scholarly journals Highly Glycolytic Immortalized Human Dermal Microvascular Endothelial Cells are Able to Grow in Glucose-Starved Conditions

Biomolecules ◽  
2019 ◽  
Vol 9 (8) ◽  
pp. 332 ◽  
Author(s):  
Ocaña ◽  
Martínez-Poveda ◽  
Quesada ◽  
Medina
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Metabolism ◽  
Monocarboxylate Transporter ◽  
Microvascular Endothelial Cells ◽  
Substrate Uptake ◽  
Monocarboxylate Transporter 1 ◽  
Extracellular Lactate ◽  
Microvascular Endothelial ◽  
Reported Data

Endothelial cells form the inner lining of blood vessels, in a process known as angiogenesis. Excessive angiogenesis is a hallmark of several diseases, including cancer. The number of studies in endothelial cell metabolism has increased in recent years, and new metabolic targets for pharmacological treatment of pathological angiogenesis are being proposed. In this work, we wanted to address experimental evidence of substrate (namely glucose, glutamine and palmitate) dependence in immortalized dermal microvascular endothelial cells in comparison to primary endothelial cells. In addition, due to the lack of information about lactate metabolism in this specific type of endothelial cells, we also checked their capability of utilizing extracellular lactate. For fulfilling these aims, proliferation, migration, Seahorse, substrate uptake/utilization, and mRNA/protein expression experiments were performed. Our results show a high glycolytic capacity of immortalized dermal microvascular endothelial cells, but an early independence of glucose for cell growth, whereas a total dependence of glutamine to proliferate was found. Additionally, in contrast with reported data in other endothelial cell lines, these cells lack monocarboxylate transporter 1 for extracellular lactate incorporation. Therefore, our results point to the change of certain metabolic features depending on the endothelial cell line.

Isolation and characterization of human and rat cardiac microvascular endothelial cells

1993 ◽  
Vol 264 (2) ◽  
pp. H639-H652 ◽  
Author(s):  
M. Nishida ◽  
W. W. Carley ◽  
M. E. Gerritsen ◽  
O. Ellingsen ◽  
R. A. Kelly ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Microvascular Endothelial Cells ◽  
Microvascular Endothelium ◽  
Adult Rat ◽  
Isolation And Characterization ◽  
Ventricular Tissue ◽  
Microvascular Endothelial ◽  
Cardiac Microvascular Endothelial Cells

Although reciprocal intercellular signaling may occur between endocardial or microvascular endothelium and cardiac myocytes, suitable in vitro models have not been well characterized. In this report, we describe the isolation and primary culture of cardiac microvascular endothelial cells (CMEC) from both adult rat and human ventricular tissue. Differential uptake of fluorescently labeled acetylated low-density lipoprotein (Ac-LDL) indicated that primary isolates of rat CMEC were quite homogeneous, unlike primary isolates of human ventricular tissue, which required cell sorting based on Ac-LDL uptake to create endothelial cell-enriched primary cultures. The endothelial phenotype of both primary isolates and postsort subcultured CMEC and their microvascular origin were determined by characteristic histochemical staining for a number of endothelial cell-specific markers, by the absence of cells with fibroblast or pericyte-specific cell surface antigens, and by rapid tube formation on purified basement membrane preparations. Importantly, [3H]-thymidine uptake was increased 2.3-fold in subconfluent rat microvascular endothelial cells 3 days after coculture with adult rat ventricular myocytes because of release of an endothelial cell mitogen(s) into the extracellular matrix, resulting in a 68% increase in cell number compared with CMEC in monoculture. Thus biologically relevant cell-to-cell interactions can be modeled with this in vitro system.

Abstract 235: Hypoxia Enhanced Delivery of Mitochondria-Targeted Catalase Protects Choroid Retinal Microvascular Endothelial Cells from Oxidative Stress

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Christopher J Dougherty ◽  
Howard Prentice ◽  
Kathleen Dorey ◽  
Keith A Webster ◽  
Janet C Blanks
Keyword(s):  
Oxidative Stress ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
High Glucose ◽  
Endothelial Permeability ◽  
Luciferase Assay ◽  
Expression Vectors ◽  
Microvascular Endothelial Cells ◽  
Tissue Specific ◽  
Microvascular Endothelial

