Changes In Endothelial Cell Morphology Research Articles

Synergistic effects of fluid shear stress and cyclic circumferential stretch on vascular endothelial cell morphology and cytoskeleton.

The development of atherosclerosis is thought to be initiated by a dysfunctional state of the vascular endothelium. The proposal that mechanical forces play a role in the localization of this disease has led researchers to develop in vitro models to assess their effects on cultured endothelial cells. The arterial endothelium is exposed simultaneously to circumferential hoop stretch and wall shear stress, yet previous investigations have focused on the isolated effects of either cyclic stretch or shear stress. The influence of physiological levels of combined shear stress and hoop stretch on the morphology and F-actin organization of bovine aortic endothelial cells was investigated. Cells subjected for 24 hours to shear stresses higher than 2 dyne/cm2 or to hoop stretch greater than 2% elongated significantly compared with unstressed controls and oriented along the direction of flow and perpendicular to the direction of stretch. Exposure to more than 4% stretch significantly enhanced the responses to shear stress. Both shear stress and hoop stretch induced formation of stress fibers that were aligned with the cells' long axes. Simultaneous exposure to both stimuli appeared to enhance stress fiber size and alignment. These results indicate that shear stress and hoop stretch synergistically induce morphological changes in endothelial cells, which suggests that circumferential strain might modulate sensitivity of endothelial cells towards shear stress.

Induction of angiogenesis by smooth muscle cell‐derived factor: Possible role in neovascularization in atherosclerotic plaque

The development of atherosclerotic plaque is associated with neovascularization in the thickened intima and media of vascular walls. Neovascularization may have a role in the progression of atherosclerotic plaque as well as in the development of intraplaque hemorrhage. However, the mechanism and stimulus for neovascularization in atherosclerotic plaque are unknown. We postulated that smooth muscle cells (SMCs), a major cellular component in the vascular wall, might contribute to the induction of neovascularization in atherosclerotic plaque through the secretion of an angiogenic factor. We observed that endothelial cells (ECs) cultured on collagen gel with SMC-conditioned medium became spindle shaped, invaded the underlying collagen gel, and organized a capillary-like branching cord structure in the collagen gel. The conditioned medium also stimulated EC proliferation and increased the EC-associated plasminogen activator activity. The angiogenic factor in SMC-conditioned medium was retained in a heparin-Sepharose column and eluted with 0.9 M NaCl. Neutralizing anti-vascular endothelial growth factor (VEGF) antibody attenuated the angiogenic activity in the conditioned medium, including the induction of morphologic changes in ECs, mitogenic activity, and increased plasminogen activator activity associated with ECs. Immunoblotting analysis confirmed the secretion of VEGF from SMCs. These observations indicate that SMC may be responsible for the neovascularization in atherosclerotic plaque through the secretion of VEGF.

31P nuclear magnetic resonance evaluation of rat liver preserved in UW solution.

A persistent problem in orthotopic liver transplantation is primary nonfunction (PNF) of the hepatic allograft. In an attempt to reduce the incidence of graft failure, the feasibility of pretransplant assessment of graft viability was investigated by 31P nuclear magnetic resonance (NMR) spectroscopy. The level of adenosine triphosphate (ATP) was measured as an indicator of liver function by 31P NMR spectroscopy after a 30 min normothermic reperfusion following cold-storage in University of Wisconsin (UW) solution. The mean +/- SD beta-ATP/Pi ratio after preservation for 0, 12, 24 or 48 h was 1.40 +/- 0.34, 0.85 +/- 0.27, 0.64 +/- 0.14 and 0.38 +/- 0.09, respectively. Significance was observed between 12h and 24h and between 12h and 48h of preservation. These results correlated well with the morphological changes in endothelial cells and sinusoidal lining cells examined by transmission electron microscopy. It is suggested strongly that microcirculatory disturbances due to endothelial cell injury impairs the recovery of ATP levels after reperfusion, and that ATP determination by 31P NMR spectroscopy, as a non-invasive modality, may help in the prediction of PNF after liver transplantation.

Contraction of human brain endothelial cells induced by thrombogenic and fibrinolytic factors. An in vitro cell culture model.

