Arterial ◽

Pentobarbital Sodium Anesthesia ◽

The Right ◽

Potential Interactions

Chronic hypoxic pulmonary hypertension (PH), associated with increased pulmonary arterial pressure (PPA) and right ventricular hypertrophy (RVH), correlates significantly with calcitonin gene-related peptide (CGRP) and somatostatin (SOM) levels in lung and blood. CGRP's role in regulation of PPA in chronic hypoxia and its potential interactions with SOM were investigated. CGRP, its antibody (ab) and blocker, CGRP-(8–37), SOM-14, SOM-28, and SOM-ab, respectively, were infused into the pulmonary circulation of hypobaric hypoxia rats for 4, 8, and 16 days. Thereafter, under pentobarbital sodium anesthesia, PPA was measured in the right ventricle and main pulmonary artery. Chronic CGRP infusion prevented PH at all times, whereas immunoneutralization and receptor blocking exacerbated PH. SOM-28 also exacerbated while SOM-14 and SOM-ab decreased PH. RVH generally reflected the PPA. Radioimmunoassay confirmed successful infusion of the peptides with negligible peptide degradation in the pumps throughout 16 days and showed complete immunoneutralization of CGRP with its ab. Peptide levels in lung tissue suggest inhibition of CGRP release by SOM-28 and increased plasma SOM with CGRP infusion. In vitro pharmacological studies suggest that CGRP exerts a receptor-mediated nonadrenergic, nonmuscarinic vasodilatory effect in the lung which is independent of endothelium-derived relaxing factor and does not involve ATP-dependent potassium channels. We conclude that endogenous CGRP plays an important role in pulmonary pressure homeostasis during hypoxia, by directly dilating pulmonary vasculature, thus ameliorating the development of chronic hypoxic pulmonary hypertension in rats.

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Ginkgolic acid induces apoptosis and autophagy of endometrial carcinoma cells via inhibiting PI3K/Akt/mTOR pathway in vivo and in vitro

Human & Experimental Toxicology ◽

10.1177/09603271211023789 ◽

2021 ◽

pp. 096032712110237

Author(s):

L Zhou ◽

S Li ◽

J Sun

Keyword(s):

Endometrial Cancer ◽

B Cells ◽

Western Blot ◽

Signal Pathway ◽

Mtor Pathway ◽

Related Protein ◽

Western Blot Assay ◽

Ginkgolic Acid

Endometrial cancer (EC) is the fourth most common malignancy in women in developed countries. The prognosis of EC is extremely poor, and it is an important factor that contributes to the death of patients. Therefore, studying EC pathogenesis and therapeutic targets, and exploring effective drugs are the primary tasks to improve the prognosis of EC. In the present study, we aimed to explore the function of ginkgolic acid (GA) in EC cell apoptosis and autophagy through PI3K/Akt/mTOR signal pathway in vitro and in vivo. Firstly, MTT assay and clone formation assay were employed to analyze the Ishikawa and HEC-1-B cell viabilities and proliferation after treatment with GA. The results showed that GA inhibited endometrial cancer cell survival. Flow cytometry assay and western blot assay were applied to examine the apoptosis and apoptosis related protein Bcl-2, Bax, Cleaved caspase-3 expression levels of Ishikawa and HEC-1-B cells after treatment with GA. Next, we applied western blot assay to analyze the autophagy associated proteins LC3I, LC3II, p62 and Beclin-1 in GA treated Ishikawa and HEC-1-B cells. We found that GA promoted apoptosis and induced autophagy of endometrial cancer cells. Meanwhile, western blot assay was also used to determine the expression levels of the PI3K/Akt/mTOR signal pathway related protein and the results revealed that GA inhibited the activity of PI3K/Akt/mTOR pathway. Finally, we found that GA inhibited tumor growth in vivo through immunohistochemistry assay. In conclusion, GA induces apoptosis and autophagy of EC cells via inhibiting PI3K/Akt/mTOR pathway in vivo and vitro.

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MYL6B drives the capabilities of proliferation, invasion, and migration in rectal adenocarcinoma through the EMT process

Open Life Sciences ◽

10.1515/biol-2020-0031 ◽

2020 ◽

Vol 15 (1) ◽

pp. 522-531

Author(s):

Jin-Liang Li ◽

Zai-Qiu Wang ◽

Xiao-Li Sun

Keyword(s):

