Changes In Endothelial Cell Morphology Research Articles

Effect of spatial gradient in fluid shear stress on morphological changes in endothelial cells in response to flow

Arterial bifurcations are common sites for development of cerebral aneurysms. Although this localization of aneurysms suggests that high shear stress (SS) and high spatial SS gradient (SSG) occurring at the bifurcations may be crucial factors for endothelial dysfunction involved in aneurysm formation, the details of the relationship between the hemodynamic environment and endothelial cell (EC) responses remain unclear. In the present study, we sought morphological responses of ECs under high-SS and high-SSG conditions using a T-shaped flow chamber. Confluent ECs were exposed to SS of 2–10 Pa with SSG of up to 34 Pa/mm for 24 and 72 h. ECs exposed to SS without spatial gradient elongated and oriented to the direction of flow at 72 h through different processes depending on the magnitude of SS. In contrast, cells did not exhibit preferred orientation and elongation under the combination of SS and SSG. Unlike cells aligned to the flow by exposure to only SS, development of actin stress fibers was not observed in ECs exposed to SS with SSG. These results indicate that SSG suppresses morphological changes of ECs in response to flow.

Regulation of Vascular Endothelial Function by Procyanidin-Rich Foods and Beverages

Flavonoid-rich diets are associated with a lower mortality from cardiovascular disease. This has been linked to improvements in endothelial function. However, the specific flavonoids, or biologically active metabolites, conferring these beneficial effects have yet to be fully defined. In this experimental study of the effect of flavonoids on endothelial function cultured endothelial cells have been used as a bioassay with endothelin-1 (ET-1) synthesis being measured an index of the response. Evaluation of the relative effects of extracts of cranberry juice compared to apple, cocoa, red wine, and green tea showed inhibition of ET-1 synthesis was dependent primarily on their oligomeric procyanidin content. Procyanidin-rich extracts of cranberry juice triggered morphological changes in endothelial cells with reorganization of the actin cytoskeleton and increased immunostaining for phosphotyrosine residues. These actions were independent of antioxidant activity. Comparison of the effects of apple procyanidin monomers through heptamer showed a clear structure-activity relationship. Although monomer, dimer, and trimer had little effect on ET-1 synthesis, procyanidin tetramer, pentamer, hexamer, and heptamer produced concentration-dependent decreases with IC(50) values of 5.4, 1.6, 0.9, and 0.7 microM, respectively. Levels of ET-1 mRNA showed a similar pattern of decreases, which were inversely correlated with increased expression of Kruppel-like factor 2 (KLF2), a key endothelial transcription factor with a broad range of antiatherosclerotic actions including suppression of ET-1 synthesis. Future investigations of procyanidin-rich products should assess the role KLF2 induction plays in the beneficial vascular effects of high flavonoid consumption.

Light and electron microscopic evaluation of canine corneal endothelium following CO2 photokeratotomy

To determine using light and scanning electron microscopy if treatment with CO2 photokeratotomy alters the corneal endothelium in healthy dogs. Eight surgery laboratory dogs were determined to be free of ocular abnormalities. Under general anesthesia, the left eye of each dog was treated in a quadrant from 12 to 3 o'clock with the CO(2) laser in a defocused mode. The right eye served as a control. There were four treatment groups, each with 2 dogs: group 1 (2 W, 0.1 J/s, 0.8 mm tip), group 2 (3 W, 0.3 J/s, 0.8 mm tip), group 3 (2 W, 0.04 J/s NovaScan), group 4 (3 W, 0.06 J/s, NovaScan). The 0.8 mm tip delivered a power density of 382 W/cm(2) or 573 W/cm(2), at 2 or 3 watts respectively. The NovaScan handpiece delivered a power density of 30 W/cm(2) or 40 W/cm(2), at 2 or 3 Watts respectively. Following euthanasia, right and left corneas including a 2-mm scleral rim were harvested and fixed in commercial grade Karnovsky's fixative. One piece of cornea was processed routinely, embedded in Embed 812 resin, sectioned at 1 um, stained with toluidine blue and evaluated with the light microscope. A separate piece of each cornea was routinely processed and examined with a JEOL 6400 scanning electron microscope (SEM) at 20 KV. No changes in endothelial cell morphology were detected by light microscopy in the sections examined. SEM indicated normal endothelial cell morphology in control eyes with presumed artifactual changes at the corneal free margin (4/8 eyes). Multiple punctate to linear regions of endothelial cell destruction were observed in 6/8 laser-treated corneas. A significant increase in corneal thickness ranging from 1.90 to 37.28% was observed in all laser treated corneas. This increase in thickness correlated linearly with the degree of endothelial damage. Ultrastructural findings also correlated with postoperative clinical findings. CO2 laser photokeratotomy alters corneal endothelial cell morphology and thickness.

