Lateral boundary mechanosensing by adherent cells in a collagen gel system

Cell adhesion responses to in-depth physical properties such as substrate roughness and topography are well described but little is known about the influence of lateral physical cues such as tissue boundaries on the function of adherent cells. Accordingly, we developed a model system to examine remote cell sensing of lateral boundaries. The model employs floating thin collagen gels supported by rigid grids of varying widths. The dynamics, lengths, and numbers of cell extensions were regulated by grid opening size, which in turn determined the distance of cells from rigid physical boundaries. In smaller grids (200 μm and 500 μm wide), cell-induced deformation fields extended to, and were resisted by, the grid boundaries. However, in larger grids (1700 μm wide), the deformation field did not extend to the grid boundaries, which strongly affected the mean length and number of cell extensions (∼60% reduction). The generation of cell extensions in collagen gels required expression of the β1 integrin, focal adhesion kinase and actomyosin activity. We conclude that the presence of physical boundaries interrupts the process of cell-mediated collagen compaction and fiber alignment in the collagen matrix and enhances the formation of cell extensions. This new cell culture platform provides a geometry that more closely approximates the native basement membrane and will help to elucidate the roles of cell extensions and lateral mechanosensing on extracellular matrix remodeling by invasion and degradation.     

Neurite branching on deformable substrates

The mechanical properties of substrates underlying cells can have profound effects on cell structure and function. To examine the effect of substrate deformability on neuronal cell growth, protein-laminated polyacrylamide gels were prepared with differing amounts of bisacrylamide to generate substrates of varying deformability with elastic moduli ranging from 500 to 5500 dyne/cm. Mouse spinal cord primary neuronal cells were plated on the gels and allowed to grow and extend neurites for several weeks in culture. While neurons grew well on the gels, glia, which are normally co-cultured with the neurons, did not survive on these deformable substrates even though the chemical environment was permissive for their growth. Substrate flexibility also had a significant effect on neurite branching. Neurons grown on softer substrates formed more than three times as many branches as those grown on stiffer gels. These results show that mechanical properties of the substrate specifically direct the formation of neurite branches, which are critical for appropriate synaptic connections during development and regeneration.     

Purification of salmon clotting factors and their use as tissue sealants

Fibrin sealant prepared from the blood of farmed Atlantic salmon (Salmo salar) represents a potential source of well-controlled natural material with utility in a variety of clinical settings. A potential advantage of this material is a lower probability of viral or bacterial infection that has limited general approval of fibrin glues made from human or bovine proteins. This report describes the purification of fibrinogen from salmon blood, the use of fibrin glues derived from this material to promote wound healing in rats, and the antigenic response to this material. While the low ambient temperature of these cold water fish significantly lessens the probability of infectious transmission to humans, fibrinogen and factor XIII derived from S. salar are activated by human thrombin at 25°C and 37°C to form clots equivalent to those formed by human fibrin. We compare the reactivity of salmon and human fibrinogen with human and bovine thrombin and the structure and viscoelastic properties of the resulting fibrin gels over a range of pH and salt concentrations. The efficacy of salmon fibrin glues in a wound healing assay and the low antigenic response to salmon fibrinogen suggest that this material may substitute for proteins derived from mammalian sources with lower probability of infections.     

The antimicrobial activity of the cathelicidin LL37 is inhibited by F-actin bundles and restored by gelsolin

Antimicrobial peptides are part of the innate host defense system, and inactivation of these peptides is implicated in airway infections in cystic fibrosis (CF). The sputum of patients with CF contains anionic polyelectrolytes, including F-actin and DNA not found in normal airway fluid. These anionic filaments aggregate to contribute to the altered viscoelastic properties of CF sputum. We hypothesized that the airway components stabilizing bundles of F-actin and DNA are in part cationic antimicrobial agents, and that appropriate modification of diseased airway fluid of patients with CF might dissociate these bundles and restore antimicrobial activity. We demonstrate that the human cathelicidin peptide LL37 forms bundles with F-actin and DNA, which are dissolved by gelsolin and DNase, respectively. Coincident with bundle formation, antimicrobial activity of LL37 is inhibited by F-actin and DNA. Pseudomonas bacteria were killed by low concentrations of LL37, but killing was significantly reduced in the presence of F-actin. The actin filament-fragmenting protein gelsolin restored bactericidal activity nearly completely. In a growth inhibition assay, the effects of F-actin were confirmed, and DNA was also shown to inhibit the activity of LL37. When added to CF sputum, gelsolin significantly reduced the growth of bacteria, suggesting activation of endogenous antimicrobial factors. These findings may have therapeutic implications for treatments previously thought to alter only the viscoelastic properties of airway secretions and amplify the possible advantage of gelsolin in CF treatment.     

