Ferrostatin-1 Reversed Chronic Intermittent Hypoxia-Induced Ferroptosis in Aortic Endothelial Cells via Reprogramming Mitochondrial Function.

Purpose: Chronic intermittent hypoxia (CIH) related arterial endothelium injury is a common cause of cardiovascular system injury. However, the mechanism still needs to be clarified. In this study, we aimed to clarify the role and mechanism of ferrostatin-1 (Fer-1) in CIH-related rat arterial endothelial cells (ROAEC) ferroptosis. Methods: ROAEC was divided into control group, CIH group, and CIH+ Fer-1 group. Cell viability was detected by cell counting kit 8 kits (CCK8). The apoptotic rate, reactive oxygen species (ROS) levels, Fe2+ levels, and lipid ROS levels were detected by flow cytometry. Malondialdehyde (MDA) levels and nicotinamide adenine dinucleotide (NAD+)/NADH ratio were detected via Elisa kits. The mRNA and protein levels of cystine/glutamate antiporter solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) were detected by qRT-PCR and Western blot. Mitochondrial structure and function were observed by transmission electron microscope (TEM) and mitochondrial membrane potential (MMP). Central carbon metabolism was measured to compare metabolites among each group. Results: After the CIH exposure, ROAEC cell viability decreased; The levels of cell apoptosis, ROS, Fe2+, MDA, and lip ROS increased; The levels of NAD+/NADP ratio decreased; The mRNA and protein levels of GPX4 and SLC7A11 decreased (all p<0.05). Co-cultured with Fer-1 reversed the levels of apoptosis rate, cell viability, ROS, Fe2+, MAD, lipid ROS, NAD+/NADH ratio and the mRNA and protein expression of GPX4 and SLC7A11 (all p<0.05). The TEM results showed that damaged mitochondrial membrane and the matrix spillover in the CIH group. The results of the JC-1 assay showed decreased MMP in the CIH group. Fer-1 treatment ameliorated the mitochondrial injury. The results of central carbon metabolism found that CIH altered the metabolites in the TCA cycle, which were reversed by Fer-1 treatment. Conclusion: CIH-induced ferroptosis in ROAEC, which were reversed by Fer-1 via reprogramming mitochondrial function.

High-efficient serum-free differentiation of endothelial cells from human iPS cells.

INTRODUCTION: Endothelial cells (ECs) form the inner lining of all blood vessels of the body play important roles in vascular tone regulation, hormone secretion, anticoagulation, regulation of blood cell adhesion and immune cell extravasation. Limitless ECs sources are required to further in vitro investigations of ECs' physiology and pathophysiology as well as for tissue engineering approaches. Ideally, the differentiation protocol avoids animal-derived components such as fetal serum and yields ECs at efficiencies that make further sorting obsolete for most applications. METHOD: Human induced pluripotent stem cells (hiPSCs) are cultured under serum-free conditions and induced into mesodermal progenitor cells via stimulation of Wnt signaling for 24 h. Mesodermal progenitor cells are further differentiated into ECs by utilizing a combination of human vascular endothelial growth factor A165 (VEGF), basic fibroblast growth factor (bFGF), 8-Bromoadenosine 3',5'-cyclic monophosphate sodium salt monohydrate (8Bro) and melatonin (Mel) for 48 h. RESULT: This combination generates hiPSC derived ECs (hiPSC-ECs) at a fraction of 90.9 ± 1.5% and is easily transferable from the two-dimensional (2D) monolayer into three-dimensional (3D) scalable bioreactor suspension cultures. hiPSC-ECs are positive for CD31, VE-Cadherin, von Willebrand factor and CD34. Furthermore, the majority of hiPSC-ECs express the vascular endothelial marker CD184 (CXCR4). CONCLUSION: The differentiation method presented here generates hiPSC-ECs in only 6 days, without addition of animal sera and at high efficiency, hence providing a scalable source of hiPSC-ECs.

Targeted Delivery of Salusin-α Into Rabbit Carotid Arterial Endothelium Using SonoVue.

