Dense Peripheral Band Research Articles

Maxillofacial Radiology 194

CBCT imaging and analysis was performed. Observe unilateral expansion by the distending soft tissue outline illustrated through 3D rendering (Figure 2). Sagittal oblique (Figure 3) and axial (Figure 4) slices depict a round heterogenous predominantly high-density lesion with an encompassing thin uniform less dense peripheral band. Irregular root resorption, displacement of the inferior alveolar nerve canal, buccal-lingual cortical expansion, thinning, and interruption was apparent. Irregular thickening at the inferior border and surrounding osteosclerosis were noted. A macroscopic view (Figure 5), photomicrograph (Figure 6), and conventional radiograph (Figure 7) of vertically sectioned surgical specimens of similar lesions. Note the intimate relationship with the tooth roots.

Regulation of Hyperoxia-induced NADPH Oxidase Activation in Human Lung Endothelial Cells by the Actin Cytoskeleton and Cortactin

Although the actin cytoskeleton has been implicated in the control of NADPH oxidase in phagocytosis, very little is known about the cytoskeletal regulation of endothelial NADPH oxidase assembly and activation. Here, we report a role for cortactin and the tyrosine phosphorylation of cortactin in hyperoxia-induced NADPH oxidase activation and ROS production in human pulmonary artery ECs (HPAECs). Exposure of HPAECs to hyperoxia for 3 h induced NADPH oxidase activation, as demonstrated by enhanced superoxide production. Hyperoxia also caused a thickening of the subcortical dense peripheral F-actin band and increased the localization of cortactin in the cortical regions and lamellipodia at cell-cell borders that protruded under neighboring cells. Pretreatment of HPAECs with the actin-stabilizing agent phallacidin attenuated hyperoxia-induced cortical actin thickening and ROS production, whereas cytochalasin D and latrunculin A enhanced basal and hyperoxia-induced ROS formation. In HPAECs, a 3-h hyperoxic exposure enhanced the tyrosine phosphorylation of cortactin and interaction between cortactin and p47(phox), a subcomponent of the EC NADPH oxidase, when compared with normoxic cells. Furthermore, transfection of HPAECs with cortactin small interfering RNA or myristoylated cortactin Src homology domain 3 blocking peptide attenuated ROS production and the hyperoxia-induced translocation of p47(phox) to the cell periphery. Similarly, down-regulation of Src with Src small interfering RNA attenuated the hyperoxia-mediated phosphorylation of cortactin tyrosines and blocked the association of cortactin with actin and p47(phox). In addition, the hyperoxia-induced generation of ROS was significantly lower in ECs expressing a tyrosine-deficient mutant of cortactin than in vector control or wild-type cells. These data demonstrate a novel function for cortactin and actin in hyperoxia-induced activation of NADPH oxidase and ROS generation in human lung endothelial cells.

Proinflammatory Reaction and Cytoskeletal Alterations in Endothelial Cells after Shock Wave Exposure

BackgroundAlthough the effects on human organs by shock waves (SWs) induced by medical treatments or high-energy trauma are well recognized, little is known about the effects on the cellular level....

Actin structure in the outflow tract of normal and glaucomatous eyes

Purpose. To characterize the in situ distribution of actin in Schlemm's canal endothelium (SCE) and juxtacanalicular tissue (JCT) cells in glaucomatous human eyes, and compare to the distribution in normal eyes. Methods. Fresh human eye bank eyes were perfused and fixed at pressure ( n=27 normal eyes and 22 confirmed glaucomatous eyes). Schlemm's canal was opened by microdissection and outflow tissues were labelled for confocal microscopy to visualize F-actin, nuclei, laminin and/or CD31. Images were acquired in Z-series from the inner wall of Schlemm's canal, juxtacanalicular tissue and outer corneoscleral meshwork. Results. In normal eyes, inner wall Schlemm's canal endothelial (SCE) cells showed a dense peripheral F-actin band, as previously described. JCT cells showed a more random and amorphous F-actin distribution. In glaucoma eyes, peripheral F-actin bands were less common in inner wall SCE cells; instead, F-actin was more centrally located within the cell and appeared ‘tangled’. These actin tangles were also prominent in JCT cells of glaucoma eyes. Glaucoma eyes also demonstrated structures with features of cross-linked actin networks (CLANs), and more frequent occurrence of punctuate actin concentrations. There was a significant degree of heterogeneity, with some regions from glaucomatous eyes appearing normal and vice versa. Conclusion. F-actin architecture in human outflow pathway cells in situ differs between normal and glaucoma eyes, with glaucomatous tissue showing a more ‘disordered’ actin architecture overall. Some of these changes are likely due to effects secondary to administration of anti-glaucoma medications. Most of the changes that we observed could potentially affect the biomechanical properties of the outflow pathway tissues in glaucoma, but their role in the pathogenesis of ocular hypertension remains unclear.