Loss of pericytes is a critical event early in the progression of microvascular dysfunction in diabetic retinopathy. Pericyte loss may be linked to high glucose mediated reactive oxygen species generation, blocking N-cadherin trafficking to the endothelial cell surface preventing pericyte recruitment and vessel stabilization. Hydrogen peroxide has been identified as a major free radical produced during high glucose exposure in endothelial cells. The goal of this research is to determine if tissue-specific hypoxia-regulated expression of a mitochondria-targeted catalase can prevent or limit RF/6A microvascular endothelial cell apoptosis and decrease vascular permeability by limiting cellular oxidative stress. For the development of tissue-specific and hypoxia-enhanced expression vectors, promoters were constructed with nine tandem combinations of HREs. This 9x HRE oligomer enhancer was inserted together into a pGL3 firefly luciferase plasmid with the Tie2( short ) promoter for endothelial-specific expression. The 9xHRE-Tie2( sh ) promoter construct was highly selective for RF/6A cells producing a basal amount of mitochondria-targeted catalase equivalent to the Tie2( short ) promoter alone. In response to hypoxia ( pO 2 = 1% ), the 9xHRE-Tie2( short ) promoter showed a 21-fold hypoxia-inducible activation similar in strength to the CMV promoter , measured by dual luciferase assay. The hybrid promoters were incorporated into a replication deficient AAV delivery system for apoptosis and cell culture based endothelial permeability assays. In preliminary assays using RF/6A microvascular endothelial cells, apoptosis was reduced by 58% and permeability was reduced by 46%. The results suggest that mitochondria-targeted catalase protects RF/6A microvascular endothelial cells from apoptosis and reduces endothelial permeability in a high-glucose, low-oxygen environment.

Characterization of adhesion receptors on cultured microvascular endothelial cells derived from the retroorbital connective tissue of patients with Graves' ophthalmopathy

1996 ◽  
Vol 134 (1) ◽  
pp. 51-60 ◽  
Author(s):  
Armin E Heufelder ◽  
Peter C Scriba
Keyword(s):  
T Cells ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Connective Tissue ◽  
Adhesion Molecule ◽  
Microvascular Endothelial Cells ◽  
Cell Populations ◽  
Graves Ophthalmopathy ◽  
Microvascular Endothelial

Heufelder AE, Scriba PC. Characterization of adhesion receptors on cultured microvascular endothelial cells derived from the retroorbital connective tissue of patients with Graves' ophthalmopathy. Eur J Endocrinol 1996:134:51–60 T lymphocytes have been demonstrated recently to play an important role in the pathogenesis and propagation of Graves' ophthalmopathy (GO). Recruitment of T cells to the retroorbital tissue in GO involves the activation of certain adhesion molecules both in the vascular endothelium and in the extravascular connective tissue within the retroorbital space. To characterize the interactions between orbital endothelial cells (OECs) and circulating T cells in vitro, we designed a two-step immunopurification procedure with bead-immobilized Ulex europaeus I lectin and anti-human endothelial cell antigen (CD3I) monoclonal antibody for rapid and reproducible isolation of highly pure microvascular endothelial cell populations from small quantities of retroorbital connective tissue. Endothelial origin of the resulting cell populations was confirmed by positive immunoreactivity for von Willebrand factor. CD 3 I and thrombomodulin. Under baseline conditions, GO-OECs, but not normal OECs, expressed intercellular adhesion molecule 1 (ICAM-1) and CD44 immunoreactivity but no immunoreactivity for endothelial leukocyte adhesion molecule I (ELAM-1) and vascular cell adhesion molecule I (VCAM-1) was detected. Exposure of GO-OEC and normal OEC monolayers to interferon γ, interleukin 1 α and tumor necrosis factor α resulted in marked up-regulation of immunoreactivity for ICAM-1 and in induction of ELAM-1 and VCAM-1. Blocking experiments using monoclonal antibodies directed against various adhesion molecules demonstrated that interactions between matched activated T lymphocytes and OECs were mediated by integrin-dependent ICAM-1/leukocyte function-associated antigen 1 (LFA-1): VCAM-1/very late antigen 4 (VLA-4)) and integrin-independent (CD44) pathways, and revealed marked differences when comparing GO-OECs and normal OECs. In conclusion, the availability of OECs from affected retroorbital tissue of patients with GO provides a valuable tool for studying further the mechanisms responsible for orbit-specific lymphocyte recruitment in GO. Armin E Heufelder, Molecular Thyroid Research Unit, Medizinische Klinik, Klinikum Innenstadt, Ziemssenstrasse 1, 80336 München, Germany

scholarly journals EPAC2 acts as a negative regulator in Matrigel-driven tubulogenesis of human microvascular endothelial cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Takayuki Ikeda ◽  
Yoshino Yoshitake ◽  
Yasuo Yoshitomi ◽  
Hidehito Saito-Takatsuji ◽  
Yasuhito Ishigaki ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Morphology ◽  
Molecular Mechanisms ◽  
Network Formation ◽  
Tissue Growth ◽  
Negative Regulator ◽  
Endothelial Cell Migration ◽  
Microvascular Endothelial Cells ◽  
Microvascular Endothelial

