Abstract P419: Revealing The Dynamics Of Vesicle Formation During Clathrin-mediated Endocytosis In Living Endothelial Cells

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Tomasz Nawara ◽  
Tejeshwar Rao ◽  
Alexa Mattheyses
Keyword(s):  
Endothelial Cells ◽  
Osmotic Pressure ◽  
De Novo ◽  
Umbilical Vein ◽  
Living Cells ◽  
Future Research ◽  
Vesicle Formation ◽  
Test Bed ◽  
High Throughput Analysis ◽  
Physiological Conditions

Clathrin-mediated endocytosis (CME) is an essential cellular process for internalizing nutrients and therapeutics at endothelial cell barriers. Studying the formation of cargo containing endocytic vesicles in living cells is challenging due to the limited resolution of fluorescence microscopy and the highly dynamic nature of CME. Moreover, it is currently unknown how the physiological conditions present in vasculature affect CME in endothelial cells. To address this challenge we used a novel microscopy approach, Simultaneous Two-wavelength Axial Ratiometry (STAR), to image vesicle formation dynamics with nanometer axial resolution in living cells. High-throughput analysis revealed that 80% of de novo clathrin accumulations contributed to endocytosis while 20% remained flat, consistent in both human umbilical vein endothelial cells (HUVECs) and our test-bed model green monkey kidney fibroblast-like (Cos-7) cells. We next investigated the interplay between coat curvature and clathrin accumulation in vesicle initiation to identify the mechanism of vesicle formation. Our results support the flexible model of vesicle formation with curvature and clathrin accumulation initiating together at shorter-lived vesicles (<20s) and through a flat-to-curved transition of clathrin lattices at longer-lived vesicles (>20s). Finally, we addressed if physiological conditions present in vasculature alter the dynamics of vesicle formation. We show that increasing osmotic pressure decreased the total number of internalizations but had no impact on the number of flat clathrin accumulations or the mechanism of vesicle formation in Cos-7 cells. In future research, we will test the hypothesis that HUVECs have distinct mechanisms to retain vesicle formation under osmotic pressure or shear stress conditions similar to their native environment. Additionally targeted drug delivery to vascular endothelial cells, for example nanocarriers binding to flat lattices or CCVs leading to different therapeutic outcomes or bioavailability, can potentially be informed by identifying clathrin morphology and dynamics and the mechanisms of endocytosis using STAR microscopy.

Platelets Stimulate Thromboplastin Synthesis in Human Endothelial Cells

1983 ◽  
Vol 49 (02) ◽  
pp. 069-072 ◽  
Author(s):  
U L H Johnsen ◽  
T Lyberg ◽  
K S Galdal ◽  
H Prydz
Keyword(s):  
Endothelial Cells ◽  
De Novo ◽  
Umbilical Vein ◽  
Time Dependent ◽  
De Novo Synthesis ◽  
Human Endothelial Cells ◽  
Tumor Promotor ◽  
Coagulation Assay ◽  
Platelet Aggregates ◽  
Umbilical Vein Endothelial Cells

SummaryHuman umbilical vein endothelial cells in culture synthesize thromboplastin upon stimulation with phytohaemagglutinin (PHA) or the tumor promotor 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The thromboplastin activity is further strongly enhanced in a time dependent reaction by the presence of gel-filtered platelets or platelet aggregates. This effect was demonstrable at platelet concentrations lower than those normally found in plasma, it may thus be of pathophysiological relevance. The thromboplastin activity increased with increasing number of platelets added. Cycloheximide inhibited the increase, suggesting that de novo synthesis of the protein component of thromboplastin, apoprotein III, is necessary.When care was taken to remove monocytes no thromboplastin activity and no apoprotein HI antigen could be demonstrated in suspensions of gel-filtered platelets, platelets aggregated with thrombin or homogenized platelets when studied with a coagulation assay and an antibody neutralization technique.

scholarly journals Alpha-thrombin induces release of platelet-derived growth factor-like molecule(s) by cultured human endothelial cells.

