Indazole-Cl inhibits hypoxia-induced cyclooxygenase-2 expression in vascular smooth muscle cells

in Journal of Molecular Endocrinology
View More View Less
  • 1 Department of Integrative Bioscience and Biotechnology, College of Life Science, Sejong University, Seoul, Korea
  • 2 Division of Functional Food Research, Korea Food Research Institute, Jeollabuk-do, Korea
  • 3 Department of Obstetrics, Gynecology and Women’s Health, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
  • 4 Department of Biomedical Engineering, College of Medicine, Kyung Hee University, Seoul, Korea

Correspondence should be addressed to Y Lee: yjlee@sejong.ac.kr

Atherosclerosis is the most common root cause of arterial disease, such as coronary artery disease and carotid artery disease. Hypoxia is associated with the formation of macrophages and increased inflammation and is known to be present in lesions of atherosclerotic. Vascular smooth muscle cells (VSMCs) are one of the major components of blood vessels, and hypoxic conditions affect VSMC inflammation, proliferation and migration, which contribute to vascular stenosis and play a major role in the atherosclerotic process. Estrogen receptor (ER)-β is thought to play an important role in preventing the inflammatory response in VSMCs. In this report, we studied the anti-inflammatory effect of indazole (In)-Cl, an ERβ-specific agonist, under conditions of hypoxia. Expression of cyclooxygenase-2 reduced by hypoxia was inhibited by In-Cl treatment in VSMCs, and this effect was antagonized by an anti-estrogen compound. Additionally, the production of reactive oxygen species induced under conditions of hypoxia was reduced by treatment with In-Cl. Increased cell migration and invasion by hypoxia were also dramatically decreased following treatment with In-Cl. The increase in cell proliferation following treatment with platelet-derived growth factor was attenuated by In-Cl in VSMCs. RNA sequencing analysis was performed to identify changes in inflammation-related genes following In-Cl treatment in the hypoxic state. Our results suggest that ERβ is a potential therapeutic target for the suppression of hypoxia-induced inflammation in VSMCs.

Abstract

Atherosclerosis is the most common root cause of arterial disease, such as coronary artery disease and carotid artery disease. Hypoxia is associated with the formation of macrophages and increased inflammation and is known to be present in lesions of atherosclerotic. Vascular smooth muscle cells (VSMCs) are one of the major components of blood vessels, and hypoxic conditions affect VSMC inflammation, proliferation and migration, which contribute to vascular stenosis and play a major role in the atherosclerotic process. Estrogen receptor (ER)-β is thought to play an important role in preventing the inflammatory response in VSMCs. In this report, we studied the anti-inflammatory effect of indazole (In)-Cl, an ERβ-specific agonist, under conditions of hypoxia. Expression of cyclooxygenase-2 reduced by hypoxia was inhibited by In-Cl treatment in VSMCs, and this effect was antagonized by an anti-estrogen compound. Additionally, the production of reactive oxygen species induced under conditions of hypoxia was reduced by treatment with In-Cl. Increased cell migration and invasion by hypoxia were also dramatically decreased following treatment with In-Cl. The increase in cell proliferation following treatment with platelet-derived growth factor was attenuated by In-Cl in VSMCs. RNA sequencing analysis was performed to identify changes in inflammation-related genes following In-Cl treatment in the hypoxic state. Our results suggest that ERβ is a potential therapeutic target for the suppression of hypoxia-induced inflammation in VSMCs.

Keywords: hypoxia; ER beta; COX-2; VSMC

Introduction

Oxygen homeostasis in blood vessels has been linked to atherosclerosis (Sluimer et al. 2008). Hypoxia is a major regulator of physiological processes and diseases of chronic inflammation, including angiogenesis, glycolysis and erythropoiesis (Guillemin & Krasnow 1997). In the cardiovascular system, for example, the blood diffusion capacity decreases, and the arterial walls thicken in atherosclerotic lesions under conditions of hypoxia, and hypoxia plays an important role in the progression of atherosclerosis (Nakano et al. 2005, Sluimer et al. 2008). VSMCs are one of the main components of vessels. The proliferation and migration of VSMCs contribute to vascular stenosis and play a major role in atherosclerosis, and hypoxia has been reported to induce VSMCs proliferation and migration (Osada-Oka et al. 2008, Zhang et al. 2009). The mechanism by which hypoxic conditions regulate VSMC growth includes direct cell cycle-specific effects and indirect effects through the regulation of VSMC mitogen production (Kourembanas et al. 1998).

Hypoxia upregulates the expression of macrophage migration inhibitory factor in VSMCs through the hypoxia-inducible factor (HIF)-1-α-dependent pathway (Fu et al. 2010). Hypoxia and cellular injury induce cyclooxygenase (COX)-2 in many cell types (Chida & Voelkel 1996, Schmedtje et al. 1997). COX-2 is a significant mediator of the vascular response to injury; COX-2 is increased in response to lipopolysaccharides and ischemia–reperfusion (Davidge 2001). Upregulation of COX-2 is important in vascular smooth muscle hyperreactivity (Guo et al. 2005). Moreover, the promotion of COX-2 expression, along with increased levels of prostanoids, mainly prostaglandin E2 (PGE2), are hallmarks of inflammation in many tissues (Dubois et al. 1998, Vane et al. 1998). In hypoxia, both nuclear factor kappa B (NF-κB) and HIF-1 are important in transducing inflammatory signals, and these transcription factors appear to regulate COX-2 expression and PGE2 secretion both directly and indirectly (Lukiw et al. 2003). These processes result in smooth muscle cell hypertrophy (Orton et al. 1992), hyperplasia and migration. Reducing COX-2 expression in VSMCs under conditions of hypoxia is expected to help prevent or mitigate vascular injury.

Estrogen has been widely used as an atheroprotective agent. According to some reports, premenopausal women have a significantly lower risk of cardiovascular disease than postmenopausal women of the same age (Kannel & Wilson 1995, Mendelsohn & Karas 1999, Reckelhoff 2001), and women with ovarian dysfunction have a higher risk of cardiovascular disease (Punnonen et al. 1997). Additionally, menopausal women undergoing estrogen replacement have been reported to have a reduced risk of coronary heart disease (Stampfer et al. 1991, Grady et al. 1992, Grodstein et al. 1996). The biological function of estrogen is mediated primarily by nuclear estrogen receptors (ERs), which function as ligand-activated transcription factors in the regulation of gene expression. Two ER isoforms exist: ERα and ERβ (Mangelsdorf et al. 1995, Kuiper et al. 1996). Whereas the physiological effects of ERα have been studied extensively, the effects of ERβ are less well defined (Harris 2007). Estrogen exhibits protective effects via the reduction of inflammatory mediators, such as cytokines, and has been shown to suppress inflammation in experimental models of carrageenan-induced pleurisy in the lungs (Cuzzocrea et al. 2000, 2001) as well as adjuvant-induced arthritis (Badger et al. 1999). However, estrogen also stimulates edema (Tchernitchin & Galand 1983), increases prostatitis (Naslund et al. 1988), promotes an influx of macrophages and increases vascular permeability in the uterus (De & Wood 1990, Kaushic et al. 1998). On the other hand, ERβ ligands are more effective in attenuating the inflammatory response in neural cells and may therefore be promising therapeutic candidates for neural anti-inflammatory therapies (Lewis et al. 2008).

Previous studies have shown that estrogen suppressed NF-κB-dependent inflammation by cytokines in incubated cerebral endothelial cells (Galea et al. 2002). However, to the best of our knowledge, no study has shown the direct effects of COX-2 regulation by ERβ-specific ligands under conditions of hypoxia. We hypothesized that, in the hypoxic state, ERβ-specific ligands reduce the expression of inflammatory mediators in VSMCs and aimed to investigate the vasoprotective effects of the ERβ-specific agonist indazole (In)-Cl through the regulation of inflammatory responses under hypoxia using COX-2 as an indicator of vascular inflammation.

Materials and methods

Materials

Celecoxib, 17β-estradiol (E2) and In-Cl were purchased from Sigma. Diarylproprionitrile (DPN) and ICI 182, 780 (ICI) were purchased from Tocris. Platelet-derived growth factor (PDGF)-BB (520-BB/CF) was purchased from R&D System.

Cell culture and hypoxic conditions

The thoracic aortas from 3-month-old Sprague–Dawley rats (160–180 g) were removed, and VSMCs were enzymatically isolated as previously described (Yoon et al. 2012). This study protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Sungkyunkwan University School of Medicine (Permission No. H-A9-003). VSMCs were cultured with phenol red-free Dulbecco’s modified Eagle medium: Nutrient Mixture F-12 (DMEM/F12) (WelGENE, Daegu, South Korea) containing 10% fetal bovine serum (FBS) (WelGENE, Daegu, South Korea) and 1% penicillin/streptomycin (GIBCO Invitrogen). Cells were incubated at 37°C in a humidified atmosphere of 5% CO2. All treatments were done in media containing 5% charcoal–dextran stripped (CD)-FBS. For the hypoxic state, cells were placed in a forma anaerobic system (Thermo Fisher Scientific) at 37°C containing 5% CO2/1% O2/94% N2 atmosphere.

Transfection and luciferase assays

For reporter assays, VSMCs were transiently transfected with 3x(NF-κB)tk-luciferase and COX-2-luciferase reporter plasmids (Lim et al. 2014) using the polyethylenimine method (Polysciences, Warrington, PA, USA). Approximately 16 h after transfection, the cell medium was changed and treated with In-Cl 10 μM for 24 h. Luciferase activity was measured with an AutoLumat LB9507 luminometer (EG & G Berthold, Bad Widbad, Germany) using the luciferase assay system (Promega). The activities were expressed as relative light units.

