Suppression of Hypoxia-Inducible Factor 1α by Low-Molecular-Weight Heparin Mitigates Ventilation-Induced Diaphragm Dysfunction in a Murine Endotoxemia Model

Mechanical ventilation (MV) is required to maintain life for patients with sepsis-related acute lung injury but can cause diaphragmatic myotrauma with muscle damage and weakness, known as ventilator-induced diaphragm dysfunction (VIDD). Hypoxia-inducible factor 1α (HIF-1α) plays a crucial role in inducing inflammation and apoptosis. Low-molecular-weight heparin (LMWH) was proven to have anti-inflammatory properties. However, HIF-1α and LMWH affect sepsis-related diaphragm injury has not been investigated. We hypothesized that LMWH would reduce endotoxin-augmented VIDD through HIF-1α. C57BL/6 mice, either wild-type or HIF-1α–deficient, were exposed to MV with or without endotoxemia for 8 h. Enoxaparin (4 mg/kg) was administered subcutaneously 30 min before MV. MV with endotoxemia aggravated VIDD, as demonstrated by increased interleukin-6 and macrophage inflammatory protein-2 levels, oxidative loads, and the expression of HIF-1α, calpain, caspase-3, atrogin-1, muscle ring finger-1, and microtubule-associated protein light chain 3-II. Disorganized myofibrils, disrupted mitochondria, increased numbers of autophagic and apoptotic mediators, substantial apoptosis of diaphragm muscle fibers, and decreased diaphragm function were also observed (p < 0.05). Endotoxin-exacerbated VIDD and myonuclear apoptosis were attenuated by pharmacologic inhibition by LMWH and in HIF-1α–deficient mice (p < 0.05). Our data indicate that enoxaparin reduces endotoxin-augmented MV-induced diaphragmatic injury, partially through HIF-1α pathway inhibition.     

Marliolide Derivative Induces Melanosome Degradation via Nrf2/p62-Mediated Autophagy

Nuclear factor erythroid 2-related factor 2 (Nrf2), which is linked to autophagy regulation and melanogenesis regulation, is activated by marliolide. In this study, we investigated the effect of a marliolide derivative on melanosome degradation through the autophagy pathway. The effect of the marliolide derivative on melanosome degradation was investigated in α-melanocyte stimulating hormone (α-MSH)-treated melanocytes, melanosome-incorporated keratinocyte, and ultraviolet (UV)B-exposed HRM-2 mice (melanin-possessing hairless mice). The marliolide derivative, 5-methyl-3-tetradecylidene-dihydro-furan-2-one (DMF02), decreased melanin pigmentation by melanosome degradation in α-MSH-treated melanocytes and melanosome-incorporated keratinocytes, evidenced by premelanosome protein (PMEL) expression, but did not affect melanogenesis-associated proteins. The UVB-induced hyperpigmentation in HRM-2 mice was also reduced by a topical application of DMF02. DMF02 activated Nrf2 and induced autophagy in vivo, evidenced by decreased PMEL in microtubule-associated proteins 1A/1B light chain 3B (LC3)-II-expressed areas. DMF02 also induced melanosome degradation via autophagy in vitro, and DMF02-induced melanosome degradation was recovered by chloroquine (CQ), which is a lysosomal inhibitor. In addition, Nrf2 silencing by siRNA attenuated the DMF02-induced melanosome degradation via the suppression of p62. DMF02 induced melanosome degradation in melanocytes and keratinocytes by regulating autophagy via Nrf2-p62 activation. Therefore, Nrf2 activator could be a promising therapeutic agent for reducing hyperpigmentation.     

