The distribution of centrosomes in migrating endothelial cells during wound healing in situ

1992 ◽  
Vol 70 (10-11) ◽  
pp. 1135-1141 ◽  
Author(s):  
Kem A. Rogers ◽  
Martin Sandig ◽  
Nancy H. McKee ◽  
Vitauts I. Kalnins
Keyword(s):  
Wound Healing ◽  
Endothelial Cells ◽  
Cell Migration ◽  
Balloon Catheter ◽  
Rabbit Aorta ◽  
Adult Rabbit ◽  
Cell Organelles ◽  
Wound Edge ◽  
In Cells

We have examined the distribution of centrioles in rabbit thoracic aortic endothelial cells induced to migrate by wounding the endothelium in situ. Following denudation of the endothelium from a segment of the aorta with a balloon catheter, a wound edge was created from which endothelial cells began to migrate onto the denuded surface. In this in situ model of cell migration, the position of centrioles was determined in cells along the wound edge by immunofluorescence and antibodies which specifically label these cell organelles, and then they were classified in relation to the nucleus and the direction of cell migration as being oriented toward the wound, in the center, or away from wound. At time 0, as in normal unwounded adult rabbit aorta, no preferential orientation of centrioles was evident. Within 12 h after wounding, the centrioles in about 53% of endothelial cells near the wound edge were oriented toward the wound, while in less than 20% of the cells they were oriented away from wound. At 24 h, in cells up to 800 μm from the wound edge, centrioles in only about 10% of the endothelial cells were oriented away from wound, while in about 52% of cells they were found in the center and in 38% of the cells they remained oriented toward the wound. At 48 h, up to 2000 μm from the wound edge, the majority of endothelial cells had their centrioles in the center, possibly as a result of an increase in mitotic index as cells replicate to reestablish an intact endothelium. The results of this study demonstrate that, in endothelial cells starting to migrate on a natural substratum in situ in response to wounding, most centrioles reorient toward the wound edge. This observation is consistent with the hypothesis that the centrosome is involved in defining the direction of cell migration in endothelial cells.Key words: centriole, in situ, endothelium, wound healing, aorta.

The distribution of microfilament bundles in rabbit endothelial cells in the intact aorta and during wound healing in situ

1989 ◽  
Vol 67 (9) ◽  
pp. 553-562 ◽  
Author(s):  
Kem A. Rogers ◽  
Martin Sandig ◽  
Nancy H. McKee ◽  
Vitauts I. Kalnins
Keyword(s):  
Wound Healing ◽  
Endothelial Cells ◽  
Blood Flow ◽  
Vessel Wall ◽  
Balloon Catheter ◽  
Wound Edge ◽  
Microfilament Bundles

The distribution of microfilament (MF) bundles in rabbit thoracic aortic endothelial cells (EC) fixed in situ was examined using en face preparations and the fluorescent probe 7-nitrobenz-2-oxa-1,3-diazole-phallacidin. In the normal aorta, prominent peripheral MF bundles are seen near the cell borders running the full length of each cell, parallel to the direction of blood flow, while shorter less prominent bundles are seen in the more central regions. In EC covering the flow dividers at intercostal ostia, the central MF bundles are more prominent, longer, and more numerous than in the other regions of the aorta examined. This increase in the number, size, and length of central MF bundles may result from the response of the cells to the higher shear forces present in this region of the vessel wall. Following denudation of the endothelium from a segment of the aorta with a balloon catheter, there is an initial reduction in the size of all of the MF bundles in cells near the wound edge. This is followed by an increase in the number and size of the central MF bundles. At 48 h after wounding, strongly stained central MF bundles could be detected in EC up to 0.75 mm from the wound edge. Adjacent to the wounds that had failed to reendothelialize 10 months after denudation, some regions had EC with prominent peripheral MF bundles and others, EC with prominent central MF bundles. At the very edge of the wound, the EC and their MF bundles were oriented with their long axes parallel to the wound edge and perpendicular to the direction of blood flow. The failure of the wounded vessel wall to become fully reendothelialized may be related to the orientation of EC at the wound edge. These results show that EC migration in situ is accompanied by a dramatic change in the organization of MF in which different stages can be identified. Microfilament bundles in rapidly migrating cells in vivo, 24 and 48 h after wounding, resemble stress fibers seen in EC migrating in vitro and in slowly migrating fibroblasts and epithelial cells.Key words: microfilaments, in situ, endothelium, wound healing, aorta.