Microtubules regulate aortic endothelial cell actin microfilament reorganization in intact and repairing monolayers.

To understand the role of microtubules and microfilaments in regulating endothelial monolayer integrity and repair, and since microtubules and microfilaments show some co-alignment in endothelial cells, we tested the hypothesis that microtubules organize microfilament distribution. Disruption of microtubules with colchicine in resting confluent aortic endothelial monolayers resulted in disruption of microfilament distribution with a loss of dense peripheral bands, an increase in actin microfilament bundles, and an associated increase of focal adhesion proteins at the periphery of the cells. However, when microfilaments were disrupted with cytochalasin B, microtubule distribution did not change. During the early stages of wound repair of aortic endothelial monolayers, microtubules and microfilaments undergo a sequential series of changes in distribution prior to cell migration. They are initially distributed randomly relative to the wound edge, then align parallel to the wound edge and then elongate perpendicular to the wound edge. When microtubules in wounded cultures were disrupted, dense peripheral bands and lamellipodia formation were lost with increases in central stress fibers. However, following microfilament disruption, microtubule redistribution was not disrupted and the microtubules elongated perpendicular to the wound edge similar to non-treated cultures. Microtubules may organize independently of microfilaments while microfilaments require microtubules to maintain normal organization in confluent and repairing aortic endothelial monolayers.

Receptor Protein Tyrosine Phosphatase μ Regulates the Paracellular Pathway in Human Lung Microvascular Endothelia

The pulmonary vascular endothelial paracellular pathway and zonula adherens (ZA) integrity are regulated, in part, through protein tyrosine phosphorylation. ZA-associated protein tyrosine phosphatase (PTP)s are thought to counterregulate tyrosine phosphorylation events within the ZA multiprotein complex. One such receptor PTP, PTPmu, is highly expressed in lung tissue and is almost exclusively restricted to the endothelium. We therefore studied whether PTPmu, in pulmonary vascular endothelia, associates with and/or regulates both the tyrosine phosphorylation state of vascular endothelial (VE)-cadherin and the paracellular pathway. PTPmu was expressed in postconfluent human pulmonary artery and lung microvascular endothelial cells (ECs) where it was almost exclusively restricted to EC-EC boundaries. In human lung microvascular ECs, knockdown of PTPmu through RNA interference dramatically impaired barrier function. In immortalized human microvascular ECs, overexpression of wild-type PTPmu enhanced barrier function. PTPmu-VE-cadherin interactions were demonstrated through reciprocal co-immunoprecipitation assays and co-localization with double-label fluorescence microscopy. When glutathione S-transferase-PTPmu was incubated with purified recombinant VE-cadherin, and when glutathione S-transferase-VE-cadherin was incubated with purified recombinant PTPmu, PTPmu directly bound to VE-cadherin. Overexpression of wild-type PTPmu decreased tyrosine phosphorylation of VE-cadherin. Therefore, PTPmu is expressed in human pulmonary vascular endothelia where it directly binds to VE-cadherin and regulates both the tyrosine phosphorylation state of VE-cadherin and barrier integrity.