Quantitative real-time PCR

Total RNA was extracted and the first-strand cDNA was synthesized according to a previously reported method (Park & Lee 2014). Quantitative real-time PCR was performed using the Power SYBR® Green PCR Master Mix (Applied Biosystems). The primers used were GAPDH sense primer, 5′-AGTTCAACGGCACAGTCAAG-3′; GAPDH anti-sense primer, 5′-TACTCAGCACCAGCATCACC-3′; COX-2 sense primer, 5′-GCAGGAAGTCTTTGGTCTGG-3′; COX-2 anti-sense primer, 5′-CTCGTCATCCCACTCAGGAT-3′. Real-time PCR was conducted using a StepOnePlus Real-Time PCR System (Life Technologies). The amplification data were analyzed using the 2.3 version of StepOne optical system software and calculated by the ΔΔCT method.

Western blot analysis

Protein isolation and western blot analysis were performed according to previously reported methods (Park & Lee 2014). Anti-COX-2 (sc-1745, Santa Cruz Biotechnology) was diluted to 1:1000, and blots were incubated overnight. Anti-β-actin (A5441, Sigma) was diluted to 1:5000, and blots were incubated. Then, blots were incubated with the secondary antibody diluted 1: 5000. Immunoreactive bands were detected using enhanced chemiluminescence blotting detection reagents (Amersham Pharmacia Biotech, Buckinghamshire, UK).

Reactive oxygen species (ROS) measurement by flow cytometry

VSMCs were pretreated with In-Cl 0.1 μM for 6 h and cultured with or without PDGF-BB (20 ng/mL). Measurement of intracellular ROS levels were according to previously reported methods (Song et al. 2017). The data were analyzed by FlowJo v.10 (FlowJo LLC, Ashland, OR, USA).

Cell invasion and migration assays

Cells were treated with In-Cl 0.1 μM and/or 1 μM ICI. After 24 h or 48 h of incubation under normoxic or hypoxic conditions, migration and invasion assays were performed according to previously reported methods (Park & Lee 2014).

Cell proliferation assay

VSMC proliferation was determined with a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (Sigma) assay. In brief, cells were plated in 96-well culture media and incubated for 48 h. Later, cells were pretreated with or without In-Cl and treated with PDGF-BB for 24, 48 and 72 h. At the indicated time points, cells were incubated with MTT (0.5 mg/mL) at 37°C. Then the medium was discarded and DMSO was added for 15 min. The optical density was measured for each well at 590 nm, with a reference wavelength of 620 nm using the Microplate Autoreader (Bio-Tek Instruments Inc.).

mRNA library preparation and sequencing

RNA purity and mRNA sequencing libraries were determined according to previously reported methods (Kim et al. 2018). And then the flow cell loaded on HISEQ 2500 sequencing system (Illumina), performed sequencing with 2 × 100 bp read length.

Transcriptome data analysis

QuantSeq 3′ mRNA-Seq reads were aligned using Bowtie2 (Langmead & Salzberg 2012). Differentially expressed genes were determined based on counts from unique and multiple alignments using coverage in Bedtools (Quinlan & Hall 2010). The RT (Read Count) data were processed based on quantile normalization method using EdgeR within R using Bioconductor. Gene classification was based on searches performed by Medline databases (http://www.ncbi.nlm.nih.gov/) and DAVID (http://david.abcc.ncifcrf.gov/). High-throughput sequencing data have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE113940.

The functional annotation of differentially transcribed genes was analyzed using the DAVID web-server v.6.8. Gene Ontology (GO) terms for biological processes included in the DAVID knowledgebase were considered. We used a P value <0.05 as a cut-off. GO terms were submitted to REVIGO, a web server that takes long lists of GO terms and summarizes them in categories and clusters of differentially expressed genes by removing redundant entries.

Statistical analysis

Data shown in this study are expressed as means ± s.d., and statistical analysis for single comparison was performed using the Student’s t test. The criterion for statistical significance was P < 0.05.

Results

Inhibition of hypoxia-induced COX-2 expression by ERβ agonist

In-Cl is an ERβ agonist with a phenyl-2H-indazole core that was synthesized by the Katzenellenbogen group from a series of nonsteroidal compounds (De Angelis et al. 2005). COX-2 is induced by hypoxic conditions and has been implicated in angiogenesis, inflammation and cell cycle progression (Greenhough et al. 2009). ERβ has been shown to exhibit anti-inflammatory effects in inflammatory diseases (Harris et al. 2003, Cristofaro et al. 2006, Follettie et al. 2006). Recent studies have shown that In-Cl suppresses inflammation in a model of endometriosis (Madak-Erdogan et al. 2016). Therefore, we determined the effects of In-Cl on regulation of COX-2 under conditions of hypoxia in VSMCs. To assess the role of ERβ in the hypoxic induction of COX-2 in VSMCs, we pre-incubated cells with In-Cl and/or the ER antagonist ICI for 1 h and subjected the cells to hypoxia. We confirmed the inhibitory effect of 100 nM In-Cl on the induction of COX-2 in hypoxia, which was blocked following treatment with 1 μM ICI (Fig. 1A).

Figure 1
Figure 1

Indazole (In)-Cl inhibits hypoxia-induced cyclooxygenase (COX)-2 transcriptional activity in vascular smooth muscle cells (VSMCs). (A) VSMCs were pretreated with In-Cl (0.1 μM) and/or ICI (1 μM) for 1 h prior to treatment under hypoxic conditions for 24 h and analysis by western blotting. (B) VSMCs were pretreated with In-Cl (0.1 μM) for 1 h before treatment under hypoxic conditions for 16 h and analysis by real-time PCR. (C) VSMCs were transfected with a COX-2-Luc reporter and treated with In-Cl (10 μM) for 24 h. (D) VSMCs were transfected with an NF-κB-Luc reporter. Cells were treated with In-Cl (10 μM) and incubated for 24 h. All experiments were repeated at least three times.

Citation: Journal of Molecular Endocrinology 63, 1; 10.1530/JME-19-0018

To further elucidate the molecular mechanism underpinning the inhibition of hypoxia-induced COX-2 by In-Cl, VSMCs treated with In-Cl were exposed to hypoxic conditions. The hypoxia-dependent increase in COX-2 mRNA expression was significantly reduced by In-Cl (Fig. 1B). To determine whether transcription of the COX-2 gene was regulated by In-Cl, the effect of In-Cl on COX-2 promoter activity was examined using a COX-2 reporter gene. Under hypoxic conditions, COX-2 promoter activity was effectively suppressed by In-Cl, suggesting that In-Cl inhibits hypoxia-induced COX-2 expression (Fig. 1C). The production of proinflammatory cytokines and proteins is regulated primarily by NF-κB (Li & Verma 2002), and the promoter region of the COX-2 gene includes a binding site for NF-κB (Crofford et al. 1997). Next, the effect of In-Cl on NF-κB-mediated gene transcription was investigated using an NF-κB reporter gene. The results indicated that transcriptional activity of NF-κB induced by hypoxia was reduced following treatment with In-Cl (Fig. 1D).

Attenuation of ROS by In-Cl under conditions of hypoxia

Increased levels of ROS are important in the development of vascular pathologies and atherosclerosis, and hypoxia increases the accumulation of ROS via inflammation (Singh & Jialal 2006, Tafani et al. 2016). We assessed whether In-Cl decreased ROS accumulation under hypoxia in VSMCs. VSMCs were pretreated with In-Cl for 6 h and then incubated under conditions of hypoxia for 24 h to generate ROS. ROS levels were attenuated by In-Cl (Fig. 2), indicating that In-Cl can reduce the levels of ROS in cultured VSMCs.

Figure 2
Figure 2

In-Cl decreases reactive oxygen species (ROS) levels under conditions of hypoxia. VSMCs were incubated under conditions of hypoxia following pre-treatment with In-Cl (0.1 μM) for 3 h. ROS levels were measured by flow cytometry. VSMCs were treated with 1 μM DCF-DA for 15 min. All experiments were repeated at least three times.

Citation: Journal of Molecular Endocrinology 63, 1; 10.1530/JME-19-0018

Prevention of hypoxia-induced cellular migration and invasion by In-Cl

Vascular lesions are formed by migration and invasion of VSMCs and contribute to the progression of hyperplasia diseases (Park et al. 2015). To demonstrate the biological relevance of the decreased COX-2 expression caused by In-Cl in VSMCs, we confirmed the effect of In-Cl on VSMC migration and invasion under conditions of hypoxia. The effect of In-Cl on hypoxia-induced VSMC migration was determined by transwell assay. Hypoxic stimulation for 24 h induced VSMC migration potential compared with the control group, and the stimulatory effect of hypoxia on VSMC migration was effectively inhibited by In-Cl. However, the effect of In-Cl was not blocked by the ER antagonist ICI (Fig. 3A), likely because migration was not a direct cause of ER activation.

Figure 3
Figure 3

In-Cl inhibits hypoxia-induced cellular migration and invasion of VSMCs. (A) VSMCs were treated with 1 μM In-Cl and/or 1 μM ICI. Transwell migration assays were conducted under conditions of hypoxia or normoxia. (B) VSMCs were treated with 5 μM celecoxib and/or 0.1 μM In-Cl and incubated under hypoxia for 24 h. Cell migration was determined using a transwell migration assay. (C) VSMCs were untreated or treated with In-Cl (0.1 μM), incubated for 48 h under hypoxic conditions, and invasion was assessed using Matrigel-coated transwell chambers. Translocated cells are visible on the lower surface of the filter. Migrating and invading cells were counted and are shown in the graph. All experiments were repeated at least two times.

Citation: Journal of Molecular Endocrinology 63, 1; 10.1530/JME-19-0018

Celecoxib, a selective inhibitor of COX-2, reduces gastrointestinal bleeding compared with typical nonsteroidal anti-inflammatory agents (NSAIDs; e.g., acetylsalicylic acid and indomethacin) (Goldenberg 1999). Figure 3B shows representative images of VSMCs treated with 5 μM celecoxib. The migratory potential of VSMCs in the hypoxic state was significantly inhibited by treatment with celecoxib and In-Cl, suggesting that the decrease in cell migration is related to the suppression in COX-2 expression. The effect of In-Cl on the invasion potential of VSMCs induced by hypoxia was investigated using a Matrigel invasion assay. VSMC invasiveness was induced under hypoxia compared with normoxia (Fig. 3C). In the hypoxic state, VSMC invasiveness was significantly reduced by In-Cl, indicating that In-Cl induces anti-invasion and anti-migratory properties in VSMCs under hypoxic conditions.

