Centrosome reorientation in regenerating endothelial monolayers requires bFGF

Monolayers of endothelial cells respond to physical denudation with a characteristic sequence of lamellipodia extrusion, cell migration, and cell proliferation. Basic fibroblast growth factor (bFGF) has been implicated as a necessary component of this process: addition of exogenous bFGF enhances monolayer regeneration both in vitro and in vivo, and monolayer regeneration can be inhibited in vitro by treatment with neutralizing antibodies raised against bFGF. Centrosome reorientation from a random location to one preferentially situated between the nucleus and the denudation edge has been postulated as a mechanism essential for cell polarization and subsequent migration. This present study examined the effects of a polyclonal antibody to bFGF and suramin on monolayer regeneration, actin microfilament staining, and centrosome orientation at the wound edge of partially denuded bovine large vessel endothelial monolayers. Treatment with anti-bFGF or suramin abolished monolayer repair in these cultures. Cells at the denudation edge showed altered actin staining patterns and reduced lamellipodia extrusion, and there was complete inhibition of centrosome reorientation in treated cultures. Monolayer repair and centrosome reorientation could be restored by addition of exogenous bFGF in antibody but not suramin treated cultures. Recent evidence suggests that preferential centrosome location in migrating cells may be a consequence of lamellipodia protrusion and cell spreading, rather than an indication of cell polarization. However, these results indicate that agents which interfere with bFGF availability prevent endothelial monolayer regeneration via mechanisms involving cell spreading and/or centrosome reorientation.     

正常及损伤后修复的内皮细胞合成的蛋白聚糖

用自制尼龙刷将培养至汇合的人脐静脉内皮细胞刮伤后,造成规则的内皮细胞缺失区。继续培养可见,原有的内皮钿胞很快迁移到缺失区,并分裂增殖。约48小时新生的内皮细胞即将缺失区全部修复而形成新的汇合单层。以DEAE-Sephacel离子交换及Sepharose 6B凝胶过滤柱层析分析损伤后修复的内皮细胞合成的蛋白聚糖时发现所合成的蛋白聚糖总量减少;硫酸乙酰肝素蛋白聚糖合成相对减少、而硫酸软骨素及/或硫酸皮肤素蛋白聚糖合成相对增多。说明:伴随着内皮细胞的损伤后修复其蛋白聚糖的合成也有质和量的改变。     

Cytoskeleton in TFG-beta- and bFGF-modulated endothelial monolayer repair

Endothelial cell proliferation and migration in vitro is depressed by transforming growth factor beta (TFG-beta) and enhanced by basic fibroblast growth factor (bFGF) treatment. This study examines interactions between cytoskeletal changes and cell proliferation in regenerating endothelial monolayers treated with bFGF, TFG-beta, and both factors. As previously described by others, monolayer regeneration is enhanced by bFGF and reduced by TFG-beta. Endothelial cell morphology is altered by TFG-beta treatment. Cells lose their cobblestone appearance and assume a pleomorphic shape. Actin microfilament staining is modified in both intact and regenerating TFG-beta-treated monolayers as well. There is a loss of dense peripheral band staining and an enhancement in staining intensity of cytoplasmic stress fibers. No such alterations are seen in bFGF-treated cultures. Cell proliferation at the wound edge, as indicated by bromodeoxyuridine incorporation, is inhibited by TGF-beta. Although monolayer repair is modulated by growth factor treatment, centrosome reorientation and microtubule staining patterns are not altered by either factor. Thus these factors appear to have effects on a mechanism(s) other than centrosome reorientation which may be involved in repair of denuded endothelial monolayers.     

