BACKGROUND:The angiogenic function of endothelial cells is regulated by numerous mechanisms, but the impact of long noncoding RNAs (lncRNAs) has hardly been studied. We set out to identify novel and ...functionally important endothelial lncRNAs.
METHODS:Epigenetically controlled lncRNAs in human umbilical vein endothelial cells were searched by exon-array analysis after knockdown of the histone demethylase JARID1B. Molecular mechanisms were investigated by RNA pulldown and immunoprecipitation, mass spectrometry, microarray, several knockdown approaches, CRISPR-Cas9, assay for transposase-accessible chromatin sequencing, and chromatin immunoprecipitation in human umbilical vein endothelial cells. Patient samples from lung and tumors were studied for MANTIS expression.
RESULTS:A search for epigenetically controlled endothelial lncRNAs yielded lncRNA n342419, here termed MANTIS, as the most strongly regulated lncRNA. Controlled by the histone demethylase JARID1B, MANTIS was downregulated in patients with idiopathic pulmonary arterial hypertension and in rats treated with monocrotaline, whereas it was upregulated in carotid arteries of Macaca fascicularis subjected to atherosclerosis regression diet, and in endothelial cells isolated from human glioblastoma patients. CRISPR/Cas9-mediated deletion or silencing of MANTIS with small interfering RNAs or GapmeRs inhibited angiogenic sprouting and alignment of endothelial cells in response to shear stress. Mechanistically, the nuclear-localized MANTIS lncRNA interacted with BRG1, the catalytic subunit of the switch/sucrose nonfermentable chromatin-remodeling complex. This interaction was required for nucleosome remodeling by keeping the ATPase function of BRG1 active. Thereby, the transcription of key endothelial genes such as SOX18, SMAD6, and COUP-TFII was regulated by ensuring efficient RNA polymerase II machinery binding.
CONCLUSION:MANTIS is a differentially regulated novel lncRNA facilitating endothelial angiogenic function.
Oxidized phospholipids (oxPAPC) induce endothelial dysfunction and atherosclerosis. Here we show that oxPAPC induce a gene network regulating serine-glycine metabolism with the mitochondrial ...methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2) as a causal regulator using integrative network modeling and Bayesian network analysis in human aortic endothelial cells. The cluster is activated in human plaque material and by atherogenic lipoproteins isolated from plasma of patients with coronary artery disease (CAD). Single nucleotide polymorphisms (SNPs) within the MTHFD2-controlled cluster associate with CAD. The MTHFD2-controlled cluster redirects metabolism to glycine synthesis to replenish purine nucleotides. Since endothelial cells secrete purines in response to oxPAPC, the MTHFD2-controlled response maintains endothelial ATP. Accordingly, MTHFD2-dependent glycine synthesis is a prerequisite for angiogenesis. Thus, we propose that endothelial cells undergo MTHFD2-mediated reprogramming toward serine-glycine and mitochondrial one-carbon metabolism to compensate for the loss of ATP in response to oxPAPC during atherosclerosis.
Sphingosine-1-Phosphate (S1P) is a potent signaling lipid. The effects of S1P are mediated by the five S1P receptors (S1PR). In the endothelium S1PR1 is the predominant receptor and thus S1PR1 ...abundance limits S1P signaling. Recently, lncRNAs were identified as a novel class of molecules regulating gene expression. Interestingly, the lncRNA NONHSAT004848 (LISPR1, Long intergenic noncoding RNA antisense to S1PR1), is closely positioned to the S1P1 receptors gene and in part shares its promoter region. We hypothesize that LISPR1 controls endothelial S1PR1 expression and thus S1P-induced signaling in endothelial cells. In vitro transcription and translation as well as coding potential assessment showed that LISPR1 is indeed noncoding. LISPR1 was localized in both cytoplasm and nucleus and harbored a PolyA tail at the 3’end. In human umbilical vein endothelial cells, as well as human lung tissue, qRT-PCR and RNA-Seq revealed high expression of LISPR1. S1PR1 and LISPR1 were downregulated in human pulmonary diseases such as COPD. LISPR1 but also S1PR1 were induced by inflammation, shear stress and statins. Knockdown of LISPR1 attenuated endothelial S1P-induced migration and spheroid outgrowth of endothelial cells. LISPR1 knockdown decreased S1PR1 expression, which was paralleled by an increase of the binding of the transcriptional repressor ZNF354C to the S1PR1 promoter and a reduction of the recruitment of RNA Polymerase II to the S1PR1 5’end. This resulted in attenuated S1PR1 expression and attenuated S1P downstream signaling. Collectively, the disease relevant lncRNA LISPR1 acts as a novel regulatory unit important for S1PR1 expression and endothelial cell function.