Effects of In-Cl on the proliferation of PDGF-BB-stimulated VSMCs

VSMC growth and proliferation are key events in inflammation and atherogenesis, which can cause thickening of the arteries. VSMC proliferation was induced using PDGF-BB, a growth factor for VSMCs that is produced under conditions of hypoxia (Lu et al. 2015). The effect of In-Cl on VSMC proliferation was confirmed by MTT assay (Fig. 4A). Compared with that in the control group, proliferation was increased by 1.7-fold in the PDGF-BB (20 ng/mL) 48-h-treated group and by 1.4-fold in the 72-h-treated group. Increased VSMC proliferation following treatment with PDGF-BB tended to be reduced by treatment with In-Cl, suggesting that In-Cl attenuates VSMC proliferation.

Figure 4
Figure 4

In-Cl regulates proliferation of VSMCs. (A) VSMCs were plated on a 96-well plate. Cells were pretreated with In-Cl (0.1 μM) for 6 h and cultured with or without platelet-derived growth factor (PDGF)-BB (20 ng/mL). Cell proliferation was measured using the MTT assay. All experiments were repeated at least three times. (B) Heat map analysis of known cell proliferation-related genes regulated by ERβ agonists (In-Cl and DPN) in VSMCs. The blue bands indicate reduced gene expression; the red bands indicate increased gene expression.

Citation: Journal of Molecular Endocrinology 63, 1; 10.1530/JME-19-0018

To further confirm the regulation of VSMC proliferation by In-Cl, we constructed a heat map consisting of genes related to cell proliferation from RNA sequencing data (Fig. 4B). Downregulated and upregulated genes are shown in blue series and red series, respectively. Overall, proliferation-related genes downregulated in the hypoxic state were upregulated when treated with ERβ agonists, whereas proliferation-related genes upregulated in the hypoxic state were downregulated when treated with ERβ agonists. PDGF is a factor that alters the phenotype of VSMCs (Owens et al. 2004, Zimna & Kurpisz 2015); the phenotype conversion of VSMCs plays a major role in atherosclerosis. In particular, the PDGFB gene was increased by 1.9-fold in the hypoxic state and decreased by 0.78-fold in the hypoxic state following treatment with In-Cl. These results demonstrate that In-Cl treatment of VSMCs alters the expression of proliferation-related genes in hypoxic states, confirming our results that increased cell proliferation by PDGF-BB is reduced by In-Cl.

Differentially expressed genes (DEGs) under conditions of hypoxia

Because our results suggested that COX-2 is reduced by ERβ agonists in VSMCs, we performed RNA sequencing analysis to identify the transcripts that were differentially regulated in VSMCs following treatment with the ERβ agonists In-Cl and DPN, as well as the ER agonist E2. The Venn diagram using the standard two-fold change in expression as the threshold criterion shows that 578, 158 and 198 genes were differentially expressed in the hypoxic, DPN-treated and In-Cl-treated states, respectively (Fig. 5A). We confirmed that 50 and 53 genes following DPN and In-Cl treatment, respectively, exhibited different levels of expression under the hypoxic condition compared with normoxia. On the other hand, we identified 18 upregulated and 10 downregulated genes with the same regulatory pattern in VSMCs treated with DPN or In-Cl under hypoxic conditions. GO was analyzed by selecting genes with a more than 1.5-fold difference in the hypoxic and hypoxic groups treated with reagents (In-Cl or E2). DEGs based on GO and grouped by REVIGO could be characterized in clusters according to biological processes (Fig. 5B). With the In-Cl treatment in hypoxia, the clusters included peptidyl-tyrosine dephosphorylation, immune system process, cytokine-mediated signaling pathway, cell-matrix adhesion, negative regulation of cell proliferation and positive regulation of interleukin-6 production. With the E2 treatment in hypoxia, the clusters included peptidyl-tyrosine dephosphorylation, metabolism, immune response, negative regulation of JAK-STAT cascade, cytokine-mediated signaling pathway and positive regulation of circadian rhythm. The expression patterns of 87 DEGs related to inflammation under hypoxia compared with hypoxia + ER agonist treatment are presented in Fig. 5C. RNA sequencing data confirmed our data showing that expression of prostaglandin H synthase 2 (also known as COX-2) was increased under hypoxia; this effect was counteracted by DPN and In-Cl but strengthened by E2. Interleukin (IL)-17b, which has been reported to stimulate the release of tumor necrosis factor α and IL-1B, was present at higher levels in the hypoxic state; this effect was reversed by treatment with DPN, In-Cl and E2. The expression patterns of the ERβ-specific agonists DPN and In-Cl were more similar to one another than to those of the ER agonist E2. These results showed that the ERβ-specific agonists affect the expression of genes related to inflammation under hypoxic conditions.

Figure 5
Figure 5

Hierarchical clustering and Venn diagram of differentially expressed genes (DEGs) following treatment with ER ligands under hypoxic conditions. (A) Venn diagram showing the overlap between DEGs following treatment with ERβ ligand under hypoxic conditions. (B) Enriched GO terms among the transcripts significantly regulated by In-Cl or E2 in hypoxia. REVIGO uses multi-dimensional scaling of the dimensionality of a matrix of GO terms with pairwise semantic similarities. This results in semantically similar GO terms remaining close together in the plot. (C) Hierarchical clustering of DEGs using RNA sequencing data derived from VSMCs treated with ER ligands (In-Cl, DPN and E2) in a hypoxic state.

Citation: Journal of Molecular Endocrinology 63, 1; 10.1530/JME-19-0018

Discussion

The results presented in this study show how the activation of ERβ by In-Cl contributes to the reduction of inflammatory response in VSMCs. COX-2 expression in VSMCs was increased in the hypoxic state, which increased proliferation, inflammation and migration. COX-2 contributes to vascular diseases, such as atherosclerosis. Recent studies have suggested that, in both brain and coronary VSMCs, dihydrotestosterone decreases cytokine- or hypoxia-induced COX-2 protein expression via an ERβ-dependent mechanism (Osterlund et al. 2010, Zuloaga & Gonzales 2011). Using COX-2 protein expression as a marker of vascular inflammation, we examined the effects of In-Cl on COX-2 expression in VSMCs. Our results showed that In-Cl decreased hypoxia-induced COX-2 expression in VSMCs and that this was mediated by ERβ.

Hypoxia stimulates excessive growth of VSMCs, which contributes to vascular remodeling (Tan et al. 2017). Vascular remodeling is a common feature of cardiovascular diseases, such as atherosclerosis and pulmonary arterial hypertension (Jeffery & Wanstall 2001, Stenmark et al. 2006, Hänze et al. 2007). Long-term hypoxia enhances the induction of COX-2 in human pulmonary artery cells (Bradbury et al. 2002). COX-2 is associated with proliferation, inflammation and tumor growth. These studies have indicated the importance of decreasing COX-2 expression to prevent hypoxia-induced cell migration and invasion. Celecoxib is among the new generation of currently approved NSAIDs that characteristically inhibit COX-2 activity. Recent studies have suggested that celecoxib downregulates HIF-1α, PI3K and p-Akt expression in a dose-dependent manner (Sui et al. 2014). Our data demonstrate that hypoxia-induced cell migration is reduced by the COX-2 inhibitor celecoxib and the ERβ agonist In-Cl. These results suggest that In-Cl has significant effects on the migration and invasion induced by COX-2 under conditions of hypoxia. It should be noted that ICI, which is an ER antagonist and breast cancer drug, did not prevent the inhibition of cell migration by In-Cl, supporting the validity of ICI as an anticancer agent. Lau and To (2016) showed that ICI interacts with ERβ, and this complex translocates into the nucleus to activate the hsa-miR765 promoter via an ERβ recognition site. Upon stimulation, the increase in hsa-miR765 expression suppresses HMGA1 protein expression, induces G2 cell cycle arrest and interferes with cell migration and invasion.

ROS are upregulated by hypoxia; their levels are increased in human atherosclerotic lesions, and their induction increases atherosclerosis and vascular disease. Previous studies have suggested that IL-19, an anti-inflammatory cytokine, stimulates heme oxygenase-1 and reduces ROS levels in VSMCs (Gabunia et al. 2012). Our study showed that In-Cl blocked the increased oxidative burden due to hypoxia. In-Cl attenuated the promotion of cell migration following COX-2-induced ROS production under conditions of hypoxia.

COX-2 is increased by inflammatory stimuli and other factors important for cardiovascular diseases, such as endothelin (ET)-1 or angiotensin (Ang)-II (Ohnaka et al. 2000, Álvarez et al. 2007, Montezano et al. 2007, Martín et al. 2012) and is increased in pathological conditions (Félétou et al. 2011, Hernanz et al. 2014, Ozen & Norel 2017). ET-1 and Ang-II have been shown to cause vascular stiffness (Aroor et al. 2013, Muniyappa & Sowers 2013). Increased cytokine secretion and COX-2 expression and activity led to greater vascular contractile responses in the aorta (García-Redondo et al. 2018). The contractile state of VSMCs determines resistance vessel diameter, which is critical in the regulation of blood pressure and tissue blood flow. One of the fundamental manifestation of hypertension is increased stiffness of the aorta (Zhou et al. 2017a). Previous studies have demonstrated that intrinsic VSMC stiffening was significantly increased in the aortic stiffness of hypertension and aging (Zhou et al. 2017b). VSMC stiffness was enhanced after 24 h of hypoxia treatment, but no further change was detected following treatment with In-Cl (data not shown). Further studies investigating how to decrease the VSMC stiffness increased by hypoxia will be valuable in the development of novel treatment strategies.

