Primary Human Coronary Endothelial Cells Research Articles

Atherosclerotic Plaque Epigenetic Age Acceleration Predicts a Poor Prognosis and Is Associated With Endothelial-to-Mesenchymal Transition in Humans.

Epigenetic age estimators (clocks) are predictive of human mortality risk. However, it is not yet known whether the epigenetic age of atherosclerotic plaques is predictive for the risk of cardiovascular events. Whole-genome DNA methylation of human carotid atherosclerotic plaques (n=485) and of blood (n=93) from the Athero-Express endarterectomy cohort was used to calculate epigenetic age acceleration (EAA). EAA was linked to clinical characteristics, plaque histology, and future cardiovascular events (n=136). We studied whole-genome DNA methylation and bulk and single-cell transcriptomics to uncover molecular mechanisms of plaque EAA. We experimentally confirmed our in silico findings using in vitro experiments in primary human coronary endothelial cells. Male and female patients with severe atherosclerosis had a median chronological age of 69 years. The median epigenetic age was 65 years in females (median EAA, -2.2 [interquartile range, -4.3 to 2.2] years) and 68 years in males (median EAA, -0.3 [interquartile range, -2.9 to 3.8] years). Patients with diabetes and a high body mass index had higher plaque EAA. Increased EAA of plaque predicted future events in a 3-year follow-up in a Cox regression model (univariate hazard ratio, 1.7; P=0.0034) and adjusted multivariate model (hazard ratio, 1.56; P=0.02). Plaque EAA predicted outcome independent of blood EAA (hazard ratio, 1.3; P=0.018) and of plaque hemorrhage (hazard ratio, 1.7; P=0.02). Single-cell RNA sequencing in plaque samples from 46 patients in the same cohort revealed smooth muscle and endothelial cells as important cell types in plaque EAA. Endothelial-to-mesenchymal transition was associated with EAA, which was experimentally confirmed by TGFβ-triggered endothelial-to-mesenchymal transition inducing rapid epigenetic aging in coronary endothelial cells. Plaque EAA is a strong and independent marker of poor outcome in patients with severe atherosclerosis. Plaque EAA was linked to mesenchymal endothelial and smooth muscle cells. Endothelial-to-mesenchymal transition was associated with EAA, which was experimentally validated. Epigenetic aging mechanisms may provide new targets for treatments that reduce atherosclerosis complications.

The Effects of Sirolimus and Magnesium on Primary Human Coronary Endothelial Cells: An In Vitro Study.

Drug eluting magnesium (Mg) bioresorbable scaffolds represent a novel paradigm in percutaneous coronary intervention because Mg-based alloys are biocompatible, have adequate mechanical properties and can be resorbed without adverse events. Importantly, Mg is fundamental in many biological processes, mitigates the inflammatory response and is beneficial for the endothelium. Sirolimus is widely used as an antiproliferative agent in drug eluting stents to inhibit the proliferation of smooth muscle cells, thus reducing the occurrence of stent restenosis. Little is known about the potential interplay between sirolimus and Mg in cultured human coronary artery endothelial cells (hCAEC). Therefore, the cells were treated with sirolimus in the presence of different concentrations of extracellular Mg. Cell viability, migration, barrier function, adhesivity and nitric oxide synthesis were assessed. Sirolimus impairs the viability of subconfluent, but not of confluent cells independently from the concentration of Mg in the culture medium. In confluent cells, sirolimus inhibits migration, while it cooperates with Mg in exerting an anti-inflammatory action that might have a role in preventing restenosis and thrombosis.

Low Effectiveness of the Introduction of pmaxGFP into Primary Human Coronary Endothelial Cells Using Cell-Penetrating Peptides and Nuclear-Localization Sequences in Non-Covalent Interactions

Cell-penetrating peptides (CPPs), due to their effectiveness and low cytotoxicity, are of increasing interest in the context of the transport of macromolecules to the cells. The simplest and safest method seems to be the non-covalent binding of CPP and cargo molecules. However, it requires the optimization of the reaction conditions. The study aimed to determine the effectiveness and cytotoxicity of the Pep-1, KALA, and TAT proteins as well as the NLS [47–55] and NLS [47–56] sequences derived from the Simian Vacuolating 40 (SV40) T-antigen in the context of the transport of the pmaxGFP plasmid to primary human coronary artery endothelial cells. The results are presented in the form of extensive photographic documentation, which shows significant differences in the efficiency of the transfection process between electroporation and the use of CPPs. The study presents negative results in which, despite the manipulation of various parameters (incubation time, incubation temperature, culture time, charge ratio, plasmid concentration), results similar to electroporation were not obtained.