AbstractAngiogenesis is physiologically essential for embryogenesis and development and reinitiated in adult animals during tissue growth and repair. Forming new vessels from the walls of existing vessels occurs as a multistep process coordinated by sprouting, branching, and a new lumenized network formation. However, little is known regarding the molecular mechanisms that form new tubular structures, especially molecules regulating the proper network density of newly formed capillaries. This study conducted microarray analyses in human primary microvascular endothelial cells (HMVECs) plated on Matrigel. The RAPGEF4 gene that encodes exchange proteins directly activated by cAMP 2 (EPAC2) proteins was increased in Matrigel-driven tubulogenesis. Tube formation was suppressed by the overexpression of EPAC2 and enhanced by EPAC2 knockdown in endothelial cells. Endothelial cell morphology was changed to round cell morphology by EPAC2 overexpression, while EPAC2 knockdown showed an elongated cell shape with filopodia-like protrusions. Furthermore, increased EPAC2 inhibited endothelial cell migration, and ablation of EPAC2 inversely enhanced cell mobility. These results suggest that EPAC2 affects the morphology and migration of microvascular endothelial cells and is involved in the termination and proper network formation of vascular tubes.

Effect of Thrombin on the Production of Plasminogen Activators and PA Inhibitor-1 by Human Foreskin Microvascular Endothelial Cells

1987 ◽  
Vol 57 (02) ◽  
pp. 148-153 ◽  
Author(s):  
Victor W M Van Hinsbergh ◽  
Erik D Sprengers ◽  
Teake Kooistra
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Plasminogen Activator ◽  
Polyacrylamide Gel Electrophoresis ◽  
Tissue Type ◽  
Microvascular Endothelial Cells ◽  
Inhibitor Activity ◽  
Microvascular Endothelial ◽  
High Concentrations ◽  
Microvascular Cells

SummaryHuman foreskin microvascular endothelial cells synthesize and release tissue-type plasminogen activator (t-PA) in similar amounts as do endothelial cells from umbilical cord artery and vein. Human thrombin increases the production of t-PA by these cells, which could be visualized from 8 h after addition of 0.1-5 units/ml thrombin by fibrin autography after SDS polyacrylamide gel electrophoresis of the endothelial cell conditioned media. Thrombin also increased the secretion of t-PA antigen. Together with t-PA, human microvascular cells release urokinasetype plasminogen activator (u-PA) antigen and endothelial cell-type PA inhibitor, PA inhibitor-1, which were both demonstrated by specific immunoprécipitation from radiolabeled endothelial cell conditioned medium. Thrombin increases the release of u-PA antigen, but no u-PA activity could be demonstrated. Thrombin induced a two-fold stimulation of the synthesis and secretion of PA inhibitor-1 antigen. At 0.1 unit/ml thrombin also an increase in PA inhibitor activity was found. At high concentrations of thrombin a decrease of PA inhibitor activity was found, due to the conversion of the active 46 kD PA inhibitor-1 into a 42 kD product without PA inhibitor activity. Our data indicate that interaction of thrombin with microvascular endothelial cells will shift the balance between t-PA, u-PA and PA inhibitor-1, and thus affects the regulation of fibrinolysis.

scholarly journals Type 5 phosphodiesterase expression is a critical determinant of the endothelial cell angiogenic phenotype

2009 ◽  
Vol 296 (2) ◽  
pp. L220-L228 ◽  
Author(s):  
Bing Zhu ◽  
Li Zhang ◽  
Mikhail Alexeyev ◽  
Diego F. Alvarez ◽  
Samuel J. Strada ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Network Formation ◽  
Microvascular Endothelial Cells ◽  
Critical Determinant ◽  
Angiogenic Potential ◽  
Matrigel Plug ◽  
Microvascular Endothelial

Type 5 phosphodiesterase (PDE5) inhibitors increase endothelial cell cGMP and promote angiogenesis. However, not all endothelial cell phenotypes express PDE5. Indeed, whereas conduit endothelial cells express PDE5, microvascular endothelial cells do not express this enzyme, and they are rapidly angiogenic. These findings bring into question whether PDE5 activity is a critical determinant of the endothelial cell angiogenic potential. To address this question, human full-length PDE5A1 was stably expressed in pulmonary microvascular endothelial cells. hPDE5A1 expression reduced the basal and atrial natriuretic peptide (ANP)-stimulated cGMP concentrations in these cells. hPDE5A1-expressing cells displayed attenuated network formation on Matrigel in vitro and also produced fewer blood vessels in Matrigel plug assays in vivo; the inhibitory actions of hPDE5A1 were reversed using sildenafil. To examine whether endogenous PDE5 activity suppresses endothelial cell angiogenic potential, small interfering RNA (siRNA) constructs were stably expressed in pulmonary artery endothelial cells. siRNA selectively decreased PDE5 expression and increased basal and ANP-stimulated cGMP concentrations in these conduit cells. PDE5 downregulation increased network formation on Matrigel in vitro and increased blood vessel formation in Matrigel plug assays in vivo. Collectively, our results indicate that PDE5 activity is an essential determinant of angiogenesis and suggest that PDE5 downregulation in microvascular endothelium imparts a stable, enhanced angiogenic potential to this cell type.

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