Transcriptome characterization can help elucidate the functional complexity of the genome as well as the mechanisms underlying cellular activities, such as growth, development and immune responses. We observed that inflammation-related gene expression patterns were altered under conditions of hypoxia in the presence of ERβ ligands. Hierarchical clustering showed that samples were more closely grouped based on treatment with the ERβ-specific ligands DPN and In-Cl than with ER ligands (E2) in the hypoxic state. The results of this study suggest that ERβ agonists exhibit potential therapeutic value as a promising target for suppressing inflammation.

This study indicates that hypoxia-induced COX-2, an inflammatory marker, is reduced following treatment with the ERβ agonist In-Cl. Because other environmental factors may be associated with inflammation, the combined effects of multiple exposure factors as well as inflammatory factors, such as COX-2 and ROS, should be investigated further. The findings of the present study suggest that ERβ may be targeted to suppress inflammation related to vascular diseases in a hypoxic environment.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2017R1D1A1B03034283).

References

  • Álvarez Y, Pérez-Girón JV, Hernanz R, Briones AM, García-Redondo A, Beltrán A, Alonso MJ & Salaices M 2007 Losartan reduces the increased participation of cyclooxygenase-2-derived products in vascular responses of hypertensive rats. Journal of Pharmacology and Experimental Therapeutics 321 381388. (https://doi.org/10.1124/jpet.106.115287)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Aroor AR, DeMarco VG, Jia G, Sun Z, Nistala R, Meininger GA & Sowers JR 2013 The role of tissue renin-angiotensin-aldosterone system in the development of endothelial dysfunction and arterial stiffness. Frontiers in Endocrinology 4 161. (https://doi.org/10.3389/fendo.2013.00161)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Badger AM, Blake SM, Dodds RA, Griswold DE, Swift BA, Rieman DJ, Stroup GB, Hoffman SJ & Gowen M 1999 Idoxifene, a novel selective estrogen receptor modulator, is effective in a rat model of adjuvant-induced arthritis. Journal of Pharmacology and Experimental Therapeutics 291 13801386.

    • Search Google Scholar
    • Export Citation
  • Bradbury DA, Newton R, Zhu YM, Stocks J, Corbett L, Holland ED, Pang LH & Knox AJ 2002 Effect of bradykinin, TGF-β1, IL-1β, and hypoxia on COX-2 expression in pulmonary artery smooth muscle cells. American Journal of Physiology: Lung Cellular and Molecular Physiology 283 L717L725. (https://doi.org/10.1152/ajplung.00070.2002)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Chida M & Voelkel NF 1996 Effects of acute and chronic hypoxia on rat lung cyclooxygenase. American Journal of Physiology 270 L872L878. (https://doi.org/10.1152/ajplung.1996.270.5.L872)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Cristofaro PA, Opal SM, Palardy JE, Parejo NA, Jhung J, Keith Jr JC & Harris HA 2006 WAY-202196, a selective estrogen receptor-beta agonist, protects against death in experimental septic shock. Critical Care Medicine 34 21882193. (https://doi.org/10.1097/01.CCM.0000227173.13497.56)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Crofford LJ, Tan B, McCarthy CJ & Hla T 1997 Involvement of nuclear factor kB in the regulation of cyclooxygenase-2 expression by interleukin-1 in rheumatoid synoviocytes. Arthritis and Rheumatism 40 226236. (https://doi.org/10.1002/art.1780400207)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Cuzzocrea S, Santagati S, Sautebin L, Mazzon E, Calabrò G, Serraino I, Caputi AP & Maggi A 2000 17β-Estradiol antiinflammatory activity in carrageenan-induced pleurisy. Endocrinology 141 14551463. (https://doi.org/10.1210/endo.141.4.7404)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Cuzzocrea S, Mazzon E, Sautebin L, Serraino I, Dugo L, Calabró G, Caputi AP & Maggi A 2001 The protective role of endogenous estrogens in carrageenan-induced lung injury in the rat. Molecular Medicine 7 478487. (https://doi.org/10.1007/BF03401853)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Davidge ST 2001 Prostaglandin H synthase and vascular function. Circulation Research 89 650660. (https://doi.org/10.1161/hh2001.098351)

  • De M & Wood GW 1990 Influence of oestrogen and progesterone on macrophage distribution in the mouse uterus. Journal of Endocrinology 126 417424. (https://doi.org/10.1677/joe.0.1260417)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • De Angelis M, Stossi F, Carlson KA, Katzenellenbogen BS & Katzenellenbogen JA 2005 Indazole estrogens: highly selective ligands for the estrogen receptor β. Journal of Medicinal Chemistry 48 11321144. (https://doi.org/10.1021/jm049223g)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Dubois RN, Abramson SB, Crofford L, Gupta RA, Simon LS, Van De Putte LB & Lipsky PE 1998 Cyclooxygenase in biology and disease. FASEB Journal 12 10631073. (https://doi.org/10.1096/fasebj.12.12.1063)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Félétou M, Huang Y & Vanhoutte PM 2011 Endothelium-mediated control of vascular tone: COX-1 and COX-2 products. British Journal of Pharmacology 164 894912. (https://doi.org/10.1111/j.1476-5381.2011.01276.x)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Follettie MT, Pinard M, Keith JC Jr, Wang L, Chelsky D, Hayward C, Kearney P, Thibault P, Paramithiotis E, Dorner AJ, 2006 Organ messenger ribonucleic acid and plasma proteome changes in the adjuvant-induced arthritis model: responses to disease induction and therapy with the estrogen receptor-β selective agonist ERB-041. Endocrinology 147 714723. (https://doi.org/10.1210/en.2005-0600)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Fu H, Luo F, Yang L, Wu W & Liu X 2010 Hypoxia stimulates the expression of macrophage migration inhibitory factor in human vascular smooth muscle cells via HIF-1α dependent pathway. BMC Cell Biology 11 66. (https://doi.org/10.1186/1471-2121-11-66)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Gabunia K, Ellison SP, Singh H, Datta P, Kelemen SE, Rizzo V & Autieri MV 2012 Interleukin-19 (IL-19) induces heme oxygenase-1 (HO-1) expression and decreases reactive oxygen species in human vascular smooth muscle cells. Journal of Biological Chemistry 287 24772484. (https://doi.org/10.1074/jbc.M111.312470)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Galea E, Santizo R, Feinstein DL, Adamsom P, Greenwood J, Koenig HM & Pelligrino DA 2002 Estrogen inhibits NFκB-dependent inflammationin brain endothelium without interfering withIκB degradation. NeuroReport 13 14691472. (https://doi.org/10.1097/00001756-200208070-00024)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • García-Redondo AB, Esteban V, Briones AM, del Campo LSD, González-Amor M, Méndez-Barbero N, Campanero MR, Redondo JM & Salaices M 2018 Regulator of calcineurin 1 modulates vascular contractility and stiffness through the upregulation of COX-2-derived prostanoids. Pharmacological Research 133 236249. (https://doi.org/10.1016/j.phrs.2018.01.001)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Goldenberg MM 1999 Celecoxib, a selective cyclooxygenase-2 inhibitor for the treatment of rheumatoid arthritis and osteoarthritis. Clinical Therapeutics 21 14971513; discussion 1427.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Grady D, Rubin SM, Petitti DB, Fox CS, Black D, Ettinger B, Ernster VL & Cummings SR 1992 Hormone therapy to prevent disease and prolong life in postmenopausal women. Annals of Internal Medicine 117 10161037. (https://doi.org/10.7326/0003-4819-117-12-1016)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Greenhough A, Smartt HJ, Moore AE, Roberts HR, Williams AC, Paraskeva C & Kaidi A 2009 The COX-2/PGE 2 pathway: key roles in the hallmarks of cancer and adaptation to the tumour microenvironment. Carcinogenesis 30 377386. (https://doi.org/10.1093/carcin/bgp014)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Grodstein F, Stampfer MJ, Manson JE, Colditz GA, Willett WC, Rosner B, Speizer FE & Hennekens CH 1996 Postmenopausal estrogen and progestin use and the risk of cardiovascular disease. New England Journal of Medicine 335 453461. (https://doi.org/10.1056/NEJM199608153350701)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Guillemin K & Krasnow MA 1997 The hypoxic response: huffing and HIFing. Cell 89 912. (https://doi.org/10.1016/S0092-8674(00)80176-2)

  • Guo Z, Su W, Allen S, Pang H, Daugherty A, Smart E & Gong MC 2005 COX-2 up-regulation and vascular smooth muscle contractile hyperreactivity in spontaneous diabetic db/db mice. Cardiovascular Research 67 723735. (https://doi.org/10.1016/j.cardiores.2005.04.008)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Hänze J, Weissmann N, Grimminger F, Seeger W & Rose F 2007 Cellular and molecular mechanisms of hypoxia-inducible factor driven vascular remodeling. Thrombosis and Haemostasis 97 774787. (https://doi.org/10.1160/TH06-12-0744)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Harris HA 2007 Estrogen receptor-β: recent lessons from in vivo studies. Molecular Endocrinology 21 113. (https://doi.org/10.1210/me.2005-0459)

  • Harris HA, Albert LM, Leathurby Y, Malamas MS, Mewshaw RE, Miller CP, Kharode YP, Marzolf J, Komm BS, Winneker RC, 2003 Evaluation of an estrogen receptor-β agonist in animal models of human disease. Endocrinology 144 42414249. (https://doi.org/10.1210/en.2003-0550)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Hernanz R, Briones AM, Salaices M & Alonso MJ 2014 New roles for old pathways? A circuitous relationship between reactive oxygen species and cyclo-oxygenase in hypertension. Clinical Science 126 111121. (https://doi.org/10.1042/CS20120651)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Jeffery TK & Wanstall JC 2001 Pulmonary vascular remodeling: a target for therapeutic intervention in pulmonary hypertension. Pharmacology and Therapeutics 92 120. (https://doi.org/10.1016/S0163-7258(01)00157-7)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Kannel WB & Wilson PW 1995 Risk factors that attenuate the female coronary disease advantage. Archives of Internal Medicine 155 5761.