Western Blot ◽

Cell Apoptosis ◽

Rectal Adenocarcinoma ◽

Drug Application ◽

Biological Significance ◽

Expression Level ◽

Cell Counting ◽

Migration And Invasion ◽

Promoting Effect

AbstractObjectiveThis study was designed to explore the biological significance of myosin light chain 6B (MYL6B) in rectal adenocarcinoma.MethodsProfiles on the Oncomine dataset, GEPIA website, and UALCAN-TCGA database were searched to assess the MYL6B expression level in rectal adenocarcinoma tissues and normal tissues. After MYL6B knockdown using siRNA strategy, cell counting kit-8 (CCK-8) and transwell assays were conducted to measure cell proliferation, migration and invasion, respectively. Flow cytometry analysis was conducted to assess cell apoptosis. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot were performed to detect the expression level of mRNAs and proteins.ResultsThe data showed that overexpression of MYL6B was observed in rectal adenocarcinoma tissues and correlated with a poor prognosis of patients. Functional in vitro experiments revealed that MYL6B knockdown could inhibit proliferation, migration, and invasion of rectal adenocarcinoma cells, while promote cell apoptosis. Moreover, western blot analysis suggested that increased expression of E-cadherin and decreased expression of N-cadherin and Vimentin were induced by si-MYL6B.ConclusionIn summary, this study elaborated on the promoting effect of MYL6B in rectal adenocarcinoma progression, thus providing novel insight for strategies of clinical diagnosis and drug application in the future clinical study.

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Serotonin contributes to high pulmonary vascular tone in a sheep model of persistent pulmonary hypertension of the newborn

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00043.2013 ◽

2013 ◽

Vol 304 (12) ◽

pp. L894-L901 ◽

Cited By ~ 25

Author(s):

Cassidy Delaney ◽

Jason Gien ◽

Gates Roe ◽

Nicole Isenberg ◽

Jenai Kailey ◽

...

Keyword(s):

Pulmonary Hypertension ◽

Pulmonary Artery ◽

Tryptophan Hydroxylase ◽

Late Gestation ◽

Persistent Pulmonary Hypertension ◽

Sheep Model ◽

Pulmonary Hemodynamics ◽

Fetal Sheep ◽

Pulmonary Arterial

Although past studies demonstrate that altered serotonin (5-HT) signaling is present in adults with idiopathic pulmonary arterial hypertension, whether serotonin contributes to the pathogenesis of persistent pulmonary hypertension of the newborn (PPHN) is unknown. We hypothesized that 5-HT contributes to increased pulmonary vascular resistance (PVR) in a sheep model of PPHN and that selective 5-HT reuptake inhibitor (SSRI) treatment increases PVR in this model. We studied the hemodynamic effects of 5-HT, ketanserin (5-HT2A receptor antagonist), and sertraline, an SSRI, on pulmonary hemodynamics of the late gestation fetal sheep with PPHN caused by prolonged constriction of the ductus arteriosis. Brief intrapulmonary infusions of 5-HT increased PVR from 1.0 ± 0.07 (baseline) to 1.4 ± 0.22 mmHg/ml per minute of treatment ( P < 0.05). Ketanserin decreased PVR from 1.1 ± 0.15 (baseline) to 0.82 ± 0.09 mmHg/ml per minute of treatment ( P < 0.05). Sertraline increased PVR from 1.1 ± 0.17 (baseline) to 1.4 ± 0.17 mmHg/ml per minute of treatment ( P = 0.01). In addition, we studied 5-HT production and activity in vitro in experimental PPHN. Compared with controls, pulmonary artery endothelial cells from fetal sheep with PPHN exhibited increased expression of tryptophan hydroxylase 1 and 5-HT production by twofold and 56%, respectively. Compared with controls, 5-HT2A R expression was increased in lung homogenates and pulmonary artery smooth muscle cell lysates by 35% and 32%, respectively. We concluded that increased 5-HT contributes to high PVR in experimental PPHN through activation of the 5-HT2A receptor and that SSRI infusion further increases PVR in this model.

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Silencing VEGF enhances the radiosensitivity of the radioresistant human nasopharyngeal carcinoma cell line CNE-2R by inhibiting autophagy through the activation of mTOR pathway

10.21203/rs.3.rs-26678/v1 ◽

2020 ◽

Author(s):

Li Chen ◽

Guoxiang Lin ◽

Kaihua Chen ◽

Fangzhu Wan ◽

Yongchu Sun ◽

...

Keyword(s):

Dna Damage ◽

Nasopharyngeal Carcinoma ◽

Cell Line ◽

Western Blotting ◽

Xenograft Model ◽

Mtor Pathway ◽

Angiogenic Factor ◽

Cell Counting

Abstract Background: Vascular endothelial growth factor (VEGF) is an important pro-angiogenic factor. VEGF was reported to promote the occurrence of autophagy, which enhanced to the radioresistance of tumors. The purpose of our study was to investigate the influence of VEGF silencing on the radiosensitivity of nasopharyngeal carcinoma radioresistant cell line CNE-2R and the underlying mechanisms.Methods: The radiosensitivity of CNE-2R cells after silencing VEGF was detected by cell counting kit 8 (CCK-8) and clonogenic assay, cell cycle and apoptosis was subjected to flow cytometry. DNA damage and autophagy were observed by immunofluorescence and western blotting. The interaction between VEGF and mTOR was confirmed by western blotting and co-immunoprecipitation analysis. In vivo, the effect of VEGF on radiosensitivity of NPC cells was investigated through xenograft model, furthermore, immunohistochemistry and TUNEL assay were used to further verify the relationship between autophagy and radiosensitivity in NPC after VEGF depletion.Results: Downregulation of VEGF significantly inhibited cell proliferation and induced apoptosis of CNE-2R cells after radiotherapy in vitro and in vivo. In addition, VEGF knockdown not only decreased autophagy level, but also delayed the DNA damage repair in CNE-2R cells after irradiation. Mechanistically, silencing VEGF suppressed autophagy through the activation of mTOR pathway.Conclusion: VEGF depletion increased radiosensitivity of NPC radioresistant cell CNE-2R by suppressing autophagy via the activation of mTOR pathway.

