Possible Molecular Mechanisms of Hypertension Induced by Sleep Apnea Syndrome/Intermittent Hypoxia.

Intermittent hypoxia (IH) is a central characteristic of sleep apnea syndrome (SAS), and it subjects cells in the body to repetitive apnea, chronic hypoxia, oxygen desaturation, and hypercapnia. Since SAS is linked to various serious cardiovascular complications, especially hypertension, many studies have been conducted to elucidate the mechanism of hypertension induced by SAS/IH. Hypertension in SAS is associated with numerous cardiovascular disorders. As hypertension is the most common complication of SAS, cell and animal models to study SAS/IH have developed and provided lots of hints for elucidating the molecular mechanisms of hypertension induced by IH. However, the detailed mechanisms are obscure and under investigation. This review outlines the molecular mechanisms of hypertension in IH, which include the regulation systems of reactive oxygen species (ROS) that activate the renin-angiotensin system (RAS) and catecholamine biosynthesis in the sympathetic nervous system, resulting in hypertension. And hypoxia-inducible factors (HIFs), Endotheline 1 (ET-1), and inflammatory factors are also mentioned. In addition, we will discuss the influences of SAS/IH in cardiovascular dysfunction and the relationship of microRNA (miRNA)s to regulate the key molecules in each mechanism, which has become more apparent in recent years. These findings provide insight into the pathogenesis of SAS and help in the development of future treatments.

Intermittent hypoxia increased the expression of ESM1 and ICAM-1 in vascular endothelial cells via the downregulation of microRNA-181a1.

Sleep apnea syndrome (SAS) exposes cells throughout the body to intermittent hypoxia (IH). Intermittent hypoxia is a risk factor not only for hypertension and insulin resistance but also for vascular dysfunction. We have reported correlations between IH, insulin resistance and hypertension. However, the details of why IH leads to vascular dysfunction remain unclear. In this study, we investigated inflammation-related transcripts in vascular endothelial cells (human HUEhT-1 and mouse UV2) exposed to IH by real-time RT-PCR and found that intercellular adhesion molecule-1 (ICAM-1) and endothelial cell-specific molecule-1 (ESM1) mRNAs were significantly increased. ELISA confirmed that, in the UV2 cell medium, ICAM-1 and ESM1 were significantly increased by IH. However, the promoter activities of ICAM-1 and ESM1 were not upregulated. On the other hand, IH treatment significantly decreased microRNA (miR)-181a1 in IH-treated cells. The introduction of miR-181a1 mimic but not miR-181a1 mimic NC abolished the IH-induced upregulation of Ican-1 and ESM1. These results indicated that ICAM-1 and ESM1 were upregulated by IH via the IH-induced downregulation of miR-181a1 in vascular endothelial cells and suggested that SAS patients developed atherosclerosis via the IH-induced upregulation of ICAM-1 and ESM1.

Upregulation of IL-8, osteonectin, and myonectin mRNAs by intermittent hypoxia via OCT1- and NRF2-mediated mechanisms in skeletal muscle cells.

Sleep apnoea syndrome is characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH]) and is a risk factor for insulin resistance/Type 2 diabetes. The induction of insulin resistance in skeletal muscle is a key phenomenon to develop diabetes. However, the mechanisms linking IH stress and insulin resistance remain elusive. We exposed human RD and mouse C2C12 muscle cells to normoxia or IH and measured their mRNA levels by real-time RT-PCR. We found that IH significantly increased the mRNA and protein levels of muscle-derived insulin resistance-factors (myokines) such as IL-8, osteonectin (ON), and myonectin (MN) in muscle cells. We further analysed the IH-induced expression mechanisms of IL-8, ON, and MN genes in muscle cells. Deletion analyses of the human myokine promoter(s) revealed that the regions -152 to -151 in IL-8, -105 to -99 in ON, and - 3741 to -3738 in MN promoters were responsible for the activation by IH in RD cells. The promoters contain consensus transcription factor binding sequences for OCT1 in IL-8 and MN promoters, and for NRF2 in ON promoter, respectively. The introduction of siRNA for OCT1 abolished the IH-induced expression(s) of IL-8 and MN and siRNA for NRF2 abolished the IH-induced expression of ON.