Oligosaccharides of hyaluronan induce angiogenesis through distinct CD44 and RHAMM-mediated signalling pathways involving Cdc2 and γ-adducin

We used short-interfering RNA (siRNA) to knockdown the hyaluronan (HA) receptors CD44 and the receptor for hyaluronan-mediated motility (RHAMM) in vascular endothelial cells to investigate their role in angiogenesis. We showed that CD44 and RHAMM single knockdown inhibited low molecular weight hyaluronan (o-HA)-induced endothelial cell tube formation in Matrigel, but no change in the control, epidermal growth factor-induced tube formation was observed. Using a Kinexus phosphoprotein array and confirmational Western blotting we were able to show a differential effect on HA-induced protein expression after CD44 and RHAMM knockdown. CD44 knockdown abolished o-HA-induced membrane phospho-protein kinase C-alpha (PKC-alpha) and down-stream phospho-gamma-adducin expression. Using the PKC inhibitor Go6976, we demonstrated the involvement of PKC-alpha and gamma-adducin in o-HA-induced tube formation, whilst o-HA-induced enzymatic activity of MMP9 was also reduced. This suggests that endothelial tube formation involves activation of MMP9 via PKC-alpha. Furthermore, the involvement of gamma-adducin in o-HA-induced F-actin cytoskeleton rearrangement was CD44-dependent and the reduction of CD44 expression lead to a change in endothelial cell morphology. Both RHAMM and CD44 knockdown completely inhibited o-HA-induced Cdc2 (Cdk1) phosphorylation suggesting a possible involvement in cell cycle control. Although CD44 and RHAMM are both involved in o-HA-induced endothelial tube formation in Matrigel, they mediate distinct angiogenic signalling pathway and for the first time we demonstrated the specific involvement of gamma-adducin in CD44/o-HA-induced endothelial tube formation. The data presented here extend our understanding of key stages of the processes of o-HA-induced angiogenesis which may have relevance to tumour progression.

Antivascular Actions of Microtubule-Binding Drugs

Microtubule-binding drugs (MBD) are widely used in cancer chemotherapy and also have clinically relevant antiangiogenic and vascular-disrupting properties. These antivascular actions are due in part to direct effects on endothelial cells, and all MBDs (both microtubule-stabilizing and microtubule-destabilizing) inhibit endothelial cell proliferation, migration, and tube formation in vitro, actions that are thought to correspond to therapeutic antiangiogenic actions. In addition, the microtubule-destabilizing agents cause prominent changes in endothelial cell morphology, an action associated with rapid vascular collapse in vivo. The effects on endothelial cells occur in vitro at low drug concentrations, which do not affect microtubule gross morphology, do not cause microtubule bundling or microtubule loss and do not induce cell cycle arrest, apoptosis, or cell death. Rather, it has been hypothesized that, at low concentrations, MBDs produce more subtle effects on microtubule dynamics, block critical cell signaling pathways, and prevent the microtubules from properly interacting with transient subcellular assemblies (focal adhesions and adherens junctions) whose subsequent stabilization and/or maturation are required for cell motility and cell-cell interactions. This review will focus on recent studies to define the molecular mechanisms for the antivascular actions of the MBDs, information that could be useful in the identification or design of agents whose actions more selectively target the tumor vasculature.

Influence of various radiographic contrast media on the buckling of endothelial cells

