Abstract
Aldosterone is a key factor in adverse cardiovascular remodeling by acting on the mineralocorticoid receptor (MR) in different cell types. Endothelial MR activation mediates hypertrophy, inflammation and fibrosis. Cardiovascular remodeling is often accompanied by impaired angiogenesis, which is a risk factor for the development of heart failure. In this study, we evaluated the impact of MR in endothelial cells on angiogenesis. Deoxycorticosterone acetate (DOCA)-induced hypertension was associated with capillary rarefaction in the heart of WT mice but not of mice with cell type-specific MR deletion in endothelial cells. Consistently, endothelial MR deletion prevented the inhibitory effect of aldosterone on the capillarization of subcutaneously implanted silicon tubes and on capillary sprouting from aortic ring segments. We examined MR-dependent gene expression in cultured endothelial cells by RNA-seq and identified a cluster of differentially regulated genes related to angiogenesis. We found opposing effects on gene expression when comparing activation of the mineralocorticoid receptor in ECs to treatment with vascular endothelial growth factor (VEGF), a potent activator of angiogenesis. In conclusion, we demonstrate here that activation of endothelial cell MR impaired angiogenic capacity and lead to capillary rarefaction in a mouse model of MR-driven hypertension. MR activation opposed VEGF-induced gene expression leading to the dysregulation of angiogenesis-related gene networks in endothelial cells. Our findings underscore the pivotal role of endothelial cell MR in the pathophysiology of hypertension and related heart disease.
Introduction
Aldosterone mediates its effects via the mineralocorticoid receptor (MR), a ligand-activated transcription factor. Upon binding of aldosterone, MR interacts with a distinct DNA motif to regulate transcription of its target genes (Lother et al. 2015, Jaisser & Farman 2016). The classical aldosterone target tissue is the kidney where MR in epithelial cells controls sodium and fluid balance (Rossier et al. 2015). However, MR is expressed in various other tissues including heart, vasculature, brain or skin (Jaisser & Farman 2016). In the cardiovascular system, MR is expressed in cardiac myocytes, fibroblasts, endothelial cells, smooth muscle cells and immune cells (Lother et al. 2015, Lother & Hein 2016, DuPont & Jaffe 2017). A series of studies using cell type-specific MR deletion models revealed distinct roles of MR in these different cell types for hypertension, atherosclerosis, myocardial infarction and heart failure and in particular found MR in endothelial cells to be a crucial player in cardiovascular disease (Lother et al. 2015, Young & Rickard 2015, Lother & Hein 2016, Cole & Young 2017). Activation of MR in endothelial cells impairs vascular reactivity (Schafer et al. 2013, Rickard et al. 2014, Barrett Mueller et al. 2015), promotes inflammation (Jia et al. 2015, 2016, Lother et al. 2016) and contributes to cardiac fibrosis and hypertrophy (Jia et al. 2015, Lother et al. 2016).
Cardiac hypertrophy occurs in different cardiovascular disease states including hypertension or ischemic heart disease and is a risk factor for the development of heart failure. In order to provide oxygen and nutrition supply, compensated cardiac hypertrophy requires a proportional expansion of the capillary network by angiogenesis (Oka et al. 2014). Disproportional cardiac myocyte growth may lead to myocardial ischemia and promote adverse cardiac fibrosis and failure. A key regulator of angiogenesis is vascular endothelial growth factor (VEGF) (Carmeliet & Jain 2011). In the heart, VEGF is produced by cardiac myocytes in response to hypoxia-induced factor 1α signaling (Oka et al. 2014). VEGF stimulates angiogenesis by acting on the VEGF receptor 2 (VEGFR2) on endothelial cells (Carmeliet & Jain 2011). There is some experimental evidence that aldosterone is involved in the regulation of angiogenesis (Larouche & Schiffrin 1999, Messaoudi et al. 2009, Thum et al. 2011, Fujii et al. 2012, Gravez et al. 2015) and we hypothesized that MR in endothelial cells would have a central role in this process. Thus, the aim of this study was to evaluate the specific impact of MR in endothelial cells on angiogenesis.
Materials and methods
Generation of MR mutant mice
Endothelial cell-specific deletion of the MR gene (MRCdh5Cre) was achieved using a tamoxifen-inducible Cre/loxP system under control of the cadherin 5 gene promoter as previously described (Lother et al. 2016). Cre-negative littermates served as ‘WT’ controls (MRwildtype). Both genotypes were treated with 2 mg tamoxifen (Sigma-Aldrich, 20 mg/mL in sunflower oil and 10% ethanol) i.p. per day on five consecutive days at least 2 weeks prior to any further experiment. All animal procedures were approved by the responsible animal care committee (Regierungspraesidium Freiburg, file number G13/27), and they conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (2011).
DOCA/high salt/nephrectomy model
Approximately 12-week-old male MRCdh5Cre and MR WT mice underwent unilateral nephrectomy and received 1% NaCl with drinking water (CTRL). Half of the animals in addition received deoxycorticosterone acetate (DOCA, 2.5 mg/day, Innovative Research of America, USA) from subcutaneous release pellets. Mice were killed 6 weeks after surgery.
Histology and immunohistochemistry
Tissues were fixed with 4% paraformaldehyde in phosphate-buffered saline and embedded in paraffin. Cardiac cross sections were stained using an anti-CD31 antibody (Abcam, #28364, polyclonal rabbit, 1:50 dilution) and the Vectastain ABC Kit (VectorLabs, USA) or Alexa-488 conjugated wheat germ agglutinin with DAPI (Thermo Fisher) to visualize endothelial cells or cardiac myocytes, respectively. Capillary density was quantified in five random fields per cross section and normalized to area (mm2), number of myocyte nuclei or cardiac myocyte area.
Aortic ring assay
Aortic ring segments (Baker et al. 2011) from MRCdh5Cre and MRwildtype mice were embedded in matrigel and were treated with 100 nM aldosterone (Sigma-Aldrich) or vehicle in EGM-2 medium (Lonza, Switzerland) containing 2.5% fetal calf serum and growth factor supplements without hydrocortisone. The number of capillary sprouts per aortic ring was determined on day 4.
In vivo angiogenesis
In vivo angiogenesis assay (Cultrex) was performed according to the manufacturer’s instructions. Briefly, silicon tubes containing medium with VEGF (3 ng/µL) and fibroblast growth factor 2 (9 ng/µL) and 100 nM aldosterone or vehicle were implanted subcutaneously. After 2 weeks, tubes were explanted, photographed and capillarization was determined by morphometry as described previously (Volkmann et al. 2013).
