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

Polyelectrolyte Gels Formed by Filamentous Biopolymers: Dependence of Crosslinking Efficiency on the Chemical Softness of Divalent Cations

Filamentous anionic polyelectrolytes are common in biological materials. Some examples are the cytoskeletal filaments that assemble into networks and bundled structures to give the cell mechanical resistance and that act as surfaces on which enzymes and other molecules can dock. Some viruses, especially bacteriophages are also long thin polyelectrolytes, and their bending stiffness is similar to those of the intermediate filament class of cytoskeletal polymers. These relatively stiff, thin, and long polyelectrolytes have charge densities similar to those of more flexible polyelectrolytes such as DNA, hyaluronic acid, and polyacrylates, and they can form interpenetrating networks and viscoelastic gels at volume fractions far below those at which more flexible polymers form hydrogels. In this report, we examine how different types of divalent and multivalent counterions interact with two biochemically different but physically similar filamentous polyelectrolytes: Pf1 virus and vimentin intermediate filaments (VIF). Different divalent cations aggregate both polyelectrolytes similarly, but transition metal ions are more efficient than alkaline earth ions and their efficiency increases with increasing atomic weight. Comparison of these two different types of polyelectrolyte filaments enables identification of general effects of counterions with polyelectrolytes and can identify cases where the interaction of the counterions and the filaments exhibits stronger and more specific interactions than those of counterion condensation

Salmon and human thrombin differentially regulate radicular pain, glial-induced inflammation and spinal neuronal excitability through protease-activated receptor-1.

Chronic neck pain is a major problem with common causes including disc herniation and spondylosis that compress the spinal nerve roots. Cervical nerve root compression in the rat produces sustained behavioral hypersensitivity, due in part to the early upregulation of pro-inflammatory cytokines, the sustained hyperexcitability of neurons in the spinal cord and degeneration in the injured nerve root. Through its activation of the protease-activated receptor-1 (PAR1), mammalian thrombin can enhance pain and inflammation; yet at lower concentrations it is also capable of transiently attenuating pain which suggests that PAR1 activation rate may affect pain maintenance. Interestingly, salmon-derived fibrin, which contains salmon thrombin, attenuates nerve root-induced pain and inflammation, but the mechanisms of action leading to its analgesia are unknown. This study evaluates the effects of salmon thrombin on nerve root-mediated pain, axonal degeneration in the root, spinal neuronal hyperexcitability and inflammation compared to its human counterpart in the context of their enzymatic capabilities towards coagulation substrates and PAR1. Salmon thrombin significantly reduces behavioral sensitivity, preserves neuronal myelination, reduces macrophage infiltration in the injured nerve root and significantly decreases spinal neuronal hyperexcitability after painful root compression in the rat; whereas human thrombin has no effect. Unlike salmon thrombin, human thrombin upregulates the transcription of IL-1β and TNF-α and the secretion of IL-6 by cortical cultures. Salmon and human thrombins cleave human fibrinogen-derived peptides and form clots with fibrinogen with similar enzymatic activities, but salmon thrombin retains a higher enzymatic activity towards coagulation substrates in the presence of antithrombin III and hirudin compared to human thrombin. Conversely, salmon thrombin activates a PAR1-derived peptide more weakly than human thrombin. These results are the first to demonstrate that salmon thrombin has unique analgesic, neuroprotective and anti-inflammatory capabilities compared to human thrombin and that PAR1 may contribute to these actions

Salmon thrombin does not increase pro-inflammatory cytokines in cortical cultures at early time points.

<p>(<b>A</b>) IL-1β and TNFα mRNA are significantly upregulated in cortical cultures at 4 hours after their treatment with human thrombin (HTh) at 1 U/ml compared to untreated cultures (UT) (^#p<0.001). Salmon thrombin (STh), at the same concentration, is unchanged from UT and significantly less than human thrombin treatment (HTh) (*p<0.002). (<b>B</b>) IL-6 concentration is significantly greater in cultures treated with human thrombin overall at each individual concentration (^#p<0.01) for 6 hours compared to the untreated (UT) control. IL-6 secreted from cortical cultures is significantly less after treatment with salmon thrombin (STh) compared to human thrombin (HTh) at 6 hours after stimulation overall (p<0.001) and at each individual concentration of 0.2, 0.5 and 1 U/ml (*p<0.03). Data are shown as means with standard deviations (μ ± SD).</p

Myelin basic protein (MBP) organization in the injured nerve root.

