Labeled vs. Label-Free Raman Imaging of Lipids in Endothelial Cells of Various Origins

Endothelial cells (EC) constitute a single layer of the lining of blood vessels and play an important role in maintaining cardiovascular homeostasis. Endothelial dysfunction has been recognized as a primary or secondary cause of many diseases and it manifests itself, among others, by increased lipid content or a change in the lipid composition in the EC. Therefore, the analysis of cellular lipids is crucial to understand the mechanisms of disease development. Tumor necrosis factor alpha (TNF-α)-induced inflammation of EC alters the lipid content of cells, which can be detected by Raman spectroscopy. By default, lipid detection is carried out in a label-free manner, and these compounds are recognized based on their spectral profile characteristics. We consider (3S,3′S)-astaxanthin (AXT), a natural dye with a characteristic resonance spectrum, as a new Raman probe for the detection of lipids in the EC of various vascular beds, i.e., the aorta, brain and heart. AXT colocalizes with lipids in cells, enabling imaging of lipid-rich cellular components in a time-dependent manner using laser power 10 times lower than that commonly used to measure biological samples. The results show that AXT can be used to study lipids distribution in EC at various locations, suggesting its use as a universal probe for studying cellular lipids using Raman spectroscopy. The use of labeled Raman imaging of lipids in the EC of various organs could contribute to their easier identification and to a better understanding of the development and progression of various vascular diseases, and it could also potentially improve their diagnosis and treatment.

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Human Cytomegalovirus-Specific CD4+-T-Cell Cytokine Response Induces Fractalkine in Endothelial Cells

Journal of Virology ◽

10.1128/jvi.78.23.13173-13181.2004 ◽

2004 ◽

Vol 78 (23) ◽

pp. 13173-13181 ◽

Cited By ~ 24

Author(s):

Cynthia A. Bolovan-Fritts ◽

Rodney N. Trout ◽

Stephen A. Spector

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

T Cell ◽

Mononuclear Cells ◽

Cell Injury ◽

Transplant Rejection ◽

Vascular Diseases ◽

T Cell Response ◽

Peripheral Blood Mononuclear ◽

Necrosis Factor Alpha

ABSTRACT Cytomegalovirus (CMV) infection has been linked to inflammation-related disease processes in the human host, including vascular diseases and chronic transplant rejection. The mechanisms through which CMV affects the pathogenesis of these diseases are for the most part unknown. To study the contributing role of the host immune response to CMV in these chronic inflammatory processes, we examined endothelial cell interactions with peripheral blood mononuclear cells (PBMC). Endothelial cultures were monitored for levels of fractalkine induction as a marker for initiating the host inflammatory response. Our results demonstrate that in the presence of CMV antigen PBMC from normal healthy CMV-seropositive donors produce soluble factors that induce fractalkine in endothelial cells. This was not observed in parallel assays with PBMC from seronegative donors. Examination of subset populations within the PBMC further revealed that CMV antigen-stimulated CD4+ T cells were the source of the factors, gamma interferon and tumor necrosis factor alpha, driving fractalkine induction. Direct contact between CD4+ cells and the endothelial monolayers is required for this fractalkine induction, where the endothelial cells appear to provide antigen presentation functions. These findings indicate that CMV may represent one member of a class of persistent pathogens where the antigen-specific T-cell response can result in the induction of fractalkine, leading to chronic inflammation and endothelial cell injury.

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Label-free Raman imaging of the macrophage response to the malaria pigment hemozoin

The Analyst ◽

10.1039/c4an01850h ◽

2015 ◽

Vol 140 (7) ◽

pp. 2350-2359 ◽

Cited By ~ 11

Author(s):

Alison J. Hobro ◽

Nicolas Pavillon ◽

Katsumasa Fujita ◽

Muge Ozkan ◽

Cevayir Coban ◽

...

Keyword(s):

Raman Spectroscopy ◽

Raman Imaging ◽

Biochemical Changes ◽

Label Free ◽

Macrophage Response ◽

Macrophage Cells ◽

Malaria Pigment

Raman spectroscopy highlights biochemical changes that are spectrally or spatially related to the presence of the malaria pigment, hemozoin, inside macrophage cells, during the initial stages of exposure.