The Impact of Intermittent Hypoxia on Metabolism and Cognition.

Intermittent hypoxia (IH), one of the primary pathologies of sleep apnea syndrome (SAS), exposes cells throughout the body to repeated cycles of hypoxia/normoxia that result in oxidative stress and systemic inflammation. Since SAS is epidemiologically strongly correlated with type 2 diabetes/insulin resistance, obesity, hypertension, and dyslipidemia included in metabolic syndrome, the effects of IH on gene expression in the corresponding cells of each organ have been studied intensively to clarify the molecular mechanism of the association between SAS and metabolic syndrome. Dementia has recently been recognized as a serious health problem due to its increasing incidence, and a large body of evidence has shown its strong correlation with SAS and metabolic disorders. In this narrative review, we first outline the effects of IH on the expression of genes related to metabolism in neuronal cells, pancreatic ß cells, hepatocytes, adipocytes, myocytes, and renal cells (mainly based on the results of our experiments). Next, we discuss the literature regarding the mechanisms by which metabolic disorders and IH develop dementia to understand how IH directly and indirectly leads to the development of dementia.

Upregulation of Reg IV and Hgf mRNAs by Intermittent Hypoxia via Downregulation of microRNA-499 in Cardiomyocytes.

Sleep apnea syndrome (SAS) is characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH]), and is a risk factor for cardiovascular disease (CVD) and insulin resistance/Type 2 diabetes. However, the mechanisms linking IH stress and CVD remain elusive. We exposed rat H9c2 and mouse P19.CL6 cardiomyocytes to experimental IH or normoxia for 24 h to analyze the mRNA expression of several cardiomyokines. We found that the mRNA levels of regenerating gene IV (Reg IV) and hepatocyte growth factor (Hgf) in H9c2 and P19.CL6 cardiomyocytes were significantly increased by IH, whereas the promoter activities of the genes were not increased. A target mRNA search of microRNA (miR)s revealed that rat and mouse mRNAs have a potential target sequence for miR-499. The miR-499 level of IH-treated cells was significantly decreased compared to normoxia-treated cells. MiR-499 mimic and non-specific control RNA (miR-499 mimic NC) were introduced into P19.CL6 cells, and the IH-induced upregulation of the genes was abolished by introduction of the miR-499 mimic, but not by the miR-499 mimic NC. These results indicate that IH stress downregulates the miR-499 in cardiomyocytes, resulting in increased levels of Reg IV and Hgf mRNAs, leading to the protection of cardiomyocytes in SAS patients.

Upregulation of REG IV gene in human intestinal epithelial cells by lipopolysaccharide via downregulation of microRNA-24.

The pathophysiology of inflammatory bowel diseases (IBD) reflects a balance between mucosal injury and reparative mechanisms. Some regenerating gene (Reg) family members (REG Iα, REG Iß and REG IV) are expressed in Crohn's disease (CD) and ulcerative colitis (UC) and involved as proliferative mucosal factors in IBD. We revealed that REG Iα and REG Iß were induced in cell culture system by IL-6/IL-22. Although REG IV was upregulated in IBD biopsy samples, the upregulation of REG IV was not at all induced in cell culture by autoimmune-related cytokines such as IL-6, IL-22 and TNFα. Here, we analysed REG IV expression in LS-174 T and HT-29 human intestinal epithelial cells by real-time RT-PCR and elisa. REG IV expression was induced by lipopolysaccharide (LPS). However, LPS did not activate REG IV promoter activity. As the LPS-induced upregulation of REG IV was considered to be regulated post-transcriptionally, we searched targeted microRNA (miR), which revealed that REG IV mRNA has a potential target sequence for miR-24. We measured the miR-24 level of LPS-treated cells and found that the level was significantly lower. The LPS-induced increase of REG IV mRNA was abolished by the introduction of miR-24 mimic but not by non-specific control RNA.

Downregulation of the Cd38-Cyclic ADP-Ribose Signaling in Cardiomyocytes by Intermittent Hypoxia via Pten Upregulation.