Role of the ROS-JNK Signaling Pathway in Hypoxia-Induced Atrial Fibrotic Responses in HL-1 Cardiomyocytes

By promoting atrial structural remodeling, atrial hypoxia contributes to the development of the atrial fibrillation substrate. Our study aimed to investigate the modulatory effect of hypoxia on profibrotic activity in cultured HL-1 cardiomyocytes and explore the possible signaling transduction mechanisms of profibrotic activity in vitro. Hypoxia (1% O₂) significantly and time-dependently increased the expression of hypoxia-inducible factor (HIF)-1α and fibrotic marker proteins collagen I and III (COL1A and COL3A), transforming growth factor (TGF)-β1 and α-smooth muscle actin (SMA). Western blot or immunohistochemistry analysis showed that hypoxia-induced increase in COL1A and COL3A was significantly attenuated by the addition of SP600125 (a specific c-Jun N-terminal kinase [JNK] inhibitor) or expression of dominant-negative JNK before hypoxia treatment. The inhibition of hypoxia-activated phosphorylation of JNK signal components (JNK, MKK4, nuclear c-Jun and ATF-2) by pre-treatment with SP600125 could suppress hypoxia-stimulated HIF-1α upregulation and fibrotic marker proteins expression. Hypoxia significantly increased reactive oxygen species (ROS) production in cultured HL-1 atrial cells. Pre-treatment with N-acetylcysteine significantly abrogated the expression of nuclear HIF-1α, JNK transduction components and fibrotic marker proteins. Taken together, these findings indicated that the hypoxia-induced atrial profibrotic response occurs mainly via the ROS/JNK pathway, its downstream upregulation of HIF-1α and c-Jun/ATF2 phosphorylation and nuclear translocation to up-regulate the expression of fibrosis-related proteins (COL1A, COL3A, TGF-β1 and α-SMA). Our result suggests that suppression of ROS/JNK signaling pathway is a critical mechanism for developing a novel therapeutic strategy against atrial fibrillation.     

Apigenin Induces Autophagy and Cell Death by Targeting EZH2 under Hypoxia Conditions in Gastric Cancer Cells

Hypoxia is a major obstacle to gastric cancer (GC) therapy and leads to chemoresistance as GC cells are frequently exposed to the hypoxia environment. Apigenin, a flavonoid found in traditional medicine, fruits, and vegetables and an HDAC inhibitor, is a powerful anti-cancer agent against various cancer cell lines. However, detailed mechanisms involved in the treatment of GC using APG are not fully understood. In this study, we investigated the biological activity of and molecular mechanisms involved in APG-mediated treatment of GC under hypoxia. APG promoted autophagic cell death by increasing ATG5, LC3-II, and phosphorylation of AMPK and ULK1 and down-regulating p-mTOR and p62 in GC. Furthermore, our results show that APG induces autophagic cell death via the activation of the PERK signaling, indicating an endoplasmic reticulum (ER) stress response. The inhibition of ER stress suppressed APG-induced autophagy and conferred prolonged cell survival, indicating autophagic cell death. We further show that APG induces ER stress- and autophagy-related cell death through the inhibition of HIF-1α and Ezh2 under normoxia and hypoxia. Taken together, our findings indicate that APG activates autophagic cell death by inhibiting HIF-1α and Ezh2 under hypoxia conditions in GC cells.     

Exploring the Expression of Pro-Inflammatory and Hypoxia-Related MicroRNA-20a,MicroRNA-30e,and MicroRNA-93 in Periodontitis and Gingival Mesenchymal Stem Cells under Hypoxia

Hypoxia associated with inflammation are common hallmarks observed in several diseases, and it plays a major role in the expression of non-coding RNAs, including microRNAs (miRNAs). In addition, the miRNA target genes for hypoxia-inducible factor-1α (HIF-1α) and nuclear factor of activated T cells-5 (NFAT5) modulate the adaptation to hypoxia. The objective of the present study was to explore hypoxia-related miRNA target genes for HIF-1α and NFAT5, as well as miRNA-20a, miRNA-30e, and miRNA-93 expression in periodontitis versus healthy gingival tissues and gingival mesenchymal stem cells (GMSCs) cultured under hypoxic conditions. Thus, a case-control study was conducted, including healthy and periodontitis subjects. Clinical data and gingival tissue biopsies were collected to analyze the expression of miRNA-20a, miRNA-30e, miRNA-93, HIF-1α, and NFAT5 by qRT-PCR. Subsequently, GMSCs were isolated and cultured under hypoxic conditions (1% O₂) to explore the expression of the HIF-1α, NFAT5, and miRNAs. The results showed a significant upregulation of miRNA-20a (p = 0.028), miRNA-30e (p = 0.035), and miRNA-93 (p = 0.026) in periodontitis tissues compared to healthy gingival biopsies. NFAT5 mRNA was downregulated in periodontitis tissues (p = 0.037), but HIF-1α was not affected (p = 0.60). Interestingly, hypoxic GMSCs upregulated the expression of miRNA-20a and HIF-1α, but they downregulated miRNA-93e. In addition, NFAT5 mRNA expression was not affected in hypoxic GMSCs. In conclusion, in periodontitis patients, the expression of miRNA-20a, miRNA-30e, and miRNA-93 increased, but a decreased expression of NFAT5 mRNA was detected. In addition, GMSCs under hypoxic conditions upregulate the HIF-1α and increase miRNA-20a (p = 0.049) expression. This study explores the role of inflammatory and hypoxia-related miRNAs and their target genes in periodontitis and GMSCs. It is crucial to determine the potential therapeutic target of these miRNAs and hypoxia during the periodontal immune–inflammatory response, which should be analyzed in greater depth in future studies.     