Vasogenic brain edema is a frequent complication of ischemic stroke. The mechanism of the blood-brain barrier opening that underlies the edema formation is poorly understood. In the present study we examined the response of endothelial cells cultured from adult human brain to thrombogenic and fibrinolytic factors that possibly accumulate in the occluded vascular segments in ischemic stroke. The changes in the morphology of cultured human brain microvascular endothelial cells were observed by phase-contrast light microscopy and quantified with computerized morphometry. Active proteases (eg, thrombin, plasmin, urokinase) as well as heparin and protamine, but not fibrinogen and antithrombin III, produced significant changes in endothelial cell morphology. Two shape patterns of contraction were observed: protamine treatment resulted in rounded cells with a decrease in both cell perimeter and area, whereas all other agents induced spiderlike cell morphology with increased perimeter and reduced area. The rate of contraction was dose dependent, and at comparable enzyme concentrations plasmin produced faster contraction than thrombin. The observed changes were reversed 3 hours after abrogating the treatment. In an in vitro model we have demonstrated that factors involved in thrombus formation and dissolution induce endothelial cell contraction, which could affect focally the permeability of the blood-brain barrier by opening paracellular avenues between endothelial cells in vivo. Thus, the genesis of brain edema in thromboembolic stroke or occasionally during fibrinolytic therapy can be attributed in part to the contact of these factors with the microvascular endothelium.

Donor lung preservation: effect of cold preservation fluids on cultured pulmonary endothelial cells.

Pulmonary arterial (PA) endothelial cell morphology changes during cold preservation. In the present study, the efficacy of University of Wisconsin solution (UW), UW solution without colloid (modified UW), Euro-Collins (EC), Marshall's solution (MS), and medium 199 + 10% fetal calf serum [culture medium (CM)] in maintaining and regaining the cytoskeleton of cultured porcine PA endothelial cells kept at 4 degrees C and then rewarmed was compared. Features studied were actin, microtubules, vinculin, and talin, using immunofluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting; permeability of the cell sheet; wound healing; and phagocytic capacity. When cooled, microtubules depolymerized in all fluids but EC and MS reduced depolymerization of actin. Permeability decreased at 4 degrees C (P < 0.05), and wound healing and phagocytosis ceased. When rewarmed after EC, UW, and CM preservation, wound healing and phagocytosis started within 15 min and proceeded normally. Permeability returned to normal but was excessive following UW preservation. Microtubule repolymerization was fastest following UW preservation, and actin filament repolymerization was fastest after EC preservation. Thus the type of preservation fluid used influenced the rate of loss and recovery of specific cytoskeletal components, with EC giving the fastest structural and functional recovery.

Alterations in human endothelial cell morphology, proliferation and function by a macrophage-derived factor

Changes in endothelial cell (EC) morphology occur at sites of physiological lymphocyte traffic and in areas of chronic inflammation. Previous studies have shown that EC shape changes also occur in vitro following exposure of EC monolayers to peripheral blood mononuclear cell (PBMC)-derived conditioned media (CM). In the present study, quantitative image analysis is used to define the cell of origin of the elongating factor(s), to examine changes in EC proliferation and function accompanying PBMC-induced human EC elongation and to identify the active PBMC-derived products responsible for this elongation. By separating mononuclear cells into subpopulations (macrophages, B cells and T cells) and adding conditioned media derived from these subpopulations to cultured ECs, the macrophage (M phi) is shown to be the primary cell of origin of the elongating factor(s). Furthermore, EC elongation is accompanied by both a dose-dependent decrease in cellular proliferation and an increase in prostacyclin production. These findings suggest that PBMC-induced changes in EC morphology may be associated with a shift from a proliferative state to a more secretory phase of the EC cycle. Finally, using recombinant factors it is shown that TNF alpha acting in combination with IL-1 may be the active PBMC-derived products which contribute to EC elongation.

Endothelial cell changes in endotoxin-induced uveitis.