Fluid shear stress enhanced DNA synthesis in cultured endothelial cells during repair of mechanical denudation

We have previously observed a stimulatory effect of fluid shear stress on the regeneration of cultured endothelial cell layers after mechanical denudation. In this study we examined how fluid shear stress affects endothelial cell DNA synthesis during regeneration. Following mechanical denudation of narrow linear areas, monolayers of bovine aortic endothelial cells cultured on plastic dishes were subjected to shear stress of 1.3-4.1 dynes/cm2 for 24-48 hours in a specially designed apparatus. After the application of shear stress, cells were stained with propidium iodide, and its fluorescence intensity, reflecting cellular DNA content, was measured using photometric fluorescence microscopy. The DNA content of cells exposed to shear stress increased significantly more than that of paired, static control cells (p less than 0.005 to p less than 0.001). The DNA histogram showed that cells exposed to shear stress contained a relatively high proportion of cells located in the S, G2, and M phases of the cell cycle as compared with the static control. These data suggest that fluid shear stress enhances endothelial cell DNA synthesis during the repair of mechanical denudation.     

Autocrine activities of basic fibroblast growth factor: regulation of endothelial cell movement, plasminogen activator synthesis, and DNA synthesis

We have found that the spontaneous migration of bovine aortic endothelial cells from the edge of a denuded area in a confluent monolayer is dependent upon the release of endogenous basic fibroblast growth factor (bFGF). Cell movement is blocked by purified polyclonal rabbit IgG to bFGF as well as affinity purified anti-bFGF IgG and anti-bFGF F(ab')2 fragments. The inhibitory effect of the immunoglobulins is dependent upon antibody concentration, is reversible, is overcome by the addition of recombinant bFGF, and is removed by affinity chromatography of the antiserum through a column of bFGF-Sepharose. Cell movement is also reversibly inhibited by the addition of protamine sulfate and suramin; two agents reported to block bFGF binding to its receptor. The addition of recombinant bFGF to wounded monolayers accelerates the movement of cells into the denuded area. Transforming growth factor beta which has been shown to antagonize several other effects of bFGF also inhibits cell movement. The anti-bFGF IgG prevents the movement of bovine capillary endothelial cells, BHK-21, NIH 3T3, and human skin fibroblasts into a denuded area. Antibodies to bFGF, as well as suramin and protamine sulfate also suppress the basal levels of plasminogen activator and DNA synthesis in bovine aortic endothelial cells.     

HUMAN VASCULAR ENDOTHELIAL CELLS IN CULTURE : Growth and DNA Synthesis

Human endothelial cells, obtained by collagenase treatment of term umbilical cord veins, were cultured using Medium 199 supplemented with 20% fetal calf serum. Small clusters of cells initially spread on plastic or glass, coalesced and grew to form confluent monolayers of polygonal cells by 7 days. Cells in primary and subcultures were identified as endothelium by the presence of Weibel-Palade bodies by electron microscopy. A morphologically distinct subpopulation of cells contaminating some primary endothelial cultures was selectively subcultured, and identified by ultrastructural criteria as vascular smooth muscle. Autoradiography of endothelial cells after exposure to [³H]thymidine showed progressive increases in labeling in growing cultures beginning at 24 h. In recently confluent cultures, labeling indices were 2.4% in central closely packed regions, and 53.2% in peripheral growing regions. 3 days after confluence, labeling was uniform, being 3.5 and 3.9% in central and peripheral areas, respectively. When small areas of confluent cultures were experimentally "denuded," there were localized increases in [³H]thymidine labeling and eventual reconstitution of the monolayer. Liquid scintillation measurements of [³H]thymidine incorporation in primary and secondary endothelial cultures in microwell trays showed a similar correlation of DNA synthesis with cell density. These data indicate that endothelial cell cultures may provide a useful in vitro model for studying pathophysiologic factors in endothelial regeneration.     