Endothelial cell culture and siRNA transfection
Human umbilical vein endothelial cells (HUVECs) were isolated as previously described (Heinke et al. 2008). HUVECs were cultured in endothelial cell growth medium (EGM-2, Lonza, Switzerland) with 10% fetal calf serum (FCS) and growth factor supplement without hydrocortisone and used until passage 5.
For siRNA transfection, HUVECs were seeded on six-well dishes at a density of 80,000 cells/well. siRNA against the mineralocorticoid receptor (Ambion SilencerSelect s8840, 10 nM) and control siRNA (Ambion SilencerSelect Negative Control siRNA #1, 10 nM) were transfected using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer’s instructions. After 6 h, cells were washed with PBS and medium was changed to EGM-2 with 2% FCS.
Western blot
Cells were lysed and harvested in lysis buffer containing 50 mM Tris–HCl (pH 6.7), SDS 2% and 1.0 mM sodium ortho-vanadate and proteins were separated by electrophoresis on 7.5% sodium dodecyl suphate polyacyrlamide gels and transferred to nitrocellulose membranes. Non-specific binding sites were blocked with 1% bovine serum albumin and 5% skim milk powder in phosphate-buffered saline solution for 1 h at room temperature. Membranes were incubated with primary antibodies against MR (rMR 1–18, clone 1D5-c, rat, DSHB University of Iowa, 1:500), VEGFR2 (ab39256, rabbit, Abcam, 1:1000) or the G-protein beta subunit (Gβ (T-20), rabbit, Santa Cruz Biotechnology, 1:3000) at the indicated dilutions over night at 4°C. After incubation with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature, signals were detected by chemoluminescence, visualized and processed using Fujifilm LAS-3000 (Fujifilm) and analyzed with MultiGauge software (Fujifilm).
Gene expression analysis
Three days after siRNA transfection, HUVECs were washed with PBS and medium was changed to endothelial cell basal medium (EBM-2) without growth factors containing 1% FCS and 100 nM aldosterone (Sigma-Aldrich) or ethanol as solvent control. After 18 h, total RNA was prepared from cells with the miRNeasy MiniKit (Qiagen) according to the manufacturer’s instructions. Quantitative real-time PCR was carried out using a MX3000p qPCR system (Agilent) and gene expression was normalized to the expression of ribosomal protein S29 (RPS29) by the ΔΔCt method. Primer sequences are given in Supplementary Table 1 (see section on supplementary data given at the end of this article).
Library preparation for RNAseq was conducted using the Low Input RiboMinusEukaryote System V2 (Ambion). The cDNA was fragmented into approximately 500 bp fragments using a sonication device (Bioruptor, Diagenode, USA) and 250 ng were used for library construction with the NEB Next Ultra DNA Library Prep Kit for Illumina (New England BioLabs) and amplified by fluorescence-controlled PCR. Size selection was performed with AMPure XP Beads (Beckmann Coulter). Libraries were sequenced on a HiSeq 2500 deep sequencing unit (50 bp, paired-end, Illumina) at the Max Planck Institute of Immunobiology and Epigenetics, Freiburg.
Bioinformatics analysis
RNAseq data were analyzed using the Galaxy platform (Afgan et al. 2016). Adapters were trimmed from reads using Trim Galore. Reads were mapped to the human genome (hg19) using STAR (Dobin et al. 2013) and duplicate reads were removed. Transcript abundance was estimated as fragments per kilobase of transcript per million fragments mapped using Cufflinks (Trapnell et al. 2010). Differential gene expression was determined using Cuffdiff (Trapnell et al. 2010) with q < 0.05 considered to be significant. Enrichment of molecular pathways from Gene Ontology was analyzed using ClueGO (Bindea et al. 2009) and CluePedia (Bindea et al. 2013) software. Unbiased screening for enrichment of transcription factor binding motifs within 2 kB from transcription start site was performed using findMotifs from HOMER tools (Heinz et al. 2010). Analysis was restricted to genes expressed >1 FPKM in HUVECs.
RNAseq data are provided at the NCBI Gene Expression Omnibus repository (BioProject ID PRJNA427260). Analysis of differential gene expression in HUVECs treated with VEGF for 12 h was performed accordingly using available RNAseq data (Zhang et al. 2013) (BioProject ID PRJNA176027).
Statistics
Spearman’s correlation, paired t-test, one sample t-test and 2-way ANOVA with Bonferroni multiple comparison analysis were performed using GraphPad Prism 5.04.
Results
Endothelial cell MR deletion improves capillary density in DOCA-induced hypertensive heart disease
To evaluate the role of endothelial cell MR on angiogenesis, MRwildtype and MRCdh5Cre mice were subjected to unilateral nephrectomy and high salt diet and received the MR agonist DOCA. Control mice (CTRL) underwent unilateral nephrectomy and received high salt diet but no DOCA. After 6 weeks, capillary density in cardiac cross-sections was reduced in MRwildtype mice receiving DOCA when compared to control mice (Fig. 1A, B and C). In MR-deficient mice, capillary density was slightly increased in the CTRL group and not altered by DOCA treatment (Fig. 1A, B and C). MRCdh5Cre showed less cardiac myocyte hypertrophy and a higher number of capillaries per cardiac myocyte area after DOCA treatment when compared to MRwildtype mice (Fig. 1D and E).
Capillary rarefaction in DOCA/salt/nephrectomy-induced hypertensive heart disease. WT mice and mice with endothelial cell-specific MR deletion (MRCdh5Cre) underwent unilateral nephrectomy and received high-salt diet without (CTRL) or with subcutaneous application of deoxycorticosterone acetate (DOCA). Capillary density (A, B and C) and cardiac myocyte cross sectional area (D and E) was quantified in left ventricular cross sections after immunohistochemical staining of the endothelial cell marker CD31 (A, bar 50 µm, insert bar 100 µm) or wheat germ agglutinin staining. n = 4 per group. Mean ± s.e.m. *P < 0.05, **P < 0.01 vs respective CTRL; #P < 0.05, ##P < 0.01, ###P < 0.001 vs MRwildtype.