The intra-arterial application of radiographic contrast media (RCM) can induce decreases of blood flow velocity in downstream capillaries as well as a decrease in the tissue oxygen tension. It is unclear whether changes in endothelial cell morphology contribute to the observed microcirculatory disorders. Four RCMs (Iodixanol320, Iohexol350, Iopromide370, and Imeron350) were added to the culture medium of human umbilical venous endothelial cells (HUVEC) and used for short-term incubation studies of these cells. Addition of Iohexol ( p = 0.6377) and Iodixanol ( p = 0.6309) did not affect the HUVEC height 1.5 min after incubation in the modified cell culture media supplemented with 30% v/v of the respective RCM. Strong buckling and increased endothelial height appeared after incubation in Iopromide-supplemented medium (the cell height increased by 95% compared to cells incubated under control conditions; p = 0.0065). Addition of Iomeprol-supplemented medium caused an increase by 61.6% compared to cells incubated under control conditions; p = 0.0051. After 5 min of incubation in any of the RCM-supplemented media, there was no difference in HUVEC height in comparison to incubation in control standard culture media (each p value > 0.05). The tremendous buckling caused by Iopromide and Iomeprol, coinciding with an echinocyte formation of erythrocytes might be the reason why a bolus injection of Iopromide in vivo into the left coronary artery was followed by a 50% decrease of oxygen partial pressure in the supplied tissue.

Hypoxia Inducible Factor 1 Alpha Regulates Matrigel-induced Endovascular Differentiation under Normoxia in a Human Extravillous Trophoblast Cell Line

Extravillous trophoblast (EVT) cells mimic endothelial cells during angiogenesis, inducing remodeling of the spiral arteries that increases blood flow toward the intravillous space. We have previously shown that signals involving the vascular endothelial growth factor (VEGF) axis are essential for endovascular differentiation through integrin signaling from the extracellular matrix: This was accomplished with use of the human EVT cell line TCL1, which shows tube formation that specifically recalls morphological changes in endothelial cells. To investigate endovascular differentiation in EVT further, we investigated the role of hypoxia inducible factor (HIF)1A, a subunit of HIF1 transcription factor that regulates not only adaptive responses to hypoxia, but also many cellular functions under normoxia, which was up-regulated in DNA microarray analysis during matrigel-induced endovascular differentiation under normoxia. HIF1A induces VEGF and ITGAV/ITGB3 aggregation, actions known to be important for cellular survival and endovascular differentiation in EVT. Inhibition of HIF1A up-regulation using siRNA introduction or chemical inhibition suppressed hypoxia-responsive element transcriptional activity, VEGF induction, ITGAV/ITGB3 aggregation accompanied by the inhibition of tube formation in TCL1 cells. These results suggest that HIF1A has a crucial role in regulating EVT behavior including matrigel-induced endovascular differentiation under normoxia.

Roles of Rho/ROCK and MLCK in TNF‐α‐induced changes in endothelial morphology and permeability

Tumor necrosis factor-alpha (TNF-alpha) is known to induce changes in endothelial cell morphology and permeability, but the mechanisms have not been extensively characterized. TNF-alpha rapidly induced RhoA activation and myosin light chain phosphorylation, but caused only small changes to cortical F-actin, without significantly increasing paracellular permeability up to 30 min after stimulation. TNF-alpha subsequently caused a progressive increase in permeability and in stress fiber reorganization, cell elongation, and intercellular gap formation over 8-24 h. Consistent with the increased permeability, Occludin and JAM-A were removed from tight junctions and ZO-1 was partially redistributed. Rho/ROCK but not MLCK inhibition prevented the long-term TNF-alpha-induced changes in F-actin and cell morphology, but ROCK inhibition did not affect permeability. These results suggest that the gradual increase in permeability induced by TNF-alpha does not reflect contractile mechanisms mediated by Rho, ROCK, and MLCK, but involves long-term reorganization of tight junction proteins.

Long‐term treatment with TGFβ1 impairs mechanotransduction in bovine aortic endothelial cells

Vascular endothelial cells play a role in the physiological response to mechanical stress. Transforming growth factor beta1 (TGFbeta1) induces morphological changes in endothelial cells, and this may alter their mechanosensitive responses. The aim of this study was to examine the effects of TGFbeta1 on hypotonic stress (HTS)-induced responses in bovine aortic endothelial cells (BAECs). Cultured BAECs were treated with 3 ng ml(-1) TGFbeta1 for 24 h (24h-TGFbeta1) or 7 days (7d-TGFbeta1). Cytosolic actin fibres were stained with rhodamine-phalloidin. Intracellular Ca2+ concentration was measured using fura2. Tyrosine phosphorylation and RhoA expression were assessed by Western blotting. Expression of RhoA mRNA was assessed by real-time PCR. BAECs developed pseudopod-like processes within 24 h and showed a fibroblast-like appearance after 7 days. HTS induced Ca2+ transients via endogenous ATP release in both control and 24h-TGFbeta1 BAECs but not in 7d-TGFbeta1 BAECs. We have previously shown that HTS-induced ATP release is mediated by sequential activation of RhoA and tyrosine kinases. The basal amount of membrane-bound RhoA was significantly lower in 7d-TGFbeta1 than in 24h-TGFbeta1 or control BAECs. HTS increased the membrane-bound RhoA to the same fractional level in 24h-TGFbeta1 and control BAECs, but its net maximal amount was significantly lower in 7d-TGFbeta1. HTS-induced downstream signals of RhoA activation, i.e. the tyrosine phosphorylation of FAK and paxillin, were markedly suppressed in 7d-TGFbeta1 BAECs. These results indicate that long-term treatment with TGFbeta1 does not impair mechanoreception in BAECs but impairs mechanotransduction by affecting RhoA membrane translocation.