Sleep apnea syndrome (SAS) is characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia, IH), and it is a risk factor for cardiovascular disease (CVD) and insulin resistance/type 2 diabetes. However, the mechanisms linking IH stress and CVD remain elusive. We exposed rat H9c2 and mouse P19.CL6 cardiomyocytes to experimental IH or normoxia for 24 h to analyze the mRNA expression of the components of Cd38-cyclic ADP-ribose (cADPR) signaling. We found that the mRNA levels of cluster of differentiation 38 (Cd38), type 2 ryanodine receptor (Ryr2), and FK506-binding protein 12.6 (Fkbp12.6) in H9c2 and P19.CL6 cardiomyocytes were significantly decreased by IH, whereas the promoter activities of these genes were not decreased. By contrast, the expression of phosphatase and tensin homolog deleted from chromosome 10 (Pten) was upregulated in IH-treated cells. The small interfering RNA for Pten (siPten) and a non-specific control RNA were introduced into the H9c2 cells. The IH-induced downregulation of Cd38, Ryr2, and Fkbp12.6 was abolished by the introduction of the siPten, but not by the control RNA. These results indicate that IH stress upregulated the Pten in cardiomyocytes, resulting in the decreased mRNA levels of Cd38, Ryr2, and Fkbp12.6, leading to the inhibition of cardiomyocyte functions in SAS patients.

Intermittent Hypoxia Increased the Expression of DBH and PNMT in Neuroblastoma Cells via MicroRNA-375-Mediated Mechanism.

Sleep apnea syndrome (SAS), characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia (IH)), is a risk factor for hypertension and insulin resistance. We report a correlation between IH and insulin resistance/diabetes. However, the reason why hypertension is induced by IH is elusive. Here, we investigated the effect of IH on the expression of catecholamine-metabolizing enzymes using an in vitro IH system. Human and mouse neuroblastoma cells (NB-1 and Neuro-2a) were exposed to IH or normoxia for 24 h. Real-time RT-PCR revealed that IH significantly increased the mRNA levels of dopamine ß-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) in both NB-1 and Neuro-2a. Western blot showed that the expression of DBH and PNMT in the NB-1 cells was significantly increased by IH. Reporter assays revealed that promoter activities of DBH and PNMT were not increased by IH. The miR-375 level of IH-treated cells was significantly decreased relative to that of normoxia-treated cells. The IH-induced up-regulation of DBH and PNMT was abolished by the introduction of the miR-375 mimic, but not by the control RNA. These results indicate that IH stress increases levels of DBH and PNMT via the inhibition of miR-375-mediated mRNA degradation, potentially playing a role in the emergence of hypertension in SAS patients.

Editorial to Special Issue "Sleep Apnea and Intermittent Hypoxia 2.0".

Sleep apnea syndrome (SAS) is the most common form of sleep-disordered breathing and is associated with many adverse health consequences, including increased overall mortality risk [...].

CD38-Cyclic ADP-Ribose Signal System in Physiology, Biochemistry, and Pathophysiology.

Calcium (Ca2+) is a ubiquitous and fundamental signaling component that is utilized by cells to regulate a diverse range of cellular functions, such as insulin secretion from pancreatic ß-cells of the islets of Langerhans. Cyclic ADP-ribose (cADPR), synthesized from NAD+ by ADP-ribosyl cyclase family proteins, such as the mammalian cluster of differentiation 38 (CD38), is important for intracellular Ca2+ mobilization for cell functioning. cADPR induces Ca2+ release from endoplasmic reticulum via the ryanodine receptor intracellular Ca2+ channel complex, in which the FK506-binding protein 12.6 works as a cADPR-binding regulatory protein. Recently, involvements of the CD38-cADPR signal system in several human diseases and animal models have been reported. This review describes the biochemical and molecular biological basis of the CD38-cADPR signal system and the diseases caused by its abnormalities.

Effects of Intermittent Hypoxia on Cytokine Expression Involved in Insulin Resistance.

