Malondialdehyde-Acetaldehyde Modified (MAA) Proteins Differentially Effect the Inflammatory Response in Macrophage, Endothelial Cells and Animal Models of Cardiovascular Disease

Chronic inflammation plays a critical role in the pathogenesis of atherosclerosis. Currently, the mechanism(s) by which inflammation contributes to this disease are not entirely understood. Inflammation is known to induce oxidative stress, which can lead to lipid peroxidation. Lipid peroxidation can result in the production of reactive by-products that can oxidatively modify macromolecules including DNA, proteins, and lipoproteins. A major reactive by-product of lipid peroxidation is malondialdehyde (MDA). MDA can subsequently break down to form acetaldehyde (AA). These two aldehydes can covalently interact with the epsilon (ε)-amino group of lysines within proteins and lipoproteins leading to the formation of extremely stable, highly immunogenic malondialdehyde/acetaldehyde adducts (MAA-adducts). The aim of this study was to investigate the inflammatory response to MAA-modified human serum albumin (HSA-MAA) and low-density lipoprotein (LDL-MAA). We found that animals injected with LDL-MAA generate antibodies specific to MAA-adducts. The level of anti-MAA antibodies were further increased in an animal model of atherosclerosis fed a Western diet. An animal model that combined both high fat diet and immunization of MAA-modified protein resulted in a dramatic increase in antibodies to MAA-adducts and vascular fat accumulation compared with controls. In vitro exposure of endothelial cells and macrophages to MAA-modified proteins resulted in increased fat accumulation as well as increased expression of adhesion molecules and pro-inflammatory cytokines. The expression of cytokines varied between the different cell lines and was unique to the individual modified proteins. The results of these studies demonstrate that different MAA-modified proteins elicit unique responses in different cell types. Additionally, the presence of MAA-modified proteins appears to modulate cellular metabolism leading to increased accumulation of triglycerides and further progression of the inflammatory response.

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Angiogenic properties of the chemokine RANTES/CCL5

Biochemical Society Transactions ◽

10.1042/bst20110651 ◽

2011 ◽

Vol 39 (6) ◽

pp. 1649-1653 ◽

Cited By ~ 40

Author(s):

Nadine Suffee ◽

Benjamin Richard ◽

Hanna Hlawaty ◽

Olivier Oudar ◽

Nathalie Charnaux ◽

...

Keyword(s):

Endothelial Cells ◽

Growth Factor ◽

Critical Role ◽

Cell Types ◽

Developed Countries ◽

Present Review ◽

Vasa Vasorum ◽

In Vivo Models ◽

Vitro Assay

Atherosclerosis is an inflammatory disease that is one of the leading causes of death in developed countries. This disease is defined by the formation of an atherosclerotic plaque, which is responsible for artery obstruction and affects the heart by causing myocardial infarction. The vascular wall is composed of three cell types and includes a monolayer of endothelial cells and is irrigated by a vasa vasorum. The formation of the vascular network from the vasa vasorum is a process involved in the destabilization of this plaque. Cellular and molecular approaches are studied by in vitro assay of activated endothelial cells and in in vivo models of neovascularization. Chemokines are a large family of small secreted proteins that have been shown to play a critical role in the regulation of angiogenesis during several pathophysiological processes such as ischaemia. Chemokines may exert their regulatory activity on angiogenesis directly by activating the vasa vasorum, or as a consequence of leucocyte infiltration through the endothelium, and/or by the induction of growth factor expression such as that of VEGF (vascular endothelial growth factor). The present review focuses on the angiogenic activity of the chemokines RANTES (regulated upon activation, normal T-cell expressed and secreted)/CCL5 (CC chemokine ligand 5). RANTES/CCL5 is released by many cell types such as platelets or smooth muscle cells. This chemokine interacts with GPCRs (G-protein-coupled receptors) and GAG (glycosaminoglycan) chains bound to HSPGs (heparan sulfate proteoglycans). Many studies have demonstrated, using RANTES/CCL5 mutated on their GAG or GPCR-binding sites, the involvement of these chemokines in angiogenic process. In the present review, we discuss two controversial roles of RANTES/CCL5 in the angiogenic process.

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Curcumin Alleviates Palmitic Acid-Induced LOX-1 Upregulation by Suppressing Endoplasmic Reticulum Stress in HUVECs

BioMed Research International ◽

10.1155/2021/9983725 ◽

2021 ◽

Vol 2021 ◽

pp. 1-13

Author(s):

Ruixi Luo ◽

Lifeng Zhao ◽

Shuaishuai Li ◽

Peng Chen ◽

La Wang ◽

...

