RATIONALE:The interaction of circulating cells within the vascular wall is a critical event in chronic inflammatory processes, such as atherosclerosis, but the control of the vascular inflammatory ...state is still largely unclear.
OBJECTIVE:This study was undertaken to characterize the function of the endothelial-enriched microRNA miR-100 during vascular inflammation and atherogenesis.
METHODS AND RESULTS:Based on a transcriptome analysis of endothelial cells after miR-100 overexpression, we identified miR-100 as a potent suppressor of endothelial adhesion molecule expression, resulting in attenuated leukocyte–endothelial interaction in vitro and in vivo as shown by flow cytometry and intravital imaging. Mechanistically, miR-100 directly repressed several components of mammalian target of rapamycin complex 1-signaling, including mammalian target of rapamycin and raptor, which resulted in a stimulation of endothelial autophagy and attenuated nuclear factor κB signaling in vitro and in vivo. In a low-density lipoprotein receptor–deficient atherosclerotic mouse model, pharmacological inhibition of miR-100 resulted in enhanced plaque lesion formation and a higher macrophage content of the plaque, whereas a systemic miR-100 replacement therapy had protective effects and attenuated atherogenesis, resulting in a decrease of plaque area by 45%. Finally, analysis of miR-100 expression in >70 samples obtained during carotid endarterectomy revealed that local miR-100 expression was inversely correlated with inflammatory cell content in patients.
CONCLUSIONS:In summary, we describe an anti-inflammatory function of miR-100 in the vascular response to injury and inflammation and identify an important novel modulator of mammalian target of rapamycin signaling and autophagy in the vascular system. Our findings of miR-100 as a potential protective anti-athero-miR suggest that the therapeutic replacement of this microRNA could be a potential strategy for the treatment of chronic inflammatory diseases, such as atherosclerosis, in the future.
OBJECTIVE—Endothelial cells (ECs) are a highly specialized cell type with marked diversity between different organs or vascular beds. Cardiac ECs are an important player in cardiac physiology and ...pathophysiology but are not sufficiently characterized yet. Thus, the aim of the present study was to analyze the cardiac EC transcriptome.
APPROACH AND RESULTS—We applied fluorescence-assisted cell sorting to isolate pure ECs from adult mouse hearts. RNAseq revealed 1288 genes predominantly expressed in cardiac ECs versus heart tissue including several transcription factors. We found an overrepresentation of corresponding transcription factor binding motifs within the promotor region of EC-enriched genes, suggesting that they control the EC transcriptome. Cardiac ECs exhibit a distinct gene expression profile when compared with renal, cerebral, or pulmonary ECs. For example, we found the Meox2/Tcf15, Fabp4, and Cd36 signaling cascade higher expressed in cardiac ECs which is a key regulator of fatty acid uptake and involved in the development of atherosclerosis.
CONCLUSIONS—The results from this study provide a comprehensive resource of gene expression and transcriptional control in cardiac ECs. The cardiac EC transcriptome exhibits distinct differences in gene expression compared with other cardiac cell types and ECs from other organs. We identified new candidate genes that have not been investigated in ECs yet as promising targets for future evaluation.
Diet-induced obesity can result in the development of a diverse spectrum of cardiovascular and metabolic diseases, including type 2 diabetes, dyslipidemia, non-alcoholic liver steatosis and ...atherosclerotic disease. MicroRNAs have been described to be important regulators of metabolism and disease development.
In the current study, we investigated the effects of ubiquitous miR-100 overexpression on weight gain and the metabolic phenotype in a newly generated transgenic mouse strain under normal chow and high fat diet and used microarray expression analysis to identify new potential target genes of miR-100.
While transgenic overexpression of miR-100 did not significantly affect weight and metabolism under a normal diet, miR-100 overexpressing mice showed a reduced weight gain under a high fat diet compared to wildtype mice, despite an equal calorie intake. This was accompanied by less visceral and subcutaneous fat development and lover serum LDL cholesterol. In addition, transgenic miR-100 mice were more glucose tolerant and insulin sensitive and demonstrated increased energy expenditure under high fat diet feeding. A comprehensive gene expression profiling revealed the differential expression of several genes involved in lipid storage- and metabolism, among them CD36 and Cyp4A14. Our data showed a direct regulation of CD36 by miR-100, leading to a reduced fatty acid uptake in primary hepatocytes overexpressing miR-100 and the downregulation of several downstream mediators of lipid metabolism such as ACC1, FABP4, FAS and PPARγ in the liver.