Endothelial cell confluence regulates cyclooxygenase-2 and prostaglandin E2 production that modulate motility

Endothelial cells line the vasculature and, after mechanical denudation during invasive procedures or cellular loss from natural causes, migrate to reestablish a confluent monolayer. We find confluent monolayers of human umbilical vein endothelial cells were quiescent and expressed low levels of cyclooxygenase-2, but expressed cyclooxygenase-2 at levels comparable with cytokine-stimulated cells when present in a subconfluent culture. Mechanically wounding endothelial cell monolayers stimulated rapid cyclooxygenase-2 expression that increased with the level of wounding. Cyclooxygenase-2 re-expression occurred throughout the culture, suggesting signaling from cells proximal to the wound to distal cells. Media from wounded monolayers stimulated cyclooxygenase-2 expression in confluent monolayers, which correlated with the level of wounding of the donor monolayer. Wounded monolayers and cells in subconfluent cultures secreted enhanced levels of prostaglandin (PG) E(2) that depended on cyclooxygenase-2 activity, and PGE(2) stimulated cyclooxygenase-2 expression in confluent endothelial cell monolayers. Cells from subconfluent monolayers migrated through filters more readily than those from confluent monolayers, and the cyclooxygenase-2-selective inhibitor NS-398 suppressed migration. Adding PGE(2) to NS-398-treated cells augmented migration. Endothelial cells also migrated into mechanically denuded areas of confluent monolayers, and this too was suppressed by NS-398. We conclude that endothelial cells not in contact with neighboring cells express cyclooxygenase-2 that results in enhanced release of PGE(2), and that this autocrine and paracrine loop enhances endothelial cell migration to cover denuded areas of the endothelium.     

Suramin inhibits C6 glioma-induced angiogenesis in vitro

Aspects of tumor-induced angiogenesis in vitro were examined using an assay involving collagen gel invasion by a surface monolayer of bovine endothelial cells under the influence of serum free conditioned medium produced by C6 cells, an experimentally derived rat glial tumor cell line. The effects of the polyanionic compound suramin, known to interfere with growth factor/cell signaling on this process were evaluated. Collagen gel invasion was quantified by adding C6 conditioned medium with or without various doses of suramin to monolayers of bovine aortic endothelial cells grown on type I collagen gels in transwell inserts. Cultures were monitored with phase-contrast microscopy. After various periods of incubation collagen gels were fixed, embedded in epoxy resin, and 1-μm thick sections were stained with toluidine blue. Additional cultures were used to evaluate the effects of C6 conditioned medium and suramin on endothelial cell proliferation, and on chemotaxis through 8-μm pores. C6 glioma cell conditioned medium induced large vessel endothelial cells to sprout into the underlying collagen matrix and subsequently from networks of capillary like tubes. Conditioned medium was also chemotactic and mitogenic for these cells. The addition of suramin to C6 glioma conditioned medium prevents tube formation in collagen gels, and inhibits both endothelial cell proliferation and chemotaxis in a dose dependent manner. These results suggest that glial tumor cell conditioned medium induces angiongenesis in large vessel endothelial cells in vitro via mechanisms which are disrupted by suramin, most likely involving tumor-derived growth factor release and/or endothelium-mediated matrix proteolysis.     

Synthesis of DNA and alpha-fetoprotein in the monolayer culture of mouse hepatocytes

Relation between processes of proliferation and synthesis of the embryonal serum protein alpha-fetoprotein (AFP), the influence on these processes of polyelectrolyte dextran sulfate (DS) and dimethyl-sulfoxide (DMSO) has been studied in the monolayer culture of mouse hepatocytes. In control cultures the correlations between the time of appearance and the level of DNA and AFP synthesis were observed. DS and DMSO were found to inhibit both processes. Cell proliferation could be reestablished by addition of epidermal growth factor. In case of the influence of DMSO, it wasn't followed by the induction of AFP synthesis. This the processes of DNA and AFP synthesis in monolayer cultures of mouse hepatocytes can be separated. The elongated incubation of hepatocytes with collagenase during their obtaining, abolished the effects of DS. This shows that surface components of hepatocytes, lost upon enzyme degradation, may be involved in the mechanism of DS effect.     