Keyword(s):

Endothelial Cells ◽

Endoplasmic Reticulum ◽

Palmitic Acid ◽

Er Stress ◽

Umbilical Vein ◽

Low Density Lipoprotein ◽

Critical Role ◽

Density Lipoprotein ◽

Low Density

Excessive free fatty acid- (FFA-) induced endothelial lipotoxicity is involved in the pathogenesis of atherosclerosis. Endoplasmic reticulum (ER) stress is mechanistically related to endothelial lipotoxicity. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is the major oxidatively modified low-density lipoprotein (OxLDL) receptor in endothelial cells and is highly abundant in atherosclerotic lesions. Curcumin reduces the LOX-1 expression; however, the mechanism underlying this effect remains unknown. In the current study, we explored whether curcumin ameliorates palmitic acid- (PA-) induced endothelial lipotoxicity and LOX-1 upregulation by reducing ER stress in human umbilical vein endothelial cells (HUVECs). We built endothelial lipotoxicity in vitro and found that LOX-1 was upregulated after PA stimulation, during which ER stress played an important role. Next, we observed that curcumin substantially alleviated PA-induced lipotoxicity by restoring cell viability, increasing angiogenesis, and decreasing lipid deposition. Furthermore, LOX-1 upregulation in HUVECs was blocked by curcumin, possibly via ER stress suppression. Overall, our findings demonstrated that curcumin alleviates endothelial lipotoxicity and LOX-1 upregulation, and ER stress inhibition may play a critical role in this effect.

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Morphilogy and Ultrastructure of in Vitro Cultures of Aortic Endothelial Cells Interacting with Antibodies

10.1055/s-0039-1684767 ◽

1979 ◽

Author(s):

S. Korach ◽

D. Ngo

Keyword(s):

Endothelial Cells ◽

Cell Surface ◽

Vascular Endothelial Cells ◽

Intercellular Junctions ◽

Cell Types ◽

Endothelial Damage ◽

Antibody Concentration ◽

High Yields ◽

Scanning Em

Adult pig aortas, sectioned longitudinally, were incubated in 0.1% collagenase-PBS (15 mn, 37°C). Gentle scraping of the lumenal surface resulted in high yields (3-4 x 106 cell/aorta) of viable endothelial cells, essentially devoid of other cell types by morphological and immunochemical (F VIII-antigen) criteria. Confluent monolayers were incubated for various times (5 mn to 1 wk) with decomplemented rabbit antisera raised against pig endothelial cells. Changes in cell morphology appeared to depend on antibody concentration rather than on duration of contact with antiserum. High concentrations of antiserum (5 to 20%) led to cytoplasmic shredding, bulging of cells and extensive vacuolization, whereas at lower concentrations, cells appeared almost normal. Transmission EM studies by the indirect immunoperoxydase method showed antibodies reacting with unfixed cells to be distributed all over the upper cell surface, in the outer parts of intercellular junctions, and within numerous pinocytotic vesicles. Much weaker reactions could also be seen at the lower cell surface. When viewed under the Scanning EM, antiserum-treated endothelial cells also disclosed antibody concentration-dependent bulging and release of cells from their substrate. In vitro studies of gradual modifications of vascular endothelial cells acted upon by antibodies should provide a better understanding of the structural and biochemical processes underlying endothelial damage and detachment.

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Venous and Arterial Endothelial Cells from Human Umbilical Cords: Potential Cell Sources for Cardiovascular Research

International Journal of Molecular Sciences ◽

10.3390/ijms22020978 ◽

2021 ◽

Vol 22 (2) ◽

pp. 978

Author(s):

Skadi Lau ◽

Manfred Gossen ◽

Andreas Lendlein ◽

Friedrich Jung

Keyword(s):