scholarly journals Microtubule-organizing centers and cell migration: effect of inhibition of migration and microtubule disruption in endothelial cells.

1983 ◽  
Vol 96 (5) ◽  
pp. 1266-1272 ◽  
Author(s):  
A I Gotlieb ◽  
L Subrahmanyan ◽  
V I Kalnins
Keyword(s):  
Endothelial Cells ◽  
Cell Migration ◽  
Cytochalasin B ◽  
Leading Edge ◽  
Wound Edge ◽  
Microtubule Organizing Centers ◽  
Migration Effect ◽  
Microtubule Disruption ◽  
Made In ◽  
Experimentally Induced

We have previously shown that microtubule-organizing centers (MTOC's) become preferentially oriented towards the leading edge of migrating endothelial cells (EC's) at the margin of an experimentally induced wound made in a confluent EC monolayer. To learn more about the mechanism responsible for the reorientation of MTOC's and to determine whether a similar reorientation takes place when cell migration is inhibited, we incubated the wounded cultures with colcemid (C) and cytochalasin B (CB), which disrupt microtubules (MT's) and microfilaments (MF's), respectively. The results obtained showed that the MTOC reorientation can occur independent of cell migration since MTOC's reoriented preferentially toward the wound edge in the CB-treated cultures, even though forward migration of the EC was inhibited. In addition, the MTOC reorientation is inhibited by C, indicating that it requires an intact system of MT's and/or other intracellular structures whose distribution is dependent on that of MT's.

scholarly journals Fibronectin mRNA in the developing glomerular crescent in rabbit antiglomerular basement membrane disease.

1995 ◽  
Vol 5 (12) ◽  
pp. 2087-2090
Author(s):  
S P Sady ◽  
M Goyal ◽  
P E Thomas ◽  
B L Wharram ◽  
R C Wiggins
Keyword(s):  
Wound Healing ◽  
In Situ Hybridization ◽  
Basement Membrane ◽  
Matrix Protein ◽  
Crescent Formation ◽  
Plasma Phase ◽  
Two Phases ◽  
Fibronectin Mrna ◽  
In Cells

Fibronectin is a multifunctional matrix protein important in wound healing that is markedly increased in glomerular crescents. A previous report established two phases of fibronectin metabolism in crescent formation in an anti-glomerular basement membrane model of crescentic nephritis in the rabbit. Phase I was associated with increased glomerular fibronectin content from plasma. Phase II was associated with increased fibronectin mRNA in glomeruli. To examine the hypothesis that fibronectin is synthesized in the developing crescent, rabbit fibronectin cDNA was cloned, sense and antisense riboprobes were prepared and their specificity under the conditions to be used was validated and in situ hybridization studies were performed in the model. The results showed that the cells in the developing glomerular crescent express an intense fibronectin mRNA signal at Day 7 and that this signal persisted in cells of the crescent at Day 14. This result shows that fibronectin synthesis does indeed take place in cells of the developing crescent in this model and supports the hypothesis that fibronectin may be an important agent regulating crescent formation and fibrosis.

scholarly journals Zinc deficiency promotes endothelin secretion and endothelial cell migration through nuclear hypoxia-inducible factor-1 translocation