Sleep apnea syndrome (SAS) is a prevalent disorder characterized by recurrent apnea or hypoxia episodes leading to intermittent hypoxia (IH) and arousals during sleep. Currently, the relationship between SAS and metabolic diseases is being actively analyzed, and SAS is considered to be an independent risk factor for the development and progression of insulin resistance/type 2 diabetes (T2DM). Accumulating evidence suggests that the short cycles of decreased oxygen saturation and rapid reoxygenation, a typical feature of SAS, contribute to the development of glucose intolerance and insulin resistance. In addition to IH, several pathological conditions may also contribute to insulin resistance, including sympathetic nervous system hyperactivity, oxidative stress, vascular endothelial dysfunction, and the activation of inflammatory cytokines. However, the detailed mechanism by which IH induces insulin resistance in SAS patients has not been fully revealed. We have previously reported that IH stress may exacerbate insulin resistance/T2DM, especially in hepatocytes, adipocytes, and skeletal muscle cells, by causing abnormal cytokine expression/secretion from each cell. Adipose tissues, skeletal muscle, and the liver are the main endocrine organs producing hepatokines, adipokines, and myokines, respectively. In this review, we focus on the effect of IH on hepatokine, adipokine, and myokine expression.

Okamoto model for necrosis and its expansions, CD38-cyclic ADP-ribose signal system for intracellular Ca2+ mobilization and Reg (Regenerating gene protein)-Reg receptor system for cell regeneration.

In pancreatic islet cell culture models and animal models, we studied the molecular mechanisms involved in the development of insulin-dependent diabetes. The diabetogenic agents, alloxan and streptozotocin, caused DNA strand breaks, which in turn activated poly(ADP-ribose) polymerase/synthetase (PARP) to deplete NAD+, thereby inhibiting islet ß-cell functions such as proinsulin synthesis and ultimately leading to ß-cell necrosis. Radical scavengers protected against the formation of DNA strand breaks and inhibition of proinsulin synthesis. Inhibitors of PARP prevented the NAD+ depletion, inhibition of proinsulin synthesis and ß-cell death. These findings led to the proposed unifying concept for ß-cell damage and its prevention (the Okamoto model). The model met one proof with PARP knockout animals and was further extended by the discovery of cyclic ADP-ribose as the second messenger for Ca2+ mobilization in glucose-induced insulin secretion and by the identification of Reg (Regenerating gene) for ß-cell regeneration. Physiological and pathological events found in pancreatic ß-cells have been observed in other cells and tissues.

Intermittent Hypoxia Upregulates the Renin and Cd38 mRNAs in Renin-Producing Cells via the Downregulation of miR-203.

Sleep apnea syndrome is characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH]), and it is a known risk factor for hypertension. The upregulation of the renin-angiotensin system has been reported in IH, and the correlation between renin and CD38 has been noted. We exposed human HEK293 and mouse As4.1 renal cells to experimental IH or normoxia for 24 h and then measured the mRNA levels using a real-time reverse transcription polymerase chain reaction. The mRNA levels of Renin (Ren) and Cd38 were significantly increased by IH, indicating that they could be involved in the CD38-cyclic ADP-ribose signaling pathway. We next investigated the promotor activities of both genes, which were not increased by IH. Yet, a target mRNA search of the microRNA (miRNA) revealed both mRNAs to have a potential target sequence for miR-203. The miR-203 level of the IH-treated cells was significantly decreased when compared with the normoxia-treated cells. The IH-induced upregulation of the genes was abolished by the introduction of the miR-203 mimic, but not the miR-203 mimic NC negative control. These results indicate that IH stress downregulates the miR-203 in renin-producing cells, thereby resulting in increased mRNA levels of Ren and Cd38, which leads to hypertension.

27-Hydroxycholesterol regulates human SLC22A12 gene expression through estrogen receptor action.

The excretion and reabsorption of uric acid both to and from urine are tightly regulated by uric acid transporters. Metabolic syndrome conditions, such as obesity, hypercholesterolemia, and insulin resistance, are believed to regulate the expression of uric acid transporters and decrease the excretion of uric acid. However, the mechanisms driving cholesterol impacts on uric acid transporters have been unknown. Here, we show that cholesterol metabolite 27-hydroxycholesterol (27HC) upregulates the uric acid reabsorption transporter URAT1 encoded by SLC22A12 via estrogen receptors (ER). Transcriptional motif analysis showed that the SLC22A12 gene promoter has more estrogen response elements (EREs) than other uric acid reabsorption transporters such as SLC22A11 and SLC22A13, and 27HC-activated SLC22A12 gene promoter via ER through EREs. Furthermore, 27HC increased SLC22A12 gene expression in human kidney organoids. Our results suggest that in hypercholesterolemic conditions, elevated levels of 27HC derived from cholesterol induce URAT1/SLC22A12 expression to increase uric acid reabsorption, and thereby, could increase serum uric acid levels.