Role of Ligand-Specific Integrins in Endothelial Cell Alignment and Elongation Induced by Cyclic Strain

Ligand-specific integrins are thought to play a critical role in regulating multiple biological processes. However, the mechanisms by which ligand-specific integrins mediate external stimuli and activate intracellular signaling pathways remain to be elucidated. The aim of this study was to clarify the role of ligand-specific integrins in the morphological changes induced by cyclic strain (CS) via the p38 mitogen-activated protein kinase (p38 MAPK) pathway. Endothelial cells (ECs) were cultured on collagen (a ligand for integrin alpha 2 beta1, but not for alpha 5 and beta 4)-coated flexible plates and incubated for 24 h with or without anti-alpha2 integrin antibody (anti-alpha2), anti-alpha5, anti-beta1, or anti-beta4. ECs were then subjected to 15.6% average CS at 60 cycles/min up to 24 h. After exposure to CS, the cell shape index (defined as (4pi x cell area)/(cell perimeter)(2)), the cell orientation angle, and activation of p38 MAPK were assessed. ECs in the absence of integrin-blocking antibodies were elongated and aligned in response to CS. Anti-alpha 2 and anti-beta1 abolished both morphological changes of ECs as well as the activation of p38 MAPK. In contrast, anti-alpha 5 and anti-beta 4 inhibited neither morphological changes of ECs nor the activation of p38 MAPK. Our results indicate that ligand-specific integrins play a crucial role in the morphological changes of ECs induced by CS via the p38 MAPK pathway.

Endothelialization and Flow Conditioning of Fibrin-Based Media-Equivalents

It is generally accepted that endothelialization and subsequent development of a functional endothelium are of paramount importance to the success of any bioartificial artery. In this study, we aimed to assess the ability of smooth muscle cell-remodeled, fibrin-based media-equivalents (MEs) to be endothelialized, examine the morphological changes of endothelial cells (ECs) associated with exposure to physiologically-relevant shear stress in a custom-built bioreactor, and determine if adherent ECs are capable of withstanding average physiological shear stresses. It was found that MEs could be readily endothelialized with surface coverages of 98.8 +/- 0.9% after two days, and the ECs expressed von Willebrand factor. Furthermore, EC retention remained high (steady: 96.5 +/- 4.4%, pulsatile: 94.3 +/- 4.3%) under exposure to physiologically relevant shear stresses for 48 h. The results indicate that these MEs are conducive to generating an EC monolayer, with the ECs possessing adhesion strength sufficient to withstand physiological shear stress and maintain a normal phenotype.

A role for soluble HLA‐G1 in the induction of vascular cell apoptosis

Uterine spiral artery remodelling is a crucial adaptation in early pregnancy that allows a sufficient blood supply to reach the developing fetus. We have shown that this vascular remodelling involves endothelial and vascular smooth muscle cell apoptosis triggered by the fetal-derived trophoblast cells. Extravillous trophoblasts produce soluble HLA-G1. We have investigated the hypothesis that soluble HLA-G1 is an important regulator of vascular cell apoptosis. Endothelial cells (primary HUVEC or a cell line, SGHEC-7), vascular smooth muscle cells (human aortic SMC line) or extravillous trophoblasts were incubated with recombinant sHLA-G1 and apoptosis was monitored by time-lapse microscopy and western blot analysis of apoptotic markers. Soluble HLA-G1 induced apoptotic morphological changes in endothelial cells. Apoptosis was confirmed using the broad-spectrum caspase inhibitor zVAD-fmk, which abrogated sHLA-G1 induced cell death. In addition, western blot analysis showed that sHLA-G1 treated endothelial cells had increased production of the apoptotic marker cleaved PARP. Soluble HLA-G1-induced endothelial apoptosis was almost completely blocked in the presence of an antibody that prevents Fas/FasL interactions. Soluble HLA-G1 also induced vascular smooth muscle cell apoptosis but had no effect on trophoblast cell survival. In conclusion, we have shown that sHLA-G1 induces vascular cell apoptosis and that for endothelial cells this effect may be partly through the Fas/FasL system. Soluble HLA-G1 may therefore have a role in the induction of uterine spiral artery remodelling seen in early pregnancy.