Citation: Journal of Endocrinology 240, 1; 10.1530/JOE-18-0494
The anti-angiogenic effect of aldosterone directly depends on MR in endothelial cells
To further validate the role of endothelial cell MR on angiogenesis, we compared the effect of aldosterone on growth factor-mediated vascularization of subcutaneously implanted angioreactors and ex vivo on capillary formation from aortic ring segments. Aldosterone inhibited growth-factor-mediated vascularization of angioreactors in MRwildtype mice (Fig. 2A and B). MR deletion from endothelial cells strongly attenuated this effect (Fig. 2A and B). Similarly, aldosterone significantly reduced the number of capillary tubes forming from aortic ring segments of MRwildtype mice, while this effect was prevented by MR deletion (Fig. 2C and D).
Activation of MR in endothelial cells impairs angiogenesis. The impact of endothelial cell mineralocorticoid receptor (MR) activation on angiogenesis was examined in MRwildtype mice and mice with endothelial cell-specific MR deletion (MRCdh5Cre) using silicon angioreactor tubes containing matrix, growth factors and ALDO or SOLV (n = 4 per group, A and B). Aortic ring segments were taken from MRwildtype and MRCdh5Cre mice, embedded in matrigel and stimulated with aldosterone or solvent control (SOLV, n = 12–14 per group, C and D, bar 1 mm). Mean ± s.e.m. **P < 0.01, ***P < 0.001 vs respective SOLV control; ###P < 0.001 vs MRwildtype.
Citation: Journal of Endocrinology 240, 1; 10.1530/JOE-18-0494
MR-dependent gene expression in endothelial cells
We hypothesized that the antiangiogenic effect of aldosterone was associated with MR-dependent changes in endothelial cell gene expression. We transfected HUVECs with mineralocorticoid receptor siRNA (siMR) or control siRNA (siCTRL) and stimulated the cells with aldosterone. Knockdown of MR mRNA and protein after siRNA treatment was confirmed by qRT-PCR and immunoblotting (Fig. 3A and B). Genome-wide gene expression was analyzed by RNAseq. Among the genes that were up- or downregulated by aldosterone in siCTRL but not in siMR cells were serum and glucocorticoid-regulated kinase 1 (SGK1, Fig. 3C and D) or vascular cell adhesion molecule 1 (VCAM1, Fig. 3C and E). With this unbiased approach we found 519 genes differentially regulated by MR knockdown or aldosterone treatment (Fig. 3F and Supplementary Tables 2, 3). We focused on 133 genes in which expression was up- or downregulated by aldosterone and reverted by MR knockdown (Fig. 3F and Supplementary Table 2). Analysis of the promoter regions of these genes revealed a significant enrichment of the MR-binding motif when compared to all endothelial cell-expressed genes (Supplementary Fig. 1A). Eight genes out of 133 genes regulated by aldosterone contained a MR-binding motif in their promotor region and were considered as putative direct MR targets, including the previously described MR target gene SGK1 (Supplementary Fig. 1B). In addition, we found the nuclear factor kappa B (NFkB-p65) and interferon regulatory factor 1 (IRF1) transcription factor-binding motifs enriched (Supplementary Fig. 1A).
Aldosterone-induced changes in endothelial cell gene expression. Human umbilical vein endothelial cells were transfected with siRNA vs the mineralocorticoid receptor (siMR) or control siRNA (siCTRL) and stimulated with aldosterone (ALDO) or solvent control (SOLV). Knock-down of the MR gene was validated by qRT-PCR (A, n = 5 per group) and by Western blot (B). mRNA expression was determined by RNAseq. Representative traces from RNAseq showing the serum and glucocorticoid-regulated kinase 1 (SGK1) or vascular cell adhesion molecule 1 (VCAM1) locus (D, 3 replicates per condition merged). mRNA expression of SGK1 (E) or VCAM1 (F) was validated by qRT-PCR (n = 5 per group). We identified genes that were differentially regulated after MR knockdown either in aldosterone-treated or untreated cells (q < 0.05, n = 3 per group, C). Mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 vs siCTRL SOLV; #P < 0.05, ##P < 0.01, ###P < 0.001 vs siCTRL ALDO. FPKM, fragments per kilobase mapped; Gβ, G-protein beta subunit; RPS29, ribosomal protein S29.
Citation: Journal of Endocrinology 240, 1; 10.1530/JOE-18-0494
To assess the biological function of the aldosterone up- or downregulated genes, we performed molecular pathway analysis using the gene ontology database (Fig. 4). We found genes related to ‘regulation of angiogenesis’ (15 genes up-, 7 genes downregulated) and ‘regulation of leukocyte migration’ among the most enriched gene networks (Fig. 4). At serum conditions, independently of additional aldosterone, MR knockdown led to differential regulation of genes related to ‘blood vessel development’ (Supplementary Fig. 2). We found key regulators of angiogenesis such as vascular endothelial cells growth factor A (VEGFA, Fig. 5A) and VEGF receptor 2 (VEGFR2, Fig. 5B and C) up- or downregulated, respectively, by MR knockdown after aldosterone treatment.
Impact of aldosterone on biological processes in endothelial cells. We identified biological processes enriched (P < 0.01) among genes that were concordantly up- or downregulated by aldosterone (ALDO) in siCTRL cells compared to SOLV-treated siCTRL cells and ALDO-treated siMR cells (q < 0.05).
Citation: Journal of Endocrinology 240, 1; 10.1530/JOE-18-0494
Comparison of aldosterone- and VEGF-induced changes in gene expression. mRNA expression of vascular endothelial growth factor A (VEGFA, A) or vascular endothelial growth factor receptor 2 (VEGFR2, B) was validated by qRT-PCR (n = 5 per group). VEGFR2 protein expression was determined by Western blot (C). Expression changes in genes up- or downregulated by ALDO (q < 0.05) were compared to genes that were up- or downregulated by treatment with vascular endothelial cell growth factor (VEGF, q < 0.05, D and E). Mean ± s.e.m. ###P < 0.001 vs VEGF (paired t-test), §§§P < 0.001 vs zero (one sample t-test). ALDO, aldosterone; Gβ, G-protein beta subunit; SOLV, solvent control.