1986 ◽  
Vol 103 (3) ◽  
pp. 1129-1133 ◽  
Author(s):  
J M Harlan ◽  
P J Thompson ◽  
R R Ross ◽  
D F Bowen-Pope
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Time Course ◽  
De Novo ◽  
Umbilical Vein ◽  
Active Form ◽  
Platelet Derived Growth Factor ◽  
Blue Dye ◽  
Serine Esterase ◽  
Freeze Thaw

Cultured endothelial cells secrete a platelet-derived growth factor-like molecule (PDGFc). We examined the effects of purified human alpha-thrombin on the production of PDGFc in cultures of human umbilical vein endothelial cells (HUVE) using a specific radioreceptor assay for PDGF. Addition of physiologically relevant concentrations of alpha-thrombin (0.1 to 10 U/ml) induced a time- and dose-dependent increase in the release of PDGFc into the culture medium. Significant stimulation of PDGFc release was observed as early as 1.5 h after addition of alpha-thrombin (10 U/ml) with a 4.9 +/- 1.1 fold increase at 24 h (mean +/- SEM of nine experiments, P less than 0.01). alpha-Thrombin treatment of HUVE did not affect cell viability as assessed by trypan blue dye exclusion. The receptor binding of PDGFc secreted by HUVE in response to alpha-thrombin was inhibited by monospecific antibody to purified human PDGF indicating that the molecule(s) is closely related to PDGF. alpha-Thrombin inactivated with diisopropylfluorophosphate was without stimulatory effect. Lysis of HUVE by repeated cycles of freeze/thaw released minimal PDGFc (less than 0.3 ng per 10(6) cells) compared to levels of PDGFc released into supernatant medium in response to alpha-thrombin (greater than 5.0 ng per 10(6) cells after a 24-h incubation with 10 U/ml alpha-thrombin). Moreover, incubation of freeze/thaw lysates of HUVE with alpha-thrombin failed to release PDGFc. Over a 3-h time course, however, alpha-thrombin-induced secretion of PDGFc was not prevented by cycloheximide. We conclude that alpha-thrombin induces secretion of PDGFc from HUVE by a nonlytic mechanism requiring the serine esterase activity of the enzyme. Although this effect does not initially require de novo protein synthesis, it does require cell-mediated conversion of PDGFc from an inactive to an active form.

Refractory period phenomenon in the induction of tissue factor expression on endothelial cells

Blood ◽  
1991 ◽  
Vol 78 (8) ◽  
pp. 2027-2035 ◽  
Author(s):  
N Busso ◽  
S Huet ◽  
E Nicodeme ◽  
J Hiernaux ◽  
F Hyafil
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Refractory Period ◽  
Messenger Rna ◽  
De Novo ◽  
Umbilical Vein ◽  
Interleukin 1 ◽  
Tnf Alpha ◽  
Extrinsic Pathway ◽  
Necrosis Factor Alpha

Tissue factor (TF) is the first factor of the extrinsic pathway of coagulation. Normally, TF is not expressed on the surface of endothelial cells. However, expression of TF can be induced in these cells in response to stimulation by diverse inflammatory mediators such as interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF- alpha), lipopolysaccharide (LPS), and phorbol 12-myristate 13-acetate (PMA). We have studied the effect of these mediators on the kinetics of the induction of TF-related procoagulant activity (PCA) on human umbilical vein endothelial cells (HUVECs). PCA is transiently induced on HUVECs, attaining a peak some 4 to 8 hours after addition of inflammatory agents, with maximal accumulation of TF messenger RNA (mRNA) occurring 3 to 5 hours earlier. Because the expression of PCA by treated HUVECs returns to basal levels by 20 to 30 hours, we examined the response of these cells to a second inflammatory stimulus. Continuous incubation of cells with a single inflammatory agent for 24 to 48 hours induces a hyporesponsive state with respect to the reinduction of TF expression by the same agent (14% of the initial stimulation for IL-1 beta, 39% for TNF-alpha 30% for LPS, and 7% for PMA). Such a diminution in PCA was also observed in the levels of TF mRNA. By contrast, pretreatment of HUVECs with one agent did not dramatically affect the reinduction of TF by any of the three other factors. We subsequently focused our attention on the induction of the autologous refractory period by IL-1 beta. De novo protein synthesis was not required during the preincubation of ECs for hyporesponsiveness to be observed. The establishment of the refractory state did not depend on the downmodulation of IL-1 beta receptor affinity or expression. Moreover, pretreatment of HUVECs with IL-1 beta increased prostacyclin (PGI2) production in response to a second stimulation by IL-1 beta, although such cells were unable to reexpress TF under the same conditions. This result suggests that distinct secondary messenger pathways are involved in TF induction and PGI2 synthesis by IL-1 beta in HUVECs.