•LISPR1 and S1PR1 are highly expressed in endothelial cells and reduced in COPD.•LISPR1 is important for migration and sprouting of endothelial cells.•S1PR1 gene expression requires LISPR1 for RNA Pol II recruitment to S1PR1 5’UTR.•LISPR1 may block transcriptional repressor ZNF354C binding to S1PR1 promoter.•LISPR1 controls S1P targets ICAM1, PECAM1 and ANGPT2 through S1PR1.
OBJECTIVE—Altering endothelial biology through epigenetic modifiers is an attractive novel concept, which is, however, just in its beginnings. We therefore set out to identify chromatin modifiers ...important for endothelial gene expression and contributing to angiogenesis.
APPROACH AND RESULTS—To identify chromatin modifying enzymes in endothelial cells, histone demethylases were screened by microarray and polymerase chain reaction. The histone 3 lysine 4 demethylase JARID1B was identified as a highly expressed enzyme at the mRNA and protein levels. Knockdown of JARID1B by shRNA in human umbilical vein endothelial cells attenuated cell migration, angiogenic sprouting, and tube formation. Similarly, pharmacological inhibition and overexpression of a catalytic inactive JARID1B mutant reduced the angiogenic capacity of human umbilical vein endothelial cells. To identify the in vivo relevance of JARID1B in the vascular system, Jarid1b knockout mice were studied. As global knockout results in increased mortality and developmental defects, tamoxifen-inducible and endothelial-specific knockout mice were generated. Acute knockout of Jarid1b attenuated retinal angiogenesis and endothelial sprout outgrowth from aortic segments. To identify the underlying mechanism, a microarray experiment was performed, which led to the identification of the antiangiogenic transcription factor HOXA5 to be suppressed by JARID1B. Importantly, downregulation or inhibition of JARID1B, but not of JARID1A and JARID1C, induced HOXA5 expression in human umbilical vein endothelial cells. Consistently, chromatin immunoprecipitation revealed that JARID1B occupies and reduces the histone 3 lysine 4 methylation levels at the HOXA5 promoter, demonstrating a direct function of JARID1B in endothelial HOXA5 gene regulation.
CONCLUSIONS—JARID1B, by suppressing HOXA5, maintains the endothelial angiogenic capacity in a demethylase-dependent manner.
OBJECTIVE—Reactive oxygen species generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases contribute to angiogenesis and vascular repair. NADPH oxidase organizer 1 (NoxO1) is a ...cytosolic protein facilitating assembly of constitutively active NADPH oxidases. We speculate that NoxO1 also contributes to basal reactive oxygen species formation in the vascular system and thus modulates angiogenesis.
APPROACH AND RESULTS—A NoxO1 knockout mouse was generated, and angiogenesis was studied in cultured cells and in vivo. Angiogenesis of the developing retina and after femoral artery ligation was increased in NoxO1 when compared with wild-type animals. Spheroid outgrowth assays revealed greater angiogenic capacity of NoxO1 lung endothelial cells (LECs) and a more tip-cell–like phenotype than wild-type LECs. Usually signaling by the Notch pathway switches endothelial cells from a tip into a stalk cell phenotype. NoxO1 LECs exhibited attenuated Notch signaling as a consequence of an attenuated release of the Notch intracellular domain on ligand stimulation. This release is mediated by proteolytic cleavage involving the α-secretase ADAM17. For maximal activity, ADAM17 has to be oxidized, and overexpression of NoxO1 promoted this mode of activation. Moreover, the activity of ADAM17 was reduced in NoxO1 LECs when compared with wild-type LECs.
CONCLUSIONS—NoxO1 stimulates α-secretase activity probably through reactive oxygen species–mediated oxidation. Deletion of NoxO1 attenuates Notch signaling and thereby promotes a tip-cell phenotype that results in increased angiogenesis.
We have shown previously that during myocardial ischemia/reperfusion (MI/R), toll-like receptor 2 (TLR2) signaling regulates connexin 43 (Cx43) subcellular localization and function and dampens ...arrhythmia formation. We aimed to identify sites capable of TLR2-dependent redox modification within Cx43. Post-ischemic TLR2
−/−
or wild-type (WT) mouse hearts were analyzed by OxICAT. Cx43 was mutated to exclude redox modification and transfected into HL-1 cardiomyocytes (CM) that were challenged with a TLR2 agonist. We identified Cys260 of Cx43 to be susceptible to reversible oxidation MI/R; TLR2
−/−
leads to reduced H
2
O
2
production in post-ischemic isolated mitochondria and subsequently reduced oxidation of Cx43 at Cys260. Cx43 was dephosphorylated in WT, while phosphorylation was preserved in TLR2
−/−
. Mutation of Cx43 (C260A) and lentiviral transfection in HL-1 CM accelerated pacemaker activity and reduced activity after TLR2 ligand stimulation. We here provide evidence for TLR2-dependent reversible oxidation of Cx43 at Cys260, which led to decreased Cx43 phosphorylation and affected CM pacemaker frequency and intercellular communication.