Citation: Journal of Endocrinology 240, 1; 10.1530/JOE-18-0494
Impact of MR on VEGF-dependent gene expression
To better understand the role of MR-dependent gene expression on VEGF-induced angiogenesis we compared our results to data derived from HUVECs treated with VEGF (Zhang et al. 2013). We found opposing effects of MR-activation and VEGF on endothelial cell gene expression (Fig. 5D). Genes that were significantly changed in both conditions showed an inverse correlation of expression (R 2 = 0.56, P < 0.001; Fig. 5E). We aimed to identify biological processes that were affected by MR or VEGF and performed an integrated analysis of those genes that were regulated by either one or both factors (Fig. 6). Biological processes related to inflammation contained primarily MR-regulated genes (Fig. 6A, red discs). Most of the processes that showed a high degree of interaction between MR and VEGF were related to blood vessel development (Fig. 6A, violet discs). We closer looked at those processes to identify putative key factors involved and found a network of genes that were regulated by MR or VEGF including interleukins and CXC chemokine family members (Fig. 6B).
Impact of aldosterone on VEGF-dependent biological processes. Genes up- or downregulated by aldosterone (ALDO, q < 0.05) and genes up- or downregulated by treatment with vascular endothelial cell growth factor (VEGF, q < 0.05) were analyzed side by side to identify biological processes affected by both factors (P < 0.05, A). Closer analysis of putative key gene networks involved in biological processes associated with angiogenesis (grey area in panel A) were identified using CluePedia software (B). Color code indicates genes or biological processes altered by aldosterone (red), VEGF (blue) or both factors (violet).
Citation: Journal of Endocrinology 240, 1; 10.1530/JOE-18-0494
Discussion
The key findings of our study are that (1) the anti-angiogenic effect of aldosterone directly depends on MR in endothelial cells and (2) MR leads to dysregulation of VEGFR2 and opposes the effects of VEGF on endothelial cell gene expression.
The impact of aldosterone and MR on angiogenesis is controversial. Other studies have shown that aldosterone diminishes capillary tube formation of cultured endothelial cells (Fujii et al. 2012), inhibits the formation of capillaries from aortic rings (Thum et al. 2011) and impairs vascularization of matrigel plugs (Thum et al. 2011, Fujii et al. 2012). These effects were prevented by treatment with the MR antagonist eplerenone (Thum et al. 2011, Fujii et al. 2012). In addition, eplerenone improves capillary density in hindlimb ischemia in rats (Kobayashi et al. 2010) and in an experimental model of ischemic heart failure in dogs (Suzuki et al. 2002). However, there are also studies reporting neutral or even pro-angiogenic effects of MR on angiogenesis in different models of disease (Messaoudi et al. 2009, Gravez et al. 2015, Salvador et al. 2017). In a recent study, MR deletion from endothelial cells improved left ventricular function after pressure overload. Intriguingly, the functional improvement was irrespective of cardiac hypertrophy or fibrosis. In particular, endothelial MR deletion did not affect the decrease in left ventricular capillary density (Salvador et al. 2017). In another study, treatment with the MR antagonist eplerenone dampened endothelial cell proliferation in pressure overloaded hearts (Gravez et al. 2015). However, pressure overload is not specific for aldosterone but augments angiotensin II and adrenergic signaling, too. This serves as compensatory mechanism in the acute phase and affects cardiovascular remodeling in the long term. Cross-talk of MR with other signaling pathways, in particular angiotensin II receptors, is well-described (Ruhs et al. 2017), and it has been shown that these signaling pathways have synergistic effects on angiogenesis. For example, neovascularization after hindlimb ischemia in mice was improved by aldosterone treatment but this was reverted by co-application of the AT1 angiotensin II receptor blocker valsartan (Michel et al. 2004). This suggests that activation of angiotensin II receptors might be necessary for a pro-angiogenic effect of aldosterone.
To dissect the particular role for MR in endothelial cells on angiogenesis, we evaluated mice lacking MR in endothelial cells in the primarily MR-driven, low-renin, low-angiotensin and (more controversial) low-norepinephrin DOCA/salt/nephrectomy model (Gavras et al. 1975, Wang et al. 2002, Wehrwein et al. 2013). Our study builds on the earlier finding that DOCA treatment leads to capillary rarefaction in the heart (Larouche & Schiffrin 1999). Of note, DOCA can activate both, MR and GR (Vinson 2011). Taking advantage of a cell type-specific deletion mouse model we could demonstrate that capillary rarefaction in DOCA-treated mice directly depends on MR in endothelial cells. Importantly, lack of endothelial cell MR does not affect blood pressure response to DOCA, which implies that these effects are not due to altered hemodynamics (Rickard et al. 2014, Lother et al. 2016). As reported previously (Lother et al. 2016), endothelial cell MR deletion attenuated cardiac myocyte hypertrophy after DOCA treatment. Thus, improved capillary density might result from both, improved angiogenesis or smaller cardiac myocyte size. In addition, not only endothelial cells but also cardiac myocytes, fibroblasts and immune cells are involved in the process of angiogenesis by interacting with each other, secreting pro- and anti-angiogenic factors or shaping the microenvironment (Oka et al. 2014, Rienks et al. 2014, Kamo et al. 2015). DOCA increases the number of inflammatory macrophages in the heart which is prevented by endothelial cell MR deletion (Rickard et al. 2014), and we cannot completely rule out that this contributes to the beneficial effect on capillary density. To address these points, we used in vivo and ex vivo angiogenesis assays that are largely independent from cardiac hypertrophy or inflammation. In both assays, MR deletion consistently prevented the effect of aldosterone which confirms the central role of MR in endothelial cells for angiogenesis.
We aimed to determine the effect of MR on endothelial cell gene expression and found 133 genes that were up- or downregulated by aldosterone in MR-expressing endothelial cells but not after MR knockdown. Several other studies have investigated MR-dependent gene expression in the vasculature (Jaffe & Mendelsohn 2005, Newfell et al. 2011, Sekizawa et al. 2011), the heart (Fejes-Toth & Naray-Fejes-Toth 2007, Latouche et al. 2010, Lother et al. 2011) or other tissues (Ueda et al. 2014). A number of genes that are regulated by MR in other cells or tissues were up- or downregulated in endothelial cells, including SGK1 (Fejes-Toth & Naray-Fejes-Toth 2007, Latouche et al. 2010, Lother et al. 2011, Ueda et al. 2014), TSC22D3 (Sekizawa et al. 2011, Ueda et al. 2014), ADAMTS1 (Fejes-Toth & Naray-Fejes-Toth 2007, Latouche et al. 2010), FKBP5 (Latouche et al. 2010, Sekizawa et al. 2011, Ueda et al. 2014) or KLF9 (Newfell et al. 2011, Sekizawa et al. 2011, Ueda et al. 2014). This indicates that there is a common signature of MR-regulated genes in different cell types.