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Sustained ERK phosphorylation is necessary but not sufficient for MMP-9 regulation in endothelial cells: involvement of Ras-dependent and -independent pathways

Journal of Cell Science ◽

10.1242/jcs.113.23.4319 ◽

2000 ◽

Vol 113 (23) ◽

pp. 4319-4330 ◽

Cited By ~ 1

Author(s):

E. Genersch ◽

K. Hayess ◽

Y. Neuenfeld ◽

H. Haller

Keyword(s):

Endothelial Cells ◽

Umbilical Vein ◽

Signal Transduction Pathway ◽

Cell Types ◽

Tnf Alpha ◽

Dependent Manner ◽

Necrosis Factor Alpha ◽

Type Iv ◽

Erk Phosphorylation ◽

Umbilical Vein Endothelial Cells

Endothelial expression of matrix metalloproteinase-9 (MMP-9), which degrades native type IV collagen, was implicated as a prerequisite for angiogenesis. Therefore, the aim of this study was to determine signaling requirements that regulate MMP-9 expression in endothelial cells. Both, primary and permanent human umbilical vein endothelial cells (HUVEC and ECV304, respectively) were stimulated with phorbol 12-myristate 13-acetate (PMA) and the cytokine tumor necrosis factor-(alpha) (TNF(alpha)) to induce MMP-9 expression. While both cell types responded to PMA at the protein, mRNA and promoter level by induction of MMP-9, TNF(alpha) caused this response only in ECV304. Inhibitors specific for mitogen-activated protein/ERK kinase 1/2 (MEK1/2), protein kinase C (PKC), and Ras and co-transfections of wild-type and mutant Raf were used to elucidate the signaling cascades involved. Thus, we could show that the Raf/MEK/ERK cascade is mainly responsible for MMP-9 induction in endothelial cells and that this cascade is regulated independently of PKC and Ras subsequent to TNF(alpha) stimulation and in a PKC-dependent manner as a result of PMA treatment. In addition, PMA triggers a Ras-dependent signal transduction pathway bypassing the phosphorylation of ERK. Finally, we provide evidence that sustained phosphorylation of ERK1/2 is necessary but not sufficient for expression of MMP-9.

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Coordinate regulation of endothelin and adrenomedullin secretion by oxidative stress in endothelial cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2001.281.3.h1364 ◽

2001 ◽

Vol 281 (3) ◽

pp. H1364-H1371 ◽

Cited By ~ 23

Author(s):

Takatoshi Saito ◽

Hiroshi Itoh ◽

Tae-Hwa Chun ◽

Yasutomo Fukunaga ◽

Jun Yamashita ◽

...

Keyword(s):

Oxidative Stress ◽

Endothelial Cells ◽

Mrna Expression ◽

Vascular Diseases ◽

Transcriptional Level ◽

Dependent Manner ◽

Camp Pathway ◽

Intracellular Ca ◽

Dose Dependent Fashion ◽

Dose Dependent

To elucidate the significance of oxidative stress in the modulation of endothelial functions, we examined the effects of H2O2 on the expression of two endothelium-derived vasoactive peptides, endothelin (ET) and adrenomedullin (Am), and their interaction. H2O2 dose dependently suppressed ET secretion and ET-1 mRNA expression in bovine carotid endothelial cells (ECs). Menadion sodium bisulfate, a redox cycling drug, also decreased ET secretion in a dose-dependent manner. Catalase, a H2O2 reductase, and dl-α-tocopherol (vitamin E) significantly inhibited H2O2-induced suppression of ET secretion. Downregulation of ET-1 mRNA under oxidative stress was regulated at the transcriptional level. In contrast, H2O2increased Am secretion (and its mRNA expression) accompanied by the augmentation of cAMP production. Am, as well as 8-bromo-cAMP and forskolin decreased ET secretion in a dose-dependent fashion. Furthermore, an anti-Am monoclonal antibody that we developed abolished H2O2-induced suppression of ET secretion at 6–24 h after the addition of H2O2. H2O2 increased the intracellular Ca2+ concentration ([Ca2+]i). Moreover, treatment with ionomycin, a Ca2+ ionophore, and thapsigargin, an inhibitor of endoplasmic reticulum ATPase, decreased ET secretion dose dependently for 3 h. These results suggest that the production of ET was decreased via activation of the Am-cAMP pathway and by the elevation of [Ca2+]i under oxidative stress. These findings elucidate the coordinate expression of two local vascular hormones, ET and Am, under oxidative stress, which may protect against vascular diseases.