The role of the glycocalyx in reorganization of the actin cytoskeleton under fluid shear stress: A “bumper-car” model

We propose a conceptual model for the cytoskeletal organization of endothelial cells (ECs) based on a major dichotomy in structure and function at basal and apical aspects of the cells. Intracellular distributions of filamentous actin (F-actin), vinculin, paxillin, ZO-1, and Cx43 were analyzed from confocal micrographs of rat fat-pad ECs after 5 h of shear stress. With intact glycocalyx, there was severe disruption of the dense peripheral actin bands (DPABs) and migration of vinculin to cell borders under a uniform shear stress (10.5 dyne/cm2; 1 dyne = 10 microN). This behavior was augmented in corner flow regions of the flow chamber where high shear stress gradients were present. In striking contrast, no such reorganization was observed if the glycocalyx was compromised. These results are explained in terms of a "bumper-car" model, in which the actin cortical web and DPAB are only loosely connected to basal attachment sites, allowing for two distinct cellular signaling pathways in response to fluid shear stress, one transmitted by glycocalyx core proteins as a torque that acts on the actin cortical web (ACW) and DPAB, and the other emanating from focal adhesions and stress fibers at the basal and apical membranes of the cell.

Finite Element Analysis for Sheared Vascular Endothelial Cells Using Structural Optimization Method

A finite element analysis using structural optimization method was performed to simulate the remodeling of bovine aortic endothelial cells (BAECs) under flow condition. BAECs showed marked elongation and aligned in the flow direction by exposing to a steady shear stress of 2 Pa for 24 h. An atomic force microscope (AFM) system was used to obtain cell surface topography, showing that the cell height decreased significantly from 2.8±1.0μm to 1.4±0.5μm with exposure to fluid flow. The fluorescent images of cells stained by rhodamine-phalloidin showed that control cells exhibited dense peripheral bands of actin filaments, while sheared cells exhibited centrally located actin stress fibers parallel to the flow direction. In the analysis, the two-dimensional mesh was generated based on the cell surface data measured by AFM and then elastic modulus of each element was changed in accordance with an object stress, together with update of cell shape. Numerical results showed that the cell height decreased with fluid flow and the higher elastic modulus appeared in the upstream region of the nucleus at the final step, which may correspond with cytoskeletal structure. The present analysis should be effective for elucidating the remodeling of endothelial cells.

Remodeling of Vascular Endothelial Cells Exposed to Mechanical Stimuli.

Morphology and local mechanical properties of sheared endothelial cells were measured by using atomic force microscope. After applying a steady shear stress of 2 Pa for 6 hours, bovine aortic endothelial cells showed marked elongation and aligned in the flow direction. The peak cell height decreased significantly with exposure to fluid flow. The fluorescent images showed that control cells exhibited dense peripheral bands of F-actin filaments, while sheared cells exhibited F-actin stress fibers which were thick and centrally located parallel to the flow direction. Elastic modulus estimated by the Hertz model significantly increased with fluid shear stress. Change in mechanical properties might be closely correlated to the development of cytoskeletal structure.

Effect of fluid shear stress on the permeability of the arterial endothelium.

The localization of atherosclerotic lesions is due, in part, to regional variations in the permeability of arterial endothelium to macromolecules. In turn, endothelial permeability may be influenced by fluid shear stresses. The spatial variation in endothelial permeability is reviewed and evidence for shear stress dependence upon permeability is presented. These results are examined in light of various signaling mechanisms that increase permeability by increasing the transport of water and macromolecules through the junctions separating endothelial cells. Signaling pathways cause a change in the dense peripheral band of actin and actin stress fibers or alter the phosphorylation of junction proteins which affects their ability to localize in junctions. Future directions to clarify the effect of shear stress on permeability are considered.

Changes in corneal endothelial apical junctional protein organization after corneal cold storage.