2019 ◽  
Vol 317 (2) ◽  
pp. C270-C276 ◽  
Author(s):  
Jessica Morand ◽  
Anne Briançon-Marjollet ◽  
Emeline Lemarie ◽  
Brigitte Gonthier ◽  
Josiane Arnaud ◽  
...  
Keyword(s):  
Wound Healing ◽  
Endothelial Cells ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Zinc Deficiency ◽  
Hypoxia Inducible Factor ◽  
Endothelial Cell Migration ◽  
Hypoxia Inducible Factor 1 ◽  
Obstructive Sleep ◽  
And Function

Zinc is involved in the expression and function of various transcription factors, including the hypoxia-inducible factor-1 (HIF-1). HIF-1 and its target gene endothelin-1 (ET-1) are activated by intermittent hypoxia (IH), one of the main consequences of obstructive sleep apnea (OSA), and both play a key role in the cardiovascular consequences of IH. Because OSA and IH are associated with zinc deficiency, we investigated the effect of zinc deficiency caused by chelation on the HIF-1/ET-1 pathway and its functional consequences in endothelial cells. Primary human microvascular endothelial cells (HMVEC) were incubated with submicromolar doses of the zinc-specific membrane-permeable chelator N, N, N′, N′-tetrakis(2-pyridylmethyl)-ethylene diamine (TPEN, 0.5 µM) or ET-1 (0.01 µM) with or without bosentan, a dual ET-1-receptor antagonist. HIF-1α expression was silenced by transfection with specific siRNA. Nuclear HIF-1 content was assessed by immunofluorescence microscopy and Western blot. Migratory capacity of HMVEC was evaluated with a wound-healing scratch assay. Zinc chelation by TPEN exposure induced the translocation of the cytosolic HIF-1α subunit of HIF-1 to the nucleus as well as an HIF-1-mediated ET-1 secretion by HMVEC. Incubation with either TPEN or ET-1 increased endothelial wound-healing capacity. Both HIF-1α silencing or bosentan abolished this effect. Altogether, these results suggest that zinc deficiency upregulates ET-1 signaling through HIF-1 activation and stimulates endothelial cell migration, suggesting an important role of zinc in the vascular consequences of IH and OSA mediated by HIF-1-ET- signaling.

scholarly journals Effects of IL-1β and TNF-α on the Expression of P311 in Vascular Endothelial Cells and Wound Healing in Mice

2020 ◽  
Vol 11 ◽  
Author(s):  
Daijun Zhou ◽  
Tengfei Liu ◽  
Song Wang ◽  
Weifeng He ◽  
Wei Qian ◽  
...  
Keyword(s):  
Wound Healing ◽  
Endothelial Cells ◽  
Cell Migration ◽  
Protein Expression ◽  
Vascular Endothelial Cells ◽  
Control Group ◽  
Vascular Endothelial ◽  
Expression Levels ◽  
Relative Expression ◽  
Tnf Α

ObjectiveThis study aimed to define the role of interleukine-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the expression of P311 in vascular endothelial cells (VECs) and in wound healing.MethodsDAPI staining, a CCK-8 assay, cell migration assay, and an angiogenesis assay were used to assess the effects exerted by TNF-α and IL-1β at various concentrations on morphology, proliferation, migration, and angiogenesis of VECs. Western blot (WB) and reverse transcription-polymerase chain reaction (RT-PCR) models were employed to observe the effects exerted by proteins related to the nuclear factor-kappa B (NF-κB) signaling pathway and P311 mRNA expression. Bioinformatics analysis was performed on the binding sites of P311 and NF-κB. Finally, to investigate the effects of IL-1β and TNF-α on wound healing and the length of new epithelium in mice, we established a full-thickness wound defect model in mice. Immunohistochemistry was used to measure changes in P311, proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF), CD31 (platelet endothelial cell adhesion molecule-1, PECAM-1/CD31), as well as other related proteins.ResultsWhen levels of TNF-α and IL-1β were both 20 ng/ml, their effects on cell proliferation, cytoskeleton protein expression, cell migration, and angiogenesis were the greatest (P < 0.05). IL-1β and TNF-α at moderate concentrations effectively promoted P311 mRNA and p-NF-κB protein expression (P < 0.05), while p-NF-K b protein expression was decreased (P < 0.05). Luciferase assays showed that P311 expression was also relatively greater when stimulated at moderate concentrations (P < 0.05), while relative expression was significantly lower when the p-NF-K b inhibitor CAPE was added (P < 0.05). On 7-day wound healing rate comparison, the control, IL-1β, IL-1βab, TNF-α, and TNF-αab groups were 18, 37, 35, 39, and 36%, respectively, while control group + P311 siRNA was 31% (P < 0.05). New epithelial length, granulation tissue thickness, and number of blood vessels trends were also the same. In the control group, P311 showed lower relative expression levels than the others (P < 0.05). P311 relative expression levels trended as follows: control group > IL-1βab > IL-1β > TNF-αab > TNF-α (P < 0.05).ConclusionWhen IL-1β and TNF-α concentrations are moderate, they effectively promote the proliferation, expression, migration, and angiogenesis of VECs, possibly by promoting the expression of the NF-K b pathway and thereby promoting the expression of P311. In vitro experiments on mice suggest that P311 effectively promotes wound healing, and its mechanism may be closely related to PCNA, CD31, and VEGF.