OBJECTIVES: A new method based on the adhesion of SonoVue to plasmids was assessed to achieve targeted gene delivery into the vascular endothelium. METHODS: pEGFP-Salusin-α and pcDNA3.1-Salusin-α plasmids were transfected into the arterial endothelium of different rabbit groups. Western blotting was performed to analyze the expression of EGFP and salusin-α in the common carotid arteries of rabbits from different groups, and ELISA was performed to detect plasma salusin-α levels in rabbits from each group; simultaneously, blood parameters of different groups of rabbits were measured. RESULTS: Green fluorescence was observed in the right common carotid artery of rabbits transfected with pEGFP-Salusin-α, but not in the endothelial cells of not-transfected control rabbits. The expression of salusin-α in the transfected animals was higher than that in the control not-transfected animals (P < .05). In rabbits transfected with pcDNA3.1-Salusin-α plasmid, salusin-α expression was higher than in the not-transfected control animals (P < .05). However, there was no significant difference in plasma salusin-α levels between transfected animals and controls (P > .05). Blood parameters were also measured in both groups. CONCLUSIONS: Our data confirm the establishment of a new method using SonoVue for targeted gene delivery into the arterial endothelium. Our study outcomes propose a new method of intervention in atherosclerosis and a new tool for targeted gene delivery.

Tribbles Homolog 3-Mediated Vascular Insulin Resistance Contributes to Hypoxic Pulmonary Hypertension in Intermittent Hypoxia Rat Model.

This study aimed to investigate the role of vascular insulin resistance (VIR) and Tribbles homolog 3 (TRIB3) in the pathogenesis of hypoxia-induced pulmonary hypertension (HPH). Rats were subjected to low air pressure and low oxygen intermittently for 4 weeks to induce HPH. The mean right ventricular pressure (mRVP), mean pulmonary arterial pressure (mPAP), and right ventricular index (RVI) were significantly increased in HPH rats. Pulmonary arteries from HPH rats showed VIR with reduced vasodilating effect of insulin. The protein levels of peroxisome proliferator-activated receptor gamma (PPARγ), phosphoinositide 3-kinase (PI3K), phosphorylations of Akt, and endothelial nitric oxide (NO) synthase (eNOS) were decreased, and TRIB3 and phosphorylated extracellular signal-regulated protein kinases (ERK1/2) were increased in pulmonary arteries of HPH rats. Early treatment of pioglitazone (PIO) partially reversed the development of HPH, improved insulin-induced vasodilation, and alleviated the imbalance of the insulin signaling. The overexpression of TRIB3 in rat pulmonary arterial endothelial cells (PAECs) reduced the levels of PPARγ, PI3K, phosphorylated Akt (p-Akt), and phosphorylated eNOS (p-eNOS) and increased p-ERK1/2 and the synthesis of endothelin-1 (ET-1), which were further intensified under hypoxic conditions. Moreover, TRIB3 knockdown caused significant improvement in Akt and eNOS phosphorylations and, otherwise, a reduction of ERK1/2 activation in PAECs after hypoxia. In conclusion, impaired insulin-induced pulmonary vasodilation and the imbalance of insulin-induced signaling mediated by TRIB3 upregulation in the endothelium contribute to the development of HPH. Early PIO treatment improves vascular insulin sensitivity that may help to limit the progression of hypoxic pulmonary hypertension.

Sphingosine 1-phosphate-regulated transcriptomes in heterogenous arterial and lymphatic endothelium of the aorta.