  • Kaushic C, Frauendorf E, Rossoll RM, Richardson JM & Wira CR 1998 Influence of the estrous cycle on the presence and distribution of immune cells in the rat reproductive tract. American Journal of Reproductive Immunology 39 209216. (https://doi.org/10.1111/j.1600-0897.1998.tb00355.x)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Kim K, Kim JH, Kim YH, Hong SE & Lee SH 2018 Pathway profiles based on gene-set enrichment analysis in the honey bee Apis mellifera under brood rearing-suppressed conditions. Genomics 110 4349. (https://doi.org/10.1016/j.ygeno.2017.08.004)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kourembanas S, Morita T, Christou H, Liu Y, Koike H, Brodsky D, Arthur V & Mitsial SA 1998 Hypoxic responses of vascular cells. Chest 114 25S28S. (https://doi.org/10.1378/chest.114.1_Supplement.25S-a)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S & Gustafsson JA 1996 Cloning of a novel receptor expressed in rat prostate and ovary. PNAS 93 59255930. (https://doi.org/10.1073/pnas.93.12.5925)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Langmead B & Salzberg SL 2012 Fast gapped-read alignment with Bowtie 2. Nature Methods 9 357359. (https://doi.org/10.1038/nmeth.1923)

  • Lau KM & To KF 2016 Importance of estrogenic signaling and its mediated receptors in prostate cancer. International Journal of Molecular Sciences 17 1434. (https://doi.org/10.3390/ijms17091434)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Lewis DK, Johnson AB, Stohlgren S, Harms A & Sohrabji F 2008 Effects of estrogen receptor agonists on regulation of the inflammatory response in astrocytes from young adult and middle-aged female rats. Journal of Neuroimmunology 195 4759. (https://doi.org/10.1016/j.jneuroim.2008.01.006)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Li Q & Verma IM 2002 NF-κB regulation in the immune system. Nature Reviews: Immunology 2 725734. (https://doi.org/10.1038/nri910)

  • Lim W, Park C, Shim MK, Lee YH, Lee YM & Lee Y 2014 Glucocorticoids suppress hypoxia-induced COX-2 and hypoxia inducible factor-1α expression through the induction of glucocorticoid-induced leucine zipper. British Journal of Pharmacology 171 735745. (https://doi.org/10.1111/bph.12491)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lu Y, Lin N, Chen Z & Xu R 2015 Hypoxia-induced secretion of platelet-derived growth factor-BB by hepatocellular carcinoma cells increases activated hepatic stellate cell proliferation, migration and expression of vascular endothelial growth factor-A. Molecular Medicine Reports 11 691697. (https://doi.org/10.3892/mmr.2014.2689)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lukiw WJ, Ottlecz A, Lambrou G, Grueninger M, Finley J, Thompson HW & Bazan NG 2003 Coordinate activation of HIF-1 and NF-κB DNA binding and COX-2 and VEGF expression in retinal cells by hypoxia. Investigative Ophthalmology and Visual Science 44 41634170. (https://doi.org/10.1167/iovs.02-0655)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Madak-Erdogan Z, Kim SH, Gong P, Zhao YC, Zhang H, Chambliss KL, Carlson KE, Mayne CG, Shaul PW, Korach KS, 2016 Design of pathway preferential estrogens that provide beneficial metabolic and vascular effects without stimulating reproductive tissues. Science Signaling 9 ra53. (https://doi.org/10.1126/scisignal.aad8170)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schütz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, 1995 The nuclear receptor superfamily: the second decade. Cell 83 835839. (https://doi.org/10.1016/0092-8674(95)90199-X)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Martín A, Pérez-Girón JV, Hernanz R, Palacios R, Briones AM, Fortuño A, Zalba G, Salaices M & Alonso MJ 2012 Peroxisome proliferator-activated receptor-γ activation reduces cyclooxygenase-2 expression in vascular smooth muscle cells from hypertensive rats by interfering with oxidative stress. Journal of Hypertension 30 315326. (https://doi.org/10.1097/HJH.0b013e32834f043b)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Mendelsohn ME & Karas RH 1999 The protective effects of estrogen on the cardiovascular system. New England Journal of Medicine 340 18011811. (https://doi.org/10.1056/NEJM199906103402306)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Montezano AC, Amiri F, Tostes RC, Touyz RM & Schiffrin EL 2007 Inhibitory effects of PPAR-γ on endothelin-1-induced inflammatory pathways in vascular smooth muscle cells from normotensive and hypertensive rats. Journal of the American Society of Hypertension 1 150160. (https://doi.org/10.1016/j.jash.2007.01.005)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Muniyappa R & Sowers JR 2013 Role of insulin resistance in endothelial dysfunction. Reviews in Endocrine and Metabolic Disorders 14 512. (https://doi.org/10.1007/s11154-012-9229-1)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Nakano D, Hayashi T, Tazawa N, Yamashita C, Inamoto S, Okuda N, Mori T, Sohmiya K, Kitaura Y, Okada Y, 2005 Chronic hypoxia accelerates the progression of atherosclerosis in apolipoprotein E-knockout mice. Hypertension Research 28 837845. (https://doi.org/10.1291/hypres.28.837)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Naslund MJ, Strandberg JD & Coffey DS 1988 The role of androgens and estrogens in the pathogenesis of experimental nonbacterial prostatitis. Journal of Urology 140 10491053. (https://doi.org/10.1016/S0022-5347(17)41924-0)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Ohnaka K, Numaguchi K, Yamakawa T & Inagami T 2000 Induction of cyclooxygenase-2 by angiotensin II in cultured rat vascular smooth muscle cells. Hypertension 35 6875.

  • Orton EC, LaRue SM, Ensley B & Stenmark K 1992 Bromodeoxyuridine labeling and DNA content of pulmonary arterial medial cells from hypoxia-exposed and nonexposed healthy calves. American Journal of Veterinary Research 53 19251930.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Osada-Oka M, Ikeda T, Akiba S & Sato T 2008 Hypoxia stimulates the autocrine regulation of migration of vascular smooth muscle cells via HIF-1α-dependent expression of thrombospondin-1. Journal of Cellular Biochemistry 104 19181926. (https://doi.org/10.1002/jcb.21759)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Osterlund KL, Handa RJ & Gonzales RJ 2010 Dihydrotestosterone alters cyclooxygenase-2 levels in human coronary artery smooth muscle cells. American Journal of Physiology: Endocrinology and Metabolism 298 E838E845. (https://doi.org/10.1152/ajpendo.00693.2009)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Owens GK, Kumar MS & Wamhoff BR 2004 Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiological Reviews 84 767801. (https://doi.org/10.1152/physrev.00041.2003)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ozen G & Norel X 2017 Prostanoids in the pathophysiology of human coronary artery. Prostaglandins and Other Lipid Mediators 133 2028. (https://doi.org/10.1016/j.prostaglandins.2017.03.003)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Park C & Lee Y 2014 Overexpression of ERβ is sufficient to inhibit hypoxia-inducible factor-1 transactivation. Biochemical and Biophysical Research Communications 450 261266. (https://doi.org/10.1016/j.bbrc.2014.05.107)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Park SL, Won SY, Song JH, Kambe T, Nagao M, Kim WJ & Moon SK 2015 EPO gene expression promotes proliferation, migration and invasion via the p38MAPK/AP-1/MMP-9 pathway by p21WAF1 expression in vascular smooth muscle cells. Cellular Signalling 27 470478. (https://doi.org/10.1016/j.cellsig.2014.12.001)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Punnonen R, Jokela H, Aine R, Teisala K, Salomäki A & Uppa H 1997 Impaired ovarian function and risk factors for atherosclerosis in premenopausal women. Maturitas 27 231238. (https://doi.org/10.1016/S0378-5122(97)00040-6)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Quinlan AR & Hall IM 2010 BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26 841842. (https://doi.org/10.1093/bioinformatics/btq033)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Reckelhoff JF 2001 Gender differences in the regulation of blood pressure. Hypertension 37 11991208. (https://doi.org/10.1161/01.HYP.37.5.1199)

  • Schmedtje JF, Ji YS, Liu WL, DuBois RN & Runge MS 1997 Hypoxia induces cyclooxygenase-2 via the NF-κB p65 transcription factor in human vascular endothelial cells. Journal of Biological Chemistry 272 601608. (https://doi.org/10.1074/jbc.272.1.601)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Singh U & Jialal I 2006 Oxidative stress and atherosclerosis. Pathophysiology 13 129142. (https://doi.org/10.1016/j.pathophys.2006.05.002)