EMMA-4 analysis of iron in cells of the thymic cortex of a weaver-bird ( Quelea quelea )

1975 ◽  
Vol 273 (923) ◽  
pp. 79-82 ◽  
Keyword(s):  
Electron Microscope ◽  
Erythroid Cells ◽  
Cell Organelles ◽  
Analytical Studies ◽  
Analytical Electron ◽  
Analytical Electron Microscope ◽  
Avian Thymus ◽  
Reticular Cells ◽  
In Cells

Combined morphological and analytical studies with the EMMA-4 analytical electron microscope have enabled very early erythroid cells to be identified within the cortex of enlarging thymic lobes of Quelea quelea . These early erythroid cells have pale cytoplasm (sometimes with ferritin-like crystals present), slightly pachychromatic nuclei and have fewer cell organelles (mitochondria) than lymphocytes. Counts for iron were approximately 70% lower than counts from mature erythrocytes found free in the cortex. Iron was also recorded from some epithelial reticular cells and pyknotic nuclei; no iron was recorded from small lymphocytes (thymocytes) in the cortex. The presence of very early erythroid cells is a further indication that erythropoiesis occurs in situ in the avian thymus.

scholarly journals Beta actin and its mRNA are localized at the plasma membrane and the regions of moving cytoplasm during the cellular response to injury.

1991 ◽  
Vol 112 (4) ◽  
pp. 653-664 ◽  
Author(s):  
T C Hoock ◽  
P M Newcomb ◽  
I M Herman
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
In Situ Hybridization ◽  
Cellular Response ◽  
Stress Fibers ◽  
Wound Edge ◽  
Actin Isoforms ◽  
Beta Actin ◽  
Cell Biol