Quantitative measurement of plasma gelsolin and its incorporation into fibrin clots

Gelsolin is a newly recognized actin-binding protein of plasma that severs actin filaments. The concentration of plasma gelsolin was measured by three independent methods: an enzyme-linked immunosorbent assay (ELISA) and two functional assays based on the ability of gelsolin to accelerate the polymerization of actin (a "nucleating" assay) or to sever preformed actin filaments (a "cutting" assay). The gelsolin level in 56 samples of human plasma ranged between 100 and 330 micrograms/ml. The mean plasma concentrations measured by the different assays were ELISA, 207 micrograms/ml; nucleating assay, 233 micrograms/ml; cutting assay, 247 micrograms/ml. The mean serum level of gelsolin was found to be 24% lower than that of plasma when paired samples were examined (183 micrograms/ml). The difference between plasma and serum gelsolin concentrations can be accounted for by a direct interaction between gelsolin and fibrin as shown by the binding of radiolabeled gelsolin to clots made from purified fibrinogen. Further, unlabeled gelsolin inhibits the binding of the labeled species to fibrin, but hemoglobin and albumin do not. Fibrin oligomers, but not fibrinogen, alter the sedimentation characteristics of radiolabeled gelsolin in sucrose gradients. The amount of gelsolin incorporated into the clot is increased if the clots are made in the presence of actin filaments or fibronectin. Thus, serum levels of gelsolin are lower than plasma levels because of an interaction between gelsolin and fibrin clots.     

Effects of actin filaments on fibrin clot structure and lysis.

The muscle and cytoskeletal protein actin is released from cells as a consequence of cell death and interacts with components of the hemostatic and fibrinolytic systems, including platelets, plasmin, and fibrin. We report here that incorporation of actin filaments into fibrin clots changes their viscoelastic properties by increasing their shear modulus at low deforming stresses and by nearly eliminating their tendency to become more rigid with increasing deformation (ie, exhibit strain-hardening). The viscoelastic effects depended on the length of the actin filaments as shown by the effects of the plasma filament-severing protein, gelsolin. Binding of actin to fibrin clots also varied with actin filament length. The plasma actin-binding proteins gelsolin and vitamin D-binding protein reduced, but did not eliminate, the incorporation of actin in the clot. Fluorescence microscopy showed a direct association of rhodamine-labeled actin filaments with the fibrin network. Incubation of clots containing long actin filaments in solutions containing physiologic concentrations of gelsolin (2 mumol/L) released 60% of the actin trapped in the clot. Reduction of the actin content of a fibrin clot by incubation in a gelsolin-containing solution resulted in an increased rate of clot lysis. The ability of plasma gelsolin to shorten actin filaments may therefore be of physiologic and potentially of therapeutic importance insofar as gelsolin-mediated diffusion of actin from the clot may restore the clot's rheologic properties and render it more sensitive to the lytic action of plasmin.     

Structural and functional damage sustained by mitochondria after traumatic brain injury in the rat: evidence for differentially sensitive populations in the cortex and hippocampus.

The cellular and molecular pathways initiated by traumatic brain injury (TBI) may compromise the function and structural integrity of mitochondria, thereby contributing to cerebral metabolic dysfunction and cell death. The extent to which TBI affects regional mitochondrial populations with respect to structure, function, and swelling was assessed 3 hours and 24 hours after lateral fluid-percussion brain injury in the rat. Significantly less mitochondrial protein was isolated from the injured compared with uninjured parietotemporal cortex, whereas comparable yields were obtained from the hippocampus. After injury, cortical and hippocampal tissue ATP concentrations declined significantly to 60% and 40% of control, respectively, in the absence of respiratory deficits in isolated mitochondria. Mitochondria with ultrastructural morphologic damage comprised a significantly greater percent of the population isolated from injured than uninjured brain. As determined by photon correlation spectroscopy, the mean mitochondrial radius decreased significantly in injured cortical populations (361 +/- 40 nm at 24 hours) and increased significantly in injured hippocampal populations (442 +/- 36 at 3 hours) compared with uninjured populations (Ctx: 418 +/- 44; Hipp: 393 +/- 24). Calcium-induced deenergized swelling rates of isolated mitochondrial populations were significantly slower in injured compared with uninjured samples, suggesting that injury alters the kinetics of mitochondrial permeability transition (MPT) pore activation. Cyclosporin A (CsA)-insensitive swelling was reduced in the cortex, and CsA-sensitive and CsA-insensitive swelling both were reduced in the hippocampus, demonstrating that regulated MPT pores remain in mitochondria isolated from injured brain. A proposed mitochondrial population model synthesizes these data and suggests that cortical mitochondria may be depleted after TBI, with a physically smaller, MPT-regulated population remaining. Hippocampal mitochondria may sustain damage associated with ballooned membranes and reduced MPT pore calcium sensitivity. The heterogeneous mitochondrial response to TBI may underlie posttraumatic metabolic dysfunction and contribute to the pathophysiology of TBI.     