Biochemical and morphological changes in endothelial cells in response to hypoxic interstitial edema

BackgroundA correlation between interstial pulmonary matrix disorganization and lung cellular response was recently documented in cardiogenic interstitial edema as changes in the signal-cellular transduction platforms (lipid microdomains: caveoale and lipid rafts). These findings led to hypothesize a specific "sensing" function by lung cells resulting from a perturbation in cell-matrix interaction. We reason that the cell-matrix interaction may differ between the cardiogenic and the hypoxic type of lung edema due to the observed difference in the sequential degradation of matrix proteoglycans (PGs) family. In cardiogenic edema a major fragmentation of high molecular weight PGs of the interfibrillar matrix was found, while in hypoxia the fragmentation process mostly involved the PGs of the basement membrane controlling microvascular permeability. Based on these considerations, we aim to describe potential differences in the lung cellular response to the two types of edema.MethodsWe analysed the composition of plasma membrane and of lipid microdomains in lung tissue samples from anesthetized rabbits exposed to mild hypoxia (12 % O2 for 3–5 h) causing interstitial lung edema. Lipid analysis was performed by chromatographic techniques, while protein analysis by electrophoresis and Western blotting. Lipid peroxidation was assessed on total plasma membranes by a colorimetric assay (Bioxytech LPO-586, OxisResearch). Plasma membrane fluidity was also assessed by fluorescence. Lipid microdomains were isolated by discontinuous sucrose gradient. We also performed a morphometric analysis on lung cell shape on TEM images from lung tissue specimen.ResultsAfter hypoxia, phospholipids content in plasma membranes remained unchanged while the cholesterol/phospholipids ratio increased significantly by about 9% causing a decrease in membrane fluidity. No significant increase in lipid peroxidation was detected. Analysis of lipid microdomains showed a decrease of caveolin-1 and AQP1 (markers of caveolae), and an increase in CD55 (marker of lipid rafts). Morphometry showed a significant decrease in endothelial cell volume, a marked increase in the cell surface/volume ratio and a decrease in caveolar density; epithelial cells did not show morphological changes.ConclusionThe biochemical, signaling and morphological changes observed in lung endothelial cell exposed to hypoxia are opposite to those previously described in cardiogenic edema, suggesting a differential cellular response to either type of edema.

Continuous Visualization of Morphological Changes in Endothelial Cells in Response to Cyclic Stretch

Endothelial cells are known to change their shapes in alignment with the direction perpendicular to that of cyclic stretch. However, the mechanisms are still not clear. In this study, we developed a new cell-stretching device which allows continuous observation of cyclically stretched/relaxed cells. Using this apparatus, morphological changes of single cells in response to cyclic stretch (1Hz, 10%, 2 hours) were visualized and analyzed. The data showed that there were two major patterns of morphological changes of ECs, depending on their initial shapes. While ECs with round shapes at the initial state changed their shapes to become more elongated in a direction perpendicular to that of stretch, cells oriented and elongated in the stretch direction at the initial state changed their shapes to first become rounder and then elongated to align perpendicular to the stretch direction at the second step. These results suggest that cell retraction as well as elongation is important in the stretch-induced endothelial morphological changes, and that the elucidation of the mechanisms of the morphological changes requires studies of cell retraction, elongation, and their coordination at the single-cell level.