Despite the medical importance of G protein-coupled receptors (GPCRs), in vivo cellular heterogeneity of GPCR signaling and downstream transcriptional responses are not understood. We report the comprehensive characterization of transcriptomes (bulk and single-cell) and chromatin domains regulated by sphingosine 1-phosphate receptor-1 (S1PR1) in adult mouse aortic endothelial cells. First, S1PR1 regulates NFκB and nuclear glucocorticoid receptor pathways to suppress inflammation-related mRNAs. Second, S1PR1 signaling in the heterogenous endothelial cell (EC) subtypes occurs at spatially-distinct areas of the aorta. For example, a transcriptomically distinct arterial EC population at vascular branch points (aEC1) exhibits ligand-independent S1PR1/ß-arrestin coupling. In contrast, circulatory S1P-dependent S1PR1/ß-arrestin coupling was observed in non-branch point aEC2 cells that exhibit an inflammatory gene expression signature. Moreover, S1P/S1PR1 signaling regulates the expression of lymphangiogenic and inflammation-related transcripts in an adventitial lymphatic EC (LEC) population in a ligand-dependent manner. These insights add resolution to existing concepts of endothelial heterogeneity, GPCR signaling and S1P biology.

Different Effect of Hemodialysis on Function of Human Arterial and Venous Endothelial Cells.

BACKGROUND/AIMS: Hemodialysis causes the systemic inflammatory response, which may affect the function of endothelial cells. METHODS: We studied the effect of the serum obtained after a hemodialysis session, compared to serum collected before the start of the treatment, on the gene expression and secretory activity of arterial endothelial cells (AECs) and venous endothelial cells (VECs) in in vitro culture. RESULTS: Serum collected at the end of the hemodialysis session increased expression of the studied genes in VECs, and at the same time decreased their expression in AECs. Secretory activity was increased in VEC: (interleukin-6 [IL-6] +29%, p < 0.05, von Willebrand factor +23%, p < 0.02; tissue plasminogen activator [t-PA] +35%, p < 0.002, t-PA/plasminogen activator inhibitor-1 [PAI-1] ratio + 57%, p < 0.005). In AEC, synthesis of IL-6 and vascular endothelial growth factor were reduced (-36%, p < 0.02, -34%, p < 0.05, respectively) and the tPA/PAI-1 ratio was increased (+22%, p < 0.01). CONCLUSIONS: Hemodialysis induces the inflammatory, procoagulant, and profibrinolytic activity of VEC, whereas suppression of AEC is observed at the same time. Video Journal Club 'Cappuccino with Claudio Ronco' at https://www.karger.com/Journal/ArticleNews/223997?sponsor=52.

Differential effects of short chain fatty acids on endothelial Nlrp3 inflammasome activation and neointima formation: Antioxidant action of butyrate.

Short chain fatty acids (SCFAs), a family of gut microbial metabolites, have been reported to promote preservation of endothelial function and thereby exert anti-atherosclerotic action. However, the precise mechanism mediating this protective action of SCFAs remains unknown. The present study investigated the effects of SCFAs (acetate, propionate and butyrate) on the activation of Nod-like receptor pyrin domain 3 (Nlrp3) inflammasome in endothelial cells (ECs) and associated carotid neointima formation. Using a partial ligated carotid artery (PLCA) mouse model fed with the Western diet (WD), we found that butyrate significantly decreased Nlrp3 inflammasome formation and activation in the carotid arterial wall of wild type mice (Asc+/+), which was comparable to the effect of gene deletion of the adaptor protein apoptosis-associated speck-like protein gene (Asc-/-). Nevertheless, both acetate and propionate markedly enhanced the formation and activation of the Nlrp3 inflammasome as well as carotid neointima formation in the carotid arteries with PLCA in Asc+/+, but not Asc-/- mice. In cultured ECs (EOMA cells), butyrate was found to significantly decrease the formation and activation of Nlrp3 inflammasomes induced by 7-ketocholesterol (7-Ket) or cholesterol crystals (CHC), while acetate did not inhibit Nlrp3 inflammasome activation induced by either 7-Ket or CHC, but itself even activated Nlrp3 inflammsomes. Mechanistically, the inhibitory action of butyrate on the Nlrp3 inflammasome was attributed to a blockade of lipid raft redox signaling platforms to produce O2•- upon 7-Ket or CHC stimulations. These results indicate that SCFAs have differential effects on endothelial Nlrp3 inflammasome activation and associated carotid neointima formation.