  • Sluimer JC, Gasc JM, van Wanroij JL, Kisters N, Groeneweg M, Gelpke MDS, Cleutjens JP, van den Akker LH, Corvol P, Wouters BG, 2008 Hypoxia, hypoxia-inducible transcription factor, and macrophages in human atherosclerotic plaques are correlated with intraplaque angiogenesis. Journal of the American College of Cardiology 51 12581265. (https://doi.org/10.1016/j.jacc.2007.12.025)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Song H, Park J, Bui PTC, Choi K, Gye MC, Hong YC, Kim JH & Lee YJ 2017 Bisphenol A induces COX-2 through the mitogen-activated protein kinase pathway and is associated with levels of inflammation-related markers in elderly populations. Environmental Research 158 490498. (https://doi.org/10.1016/j.envres.2017.07.005)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Stampfer MJ, Colditz GA, Willett WC, Manson JE, Rosner B, Speizer FE & Hennekens CH 1991 Postmenopausal estrogen therapy and cardiovascular disease: ten-year follow-up from the Nurses’ Health Study. New England Journal of Medicine 325 756762. (https://doi.org/10.1056/NEJM199109123251102)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Stenmark KR, Fagan KA & Frid MG 2006 Hypoxia-induced pulmonary vascular remodeling: cellular and molecular mechanisms. Circulation Research 99 675691. (https://doi.org/10.1161/01.RES.0000243584.45145.3f)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Sui W, Zhang Y, Wang Z, Wang Z, Jia Q, Wu L & Zhang W 2014 Antitumor effect of a selective COX-2 inhibitor, celecoxib, may be attributed to angiogenesis inhibition through modulating the PTEN/PI3K/Akt/HIF-1 pathway in an H22 murine hepatocarcinoma model. Oncology Reports 31 22522260. (https://doi.org/10.3892/or.2014.3093)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Tafani M, Sansone L, Limana F, Arcangeli T, De Santis E, Polese M, Fini M & Russo MA 2016 The interplay of reactive oxygen species, hypoxia, inflammation, and sirtuins in cancer initiation and progression. Oxidative Medicine and Cellular Longevity 2016 3907147. (https://doi.org/10.1155/2016/3907147)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Tan X, Feng L, Huang X, Yang Y, Yang C & Gao Y 2017 Histone deacetylase inhibitors promote eNOS expression in vascular smooth muscle cells and suppress hypoxia-induced cell growth. Journal of Cellular and Molecular Medicine 21 20222035. (https://doi.org/10.1111/jcmm.13122)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Tchernitchin AN & Galand P 1983 Oestrogen levels in the blood, not in the uterus, determine uterine eosinophilia and oedema. Journal of Endocrinology 99 123130. (https://doi.org/10.1677/joe.0.0990123)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Vane JR, Bakhle YS & Botting RM 1998 Cyclooxygenases 1 and 2. Annual Review of Pharmacology and Toxicology 38 97120. (https://doi.org/10.1146/annurev.pharmtox.38.1.97)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Yoon BK, Kang YH, Oh WJ, Park K, Lee DY, Choi D, Kim DK, Lee Y & Rhyu MR 2012 Impact of lysophosphatidylcholine on the plasminogen activator system in cultured vascular smooth muscle cells. Journal of Korean Medical Science 27 803810. (https://doi.org/10.3346/jkms.2012.27.7.803)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Zhang R, Wu Y, Zhao M, Liu C, Zhou L, Shen S, Liao S, Yang K, Li Q & Wan H 2009 Role of HIF-1α in the regulation ACE and ACE2 expression in hypoxic human pulmonary artery smooth muscle cells. American Journal of Physiology: Lung Cellular and Molecular Physiology 297 L631L640. (https://doi.org/10.1152/ajplung.90415.2008)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Zhou N, Lee JJ, Stoll S, Ma B, Costa KD & Qiu H 2017a Rho kinase regulates aortic vascular smooth muscle cell stiffness via actin/SRF/myocardin in hypertension. Cellular Physiology and Biochemistry 44 701715. (https://doi.org/10.1159/000485284)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Zhou N, Lee JJ, Stoll S, Ma B, Wiener R, Wang C, Costa KD & Qiu H 2017b Inhibition of SRF/myocardin reduces aortic stiffness by targeting vascular smooth muscle cell stiffening in hypertension. Cardiovascular Research 113 171182. (https://doi.org/10.1093/cvr/cvw222)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Zimna A & Kurpisz M 2015 Hypoxia-inducible factor-1 in physiological and pathophysiological angiogenesis: applications and therapies. BioMed Research International 2015 549412. (https://doi.org/10.1155/2015/549412)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Zuloaga KL & Gonzales RJ 2011 Dihydrotestosterone attenuates hypoxia inducible factor-1α and cyclooxygenase-2 in cerebral arteries during hypoxia or hypoxia with glucose deprivation. American Journal of Physiology: Heart and Circulatory Physiology 301 H1882H1890. (https://doi.org/10.1152/ajpheart.00446.2011)

    • Crossref
    • Search Google Scholar
    • Export Citation

If the inline PDF is not rendering correctly, you can download the PDF file here.

 

      Society for Endocrinology

Sept 2018 onwards Past Year Past 30 Days
Abstract Views 1574 574 0
Full Text Views 272 239 38
PDF Downloads 82 64 9
  • View in gallery

    Indazole (In)-Cl inhibits hypoxia-induced cyclooxygenase (COX)-2 transcriptional activity in vascular smooth muscle cells (VSMCs). (A) VSMCs were pretreated with In-Cl (0.1 μM) and/or ICI (1 μM) for 1 h prior to treatment under hypoxic conditions for 24 h and analysis by western blotting. (B) VSMCs were pretreated with In-Cl (0.1 μM) for 1 h before treatment under hypoxic conditions for 16 h and analysis by real-time PCR. (C) VSMCs were transfected with a COX-2-Luc reporter and treated with In-Cl (10 μM) for 24 h. (D) VSMCs were transfected with an NF-κB-Luc reporter. Cells were treated with In-Cl (10 μM) and incubated for 24 h. All experiments were repeated at least three times.

  • View in gallery

    In-Cl decreases reactive oxygen species (ROS) levels under conditions of hypoxia. VSMCs were incubated under conditions of hypoxia following pre-treatment with In-Cl (0.1 μM) for 3 h. ROS levels were measured by flow cytometry. VSMCs were treated with 1 μM DCF-DA for 15 min. All experiments were repeated at least three times.

  • View in gallery

    In-Cl inhibits hypoxia-induced cellular migration and invasion of VSMCs. (A) VSMCs were treated with 1 μM In-Cl and/or 1 μM ICI. Transwell migration assays were conducted under conditions of hypoxia or normoxia. (B) VSMCs were treated with 5 μM celecoxib and/or 0.1 μM In-Cl and incubated under hypoxia for 24 h. Cell migration was determined using a transwell migration assay. (C) VSMCs were untreated or treated with In-Cl (0.1 μM), incubated for 48 h under hypoxic conditions, and invasion was assessed using Matrigel-coated transwell chambers. Translocated cells are visible on the lower surface of the filter. Migrating and invading cells were counted and are shown in the graph. All experiments were repeated at least two times.

  • View in gallery

    In-Cl regulates proliferation of VSMCs. (A) VSMCs were plated on a 96-well plate. Cells were pretreated with In-Cl (0.1 μM) for 6 h and cultured with or without platelet-derived growth factor (PDGF)-BB (20 ng/mL). Cell proliferation was measured using the MTT assay. All experiments were repeated at least three times. (B) Heat map analysis of known cell proliferation-related genes regulated by ERβ agonists (In-Cl and DPN) in VSMCs. The blue bands indicate reduced gene expression; the red bands indicate increased gene expression.

  • View in gallery

    Hierarchical clustering and Venn diagram of differentially expressed genes (DEGs) following treatment with ER ligands under hypoxic conditions. (A) Venn diagram showing the overlap between DEGs following treatment with ERβ ligand under hypoxic conditions. (B) Enriched GO terms among the transcripts significantly regulated by In-Cl or E2 in hypoxia. REVIGO uses multi-dimensional scaling of the dimensionality of a matrix of GO terms with pairwise semantic similarities. This results in semantically similar GO terms remaining close together in the plot. (C) Hierarchical clustering of DEGs using RNA sequencing data derived from VSMCs treated with ER ligands (In-Cl, DPN and E2) in a hypoxic state.

  • Álvarez Y, Pérez-Girón JV, Hernanz R, Briones AM, García-Redondo A, Beltrán A, Alonso MJ & Salaices M 2007 Losartan reduces the increased participation of cyclooxygenase-2-derived products in vascular responses of hypertensive rats. Journal of Pharmacology and Experimental Therapeutics 321 381388. (https://doi.org/10.1124/jpet.106.115287)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Aroor AR, DeMarco VG, Jia G, Sun Z, Nistala R, Meininger GA & Sowers JR 2013 The role of tissue renin-angiotensin-aldosterone system in the development of endothelial dysfunction and arterial stiffness. Frontiers in Endocrinology 4 161. (https://doi.org/10.3389/fendo.2013.00161)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Badger AM, Blake SM, Dodds RA, Griswold DE, Swift BA, Rieman DJ, Stroup GB, Hoffman SJ & Gowen M 1999 Idoxifene, a novel selective estrogen receptor modulator, is effective in a rat model of adjuvant-induced arthritis. Journal of Pharmacology and Experimental Therapeutics 291 13801386.

    • Search Google Scholar
    • Export Citation
  • Bradbury DA, Newton R, Zhu YM, Stocks J, Corbett L, Holland ED, Pang LH & Knox AJ 2002 Effect of bradykinin, TGF-β1, IL-1β, and hypoxia on COX-2 expression in pulmonary artery smooth muscle cells. American Journal of Physiology: Lung Cellular and Molecular Physiology 283 L717L725. (https://doi.org/10.1152/ajplung.00070.2002)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Chida M & Voelkel NF 1996 Effects of acute and chronic hypoxia on rat lung cyclooxygenase. American Journal of Physiology 270 L872L878. (https://doi.org/10.1152/ajplung.1996.270.5.L872)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Cristofaro PA, Opal SM, Palardy JE, Parejo NA, Jhung J, Keith Jr JC & Harris HA 2006 WAY-202196, a selective estrogen receptor-beta agonist, protects against death in experimental septic shock. Critical Care Medicine 34 21882193. (https://doi.org/10.1097/01.CCM.0000227173.13497.56)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Crofford LJ, Tan B, McCarthy CJ & Hla T 1997 Involvement of nuclear factor kB in the regulation of cyclooxygenase-2 expression by interleukin-1 in rheumatoid synoviocytes. Arthritis and Rheumatism 40 226236. (https://doi.org/10.1002/art.1780400207)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Cuzzocrea S, Santagati S, Sautebin L, Mazzon E, Calabrò G, Serraino I, Caputi AP & Maggi A 2000 17β-Estradiol antiinflammatory activity in carrageenan-induced pleurisy. Endocrinology 141 14551463. (https://doi.org/10.1210/endo.141.4.7404)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Cuzzocrea S, Mazzon E, Sautebin L, Serraino I, Dugo L, Calabró G, Caputi AP & Maggi A 2001 The protective role of endogenous estrogens in carrageenan-induced lung injury in the rat. Molecular Medicine 7 478487. (https://doi.org/10.1007/BF03401853)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Davidge ST 2001 Prostaglandin H synthase and vascular function. Circulation Research 89 650660. (https://doi.org/10.1161/hh2001.098351)

