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

Abstract 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.

0][11][12][13][14] Therefore, understanding the mechanism behind vascular endothelial dysfunction in SAS is critical to the understanding and treatment of the cardiovascular consequences of SAS.
Intercellular adhesion molecule-1 (ICAM-1) is a cell surface glycoprotein known as an adhesion receptor that directs leukocytes from circulation to sites of inflammation.ICAM-1 is expressed at low levels in immune cells, endothelial cells and epithelial cells but is known to be upregulated in response to inflammatory stimuli.The function of ICAM-1 has been best studied in leukocyte trans-endothelial migration, where ICAM-1 regulates leukocyte rolling and adhesive interactions with the vessel wall and guides leukocyte crossing of the endothelial layer. 15Endothelial cell-specific molecule 1 (ESM1) is an endothelial cell-associated proteoglycan and is upregulated by proangiogenic molecules and pro-inflammatory cytokine stimulation.ESM1 is considered a novel tissue-and blood-based relevant biomarker as it reflects endothelial activation and dysfunction.Recently, serum ESM1 level was revealed to be associated with the severity of SAS and endothelial dysfunction. 16MicroRNAs (miRs) are short RNAs of 19 to 25 nucleotides that regulate post-transcriptional silencing of target gens.A single miR can target hundreds of mRNAs and influence the expression of many genes often involved in a functional interacting pathway. 17 the present study, we investigated the direct effects of IH on vascular endothelial cells using human HUEhT-1 and mouse UV2 vascular endothelial cells and an in vitro IH system.An in vitro IH system is a controlled gas-delivery system that regulates the flow of nitrogen and oxygen to generate IH. 18,19 We found that IH significantly increased the mRNA levels of intercellular adhesion molecule-1 (ICAM-1) and endothelial cell-specific molecule-1 (ESM1) through the downregulation of microRNA (miR)-181a1.

| Cell culture
The human vascular endothelial HUEhT-1 cells, which were established from human umbilical vein endothelial cell line by electroporation of pIRES-hTERT-hygr, [20][21][22] and mouse vascular endothelial UV2 cells [23][24][25] were purchased from the Japanese Collection of Research Bioresources Cell Bank (Ibaraki, Japan) and RIKEN BioResorce Research Center, respectively.The HUEhT-1 cells were grown in MCDB131 medium (Gibco) containing 10% (v/v) foetal calf serum (FCS), 10 mM L-glutamine, 5 μg/mL heparin, 30 mg/L endothelial cell growth supplement (Corning, Corning, NY), 100 units/mL penicillin G (FUJIFILM Wako) and 100 μg/mL streptomycin (FUJIFILM Wako).The mouse vascular endothelial UV2 cells were grown in DMEM medium (FUJIFILM Wako) containing 10% (v/v) FCS, 100 units/ mL penicillin G (FUJIFILM Wako) and 100 μg/mL streptomycin (FUJIFILM Wako).7][28][29][30][31][32] We used this in vitro model of IH, which resulted in fluctuations in the pressure of oxygen similar to the IH condition observed in patients with severe SAS, to repeatedly expose the cells to severe hypoxemia followed by mild hypoxemia or normoxia (i.e.IH). 33We have previously reported that the magnitude of the IH expressed by SpO 2 fluctuated between 75% and 98% and between 50% and 80% in patients with SAS, 18,19 which was nearly equivalent to the medium condition in the present study.

| Real-time RT-PCR
[29][30][31][32]34,35 Total RNA, including miRNA, was isolated from UV2 cells using the miRNeasy mini kit (Qiagen) according to the manufacturer's instructions.An equal amount of DNase-treated RNA was poly-A-tailed using a Mir-X™ miRNA first-strand synthesis kit (Clontech Laboratories, Inc., Mountain View, CA) according to the manufacturer's protocol.A real-time PCR was performed using an to the U6 RNA level.

| Measurements of ICAM-1 and ESM1 in the culture medium by enzyme-linked immunosorbent assay (ELISA)
The mouse UV2 vascular epithelial cells were exposed to either normoxia or IH for 24 h.Then, the culture medium was collected, and the ICAM-1 and ESM1 concentrations were determined by using a Mouse ICAM-1/CD54 Sandwich ELISA Kit (Proteintech) and Mouse ESM1 ELISA Kit (Wuhan Fine Biotech Co., Ltd.) according to the supplier's instructions.