Our findings demonstrate a protective role of miR-100 in high fat diet induced metabolic syndrome and liver steatosis, partially mediated by the direct repression of CD36 and attenuation of hepatic lipid storage, implicating miR-100 as a possible therapeutic target in liver steatosis.
Summary
MicroRNAs are small non-coding RNAs that negatively regulate posttranscriptional gene expression. Several microRNAs have been described to regulate the process of angiogenesis. Previously, we ...have shown that bone morphogenetic protein 4 (BMP4) increased the proangiogenic activity of endothelial cells. In this project, we now investigated how the pro-angiogenic BMP4 effect is mediated by microRNAs. First, we performed a microRNA array with BMP4-stimulated human umbilical vein endothelial cells (HUVECs). Among the topregulated microRNAs, we detected a decreased expression of miR-494 and increased expression of miR-126–5p. Next, we analysed the canonical Smad and alternative signalling pathways, through which BMP4 would regulate miR-126–5p and miR-494 expression. Furthermore, the functional effect of miR-494 and miR-126–5p on endothelial cells was investigated. MicroRNA-494 overexpression decreased endothelial cell proliferation, migration and sprout formation. Consistently, miR-494 inhibition increased endothelial cell function. As potential miR-494 targets, bFGF and BMP endothelial cell precursorderived regulator (BMPER) were identified and confirmed by western blot. Luciferase assays showed direct miR-494 binding in BMPER 3’UTR. In contrast, miR-126–5p overexpression increased pro-angiogenic endothelial cell behaviour and, accordingly, miR-126–5p inhibition decreased endothelial cell function. As a direct miR-126–5p target we identified the anti-angiogenic thrombospondin-1 which was confirmed by western blot analysis and luciferase assays. In the Matrigel plug assay application of antagomiR-494 increased endothelial cell ingrowth, whereas antagomiR-126–5p treatment decreased cell ingrowth
in vivo
. Taken together, through differential regulation of the anti-angiomiR-494 and the angiomiR-126–5p by BMP4 both microRNAs contribute to the pro-angiogenic BMP4 effect on endothelial cells.
Supplementary Material to this article is available online at www.thrombosis-online.com.
BACKGROUND—Adaptive neovascularization after arterial occlusion is an important compensatory mechanism in cardiovascular disease and includes both the remodeling of pre-existing vessels to collateral ...arteries (arteriogenesis) and angiogenic capillary growth. We now aimed to identify regulatory microRNAs involved in the modulation of neovascularization after femoral artery occlusion in mice.
METHODS AND RESULTS—Using microRNA-transcriptome analysis, we identified miR-155 as a downregulated microRNA during hindlimb ischemia. Correspondingly, inhibition of miR-155 in endothelial cells had a stimulatory effect on proliferation and angiogenic tube formation via derepression of its direct target gene angiotensin II type 1 receptor. Surprisingly, miR-155–deficient mice showed an unexpected phenotype in vivo, with a strong reduction of blood flow recovery after femoral artery ligation (arteriogenesis) dependent on the attenuation of leukocyte-endothelial interaction and a reduction of proarteriogenic cytokine expression. Consistently, miR-155–deficient macrophages exhibit a specific alteration of the proarteriogenic cytokine expression profile, which is partly mediated by the direct miR-155 target gene SOCS-1.
CONCLUSIONS—Our data demonstrate that miR-155 exerts an antiangiogenic but proarteriogenic function in the regulation of neovascularization via the suppression of divergent cell-specific target genes and that its expression in both endothelial and bone marrow–derived cells is essential for arteriogenesis in response to hindlimb ischemia in mice.