  • De M & Wood GW 1990 Influence of oestrogen and progesterone on macrophage distribution in the mouse uterus. Journal of Endocrinology 126 417424. (https://doi.org/10.1677/joe.0.1260417)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • De Angelis M, Stossi F, Carlson KA, Katzenellenbogen BS & Katzenellenbogen JA 2005 Indazole estrogens: highly selective ligands for the estrogen receptor β. Journal of Medicinal Chemistry 48 11321144. (https://doi.org/10.1021/jm049223g)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Dubois RN, Abramson SB, Crofford L, Gupta RA, Simon LS, Van De Putte LB & Lipsky PE 1998 Cyclooxygenase in biology and disease. FASEB Journal 12 10631073. (https://doi.org/10.1096/fasebj.12.12.1063)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Félétou M, Huang Y & Vanhoutte PM 2011 Endothelium-mediated control of vascular tone: COX-1 and COX-2 products. British Journal of Pharmacology 164 894912. (https://doi.org/10.1111/j.1476-5381.2011.01276.x)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Follettie MT, Pinard M, Keith JC Jr, Wang L, Chelsky D, Hayward C, Kearney P, Thibault P, Paramithiotis E, Dorner AJ, 2006 Organ messenger ribonucleic acid and plasma proteome changes in the adjuvant-induced arthritis model: responses to disease induction and therapy with the estrogen receptor-β selective agonist ERB-041. Endocrinology 147 714723. (https://doi.org/10.1210/en.2005-0600)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Fu H, Luo F, Yang L, Wu W & Liu X 2010 Hypoxia stimulates the expression of macrophage migration inhibitory factor in human vascular smooth muscle cells via HIF-1α dependent pathway. BMC Cell Biology 11 66. (https://doi.org/10.1186/1471-2121-11-66)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Gabunia K, Ellison SP, Singh H, Datta P, Kelemen SE, Rizzo V & Autieri MV 2012 Interleukin-19 (IL-19) induces heme oxygenase-1 (HO-1) expression and decreases reactive oxygen species in human vascular smooth muscle cells. Journal of Biological Chemistry 287 24772484. (https://doi.org/10.1074/jbc.M111.312470)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Galea E, Santizo R, Feinstein DL, Adamsom P, Greenwood J, Koenig HM & Pelligrino DA 2002 Estrogen inhibits NFκB-dependent inflammationin brain endothelium without interfering withIκB degradation. NeuroReport 13 14691472. (https://doi.org/10.1097/00001756-200208070-00024)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • García-Redondo AB, Esteban V, Briones AM, del Campo LSD, González-Amor M, Méndez-Barbero N, Campanero MR, Redondo JM & Salaices M 2018 Regulator of calcineurin 1 modulates vascular contractility and stiffness through the upregulation of COX-2-derived prostanoids. Pharmacological Research 133 236249. (https://doi.org/10.1016/j.phrs.2018.01.001)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Goldenberg MM 1999 Celecoxib, a selective cyclooxygenase-2 inhibitor for the treatment of rheumatoid arthritis and osteoarthritis. Clinical Therapeutics 21 14971513; discussion 1427.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Grady D, Rubin SM, Petitti DB, Fox CS, Black D, Ettinger B, Ernster VL & Cummings SR 1992 Hormone therapy to prevent disease and prolong life in postmenopausal women. Annals of Internal Medicine 117 10161037. (https://doi.org/10.7326/0003-4819-117-12-1016)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Greenhough A, Smartt HJ, Moore AE, Roberts HR, Williams AC, Paraskeva C & Kaidi A 2009 The COX-2/PGE 2 pathway: key roles in the hallmarks of cancer and adaptation to the tumour microenvironment. Carcinogenesis 30 377386. (https://doi.org/10.1093/carcin/bgp014)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Grodstein F, Stampfer MJ, Manson JE, Colditz GA, Willett WC, Rosner B, Speizer FE & Hennekens CH 1996 Postmenopausal estrogen and progestin use and the risk of cardiovascular disease. New England Journal of Medicine 335 453461. (https://doi.org/10.1056/NEJM199608153350701)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Guillemin K & Krasnow MA 1997 The hypoxic response: huffing and HIFing. Cell 89 912. (https://doi.org/10.1016/S0092-8674(00)80176-2)

  • Guo Z, Su W, Allen S, Pang H, Daugherty A, Smart E & Gong MC 2005 COX-2 up-regulation and vascular smooth muscle contractile hyperreactivity in spontaneous diabetic db/db mice. Cardiovascular Research 67 723735. (https://doi.org/10.1016/j.cardiores.2005.04.008)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Hänze J, Weissmann N, Grimminger F, Seeger W & Rose F 2007 Cellular and molecular mechanisms of hypoxia-inducible factor driven vascular remodeling. Thrombosis and Haemostasis 97 774787. (https://doi.org/10.1160/TH06-12-0744)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Harris HA 2007 Estrogen receptor-β: recent lessons from in vivo studies. Molecular Endocrinology 21 113. (https://doi.org/10.1210/me.2005-0459)

  • Harris HA, Albert LM, Leathurby Y, Malamas MS, Mewshaw RE, Miller CP, Kharode YP, Marzolf J, Komm BS, Winneker RC, 2003 Evaluation of an estrogen receptor-β agonist in animal models of human disease. Endocrinology 144 42414249. (https://doi.org/10.1210/en.2003-0550)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Hernanz R, Briones AM, Salaices M & Alonso MJ 2014 New roles for old pathways? A circuitous relationship between reactive oxygen species and cyclo-oxygenase in hypertension. Clinical Science 126 111121. (https://doi.org/10.1042/CS20120651)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Jeffery TK & Wanstall JC 2001 Pulmonary vascular remodeling: a target for therapeutic intervention in pulmonary hypertension. Pharmacology and Therapeutics 92 120. (https://doi.org/10.1016/S0163-7258(01)00157-7)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Kannel WB & Wilson PW 1995 Risk factors that attenuate the female coronary disease advantage. Archives of Internal Medicine 155 5761.

  • Kaushic C, Frauendorf E, Rossoll RM, Richardson JM & Wira CR 1998 Influence of the estrous cycle on the presence and distribution of immune cells in the rat reproductive tract. American Journal of Reproductive Immunology 39 209216. (https://doi.org/10.1111/j.1600-0897.1998.tb00355.x)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Kim K, Kim JH, Kim YH, Hong SE & Lee SH 2018 Pathway profiles based on gene-set enrichment analysis in the honey bee Apis mellifera under brood rearing-suppressed conditions. Genomics 110 4349. (https://doi.org/10.1016/j.ygeno.2017.08.004)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kourembanas S, Morita T, Christou H, Liu Y, Koike H, Brodsky D, Arthur V & Mitsial SA 1998 Hypoxic responses of vascular cells. Chest 114 25S28S. (https://doi.org/10.1378/chest.114.1_Supplement.25S-a)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S & Gustafsson JA 1996 Cloning of a novel receptor expressed in rat prostate and ovary. PNAS 93 59255930. (https://doi.org/10.1073/pnas.93.12.5925)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Langmead B & Salzberg SL 2012 Fast gapped-read alignment with Bowtie 2. Nature Methods 9 357359. (https://doi.org/10.1038/nmeth.1923)

  • Lau KM & To KF 2016 Importance of estrogenic signaling and its mediated receptors in prostate cancer. International Journal of Molecular Sciences 17 1434. (https://doi.org/10.3390/ijms17091434)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Lewis DK, Johnson AB, Stohlgren S, Harms A & Sohrabji F 2008 Effects of estrogen receptor agonists on regulation of the inflammatory response in astrocytes from young adult and middle-aged female rats. Journal of Neuroimmunology 195 4759. (https://doi.org/10.1016/j.jneuroim.2008.01.006)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Li Q & Verma IM 2002 NF-κB regulation in the immune system. Nature Reviews: Immunology 2 725734. (https://doi.org/10.1038/nri910)