Understanding the mechanisms regulating corneal endothelial permeability during storage and recovery is of critical importance both to improving Eye Banking practices and preventing corneal transplant failure. The goal of this study was to determine the effects of cold storage on the organization of apical junctional complex (AJC) proteins and their relationship to F-actin in corneal endothelium. Immunostaining using antibodies to the AJC proteins, ZO-1, cadherin, and alpha- and beta-catenin was performed on 16 eye bank corneas and four cat corneas after 2-8 days of storage at 4 degrees C in Optisol-GS, and compared with fresh corneas. The 3-D in situ localization of the AJC proteins was then determined by using laser confocal microscopy. AJC organization also was assessed after stored human corneas were further incubated at 37 degrees C in Optisol-GS or in serum-free media. In normal human and cat corneas, F-actin was organized into dense peripheral bands (DPBs) along the apical cell border. The tight-junction protein, ZO-1, and the adherens junction proteins, cadherin and alpha- and beta-catenin, each formed a uniquely discontinuous hexagonal apical band with the largest gaps occurring at the Y-junctions between adjacent endothelial cells. In stored eye bank and cat corneas, cells lost their normal hexagonal F-actin staining pattern and appeared rounded and distorted, with increased cytoplasmic staining and incomplete and condensed DPBs. Similar distortions were observed in the apical bands of cadherin, catenin, and ZO-1 staining between endothelial cells. Gaps in staining at the endothelial Y-junctions were significantly enlarged; corresponding gaps also were observed with phalloidin staining. These changes were reversed after overnight incubation at 37 degrees C in either serum-free media or Optisol-GS. Quantitative analysis demonstrated a significant increase in the size of the Y-junctional gaps (p < 0.0001) after cold storage of cat corneas as compared with fresh corneas. These results suggest that disruption of the F-actin cytoskeleton and AJC may explain, in part, the loss of function (corneal swelling) after prolonged cold storage.

The spatial organization of apical junctional complex-associated proteins in feline and human corneal endothelium

Purpose. Previous studies suggest that proteins associated with the apical junctional complex (AJC) play essential roles in the development, maintenance and regulation of barrier function in transport epithelium and vascular endothelium. The goal of this study is to identify and determine the spatial organization of several major AJC-associated proteins in normal human and feline corneal endothelium. Methods. Fresh corneal tissue was obtained from 4 recipient buttons removed during penetrating keratoplasty (two from keratoconus patients, and two from patients with post-traumatic stromal scarring) as well as from 16 cat eyes. En bloc double- and triple-labeling of corneas was performed using phalloidin, and mouse, rat or rabbit antibodies to ZO-1, occludin, pan-cadherin, a-catenin, ß-catenin and plakoglobin (?-catenin). The 3-D localization of the proteins was then determined in situ using laser confocal microscopy. Results. Similar staining patterns were obtained for the corneal endothelium of normal cat corneas and fresh human buttons. Apically, f-actin was arranged into dense peripheral bands (DPB) in individual cells that were separated from those in adjacent cells. Diffuse phalloidin staining also extended from the DPB into the cytoplasm apically. Although weaker, phalloidin staining also appeared to be associated with the basolateral cell borders. The adherens junction protein, cadherin, formed a thin pericellular band at the apical cell junctions between the DPB. In addition, cadherin staining also appeared to extend along the basolateral cell borders in a convoluted pattern. Staining for a-catenin, ß-catenin and plakoglobin each showed a nearly identical organization as cadherin. ZO-1 formed a single apical band that was localized between the DPB; however, no basolateral ZO-1 staining was detected. Interestingly, the distribution of ZO-1 was discontinuous around the cell, with the largest gaps occurring at the Y-junctions between adjacent endothelial cells. Positive staining for occludin was not detected in either human or feline corneal endothelium. Conclusions. The composition and organization of the AJC of corneal endothelium appears to be different from that of classical transport epithelia; these findings may be related to functional differences between these two cell types.

