scholarly journals Massively calcified endosomal death (MCED) of endothelial cells

2014 ◽  
Author(s):  
Larry Weisenthal
Keyword(s):  
Endothelial Cells ◽  
Kinase Inhibitors ◽  
Culture Media ◽  
Vascular Inflammation ◽  
Primary Cultures ◽  
Human Tumor ◽  
Inflammatory Cells ◽  
Different Types ◽  
Human Sera ◽  
Contrast Exposure

We have discovered a novel and specific mechanism of endothelial cell death.We refer to this novel death mechanism as massively calcified endosomal death, or MCED. Exposure of endothelial cells to non-specific toxins or other physical stresses induces death by traditional apoptotic and non-apoptotic mechanisms, common to most different types of cells. In contrast, exposure of endothelial cells (but not other types of nucleated cells) to specific insults, such as oxidized pathogenic lipids (e.g. 7-ketocholesterol) or agents with known anti-angiogenic activity (e.g. bevacizumab, certain tyrosine kinase inhibitors, etc.) triggers cell death via a novel pathway, which involves the formation of massively calcified endosomes, which, in turn, escape from the dying endothelial cells as massively calcified exosomes. These endosomes/exosomes appear capable of provoking an inflammatory response, characterized by physical association of calcified microparticles with inflammatory cells (monocytes, lymphocytes, neutrophils) with resulting increased release of an inflammatory mediator (TNF) into the culture medium. Traditional media for the culture of endothelial cells are profoundly inhibitory to MCED, as are some mammalian sera and many human sera, explaining why MCED had not been previously discovered and reported. The present discovery of MCED was accidental, resulting from work with primary cultures of fresh human tumor cell clusters, which invariably contain microcapillary cells Our culture media are optimized for the tumor cells and not for the endothelial cells and, thus, are permissive of MCED. I propose MCED as the central mechanism underlying both intimal calcification and vascular inflammation in atherosclerosis.

scholarly journals SDPR/Cavin-2 loss inhibits monocyte adhesion to endothelial cells in abdominal aortic aneurysm via suppressing the expression of adhesion molecules

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
A Sakamoto ◽  
T Ogata ◽  
N Nakanishi ◽  
Y Higuchi ◽  
Y Tsuji ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Adhesion Molecules ◽  
Cell Adhesion Molecule ◽  
Vascular Inflammation ◽  
Inflammatory Cells ◽  
Elastin Degradation ◽  
Abdominal Aortic ◽  
Cell Adhesion Molecule 1

Abstract Background Abdominal aortic aneurysm (AAA) is a common and life-threatening vascular disease. The initial phase of AAA progression is vascular inflammation. Inflammation sites present adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1). These molecules play a crucial role in recruiting inflammatory cells to endothelial cells through NF-κB signaling. Endothelial cells express serum deprivation response (SDPR)/Cavin-2 localized in caveolae on the cell membrane. Although Cavin-2 is involved in such as cell proliferation, migration, and signal transduction, the role of Cavin-2 in vascular inflammation in the development of AAA is still unclear. Purpose To assess the influence of Cavin-2 deficiency in AAA development and clarify the role of Cavin-2 in the regulation of inflammatory cell adhesion in endothelial cells. Methods CaCl2-induced AAAs were induced by the periaortic application of 0.5 M CaCl2 in male SDPR-knockout (KO) and wild-type (WT) mice at 8–10 weeks of age. Angiotensin II (Ang II)-induced AAAs were created by 4-week-subcutaneous drug infusion in male ApoE-KO and ApoE/Cavin-2-double KO (DKO) mice at 24 weeks of age. Inflammatory response and cell adhesion were evaluated using human aortic endothelial cells (HAECs) and human monocytes (THP-1 cells). Results Six weeks after CaCl2 treatment, Cavin-2 deficiency significantly attenuated the development of AAAs. Elastin degradation was markedly suppressed and F4/80-positive macrophages infiltration in aortic walls were decreased in Cavin-2-KO mice. Although Ang II infusion for 4 weeks formed AAAs in ApoE KO mice and ApoE/Cavin-2-DKO mice, ApoE/Cavin-2-DKO mice exhibited the suppression of AAA formation independently of blood pressure. Immunohistochemical staining showed VCAM-1 expression on endothelial cells was suppressed in ApoE/Cavin-2-DKO mice. Further, in vitro co-culture experiment, the number of THP-1 cells adhered to TNF-treated SDPR-knockdown HAECs was decreased compared with that to control HAECs. Moreover, mRNA expression of VCAM-1 and ICAM-1 was decreased in TNFα-treated SDPR-knockdown HAECSs. Protein expression of VCAM-1 was also suppressed in TNFα-treated SDPR-knockdown HAECSs. The activity of NF-κB p65, an upstream regulator of VCAM-1 and ICAM-1,tended to be suppressed in TNFα-treated SDPR-knockdown HAECs. Conclusion In this study, we revealed that SDPR/Cavin-2 loss attenuated AAA development with the suppression of elastin degradation and macrophage infiltration. Our findings suggest that SDPR/Cavin-2 in the endothelial cells regulates the expression of adhesion molecules via NF-κB signaling and promotes the adhesion and infiltration of inflammatory cells to the aortic wall. Funding Acknowledgement Type of funding source: None