| Data analysis
The results are expressed as mean ± SE.Statistical significance was determined via Student's t-test using GraphPad Prism version 8.4.3 (GraphPad Software).

| The gene expression of ESM1 and ICAM-1 in vascular endothelial cells were increased by IH
Human vascular endothelial HUEhT-1 cells and mouse vascular endothelial UV2 cells were exposed to normoxia or IH for 24 h.We further measured the protein levels of ICAM-1 and ESM1 in mouse UV2 cell culture medium by ELISA and found that the ICAM-1 and ESM1 levels were significantly increased by IH: ICAM-1 (p = 0.0105) and ESM1 (p < 0.0001) (Figure 2).

| The promoter activities of ICAM-1 and ESM1 were not increased by IH
In order to determine whether the IH-induced increases in ICAM-  There could be several reasons as to why miR-181a1 was specifically decreased among miR-181 family by IH.One is that the enzymes involved in miRNA biogenesis are decreased by IH, and as a result the level of miR-181a was decreased.Another is that the level of miR-181a1 was specifically decreased by IH.We measured the mRNAs of endoribonuclease Dicer (Dicer) and ribonuclease type III (Drosha), which are involved in the biosynthesis of miRNAs 36,37 and found that their expression levels were unchanged by IH (
Five members of the Icam immunoglobulin superfamily have been identified: ICAM-1 to −5.3][44] Moreover, ICAM-1 expression is induced by various inflammatory stimuli. 43An TNFα treatment markedly upregulates ICAM-1 expression on the surface of endothelial cells. 45Pressure overload induces ICAM-1 expression in the endothelial cells of myocardial arterioles. 43edictors of vascular risk, including obesity, hyperlipidemia, metabolic syndrome, diabetes mellitus and hypertension, are indeed associated with the presence of vascular endothelial dysfunction.
ESM1, also called endocan, is a novel inflammatory marker, 46 and elevated levels have been reported in some endothelial dysfunction-related diseases. 47ESM1 is a soluble proteoglycan of 50 kDa that is secreted by the vascular endothelium and can be detected in the blood. 48ESM1 is involved in molecular interactions with a wide range of biologically active moieties, which are essential for the regulation of biological processes such as cell adhesion, migration Effects of the siRNAs against ESM1 and ICAM-1 on the IH-induced gene expression of ICAM-1 and ESM1.The siRNAs for ESM1 and ICAM-1 were transfected into the UV2 vascular endothelial cells, and the cells were then subjected to IH or normoxia for 24 h.The levels of the ESM1 and ICAM-1 mRNAs were measured via a real-time RT-PCR using Rig/RpS15 as an endogenous control.The mRNA levels exposed to normoxia were set to 1.0.The data are expressed as the mean ± SE for each group of six independent experiments (n = 6).The statistical analyses were performed using Student's t-test.and proliferation.ESM1 also plays a role in endothelium-dependent pathological disorders and may be a surrogate endothelial dysfunction marker. 49Clinical data indicate that ESM1 is positively correlated with hypertension.For every 1 pg/mL increase in ESM1, the incidence of hypertension increased by 32.2%. 50In the early stages of hypertension, the ESM1 concentration in plasma is significantly increased.ESM1 levels are positively correlated with renal enzymes, norepinephrine, 51 carotid intima-media thickness and high-sensitivity C-reactive protein, as well as being negatively correlated with leukocyte count. 51There is a correlation between the ESM1 level and the occurrence of heart failure. 52ESM1 is an independent predictor of heart failure-related events in chronic heart disease patients.Serum ESM1 levels in patients with aortic atherosclerosis strokes are significantly increased.Higher ESM1 levels in the serum of patients with aortic atherosclerotic stroke can help predict shortterm adverse outcomes. 53 investigated the mechanisms by which IH upregulates the mRNA levels of ICAM-1 and ESM1 and found that the promoter activities of the genes were not increased by IH, suggesting that the IH-induced upregulation of the ICAM-1 and ESM1 is regulated during the post-transcriptional step.MiRNAs are small non-coding RNAs (~22 nucleotides in length) that modulate gene expression via either translational suppression or the degradation of the mRNA through binding to the 3′-untranslated regions of the target genes in a base-pairing manner. 54They affect the stability of the target mRNAs, resulting in changes in the target mRNA, which is  diseases and cancer. 55Several studies addressed the correlation between miRNAs and vascular endothelial dysfunction in SAS patients.For example, circulating exosomal miR-630 was reported to be a key mediator of vascular dysfunction, 56 and circulating extracellular vesicles containing miR-144 have been found to regulate endothelial function, reducing nuclear factor erythroid 2-related factor 2 expression. 57Concerning the roles of miR-181a1, Cheng et al. reported that miR-181a1 was a novel inhibitor of mitophagy. 58Cao et al. reported that miR-181 impeded IL-17-induced non-small cell lung cancer proliferation and migration through targeting VCAM-1 expression. 59Lin et al. 60