Thrombin Induces Thromboplastin Synthesis in Cultured Vascular Endothelial Cells

1985 ◽  
Vol 54 (02) ◽  
pp. 373-376 ◽  
Author(s):  
K S Galdal ◽  
T Lyberg ◽  
S A Evensen ◽  
E Nilsen ◽  
H Prydz
Keyword(s):  
Endothelial Cells ◽  
De Novo ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Calf Serum ◽  
Phosphodiesterase Inhibitors ◽  
Fold Increase ◽  
Homocysteine Thiolactone ◽  
Umbilical Vein Endothelial Cells

SummaryCultured human umbilical vein endothelial cells responded to thrombin (10−2 – 10 NIH u/ml) with a 2-5 fold increase in thromboplastin activity. The maximum response was reached after 4 hr in serum-free medium. The effect of thrombin was fully inhibited by the presence of 50% (v/v) fetal calf serum or more in the medium, by preincubation of thrombin with hirudin or by treatment of thrombin with N-bromosuccinimide or phenylmethylsulfonyl fluoride. The thrombin-induced thromboplastin activity was inhibited by incubation of the cells with cycloheximide (2 μg/ml) or actinomycin D (2 μg/ml) showing that the response depended on de novo protein and RNA synthesis. It was also suppressed by exposure of the cells to two different phosphodiesterase inhibitors, 3-butyl-l-methyl-xanthine (5 · 10−4 M) and rac-4 (3-butoxy-4-methoxybenzyl)-2-imidazole (5 · 10−4 M), to the transmethylation inhibitors 3-deazaadenosine (10−5 M) and 1-homocysteine thiolactone (2 · 10−5 M) in combination and to the intracellular calcium antagonist 8-(N,N-diethylamino)-octyl 3,4,5,-tri-methoxybenzoate hydrochloride (8 · 10−5 M). Our results suggest that small amounts of thrombin can induce thromboplastin synthesis in endothelial cells in vitro and that this synthesis probably is regulated by the intracellular level of cAMP, by cytoplasmic Ca2+ and possibly also by transmethylation reactions.

Fluvastatin Inhibits Basal and Stimulated Plasminogen Activator Inhibitor 1, but Induces Tissue Type Plasminogen Activator in Cultured Human Endothelial Cells

2000 ◽  
Vol 84 (07) ◽  
pp. 59-64 ◽  
Author(s):  
Luciana Mussoni ◽  
Cristina Banfi ◽  
Luigi Sironi ◽  
Magda Arpaia ◽  
Elena Tremoli
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
De Novo ◽  
Umbilical Vein ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Mrna Levels ◽  
Plasminogen Activator Inhibitor 1 ◽  
Activator Inhibitor ◽  
Pai 1

SummaryThe effects of fluvastatin, a synthetic hydroxymethylglutaryl coenzyme A (HMG-CoA) inhibitor, on the biosynthesis of tissue plasminogen activator (t-PA) and of its major physiological inhibitor (plasminogen activator inhibitor type 1, PAI-1) were investigated in cultured human umbilical vein endothelial cells (HUVEC). Fluvastatin (0.1 to 2.5 µM), concentration-dependently reduced the release of PAI-1 antigen by unstimulated HUVEC, subsequent to a reduction in PAI-1 steady-state mRNA levels and de novo protein synthesis. In contrast, it increased t-PA secretion.The drug also reduced PAI-1 antigen secreted in response to 10 µg/ml bacterial lipopolysaccharide (LPS), 100 U/ml tumour necrosis factor α (TNFα) or 0.1 µM phorbol myristate acetate (PMA).Mevalonate (100 µM), a precursor of isoprenoids, added to cells simultaneously with fluvastatin, suppressed the effect of the drug on PAI-1 both in unstimulated and stimulated cells as well as on t-PA antigen. Among intermediates of the isoprenoid pathway, all-trans-geranylgeraniol (5 µM) but not farnesol (10 µM) prevented the effect of 2.5 µM fluvastatin on PAI-1 antigen, which suggests that the former intermediate of the isoprenoid synthesis is responsible for the observed effects.

scholarly journals Refractory period phenomenon in the induction of tissue factor expression on endothelial cells