Modifications in endothelial cells density and variations in endothelial pleomorphism during experimental uveitis were studied in 20 albino rabbits after intravitreal injection of 2,000 ng Salmonella typhi endotoxin into 10 of them. The other 10 rabbits were injected with sterile saline serum. Morphologic changes in endothelial cells were also determined, using a method of endothelial vital staining. For the first time, a significant reduction in endothelial density and a significant increase in endothelial pleomorphism during endotoxin-induced uveitis is reported. The control group eyes showed an endothelial density of 3,959 cells/mm2, while the uveitis eyes showed 3,667 cells/mm2 (p < 0.05, Student's t test). A reduction in the percentage of hexagonal endothelial cells was found, from 70.4% in the control group to 64.4% in the eyes with anterior uveitis (p < 0.001, Student's t test). An increase in the percentage of pentagonal endothelial cells was also found, from 14.5% in the control group to 20.1% in the uveitis eyes (p < 0.001, Student's t test). These modifications are correlated with the morphologic appearance of the stained corneal surface, and functional implications are discussed.

Morphological changes of endothelial cells after exposure to fluid-imposed shear stress: differential responses induced by extracellular matrices.

Effects of extracellular matrices (ECMs) and fluid-imposed shear stress on the cell shape and the cytoskeletal structure of microfilaments were studied in cultured porcine aortic endothelial cells (PAECs). The PAECs were cultured until confluent on non-coated or on ECM-coated glass coverslips. The components of ECM used were type IV collagen, heparan sulfate, chondroitin sulfate and dermatan sulfate. The PAECs cultured on the mixed ECMs showed marked elongation and segmental orientation with randomly distributed cell axis even under a no-flow static condition, and the microfilaments were mostly observed in parallel with the cell axis. After shear flow exposure (2 Pa, 24-48 hrs), the PAECs on non-coated glass were significantly elongated and oriented to the flow direction, however the PAECs on ECM-coated glass showed more retarded responses than the ones on non-coated glass, indicating that the anchorage to the substrate was enhanced by ECMs. The stress fibers were reorganized in accordance with the cell shape and oriented to the flow direction. These findings suggest that ECM may act together with shear stress to modify and maintain the endothelial cell configuration.

The cell wall mediates pneumococcal attachment to and cytopathology in human endothelial cells

Streptococcus pneumoniae interacts with vascular endothelial cells during the course of bacteremia. In this study, we characterized the initial attachment of pneumococci to human endothelial cells (EC) and the response of the endothelium to this interaction. Pneumococci adhered to EC in a dose-dependent fashion. Attachment was rapid, with the majority of bacteria attached by 30 min. No difference was found between the attachment of unencapsulated (R6) and encapsulated (SIII) strains. Purified pneumococcal cell wall components competitively inhibited attachment of R6 by a maximum of 60% in a dose-dependent manner. Following attachment of pneumococci or exposure of EC to pneumococcal cell wall, pronounced changes in EC morphology ensued, resulting in striking separation of the cells of the monolayer and, eventually, destruction of the cells. The cytopathic effects of the cell wall were inhibited by antibodies to interleukin-1 but not to tumor necrosis factor. Both antibodies were required to neutralize the cytopathology caused by intact pneumococci. We conclude that pneumococci attach rapidly to human EC and that the cell wall is important in this interaction. Intact pneumococci and pneumococcal cell wall induce profound morphologic changes in human EC, leading to loss of barrier integrity. These cytopathic effects are likely to be cytokine mediated.

Rapid Morphological Changes of Bovine Brain Microvascular Endothelial Cells on Extracellular Matrices

The morphological changes in endothelial cells derived from bovine brain microvasculature, carotid artery, and aorta during growth on extracellular matrices were compared. All cells formed tubular structures on a basement membrane. Ultrastructural studies showed that the tubular structures had lumens surrounded by many endothelial cells. On type I collagen gel, brain microvascular endothelial cells still formed tubular structures, but the other two cell types formed confluent monolayers. However, when a second layer of collagen gel was laid over these cells, tubular structures developed within 2-3 days. Brain microvascular endothelial cells form tubular structures more readily than endothelial cells derived from large vessels on both basement membrane and type I collagen gel.