Endothelial Cells ◽

Umbilical Cord ◽

Membrane Integrity ◽

Cell Types ◽

Cell Number ◽

Human Umbilical Cord ◽

Cardiovascular Research ◽

Cardiovascular Devices ◽

Arterial Endothelial Cells

Although cardiovascular devices are mostly implanted in arteries or to replace arteries, in vitro studies on implant endothelialization are commonly performed with human umbilical cord-derived venous endothelial cells (HUVEC). In light of considerable differences, both morphologically and functionally, between arterial and venous endothelial cells, we here compare HUVEC and human umbilical cord-derived arterial endothelial cells (HUAEC) regarding their equivalence as an endothelial cell in vitro model for cardiovascular research. No differences were found in either for the tested parameters. The metabolic activity and lactate dehydrogenase, an indicator for the membrane integrity, slightly decreased over seven days of cultivation upon normalization to the cell number. The amount of secreted nitrite and nitrate, as well as prostacyclin per cell, also decreased slightly over time. Thromboxane B2 was secreted in constant amounts per cell at all time points. The Von Willebrand factor remained mainly intracellularly up to seven days of cultivation. In contrast, collagen and laminin were secreted into the extracellular space with increasing cell density. Based on these results one might argue that both cell types are equally suited for cardiovascular research. However, future studies should investigate further cell functionalities, and whether arterial endothelial cells from implantation-relevant areas, such as coronary arteries in the heart, are superior to umbilical cord-derived endothelial cells.

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Regulation of capillary tubules and lipid formation in vascular endothelial cells and macrophages via extracellular vesicle-mediated microRNA-4306 transfer

Journal of International Medical Research ◽

10.1177/0300060518809255 ◽

2018 ◽

Vol 47 (1) ◽

pp. 453-469 ◽

Cited By ~ 1

Author(s):

Ying Yang ◽

Hui Luo ◽

Can Zhou ◽

Rongyi Zhang ◽

Si Liu ◽

...

Keyword(s):

Endothelial Cells ◽

Coronary Artery ◽

Vascular Endothelial Cells ◽

Density Lipoprotein ◽

Extracellular Vesicle ◽

Luciferase Reporter ◽

Vascular Endothelial ◽

Human Coronary Artery ◽

Lipid Formation

Objective This study aimed to examine regulation of capillary tubules and lipid formation in vascular endothelial cells and macrophages via extracellular vesicle-mediated microRNA (miRNA)-4306 transfer Methods Whole blood samples (12 mL) were collected from 53 patients, and miR-4306 levels in extracellular vesicles (EVs) were analyzed by reverse transcription-polymerase chain reaction. Human coronary artery vascular endothelial cells (HCAECs) and human monocyte-derived macrophages (HMDMs) were transfected with a scrambled oligonucleotide, an miR-4306 mimic, or an anti-miR-4306 inhibitor. The direct effect of miR-4306 on the target gene was analyzed by a dual-luciferase reporter assay. Results EV-contained miR-4306 released from HMDMs was significantly upregulated in coronary artery disease. Oxidized low-density lipoprotein (ox-LDL)-stimulated HMDM-derived EVs inhibited proliferation, migration, and angiogenesis abilities of HCAECs in vitro. However, ox-LDL-stimulated HCAEC-derived EVs enhanced lipid formation of HMDMs. The possible mechanism of these findings was partly due to EV-mediated miR-4306 upregulation of the Akt/nuclear factor kappa B signaling pathway. Conclusions Paracrine cellular crosstalk between HCAECs and HMDMs probably supports the pro-atherosclerotic effects of EVs under ox-LDL stress.

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Hydrogel-Coated Textile Scaffolds as Three-Dimensional Growth Support for Human Umbilical Vein Endothelial Cells (HUVECs): Possibilities as Coculture System in Liver Tissue Engineering

Cell Transplantation ◽

10.3727/000000002783985837 ◽

2002 ◽

Vol 11 (4) ◽

pp. 369-377 ◽

Cited By ~ 24

Author(s):

Makarand V. Risbud ◽

Erdal Karamuk ◽

René Moser ◽

Joerg Mayer

Keyword(s):

Endothelial Cells ◽

Umbilical Vein ◽

Three Dimensional ◽

Mesh Size ◽

Cell Types ◽

Cell Number ◽

Human Umbilical Vein ◽

Hydrogel Matrix ◽

Umbilical Vein Endothelial Cells

Three-dimensional (3-D) scaffolds offer an exciting possibility to develop cocultures of various cell types. Here we report chitosan–collagen hydrogel-coated fabric scaffolds with defined mesh size and fiber diameter for 3-D culture of human umbilical vein endothelial cells (HUVECs). These scaffolds did not require pre-coating with fibronectin and they supported proper HUVEC attachment and growth. Scaffolds preserved endothelial cell-specific cobblestone morphology and cells were growing in compartments defined by the textile mesh. HUVECs on the scaffold maintained the property of contact inhibition and did not exhibit overgrowth until the end of in vitro culture (day 6). MTT assay showed that cells had preserved mitochondrial functionality. It was also noted that cell number on the chitosan-coated scaffold was lower than that of collagen-coated scaffolds. Calcein AM and ethidium homodimer (EtD-1) dual staining demonstrated presence of viable and metabolically active cells, indicating growth supportive properties of the scaffolds. Actin labeling revealed absence of actin stress fibers and uniform distribution of F-actin in the cells, indicating their proper attachment to the scaffold matrix. Confocal microscopic studies showed that HUVECs growing on the scaffold had preserved functionality as seen by expression of von Willebrand (vW) factor. Observations also revealed that functional HUVECs were growing at various depths in the hydrogel matrix, thus demonstrating the potential of these scaffolds to support 3-D growth of cells. We foresee the application of this scaffold system in the design of liver bioreactors wherein hepatocytes could be cocultured in parallel with endothelial cells to enhance and preserve liver-specific functions.