Blood ◽  
1991 ◽  
Vol 78 (8) ◽  
pp. 2027-2035 ◽  
Author(s):  
N Busso ◽  
S Huet ◽  
E Nicodeme ◽  
J Hiernaux ◽  
F Hyafil
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Refractory Period ◽  
Messenger Rna ◽  
De Novo ◽  
Umbilical Vein ◽  
Interleukin 1 ◽  
Tnf Alpha ◽  
Extrinsic Pathway ◽  
Necrosis Factor Alpha

Abstract Tissue factor (TF) is the first factor of the extrinsic pathway of coagulation. Normally, TF is not expressed on the surface of endothelial cells. However, expression of TF can be induced in these cells in response to stimulation by diverse inflammatory mediators such as interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF- alpha), lipopolysaccharide (LPS), and phorbol 12-myristate 13-acetate (PMA). We have studied the effect of these mediators on the kinetics of the induction of TF-related procoagulant activity (PCA) on human umbilical vein endothelial cells (HUVECs). PCA is transiently induced on HUVECs, attaining a peak some 4 to 8 hours after addition of inflammatory agents, with maximal accumulation of TF messenger RNA (mRNA) occurring 3 to 5 hours earlier. Because the expression of PCA by treated HUVECs returns to basal levels by 20 to 30 hours, we examined the response of these cells to a second inflammatory stimulus. Continuous incubation of cells with a single inflammatory agent for 24 to 48 hours induces a hyporesponsive state with respect to the reinduction of TF expression by the same agent (14% of the initial stimulation for IL-1 beta, 39% for TNF-alpha 30% for LPS, and 7% for PMA). Such a diminution in PCA was also observed in the levels of TF mRNA. By contrast, pretreatment of HUVECs with one agent did not dramatically affect the reinduction of TF by any of the three other factors. We subsequently focused our attention on the induction of the autologous refractory period by IL-1 beta. De novo protein synthesis was not required during the preincubation of ECs for hyporesponsiveness to be observed. The establishment of the refractory state did not depend on the downmodulation of IL-1 beta receptor affinity or expression. Moreover, pretreatment of HUVECs with IL-1 beta increased prostacyclin (PGI2) production in response to a second stimulation by IL-1 beta, although such cells were unable to reexpress TF under the same conditions. This result suggests that distinct secondary messenger pathways are involved in TF induction and PGI2 synthesis by IL-1 beta in HUVECs.

Tissue Kallikrein KLK1 Is Expressed de Novo in Endothelial Cells and Mediates Relaxation of Human Umbilical Veins

2001 ◽  
Vol 382 (10) ◽  
pp. 1483-1490 ◽  
Author(s):  
J. Dedio ◽  
G. Wiemer ◽  
H. Rütten ◽  
A. Dendorfer ◽  
B.A. Schölkens ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
De Novo ◽  
Umbilical Vein ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Tissue Kallikrein ◽  
Kinin System ◽  
Western Blots ◽  
Cell Extracts ◽  
Umbilical Vein Endothelial Cells

AbstractBradykinin released by the endothelium is thought to play an important local role in cardiovascular regulation. However, the molecular identity of endothelial proteases liberating bradykinin from its precursors remained unclear. Using RTPCR and Southern blotting techniques we detected mRNA for tissue kallikrein (KLK1) in human umbilical vein endothelial cells and in bovine aortic endothelial cells. Protein expression was confirmed by precipitation of KLK1 from lysates of endothelial cells prelabeled with [35S]cysteine/methionine. Partial purification of tissue kallikrein from total endothelial cell extracts resulted in a protein triplet of about 50 kDa in Western blots using specific antiKLK1 antibodies. The immunodetection of tissue kallikrein antigen in the fractions from ion exchange chromatography correlated with the presence of amidolytic tissue kallikrein activity. Stimulation of endothelial cells with angiotensin II (ANGII), which recently has been shown to activate the vascular kinin system and to cause vasodilation, resulted in the release of bradykinin and kallidin. ANGIIdependent relaxation of preconstricted rings from human umbilical veins was abolished in the presence of a specific tissue kallikrein inhibitor. We conclude that endothelial cells de novo express significant amounts of tissue kallikrein, which likely serves in the local generation of vasoactive kinins.

scholarly journals MicroRNA-494 Regulates Endoplasmic Reticulum Stress in Endothelial Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Namita Chatterjee ◽  
Eugenia Fraile-Bethencourt ◽  
Adrian Baris ◽  
Cristina Espinosa-Diez ◽  
Sudarshan Anand
Keyword(s):  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Stress Responses ◽  
De Novo ◽  
Umbilical Vein ◽  
Critical Role ◽  
Umbilical Vein Endothelial Cells ◽  
And Control