The mineralocorticoid aldosterone is a key regulator of water and electrolyte homeostasis. Numerous recent developments have advanced the field of mineralocorticoid pharmacology-namely, clinical ...trials have shown the beneficial effects of aldosterone antagonists in chronic heart failure and post-myocardial infarction treatment. Experimental studies using cell type-specific gene targeting of the mineralocorticoid receptor (MR) gene in mice have revealed the importance of extrarenal aldosterone signaling in cardiac myocytes, endothelial cells, vascular smooth cells, and macrophages. In addition, several molecular pathways involving signal transduction via the classical MR as well as the G protein-coupled receptor GPER mediate the diverse spectrum of effects of aldosterone on cells. This knowledge has initiated the development of new pharmacological ligands to specifically interfere with targets on different levels of aldosterone signaling. For example, aldosterone synthase inhibitors such as LCI699 and the novel nonsteroidal MR antagonist BAY 94-8862 have been tested in clinical trials. Interference with the interaction between MR and its coregulators seems to be a promising strategy toward the development of selective MR modulators.
The adaptive growth of blood vessels is an important protective mechanism in cardiovascular disease. However, the underlying regulatory mechanisms of this process are only partly understood. ...Recently, small endogenous RNAs (microRNAs miRNAs) were found to play an important role in embryonic and postnatal vascular development. Here, we used miRNA transcriptome analysis after induction of hind-limb ischemia in mice to screen for miRNAs involved in adaptive blood vessel growth following arterial occlusion.
Using miRNA arrays, we explored the miRNA expression profile during adaptive neovascularization. We describe specific changes in miRNA expression patterns and show that miRNA-100 is significantly downregulated after induction of hind-limb ischemia in mice. Our data demonstrate that miR-100 modulates proliferation, tube formation, and sprouting activity of endothelial cells and migration of vascular smooth muscle cells and functions as an endogenous repressor of the serine/threonine protein kinase mammalian target of rapamycin (mTOR). Whereas miR-100 inhibition increased mTOR levels in endothelial cells, overexpression of miR-100 reduced mTOR expression and consequently attenuated cellular proliferation. Supporting this notion, overexpression of an mTOR construct lacking the miRNA binding site rescued the inhibitory effect of miR-100 on cell proliferation. Accordingly, miR-100 inhibition by specific antagomirs in vivo stimulated angiogenesis and resulted in functional improvement of perfusion after femoral artery occlusion in mice. In contrast, treatment with the mTOR inhibitor rapamycin had the opposite effect.
Our data demonstrate that miR-100 has an antiangiogenic function and represses mTOR signaling in endothelial and vascular smooth muscle cells. Inhibition of miR-100 could be a novel approach for the modulation of blood vessel growth and other mTOR-dependent processes.
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.
Abstract Background The prognosis of immediate survivors of cardiac arrest remains poor, as the majority of these patients develops an inflammatory disorder known as the post-cardiac arrest syndrome ...(PCAS). Recently, the endothelial glycocalyx has been shown to be a key modulator of vascular permeability and inflammation, but its role in PCAS remains unknown. Methods Plasma levels of the glycocalyx components syndecan-1, heparan sulfate and hyaluronic acid were measured in 25 patients after immediate survival of cardiac arrest during different phases of PCAS. Twelve hemodynamically stable patients with acute coronary syndrome served as controls. Results Cardiac arrest resulted in a significant increase in syndecan-1, heparan sulfate and hyaluronic acid levels compared to controls, indicating a shedding of the endothelial glycocalyx as a pathophysiological component of the post cardiac arrest syndrome. The time course differed between the individual glycocalyx components, with a higher increase of syndecan-1 in the early phase of PCAS (2.8-fold increase vs. controls) and a later peak of heparan sulfate (1.7-fold increase) and hyaluronic acid (2-fold increase) in the intermediate phase. Only the plasma levels of syndecan-1 correlated positively with the duration of CPR and negatively with the glycocalyx-protective protease inhibitor antithrombin III. Plasma levels of both syndecan-1 and heparan sulfate were higher in eventual non-survivors than in survivors of cardiac arrest. Conclusion Our data for the first time demonstrates a perturbation of the endothelial glycocalyx in immediate survivors of cardiac arrest and indicate a potential important role of this endothelial surface layer in the development of post-cardiac arrest syndrome.
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