Change in endothelial cell morphology at arterial branch sites caused by a reduction of intramural stress

Arterial branch sites have very high intramural stresses at physiologic intraluminal pressures; the same sites have a predilection for atherosclerosis. The effect of intramural stress on endothelial cell morphology was investigated. Five rabbits had permanent casts placed around a segment of the abdominal aorta-left renal artery branch area during controlled hypotension, thus reducing intramural stress without narrowing the lumen. These five animals, and three normal rabbits, were sacrificed after 4–8 weeks, and the vessels were perfused with buffered 2.5% glutaraldehyde for 2 h at 100 mm Hg pressure. The aortas were examined by scanning electron microscopy. In normal aortas, the distal region of the ostia of the left renal and celiac arteries just beyond the flow divider displayed many morphologically altered endothelial cells ranging from spindle shape to cobble-stone shape. The same aortic area of casted rabbits, as well as the straight abdominal aorta in all rabbits, showed a smooth surface of endothelial cells with intact cell borders and no morphologically altered cells. At branch sites, the occurrence of morphologically altered endothelial cells may be due to increased intramural stress. When intramural stress is reduced, the morphology of branch endothelial cells changes to resemble that of the unbranched regions. In conclusion, endothelial cell morphology changes in response to changes in intramural stress.

Mechanism of endothelial cell shape change in oxidant injury

Changes in endothelial cell morphology induced by neutrophil-generated hydrogen peroxide (H 2O 2) may account for the capillary leak of the adult respiratory distress syndrome (ARDS). The relationship of H 2O 2 effects on the concentration of intracellular Ca 2+([Ca 2+] i) and ATP to changes in microfilaments and microtubules, important determinants of cell shape, was examined. Bovine pulmonary artery endothelial cells were injured over a 2-hr time course with a range of H 2O 2 doses (0–20 m M). The higher concentrations of H 2O 2 consistently produced contraction and rounding of >50–75% of cells by 1–2 hr. The range of 1–20 m M H 2O 2 produced rapid, significant reductions in endothelial ATP levels over the time course of injury. Although there were significant increases in mean endothelial [Ca 2+] i in response to 5, 10, and 20 m M H 2O 2, 1 m MH 2O 2 did not affect the [Ca 2+] i. Fluorescence microscopy revealed that microfilament disruption occurred as ATP levels fell and preceded depolymerization of microtubules which developed after [Ca 2+] 1 approached 1 × 10 −6 M. H 2O 2 at 1 m M injury caused microfilament disruption but did not depolymerize microtubules. Microfilament disruption occurred without oxidant exposure, when ATP levels were reduced by glucose depletion and mitochondrial inhibition with oligomycin (650 n M). If a Ca 2+ ionophore, ionomycin (5 μ M), was then added, [Ca 2+] i rose to > 1 × 10 −6 M, microtubules fragmented and depolymerized, and cell contraction and rounding very similar to that induced by H 2O 2 occurred. These results suggest that endothelial cell dysfunction and capillary leak in ARDS may be due to H 2O 2-mediated changes in cellular ATP and [Ca 2+] i.

Quantitative studies of human monokine-induced endothelial cell elongation.

Leucocyte-endothelial cell interactions are important in the inflammatory response. In this study, the effect of peripheral blood mononuclear cell (PBMC) products on endothelial cell (EC) shape was examined and quantified. PBMC were obtained from normal donors by Ficoll-Hypaque separation of heparinized whole blood and cultured for 72 hr in media containing 10% fetal calf serum with and without concanavalin A (Con A). Media conditioned by PBMC or control, nonconditioned media were then added to preconfluent, first passage EC cultures derived from human umbilical veins. Conditioned media from Con A-stimulated PBMC resulted in a dose-dependent, marked elongation and whorling of cultured EC. The minimum effective concentration found to elicit a response was 1.25%, with a maximum response occurring at 10%. Quantitative morphometric analyses of treated EC indicated that the elongation was highly significant (p less than 0.001) when compared to EC incubated with control, nonconditioned media. In addition, EC elongation was accompanied by a highly significant (p less than 0.001) increase in cell area. Although less dramatic, conditioned media from unstimulated PBMC also elicited a similar, significant dose-dependent change in EC shape. Significant changes in EC shape were evident within 6 hr and continued over the time course of the experiment (40 h). Cell shape changes were partially reversible at 18 h after removal of the PBMC-conditioned media and replacement with control, nonconditioned media. The change in EC morphology induced by a PBMC-derived factor(s) suggests a mechanism by which activated leucocytes may modulate cellular traffic at the blood-vessel wall interface.