Systematic analysis of biological processes regulated by MR in endothelial cells revealed an overrepresentation of genes associated with angiogenesis. Differential regulation of angiogenesis-related genes by aldosterone has been demonstrated before in mouse aorta (Newfell et al. 2011) and MR-overexpressing cultured endothelial cells (Sekizawa et al. 2011). One of the most potent pro-angiogenic factors is VEGF (Carmeliet & Jain 2011), which plays an important role in adaptive angiogenesis during cardiac hypertrophic growth (Oka et al. 2014). It has previously been suggested that aldosterone impairs angiogenesis via downregulation of VEGF receptor 2 (Fujii et al. 2012, Tiberio et al. 2013). In line with that, we found an upregulation of VEGFR2 in endothelial cells after MR knockdown. We observed opposing effects on endothelial cell gene expression when comparing MR activation to treatment with VEGF (Zhang et al. 2013). It is noteworthy that MR affects the expression of VEGF family members in other cell types. For example, aldosterone induced the expression of placental growth factor (PGF) in vascular smooth muscle cells, hereby promoting smooth muscle cell proliferation and perivascular fibrosis (Jaffe et al. 2010, Newfell et al. 2011, Pruthi et al. 2014).
Angiogenesis is a highly complex process that involves the interaction of numerous molecules. The role of gene networks in this process has been recognized and is under intensive investigation (Montanez et al. 2011, Glass et al. 2015, Fish et al. 2017, Weinstein et al. 2017). Side-by-side analysis of MR- and VEGF-dependent gene expression revealed a high degree of interaction in processes related to vasculature development, including interleukins and CXC chemokine family members (Owen & Mohamadzadeh 2013), indicating that both factors act on overlapping gene networks. Accordingly, aldosterone dampened VEGF-induced angiogenesis in vivo and in vitro.
Interestingly, it has been reported that the synthetic glucocorticoid triamcinolone acetonide decreased expression of VEGFA and increased expression of VEGFR2 (Ozmen et al. 2016) similarly to what we observed after MR knockdown in this study. Aldosterone and glucocorticoids can bind to the MR with similar affinity and MR is occupied by glucocorticoids in many tissues (Jaisser & Farman 2016, Funder 2017). In endothelial cells, MR is co-expressed with 11beta-hydroxysteroid dehydrogenase isoform 1 and 2 that convert glucocorticoids into active or inactive metabolites (Gong et al. 2008, Liu et al. 2009, Lother et al. 2018) and therefor may be bound by glucocorticoids or aldosterone. We found a remarkable number of genes differentially regulated after MR knockdown at serum conditions indicating that endothelial MR is activated in absence of excess aldosterone. 67 out of 133 aldosterone-responsive genes showed differences in expression at serum conditions that were further augmented at high doses of aldosterone. Bioinformatic analysis revealed an enrichment of the MR binding motif within the promoter region of these genes. However, both MR and the glucocorticoid receptor (GR) can bind to similar DNA motifs, either as homodimers or heterodimers of both, and may differently influence gene expression (Funder 1997, Le Billan et al. 2018). This is of importance since DOCA is not selective for MR. In particular, transcriptional repression by GR is well described (Weikum et al. 2017) while MR is assumed to be predominantly a transcriptional activator. Chromatin-immunoprecipitation experiments on renal epithelial cells indicate that only a minority of MR binding occurs in promoter regions (Le Billan et al. 2015). Thus, further studies assessing MR and GR protein binding to promoter and distal enhancer sites, their interaction with co-regulator proteins and the impact of chromatin structure will be required to further understand the mechanisms of transcriptional regulation by MR in endothelial cells.
In conclusion, we demonstrate here that activation of endothelial cell MR impairs angiogenic capacity and leads to capillary rarefaction in a mouse model of MR-driven hypertension. MR activation opposes VEGF-induced gene expression leading to the dysregulation of angiogenesis-related gene networks in endothelial cells. Our findings underscore the impact of MR as important transcriptional regulator in endothelial cells and its pivotal role in the pathophysiology of cardiovascular disease.
Supplementary data
This is linked to the online version of the paper at https://doi.org/10.1530/JOE-18-0494.
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 work was supported by the European Section of the Aldosterone Council – ESAC Germany (to A Lother), the Förderkreis Dresdner Herz-Kreislauf-Tage (to A Lother) the Else-Kröner-Fresenius-Stiftung (2016_A163 to A Lother), the German Cardiac Society (Otto-Hess-Scholarships to D Fürst and J Kowalski), and the Innovationsfonds des Landes Baden-Württemberg (to L Hein). The Freiburg Galaxy Team receives funding from the Collaborative Research Centre 992 Medical Epigenetics (DFG grant SFB 992/2 2016) and the German Federal Ministry of Education and Research (BMBF grant 031 A538A RBC (de.NBI)).
Acknowledgements
The authors thank the Deep Sequencing Facility, MPI of Immunobiology and Epigenetics (Freiburg, Germany) and the Freiburg Galaxy Team, Björn Grüning and Rolf Backofen, Bioinformatics, University of Freiburg, Germany. They thank Claudia Domisch, Birgit Scherer and Ute Wering for technical assistance.
