Low-density lipoprotein induced actin cytoskeleton reorganization in endothelial cells: mechanisms of action.

The inhibitory effects of the specific NADPH oxidase inhibitor, apocynin, and non-specific NADPH oxidase inhibitors, nordihydroguaiaretic acid (NDGA) and SKF525A, on the disruption of dense peripheral bands and formation of stress fibers in cultured human umbilical vein endothelial cells exposed to atherogenic low-density lipoprotein (LDL) levels has been investigated. Endothelial cells (EC) in vitro and in vivo exposed to high LDL-cholesterol levels have cytoskeletal remodeling with stress fiber formation and loss of dense peripheral bands. Cultured EC incubated with exogenously applied hydrogen peroxide (H2O2: 1 mM) have cytoskeletal structural changes much similar to those observed with high LDL exposure. Previous studies have 1) demonstrated that exposure to atherogenic LDL levels causes heightened EC H2O2 production, 2) identified the reactive oxygen species source, NADPH oxidase, in EC, and 3) shown that the specific NADPH oxidase inhibitor, apocynin, and non-specific NADPH oxidase inhibitors, NDGA and SKF525A, suppress H2O2 production increases in high LDL-perturbed EC. In the present study, the cytoskeletal structure of EC exposed to 330 mg/dl LDL-cholesterol, and incubated with or without apocynin, NDGA and SKF525A, was examined. Each of these compounds promoted the retention of dense peripheral bands and minimized stress fiber formation. These findings are consistent with NADPH oxidase and it's reactive oxygen species byproducts modulating the cytoskeleton reorganization observed in high LDL-induced EC perturbation.

NF-kappa B-dependent inhibition of apoptosis is essential for host cellsurvival during Rickettsia rickettsii infection.

The possibility that bacteria may have evolved strategies to overcome host cell apoptosis was explored by using Rickettsia rickettsii, an obligate intracellular Gram-negative bacteria that is the etiologic agent of Rocky Mountain spotted fever. The vascular endothelial cell, the primary target cell during in vivo infection, exhibits no evidence of apoptosis during natural infection and is maintained for a sufficient time to allow replication and cell-to-cell spread prior to eventual death due to necrotic damage. Prior work in our laboratory demonstrated that R. rickettsii infection activates the transcription factor NF-kappa B and alters expression of several genes under its control. However, when R. rickettsii-induced activation of NF-kappa B was inhibited, apoptosis of infected but not uninfected endothelial cells rapidly ensued. In addition, human embryonic fibroblasts stably transfected with a superrepressor mutant inhibitory subunit Ikappa B that rendered NF-kappa B inactivatable also underwent apoptosis when infected, whereas infected wild-type human embryonic fibroblasts survived. R. rickettsii, therefore, appeared to inhibit host cell apoptosis via a mechanism dependent on NF-kappa B activation. Apoptotic nuclear changes correlated with presence of intracellular organisms and thus this previously unrecognized proapoptotic signal, masked by concomitant NF-kappa B activation, likely required intracellular infection. Our studies demonstrate that a bacterial organism can exert an antiapoptotic effect, thus modulating the host cell's apoptotic response to its own advantage by potentially allowing the host cell to remain as a site of infection.

Ultrasound reversibly disaggregates fibrin fibers.

Ultrasound accelerates fibrinolysis in vitro and in vivo, primarily through non-thermal mechanisms including cavitation. We have previously observed that ultrasound reversibly increases flow through fibrin gels, a property primarily determined by the structure of the fibrin matrix. Therefore, the effect of ultrasound on the ultrastructure of fibrin gels was examined using scanning electron microscopy. Non-crosslinked fibrin gels were fixed and prepared for microscopy before, during and after exposure to 1 MHz ultrasound, and quantitative analysis of fiber population density and diameter was performed. Gels exposed and fixed in the presence of ultrasound exhibited an increase in density of 65 +/- 26% (mean +/- SD) at 4 W/cm2 (p <0.000001) accompanied by a decrease in fiber diameter of 27 +/- 9% (p <0.000001). Gels fixed 15 min following ultrasound exposure showed no significant change in either density or diameter compared to unexposed gels, indicating that the ultrasound-induced change in fiber structure was reversible. Factor XIII-crosslinked fibrin gels exhibited no change in population density or diameter when exposed to ultrasound. These results indicate that ultrasound exposure causes reversible disaggregation of uncrosslinked fibrin fibers into smaller fibers, an effect that may alter flow resistance and create additional binding sites for fibrinolytic components, improving fibrinolytic efficacy.

Polarized secretion of fibrinogen by lung epithelial cells.

The lung epithelium has recently been identified as a novel site of fibrinogen (FBG) biosynthesis. A coordinated upregulation of A alpha, B beta, and gamma chain FBG gene transcription occurs upon stimulation of A549 lung epithelial cells with dexamethasone (DEX) and the proinflammatory mediator interleukin-6 (IL-6). Subsequently, the cells synthesize and secrete fully assembled FBG. This study addresses the polarity of such FBG secretion by A549 cells cultured on polycarbonate membrane filters. After induction with IL-6 and DEX, cells were metabolically labeled, and FBG was immunopurified from the apical and basolateral chambers. Analysis by gel electrophoresis revealed that A549 cells secreted newly synthesized FBG in a polarized manner, with the majority (80%) of FBG secreted basolaterally. Consistent with this observation, immunoelectron microscopy using Protein A-gold labeling showed FBG within secretory vesicles in close proximity to the basolateral aspect of the A549 cell membrane. Polarized secretion was microtubule-dependent since depolymerization using colchicine significantly reduced the basolateral component of secretion, causing intracellular retention of FBG. These data provide evidence that FBG is secreted by lung alveolar epithelial cells vectorially toward the basement membrane, which may reflect in vivo processes associated with local injury, inflammation, and repair mechanisms.

cis-Polyisoprene Synthesis in Guayule Plants (Parthenium argentatum Gray) Exposed to Low, Nonfreezing Temperatures.

Exposure of guayule plants (Parthenium argentatum Gray) to 6 months of a night temperature of 7 degrees C results in a 2-fold stimulation of cis-polyisoprene (rubber) formation over that of control plants exposed to 21 to 24 degrees C night temperature. Control and cold-treated plants contained 2.18% and 5.69% rubber, respectively. Examination of the stem apices by transmission electron microscopy showed extensive formation of rubber particles in the cold-treated plants compared to the control plants. The rubber particles in guayule are formed in the cytoplasm and fuse to form large globular deposits. The surface area of the rubber particles and globules range from 4 x 10(-6) to 2.9 x 10(-3) square micrometers. The deposition of rubber in the cytoplasm of the cortical parenchyma cells differs from rubber deposition in the vacuoles of laticifers of Asclepias syriaca. Electron micrographs of stem cortical parenchyma in control plants show mature cells with large central vacuoles, thin layers of parietal cytoplasm, and smaller numbers of rubber particles. Radioactive acetate and mevalonate are incorporated into rubber at a faster rate in stem slices from cold-treated plants compared to slices from control plants. A faster rate of these reactions may account for the increase in rubber synthesis in the cold-treated plants.