Inhibition of endothelial cell proliferation by gamma-interferon.

Endothelial cell growth factor (ECGF) is a potent polypeptide mitogen for endothelial cells and fibroblasts. The mitogenic effects of ECGF are inhibited by the lymphokine gamma-interferon (gamma-IFN) in a dose-dependent manner. Gamma-IFN also induces a unique change in endothelial cell morphology which is maximally expressed in the presence of ECGF. The antiproliferative and phenotypic modulatory effects of gamma-IFN on endothelial cells are reversible. Inhibition of ECGF-induced endothelial cell proliferation by gamma-IFN is accompanied by a concentration- and time-dependent decrease in binding of 125I-ECGF to the endothelial cell surface. Scatchard analyses of the binding data in the presence and absence of gamma-IFN demonstrate a decrease in the number of ECGF-binding sites rather than a decrease in ligand affinity for the receptor. Cross-linking experiments with disuccinimidyl suberate demonstrate a decrease in the 170,000 Mr cross-linked receptor-ligand complex. These data suggest that gamma-IFN inhibits endothelial cell proliferation by a mechanism which involves growth factor receptor modulation.

Stimulation of endothelial cell proliferation by rat granulosa cell-conditioned medium.

The ability of granulosa cells to produce mitogenic factors for vascular endothelial cells, factors which could potentially mediate angiogenesis in the ovary, was examined. Granulosa cells were obtained from preovulatory follicles of immature rats 48 h after priming with PMSG (20 IU). The cells (1 X 10(6)/well) were cultured in 3 ml serum-free medium 199 (M199) at 37 C without further treatment or in the presence of LH (100 ng/ml) or FSH (20 ng/ml). Since oxygen tension has been shown to regulate the production of angiogenic factors by other cell types, the cultures were carried out with either a high (20%) or a low (2%) oxygen concentration in the culture chamber. After 48 h, the medium was collected, filtered (0.2 micron), and frozen until tested for mitogenic effects on sparsely plated fetal bovine aortic endothelial cells. A 1:1 mixture of granulosa cell-conditioned M199 with fresh M199 plus 1% dialyzed fetal bovine serum resulted in 7- to 8-fold increases in endothelial cell numbers over the 4-day test period compared to controls (fresh M199 + 1% dialyzed fetal bovine serum only). Neither gonadotropin treatment nor the oxygen concentration during the conditioning period influenced the proliferation-stimulating activity of the medium. Medium conditioned by granulosa cells in 2% oxygen, however, did have an additional effect on endothelial cell morphology; the cells were more elongated and aligned than those treated with medium conditioned by granulosa cells in 20% oxygen, which showed a typical cobblestone morphology. Preliminary characterization studies indicate that both high (greater than 30,000) and low (less than 10,000) mol wt mitogenic factors are present. The mitogenic activity is heat resistant but partially destroyed by trypsin. The morphology-altering activity is confined to high mol wt fractions (greater than 30,000). These studies demonstrate that granulosa cells from preovulatory follicles release one or more factors in vitro which are mitogenic for endothelial cells. Furthermore, conditioned medium from granulosa cells cultured in low oxygen induces morphological changes in endothelial cells which suggest an increased propensity for migration.