References
Afgan E, Baker D, van den Beek M, Blankenberg D, Bouvier D, Cech M, Chilton J, Clements D, Coraor N, Eberhard C, et al.2016 The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update. Nucleic Acids Research 44 W3–W10. (https://doi.org/10.1093/nar/gkw343)
Baker M, Robinson SD, Lechertier T, Barber PR, Tavora B, D'Amico G, Jones DT, Vojnovic B & Hodivala-Dilke K 2011 Use of the mouse aortic ring assay to study angiogenesis. Nature Protocols 7 89–104. (https://doi.org/10.1038/nprot.2011.435)
Barrett Mueller K, Bender SB, Hong K, Yang Y, Aronovitz M, Jaisser F, Hill MA & Jaffe IZ 2015 Endothelial mineralocorticoid receptors differentially contribute to coronary and mesenteric vascular function without modulating blood pressure. Hypertension 66 988–997. (https://doi.org/10.1161/HYPERTENSIONAHA.115.06172)
Bindea G, Mlecnik B, Hackl H, Charoentong P, Tosolini M, Kirilovsky A, Fridman WH, Pages F, Trajanoski Z & Galon J 2009 ClueGO: a cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks. Bioinformatics 25 1091–1093. (https://doi.org/10.1093/bioinformatics/btp101)
Bindea G, Galon J & Mlecnik B 2013 CluePedia cytoscape plugin: pathway insights using integrated experimental and in silico data. Bioinformatics 29 661–663. (https://doi.org/10.1093/bioinformatics/bbib19)
Carmeliet P & Jain RK 2011 Molecular mechanisms and clinical applications of angiogenesis. Nature 473 298–307. (https://doi.org/10.1038/nature10144)
Cole TJ & Young MJ 2017 30 years of the mineralocorticoid receptor: mineralocorticoid receptor null mice: informing cell-type-specific roles. Journal of Endocrinology 234 T83–T92. (https://doi.org/10.1530/JOE-17-0155)
Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M & Gingeras TR 2013 STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29 15–21. (https://doi.org/10.1093/bioinformatics/bts635)
DuPont JJ & Jaffe IZ 2017 30 years of the mineralocorticoid receptor: the role of the mineralocorticoid receptor in the vasculature. Journal of Endocrinology 234 T67–T82. (https://doi.org/10.1530/JOE-17-0009)
Fejes-Toth G & Naray-Fejes-Toth A 2007 Early aldosterone-regulated genes in cardiomyocytes early aldosterone-regulated genes in cardiomyocytes: clues to cardiac remodeling? Endocrinology 148 1502–1510. (https://doi.org/10.1210/en.2006-1438)
Fish JE, Cantu Gutierrez M, Dang LT, Khyzha N, Chen Z, Veitch S, Cheng HS, Khor M, Antounians L, Njock MS, et al.2017 Dynamic regulation of VEGF-inducible genes by an ERK/ERG/p300 transcriptional network. Development 144 2428–2444. (https://doi.org/10.1242/dev.146050)
Fujii M, Inoki I, Saga M, Morikawa N, Arakawa K, Inaba S, Yoshioka K, Konoshita T & Miyamori I 2012 Aldosterone inhibits endothelial morphogenesis and angiogenesis through the downregulation of vascular endothelial growth factor receptor-2 expression subsequent to peroxisome proliferator-activated receptor gamma. Journal of Steroid Biochemistry and Molecular Biology 129 145–152. (https://doi.org/10.1016/j.jsbmb.2011.12.014)
Funder JW 1997 Glucocorticoid and mineralocorticoid receptors: biology and clinical relevance. Annual Review of Medicine 48 231–240. (https://doi.org/10.1146/annurev.med.48.1.231)
Funder J 2017 30 years of the mineralocorticoid receptor: mineralocorticoid receptor activation and specificity-conferring mechanisms: a brief history. Journal of Endocrinology 234 T17–T21. (https://doi.org/10.1530/JOE-17-0119)
Gavras H, Brunner HR, Laragh JH, Vaughan ED Jr, Koss M, Cote LJ & Gavras I 1975 Malignant hypertension resulting from deoxycorticosterone acetate and salt excess: role of renin and sodium in vascular changes. Circulation Research 36 300–309. (https://doi.org/10.1161/01.RES.36.2.300)
Glass K, Quackenbush J, Spentzos D, Haibe-Kains B & Yuan GC 2015 A network model for angiogenesis in ovarian cancer. BMC Bioinformatics 16 115. (https://doi.org/10.1186/s12859-015-0551-y)
Gong R, Morris DJ & Brem AS 2008 Variable expression of 11beta hydroxysteroid dehydrogenase (11beta-HSD) isoforms in vascular endothelial cells. Steroids 73 1187–1196. (https://doi.org/10.1016/j.steroids.2008.05.009)
Gravez B, Tarjus A, Pelloux V, Ouvrard-Pascaud A, Delcayre C, Samuel J, Clement K, Farman N, Jaisser F & Messaoudi S 2015 Aldosterone promotes cardiac endothelial cell proliferation in vivo. Journal of the American Heart Association 4 e001266. (https://doi.org/10.1161/JAHA.114.001266)
Heinke J, Wehofsits L, Zhou Q, Zoeller C, Baar KM, Helbing T, Laib A, Augustin H, Bode C, Patterson C, et al.2008 BMPER is an endothelial cell regulator and controls bone morphogenetic protein-4-dependent angiogenesis. Circulation Research 103 804–812. (https://doi.org/10.1161/CIRCRESAHA.108.178434)
Heinz S, Benner C, Spann N, Bertolino E, Lin YC, Laslo P, Cheng JX, Murre C, Singh H & Glass CK 2010 Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Molecular Cell 38 576–589. (https://doi.org/10.1016/j.molcel.2010.05.004)
Jaffe IZ & Mendelsohn ME 2005 Angiotensin II and aldosterone regulate gene transcription via functional mineralocortocoid receptors in human coronary artery smooth muscle cells. Circulation Research 96 643–650. (https://doi.org/10.1161/01.RES.0000159937.05502.d1)
Jaffe IZ, Newfell BG, Aronovitz M, Mohammad NN, McGraw AP, Perreault RE, Carmeliet P, Ehsan A & Mendelsohn ME 2010 Placental growth factor mediates aldosterone-dependent vascular injury in mice. Journal of Clinical Investigation 120 3891–3900. (https://doi.org/10.1172/JCI40205)