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Abstract 11710: Poloxamer 188 Protects Coronary Artery Endothelial Cells After Extended Hypoxia and Reoxygenation in an in vitro Model of Simulated Ischemia/Reperfusion Injury

Circulation ◽

10.1161/circ.144.suppl_2.11710 ◽

2021 ◽

Vol 144 (Suppl_2) ◽

Author(s):

zhu li ◽

Matthew J Hampton ◽

Matthew B Barajas ◽

Matthias L Riess

Keyword(s):

Endothelial Cells ◽

Coronary Artery ◽

Myocardial Ischemia ◽

Ischemia Reperfusion Injury ◽

Calcium Influx ◽

Ischemia Reperfusion ◽

Cell Types ◽

Membrane Function ◽

Ldh Release

Reperfusion restores blood flow after myocardial ischemia but can cause additional cellular injury by the sudden reintroduction of oxygen and nutrients. There is still no effective remedy for myocardial ischemia/reperfusion (IR) injury. Our previous study using cardiomyocytes (CMs) found that, after 3 hrs hypoxia followed by 2 hrs reoxygenation, viability decreased, and release of lactate dehydrogenase (LDH), calcium influx, membrane leakage (insertion of fluorescent probe FM1-43) significantly increased, indicating that cell membrane function was negatively affected. This was attenuated by the triblock copolymer Poloxamer (P)188. Here, we first hypothesized that endothelial cells are also susceptible to simulated IR injury, albeit requiring longer hypoxia times. We further hypothesized that P188 can also attenuate simulated IR injury in endothelial cells when given upon reoxygenation. Mouse coronary artery endothelial cells (MCAECs) were exposed to different durations of hypoxia (2, 3, 12 and 24 hrs) in serum- and glucose-free media +/- reoxygenation for 2 hrs in regular media. P188 was administered upon reoxygenation at 0, 100, 300 or 1,000 μM in experiments of 24 hrs hypoxia / 2 hrs reoxygenation. LDH release was measured and compared to appropriately timed normoxic control experiments. Reoxygenation and hypoxia times significantly longer than 3 hrs were required to elicit sufficient injury (panel A). When P188 was given upon reoxygenation after 24 hrs hypoxia, it dose-dependently attenuated LDH release (panel B). These findings contrast to the higher susceptibility of CMs to IR injury that only allowed shorter hypoxia durations. They also confirm a protective effect of P188 on the endothelium, not just on CMs. These findings have important implications for co-culture models with MCAECs and CMs to elucidate the interplay of both cell types on each other when studying mechanisms of cardioprotective strategies and compounds like P188.

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Disruption of endothelial Pfkfb3 ameliorates diet-induced murine insulin resistance

Journal of Endocrinology ◽

10.1530/joe-20-0524 ◽

2021 ◽

Author(s):

Qiuhua Yang ◽

Jiean Xu ◽

Qian Ma ◽

Zhiping Liu ◽

Yaqi Zhou ◽

...

Keyword(s):