HIF-1α Negatively Regulates Irisin Expression Which Involves in Muscle Atrophy Induced by Hypoxia

Exposure to high altitude environment leads to skeletal muscle atrophy. As a hormone secreted by skeletal muscles after exercise, irisin contributes to promoting muscle regeneration and ameliorating skeletal muscle atrophy, but its role in hypoxia-induced skeletal muscle atrophy is still unclear. Our results showed that 4 w of hypoxia exposure significantly reduced body weight and gastrocnemius muscle mass of mice, as well as grip strength and the duration time of treadmill exercise. Hypoxic treatment increased HIF-1α expression and decreased both the circulation level of irisin and its precursor protein FNDC5 expression in skeletal muscle. In in vitro, CoCl₂-induced chemical hypoxia and 1% O₂ ambient hypoxia both reduced FNDC5, along with the increase in HIF-1α. Moreover, the decline in the area and diameter of myotubes caused by hypoxia were rescued by inhibiting HIF-1α via YC-1. Collectively, our research indicated that FNDC5/irisin was negatively regulated by HIF-1α and could participate in the regulation of muscle atrophy caused by hypoxia.     

Human Sarcopenic Myoblasts Can Be Rescued by Pharmacological Reactivation of HIF-1α

Sarcopenia, an age-related decline in muscle mass and strength, is associated with metabolic disease and increased risk of cardiovascular morbidity and mortality. It is associated with decreased tissue vascularization and muscle atrophy. In this work, we investigated the role of the hypoxia inducible factor HIF-1α in sarcopenia. To this end, we obtained skeletal muscle biopsies from elderly sarcopenic patients and compared them with those from young individuals. We found a decrease in the expression of HIF-1α and its target genes in sarcopenia, as well as of PAX7, the major stem cell marker of satellite cells, whereas the atrophy marker MURF1 was increased. We also isolated satellite cells from muscle biopsies and cultured them in vitro. We found that a pharmacological activation of HIF-1α and its target genes caused a reduction in skeletal muscle atrophy and activation of PAX7 gene expression. In conclusion, in this work we found that HIF-1α plays a role in sarcopenia and is involved in satellite cell homeostasis. These results support further studies to test whether pharmacological reactivation of HIF-1α could prevent and counteract sarcopenia.     

Paricalcitol Improves Hypoxia-Induced and TGF-β1-Induced Injury in Kidney Pericytes