Cell growth in vitro directed by handmade patterns

Summary In this report, we show how the in vitro model of mechanically injured confluent monolayers of cultured mammalian cells, consisting in denudation by gentle scraping of areas in the monolayer, can be extended to obtain patterned cell cultures without using preadded attaching matrices. This work was done with a sinusoidal endothelial liver cell line. Patterns for cell growth were drawn in confluent monolayers by cell detaching with the aid of pipette tips followed by reincubation of the culture. In one or some d, acellular patterns were closed by cell migration and proliferation. For unveiling the pattern formed by migration and cell duplication, an enzymatic digestion with trypsin-collagenase solution was applied; after some min, only the migrating and younger cells filling the previous acellular pattern remained attached to the dish, and the now cellular pattern was clearly depicted. After stopping and recovering from the enzymatic treatment, the culture was ready for starting studies such as those related to migration, proliferation, cell-cell interactions. This method allows us to create simple and complex patterns, does not require preparation of the dishes with attaching matrices, and extracellular matrices in acellular areas are absent because of the enzymatic treatment, thus, potentially having many applications in cell culture techniques.     

SPARC inhibits endothelial cell adhesion but not proliferation through a tyrosine phosphorylation-dependent pathway

SPARC, a counteradhesive matricellular protein, inhibits endothelial cell adhesion and proliferation, but the pathways through which these activities are blocked are not known. In this study, we used inhibitors of major signaling proteins to identify mediators through which SPARC exerts its counteradhesive and antiproliferative functions. Pretreatments with the general protein tyrosine kinase (PTK) inhibitors, herbimycin A and genistein, protected against the inhibitory effect of SPARC on bovine aortic endothelial (BAE) cell spreading by more than 60 %. Similar pretreatments with PTK inhibitors significantly blocked the diminishment of focal adhesions by SPARC in confluent BAE cell monolayers, as determined by the formation of actin stress-fibers and the distribution of vinculin in focal adhesion plaques. Inhibition of endothelial cell cycle progression by SPARC and a calcium-binding SPARC peptide, however, was not affected by PTK inhibitors. Inhibition of DNA synthesis by SPARC was not reversed by inhibitors of the activity of protein kinase C (PKC), or of cAMP-dependent protein kinase (PKA), but was sensitive to pertussis (and to a lesser extent, cholera) toxin. The counteradhesive effect of SPARC on endothelial cells is, therefore, mediated through a tyrosine phosphorylation-dependent pathway, whereas its antiproliferative function is dependent, in part, on signal transduction via a G protein-coupled receptor. J. Cell. Biochem. 70:543–552, 1998. © 1998 Wiley-Liss, Inc.     

Hyaluronate coat formation and cell spreading in rat fibrosarcoma cells

Hyaluronate-containing pericellular coats have been demonstrated around rat fibrosarcoma cells by exclusion of particles (fixed red blood cells). The cell coats normally form during spreading of the rat fibrosarcoma cells subsequent to subculturing. Monensin, a drug which disrupts the Golgi and which also inhibits hyaluronate synthesis in these cells, inhibits the regeneration of these coats after hyaluronidase or trypsin treatment but does not inhibit cell spreading. Cycloheximide, a drug which inhibits protein but not hyaluronate synthesis does not prevent coat regeneration but partially inhibits cell spreading. Thus by exploiting the opposing effects of cycloheximide and monensin on coat regeneration and cell spreading, we have been able to dissociate these two phenomena.     

Stimulation of “nude” spleen cells in vitro under the influence of thymus lymphocytes

Spleen cells from “nude” mice (nu/nu) were stimulated in vitro with sheep erythrocytes. After 4 days of in vitro incubation only a few plaque-forming cells were found in these cultures. This immunological deficit can be restored by adding thymus lymphocytes from allogeneic donors to nu/nu spleen cultures. The restoration is not successful when thymocytes are prevented from proliferating, by irradiation or with Mitomycin C, or when their DNA dependent RNA synthesis is suppressed by actinomycin D.     