Anorexigenic Effects of Intermittent Hypoxia on the Gut-Brain Axis in Sleep Apnea Syndrome.

Sleep apnea syndrome (SAS) is a breathing disorder characterized by recurrent episodes of upper-airway collapse, resulting in intermittent hypoxia (IH) during sleep. Experimental studies with animals and cellular models have indicated that IH leads to attenuation of glucose-induced insulin secretion from pancreatic ß cells and to enhancement of insulin resistance in peripheral tissues and cells, such as the liver (hepatocytes), adipose tissue (adipocytes), and skeletal muscles (myocytes), both of which could lead to obesity. Although obesity is widely recognized as a major factor in SAS, it is controversial whether the development of SAS could contribute directly to obesity, and the effect of IH on the expression of appetite regulatory genes remains elusive. Appetite is regulated appropriately by both the hypothalamus and the gut as a gut-brain axis driven by differential neural and hormonal signals. In this review, we summarized the recent epidemiological findings on the relationship between SAS and feeding behavior and focused on the anorexigenic effects of IH on the gut-brain axis by the IH-induced up-regulation of proopiomelanocortin and cocaine- and amphetamine-regulated transcript in neuronal cells and the IH-induced up-regulation of peptide YY, glucagon-like peptide-1 and neurotensin in enteroendocrine cells and their molecular mechanisms.

Involvement of Receptor for Advanced Glycation Endproducts in Hypertensive Disorders of Pregnancy.

Preeclampsia/hypertensive disorders of pregnancy (PE/HDP) is a serious and potentially life-threatening disease. Recently, PE/HDP has been considered to cause adipose tissue inflammation, but the detailed mechanism remains unknown. We exposed human primary cultured adipocytes with serum from PE/HDP and healthy controls for 24 h, and analyzed mRNA expression of several adipokines, cytokines, and ligands of the receptor for advanced glycation endproducts (RAGE). We found that the mRNA levels of interleukin-6 (IL-6), C-C motif chemokine ligand 2 (CCL2), high mobility group box 1 (HMGB1), and RAGE were significantly increased by the addition of PE/HDP serum. Among RAGE ligands, advanced glycation endproducts (AGE) and HMGB1 increased mRNA levels of IL-6 and CCL2 in SW872 human adipocytes and mouse 3T3-L1 cells. The introduction of small interfering RNA for RAGE (siRAGE) into SW872 cells abolished the AGE- and HMGB1-induced up-regulation of IL-6 and CCL2. In addition, lipopolysaccharide (LPS), a ligand of RAGE, increased the expression of IL-6 and CCL2 and siRAGE attenuated the LPS-induced expression of IL-6 and CCL2. These results strongly suggest that the elevated AGE, HMGB1, and LPS in pregnant women up-regulate the expression of IL-6 and CCL2 via the RAGE system, leading to systemic inflammation such as PE/HDP.

Relationship Between Intermittent Hypoxia and Type 2 Diabetes in Sleep Apnea Syndrome.

Sleep apnea syndrome (SAS) is a very common disease involving intermittent hypoxia (IH), recurrent symptoms of deoxygenation during sleep, strong daytime sleepiness, and significant loss of quality of life. A number of epidemiological researches have shown that SAS is an important risk factor for insulin resistance and type 2 diabetes mellitus (DM), which is associated with SAS regardless of age, gender, or body habitus. IH, hallmark of SAS, plays an important role in the pathogenesis of SAS and experimental studies with animal and cellular models indicate that IH leads to attenuation of glucose-induced insulin secretion from pancreatic ß cells and to enhancement of insulin resistance in peripheral tissues and cells, such as liver (hepatocytes), adipose tissue (adipocytes), and skeletal muscles (myocytes). In this review, we focus on IH-induced dysfunction in glucose metabolism and its underlying molecular mechanisms in several cells and tissues related to glucose homeostasis.