SIGNALING PATHWAYS IN THROMBIN-INDUCED ACTIN REORGANIZATION IN PULMONARY ARTERY ENDOTHELIAL CELLS

The actin-based cytoskeleton of endothelial cells plays an important role in regulating cell function. Both thrombin and phorbol 12-myristate 13-acetate (PMA) (an activator of protein kinase C; PKC) cause rearrangement of actin and increased permeability of endothelial monolayers. Conversely, thrombin, but not PMA, induces phosphorylation of myosin light chains (MLC), a process considered essential for cellular contraction. We, therefore, decided to investigate which signaling pathways are involved in thrombin-induced actin reorganization in pulmonary artery endothelial cells. Thrombin induced a rapid and transient increase in cytoskeletal actin that paralleled MLC phosphorylation. Antagonism of the +Ca2-binding protein, calmodulin (CaM), or inhibition of the CaM-dependent MLC kinase (MLCK) abolished the elevation in cytoskeletal actin whereas inhibition of PKC did not. In contrast, PMA+ decreased cytoskeleton-associated actin without affecting phosphorylation of MLC. A23187, a Ca2-+ ionophore, or thapsigargin, an inhibitor of endoplasmic Ca2+-ATPase, either in the presence or absence of PMA, did not increase cytoskeletal actin. Therefore, increased intracellular Ca2+, even with concurrent activation of PKC, is insufficient for redistribution of actin to the cytoskeleton, indicating that thrombin recruits yet another signaling pathway. Both thrombin and PMA caused extensive rearrangement of filamentous actin with a disappearance of the dense peripheral band and an increase in stress fibers, but each agent induced a distinct morphology. Thrombin-induced rearrangement of actin filaments was attenuated by inhibitors of either PKC or MLCK. These data suggest that both PKC- and MLCK-dependent pathways are involved in thrombin-induced endothelial cell actin rearrangement, but that recruitment of actin to the cytoskeleton is not necessary for this rearrangement. Recruitment of actin and myosin to the cytoskeleton does not require PKC but does involve MLCK- catalyzed phosphorylation of MLC.

Structural and functional aspects of intercellular junctions in vascular endothelium.

Cell-to-cell-junctions of endothelial cells are specialized and differentiated areas of the plasma membrane. The main functions include the separation of the intravascular and extravascular compartments, the mechanical connection of the cells, and the maintenance of the cell polarity. Although a wide heterogeneity of endothelial cell-to-cell junctions exists in situ, they should be considered in general as adherens type junctions in which gap and tight junctions are morphologically inserted. Under certain pathological conditions, such as wound healing, angiogenesis and many types of inflammation, the interendothelial junctions have to be dissociated and reorganized in which proteins of the junctions are crucially involved. These important mechanisms predict a sophisticated regulation of junctional proteins. The present paper describes the organization and functional aspects of the occludin/ZO-1 complex typically found in tight junctions, the cadherin/catenin complex of the adherens junctions and the connection of these protein complexes to the dense peripheral band via actin filaments. In addition, special attention has been drawn on the function of junction-associated proteins with respect to their role under fluid shear stress and interendothelial gap formation during inflammation.

Hydrogen peroxide-induced cytoskeletal rearrangement in cultured pulmonary endothelial cells.

Although the signaling pathways leading to hydrogen peroxide (H2O2)-induced endothelial monolayer permeability remain ambiguous, cytoskeletal proteins are known to be essential for maintaining endothelial integrity and regulating solute flux through the monolayer. We have recently demonstrated that thrombin-induced actin reorganization in bovine pulmonary artery endothelial cells (BPAEC) requires activation of both myosin light chain kinase (MLCK) and protein kinase C (PKC). Therefore, the present study was designed to investigate the effects of H2O2 on actin reorganization in BPAEC. H2O2 initiated sustained recruitment of actin to the cytoskeleton and transient myosin recruitment in a time- and concentration-dependent manner. The H2O2-induced actin recruitment was significantly inhibited by the calmodulin antagonists, W7 and TFP, but not by the MLCK inhibitor, KT5926, nor the PKC inhibitors, H7 and calphostin C. H2O2 also caused actin filament rearrangement in BPAEC with disruption of the dense peripheral bands and formation of stress fibers. These alterations occurred prior to actin translocation to the cytoskeleton and are prevented by inhibition of either MLCK or PKC. High concentrations of H2O2 transiently attenuated PKC activity but slightly increased the phosphorylation of the prominent PKC substrate and actin-binding protein, myristoylated alanine-rich C kinase substrate (MARCKS), by 5 min. However, MARCKS phosphorylation was reduced to below basal levels by 30 min. On the other hand, H2O2 induced a time- and dose-dependent phosphorylation of myosin light chains which was eliminated by both MLCK and PKC inhibitors. These data suggest that MLCK contributes to H2O2-induced myosin light chain phosphorylation and actin rearrangement and that PKC may play a permissive role. Neither of these enzymes appears to be involved in the H2O2-induced recruitment of actin to the cytoskeleton.