Jaisser F & Farman N 2016 Emerging roles of the mineralocorticoid receptor in pathology: toward new paradigms in Clinical Pharmacology. Pharmacological Reviews 68 49–75. (https://doi.org/10.1124/pr.115.011106)
Jia G, Habibi J, DeMarco VG, Martinez-Lemus LA, Ma L, Whaley-Connell AT, Aroor AR, Domeier TL, Zhu Y, Meininger GA, et al.2015 Endothelial mineralocorticoid receptor deletion prevents diet-induced cardiac diastolic dysfunction in females. Hypertension 66 1159–1167. (https://doi.org/10.1161/HYPERTENSIONAHA.115.06015)
Jia G, Habibi J, Aroor AR, Martinez-Lemus LA, DeMarco VG, Ramirez-Perez FI, Sun Z, Hayden MR, Meininger GA, Mueller KB, et al.2016 Endothelial mineralocorticoid receptor mediates diet-induced aortic stiffness in females. Circulation Research 118 935–943. (https://doi.org/10.1161/CIRCRESAHA.115.308269)
Kamo T, Akazawa H & Komuro I 2015 Cardiac nonmyocytes in the hub of cardiac hypertrophy. Circulation Research 117 89–98. (https://doi.org/10.1161/CIRCRESAHA.117.305349)
Kobayashi N, Fukushima H, Takeshima H, Koguchi W, Mamada Y, Hirata H, Machida Y, Suzuki N, Yokotsuka F, Tabei K, et al.2010 Effect of eplerenone on endothelial progenitor cells and oxidative stress in ischemic hindlimb. American Journal of Hypertension 23 1007–1013. (https://doi.org/10.1038/ajh.2010.91)
Larouche I & Schiffrin EL 1999 Cardiac microvasculature in DOCA-salt hypertensive rats: effect of endothelin ET(A) receptor antagonism. Hypertension 34 795–801. (https://doi.org/10.1161/01.HYP.34.4.795)
Latouche C, Sainte-Marie Y, Steenman M, Castro Chaves P, Naray-Fejes-Toth A, Fejes-Toth G, Farman N & Jaisser F 2010 Molecular signature of mineralocorticoid receptor signaling in cardiomyocytes: from cultured cells to mouse heart. Endocrinology 151 4467–4476. (https://doi.org/10.1210/en.2010-0237)
Le Billan F, Khan JA, Lamribet K, Viengchareun S, Bouligand J, Fagart J & Lombes M 2015 Cistrome of the aldosterone-activated mineralocorticoid receptor in human renal cells. FASEB Journal 29 3977–3989. (https://doi.org/10.1096/fj.15-274266)
Le Billan F, Amazit L, Bleakley K, Xue QY, Pussard E, Lhadj C, Kolkhof P, Viengchareun S, Fagart J & Lombes M 2018 Corticosteroid receptors adopt distinct cyclical transcriptional signatures. FASEB Journal 32 5626–5639. (https://doi.org/10.1096/fj.201800391RR)
Liu Y, Mladinov D, Pietrusz JL, Usa K & Liang M 2009 Glucocorticoid response elements and 11 beta-hydroxysteroid dehydrogenases in the regulation of endothelial nitric oxide synthase expression. Cardiovascular Research 81 140–147. (https://doi.org/10.1093/cvr/cvn231)
Lother A & Hein L 2016 Vascular mineralocorticoid receptors: linking risk factors, hypertension, and heart disease. Hypertension 68 6–10. (https://doi.org/10.1161/HYPERTENSIONAHA.116.07418)
Lother A, Berger S, Gilsbach R, Rosner S, Ecke A, Barreto F, Bauersachs J, Schutz G & Hein L 2011 Ablation of mineralocorticoid receptors in myocytes but not in fibroblasts preserves cardiac function. Hypertension 57 746–754. (https://doi.org/10.1161/HYPERTENSIONAHA.110.163287)
Lother A, Moser M, Bode C, Feldman RD & Hein L 2015 Mineralocorticoids in the heart and vasculature: new insights for old hormones. Annual Review of Pharmacology and Toxicology 55 289–312. (https://doi.org/10.1146/annurev-pharmtox-010814-124302)
Lother A, Fürst D, Bergemann S, Gilsbach R, Grahammer F, Huber TB, Hilgendorf I, Bode C, Moser M & Hein L 2016 Deoxycorticosterone acetate/salt-induced cardiac but not renal injury is mediated by endothelial mineralocorticoid receptors independently from blood pressure. Hypertension 67 130–138. (https://doi.org/10.1161/HYPERTENSIONAHA.115.06530)
Lother A, Bergemann S, Deng L, Moser M, Bode C & Hein L 2018 Cardiac endothelial cell transcriptome. Arteriosclerosis, Thrombosis, and Vascular Biology 38 566–574. (https://doi.org/10.1161/ATVBAHA.117.310549)
Messaoudi S, Milliez P, Samuel JL & Delcayre C 2009 Cardiac aldosterone overexpression prevents harmful effects of diabetes in the mouse heart by preserving capillary density. FASEB Journal 23 2176–2185. (https://doi.org/10.1096/fj.08-125302)
Michel F, Ambroisine ML, Duriez M, Delcayre C, Levy BI & Silvestre JS 2004 Aldosterone enhances ischemia-induced neovascularization through angiotensin II-dependent pathway. Circulation 109 1933–1937. (https://doi.org/10.1161/01.CIR.0000127112.36796.9B)
Montanez R, Sanchez-Jimenez F, Quesada AR & Medina MA 2011 Exploring and challenging the network of angiogenesis. Scientific Reports 1 61. (https://doi.org/10.1038/srep00061)
Newfell BG, Iyer LK, Mohammad NN, McGraw AP, Ehsan A, Rosano G, Huang PL, Mendelsohn ME & Jaffe IZ 2011 Aldosterone regulates vascular gene transcription via oxidative stress-dependent and -independent pathways. Arteriosclerosis, Thrombosis, and Vascular Biology 31 1871–1880. (https://doi.org/10.1161/ATVBAHA.111.229070)
Oka T, Akazawa H, Naito AT & Komuro I 2014 Angiogenesis and cardiac hypertrophy: maintenance of cardiac function and causative roles in heart failure. Circulation Research 114 565–571. (https://doi.org/10.1161/CIRCRESAHA.114.300507)
Owen JL & Mohamadzadeh M 2013 Macrophages and chemokines as mediators of angiogenesis. Frontiers in Physiology 4 159. (https://doi.org/10.3389/fphys.2013.00159)
Ozmen A, Unek G, Kipmen-Korgun D, Mendilcioglu I, Sanhal C, Sakinci M & Korgun ET 2016 Glucocorticoid effects on angiogenesis are associated with mTOR pathway activity. Biotechnic and Histochemistry 91 296–306. (https://doi.org/10.3109/10520295.2016.1161234)
Pruthi D, McCurley A, Aronovitz M, Galayda C, Karumanchi SA & Jaffe IZ 2014 Aldosterone promotes vascular remodeling by direct effects on smooth muscle cell mineralocorticoid receptors. Arteriosclerosis, Thrombosis, and Vascular Biology 34 355–364. (https://doi.org/10.1161/ATVBAHA.113.302854)