Effects of Peroxynitrite and Superoxide Radicals on Endothelial Monolayer Permeability: Potential Role of Peroxynitrite in Preeclampsia

Increased endothelial permeability is associated with increased oxidative stress in the maternal vasculature in women with preeclampsia. This study was to determine if oxidative stress elicited by peroxynitrite could lead to an increase in endothelial permeability. Endothelial oxidative stress was produced by adding 3-morpholinosydnonimine (SIN-1, a peroxynitrite generator) to the cell culture. Confluent endothelial cells (ECs) grown in cell culture inserts were treated with SIN-1 at a concentration of 0.5 mM alone or in combination with MnTMPyP (a peroxynitrite scavenger) or superoxide dismutase (SOD). EC permeability was determined by measuring EC electrical resistance (ER) and horseradish peroxide (HRP) leakage. Data are presented as means +/- SE and analyzed by analysis of variance (ANOVA). Junctional protein expression and distribution for vascular endothelial (VE)-cadherin, occludin, and phosphorylated focal adhesion kinase (FAK) at tyrosine 397 [pY397] were examined by fluorescent staining of ECs. First, ER was significantly reduced and HRP leakage was significantly increased in ECs treated with SIN-1 compared to those in control cells, ER: 26.97 +/- 1.41 versus 42.27 +/- 0.40 Omega.cm2, P <.01; HRP: 0.26 +/- 0.07 versus 0.02 +/- 0.01 OD 470 nm, P <.01, respectively. Second, cells treated with SIN-1 showed formation of gaps and disorganized VE-cadherin and occludin distribution at cell contact regions. FAK[pY397] expression was completely lost in cells treated with SIN-1. Finally, these functional and morphologic changes in ECs induced by SIN-1 were blocked in cells pretreated with MnTMPyP and SOD. Disorganization of junctional proteins and dephosphorylation of FAK[pY397] may account for the increased endothelial permeability induced by oxidative stress associated with preeclampsia.

Attenuation of Endotoxin-Induced Acute Lung Injury by the Rho-Associated Kinase Inhibitor, Y-27632

A small GTPase, Rho, plays key roles in cell adhesion, motility, and contraction after stimulation. Among Rho effectors isolated, the family of Rho-associated coiled-coil-forming protein kinases (ROCK) is implicated in Rho-mediated cell adhesion and smooth muscle contraction. The effect of a specific inhibitor of ROCK, Y-27632, was evaluated in a murine model of acute lung injury induced by intravenous injection of Escherichia coli endotoxin (lipopolysaccharide [LPS]). Lung edema was evaluated by measuring extravascular leakage of radio-labeled serum albumin, and neutrophil emigration into the lung parenchyma by morphometric observation and measuring myeloperoxidase activity. Pretreatment with Y-27632 attenuated both lung edema and neutrophil emigration after LPS. We also measured albumin transfer through cultured endothelial cell monolayers on a porous filter. Tumor necrosis factor-alpha significantly increased albumin transfer, which was attenuated by pretreatment with Y-27632. Fluorescence microscopy revealed that morphologic changes in endothelial cells induced by tumor necrosis factor-alpha were inhibited by Y-27632. In contrast, the increased fraction of neutrophils with polymerized actin after formyl-methionyl-leucyl-phenylalanine was not altered by Y-27632. These data suggest that ROCK may play an important role in the pathogenesis of LPS-induced lung injury and that ROCK inhibition could attenuate cytoskeletal rearrangement of endothelial cells, leading to decreased neutrophil emigration into the lung parenchyma.

Potential Antagonism of Tubulin-Binding Anticancer Agents in Combination Therapies

ZD6126 is a vascular targeting agent, developed for the treatment of solid tumors. In vivo, ZD6126 is rapidly converted into the tubulin-binding agent N-acetylcolchinol. We have previously reported that in vitro N-acetylcolchinol disrupts microtubules and induces rapid changes in endothelial cell morphology, which in a tumor would lead to a rapid loss of tumor vessel integrity and subsequent extensive tumor necrosis. The aim of this study was to investigate the effect of cytotoxic antineoplastic drugs-cisplatin, doxorubicin, vincristine, paclitaxel, and docetaxel-on endothelial cell response to N-acetylcolchinol. We found that cisplatin and doxorubicin did not interfere with the ability of N-acetylcolchinol to cause morphologic changes in human umbilical vein endothelial cells, whereas vincristine showed additive effects. In contrast, the microtubule-stabilizing agents paclitaxel (1-10 micromol/L) and docetaxel (0.1-1 micromol/L) prevented the morphologic changes induced by N-acetylcolchinol in human umbilical vein endothelial cells. The effect was observed when cells were exposed to paclitaxel and N-acetylcolchinol together or when paclitaxel was given shortly before N-acetylcolchinol. Paclitaxel and N-acetylcolchinol interacted at the level of microtubule organization, as shown in immunofluorescence analysis of the cytoskeleton. The protective effect was reversible because 4 hours after paclitaxel wash out, cells recovered the sensitivity to N-acetylcolchinol. In vivo, pretreatment of mice with paclitaxel inhibited the vascular targeting activity of ZD6126 on newly formed vessels in the Matrigel plug assay and ZD6126-induced necrosis in tumors. These findings indicate that paclitaxel, depending on the timing and schedule of administration, can affect the vascular targeting activity of ZD6126, which may have an effect on the optimal scheduling of therapies based on the combined use of microtubule-stabilizing and microtubule-destabilizing agents.

