Ephrin B2 mediates high glucose induced endothelial-to-mesenchymal transition in human aortic endothelial cells.

BACKGROUND: Previous study revealed that high glucose (HG) induced endothelial cell (EC) damage via endothelial-to-mesenchymal transition (EndMT). Recent studies suggested the role of Ephrin B2 in mediate ECs damage. However, the underlying mechanism remains unclear. The aim of the present study was to investigate whether Ephrin B2 mediates HG-induced EndMT in human aortic ECs (HAECs) and to determine the possible downstream signaling effector. METHODS: Primary HAECs were exposed to normal glucose (NG, 5.5 mM), HG (30 mM) and HG+Ephrin B2 small interfering RNA (siRNA), respectively. The pathological changes were investigated by light microscope and confocal microscopy. To study the effects of focal adhesion kinase (FAK) activation on Ephrin B2 in HAECs, cells were incubated with FAK siRNA in HG group. The expression of EndMT-related markers (CD31 and FSP1), Ephrin B2 and FAK were detected by qRT-PCR and western blot. RESULTS: The results showed that HG significantly inhibited the expression of CD31 and increased FSP1 compared with NG group. Moreover, Ephrin B2 was increased after HG incubation. Ephrin B2 siRNA attenuated HG-induced expression of EndMT-related markers. Furthermore, HG increased the expression of FAK and phosphorylated FAK (pho-FAK) in HAECs. In contrast, blocking Ephrin B2 could partially attenuate HG-induced FAK activation. And FAK siRNA further inhibited the EndMT-related markers in HAECs treated with HG. CONCLUSIONS: HG-induced EndMT in HAECs might be partially mediated by Ephrin B2 and the downstream FAK pathway.

Therapeutic Antibody Against Phosphorylcholine Preserves Coronary Function and Attenuates Vascular 18F-FDG Uptake in Atherosclerotic Mice.

This study showed that treatment with a therapeutic monoclonal immunoglobulin-G1 antibody against phosphorylcholine on oxidized phospholipids preserves coronary flow reserve and attenuates atherosclerotic inflammation as determined by the uptake of 18F-fluorodeoxyglucose in atherosclerotic mice. The noninvasive imaging techniques represent translational tools to assess the efficacy of phosphorylcholine-targeted therapy on coronary artery function and atherosclerosis in clinical studies.

Calcium-Sensing Receptor Participates in High Glucose-Induced EndMT in Primary Human Aortic Endothelial Cells.

OBJECTIVE: Previous studies have shown that high glucose (HG) induces endothelial cell (EC) damage via endothelial-to-mesenchymal transition (EndMT). Although the underlying mechanisms are still unclear, recent studies have demonstrated the role of calcium-sensing receptor (CaSR) in mediating EC damage. Therefore, the aim of our study was to investigate whether CaSR mediates HG-induced EndMT and to determine the underlying mechanism. METHODS: Bioinformatics analysis of microarray profiles (GSE30780) and protein-protein interaction (PPI) analyses were performed to select the hub genes. As for in vitro research, the human aortic ECs (HAECs) were exposed to HG to induce EndMT. The expression of CaSR and ß-catenin was determined, as well as their effects on EndMT (endothelial marker CD31, mesenchymal marker FSP1, and α-SMA). RESULTS: The bioinformatics analysis indicated CaSR was significantly increased in HG-treated HAECs and was one of the hub genes. The in vitro results showed that HG significantly inhibited the expression of CD31 and increased FSP1 and α-SMA in a concentration- and time-dependent manner. Moreover, CaSR was increased in HAECs after HG treatment. The CaSR antagonist attenuated HG-induced expression of EndMT-related markers. Furthermore, HG treatment increased the nuclear translocation of ß-catenin in HAECs. In contrast, blocking the nuclear translocation of ß-catenin by DKK1 could attenuate HG-induced EndMT (increased the protein expression of CD31 by 30% and decreased the protein expression of FSP1 by 15% and α-SMA by 25%). CaSR siRNA further inhibited the HG-induced nuclear translocation of ß-catenin in HAECs. CONCLUSION: Our research demonstrated that HG-induced EndMT in HAECs might be mediated by CaSR and the downstream nuclear translocation of ß-catenin.

Isolation of intimal endothelial cells from the human thoracic aorta: Study protocol.

Background: Vessel endothelial cells are extensively applied to study the mechanism of atherosclerosis. Some cellular sources including human umbilical artery endothelial cells (HUAECs) and human umbilical vein endothelial cells (HUVECs) are mostly applied in the experimental studies. We described a method for isolating the human endothelial cells from the human thoracic aorta. Methods: Normal aortic samples were prepared from subjects with brain death in Masih Daneshvari Hospital. The endothelial cells were isolated using collagenase and were evaluated by the measurement of CD31 marker. Furthermore, the digestion efficacy was studied by vessel histological analysis, and the adhesion mechanism was investigated by leukocyte endothelial adhesion assay kit. Results and Conclusion: The isolation protocol is found as a fast and simple technique with a proper cellular load to separate the endothelial cells from the human aorta.