Cigarette Smoke Increases Endothelial CXCL16-Leukocyte CXCR6 Adhesion In Vitro and In Vivo. Potential Consequences in Chronic Obstructive Pulmonary Disease.

Cardiovascular disease (CVD) is a major comorbidity in chronic obstructive pulmonary disease (COPD). Although the mechanism of its development remains largely unknown, it appears to be associated with cigarette consumption and reduced lung function. Therefore, the aim of this study was to investigate the potential link between water-soluble cigarette smoke extract (CSE)-induced endothelial dysfunction and the function of CXCL16/CXCR6 axis on the initial attachment of leukocytes, in addition to its possible impact on COPD-associated systemic inflammation. To do this, we employed several experimental approaches, including RNA silencing and flow cytometry analysis, the dynamic flow chamber technique, and intravital microscopy in the cremasteric arterioles of animals exposed to cigarette smoke (CS). CSE-induced arterial CXCL16 expression, leading to increased platelet-leukocyte and mononuclear cell adhesiveness. CSE-induced CXCL16 expression was dependent on Nox5 expression and subsequent RhoA/p38 MAPK/NF-κB activation. Flow cytometry analysis revealed that COPD patients (n = 35) presented greater numbers of activated circulating platelets (PAC-1+ and P-selectin+) expressing CXCL16 and CXCR6 as compared with age-matched controls (n = 17), with a higher number of CXCR6+-platelets in the smoking COPD group than in ex-smokers. This correlated with enhanced circulating CXCR6+-platelet-leukocyte aggregates in COPD patients. The increase in circulating numbers of CXCR6-expressing platelets and mononuclear cells resulted in enhanced platelet-leukocyte and leukocyte adhesiveness to CSE-stimulated arterial endothelium, which was greater than that found in age-matched controls and was partly dependent on endothelial CXCL16 upregulation. Furthermore, CS exposure provoked CXCL16-dependent leukocyte-arteriolar adhesion in cremasteric arterioles, which was significantly reduced in animals with a nonfunctional CXCR6 receptor. In conclusion, we provide the first evidence that increased numbers of CXCR6-expressing circulating platelets and mononuclear leukocytes from patients with COPD might be a marker of systemic inflammation with potential consequences in CVD development. Accordingly, CXCL16/CXCR6 axis blockade might constitute a new therapeutic approach for decreasing the risk of CVD in COPD patients.

Structural determinants and proliferative consequences of connexin 37 hemichannel function in insulinoma cells.

Connexin (Cx) 37 suppresses vascular and cancer cell proliferation. The C terminus and a channel able to function are necessary, and neither by itself is sufficient, for Cx37 to mediate growth suppression. Cx37 supports transmembrane and intercellular signaling by forming functional hemichannels (HCs) and gap junction channels (GJCs), respectively. Here we determined whether Cx37 with HC, but not GJC, functionality would suppress proliferation of rat insulinoma (Rin) cells comparably to wild-type Cx37 (Cx37-WT). We mutated extracellular loop residues hypothesized to compromise HC docking but not HC function (six cysteines mutated to alanine, C54A,C61A,C65A, C187A,C192A,C198A (designated as C6A); N55I; and Q58L). All three mutants trafficked to the plasma membrane and formed protein plaques comparably to Cx37-WT. None of the mutants formed functional GJCs, and Cx37-C6A did not form functional HCs. Cx37-N55I and -Q58L formed HCs with behavior and permeation properties similar to Cx37-WT (especially Q58L), but none of the mutants suppressed Rin cell proliferation. The data indicate that determinants of Cx37 HC function differ from other Cxs and that HC functions with associated HC-supported protein-protein interactions are not sufficient for Cx37 to suppress Rin cell proliferation. Together with previously published data, these results suggest that Cx37 suppresses Rin cell proliferation only when in a specific conformation achieved by interaction of the C terminus with a Cx37 pore-forming domain able to open as a GJC.