Abstract 210: Biosynthesis of D-series Resolvin by Isolated Vascular Cells and Tissues

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Sevan R Komshian ◽  
Anuran Chatterjee ◽  
Bian Wu ◽  
Giorgio Mottola ◽  
Mian Chen ◽  
...  
Keyword(s):  
Vascular Tissue ◽  
De Novo ◽  
Culture Media ◽  
Primary Cultures ◽  
Rabbit Aorta ◽  
Inflammatory Cells ◽  
Local Source ◽  
Vascular Cells ◽  
Resolvin D1

Introduction: Resolvin-D1 (RvD1) and other specialized pro-resolving lipid mediators (SPM) are synthesized in-vivo from docosahexaenoic acid (DHA) through transcellular pathways involving leukocytes. We investigated if vascular tissues, in the absence of inflammatory cells, can contribute to the local production of SPM. Methods: Primary cultures of human saphenous vein endothelial (EC) and smooth muscle (SMC) cells were supplemented with DHA in cell culture media (10% serum) for 4h-24h. Freshly harvested rabbit aorta was incubated intact or following gentle EC denudation in medium with or without DHA for 48h. RvD1 levels were quantified by ELISA, and lipoxygenase (LO) expression by western blotting. Results: In the absence of DHA supplementation, EC and SMC produced undetectable levels of RvD1. DHA treatment produced a dose and time-dependent increase in RvD1 production by EC and SMC (10.1 ±1.0 pg, 7.4 ±0.2 pg respectively; 1000nM DHA; 24h; Fig A, B). 5-LO expression was demonstrated in both cell types, however DHA induced increased 5-LO expression in EC (Fig C) but not in SMC. DHA-treated intact rabbit aorta segments produced 0.24±0.05 pg RvD1/mg tissue versus 0.13±0.01 pg RvD1/mg tissue in media alone. Moreover, EC-denuded aortas produced significantly less RvD1 (Fig D). Conclusions: Human vascular cells and rabbit vascular tissue can biosynthesize RvD1 de novo from its precursor DHA, signifying a potentially important local source of SPM in the vasculature.

Inhibition of Pro-Inflammatory Cytokine Secretion by Select Antioxidants in Human Coronary Artery Endothelial Cells

2020 ◽  
Vol 90 (1-2) ◽  
pp. 103-112 ◽  
Author(s):  
Michael J. Haas ◽  
Marilu Jurado-Flores ◽  
Ramadan Hammoud ◽  
Victoria Feng ◽  
Krista Gonzales ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Ascorbic Acid ◽  
Coronary Artery ◽  
Inflammatory Cytokines ◽  
Inflammatory Cytokine ◽  
Culture Media ◽  
Cytokine Secretion ◽  
Human Coronary Artery ◽  
Tnf Α ◽  
Pro Inflammatory Cytokines

Abstract. Inflammatory and oxidative stress in endothelial cells are implicated in the pathogenesis of premature atherosclerosis in diabetes. To determine whether high-dextrose concentrations induce the expression of pro-inflammatory cytokines, human coronary artery endothelial cells (HCAEC) were exposed to either 5.5 or 27.5 mM dextrose for 24-hours and interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor α (TNF α) levels were measured by enzyme immunoassays. To determine the effect of antioxidants on inflammatory cytokine secretion, cells were also treated with α-tocopherol, ascorbic acid, and the glutathione peroxidase mimetic ebselen. Only the concentration of IL-1β in culture media from cells exposed to 27.5 mM dextrose increased relative to cells maintained in 5.5 mM dextrose. Treatment with α-tocopherol (10, 100, and 1,000 μM) and ascorbic acid (15, 150, and 1,500 μM) at the same time that the dextrose was added reduced IL-1β, IL-6, and IL-8 levels in culture media from cells maintained at 5.5 mM dextrose but had no effect on IL-1β, IL-6, and IL-8 levels in cells exposed to 27.5 mM dextrose. However, ebselen treatment reduced IL-1β, IL-6, and IL-8 levels in cells maintained in either 5.5 or 27.5 mM dextrose. IL-2 and TNF α concentrations in culture media were below the limit of detection under all experimental conditions studied suggesting that these cells may not synthesize detectable quantities of these cytokines. These results suggest that dextrose at certain concentrations may increase IL-1β levels and that antioxidants have differential effects on suppressing the secretion of pro-inflammatory cytokines in HCAEC.