Shear stress induces spatial reorganization of the endothelial cell cytoskeleton

The morphology of endothelial cells in vivo depends on the local hemodynamic forces. Cells are polygonal and randomly oriented in areas of low shear stress, but they are elongated and aligned in the direction of fluid flow in regions of high shear stress. Endothelial cells in vitro also have a polygonal shape, but the application of shear stress orients and elongates the cells in the direction of fluid flow. The corresponding spatial reorganization of the cytoskeleton in response to the applied hemodynamic forces is unknown. In this study, we determined the spatial reorganization of the cytoskeleton throughout the volume of cultured bovine aortic endothelial cells after the cells had been exposed to a physiological level of shear stress for 0, 1.5, 3, 6, 12, or 24 h. The response of the monolayer to shear stress was not monotonic; it had three distinct phases. The first phase occurred within 3 h. The cells elongated and had more stress fibers, thicker intercellular junctions, and more apical microfilaments. After 6 h of exposure, the monolayer entered the second phase, where the cells exhibited characteristics of motility. The cells lost their dense peripheral bands and had more of their microtubule organizing centers and nuclei located in the upstream region of the cell. The third phase began after 12 h of exposure and was characterized by elongated cells oriented in the direction of fluid flow. The stress fibers in these cells were thicker and longer, and the heights of the intercellular junctions and microfilaments were increased. These results suggest that endothelial cells initially respond to shear stress by enhancing their attachments to the substrate and neighboring cells. The cells then demonstrate characteristics of motility as they realign. The cells eventually thicken their intercellular junctions and increase the amount of apical microfilaments. The time course of rearrangement can be described as a constrained motility that produces a new cytoskeletal organization that alters how the forces produced by fluid flow act on the cell and how the forces are transmitted to the cell interior and substrate. Cell Motil. Cytoskeleton 40:317–330, 1998. © 1998 Wiley-Liss, Inc.

Shear stress induces spatial reorganization of the endothelial cell cytoskeleton.

The morphology of endothelial cells in vivo depends on the local hemodynamic forces. Cells are polygonal and randomly oriented in areas of low shear stress, but they are elongated and aligned in the direction of fluid flow in regions of high shear stress. Endothelial cells in vitro also have a polygonal shape, but the application of shear stress orients and elongates the cells in the direction of fluid flow. The corresponding spatial reorganization of the cytoskeleton in response to the applied hemodynamic forces is unknown. In this study, we determined the spatial reorganization of the cytoskeleton throughout the volume of cultured bovine aortic endothelial cells after the cells had been exposed to a physiological level of shear stress for 0, 1.5, 3, 6, 12, or 24 h. The response of the monolayer to shear stress was not monotonic; it had three distinct phases. The first phase occurred within 3 h. The cells elongated and had more stress fibers, thicker intercellular junctions, and more apical microfilaments. After 6 h of exposure, the monolayer entered the second phase, where the cells exhibited characteristics of motility. The cells lost their dense peripheral bands and had more of their microtubule organizing centers and nuclei located in the upstream region of the cell. The third phase began after 12 h of exposure and was characterized by elongated cells oriented in the direction of fluid flow. The stress fibers in these cells were thicker and longer, and the heights of the intercellular junctions and microfilaments were increased. These results suggest that endothelial cells initially respond to shear stress by enhancing their attachments to the substrate and neighboring cells. The cells then demonstrate characteristics of motility as they realign. The cells eventually thicken their intercellular junctions and increase the amount of apical microfilaments. The time course of rearrangement can be described as a constrained motility that produces a new cytoskeletal organization that alters how the forces produced by fluid flow act on the cell and how the forces are transmitted to the cell interior and substrate.

Alterations in endothelial F-actin microfilaments in rabbit aorta in hypercholesterolemia.