ICAM- 1
were synthesised by NGRL.The sense sequence of the siR-NAs for the mouse ESM1 and ICAM-1 were 5′-UUCUC CAG CAC CCA UAUGUtt-3′ (corresponding to the 1512-1530 of NM_023612.3)and 5′-CCAAC UGG AAG CUG UUUGAtt-3′ (corresponding to the 313-331 of NM_010493.3),respectively.The Silencer® Select scrambled siRNA Ambion was used as a control.The transfection of the siRNA into the UV2 vascular endothelial cells was carried out using the Lipofectamine® RNAiMAX Transfection Reagent Following the IH/normoxia exposure, cellular RNA was prepared, and the mRNA levels of endothelial inflammatory transcripts were measured.Several mRNAs encoding ICAM-1, interleukin IL-8, CCL5, ESM1 and NOS3 were upregulated in response to IH in human HUEhT-1 vascular endothelial cells (Figure 1A), and the mRNAs of Vcam-1, ICAM-1, ESM1 and Edn-1 were upregulated by IH in mouse UV2 endothelial cells (Figure 1B).ICAM-1 and ESM1 were commonly upregulated by IH in human HUEhT-1 and mouse UV2 vascular endothelial cells.

1 3 . 3 | 3 . 4 |F I G U R E 1
and ESM1 mRNAs were caused by their transcription, the human ICAM-1 promoter (−1795 ~ +43) and the human ESM1 promoter (−1180 ~ +37) were fused to the luciferase gene of pGL4.17 and transfected them into mouse UV2 vascular endothelial cells.After IH stimulation, we measured promoter activities and found that ICAM-1 and ESM1 promoter activities were not increased but, rather, decreased by IH in UV2 cells (Figure3: p = 0.0015 in ICAM-1 promoter and p < 0.0001 in ESM1 promoter).These results suggested that the gene expression of ICAM-1 and ESM1, overexpressed by IH, was not regulated by transcription.Downregulation of ESM1 by siRNA for ESM1 attenuated the ICAM-1 increase in UV2 cells treated with IH To see the mechanism of expressions of ICAM-1 and ESM1 in the UV2 cells, the ICAM-1 and ESM1 genes were knocked down using siRNA.The expression of ICAM-1 and ESM1 was significantly increased by IH in the presence of scrambled RNA (Figure 4, left panel).In contrast, the introduction of the siRNA for ESM1 (siESM1) inhibited not only the IH-induced increases in the mRNAs for ESM1 but also the IH-induced increase in ICAM-1 levels in the UV2 cells (Figure 4, middle panel).However, the introduction of siRNA for ICAM-1 (siI-cam1) clearly inhibited the IH-induced increase in ICAM-1 mRNA but unchanged the IH-induced increase in ESM1 mRNA (Figure 4 right panel).These results strongly suggested that the increases in the ICAM-1 by IH (Figure 1A,B) were caused by the increased ESM1 expression.The miR-181a1 level was significantly decreased by IHWe considered a potential explanation for the IH-induced upregulation of ICAM-1, and ESM1 was under the post-transcriptional control via ESM1 expression.Therefore, we searched for targeted miRNA using the Micro RNA.org programme (http:// www.micro rna.org/ micro rna/ home.do) and found that human ESM1 and mouse ESM1 mRNAs have a potential target sequence for miR-181 family.There was no other miRNA candidate targeting for both genes.We measured all the miR-181 family in IH-treated UV2 cells by real-time RT-PCR and found that the miR-181a1 level was significantly lower than that of normoxia-treated cells (Figure5: 0.7694 fold vs. normoxia, The mRNA levels of vascular endothelial inflammatory transcripts in (A) human vascular HUEhT-1 endothelial cells and (B) mouse vascular UV2 endothelial cells.Human HUEhT-1 and mouse UV2 vascular endothelial cells were treated with normoxia or IH for 24 h.The mRNA levels were measured via real-time RT-PCR and normalised using β-Actin in human HUEhT-1 cells and rat insulinoma gene (Rig)/ribosomal protein S15 (RpS15) in mouse UV2 cells as an internal standard.In HUEhT-1.The mRNAs of P-SEL, CCL5, CXCL12 and ESM1 in normoxia were not detected in HUEhT-1 cells.Therefore, the mRNA levels exposed to normoxia were set to 1.0 except for P-SEL, CCL5, CXCL12 and ESM1.Data are expressed as the mean ± SE of the samples.Statistical analyses were performed using Student's t-test.Intermittent hypoxia significantly increased the mRNA levels of ICAM-1, IL-8, CCL5, ESM1 and NOS3 in human HUEhT-1 cells.The mRNA expressions of VCAM-1, TNFα, P-SEL, CXCL12 and EDN-1 were not significantly increased by IH (p = 0.2172, p = 0.3696, p = 0.2770, p = 0.1336 and p = 0.4022, respectively).Intermittent hypoxia significantly increased the mRNA levels of Vcam-1, ICAM-1, ESM1 and Edn-1 in mouse UV2 cells.