Tumor Necrosis Factor and Vascular Endothelial Growth Factor Induce Endothelial Integrin Repertories, Regulating Endovascular Differentiation and Apoptosis in a Human Extravillous Trophoblast Cell Line1

Angiogenesis is crucial in human development. Extravillous trophoblast (EVT) cells mimic endothelial cells in angiogenesis during endovascular differentiation, inducing a remodeling of spiral arteries that increases blood flow toward the intravillous space. We have previously shown that tumor necrosis factor (TNF) alpha regulates expression of ITGA6 and ITGA1, which are involved in cell survival, in the human EVT cell line TCL1. To further investigate endovascular differentiation, we examined the effects of vascular endothelial growth factor (VEGF), TNF, and extracellular matrix (ECM) on TCL1 cells. Seeded on Matrigel, TCL1 cells show tube-like formation that specifically recalls morphological changes in endothelial cells. Anti-ITGAV/ITGB3 antibodies significantly reduced the size of the capillary network (P < 0.05) on Matrigel and also suppressed TNF-induced apoptosis (P < 0.05) in TCL1 cells. VEGF induced expression of ITGAV/ITGB3 subunits and protein aggregation, as in the case of TNF, which in turn, induces synthesis of VEGF in TCL1 cells. Soluble FLT1 suppressed these activities in TCL1 cells, indicating that signals involving VEGF axis are essential for endovascular differentiation. These results suggest that TNF, VEGF, and ECM collaboratively regulate EVT behavior, including cell survival and endovascular differentiation, through integrin signaling during establishment and maintenance of successful human pregnancies.

Vascular corrosion casts mirroring early morphological changes that lead to the formation of saccular cerebral aneurysm: an experimental study in rats

The formation of cerebral aneurysms involves complex processes and little is known about the mechanisms by which they originate, grow, and rupture. The purpose of this study was to identify early ultrastructural morphological changes that lead to the formation of experimental cerebral aneurysms. Twenty male Sprague-Dawley rats were subjected to cerebral aneurysm induction (renal hypertension and right common carotid artery ligation); 10 intact rats served as the control group. The animals were killed after 2 months, and a vascular corrosion cast of their cerebral arteries was prepared and screened for aneurysm development by using a scanning electron microscope. Sequential morphological changes observed at the cerebral artery bifurcation in response to hemodynamic shear stress included endothelial changes, intimal pad elevation, and saccular dilation. Endothelial cell changes were the first observed morphological changes; they were followed by various degrees of artery wall dilation. No aneurysmal changes developed in any of the control rats. Of the 20 surgically treated rats, 11 displayed aneurysmal changes. In five of these animals only changes in the endothelial cell imprints could be identified. In the other six rats morphological changes in endothelial cells were associated with different stages of aneurysmal dilation. This is the first study to demonstrate in vivo early morphological changes that lead to the formation of cerebral aneurysms. The morphological findings indicate the principal role of endothelial cells in the pathogenesis of cerebral aneurysms and suggest that hemodynamic shear stress and blood flow patterns may precipitate these early changes.

Rho GTPases and leucocyte-induced endothelial remodelling

Leucocytes in the bloodstream respond rapidly to inflammatory signals by crossing the blood vessel wall and entering the tissues. This process involves adhesion to, and subsequent transmigration across, the endothelium, mediated by a cascade of interactions between adhesion molecules and stimulation of intracellular signalling pathways in both leucocytes and endothelial cells. This leads to changes in endothelial cell morphology that assist leucocyte extravasation, including endothelial cell contraction, intercellular junction disruption, increased permeability, remodelling of the endothelial apical surface and alterations in vesicle trafficking. Rho GTPases play a central role in many of the endothelial responses to leucocyte interaction. In this review, we discuss recent findings on leucocyte-induced alterations to endothelial cells, and the roles of Rho GTPases in these responses.