Previous work in our laboratory has shown that microvascular pericytes sort muscle and nonmuscle actin isoforms into discrete cytoplasmic domains (Herman, I. M., and P. A. D'Amore. 1985. J. Cell Biol. 101:43-52; DeNofrio, D.T.C. Hoock, and I. M. Herman. J. Cell. Biol. 109:191-202). Specifically, muscle (alpha-smooth) actin is present on the stress fibers while nonmuscle actins (beta and gamma) are located on stress fibers and in regions of moving cytoplasm (e.g., ruffles, lamellae). To determine the form and function of beta actin in microvascular pericytes and endothelial cells recovering from injury, we prepared isoform-specific antibodies and cDNA probes for immunolocalization, Western and Northern blotting, as well as in situ hybridization. Anti-beta actin IgG was prepared by adsorption and release of beta actin-specific IgG from electrophoretically purified pericyte beta actin bound to nitrocellulose paper. Anti-beta actin IgGs prepared by this affinity selection procedure showed exclusive binding to beta actin present in crude cell lysates containing all three actin isoforms. For controls, we localized beta actin as a bright rim of staining beneath the erythrocyte plasma membrane. Anti-beta actin IgG, absorbed with beta actin bound to nitrocellulose, failed to stain erythrocytes. Simultaneous localization of beta actin with the entire F-actin pool was performed on microvascular pericytes or endothelial cells and 3T3 fibroblasts recovering from injury using anti-beta actin IgG in combination with fluorescent phalloidin. Results of these experiments revealed that pericyte beta actin is localized beneath the plasma membrane in association with filopods, pseudopods, and fan lamellae. Additionally, we observed bright focal fluorescence within fan lamellae and in association with the ends of stress fibers that are preferentially associated with the ventral plasmalemma. Whereas fluorescent phalloidin staining along the stress fibers is continuous, anti-beta actin IgG localization is discontinuous. When injured endothelial and 3T3 cells were stained through wound closure, we localized beta actin only in motile cytoplasm at the wound edge. Staining disappeared as cells became quiescent upon monolayer restoration. Appearance of beta actin at the wound edge correlated with a two- to threefold increase in steady-state levels of beta actin mRNA, which rose within 15-60 min after injury and returned to noninjury levels during monolayer restoration. In situ hybridization revealed that transcripts encoding beta actin were localized at the wound edge in association with the repositioned protein. Results of these experiments indicate that beta actin and its encoded mRNA are polarized at the membrane-cytoskeletal interface within regions of moving cytoplasm.

Sub-Marginal Endothelial Cells Dynamically Regulate Endothelial Sheet Migration.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3971-3971
Author(s):  
Solomon F. Ofori-Acquah
Keyword(s):  
Wound Healing ◽  
Endothelial Cells ◽  
Vascular Permeability ◽  
Cell Tracking ◽  
Time Lapse ◽  
Endothelial Cell Migration ◽  
Wound Edge ◽  
And Migration ◽  
Marginal Cells ◽  
Sheet Migration

Abstract Migration of endothelial cells as a sheet in fully differentiated blood vessels is essential for reducing vascular permeability during wound healing. Indeed, loss of collective endothelial sheet migration contributes to increased vascular permeability in tumor angiogenesis and several vascular proliferative disorders. Despite this significance, mechanisms responsible for keeping migrating endothelial cells in a monolayer, sheet or tube are poorly understood. To unravel the basis for collective endothelial cell migration, we used time-lapse video microscopy to study early events of wound closure in confluent monolayers of primary microvascular endothelial cells in a live cell chamber. Immediately after wounding (0– 20 min), endothelial cells at the margin of the wound (marginal cells) retracted away from the wounded area and showed no visible lamellipodia extensions. The next phase of early wound healing (20 min – 6 hours) revealed extensive lamellipodia formation and migration of marginal cells into the wounded region. Remarkably, sub-marginal endothelial cells that were several microns away from the wound edge protruded lamellipodia that formed dynamic cell-cell contacts with the substratum of marginal cells at the wound edge. In several instances sub-marginal cells physically and coordinately pulled back endothelial cells at wound edge to maintain regularity of the endothelial sheet front. Cell-tracking measurements revealed autonomous and yet coordinated migration of marginal and sub-marginal endothelial cells culminating in net protrusion of the endothelial sheet into the wound. This study provides in real-time evidence of retraction of endothelial cells at the wound edge by several microns prior to the initiation of forward migration. In addition, we show for the first time that endothelial cells several microns away from the wound edge actively participate in sheet migration through the extention of lamellipodia into the substratum of cells at the wound edge. These findings highlight an important role in endothelial sheet migration for the Rho family of GTPases given their intimate control of cell retraction and lamelipodia extensions. Future studies will directly evaluate the influence of Rac, Cdc42 and RhoA in retraction of marginal cells and formation of lamellipodia by sub-marginal cells in endothelial sheet migration.

scholarly journals Polymerisation of fibrin αC-domains promotes endothelial cell migration and proliferation