  • Lim W, Park C, Shim MK, Lee YH, Lee YM & Lee Y 2014 Glucocorticoids suppress hypoxia-induced COX-2 and hypoxia inducible factor-1α expression through the induction of glucocorticoid-induced leucine zipper. British Journal of Pharmacology 171 735745. (https://doi.org/10.1111/bph.12491)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lu Y, Lin N, Chen Z & Xu R 2015 Hypoxia-induced secretion of platelet-derived growth factor-BB by hepatocellular carcinoma cells increases activated hepatic stellate cell proliferation, migration and expression of vascular endothelial growth factor-A. Molecular Medicine Reports 11 691697. (https://doi.org/10.3892/mmr.2014.2689)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lukiw WJ, Ottlecz A, Lambrou G, Grueninger M, Finley J, Thompson HW & Bazan NG 2003 Coordinate activation of HIF-1 and NF-κB DNA binding and COX-2 and VEGF expression in retinal cells by hypoxia. Investigative Ophthalmology and Visual Science 44 41634170. (https://doi.org/10.1167/iovs.02-0655)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Madak-Erdogan Z, Kim SH, Gong P, Zhao YC, Zhang H, Chambliss KL, Carlson KE, Mayne CG, Shaul PW, Korach KS, 2016 Design of pathway preferential estrogens that provide beneficial metabolic and vascular effects without stimulating reproductive tissues. Science Signaling 9 ra53. (https://doi.org/10.1126/scisignal.aad8170)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schütz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, 1995 The nuclear receptor superfamily: the second decade. Cell 83 835839. (https://doi.org/10.1016/0092-8674(95)90199-X)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Martín A, Pérez-Girón JV, Hernanz R, Palacios R, Briones AM, Fortuño A, Zalba G, Salaices M & Alonso MJ 2012 Peroxisome proliferator-activated receptor-γ activation reduces cyclooxygenase-2 expression in vascular smooth muscle cells from hypertensive rats by interfering with oxidative stress. Journal of Hypertension 30 315326. (https://doi.org/10.1097/HJH.0b013e32834f043b)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Mendelsohn ME & Karas RH 1999 The protective effects of estrogen on the cardiovascular system. New England Journal of Medicine 340 18011811. (https://doi.org/10.1056/NEJM199906103402306)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Montezano AC, Amiri F, Tostes RC, Touyz RM & Schiffrin EL 2007 Inhibitory effects of PPAR-γ on endothelin-1-induced inflammatory pathways in vascular smooth muscle cells from normotensive and hypertensive rats. Journal of the American Society of Hypertension 1 150160. (https://doi.org/10.1016/j.jash.2007.01.005)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Muniyappa R & Sowers JR 2013 Role of insulin resistance in endothelial dysfunction. Reviews in Endocrine and Metabolic Disorders 14 512. (https://doi.org/10.1007/s11154-012-9229-1)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Nakano D, Hayashi T, Tazawa N, Yamashita C, Inamoto S, Okuda N, Mori T, Sohmiya K, Kitaura Y, Okada Y, 2005 Chronic hypoxia accelerates the progression of atherosclerosis in apolipoprotein E-knockout mice. Hypertension Research 28 837845. (https://doi.org/10.1291/hypres.28.837)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Naslund MJ, Strandberg JD & Coffey DS 1988 The role of androgens and estrogens in the pathogenesis of experimental nonbacterial prostatitis. Journal of Urology 140 10491053. (https://doi.org/10.1016/S0022-5347(17)41924-0)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Ohnaka K, Numaguchi K, Yamakawa T & Inagami T 2000 Induction of cyclooxygenase-2 by angiotensin II in cultured rat vascular smooth muscle cells. Hypertension 35 6875.

  • Orton EC, LaRue SM, Ensley B & Stenmark K 1992 Bromodeoxyuridine labeling and DNA content of pulmonary arterial medial cells from hypoxia-exposed and nonexposed healthy calves. American Journal of Veterinary Research 53 19251930.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Osada-Oka M, Ikeda T, Akiba S & Sato T 2008 Hypoxia stimulates the autocrine regulation of migration of vascular smooth muscle cells via HIF-1α-dependent expression of thrombospondin-1. Journal of Cellular Biochemistry 104 19181926. (https://doi.org/10.1002/jcb.21759)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Osterlund KL, Handa RJ & Gonzales RJ 2010 Dihydrotestosterone alters cyclooxygenase-2 levels in human coronary artery smooth muscle cells. American Journal of Physiology: Endocrinology and Metabolism 298 E838E845. (https://doi.org/10.1152/ajpendo.00693.2009)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Owens GK, Kumar MS & Wamhoff BR 2004 Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiological Reviews 84 767801. (https://doi.org/10.1152/physrev.00041.2003)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ozen G & Norel X 2017 Prostanoids in the pathophysiology of human coronary artery. Prostaglandins and Other Lipid Mediators 133 2028. (https://doi.org/10.1016/j.prostaglandins.2017.03.003)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Park C & Lee Y 2014 Overexpression of ERβ is sufficient to inhibit hypoxia-inducible factor-1 transactivation. Biochemical and Biophysical Research Communications 450 261266. (https://doi.org/10.1016/j.bbrc.2014.05.107)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Park SL, Won SY, Song JH, Kambe T, Nagao M, Kim WJ & Moon SK 2015 EPO gene expression promotes proliferation, migration and invasion via the p38MAPK/AP-1/MMP-9 pathway by p21WAF1 expression in vascular smooth muscle cells. Cellular Signalling 27 470478. (https://doi.org/10.1016/j.cellsig.2014.12.001)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Punnonen R, Jokela H, Aine R, Teisala K, Salomäki A & Uppa H 1997 Impaired ovarian function and risk factors for atherosclerosis in premenopausal women. Maturitas 27 231238. (https://doi.org/10.1016/S0378-5122(97)00040-6)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Quinlan AR & Hall IM 2010 BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26 841842. (https://doi.org/10.1093/bioinformatics/btq033)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Reckelhoff JF 2001 Gender differences in the regulation of blood pressure. Hypertension 37 11991208. (https://doi.org/10.1161/01.HYP.37.5.1199)

  • Schmedtje JF, Ji YS, Liu WL, DuBois RN & Runge MS 1997 Hypoxia induces cyclooxygenase-2 via the NF-κB p65 transcription factor in human vascular endothelial cells. Journal of Biological Chemistry 272 601608. (https://doi.org/10.1074/jbc.272.1.601)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Singh U & Jialal I 2006 Oxidative stress and atherosclerosis. Pathophysiology 13 129142. (https://doi.org/10.1016/j.pathophys.2006.05.002)

  • Sluimer JC, Gasc JM, van Wanroij JL, Kisters N, Groeneweg M, Gelpke MDS, Cleutjens JP, van den Akker LH, Corvol P, Wouters BG, 2008 Hypoxia, hypoxia-inducible transcription factor, and macrophages in human atherosclerotic plaques are correlated with intraplaque angiogenesis. Journal of the American College of Cardiology 51 12581265. (https://doi.org/10.1016/j.jacc.2007.12.025)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Song H, Park J, Bui PTC, Choi K, Gye MC, Hong YC, Kim JH & Lee YJ 2017 Bisphenol A induces COX-2 through the mitogen-activated protein kinase pathway and is associated with levels of inflammation-related markers in elderly populations. Environmental Research 158 490498. (https://doi.org/10.1016/j.envres.2017.07.005)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Stampfer MJ, Colditz GA, Willett WC, Manson JE, Rosner B, Speizer FE & Hennekens CH 1991 Postmenopausal estrogen therapy and cardiovascular disease: ten-year follow-up from the Nurses’ Health Study. New England Journal of Medicine 325 756762. (https://doi.org/10.1056/NEJM199109123251102)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Stenmark KR, Fagan KA & Frid MG 2006 Hypoxia-induced pulmonary vascular remodeling: cellular and molecular mechanisms. Circulation Research 99 675691. (https://doi.org/10.1161/01.RES.0000243584.45145.3f)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Sui W, Zhang Y, Wang Z, Wang Z, Jia Q, Wu L & Zhang W 2014 Antitumor effect of a selective COX-2 inhibitor, celecoxib, may be attributed to angiogenesis inhibition through modulating the PTEN/PI3K/Akt/HIF-1 pathway in an H22 murine hepatocarcinoma model. Oncology Reports 31 22522260. (https://doi.org/10.3892/or.2014.3093)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Tafani M, Sansone L, Limana F, Arcangeli T, De Santis E, Polese M, Fini M & Russo MA 2016 The interplay of reactive oxygen species, hypoxia, inflammation, and sirtuins in cancer initiation and progression. Oxidative Medicine and Cellular Longevity 2016 3907147. (https://doi.org/10.1155/2016/3907147)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Tan X, Feng L, Huang X, Yang Y, Yang C & Gao Y 2017 Histone deacetylase inhibitors promote eNOS expression in vascular smooth muscle cells and suppress hypoxia-induced cell growth. Journal of Cellular and Molecular Medicine 21 20222035. (https://doi.org/10.1111/jcmm.13122)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Tchernitchin AN & Galand P 1983 Oestrogen levels in the blood, not in the uterus, determine uterine eosinophilia and oedema. Journal of Endocrinology 99 123130. (https://doi.org/10.1677/joe.0.0990123)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Vane JR, Bakhle YS & Botting RM 1998 Cyclooxygenases 1 and 2. Annual Review of Pharmacology and Toxicology 38 97120. (https://doi.org/10.1146/annurev.pharmtox.38.1.97)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Yoon BK, Kang YH, Oh WJ, Park K, Lee DY, Choi D, Kim DK, Lee Y & Rhyu MR 2012 Impact of lysophosphatidylcholine on the plasminogen activator system in cultured vascular smooth muscle cells. Journal of Korean Medical Science 27 803810. (https://doi.org/10.3346/jkms.2012.27.7.803)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Zhang R, Wu Y, Zhao M, Liu C, Zhou L, Shen S, Liao S, Yang K, Li Q & Wan H 2009 Role of HIF-1α in the regulation ACE and ACE2 expression in hypoxic human pulmonary artery smooth muscle cells. American Journal of Physiology: Lung Cellular and Molecular Physiology 297 L631L640. (https://doi.org/10.1152/ajplung.90415.2008)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Zhou N, Lee JJ, Stoll S, Ma B, Costa KD & Qiu H 2017a Rho kinase regulates aortic vascular smooth muscle cell stiffness via actin/SRF/myocardin in hypertension. Cellular Physiology and Biochemistry 44 701715. (https://doi.org/10.1159/000485284)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Zhou N, Lee JJ, Stoll S, Ma B, Wiener R, Wang C, Costa KD & Qiu H 2017b Inhibition of SRF/myocardin reduces aortic stiffness by targeting vascular smooth muscle cell stiffening in hypertension. Cardiovascular Research 113 171182. (https://doi.org/10.1093/cvr/cvw222)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Zimna A & Kurpisz M 2015 Hypoxia-inducible factor-1 in physiological and pathophysiological angiogenesis: applications and therapies. BioMed Research International 2015 549412. (https://doi.org/10.1155/2015/549412)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Zuloaga KL & Gonzales RJ 2011 Dihydrotestosterone attenuates hypoxia inducible factor-1α and cyclooxygenase-2 in cerebral arteries during hypoxia or hypoxia with glucose deprivation. American Journal of Physiology: Heart and Circulatory Physiology 301 H1882H1890. (https://doi.org/10.1152/ajpheart.00446.2011)

    • Crossref
    • Search Google Scholar
    • Export Citation