scholarly journals Caveolae protect endothelial cells from membrane rupture during increased cardiac output

2015 ◽  
Vol 211 (1) ◽  
pp. 53-61 ◽  
Author(s):  
Jade P.X. Cheng ◽  
Carolina Mendoza-Topaz ◽  
Gillian Howard ◽  
Jessica Chadwick ◽  
Elena Shvets ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cardiac Output ◽  
Plasma Membrane ◽  
Cultured Cells ◽  
Protein Complexes ◽  
Membrane Integrity ◽  
Hemodynamic Forces ◽  
Membrane Rupture ◽  
Acute Rupture

Caveolae are strikingly abundant in endothelial cells, yet the physiological functions of caveolae in endothelium and other tissues remain incompletely understood. Previous studies suggest a mechanoprotective role, but whether this is relevant under the mechanical forces experienced by endothelial cells in vivo is unclear. In this study we have sought to determine whether endothelial caveolae disassemble under increased hemodynamic forces, and whether caveolae help prevent acute rupture of the plasma membrane under these conditions. Experiments in cultured cells established biochemical assays for disassembly of caveolar protein complexes, and assays for acute loss of plasma membrane integrity. In vivo, we demonstrate that caveolae in endothelial cells of the lung and cardiac muscle disassemble in response to acute increases in cardiac output. Electron microscopy and two-photon imaging reveal that the plasma membrane of microvascular endothelial cells in caveolin 1−/− mice is much more susceptible to acute rupture when cardiac output is increased. These data imply that mechanoprotection through disassembly of caveolae is important for endothelial function in vivo.

Chronic shear induces caveolae formation and alters ERK and Akt responses in endothelial cells

2003 ◽  
Vol 285 (3) ◽  
pp. H1113-H1122 ◽  
Author(s):  
Nolan L. Boyd ◽  
Heonyong Park ◽  
Hong Yi ◽  
Yong Chool Boo ◽  
George P. Sorescu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Cultured Cells ◽  
Signaling Molecules ◽  
Activation Patterns ◽  
Aortic Endothelial Cells ◽  
Erk Activation ◽  
Caveolin 1 ◽  
Protein Levels

Caveolae are plasmalemmal domains enriched with cholesterol, caveolins, and signaling molecules. Endothelial cells in vivo are continuously exposed to shear conditions, and their caveolae density and location may be different from that of static cultured cells. Here, we show that chronic shear exposure regulates formation and localization of caveolae and caveolin-1 in bovine aortic endothelial cells (BAEC). Chronic exposure (1 or 3 days) of BAEC to laminar shear increased the total number of caveolae by 45–48% above static control. This increase was due to a rise in the luminal caveolae density without changing abluminal caveolae numbers or increasing caveolin-1 mRNA and protein levels. Whereas some caveolin-1 was found in the plasma membrane in static-cultured cells, it was predominantly localized in the Golgi. In contrast, chronic shear-exposed cells showed intense caveolin-1 staining in the luminal plasma membrane with minimum Golgi association. The preferential luminal localization of caveolae may play an important role in endothelial mechanosensing. Indeed, we found that chronic shear exposure (preconditioning) altered activation patterns of two well-known shear-sensitive signaling molecules (ERK and Akt) in response to a step increase in shear stress. ERK activation was blunted in shear preconditioned cells, whereas the Akt response was accelerated. These results suggest that chronic shear stimulates caveolae formation by translocating caveolin-1 from the Golgi to the luminal plasma membrane and alters cell signaling responses.

scholarly journals Effects of cytoplasmic acidification on clathrin lattice morphology.

1989 ◽  
Vol 108 (2) ◽  
pp. 401-411 ◽  
Author(s):  
J Heuser
Keyword(s):  
Plasma Membrane ◽  
Cultured Cells ◽  
Membrane Receptors ◽  
The Other ◽  
Culture Dish ◽  
Initial Curvature ◽  
Internal Ph ◽  
Cytoplasmic Acidification