Differential growth factor induction and modulation of human gastric epithelial regeneration

While several autocrine/paracrine growth factors (GFs) can all stimulate epithelial regeneration in experimentally wounded primary gastric cultures, clinical relevance for their non-redundant cooperative actions in human gastric ulcer healing is suggested by the sequential pattern of GF gene induction in vivo. Using new HGE cell lines able to form a coherent monolayer with tight junctions as well as using primary human gastric epithelial cultures, we show that EGF, TGFalpha, HGF and IGFs accelerate epithelial restitution upon wounding, independently of the TGFbeta pathway (as opposed to intestinal cells). However, they differently modulate cell behavior: TGFalpha exerts strong effects (even more than EGF) on cytoplasmic spreading and non-oriented protruding activity of bordering cells whereas HGF preferentially coordinates single lamella formation, cell elongation and migration into the wound. IGF-I and IGF-II rather induce the alignment of bordering cells and maintain a compact monolayer front. The number of mitotic cells maximally increases with EGF, followed by TGFalpha and IGF-I,-II. The current study demonstrates that GFs differentially regulate the regeneration of human gastric epithelial cells through specific modulation of cell shape adaptation, migration and proliferation, further stressing that a coordination of GF activities would be necessary for the normal progression of post-wounding epithelial repair.     

Fibroblastic cell cycling in collagen gels

Abstract. Quiescent C3H10T1/2 mouse fibroblasts resume DNA synthesis and proliferation following incubation in medium containing fresh serum both when grown in monolayer and when grown in a collagen matrix. We observed that the rate of DNA synthesis is reduced at high initial cell densities and low initial collagen concentrations. In a collagen matrix, fibroblasts contract the matrix causing an increase in cell density and collagen concentration. We studied the chronological relationship between the kinetics of DNA synthesis and the collagen matrix contraction. The rate of collagen collection per cell changes in time, dependent on initial cell and collagen concentration. The kinetics of the collagen collection showed a positive correlation with the kinetics of DNA synthesis, 16 h later.     

Behavior of confluent endothelial cells after irradiation. Modulation of wound repair by heparin and acidic fibroblast growth factor

Image analysis was used to study the repair process of a circular mechanical lesion of confluent human endothelial cells in culture after irradiation (10 Gy) prior to wounding. Coverage of denuded areas 48 and 96 h after injury of endothelial cells was identical in control and irradiated cultures, although the labeling index was lowered by 80 to 95% in irradiated cultures. The cell density of non damaged irradiated areas was decreased by 50%. When cultures were submitted to increasing doses of radiation (5.0-30 Gy), the labeling index of the cells diminished rapidly between 0 and 5.0 Gy and reached a plateau at 10 Gy. The decrease in cell density (50% of control at 96 h) was identical at each dose of radiation. Thus cell migration alone could be sufficient for the repair of the lesion, while cell proliferation would mainly maintain the original cell density. The addition of heparin to the culture medium slowed down cell migration and proliferation, but the speed of repair was identical in irradiated and non-irradiated cultures. Acidic fibroblast growth factor plus heparin accelerated equally the repair process whether the cultures were irradiated or not. In irradiated cultures the presence of acidic fibroblast growth factor and heparin maintained cell density in confluent areas at a level similar to that in non-irradiated damaged control cultures without addition of mitogens. Thus heparin and acidic fibroblast growth factor play a role in cell proliferation, in the maintenance of the cell monolayer integrity and in restoring a continuous layer by rapid cell migration and elongation after irradiation.     