Natriuretic Peptides Regulate the Expression of Tissue Factor and PAI-1 in Endothelial Cells

1999 ◽  
Vol 82 (11) ◽  
pp. 1497-1503 ◽  
Author(s):  
Hajime Tsuji ◽  
Hiromi Nishimura ◽  
Haruchika Masuda ◽  
Yasushi Kunieda ◽  
Hidehiko Kawano ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Plasminogen Activator ◽  
Natriuretic Peptide ◽  
Culture Media ◽  
Tissue Type ◽  
Dependent Manner ◽  
Ang Ii ◽  
Plasminogen Activator Inhibitor 1 ◽  
Pai 1

SummaryIn the present study, we demonstrate that brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) interact with angiotensin II (Ang II) in regulative blood coagulation and fibrinolysis by suppressing the expressions of both tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) induced by Ang II. The expressions of TF and PAI-1 mRNA were analyzed by northern blotting methods, and the activities of TF on the surface of rat aortic endothelial cells (RAECs) and PAI-1 in the culture media were respectively measured by chromogenic assay.Both BNP and CNP suppressed the expressions of TF and PAI-1 mRNA induced by Ang II in a time- and concentration-dependent manner via cGMP cascade, which suppressions were accompanied by respective decrease in activities of TF and PAI-1. However, neither the expression of tissue factor pathway inhibitor (TFPI) nor tissue-type plasminogen activator (TPA) mRNA was affected by the treatment of BNP and CNP.

Studies of the Factor Xa-Dependent Inhibitor of Factor VIIa/Tissue Factor (Extrinsic Pathway Inhibitor) from Cell Supernates of Cultured Human Umbilical Vein Endothelial Cells

1989 ◽  
Vol 61 (01) ◽  
pp. 101-105 ◽  
Author(s):  
Bonnie J Warn-Cramer ◽  
Fanny E Almus ◽  
Samuel I Rapaport
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Tissue Factor ◽  
Phorbol Ester ◽  
Umbilical Vein ◽  
Primary Cultures ◽  
Factor Xa ◽  
Extrinsic Pathway ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells

SummaryCultured human umbilical vein endothelial cells (HUVEC) have been reported to produce extrinsic pathway inhibitor (EPI), the factor Xa-dependent inhibitor of factor VHa/tissue factor (TF). We examined the release of this inhibitor from HUVEC as a function of their growth state and in response to the induction of endothelial cell TF activity. HUVEC constitutively produced significant amounts of EPI at all stages of their growth in culture including the post-confluent state. Rate of release varied over a 3-fold range for primary cultures from 12 different batches of pooled umbilical cord cells. Constitutive EPI release was unaltered during a 6 hour period of induction of TF activity with thrombin or phorbol ester but slowed during longer incubation of the cells with phorbol ester. Whereas plasma contains two molecular weight forms of EPI, only the higher of these two molecular weight forms was demonstrable by Western analysis of HUVEC supernatants with 125I-factor Xa as the ligand.

Specificity of the Thrombin-Induced Release of Tissue Plasminogen Activator from Cultured Human Endothelial Cells

1986 ◽  
Vol 56 (02) ◽  
pp. 115-119 ◽  
Author(s):  
Eugene G Levin ◽  
David M Stern ◽  
Peter P Nawroth ◽  
Richard A Marlar ◽  
Daryl S Fair ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Protein C ◽  
Primary Cultures ◽  
Activated Protein C ◽  
Specific Inhibitor ◽  
Factor Xa ◽  
Human Endothelial Cells ◽  
Tissue Plasminogen