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Phorbol-stimulated influx of extracellular calcium in rat pulmonary artery endothelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1994.267.2.l145 ◽

1994 ◽

Vol 267 (2) ◽

pp. L145-L151 ◽

Cited By ~ 1

Author(s):

H. S. Murphy ◽

M. Maroughi ◽

G. O. Till ◽

P. A. Ward

Keyword(s):

Endothelial Cells ◽

Pulmonary Artery ◽

Extracellular Calcium ◽

Dependent Manner ◽

Necrosis Factor Alpha ◽

Calcium Dependent ◽

High K ◽

Voltage Dependent ◽

Dependent Inhibition ◽

Protein Kinase C Inhibitor

Stimulation of rat pulmonary artery endothelial cells (RPAEC) with phorbol 12-myristate 13-acetate (PMA) resulted in an increase in intracellular calcium ([Ca2+]i). Unlike the response to bradykinin, C5a and tumor necrosis factor-alpha (TNF-alpha) previously reported (15), the PMA-induced increase in [Ca2+]i was predominantly dependent on extracellular calcium. The PMA response paralleled the BAY K 8644-induced, extracellular calcium-dependent increase in [Ca2+]i. Pretreatment of endothelial cells with the protein kinase C inhibitor staurosporine resulted in a concentration-dependent inhibition of the increase in [Ca2+]i in response to PMA. The ability of PMA analogues to induce significant increase in [Ca2+]i paralleled their ability to induce O2- generation in neutrophils. The PMA-induced influx of extracellular Ca2+ was inhibited by the L-channel selective antagonists diltiazem, nifedipine, nicardipine, and verapamil in a dose-dependent manner. Depolarizing conditions induced by high [K+]o enhanced the calcium response to PMA. The data presented are consistent with the hypothesis that PMA-induced increases in [Ca2+]i in endothelial cells are the result of Ca2+ influx through voltage-dependent L-type Ca2+ channels.

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Primary Human Derived Blood Outgrowth Endothelial Cells: An Appropriate In Vitro Model to Study Shiga Toxin Mediated Damage of Endothelial Cells

Toxins ◽

10.3390/toxins12080483 ◽

2020 ◽

Vol 12 (8) ◽

pp. 483

Author(s):

Wouter J. C. Feitz ◽

Nicole C. A. J. van de Kar ◽

Ian Cheong ◽

Thea J. A. M. van der Velden ◽

Carolina G. Ortiz-Sandoval ◽

...

Keyword(s):

Wound Healing ◽

Endothelial Cells ◽

Protein Synthesis ◽

Cell Surface ◽

Shiga Toxin ◽

Dependent Manner ◽

Surface Binding ◽

Necrosis Factor Alpha ◽

Static Conditions

Hemolytic uremic syndrome (HUS) is a rare disease primarily characterized by hemolytic anemia, thrombocytopenia, and acute renal failure. Endothelial damage is the hallmark of the pathogenesis of HUS with an infection with the Shiga toxin (Stx) producing Escherichia coli (STEC-HUS) as the main underlying cause in childhood. In this study, blood outgrowth endothelial cells (BOECs) were isolated from healthy donors serving as controls and patients recovered from STEC-HUS. We hypothesized that Stx is more cytotoxic for STEC-HUS BOECs compared to healthy donor control BOECs explained via a higher amount of Stx bound to the cell surface. Binding of Shiga toxin-2a (Stx2a) was investigated and the effect on cytotoxicity, protein synthesis, wound healing, and cell proliferation was studied in static conditions. Results show that BOECs are highly susceptible for Stx2a. Stx2a is able to bind to the cell surface of BOECs with cytotoxicity in a dose-dependent manner as a result. Pre-treatment with tumor necrosis factor alpha (TNF-α) results in enhanced Stx binding with 20–30% increased lactate dehydrogenase (LDH) release. Endothelial wound healing is delayed in a Stx2a-rich environment; however, this is not caused by an effect on the proliferation rate of BOECs. No significant differences were found between control BOECs and BOECs from recovered STEC-HUS patients in terms of Stx2a binding and inhibition of protein synthesis.