Increased acetylation of H3K14 in the genomic regions that encode trained immunity enzymes in lysophosphatidylcholine-activated human aortic endothelial cells - Novel qualification markers for chronic disease risk factors and conditional DAMPs.

To test our hypothesis that proatherogenic lysophosphatidylcholine (LPC) upregulates trained immunity pathways (TIPs) in human aortic endothelial cells (HAECs), we conducted an intensive analyses on our RNA-Seq data and histone 3 lysine 14 acetylation (H3K14ac)-CHIP-Seq data, both performed on HAEC treated with LPC. Our analysis revealed that: 1) LPC induces upregulation of three TIPs including glycolysis enzymes (GE), mevalonate enzymes (ME), and acetyl-CoA generating enzymes (ACE); 2) LPC induces upregulation of 29% of 31 histone acetyltransferases, three of which acetylate H3K14; 3) LPC induces H3K14 acetylation (H3K14ac) in the genomic DNA that encodes LPC-induced TIP genes (79%) in comparison to that of in LPC-induced effector genes (43%) including ICAM-1; 4) TIP pathways are significantly different from that of EC activation effectors including adhesion molecule ICAM-1; 5) reactive oxygen species generating enzyme NOX2 deficiency decreases, but antioxidant transcription factor Nrf2 deficiency increases, the expressions of a few TIP genes and EC activation effector genes; and 6) LPC induced TIP genes(81%) favor inter-chromosomal long-range interactions (CLRI, trans-chromatin interaction) while LPC induced effector genes (65%) favor intra-chromosomal CLRIs (cis-chromatin interaction). Our findings demonstrated that proatherogenic lipids upregulate TIPs in HAECs, which are a new category of qualification markers for chronic disease risk factors and conditional DAMPs and potential mechanisms for acute inflammation transition to chronic ones. These novel insights may lead to identifications of new cardiovascular risk factors in upregulating TIPs in cardiovascular cells and novel therapeutic targets for the treatment of metabolic cardiovascular diseases, inflammation, and cancers. (total words: 245).

Increasing Upstream Chromatin Long-Range Interactions May Favor Induction of Circular RNAs in LysoPC-Activated Human Aortic Endothelial Cells.

Circular RNAs (circRNAs) are non-coding RNAs that form covalently closed continuous loops, and act as gene regulators in physiological and disease conditions. To test our hypothesis that proatherogenic lipid lysophosphatidylcholine (LPC) induce a set of circRNAs in human aortic endothelial cell (HAEC) activation, we performed circRNA analysis by searching our RNA-Seq data from LPC-activated HAECs, and found: (1) LPC induces significant modulation of 77 newly characterized cirRNAs, among which 47 circRNAs (61%) are upregulated; (2) 34 (72%) out of 47 upregulated circRNAs are upregulated when the corresponding mRNAs are downregulated, suggesting that the majority of circRNAs are upregulated presumably via LPC-induced "abnormal splicing" when the canonical splicing for generation of corresponding mRNAs is suppressed; (3) Upregulation of 47 circRNAs is temporally associated with mRNAs-mediated LPC-upregulated cholesterol synthesis-SREBP2 pathway and LPC-downregulated TGF-ß pathway; (4) Increase in upstream chromatin long-range interaction sites to circRNA related genes is associated with preferred circRNA generation over canonical splicing for mRNAs, suggesting that shifting chromatin long-range interaction sites from downstream to upstream may promote induction of a list of circRNAs in lysoPC-activated HAECs; (5) Six significantly changed circRNAs may have sponge functions for miRNAs; and (6) 74% significantly changed circRNAs contain open reading frames, suggesting that putative short proteins may interfere with the protein interaction-based signaling. Our findings have demonstrated for the first time that a new set of LPC-induced circRNAs may contribute to homeostasis in LPC-induced HAEC activation. These novel insights may lead to identifications of new therapeutic targets for treating metabolic cardiovascular diseases, inflammations, and cancers.

Neutrophil Subsets, Platelets, and Vascular Disease in Psoriasis.

Psoriasis is an inflammatory skin disease associated with increased cardiovascular risk and serves as a reliable model to study inflammatory atherogenesis. Because neutrophils are implicated in atherosclerosis development, this study reports that the interaction among low-density granulocytes, a subset of neutrophils, and platelets is associated with a noncalcified coronary plaque burden assessed by coronary computed tomography angiography. Because early atherosclerotic noncalcified burden can lead to fatal myocardial infarction, the low-density granulocyte-platelet interaction may play a crucial target for clinical intervention.