Defects in stress responses are important contributors in many chronic conditions including cancer, cardiovascular disease, diabetes, and obesity-driven pathologies like non-alcoholic steatohepatitis (NASH). Specifically, endoplasmic reticulum (ER) stress is linked with these pathologies and control of ER stress can ameliorate tissue damage. MicroRNAs have a critical role in regulating diverse stress responses including ER stress. Here, we show that miR-494 plays a functional role during ER stress. Pharmacological ER stress inducers (tunicamycin (TCN) and thapsigargin) and hyperglycemia robustly increase the expression of miR-494 in vitro. ATF6 impacts the primary miR-494 levels whereas all three ER stress pathways are necessary for the increase in mature miR-494. Surprisingly, miR-494 pretreatment dampens the induction and magnitude of ER stress in response to TCN in endothelial cells and increases cell viability. Conversely, inhibition of miR-494 increases ER stress de novo and amplifies the effects of ER stress inducers. Using Mass Spectrometry (TMT-MS) we identified 23 proteins that are downregulated by both TCN and miR-494 in cultured human umbilical vein endothelial cells. Among these, we found 6 transcripts which harbor a putative miR-494 binding site. We validated the anti-apoptotic gene BIRC5 (survivin) and GINS4 as targets of miR-494 during ER stress. In summary, our data indicates that ER stress driven miR-494 may act in a feedback inhibitory loop to dampen downstream ER stress signaling.

scholarly journals Invasion of Aortic and Heart Endothelial Cells byPorphyromonas gingivalis

1998 ◽  
Vol 66 (11) ◽  
pp. 5337-5343 ◽  
Author(s):  
Rajashri G. Deshpande ◽  
Mahfuz B. Khan ◽  
Caroline Attardo Genco
Keyword(s):  
Electron Microscopy ◽  
Endothelial Cells ◽  
Protein Synthesis ◽  
De Novo ◽  
Umbilical Vein ◽  
Host Cells ◽  
Periodontal Pathogen ◽  
Host Immune System ◽  
Umbilical Vein Endothelial Cells ◽  
De Novo Protein Synthesis

ABSTRACT Invasion of host cells is believed to be an important strategy utilized by a number of pathogens, which affords them protection from the host immune system. The connective tissues of the periodontium are extremely well vascularized, which allows invading microorganisms, such as the periodontal pathogen Porphyromonas gingivalis, to readily enter the bloodstream. However, the ability of P. gingivalis to actively invade endothelial cells has not been previously examined. In this study, we demonstrate that P. gingivalis can invade bovine and human endothelial cells as assessed by an antibiotic protection assay and by transmission and scanning electron microscopy. P. gingivalis A7436 was demonstrated to adhere to and to invade fetal bovine heart endothelial cells (FBHEC), bovine aortic endothelial cells (BAEC), and human umbilical vein endothelial cells (HUVEC). Invasion efficiencies of 0.1, 0.2, and 0.3% were obtained with BAEC, HUVEC, and FBHEC, respectively. Invasion of FBHEC and BAEC by P. gingivalis A7436 assessed by electron microscopy revealed the formation of microvillus-like extensions around adherent bacteria followed by the engulfment of the pathogen within vacuoles. Invasion of BAEC by P. gingivalisA7436 was inhibited by cytochalasin D, nocodazole, staurosporine, protease inhibitors, and sodium azide, indicating that cytoskeletal rearrangements, protein phosphorylation, energy metabolism, andP. gingivalis proteases are essential for invasion. In contrast, addition of rifampin, nalidixic acid, and chloramphenicol had little effect on invasion, indicating that bacterial RNA, DNA, and de novo protein synthesis are not required for P. gingivalisinvasion of endothelial cells. Likewise de novo protein synthesis by endothelial cells was not required for invasion by P. gingivalis. P. gingivalis 381 was demonstrated to adhere to and to invade BAEC (0.11 and 0.1% efficiency, respectively). However, adherence and invasion of the corresponding fimA mutant DPG3, which lacks the major fimbriae, was not detected. These results indicate thatP. gingivalis can actively invade endothelial cells and that fimbriae are required for this process. P. gingivalisinvasion of endothelial cells may represent another strategy utilized by this pathogen to thwart the host immune response.

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