Objective: Stress is known to contribute to other well-known environmental risk factors to hypertension leading to sympathetic overactivity. While usual environmental factors can be easily measured, ...exposure to stress is difficult to objectivize, especially during catastrophic events which are recognized as main stressors. There are several reports about earthquakes, blood pressure (BP) and cardiovascular events. Design and method: We are reporting a case on ambulatory blood pressure monitoring (ABPM) in 42-year-old women during Croatian earthquake. Results: A 42-year-old women with multiple genetic and environmental risk factors for hypertension was evaluated for secondary hypertension, and adult autosomal polycystic kidney disease was diagnosed. Kidney function was normal and there were no signs of target organ damage. She was treated with a fixed dose combination perindopril/amlodipine 4/5 mg (morning). ABPM revealed mildly uncontrolled systolic-diastolic hypertension with extreme nocturnal dip. Average 24-hour, daytime and nighttime BP values were 130±20,4/79±19,9 mmHg, 137±16,2 /85±17,9 mmHg, 100±11,8 /55±11,7 mmHg, respectively. White coat effect was present, and masked hypertension was ruled out. Average 24-hour, daytime and nighttime heart rate were: 88b/min, 93b/min, and 79b/min. A significant surge in BP and heart rate (200/130 mmHg,140 b/min) was recorded at 12:30 p.m. exactly at the time when earthquake occurred in city Petrinja, also felt in Zagreb city. The earthquake was clearly stated in the blood pressure diary (Figure 1). Conclusions: Although chronic stress is emphasized to affect human health more than acute, catastrophic events such as earthquakes can have deleterious effect such as enormous elevation of BP. It is necessary to have strategies for BP management in disaster-related diseases to prevent organ damage.
Objective:
The significance of multiple renal arteries (MRAs) in arterial hypertension (AH) is not fully understood. Lower perfusion pressure due to longer length and narrower caliber of MRAs could ...cause segmental hyperreninemia sufficient to result in renin-dependent systemic hypertension. Sympathetic renal nerve activity might be affected by hyperreninema as well.
The aim of the study was to examine the characteristics of young adult hypertensives in relation to the existence of MRAs that were diagnosed during evaluation for secondary arterial hypertension.
Design and method:
32 patients underwent routine evaluation for secondary hypertension and target organ damage. Patients were divided into two groups, one with MRAs and the other without MRAs. Data were presented as mean +standard deviation and compared by the Indipendent-Sample t-test.
Results:
The mean age was 38 years, and 13 patients were diagnosed with MRAs. There were more men in the MRAs group, otherwise, there were no significant differences in baseline characteristics between the groups (BMI, smoking, family history). None of them were diagnosed with secondary endocrine hypertension or renal artery stenosis. MRAs group showed significantly higher values of office SBP/DBP (p < 0,012) and 24-hour SBP/DBP (p < 0,001), and nonsignificantly higher values of heart rate. Although nonsignificant, direct renin concentrations were slightly higher in the MRAs group, while insulin levels were higher in the non-MRAs group. Dyslipidemia was diagnosed in most patients, without significant differences between groups.
Electrocardiographic and echocardiographic changes suggesting hypertensive heart remodeling were found in 55% of patients in MRAs compared to 32% in non-MRAs, there were no significant differences in NTproBNP levels. A nonsignificant reduction in glomerular filtration rate was noticed in the MRAs group, while there were no significant differences in 24-hour albuminuria (p 0,308; p 0,614).
Conclusions:
The significance of MRAs in arterial hypertension is a matter of debate. In our study of young patients with arterial hypertension and MRAs, we observed features of more severe hypertension with already existing hypertensive target organ damages. Proper medical treatment with antihypertensive agents is crucial. Future studies will answer the question about interventional therapeutic procedures’ role in preventing cardiovascular complications.
We aim to determine the relationship between bone mineral density (BMD), measured by
T-
and
Z
–score, and mortality risk in hemodialysis (HD) patients. We also investigate which are the most suitable ...skeletal sites for predicting mortality rate. We analyzed the survival of 102 patients who had been treated with chronic HD according to BMD. Patients with a
T
-score ≤2.5 at the middle, ultradistal and proximal part of the forearm had a higher mortality risk than those with a
T
-score of −2.5 or higher. Furthermore, no statistically significant association was found between loss of bone mass at other measuring points—lumbar spine (anteroposterior orientation from L1–L4) and hip (neck, trochanter, intertrochanter, total and Ward’s triangle)—and mortality risk. We were also interested in exploring the relationship between
Z
-score at different skeletal regions and mortality risk. We found that patients with a
Z
-score of −1 or lower at all three parts of the forearm had a greater mortality risk. It is also worth noting that the
Z
-score at all three parts of the forearm was a more apparent predictor of mortality, compared to the
T
-score at the same skeletal regions. This empirical analysis showed that BMD assessments should be obtained at the forearm, due to the good predictability of this skeletal site regarding mortality of HD patients. Moreover, data concerning bone density should be reported as
Z-
scores.
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