Prokineticin Receptor-1 Signaling Inhibits Dose- and Time-Dependent Anthracycline-Induced Cardiovascular Toxicity Via Myocardial and Vascular Protection.

OBJECTIVES: This study investigated how different concentrations of doxorubicin (DOX) can affect the function of cardiac cells. This study also examined whether activation of prokineticin receptor (PKR)-1 by a nonpeptide agonist, IS20, prevents DOX-induced cardiovascular toxicity in mouse models. BACKGROUND: High prevalence of heart failure during and following cancer treatments remains a subject of intense research and therapeutic interest. METHODS: This study used cultured cardiomyocytes, endothelial cells (ECs), and epicardium-derived progenitor cells (EDPCs) for in vitro assays, tumor-bearing models, and acute and chronic toxicity mouse models for in vivo assays. RESULTS: Brief exposure to cardiomyocytes with high-dose DOX increased the accumulation of reactive oxygen species (ROS) by inhibiting a detoxification mechanism via stabilization of cytoplasmic nuclear factor, erythroid 2. Prolonged exposure to medium-dose DOX induced apoptosis in cardiomyocytes, ECs, and EDPCs. However, low-dose DOX promoted functional defects without inducing apoptosis in EDPCs and ECs. IS20 alleviated detrimental effects of DOX in cardiac cells by activating the serin threonin protein kinase B (Akt) or mitogen-activated protein kinase pathways. Genetic or pharmacological inactivation of PKR1 subdues these effects of IS20. In a chronic mouse model of DOX cardiotoxicity, IS20 normalized an elevated serum marker of cardiotoxicity and vascular and EDPC deficits, attenuated apoptosis and fibrosis, and improved the survival rate and cardiac function. IS20 did not interfere with the cytotoxicity or antitumor effects of DOX in breast cancer lines or in a mouse model of breast cancer, but it did attenuate the decreases in left ventricular diastolic volume induced by acute DOX treatment. CONCLUSIONS: This study identified the molecular and cellular signature of dose-dependent, DOX-mediated cardiotoxicity and provided evidence that PKR-1 is a promising target to combat cardiotoxicity of cancer treatments.

Asiaticoside Inhibits TNF-α-Induced Endothelial Hyperpermeability of Human Aortic Endothelial Cells.

The increase in endothelial permeability often promotes edema formation in various pathological conditions. Tumor necrosis factor-alpha (TNF-α), a pro-atherogenic cytokine, impairs endothelial barrier function and causes endothelial dysfunction in early stage of atherosclerosis. Asiaticoside, one of the triterpenoids derived from Centella asiatica, is known to possess antiinflammatory activity. In order to examine the role of asiaticoside in preserving the endothelial barrier, we assessed its effects on endothelial hyperpermeability and disruption of actin filaments evoked by TNF-α in human aortic endothelial cells (HAEC). TNF-α caused an increase in endothelial permeability to fluorescein isothiocyanate (FITC)-dextran. Asiaticoside pretreatment significantly suppressed TNF-α-induced increased permeability. Asiaticoside also prevented TNF-α-induced actin redistribution by suppressing stress fiber formation. However, the increased F to G actin ratio stimulated by TNF-α was not changed by asiaticoside. Cytochalasin D, an actin depolymerizing agent, was used to correlate the anti-hyperpermeability effect of asiaticoside with actin cytoskeleton. Surprisingly, asiaticoside failed to prevent cytochalasin D-induced increased permeability. These results suggest that asiaticoside protects against the disruption of endothelial barrier and actin rearrangement triggered by TNF-α without a significant change in total actin pool. However, asiaticoside seems to work by other mechanisms to maintain the integrity of endothelial barrier rather than stabilizing the F-actin organization.

SLIT3-ROBO4 activation promotes vascular network formation in human engineered tissue and angiogenesis in vivo.

Successful implantation and long-term survival of engineered tissue grafts hinges on adequate vascularization of the implant. Endothelial cells are essential for patterning vascular structures, but they require supportive mural cells such as pericytes/mesenchymal stem cells (MSCs) to generate stable, functional blood vessels. While there is evidence that the angiogenic effect of MSCs is mediated via the secretion of paracrine signals, the identity of these signals is unknown. By utilizing two functionally distinct human MSC clones, we found that so-called "pericytic" MSCs secrete the pro-angiogenic vascular guidance molecule SLIT3, which guides vascular development by directing ROBO4-positive endothelial cells to form networks in engineered tissue. In contrast, "non-pericytic" MSCs exhibit reduced activation of the SLIT3/ROBO4 pathway and do not support vascular networks. Using live cell imaging of organizing 3D vascular networks, we show that siRNA knockdown of SLIT3 in MSCs leads to disorganized clustering of ECs. Knockdown of its receptor ROBO4 in ECs abolishes the generation of functional human blood vessels in an in vivo xenogenic implant. These data suggest that the SLIT3/ROBO4 pathway is required for MSC-guided vascularization in engineered tissues. Heterogeneity of SLIT3 expression may underlie the variable clinical success of MSCs for tissue repair applications.