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Superoxide responses of endothelial cells to C5a and TNF-alpha: divergent signal transduction pathways

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1992.263.1.l51 ◽

1992 ◽

Vol 263 (1) ◽

pp. L51-L59 ◽

Cited By ~ 14

Author(s):

H. S. Murphy ◽

J. A. Shayman ◽

G. O. Till ◽

M. Mahrougui ◽

C. B. Owens ◽

...

Keyword(s):

Signal Transduction ◽

Endothelial Cells ◽

Tnf Alpha ◽

Dependent Manner ◽

Necrosis Factor Alpha ◽

Cell Monolayers ◽

Kinase C ◽

Factor Alpha ◽

Dose Dependent ◽

Necrosis Factor

There is increasing evidence that endothelial cells respond to a variety of mediators. In the current studies rat pulmonary artery endothelial cells (RPAEC) responded to human recombinant C5a and tumor necrosis factor-alpha (TNF-alpha) with the generation of superoxide (O2-). RPAEC responsiveness was dependent on whether cells had been obtained from confluent or subconfluent cell monolayers. RPAEC responded to C5a and TNF-alpha in a dose-dependent manner, with increases in intracellular Ca2+ (Cai2+), formation of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], and generation of O2-. Optimal O2- responses occurred in cells that had been pretreated with the inhibitor of superoxide dismutase (SOD), diethyldithiocarbamate, and O2- responses were allopurinol insensitive. Pertussis toxin pretreatment abolished the ability of C5a to cause increases in Ins(1,4,5)P3 and Cai2+ and formation of O2- but did not inhibit the changes in Cai2+ and formation of O2- after addition of TNF-alpha. The O2- response to C5a but not to TNF-alpha was abolished by pretreatment with the inhibitor of protein kinase C, staurosporine. These data indicate that signal transduction events in response to C5a and TNF-alpha were fundamentally different.

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Chloroquine-Induced Accumulation of Autophagosomes and Lipids in the Endothelium

International Journal of Molecular Sciences ◽

10.3390/ijms22052401 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2401

Author(s):

Ewelina Bik ◽

Lukasz Mateuszuk ◽

Jagoda Orleanska ◽

Malgorzata Baranska ◽

Stefan Chlopicki ◽

...

Keyword(s):

Raman Spectroscopy ◽

Endothelial Cells ◽

Neutral Lipids ◽

Reference Method ◽

Label Free ◽

Fluorescence Staining ◽

Autophagy Flux ◽

Endothelial Inflammation ◽

Endothelial Response ◽

Autophagy Inhibition

Chloroquine (CQ) is an antimalarial drug known to inhibit autophagy flux by impairing autophagosome–lysosome fusion. We hypothesized that autophagy flux altered by CQ has a considerable influence on the lipid composition of endothelial cells. Thus, we investigated endothelial responses induced by CQ on human microvascular endothelial cells (HMEC-1). HMEC-1 cells after CQ exposure were measured using a combined methodology based on label-free Raman and fluorescence imaging. Raman spectroscopy was applied to characterize subtle chemical changes in lipid contents and their distribution in the cells, while the fluorescence staining (LipidTox, LysoTracker and LC3) was used as a reference method. The results showed that CQ was not toxic to endothelial cells and did not result in the endothelial inflammation at concentrations of 1- 30 µM. Notwithstanding, it yielded an increased intensity of LipidTox, LysoTracker, and LC3 staining, suggesting changes in the content of neutral lipids, lysosomotropism, and autophagy inhibition, respectively. The CQ-induced endothelial response was associated with lipid accumulation and was characterized by Raman spectroscopy. CQ-induced autophagosome accumulation in the endothelium is featured by a pronounced alteration in the lipid profile, but not in the endothelial inflammation. Raman-based assessment of CQ-induced biochemical changes offers a better understanding of the autophagy mechanism in the endothelial cells.