Behavior of confluent endothelial cells after irradiation. Modulation of wound repair by heparin and acidic fibroblast growth factor

Image analysis was used to study the repair process of a circular mechanical lesion of confluent human endothelial cells in culture after irradiation (10 Gy) prior to wounding. Coverage of denuded areas 48 and 96 h after injury of endothelial cells was identical in control and irradiated cultures, although the labeling index was lowered by 80 to 95% in irradiated cultures. The cell density of non damaged irradiated areas was decreased by 50%. When cultures were submitted to increasing doses of radiation (5.0–30 Gy), the labeling index of the cells diminished rapidly between 0 and 5.0 Gy and reached a plateau at 10 Gy. The decrease in cell density (50% of control at 96 h) was identical at each dose of radiation. Thus cell migration alone could be sufficient for the repair of the lesion, while cell proliferation would mainly maintain the original cell density. The addition of heparin to the culture medium slowed down cell migration and proliferation, but the speed of repair was identical in irradiated and non-irradiated cultures. Acidic fibroblast growth factor plus heparin accelerated equally the repair process whether the cultures were irradiated or not. In irradiated cultures the presence of acidic fibroblast growth factor and heparin maintained cell density in confluent areas at a level similar to that in non-irradiated damaged control cultures without addition of mitogens. Thus heparin and acidic fibroblast growth factor play a role in cell proliferation, in the maintenance of the cell monolayer integrity and in restoring a continuous layer by rapid cell migration and elongation after irradiation.     

c-Jun/AP-1 is required for CCK-induced pancreatic acinar cell dedifferentiation and DNA synthesis in vitro

Endogenous CCK plays an important role in pancreatic regeneration after pancreatitis. We used primary culture of mouse pancreatic acinar cells to evaluate the effect of CCK on acinar cell morphology and gene expression and to determine signaling pathways required for proliferation of acinar cells in vitro. Over 4 days in culture, cells grew out from acini and formed patches of monolayer, which displayed a reduced expression of acinar cell markers including digestive enzymes and Mist1 and an increased expression of ductal and embryonic markers, including cytokeratin 7, β-catenin, E-cadherin, pdx-1, and nestin. There was no appearance of stellate cell markers. CCK enhanced cellular spreading, DNA synthesis, and cyclin D1 expression. When signaling pathways were evaluated, CCK stimulation increased c-Jun expression, JNK and ERK activity, and AP-1 activation. Chemical inhibitors of JNK and ERK pathways, dominant-negative JNK and c-Jun, and c-Jun shRNA significantly inhibited CCK-induced DNA synthesis, CCK-induced AP-1 activation, and cyclin D1 expression. Furthermore, dominant-negative c-Jun reduced the increased expression of β-catenin and the decreased expression of amylase during culture. These results show that MAPK/c-Jun/AP-1 pathway plays an important role in pancreatic acinar cell dedifferentiation and proliferation in culture. Monolayer culture can serve as a model to study acinar cell proliferation similar to regeneration after pancreatitis in vivo.     

Differential control of cytokeratins and vimentin synthesis by cell- cell contact and cell spreading in cultured epithelial cells

The expression of cytokeratins and vimentin was investigated in Madin- Darby bovine epithelial cells (MDBK) in culture under conditions of varied cell spreading and cell-cell contact. When extensive cell-cell contact was achieved by seeding cells at high density in monolayer, or in suspension culture in which multicellular aggregates formed, the cells synthesized high levels of cytokeratins and low levels of vimentin. In contrast, in sparse monolayer and suspension cultures where cell-cell contact was minimal, the cells synthesized very low levels of cytokeratins. The level of vimentin synthesis was high in sparse monolayer culture and was low in both sparse and dense suspension cultures. The ratio of cytokeratin to vimentin synthesis was not affected during the cell cycle, or when cell growth was inhibited by ara C and in serum-starvation-stimulation experiments. The variations in the synthesis of cytokeratins and vimentin under the various culture conditions were also reflected at the level of mRNA activity in a cell-free in vitro translation system and as determined by RNA blot hybridization with cDNA to vimentin and cytokeratins. The results suggest that control of cytokeratin synthesis involves cell- cell contact, characteristic of epithelia in vivo, while vimentin synthesis responds to alterations in cell spreading.