The current study tests whether hypercholesterolemia influences the distribution of endothelial cell microfilaments during the initiation and growth of fatty streak-type lesions. We classified the lesions occurring over a 20-week period into four types based on the location and extent of macrophage infiltration observed microscopically. The earliest lesion was characterized by leukocytes adherent to the endothelial surface. Minimal lesions were characterized by a few cells in the subendothelium. Intermediate lesions consisted of numerous subendothelial leukocytes in a minimally raised lesion. Advanced fatty streak lesions were elevated, with several layers of leukocytes. The organization of peripheral junctional actin (the dense peripheral band) and of central endothelial cell actin microfilament bundles was studied in each of these lesions by using fluorescent microscopy. We found that in the aorta away from branch sites and in areas away from lesions, the central microfilament distribution was unaffected by hypercholesterolemia. The macrophages entered the wall without any identifiable reorganization in the microfilaments. During the accumulation of subendothelial macrophages in minimal and intermediate lesions, stress fibers were initially increased in comparison to lesion-free areas. In raised advanced lesions, the central microfilaments became thinner and disappeared. However, at flow dividers, where central stress fibers are normally prominent, endothelial cells on the surface of intermediate lesions showed a reduction in central fibers, and peripheral bands became prominent. This finding was associated with changes in cell shape from elongated to cobblestone type. Thus, actin microfilament bundles in endothelial cells underwent substantial changes in distribution during the accumulation of subendothelial macrophages, forming hypercholesterolemia-induced fatty streak-type lesions. These changes may influence endothelial substrate adhesion, permeability, or repair after injury.

Nitric Oxide and cGMP Regulate Endothelial Permeability and F-Actin Distribution in Hydrogen Peroxide-Treated Endothelial Cells

We have previously reported that hydrogen peroxide (H2O2) has a concentration-dependent effect on endothelial permeability and F-actin distribution. In the present study, we considered the involvement of endogenous production of nitric oxide (NO) in the indicated effect of H2O2. This was done by measuring endothelial permeability to sodium fluorescein (MW 376 Da, Na-F) and to different-sized fluorescein-isothiocynate-labeled dextrans (FITC-dextrans) and by staining F-actin with rhodamine-labeled phalloidin in cultured bovine aortic endothelial cells growing on filters. A low concentration of H2O2(10−5M) had no effect on either dense peripheral bands of F-actin (DPBs) or permeability. WhenN-nitro-l-arginine methylester (l-NAME), an inhibitor of NO production, was coadministrated with 10−5MH2O2, DPBs were disrupted and the permeability to FITC-dextran 40 and FITC-dextran 70, but not to Na-F and FITC-dextran 20, was increased. Combining of 10−5MH2O2withl-arginine, a substrate for nitric oxide synthase, caused an increase in DPBs and a decrease in permeability to FITC-dextran 40 and FITC-dextran 70.l-arginine orl-NAME alone had no effect on either F-actin structure or endothelial permeability. A 10-fold higher concentration of H2O2caused a disruption of DPBs and an increase in permeability; this could be prevented by addingl-arginine. An analogue of cGMP, i.e., 8-Br-cGMP, maintained DPBs and abolished the increase in permeability induced by the treatment with either 10−4MH2O2or a combination of H2O2andl-NAME. These results suggest that the endogenous production of NO is involved in maintaining endothelial junctions in H2O2-treated cells and that this involvement occurs via a cGMP-dependent mechanism.

Filamin Redistribution in an Endothelial Cell Reoxygenation Injury Model

Ischemia-reperfusion injury increases vascular permeability in part by generating reactive oxygen species that disassemble the endothelial cell actin dense peripheral band. This is followed by an increase in the number and diameter of intercellular gaps. Millimolar concentrations of reactive oxygen metabolites lead to nonspecific endothelial cell injury, but micromolar concentrations activate inflammatory second messenger cascades which produce distributional changes in endothelial cell cytoskeletal proteins. H 2O 2 (100 μM) causes translocation of filamin, from the membrane to the cytosol within 1 min. Subsequently, gap formation occurs within 10–25 min, which is attributed to rearrangement of the dense peripheral band of F-actin. Plasma membrane blebbing occurs after 90 min and decreases in mitochondrial activity occur after 1–2 h. Deferoxamine (iron chelator) and TEMPO (nonspecific free radical scavenger) inhibit these changes. H 2O 2 (100–1000 μM) does not increase endothelial cell intracellular Ca 2+ through 30 min and pretreating cells with a Ca 2+-calmodulin kinase inhibitor or an intracellular Ca 2+ chelator does not prevent filamin translocation. Filamin redistribution and actin rearrangement are early events in H 2O 2-mediated endothelial cell injury that appear to occur through Ca 2+-independent pathways. Copyright © 1997 Elsevier Science Inc.