Figure 6 :
p = 0.3501 in Dicer and p = 0.6784 in Drosha).These results suggest that the reduction of miR-181a1 plays a key role in the post-transcriptional upregulation of mRNA levels of ESM1, leading to the upregulation of ICAM-1 mRNA.In order to clarify whether ESM1 and ICAM-1 expression in IH is regulated by miR-181a1, miR-181a1 mimic and miR-181a1 mimic NC (non-specific control RNA) were introduced into UV2 vascular endothelial cells, and cells were exposed to IH or normoxia.We prepared RNA from the cells, and the mRNA levels of ESM1 and ICAM-1 were measured by RT-PCR.As shown in Figure 7A, we found that the IH-induced increases in the ICAM-1 and ESM1 mRNAs were abolished by the introduction of the miR-181a1 mimic but not by miR-181a1 mimic NC.The increases of ICAM-1 and ESM1 in the medium protein in response to IH were also abolished by the introduction of the miR-181a1 mimic (Figure 7B).These results indicated that IH stress downregulates the miR-181a1 in UV2 vascular endothelial cells, resulting the levels of the ESM1 mRNA are increased via the miR-181a1-mediated mechanism.

F I G U R E 3
Promoter activities of ICAM-1 (left panel) and ESM1 (right panel) in mouse UV2 vascular endothelial cells.Reporter plasmids prepared by inserting the promoter fragments of human ICAM-1 (−1792 ~ +43) and ESM1 (−1180 ~ +37) upstream of a firefly luciferase reporter gene in a pGL4.17vectors were transfected into the UV2 cells.After the cells were exposed to either IH or normoxia for 24 h, they were lysed, and the promoter activities of ICAM-1 and ESM1 were measured.All data are represented as the mean ± SE of the samples of six independent experiments.The promoter activities exposed to normoxia were set to 1.0.The statistical analyses were performed using Student's t-test. of cardiovascular diseases, has been reported in SAS patients, the mechanism explaining why the endothelial complications were induced by IH remained unclear.In the present study, we exposed human and mouse vascular endothelial cells to IH, analysed gene expression, and found that IH exposure induced increases in ICAM-1 and ESM1 mRNAs.We further examined the mechanisms how IH upregulates the mRNA levels of both ICAM-1 and ESM1 and identified potential post-transcriptional miR-181a1mediated regulated mechanisms.Recent epidemiological research has demonstrated that SAS may be associated with various metabolic dysfunctions, including dyslipidemia, cardiovascular diseases, insulin resistance and hypertension, and the dysfunctions may lead vascular dysfunction.The pathophysiology of vascular endothelial dysfunction in relation to SAS is dependent on various factors, for example, oxidative stress, lipid peroxidation, protein oxidation, DNA oxidation, nitric oxide-related compounds, and inflammatory markers.40Cell adhesion molecules are a subset of cell adhesion proteins located on the cell surface and play an important role in enabling cells to bind to other cells or with extracellular matrix molecules.The immunoglobulin superfamily is one of the largest superfamilies of proteins in the body, and it includes many diverse cell adhesion molecules, such as Icam(s) and Vcam(s), which undergo heterophilic binding with molecules such as carbohydrates and integrins.41Interestingly, both Icam(s) and Vcam(s) are endothelial cell adhesion molecules of the immunoglobulin superfamily with a crucial role in mediating the firm adhesion of leukocytes to endothelial cells that are expressed on vascular endothelial cells and interact with integrins such as CD11/18