Rickard AJ, Morgan J, Chrissobolis S, Miller AA, Sobey CG & Young MJ 2014 Endothelial cell mineralocorticoid receptors regulate deoxycorticosterone/salt-mediated cardiac remodeling and vascular reactivity but not blood pressure. Hypertension 63 1033–1040. (https://doi.org/10.1161/HYPERTENSIONAHA.113.01803)
Rienks M, Papageorgiou AP, Frangogiannis NG & Heymans S 2014 Myocardial extracellular matrix: an ever-changing and diverse entity. Circulation Research 114 872–888. (https://doi.org/10.1161/CIRCRESAHA.114.302533)
Rossier BC, Baker ME & Studer RA 2015 Epithelial sodium transport and its control by aldosterone: the story of our internal environment revisited. Physiological Reviews 95 297–340. (https://doi.org/10.1152/physrev.00011.2014)
Ruhs S, Nolze A, Hubschmann R & Grossmann C 2017 30 years of the mineralocorticoid receptor: nongenomic effects via the mineralocorticoid receptor. Journal of Endocrinology 234 T107–T124. (https://doi.org/10.1530/JOE-16-0659)
Salvador AM, Moss ME, Aronovitz M, Mueller KB, Blanton RM, Jaffe IZ & Alcaide P 2017 Endothelial mineralocorticoid receptor contributes to systolic dysfunction induced by pressure overload without modulating cardiac hypertrophy or inflammation. Physiological Reports 5 e13313. (https://doi.org/10.14814/phy2.13313)
Schafer N, Lohmann C, Winnik S, van Tits LJ, Miranda MX, Vergopoulos A, Ruschitzka F, Nussberger J, Berger S, Luscher TF, et al.2013 Endothelial mineralocorticoid receptor activation mediates endothelial dysfunction in diet-induced obesity. European Heart Journal 34 3515–3524. (https://doi.org/10.1093/eurheartj/ehbib95)
Sekizawa N, Yoshimoto T, Hayakawa E, Suzuki N, Sugiyama T & Hirata Y 2011 Transcriptome analysis of aldosterone-regulated genes in human vascular endothelial cell lines stably expressing mineralocorticoid receptor. Molecular and Cellular Endocrinology 341 78–88. (https://doi.org/10.1016/j.mce.2011.05.029)
Suzuki G, Morita H, Mishima T, Sharov VG, Todor A, Tanhehco EJ, Rudolph AE, McMahon EG, Goldstein S & Sabbah HN 2002 Effects of long-term monotherapy with eplerenone, a novel aldosterone blocker, on progression of left ventricular dysfunction and remodeling in dogs with heart failure. Circulation 106 2967–2972. (https://doi.org/10.1161/01.CIR.0000039104.56479.42)
Thum T, Schmitter K, Fleissner F, Wiebking V, Dietrich B, Widder JD, Jazbutyte V, Hahner S, Ertl G & Bauersachs J 2011 Impairment of endothelial progenitor cell function and vascularization capacity by aldosterone in mice and humans. European Heart Journal 32 1275–1286. (https://doi.org/10.1093/eurheartj/ehq254)
Tiberio L, Nascimbeni R, Villanacci V, Casella C, Fra A, Vezzoli V, Furlan L, Meyer G, Parrinello G, Baroni MD, et al.2013 The decrease of mineralcorticoid receptor drives angiogenic pathways in colorectal cancer. PLoS ONE 8 e59410. (https://doi.org/10.1371/journal.pone.0059410)
Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ & Pachter L 2010 Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nature Biotechnology 28 511–515. (https://doi.org/10.1038/nbt.1621)
Ueda K, Fujiki K, Shirahige K, Gomez-Sanchez CE, Fujita T, Nangaku M & Nagase M 2014 Genome-wide analysis of murine renal distal convoluted tubular cells for the target genes of mineralocorticoid receptor. Biochemical and Biophysical Research Communications 445 132–137. (https://doi.org/10.1016/j.bbrc.2014.01.125)
Vinson GP 2011 The mislabelling of deoxycorticosterone: making sense of corticosteroid structure and function. Journal of Endocrinology 211 3–16. (https://doi.org/10.1530/JOE-11-0178)
Volkmann I, Kumarswamy R, Pfaff N, Fiedler J, Dangwal S, Holzmann A, Batkai S, Geffers R, Lother A, Hein L, et al.2013 MicroRNA-mediated epigenetic silencing of sirtuin1 contributes to impaired angiogenic responses. Circulation Research 113 997–1003. (https://doi.org/10.1161/CIRCRESAHA.113.301702)
Wang Q, Hummler E, Nussberger J, Clement S, Gabbiani G, Brunner HR & Burnier M 2002 Blood pressure, cardiac, and renal responses to salt and deoxycorticosterone acetate in mice: role of renin genes. Journal of the American Society of Nephrology 13 1509–1516. (https://doi.org/10.1097/01.ASN.0000017902.77985.84)
Wehrwein EA, Novotny M, Swain GM, Parker LM, Esfahanian M, Spitsbergen JM, Habecker BA & Kreulen DL 2013 Regional changes in cardiac and stellate ganglion norepinephrine transporter in DOCA-salt hypertension. Autonomic Neuroscience 179 99–107. (https://doi.org/10.1016/j.autneu.2013.08.070)
Weikum ER, Knuesel MT, Ortlund EA & Yamamoto KR 2017 Glucocorticoid receptor control of transcription: precision and plasticity via allostery. Nature Reviews: Molecular Cell Biology 18 159–174. (https://doi.org/10.1038/nrm.2016.152)
Weinstein N, Mendoza L, Gitler I & Klapp J 2017 A network model to explore the effect of the micro-environment on endothelial cell behavior during angiogenesis. Frontiers in Physiology 8 960. (https://doi.org/10.3389/fphys.2017.00960)
Young MJ & Rickard AJ 2015 Mineralocorticoid receptors in the heart: lessons from cell-selective transgenic animals. Journal of Endocrinology 224 R1–R13. (https://doi.org/10.1530/JOE-14-0471)
Zhang B, Day DS, Ho JW, Song L, Cao J, Christodoulou D, Seidman JG, Crawford GE, Park PJ & Pu WT 2013 A dynamic H3K27ac signature identifies VEGFA-stimulated endothelial enhancers and requires EP300 activity. Genome Research 23 917–927. (https://doi.org/10.1101/gr.149674.112)