Stability, sterility, coagulation, and immunologic studies of salmon coagulation proteins with potential use for mammalian wound healing and cell engineering

Fibrin sealants made by polymerization of fibrinogen activated by the protease thrombin have many applications in hemostasis and wound healing. In treatments of acute injury or surgical wounds, concentrated fibrin preparations mimic the initial matrix that normally prevents bleeding and acts as a scaffold for cells that initiate tissue repair. However risks of infectious disease, immunogenic reaction, and the high cost of purified human or other mammalian blood proteins limit widespread use of these materials. Purified coagulation proteins from Atlantic salmon represent a potentially safer, equally effective, and less costly alternative in part because of the low ambient temperature of these farmed animals and the absence of endogenous agents known to be infectious in mammalian hosts. This study reports rheologic measurements of lyophilized salmon fibrinogen and thrombin that demonstrate stability to prolonged storage and gamma irradiation sufficient to reduce viral loads by over five orders of magnitude. Coagulation and immunologic studies in rats and rabbits treated intraperitoneally with salmon fibrin show no deleterious effects on coagulation profiles and no cross reactivity with host fibrinogen or thrombin. The results support the potential of salmon fibrin as an alternative to mammalian proteins in clinical applications.     

Interaction of the gelsolin-derived antibacterial PBP 10 peptide with lipid bilayers and cell membranes

PBP 10, an antibacterial, cell membrane-permeant rhodamine B-conjugated peptide derived from the polyphosphoinositide binding site of gelsolin, interacts selectively with both lipopolysaccharides (LPS) and lipoteichoic acid (LTA), the distinct components of gram-negative and gram-positive bacteria, respectively. Isolated LPS and LTA decrease the antimicrobial activities of PBP 10, as well as other antimicrobial peptides, such as cathelicidin-LL37 (LL37) and mellitin. In an effort to elucidate the mechanism of bacterial killing by PBP 10, we compared its effects on artificial lipid bilayers and eukaryotic cell membranes with the actions of the mellitin, magainin II, and LL37 peptides. This study reveals that pore formation is unlikely to be involved in PBP 10-mediated membrane destabilization. We also investigated the effects of these peptides on platelets and red blood cells (RBCs). Comparison of these antimicrobial peptides shows that only mellitin has a toxic effect on platelets and RBCs in a concentration range concomitant with its bactericidal activity. The hemolytic activities of the PBP 10 and LL37 peptides significantly increase when RBCs are osmotically swollen in hypotonic solution, indicating that these antibacterial peptides may take advantage of the more extended form of bacterial membranes in exerting their killing activities. Additionally, we found that LL37 hemolytic activity was much higher when RBCs were induced to expose phosphatidylserine to the external leaflet of their plasma membranes. This finding suggests that asymmetrical distribution of phospholipids in the external membranes of eukaryotic cells may represent an important factor in determining the specificity of antibacterial peptides for targeting bacteria rather than eukaryotic cells.     

Non-Linear Elasticity of Extracellular Matrices Enables Contractile Cells to Communicate Local Position and Orientation

Most tissue cells grown in sparse cultures on linearly elastic substrates typically display a small, round phenotype on soft substrates and become increasingly spread as the modulus of the substrate increases until their spread area reaches a maximum value. As cell density increases, individual cells retain the same stiffness-dependent differences unless they are very close or in molecular contact. On nonlinear strain-stiffening fibrin gels, the same cell types become maximally spread even when the low strain elastic modulus would predict a round morphology, and cells are influenced by the presence of neighbors hundreds of microns away. Time lapse microscopy reveals that fibroblasts and human mesenchymal stem cells on fibrin deform the substrate by several microns up to five cell lengths away from their plasma membrane through a force limited mechanism. Atomic force microscopy and rheology confirm that these strains locally and globally stiffen the gel, depending on cell density, and this effect leads to long distance cell-cell communication and alignment. Thus cells are acutely responsive to the nonlinear elasticity of their substrates and can manipulate this rheological property to induce patterning.