SummaryThe addition of thrombin (9 nM) to primary cultures of human endothelial cells induces a 6- to 7-fold increase in the rate of release of tissue plasminogen activator (tPA). Several other serine proteases which specifically interact with endothelial cells were also analyzed for their effect on tPA release. Gamma-thrombin, an autocatalytic product of α-thrombin, promoted tPA release but was less effective than α-thrombin. A maximum increase of 5.5-fold was observed, although a concentration of γ-thrombin 20 times greater than α-thrombin was required. The response to Factor Xa was similar to α-thrombin, although the stimulation was significantly reduced by the addition of hirudin or DAPA suggesting that prothrombin activation was occurring. The simultaneous addition of prothrombin with Factor Xa resulted in enhanced tPA release equal to that observed with an equimolar concentration of active α-thrombin. Thus, under these conditions, Factor Xa-cell surface mediated activation of prothrombin can lead to a secondary effect resulting from cell-thrombin interaction. Activated protein C, which has been implicated as a profibrinolytic agent, was also tested. No change in tPA release occurred after the addition of up to 325 nM activated protein C in the presence or absence of proteins. Factor IXa and plasmin were also ineffective. The effect of thrombin on the endothelial cell derived plasminogen activator specific inhibitor was also studied. Thrombin produced a small but variable release of the inhibitor with an increase of less than twice that of non-thrombin treated controls.

Nephrotoxicity Testing In Vitro: The Current Situation

1997 ◽  
Vol 25 (5) ◽  
pp. 497-503
Author(s):  
Jean-Paul Morin ◽  
Marc E. De Broe ◽  
Walter Pfaller ◽  
Gabriele Schmuck
Keyword(s):  
Proximal Tubule ◽  
Culture Media ◽  
Task Force ◽  
Primary Cultures ◽  
Phenotypic Expression ◽  
Transepithelial Transport ◽  
Specific Cell ◽  
Proximal Tubule Cells ◽  
Tubule Cells

An ECVAM task force on nephrotoxicity has been established to advise, in particular, on the follow-up to recommendations made in the ECVAM workshop report on nephrotoxicity testing in vitro. Since this workshop was held, in 1994, there have been several improvements in the techniques used. For example, the duration of renal slice viability, and the maintenance of functional activities in slices, have been improved by using dynamic incubation systems with higher oxygen tensions and more-appropriate cell culture media. Highly differentiated primary cultures of pig, human and rabbit proximal tubule cells have been established by using specific cell isolation procedures and/or selective culture media. To date, the most comparable phenotypic expression and transepithelial transport capacities to proximal tubules in vivo have been obtained with primary cultures of rabbit proximal tubule cells which are grown on bicompartmental supports; in this system, transepithelial substrate gradients are generated and the transepithelial transport of both organic anions and cations is highly active. This in vitro system has been selected by ECVAM for further evaluation and prevalidation. Industrial needs in the area of nephrotoxicity testing have been identified, and recommendations are made at the end of this report concerning possible future initiatives.

scholarly journals Vascular Inflammation Is Associated with Loss of Aquaporin 1 Expression on Endothelial Cells and Increased Fluid Leakage in SARS-CoV-2 Infected Golden Syrian Hamsters

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 639
Author(s):  
Lisa Allnoch ◽  
Georg Beythien ◽  
Eva Leitzen ◽  
Kathrin Becker ◽  
Franz-Josef Kaup ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Vascular Inflammation ◽  
Intercellular Junctions ◽  
Edema Formation ◽  
Vascular Integrity ◽  
Syrian Hamsters ◽  
Aquaporin 1 ◽  
Vascular Walls ◽  
Transmission Electron ◽  
Mock Infection

Vascular changes represent a characteristic feature of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection leading to a breakdown of the vascular barrier and subsequent edema formation. The aim of this study was to provide a detailed characterization of the vascular alterations during SARS-CoV-2 infection and to evaluate the impaired vascular integrity. Groups of ten golden Syrian hamsters were infected intranasally with SARS-CoV-2 or phosphate-buffered saline (mock infection). Necropsies were performed at 1, 3, 6, and 14 days post-infection (dpi). Lung samples were investigated using hematoxylin and eosin, alcian blue, immunohistochemistry targeting aquaporin 1, CD3, CD204, CD31, laminin, myeloperoxidase, SARS-CoV-2 nucleoprotein, and transmission electron microscopy. SARS-CoV-2 infected animals showed endothelial hypertrophy, endothelialitis, and vasculitis. Inflammation mainly consisted of macrophages and lower numbers of T-lymphocytes and neutrophils/heterophils infiltrating the vascular walls as well as the perivascular region at 3 and 6 dpi. Affected vessels showed edema formation in association with loss of aquaporin 1 on endothelial cells. In addition, an ultrastructural investigation revealed disruption of the endothelium. Summarized, the presented findings indicate that loss of aquaporin 1 entails the loss of intercellular junctions resulting in paracellular leakage of edema as a key pathogenic mechanism in SARS-CoV-2 triggered pulmonary lesions.

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