Abstract 90: Loss of Collectrin Augments Salt-sensitivity and Alters Nitric Oxide and Superoxide Balance.

Collectrin, an ACE-2 homologue, plays a major role in amino acid transport in renal epithelial cells. In the rodent kidney, the expression of collectrin is upregulated after reduction of nephron mass and during salt-sensitive hypertension. These observations raise the question of how collectrin might be functionally integrated in the ACE family in the context of blood pressure (BP) regulation. We previously reported that, on a susceptible genetic background, collectrin-deficient (KO) mice have elevated baseline systolic blood pressure (SBP) and impaired endothelium-dependent relaxation (EDR) of thoracodorsal arteries (TDA). Treatment with L-NAME, a nitric oxide synthase (NOS) inhibitor, normalized their BP and EDR of TDA. Here, to determine whether collectrin plays a role in salt-sensitivity (SS), we fed mice a high salt diet (HSD) for 2 weeks. By radiotelemetry, KO mice displayed augmented salt-sensitivity (SS) (Normal salt (NS): Wild-type (WT) 133 ± 1 mm Hg, KO 141 ± 3 mm Hg; HSD: WT 141 ± 2 mm Hg, KO 155 ± 3 mm Hg; change in SBP: WT 7.8 ± 1.4 mm Hg, KO 14.4 ± 1.5 mm Hg; p ≤ 0.02), and lower urinary sodium excretion (UNa) during the first 3 days of HSD (mean UNa over 3 days: KO: 1980 ± 193 μmol/24h, WT: 2650 ± 98 μmol/24h, p = 0.006, ANOVA; n ≥ 6). The impaired pressure natriuresis in KO mice was associated with altered urinary excretion of 8-isoprostane F 2 α (WT 3.0 ± 0.67 ng/mg, KO 7.7 ± 1.5 ng/mg creatinine, p = 0.008) and nitrates/nitrites (WT 10.2 ± 1.4 nmol/day, KO 6.2 ± 0.61 nmol/day, p = 0.03, n ≥ 4 each). We previously reported that KO mice have increased state of endothelial NOS (eNOS) uncoupling. Here, by immunostaining, we found that collectrin is expressed in endothelial cells (ECs). We next queried whether collectrin affects L-arginine (L-Arg) transport in ECs. After isolation from the pulmonary artery, primary ECs from KO mice displayed impaired [ 3 H]L-Arg uptake (WT 2.00 ± 0.19, KO 0.90 ± 0.06 pmol/mg protein/min, p=0.01) To establish direct cause and effect, we over-expressed collectrin in primary human coronary ECs which resulted in increased [ 3 H]L-Arg uptake (Control 4.20 ± 0.32; Overexpression 6.60 ± 0.59 pmol/mg protein/min, p < 0.003). In conclusion, collectrin may regulate BP and SS by modulating the balance of NO and SO through its role in L-Arg transport in ECs.

Verapamil: metabolism in cultures of primary human coronary arterial endothelial cells.

Endothelium is a metabolically active secretory tissue and an important barrier for metabolic products. Little is known about its contribution to drug oxidation. We investigated the gene and protein expression and enzyme activity of major cytochrome P450 monooxygenases in cultures of primary human coronary endothelial cells and studied its ability to metabolize verapamil, a commonly and widely prescribed calcium antagonist. Of the total 18 P450 monooxygenases investigated, transcripts for CYP1A1, CYP2A6/7, CYP2A13, CYP2B6/7, CYP2C8, CYP2E1, and CYP2J2 were expressed, albeit at different levels. Furthermore, metabolism of verapamil proceeded predominantly via N-desmethylation and/or N-desalkylation, i.e., production of D-617 [2-(3,4-dimethoxyphenyl)-5-amino-2-isopropylvaleronitrile], D-620 [2-(3,4-dimethoxyphenyl)-5-methylamino-2-isopropylvaleronitrile], and norverapamil; but additional metabolites are the O-demethylated products, D-702 [2-(3,4-dimethoxyphenyl)-8-(4-hydroxy-3-methoxyphenyl)-6-methyl-2-isopropyl-6-azaoctanitrile] and D-703 [O-demethylverapamil; 5-N-(3,4-dimethoxyphenethyl)methylamino-2-(3'-methoxy-4'-hydroxyphenyl)-2-isopropylvaleronitrile]. We show endothelium to express an array of monooxygenases, and in view of its large body distribution, endothelium should be considered in the biotransformation of drugs, particularly when tissue-specific metabolism and/or metabolic inactivation are being investigated.