Recently, the role of kidney pericytes in kidney fibrosis has been investigated. This study aims to evaluate the effect of paricalcitol on hypoxia-induced and TGF-β1-induced injury in kidney pericytes. The primary cultured pericytes were pretreated with paricalcitol (20 ng/mL) for 90 min before inducing injury, and then they were exposed to TGF-β1 (5 ng/mL) or hypoxia (1% O₂ and 5% CO₂). TGF-β1 increased α-SMA and other fibrosis markers but reduced PDGFRβ expression in pericytes, whereas paricalcitol reversed the changes. Paricalcitol inhibited the TGF-β1-induced cell migration of pericytes. Hypoxia increased TGF-β1, α-SMA and other fibrosis markers but reduced PDGFRβ expression in pericyte, whereas paricalcitol reversed them. Hypoxia activated the HIF-1α and downstream molecules including prolyl hydroxylase 3 and glucose transporter-1, whereas paricalcitol attenuated the activation of the HIF-1α-dependent molecules and TGF-β1/Smad signaling pathways in hypoxic pericytes. The gene silencing of HIF-1α vanished the hypoxia-induced TGF-β1, α-SMA upregulation, and PDGFRβ downregulation. The effect of paricalcitol on the HIF-1α-dependent changes of fibrosis markers was not significant after the gene silencing of HIF-1α. In addition, hypoxia aggravated the oxidative stress in pericytes, whereas paricalcitol reversed the oxidative stress by increasing the antioxidant enzymes in an HIF-1α-independent manner. In conclusion, paricalcitol improved the phenotype changes of pericyte to myofibroblast in TGF-β1-stimulated pericytes. In addition, paricalcitol improved the expression of fibrosis markers in hypoxia-exposed pericytes both in an HIF-1α-dependent and independent manner.     

Melatonin Suppresses Hypoxia-Induced Migration of HUVECs via Inhibition of ERK/Rac1 Activation

Melatonin, a naturally-occurring hormone, possesses antioxidant properties and ameliorates vascular endothelial dysfunction. In this study, we evaluate the impact of melatonin on the migratory capability of human umbilical vein endothelial cells (HUVECs) to hypoxia and further investigate whether ERK/Rac1 signaling is involved in this process. Here, we found that melatonin inhibited hypoxia-stimulated hypoxia-inducible factor-1α (HIF-1α) expression and cell migration in a dose-dependent manner. Mechanistically, melatonin inhibited Rac1 activation and suppressed the co-localized Rac1 and F-actin on the membrane of HUVECs under hypoxic condition. In addition, the blockade of Rac1 activation with ectopic expression of an inactive mutant form of Rac1-T17N suppressed HIF-1α expression and cell migration in response to hypoxia, as well, but constitutive activation of Rac1 mutant Rac1-V12 restored HIF-1α expression, preventing the inhibition of melatonin on cell migration. Furthermore, the anti-Rac1 effect of melatonin in HUVECs appeared to be associated with its inhibition of ERK phosphorylation, but not that of the PI3k/Akt signaling pathway. Taken together, our work indicates that melatonin exerts an anti-migratory effect on hypoxic HUVECs by blocking ERK/Rac1 activation and subsequent HIF-1α upregulation.     

Role and Regulation of Mechanotransductive HIF-1α Stabilisation in Periodontal Ligament Fibroblasts

Orthodontic tooth movement (OTM) creates compressive and tensile strain in the periodontal ligament, causing circulation disorders. Hypoxia-inducible factor 1α (HIF-1α) has been shown to be primarily stabilised by compression, but not hypoxia in periodontal ligament fibroblasts (PDLF) during mechanical strain, which are key regulators of OTM. This study aimed to elucidate the role of heparan sulfate integrin interaction and downstream kinase phosphorylation for HIF-1α stabilisation under compressive and tensile strain and to which extent downstream synthesis of VEGF and prostaglandins is HIF-1α-dependent in a model of simulated OTM in PDLF. PDLF were subjected to compressive or tensile strain for 48 h. In various setups HIF-1α was experimentally stabilised (DMOG) or destabilised (YC-1) and mechanotransduction was inhibited by surfen and genistein. We found that HIF-1α was not stabilised by tensile, but rather by compressive strain. HIF-1α stabilisation had an inductive effect on prostaglandin and VEGF synthesis. As expected, HIF-1α destabilisation reduced VEGF expression, whereas prostaglandin synthesis was increased. Inhibition of integrin mechanotransduction via surfen or genistein prevented stabilisation of HIF-1α. A decrease in VEGF expression was observed, but not in prostaglandin synthesis. Stabilisation of HIF-1α via integrin mechanotransduction and downstream phosphorylation of kinases seems to be essential for the induction of VEGF, but not prostaglandin synthesis by PDLF during compressive (but not tensile) orthodontic strain.