Reducing the internal pH of cultured cells by several different protocols that block endocytosis is found to alter the structure of clathrin lattices on the inside of the plasma membrane. Lattices curve inward until they become almost spherical yet remain stubbornly attached to the membrane. Also, the lattices bloom empty "microcages" of clathrin around their edges. Correspondingly, broken-open cells bathed in acidified media demonstrate similar changes in clathrin lattices. Acidification accentuates the normal tendency of lattices to round up in vitro and also stimulates them to nucleate microcage formation from pure solutions of clathrin. On the other hand, several conditions that also inhibit endocytosis have been found to create, instead of unusually curved clathrin lattices with extraneous microcages, a preponderance of unusually flat lattices. These treatments include pH-"clamping" cells at neutrality with nigericin, swelling cells with hypotonic media, and sticking cells to the surface of a culture dish with soluble polylysine. Again, the unusually flat lattices in such cells display a tendency to round up and to nucleate clathrin microcage formation during subsequent in vitro acidification. This indicates that regardless of the initial curvature of clathrin lattices, they all display an ability to grow and increase their curvature in vitro, and this is enhanced by lowering ambient pH. Possibly, clathrin lattice growth and curvature in vivo may also be stimulated by a local drop in pH around clusters of membrane receptors.

scholarly journals Caveolae protect endothelial cells from membrane rupture during increased cardiac output

2015 ◽  
Vol 212 (11) ◽  
pp. 21211OIA89
Author(s):  
Jade P.X. Cheng ◽  
Carolina Mendoza-Topaz ◽  
Gillian Howard ◽  
Jessica Chadwick ◽  
Elena Shvets ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cardiac Output ◽  
Membrane Rupture

scholarly journals NETosis proceeds by cytoskeleton and endomembrane disassembly and PAD4-mediated chromatin decondensation and nuclear envelope rupture

2020 ◽  
Vol 117 (13) ◽  
pp. 7326-7337 ◽  
Author(s):  
Hawa Racine Thiam ◽  
Siu Ling Wong ◽  
Rong Qiu ◽  
Mark Kittisopikul ◽  
Amir Vahabikashi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Nuclear Envelope ◽  
Time Lapse ◽  
Extracellular Dna ◽  
Sterile Inflammation ◽  
Human Neutrophils ◽  
Chromatin Decondensation ◽  
Membrane Rupture ◽  
Cellular Events

Neutrophil extracellular traps (NETs) are web-like DNA structures decorated with histones and cytotoxic proteins that are released by activated neutrophils to trap and neutralize pathogens during the innate immune response, but also form in and exacerbate sterile inflammation. Peptidylarginine deiminase 4 (PAD4) citrullinates histones and is required for NET formation (NETosis) in mouse neutrophils. While the in vivo impact of NETs is accumulating, the cellular events driving NETosis and the role of PAD4 in these events are unclear. We performed high-resolution time-lapse microscopy of mouse and human neutrophils and differentiated HL-60 neutrophil-like cells (dHL-60) labeled with fluorescent markers of organelles and stimulated with bacterial toxins or Candida albicans to induce NETosis. Upon stimulation, cells exhibited rapid disassembly of the actin cytoskeleton, followed by shedding of plasma membrane microvesicles, disassembly and remodeling of the microtubule and vimentin cytoskeletons, ER vesiculation, chromatin decondensation and nuclear rounding, progressive plasma membrane and nuclear envelope (NE) permeabilization, nuclear lamin meshwork and then NE rupture to release DNA into the cytoplasm, and finally plasma membrane rupture and discharge of extracellular DNA. Inhibition of actin disassembly blocked NET release. Mouse and dHL-60 cells bearing genetic alteration of PAD4 showed that chromatin decondensation, lamin meshwork and NE rupture and extracellular DNA release required the enzymatic and nuclear localization activities of PAD4. Thus, NETosis proceeds by a stepwise sequence of cellular events culminating in the PAD4-mediated expulsion of DNA.

scholarly journals Growth factors are released by mechanically wounded endothelial cells.

1989 ◽  
Vol 109 (2) ◽  
pp. 811-822 ◽  
Author(s):  
P L McNeil ◽  
L Muthukrishnan ◽  
E Warder ◽  
P A D'Amore
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Growth Factors ◽  
Growth Factor ◽  
Mechanical Forces ◽  
Fibroblast Growth ◽  
Growth Promoting ◽  
Transient Plasma