Postprandial lipoproteins and the molecular regulation of vascular homeostasis.

Blood levels of triglyceride-rich lipoproteins (TRL) increase postprandially, and a delay in their clearance results in postprandial hyperlipidemia, an important risk factor in atherosclerosis development. Atherosclerosis is a multifactorial inflammatory disease, and its initiation involves endothelial dysfunction, invasion of the artery wall by leukocytes and subsequent formation of foam cells. TRL are implicated in several of these inflammatory processes, including the formation of damaging free radicals, leukocyte activation, endothelial dysfunction and foam cell formation. Recent studies have provided insights into the mechanisms of uptake and the signal transduction pathways mediating the interactions of TRL with leukocytes and vascular cells, and how they are modified by dietary lipids. Multiple receptor and non-receptor mediated pathways function in macrophage uptake of TRL. TRL also induce expression of adhesion molecules, cyclooxygenase-2 and heme-oxygenase-1 in endothelial cells, and activate intracellular signaling pathways involving mitogen-activated protein kinases, NF-κB and Nrf2. Many of these effects are strongly influenced by dietary components carried in TRL. There is extensive evidence indicating that raised postprandial TRL levels are a risk factor for atherosclerosis, but the molecular mechanisms involved are only now becoming appreciated. Here, we review current understanding of the mechanisms by which TRL influence vascular cell function.

The preventive effect of uncarboxylated osteocalcin against free fatty acid-induced endothelial apoptosis through the activation of phosphatidylinositol 3-kinase/Akt signaling pathway.

OBJECTIVE: Increasing evidence suggests that osteocalcin (OC), one of the osteoblast-specific proteins, has been associated with atherosclerosis, but results are conflicting. The aim of this study was to elucidate the independent effect of uncarboxylated osteocalcin (ucOC), an active form of osteocalcin which has been suggested to have an insulin sensitizing effect, on vascular endothelial cells. MATERIALS AND METHODS: We used human aortic endothelial cells and treated them with ucOC. Linoleic acid (LA) was used as a representative free fatty acid. Apoptosis was evaluated using various methods including a terminal deoxyribonucleotide transferase-mediated deoxyuridine triphosphate nick-end labeling analysis kit and Western blotting for cleaved caspase 3, cleaved poly (ADP-ribose) polymerase and Bcl-xL. The phosphorylations of Akt and endothelial nitric oxide synthase (eNOS) as well as the level of NO were measured to confirm the effect of ucOC on insulin signaling pathway. RESULTS: Pretreatment of ucOC (30 ng/ml) prevented LA-induced apoptosis in insulin-stimulated endothelial cells; effects were abolished by pretreatment with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, wortmannin. Treatment of ucOC (ranged from 0.3 to 30 ng/ml) significantly increased the phosphorylation of Akt and eNOS and nitric oxide secretion from endothelial cells in a PI3-kinase dependent manner. CONCLUSIONS: Our study is the first to demonstrate the independent effect of ucOC on vascular endothelial cells. Our results further suggest that ucOC could have beneficial effects on atherosclerosis.

Effects of Angiotensin II and Shear Stress Interaction on Vascular Inflammation

BACKGROUND: Angiotensin II (AngII) and abnormal oscillatory shear stress are highly associated with vascular inflammation including atherosclerosis. However, it is poorly understood how interactions between AngII and shear stress in human aortic endothelial cells (HAEC) are involved in mechanisms by which cellular adhesion molecules are expressed. The purpose of this study was to improve that understanding. METHODS: AngII (10(-7)M for 6 hr) and two-types of shear stress treatments were used: laminar shear stress (LS: unidirectional, 12 dynes/cm2) and oscillatory shear stress (OS: bi-directional, 5 dynes/cm2, 1 Hz) in HAEC. Immunoblotting was used to detect expression of cellular adhesion molecules markers such as vascular cell adhesion molecule 1 (VCAM1) and intercellular adhesion molecule 1 (ICAM1). RESULTS: AngII significantly increased VCAM1 and ICAM1 expression in HAEC that had been reduced due to pretreatment with telmisartan. AngII-LS co-stimulation and AngII-OS co-stimulation significantly increased VCAM1 and ICAM1 expression in HAEC. The expression levels of VCAM1 and ICAM1 were also, significantly reduced when pretreated with telmisartan. However, VCAM1 and ICAM1 expression were significantly reduced under LS and OS stimulation. CONCLUSIONS: Telmisartan may modulate the expressions of VCAM1 and ICAM1 via different types of shear stress in HAEC that are activated by AngII.