Effects of Intermittent Hypoxia on Pulmonary Vascular and Systemic Diseases.

Obstructive sleep apnea (OSA) causes many systemic disorders via mechanisms related to sympathetic nerve activation, systemic inflammation, and oxidative stress. OSA typically shows repeated sleep apnea followed by hyperventilation, which results in intermittent hypoxia (IH). IH is associated with an increase in sympathetic activity, which is a well-known pathophysiological mechanism in hypertension and insulin resistance. In this review, we show the basic and clinical significance of IH from the viewpoint of not only systemic regulatory mechanisms focusing on pulmonary circulation, but also cellular mechanisms causing lifestyle-related diseases. First, we demonstrate how IH influences pulmonary circulation to cause pulmonary hypertension during sleep in association with sleep state-specific change in OSA. We also clarify how nocturnal IH activates circulating monocytes to accelerate the infiltration ability to vascular wall in OSA. Finally, the effects of IH on insulin secretion and insulin resistance are elucidated by using an in vitro chamber system that can mimic and manipulate IH. The obtained data implies that glucose-induced insulin secretion (GIS) in pancreatic ß cells is significantly attenuated by IH, and that IH increases selenoprotein P, which is one of the hepatokines, as well as TNF-α, CCL-2, and resistin, members of adipokines, to induce insulin resistance via direct cellular mechanisms. Clinical and experimental findings concerning IH give us productive new knowledge of how lifestyle-related diseases and pulmonary hypertension develop during sleep.

Proliferative Pathways of Vascular Smooth Muscle Cells in Response to Intermittent Hypoxia.

Obstructive sleep apnea (OSA) is characterized by intermittent hypoxia (IH) and is a risk factor for cardiovascular diseases (e.g., atherosclerosis) and chronic inflammatory diseases (CID). The excessive proliferation of vascular smooth muscle cells (VSMCs) plays a pivotal role in the progression of atherosclerosis. Hypoxia-inducible factor-1 and nuclear factor-κB are thought to be the main factors involved in responses to IH and in regulating adaptations or inflammation pathways, however, further evidence is needed to demonstrate the underlying mechanisms of this process in VSMCs. Furthermore, few studies of IH have examined smooth muscle cell responses. Our previous studies demonstrated that increased interleukin (IL)-6, epidermal growth factor family ligands, and erbB2 receptor, some of which amplify inflammation and, consequently, induce CID, were induced by IH and were involved in the proliferation of VSMCs. Since IH increased IL-6 and epiregulin expression in VSMCs, the same phenomenon may also occur in other smooth muscle cells, and, consequently, may be related to the incidence or progression of several diseases. In the present review, we describe how IH can induce the excessive proliferation of VSMCs and we develop the suggestion that other CID may be related to the effects of IH on other smooth muscle cells.

Resveratrol­induced REG III expression enhances chemo­ and radiosensitivity in head and neck cancer in xenograft mice.

Identifying the key molecules that enhance chemo­ and radiosensitivity in head and neck squamous cell carcinoma (HNSCC) as well as reliable biomarkers for predicting recurrence and metastasis would be desirable to improve the prognosis of HNSCC. Previously, we have reported that Regenerating gene III (REG III) expression was associated with an improved survival rate for patients with HNSCC. In addition, resveratrol (3,4',5­trihydroxystilbene) significantly increased REG III expression in HNSCC cells, and significantly inhibited cell growth, enhanced chemo­ and radiosensitivity, and blocked the cancer invasion of HNSCC cells in vitro. In the present study, the effect of resveratrol on cancer progression in HNSCC was investigated in vivo using a xenograft nude mouse model. The results revealed that resveratrol increased the mRNA level of REG III in vivo, which was in agreement with our previous in vitro findings. Furthermore, REG III increased the antitumor effect of radiation or cisplatin in vivo, and resveratrol sensitized HNSCC to irradiation and cisplatin in vivo. These results indicated that resveratrol could increase the efficacy of cisplatin and irradiation through the REG III expression pathway, resulting in the inhibition of HNSCC progression in vivo.