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Transgelin as a therapeutic target to prevent hypoxic pulmonary hypertension

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00327.2013 ◽

2014 ◽

Vol 306 (6) ◽

pp. L574-L583 ◽

Cited By ~ 20

Author(s):

Ruifeng Zhang ◽

Liuhong Shi ◽

Lin Zhou ◽

Gensheng Zhang ◽

Xiaohong Wu ◽

...

Keyword(s):

Pulmonary Hypertension ◽

Lentiviral Vector ◽

Hypoxia Inducible Factor ◽

Systolic Pressure ◽

Cellular Migration ◽

Regulation Mechanism ◽

Migration Ability ◽

Pulmonary Vessel ◽

Hypoxic Pulmonary Hypertension

We previously observed that transgelin was preferentially expressed in human pulmonary arterial smooth muscle cells (PAMSCs) under hypoxia and that the upregulation of transgelin was independent of hypoxia-inducible factor 1α (HIF-1α). Reduced transgelin expression was accompanied by significantly impaired migration ability in vitro. However, the regulation mechanism of transgelin and its function in preventing hypoxic pulmonary hypertension (HPH) was unclear. In the present study, RNA interference with hypoxia-inducible factor 2α (HIF-2α) was employed in human PASMCs. Transgelin expression was diminished in HIF-2α-siRNA-treated cells at both the mRNA and protein levels under hypoxia. However, HIF-2α did not transactivate the transgelin promoter directly. TGF-β1 concentration in human PASMCs culture medium was higher under hypoxia, and the accumulated TGF-β1 under hypoxia was regulated by HIF-2α. Furthermore, luciferase and chromatin immunoprecipitation assays indicated that TGF-β1/Smad3 could bind to the transgelin promoter, resulting in increased transgelin expression. In addition to nonintact cellular migration, inhibition of transgelin expression resulted in impaired proliferation in vitro under hypoxia. A lentiviral vector used to inhibit transgelin expression was constructed and intratracheally instilled in rats 3 wk prior to hypoxia treatment. Our final results indicated that inhibition of transgelin expression locally could attenuate increased right ventricular systolic pressure and its associated cardiac and pulmonary vessel remodeling under hypoxia. Our findings indicate that HIF-2α upregulates transgelin indirectly and that accumulated TGF-β1 is a mediator in the upregulation of transgelin by HIF-2α under hypoxia. Inhibition of transgelin expression locally could prevent HPH and pulmonary vascular remodeling in vivo.

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L-Arginine, a precursor of EDRF in vitro, produces pulmonary vasodilation in lambs

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1991.261.5.h1563 ◽

1991 ◽

Vol 261 (5) ◽

pp. H1563-H1569 ◽

Cited By ~ 5

Author(s):

J. R. Fineman ◽

R. Chang ◽

S. J. Soifer

Keyword(s):

Pulmonary Hypertension ◽

Arterial Pressure ◽

Pulmonary Arterial Pressure ◽

Phosphodiesterase Inhibitor ◽

Hemodynamic Effects ◽

Synthesis Inhibitor ◽

Pulmonary Vasodilation ◽

Pulmonary Arterial ◽

Rate Limiting

There is increasing evidence that resting pulmonary vascular tone is mediated in part by the release of endothelium-derived relaxing factors (EDRF). Because L-arginine may be a precursor for EDRF synthesis, we studied the pulmonary vasodilating effects of L-arginine at rest and during pulmonary hypertension in 16 intact newborn lambs. At rest, the intravenous infusions of L-arginine (150 mg/kg) had no hemodynamic effects. However, during pulmonary hypertension induced by hypoxia or the infusion of U-46619 (a thromboxane A2 mimic), L-arginine decreased pulmonary arterial pressure by 22 and 27%, respectively (P less than 0.05). The decrease in pulmonary arterial pressure produced by L-arginine was blocked by methylene blue, a guanylate cyclase inhibitor, and augmented by Zapranast, a guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase inhibitor (-17.9 vs. -31.2%, P less than 0.05). In addition, L-arginine partially reversed the pulmonary hypertension induced by N omega-nitro-L-arginine, a competitive EDRF synthesis inhibitor, but D-arginine had no hemodynamic effects. This study suggests that L-arginine produces pulmonary vasodilation by increasing cGMP concentrations, supporting the in vitro hypothesis that L-arginine is a precursor for EDRF synthesis, whose availability may become rate limiting during pulmonary hypertension.

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