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Radiation Response of Human Cardiac Endothelial Cells Reveals a Central Role of the cGAS-STING Pathway in the Development of Inflammation

Proteomes ◽

10.3390/proteomes8040030 ◽

2020 ◽

Vol 8 (4) ◽

pp. 30

Author(s):

Jos Philipp ◽

Ronan Le Gleut ◽

Christine von Toerne ◽

Prabal Subedi ◽

Omid Azimzadeh ◽

...

Keyword(s):

Cardiovascular Disease ◽

Dna Damage ◽

Endothelial Cells ◽

High Dose ◽

Dependent Manner ◽

Label Free ◽

Human Coronary Artery ◽

Free Data ◽

Radiation Induced ◽

Sting Pathway

Radiation-induced inflammation leading to the permeability of the endothelial barrier may increase the risk of cardiovascular disease. The aim of this study was to investigate potential mechanisms in vitro at the level of the proteome in human coronary artery endothelial cells (HCECest2) that were exposed to radiation doses of 0, 0.25, 0.5, 2.0 and 10 Gy (60Co-γ). Proteomics analysis was performed using mass spectrometry in a label-free data-independent acquisition mode. The data were validated using bioinformatics and immunoblotting. The low- and moderate-dose-irradiated samples (0.25 Gy, 0.5 Gy) showed only scarce proteome changes. In contrast, an activation of DNA-damage repair, inflammation, and oxidative stress pathways was seen after the high-dose treatments (2 and 10 Gy). The level of the DNA damage response protein DDB2 was enhanced early at the 10 Gy dose. The expression of proteins belonging to the inflammatory response or cGAS-STING pathway (STING, STAT1, ICAM1, ISG15) increased in a dose-dependent manner, showing the strongest effects at 10 Gy after one week. This study suggests a connection between the radiation-induced DNA damage and the induction of inflammation which supports the inhibition of the cGAS-STING pathway in the prevention of radiation-induced cardiovascular disease.

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Abstract WMP76: Clots are Potent Triggers of Inflammatory Cell Gene Expression - Indications for Timely Fibrinolysis in Stroke

Stroke ◽

10.1161/str.48.suppl_1.wmp76 ◽

2017 ◽

Vol 48 (suppl_1) ◽

Author(s):

Robert A Campbell ◽

Tammy Smith ◽

Adriana Vieira-de-Abreu ◽

Zechriah G Franks ◽

Jesse W Rowley ◽

...

Keyword(s):

Gene Expression ◽

Whole Blood ◽

Inflammatory Responses ◽

Vascular Diseases ◽

Clot Formation ◽

Clot Lysis ◽

Dependent Manner ◽

Plasma Clot ◽

Necrosis Factor Alpha

Rationale: Blood vessel wall damage often results in the formation of a fibrin clot that traps inflammatory cells, including monocytes. The effect of clot formation and subsequent lysis on the expression of monocyte-derived genes involved in the development and progression of ischemic stroke and other vascular diseases, however, is unknown. Objective: Determine if clot formation and lysis regulate the expression of human monocyte-derived genes that modulate vascular diseases. Methods and Results: We performed Next Generation RNA sequencing on monocytes extracted from whole blood clots. Thousands of mRNAs were significantly differentially expressed by monocytes from clotted versus unclotted whole blood (p>0.05), including upregulation of many inflammatory cytokines such as interleukin 8 (IL-8), IL-1β, tumor necrosis factor alpha, and monocyte chemoattractant protein-1 (MCP-1). Clotted plasma also increased expression of IL-8 and MCP-1, which far exceeded responses observed in LPS-stimulated monocytes. Upregulation of IL-8 and MCP-1 occurred in a thrombin-independent, but fibrin-dependent, manner. Fibrinolysis initiated shortly after plasma clot formation (i.e., 1-2 hours) reduced the global inflammatory response based on RNA-seq analysis and significantly reduced the synthesis of IL-8 and MCP-1 (p>0.05). Delayed fibrinolysis was far less effective in reducing inflammation. Consistent with these in vitro models, monocytes embedded in unresolved thrombi from patients undergoing thrombectomy stained positively for IL-8 and MCP-1. Conclusion: These findings demonstrate that clots are potent inducers of monocyte gene expression, and that timely fibrinolysis attenuates inflammatory responses. Dampening of inflammatory gene expression by timely clot lysis may contribute to the clinically-proven efficacy of fibrinolytic drug treatment within hours of stroke onset.

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