2014 ◽  
Vol 112 (12) ◽  
pp. 1244-1251 ◽  
Author(s):  
Sergiy Yakovlev ◽  
Irina Mikhailenko ◽  
Galina Tsurupa ◽  
Alexey Belkin ◽  
Leonid Medved
Keyword(s):  
Cell Proliferation ◽  
Wound Healing ◽  
Endothelial Cells ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Wound Closure ◽  
Signalling Pathway ◽  
Endothelial Cell Migration ◽  
Specific Antagonist ◽  
Cell Migration And Proliferation

SummaryUpon conversion of fibrinogen into fibrin, fibrinogen αC-domains containing the RGD recognition motif form ordered αC polymers. Our previous study revealed that polymerisation of these domains promotes integrin-dependent adhesion and spreading of endothelial cells, as well as integrin-mediated activation of the FAK and ERK1/2 signalling pathways. The major goal of this study was to test the impact of αC-domain polymerisation on endothelial cell migration and proliferation during wound healing, and to clarify the mechanism underlying superior activity of αC polymers toward endothelial cells. In an in vitro wound healing assay, confluent endothelial cell monolayers on tissue culture plates coated with the αC monomer or αC polymers were wounded by scratching and wound closure was monitored by timelapse videomicroscopy. Although the plates were coated with equal amounts of αC species, as confirmed by ELISA, wound closure by the cells occurred much faster on αC polymers, indicating that αC-domain polymerisation promotes cell migration and proliferation. In agreement, endothelial cell proliferation was also more efficient on αC polymers, as revealed by cell proliferation assay. Wound closure on both types of substrates was equally inhibited by the integrin-blocking GRGDSP peptide and a specific antagonist of the ERK1/2 signalling pathway. In contrast, blocking the FAK signaling pathway by a specific antagonist decreased wound closure only on αC polymers. These results indicate that polymerisation of the αC-domains enhances integrin-dependent endothelial cell migration and proliferation mainly through the FAK signalling pathway. Furthermore, clustering of integrin-binding RGD motifs in αC polymers is the major mechanism triggering these events.

Lysophosphatidic acid and sphingosine 1-phosphate stimulate endothelial cell wound healing

2000 ◽  
Vol 278 (3) ◽  
pp. C612-C618 ◽  
Author(s):  
Hsinyu Lee ◽  
Edward J. Goetzl ◽  
Songzhu An
Keyword(s):  
Wound Healing ◽  
Endothelial Cells ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Lysophosphatidic Acid ◽  
Mitogen Activated Protein Kinase ◽  
Endothelial Cell Migration ◽  
Mapk Phosphorylation ◽  
Cell Functions ◽  
Sphingosine 1 Phosphate

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are potent lipid growth factors with similar abilities to stimulate cytoskeleton-based cellular functions. Their effects are mediated by a subfamily of G protein-coupled receptors (GPCRs) encoded by endothelial differentiation genes ( edgs). We hypothesize that large quantities of LPA and S1P generated by activated platelets may influence endothelial cell functions. Using an in vitro wound healing assay, we observed that LPA and S1P stimulated closure of wounded monolayers of human umbilical vein endothelial cells and adult bovine aortic endothelial cells, which express LPA receptor Edg2, and S1P receptors Edg1 and Edg3. The two major components of wound healing, cell migration and proliferation, were stimulated individually by both lipids. LPA and S1P also stimulated intracellular Ca2+mobilization and mitogen-activated protein kinase (MAPK) phosphorylation. Pertussis toxin partially blocked the effects of both lipids on endothelial cell migration, MAPK phosphorylation, and Ca2+ mobilization, implicating Gi/o-coupled Edg receptor signaling in endothelial cells. LPA and S1P did not cross-desensitize each other in Ca2+ responses, suggesting involvement of distinct receptors. Thus LPA and S1P affect endothelial cell functions through signaling pathways activated by distinct GPCRs and may contribute to the healing of wounded vasculatures.

Sign in / Sign up

Export Citation Format

Share Document