Insulin Resistance ◽

Endothelial Cells ◽

High Fat Diet ◽

Inflammatory Responses ◽

Critical Role ◽

Endothelial Inflammation ◽

Glucose Clearance ◽

Deficient Mice

Overnutrition-induced endothelial inflammation plays a crucial role in high fat diet (HFD)-induced insulin resistance in animals. Endothelial glycolysis plays a critical role in endothelial inflammation and proliferation, but its role in diet-induced endothelial inflammation and subsequent insulin resistance has not been elucidated. PFKFB3 is a critical glycolytic regulator, and its increased expression has been observed in adipose vascular endothelium of C57BL/6J mice fed with HFD in vivo, and in palmitate (PA)-treated primary human adipose microvascular endothelial cells (HAMECs) in vitro. We generated mice with Pfkfb3 deficiency selective for endothelial cells to examine the effect of endothelial Pfkfb3 in endothelial inflammation in metabolic organs and in the development of HFD-induced insulin resistance. EC Pfkfb3-deficient mice exhibited mitigated HFD-induced insulin resistance, including decreased body weight and fat mass, improved glucose clearance and insulin sensitivity, and alleviated adiposity and hepatic steatosis. Mechanistically, cultured PFKFB3 knockdown HAMECs showed decreased NF-κB activation induced by PA, and consequent suppressed adhesion molecule expression and monocyte adhesion. Taken together, these results demonstrate that increased endothelial PFKFB3 expression promotes diet-induced inflammatory responses and subsequent insulin resistance, suggesting that endothelial metabolic alteration plays an important role in the development of insulin resistance.

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Cellular Uptake and Clearance of Oxidatively-modified Apolipoprotein E3 by Cerebral Cortex Endothelial Cells

International Journal of Molecular Sciences ◽

10.3390/ijms20184582 ◽

2019 ◽

Vol 20 (18) ◽

pp. 4582 ◽

Cited By ~ 1

Author(s):

Siobanth Cruz ◽

Vasanthy Narayanaswami

Keyword(s):

Endothelial Cells ◽

Cellular Uptake ◽

Hot Spot ◽

Oxidized Ldl ◽

Critical Role ◽

Density Lipoprotein ◽

Oxidative Modification ◽

Alternative Mechanism ◽

Type I ◽

Western Blots

Apolipoprotein E3 (apoE3) plays a critical role in the metabolism of lipoproteins and lowers plasma lipid levels by serving as a ligand for the low-density lipoprotein receptor (LDLr) family of proteins and by promoting macrophage cholesterol efflux. The current study examines the effect of acrolein (an endogenously generated metabolite and an environmental pollutant) modification on the structure and function of apoE3. Acrolein modification was confirmed in Western blots by reactivity with acrolein–lysine-specific antibody and by the presence of oligomeric species due to cross-linking. LC-MS/MS analysis revealed modification of 10 out of 12 lysines in apoE3, with Nε-(3-methylpyridinium)-lysine being the predominant form of modification, and Lys75 being a ‘hot spot’ in terms of susceptibility to oxidation. Circular dichroism spectroscopy showed no major change in overall secondary structure compared to unmodified apoE3. Reconstituted high density lipoprotein (HDL) bearing acrolein modified apoE3 showed loss of binding to soluble LDLr; however, incubation with mouse endothelioma bEnd.3 cells showed that it was internalized. Incubation with excess LDL did not abolish cellular uptake of acrolein modified apoE3, suggesting alternative mechanism(s) not involving LDLr. Incubation with anti-CD36 antibody did not show a decrease in internalization while incubation with anti- lectin-like oxidized LDL receptor 1 (LOX1) showed partial internalization. However, incubation with anti-scavenger receptor class B type I (SRB1) antibody abolished internalization of acrolein modified apoE3. Taken together, our studies suggest that acrolein modification of apoE3 at lysine residues leads to increase in net negative charge, and as a consequence, results in clearance by LOX1 and SRB1 on endothelial cells. Overall, oxidative modification of apoE3 likely impairs its role in regulating plasma cholesterol homeostasis, eventually leading to lipid disorders.

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Endocytosis of formaldehyde-treated serum albumin via scavenger pathway in liver endothelial cells

Biochemical Journal ◽

10.1042/bj2180081 ◽

1984 ◽

Vol 218 (1) ◽

pp. 81-86 ◽

Cited By ~ 72

Author(s):

R Blomhoff ◽

W Eskild ◽

T Berg

Keyword(s):

Endothelial Cells ◽

Low Density Lipoprotein ◽

Scavenger Receptor ◽

Density Lipoprotein ◽

Low Density ◽

Surface Culture ◽

Rat Liver Cells ◽

Liver Endothelial Cells ◽

Isolated Liver

Denatured or modified proteins (including albumin and low-density lipoprotein) are catabolized in vitro via scavenger receptors. We have studied the distribution of formaldehyde-denatured albumin in rat liver cells after intravenous injection of tracer doses of the protein. At 12 min after injection, most of the formaldehyde-denatured albumin (about 70% of the injected dose) was recovered in liver endothelial cells. Furthermore, isolated liver endothelial cells in suspension and in surface culture took up formaldehyde-denatured albumin by receptor-mediated endocytosis. Our data indicate that the scavenger receptor in liver is mainly located on the endothelial cells. Implications for the catabolism of low-density lipoproteins are discussed.

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