<p>−striated MBP patterning;</p><p>+mostly striated MBP with some disruption in labeling;</p><p>++mostly disorganized MBP labeling;</p><p>+++highly disorganized MBP labeling with pockets of debris.</p

Salmon and human thrombin have the same Michaelis-Menton kinetics towards human fibrinogen Aa chain.

<p>The rate of Chromozym TH cleavage by 0.03/ml of thrombin for different concentrations of substrate show no differences in the inverse of cleavage rate between salmon (STh) and human (HTh) thrombin at any concentration. The 1/Km and 1/Vmax of salmon and human thrombin for the chromogenic peptide are derived from the intercept of the X axis at 1/rate = 0 and the intercept of the Y axis at 1/S = 0, respectively. Data are shown as means with standard errors of mean (μ ± SEM).</p

Salmon and human thrombin have similar affinities for serum containing media and ATIII, but not hirudin.

<p>(<b>A</b>) Salmon thrombin (STh) and human thrombin (HTh) maintain similar enzymatic activity towards a fluorescent fibrinogen-like substrate over time in serum containing media kept at 37°C; their normalized cleavage rate is not different at any time point after thrombin addition. (<b>B</b>) The activity of salmon thrombin is inhibited significantly less (p = 0.005) than human thrombin by Antithrombin III (ATIII) overall, but not at any one individual concentration ranging from 0 to 45 nM. (<b>C</b>) Salmon thrombin activity towards fibriniogen is inhibited less than human thrombin overall (p<0.001) and at hirudin-to-thrombin ratios of 1 and 1.5 (*p<0.001). Data are shown as means with standard deviations (μ ± SD).</p

Salmon thrombin preserves nerve root health and prevents inflammation after painful compression in the rat.

<p>(<b>A</b>) Schematic depicting the spinal cord, nerve root and dorsal root ganglion (DRG); red box indicates location within the nerve root where the root was analyzed. (<b>B</b>) Uncompressed nerve roots from un-operated (normal) rats exhibit myelin basic protein (MBP; green) labeling in a striated pattern that is homogenous across the width of the root and no immunoreactivity for macrophages (Iba1; red). On day 7 after a painful root compression treated with neurobasal media (NB media), MBP is disrupted and Iba1 is more abundant. Human thrombin treated roots (HTh) have MBP and Iba1 labeling that is similar to those roots treated with NB media. Salmon thrombin treated roots (STh) exhibit the same striated MBP labeling with minimal Iba1 as normal roots and much less than roots treated with NB media or human thrombin. Scale bar is 100 µm. (<b>C</b>) Quantification of positive Iba1 labeling normalized to expression in normal un-operated tissue. Normal tissue exhibits very low levels of Iba1. Human thrombin significantly increases (^#p<0.001) Iba1 in the nerve root compared to normal. Roots treated with salmon thrombin are not different from normal levels or those treated with neurobasal media, but induces significantly less (*p = 0.035) Iba1 infiltration in the nerve root compared to human thrombin.</p

Salmon thrombin clots human fibrinogen slower than human thrombin at lower concentrations.

<p>Human thrombin (HTh) or salmon thrombin (STh) (separate assays in triplicate for 3 lots, n = 9) were used to determine clotting times of 2 mg/mg of human (<b>A</b>) or salmon (<b>B</b>) fibrinogen in PBS. STh clots human fibrinogen at a slower rate than HTh over all concentrations (p<0.001) and at thrombin concentrations of 0.25 and 0.5/ml (*p<0.043). Salmon thrombin and human thrombin have the same clotting rate for human fibrinogen at 2, 4 and 8 U/ml and over the entire concentration range for salmon fibrinogen. Data are shown as means with standard deviations (μ ± SD).</p

Salmon thrombin cleaves peptides mapping the PAR1 cleavage site slower than human thrombin.

<p>Hydrolysis of the fluorogenic PAR1 peptide including only the PAR1 cleavage site (PAR1) is significantly (*p = 0.004) slower for salmon thrombin (STh) than for human thrombin (HTh) (*p = 0.004). For the peptide sequence that also includes a hirudin-like binding site (PAR1 extended), salmon thrombin (STh) also cleaves the peptide significantly slower than human thrombin (HTh) (*p<0.001). Data are shown as means with standard deviations (μ ± SD).</p