6 5 Relative
The levels of the Dicer mRNA (left panel) and Drosha mRNA (right panel) of the UV2 mouse vascular endothelial cells subjected to normoxia or IH for 24 h.The levels of the Dicer and Drosha mRNAs were measured by means of a real-time RT-PCR using Rig/ RpS15 as an endogenous control.The data are expressed as the mean ± SE for each group of six independent experiments.The mRNA levels of cells exposed to normoxia were set to 1.0.The statistical analyses were performed using Student's t-test.Drosha mRNA (fold) one of the mechanisms associated with post-transcriptional regulation.To date, a number of studies concerning the role of the miR-181 family (miR-181a1, −181a2, −181b1, −181b2, −181c and −181d) in the development, differentiation, neurodegenerative

F I G U R E 7
reported that miR-181 functioned as an anti-oncogene via NFκB pathway by targeting Rhotekin2 in ovarian cancers.In the present study, the decline of the miR-181a1, with a target sequence in the ESM1 mRNA, could have contributed to the worsening of vascular dysfunction in the IH-condition, inducing the upregulation of the ESM1 and ICAM-1 mRNAs.We initially have some other possibility to induce vascular endothelial dysfunction by IH such as RhoA/Rho kinase-mediated Effects of miR-181a1 mimic transfection on the ICAM-1 and ESM1 expression.The miR-181a1 mimic (5´-AACAU UCA ACG CUG UCG GUGAGUtt-3′, 5´-ACUCA CCG ACA GCG UUG AAUGUUtt-3′) and the nonspecific control RNA (miR-181a1 mimic NC) (5´-UUUGU ACU ACA CAA AAG UACUGtt-3′, 5´-CAGUA CUU UUG UGU AGU ACAAAtt-3′) were synthesized by Nihon Gene Research Laboratories, Inc. (NGRL).They were introduced into the UV2 vascular endothelial cells using Lipofectamine® RNAiMAX just prior to the IH/normoxia exposure.(A) The mRNA levels of ICAM-1 and ESM1 were measured by means of a real-time RT-PCR, as described in the Materials and Methods section, using Rig/RpS15 as an endogenous control.The data are expressed as the mean ± SE for each group of six independent experiments.The mRNA levels of cells exposed to normoxia were set to 1.0.The statistical analyses were performed using Student's t-test.(B) The protein levels of ICAM-1 and ESM1 were measured using ELISA, as described in the Materials and Methods section.The data are expressed as the mean ± SE for each group of six independent experiments.In normoxiaand IH-treated cells, in the cell medium to which miR-181a1 mimic was introduced cell, ICAM-1 and ESM1 were unchanged (p = 0.5226 and p = 0.1959, respectively).The statistical analyses were performed using Student's t-test.
Concentrations of ICAM-1 and ESM1 in UV2 mouse vascular endothelial cell culture medium.Mouse UV2 vascular endothelial cells were treated with normoxia or IH for 24 h.The ICAM-1 (left panel) and ESM1 (right panel) concentrations in the medium were measured via ELISA.Data are expressed as mean ± SE for each group.Statistical analyses were performed using Student's t-test.
drial dysfunction, inflammation and hyperactivation of the sympathetic nervous system.Vascular fibrosis, characterised by reduced lumen diameter and thickened arterial wall resulting from excessive deposition of extracellular matrix, is associated with many clinical diseases and pathological progressions, including atherosclerosis.Although vascular endothelial dysfunction, the earliest predictor of F I G U R E 2