1632 HYPEROXIA DAMAGES ENDOTHELIAL CELLS IN TISSUE CULTURE

When exposed to hyperoxia, patients and animals develop significant lung injury which may lead to respiratory failure and even death. Changes seen in lungs exposed to hyperoxia indicate that endothelial cell damage with vascular leak is a prominent and early feature of the pathophysiology of pulmonary oxygen toxicity. We hypothesized that hyperoxia directly damages endothelial cells, leading to vascular leak and compromise of gas exchange. To test this hypothesis, we exposed monolayers of bovine pulmonary artery endothelial cells to hyperoxia (95% O2 - 5% CO2) or normoxia (80% N2 - 15% O2 - 5% CO2) at ambient atmospheric pressure for 24, 48, 72 and 96 hours. We found that exposure to hyperoxia caused increasingly severe morphologic changes in endothelial cells, manifested by clumping of nuclear material and gross cytoplasmic disorganization. Such changes appeared in increasing proportions of cells between 24 and 96 hours of hyperoxic exposure, but were rarely seen in cells exposed to normoxia. These changes are quite similar to those found in the lung endothelial cells of animals exposed to hyperoxia. We also found increasing release of cytoplasmic lactic dehydrogenase from the hyperoxia-exposed but not normoxia-exposed cells. These results suggest that hyperoxia alone can damage pulmonary endothelial cells and support the possibility that this mechanism may contribute to the respiratory failure seen in oxygen toxicity.

Ultrastructural changes in feline arterial endothelium following subarachnoid hemorrhage.

Scanning electron microscopy of feline basilar arterial endothelium 4 hours, and 1, 3,5, and 7 days after subarachnoid hemorrhage (SAH) showed longitudinal furrows that correlated with angiographically demonstrated vasospasm. These ridges persisted after fixation at physiological pressure, and probably reflected medial contraction with undulation of the underlying elastic lamina. No change in endothelial cell morphology or thrombogenesis was observed as long as 7 days after SAH. There is no evidence from this study to suggest that ischemia from vasospasm is a product of thromboembolism from damaged endothelial surfaces.

Changes in the fine structure of the dental pulp in human caries pulpitis. 1. Nerves and blood vessels.

Abstract. Electron microscopic investigation of human pulps manifesting caries pulpitis revealed degenerative changes in myelinated and unmyelinated nerves and blood vessels. The degenerative changes noted in nerves were generally less severe and less frequent than those noted in the blood vessels and surrounding pulp tissue. This was attributed to the presence of an abundance of collagen fibers about the nerve bundles and nerve axons. The degenerative changes in the blood vessels ranged from minor changes in endothelial cell morphology to complete necrosis and lysis. These changes were generally accompanied by alterations in the integrity of the basement membrane. Advanced changes in blood vessels of the pulp appeared to result in an increase in vascular permeability causing edema. It was suggested that some of the degenerative changes noted might be related to the humoral phase of the inflammatory response.

Contraction of Endothelial Cells as a Key Mechanism in Atherogenesis

The combined SEM and TEM observations of morphological changes in endothelial cells of the aorta induced by Anitschkow's procedure, i.e, one-shot treatment of rabbits with cholesterol (1g/kg p.o.), were subjects of the present investigation. (1) In the SEM analysis of the luminal surface of thoracic aorta, the appearance of vertical lines at regular intervals of 0.6 to 1.8μ and and depths of 0.2 to 0.3μ and of crater-like structures was observed after administration of cholesterol (p<0.01, p<0.01). PDC pretreatment reduced the appearance of both changes (p<0.01, p<0.01). The crater-like structures appeared predominantly on or close to the boundaries of endothelial cells and, on the basis of TEM observation done concomitantly, the majority of such structures were reasonably considered to reprsent blebs, while the vertical lines were considered to represent the active contraction of endothelial cells. (2) In the TEM analysis, the appearance of subendothelial edema was reconfirmed to be induced by one-shot treatment of rabbits with cholesterol (p<0.05). In such animals an unevenness of endothelial cells, accompanied by relatively strong indentations and occasionally pinches (p<0.001), which represent the active contraction of endothelial cells, 6) seen. It was also shown that the formation of blebs, especially of large ones with a diameter over 1μ, can be induced by the above mentioned challenge (p<0.01, p<0.01). Also, the sticking of platelets to blebs and the formation of fibrin by such platelets were often observed. All such changes were inhibited by PDC pretreatment (10mg/kg p.o.). (3) The characteristic response of endothelial cells of the artery to cholesterol, which was shown in the present observations, seemed to result from the active contraction of their contractile proteins.8) Such a response has been proposed as the trigger-mechanism in atherogenesis, and possibly in thrombogenesis.