Growth factors may be required at sites of mechanical injury and normal wear and tear in vivo, suggesting that the direct action of mechanical forces on cells could lead to growth factor release. Scraping of cells from the tissue culture substratum at 37 degrees C was used to test this possibility. We show that scraping closely mimics in vitro both the transient plasma membrane wounds observed in cells subject to mechanical forces in vivo (McNeil, P. L., and S. Ito. 1989. Gastroenterology. 96:1238-1248) and the transient plasma membrane wounds shown here to occur in endothelial cells under normal culturing conditions. Scraping of endothelial cells from the culturing substratum released into the culture medium a potent growth-promoting activity for Swiss 3T3 fibroblasts. Growth-promoting activity was released rapidly (within 5 min) after scraping but was not subsequently degraded by the endothelial cells for at least 24 h thereafter. A greater quantity of growth-promoting activity was released by cells scraped 4 h after plating than by those scraped 4 or 7 d afterwards. Thus release is not due to scraping-induced disruption of extracellular matrix. Release was only partially cold inhibitable, was poorly correlated with the level of cell death induced by scraping, and did not occur when cells were killed with metabolic poisons. These results suggest that mechanical disruption of plasma membrane, either transient or permanent, is the essential event leading to release. A basic fibroblast growth factor-like molecule and not platelet-derived growth factor appears to be partially responsible for the growth-promoting activity. We conclude that one biologically relevant route of release of basic fibroblast growth factor, a molecule which lacks the signal peptide sequence for transport into the endoplasmic reticulum, could be directly through mechanically induced membrane disruptions of endothelial cells growing in vivo and in vitro.

scholarly journals Glucagon receptors in endothelial and Kupffer cells of mouse liver.

1988 ◽  
Vol 36 (9) ◽  
pp. 1081-1089 ◽  
Author(s):  
J Watanabe ◽  
K Kanai ◽  
S Kanamura
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Kupffer Cells ◽  
Mouse Liver ◽  
Intracellular Transport ◽  
Coated Pits ◽  
Sinusoidal Cells ◽  
Nonparenchymal Cells ◽  
Glucagon Receptors

To determine whether hepatic sinusoidal cells contain glucagon receptors and, if so, to study the significance of the receptors in the cells, binding of [125I]-glucagon to nonparenchymal cells (mainly endothelial cells and Kupffer cells) isolated from mouse liver was examined by quantitative autoradiography and biochemical methods. Furthermore, the pathway of intracellular transport of colloidal gold-labeled glucagon (AuG) was examined in vivo. Autoradiographic and biochemical results demonstrated many glucagon receptors in both endothelial cells and Kupffer cells, and more receptors being present in endothelial cells than in Kupffer cells. In vivo, endothelial cells internalized AuG particles into coated vesicles via coated pits and transported the particles to endosomes, lysosomes, and abluminal plasma membrane. Therefore, receptor-mediated transcytosis of AuG occurs in endothelial cells. The number of particles present on the abluminal plasma membrane was constant if the amount of injected AuG increased. Therefore, the magnitude of receptor-mediated transcytosis of AuG appears to be regulated by endothelial cells. Kupffer cells internalized the ligand into cytoplasmic tubular structures via plasma membrane invaginations and transported the ligand exclusively to endosomes and lysosomes, suggesting that the ligand is degraded by Kupffer cells.

scholarly journals Lysosomal retargeting of Myoferlin mitigates membrane stress to enable pancreatic cancer growth

2021 ◽  
Author(s):  
Suprit Gupta ◽  
Julian Yano ◽  
Htet Htwe Htwe ◽  
Hijai R. Shin ◽  
Zeynep Cakir ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Plasma Membrane ◽  
Membrane Damage ◽  
Membrane Integrity ◽  
Human Pancreatic Cancer ◽  
Cancer Growth ◽  
Membrane Repair ◽  
Pancreatic Cancer Cells ◽  
Plasma Membrane Repair

AbstractLysosomes must maintain integrity of their limiting membrane to ensure efficient fusion with incoming organelles and degradation of substrates within their lumen. Pancreatic cancer cells upregulate lysosomal biogenesis to enhance nutrient recycling and stress resistance, but whether dedicated programs for maintaining lysosomal membrane integrity facilitate pancreatic cancer growth is unknown. Using proteomic-based organelle profiling, we identify the Ferlin family plasma membrane repair factor, Myoferlin, as selectively and highly enriched on the membrane of pancreatic cancer lysosomes. Mechanistically, lysosome localization of Myoferlin is necessary and sufficient for maintenance of lysosome health and provides an early-acting protective system against membrane damage that is independent from the endosomal sorting complex required for transport (ESCRT)-mediated repair network. Myoferlin is upregulated in human pancreatic cancer, predicts poor survival, and its ablation severely impairs lysosome function and tumour growth in vivo. Thus, retargeting of plasma membrane repair factors enhances pro-oncogenic activities of the lysosome.

scholarly journals Endothelial-specific gene expression directed by the tie gene promoter in vivo

Blood ◽  
1995 ◽  
Vol 86 (5) ◽  
pp. 1828-1835 ◽  
Author(s):  
J Korhonen ◽  
I Lahtinen ◽  
M Halmekyto ◽  
L Alhonen ◽  
J Janne ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transgenic Mice ◽  
Reporter Gene ◽  
Promoter Activity ◽  
Cultured Cells ◽  
Luciferase Reporter ◽  
Specific Gene ◽  
Luciferase Reporter Gene ◽  
Human And Mouse

The tie gene encodes a receptor tyrosine kinase that is expressed in the endothelium of blood vessels, particularly during embryonic development and angiogenesis in adults. We have cloned and characterized the mouse tie gene and isolated the human and mouse tie promoters. The promoter activities of human and mouse tie were analyzed using luciferase reporter gene constructs in transfected cell lines and beta-galactosidase constructs in transgenic mice. In transfection assays of cultured cells, both human and mouse promoter DNA fragments showed activity that was not restricted to endothelial cells. In contrast, in transgenic mice both promoters directed expression of the reporter gene to endothelial cells undergoing vasculogenesis and angiogenesis. In adult mice, tie promoter activity in lung and many vessels of the kidney was as high as in the vessels of the corresponding embryonic tissues, whereas in the heart, brain and liver, tie promoter activity was downregulated and restricted to coronaries, cusps, capillaries, and arteries. Our results show that the endothelial cell-type specificity of the tie promoter in vivo can be transferred to heterologous genes by using relatively short promoter fragments. The tie promoter, thus, has useful properties for potential gene therapy.

CHANGES IN ENZYMATIC ACTIVITIES IN BRAIN CAPILLARY ENDOTHELIAL CELLS INJURED BY PLATELET AGGREGATION IN VIVO

1987 ◽  
Author(s):  
T Fujimoto ◽  
B Djuricic ◽  
K Tanoue ◽  
Y Fukushima ◽  
H Yamazaki
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Platelet Aggregation ◽  
Enzymatic Activities ◽  
Gamma Glutamyl Transferase ◽  
Discontinuous Sucrose Gradient ◽  
Capillary Endothelial Cells ◽  
Mao Activity ◽  
Glutamyl Transferase

We have reported cerebrovascular injuries induced by platelet aggregation in vivo. Appearance of vacuoles in endothelial cells and eventual deendothelialization are characteristic in large cerebral arteries (Stroke, 16:245, 1985). Minor changes are observed in brain capillaries, but disturbances of blood-brain barrier (BBB) are seen. To analyse changes in BBB, enzymatic activities in capillary endothelial cells before and after ADP-induced platelet aggregation in vivo were investigated.80 mg/kg of ADP was injected through a catheter into the right internal carotid artery of 32 rabbits. One hr later, right and left cortexes freed from pial membranes were homogenized and microvessels were isolated using discontinuous sucrose gradient ultracentrifugation. Purity of microvessel fraction was ascertained microscopically. The follwing enzymatic activities in these samples were measured.; cytochrome C oxydase (CCO), monoamine oxidase (MAO), p-nitrophenyl-phosphatase transferase (pNPPase, K-dependent component of Na, K-ATPase), gamma-glutamyl transferase (GT) and adenylate cyclase (AC). The enzymatic activities did not change after a vehicle-injection and did not show any differences between capillaries of both the cortexes before the ADP-injection. One hr after the ADP, GT and CCO activities decreased significantly in the capillaries of injection side. MAO activity also reduced without significance. The other enzymes did not show significant changes in their activities. Although pNPPase and AC which are associated with inner surface of plasma membrane were preserved well, activity of GT which is associated with outer portion of the membrane decreased significantly. It suggests superficial luminal injury and that plasma membrane might be affected from the side of vascular lumen. Reduced CCO activity suggests that disturbance in BBB is probably related to the increase in vesicular transportation and/or energy failure. Reduction of MAO activity indicates that damages to mitochondria exist in the capillaries. Cerebral blood vessels are prone to damage by released substances from activated platelet in vivo.

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