Measurement of Liver Stiffness using Atomic Force Microscopy Coupled with Polarization Microscopy.

Matrix stiffening has been recognized as one of the key drivers of the progression of liver fibrosis. It has profound effects on various aspects of cell behavior such as cell function, differentiation, and motility. However, as these processes are not homogeneous throughout the whole organ, it has become increasingly important to understand changes in the mechanical properties of tissues on the cellular level.

Phenotypic drug screening in a human fibrosis model identified a novel class of antifibrotic therapeutics.

Fibrogenic processes instigate fatal chronic diseases leading to organ failure and death. Underlying biological processes involve induced massive deposition of extracellular matrix (ECM) by aberrant fibroblasts. We subjected diseased primary human lung fibroblasts to an advanced three-dimensional phenotypic high-content assay and screened a repurposing drug library of small molecules for inhibiting ECM deposition.

ADAM10 and ADAM17 regulate EGFR, c-Met and TNF RI signalling in liver regeneration and fibrosis.

ADAM10 and ADAM17 are proteases that affect multiple signalling pathways by releasing molecules from the cell surface. As their substrate specificities partially overlaps, we investigated their concurrent role in liver regeneration and fibrosis, using three liver-specific deficient mouse lines: ADAM10- and ADAM17-deficient lines, and a line deficient for both proteases. In the model of partial hepatectomy, double deficient mice exhibited decreased AKT phosphorylation, decreased release of EGFR activating factors and lower shedding of HGF receptor c-Met.

Isolation and 3D Collagen Sandwich Culture of Primary Mouse Hepatocytes to Study the Role of Cytoskeleton in Bile Canalicular Formation In Vitro.

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or more hepatocytes contribute apical membranes to form a bile canalicular network through which bile is secreted. Hepatocyte polarization is essential for correct canalicular formation and depends on interactions between the hepatocyte cytoskeleton, cell-cell contacts, and the extracellular matrix.

Plectin controls biliary tree architecture and stability in cholestasis.

Background & Aims: Plectin, a highly versatile cytolinker protein, controls intermediate filament cytoarchitecture and cellular stress response. In the present study, we investigate the role of plectin in the liver under basal conditions and in experimental cholestasis.

Methods: We generated liver-specific plectin knockout (Ple) mice and analyzed them using two cholestatic liver injury models: bile duct ligation (BDL) and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) feeding.

Liver protective effect of ursodeoxycholic acid includes regulation of ADAM17 activity.

Background: Ursodeoxycholic acid (UDCA) is used to treat primary biliary cirrhosis, intrahepatic cholestasis, and other cholestatic conditions. Although much has been learned about the molecular basis of the disease pathophysiology, our understanding of the effects of UDCA remains unclear. Possibly underlying its cytoprotective, anti-apoptotic, anti-oxidative effects, UDCA was reported to regulate the expression of TNFα and other inflammatory cytokines.

Hepatoprotective effect of MMP-19 deficiency in a mouse model of chronic liver fibrosis.

Liver fibrosis is characterized by the deposition and increased turnover of extracellular matrix. This process is controlled by matrix metalloproteinases (MMPs), whose expression and activity dynamically change during injury progression. MMP-19, one of the most widely expressed MMPs, is highly expressed in liver; however, its contribution to liver pathology is unknown.

Structural and therapeutic insights from the species specificity and in vivo antithrombotic activity of a novel alphaIIb-specific alphaIIbbeta3 antagonist.

We previously reported on a novel compound (Compound 1; RUC-1) identified by high-throughput screening that inhibits human alphaIIbbeta3. RUC-1 did not inhibit alphaVbeta3, suggesting that it interacts with alphaIIb, and flexible ligand/rigid protein molecular docking studies supported this speculation. We have now studied RUC-1's effects on murine and rat platelets, which are less sensitive than human to inhibition by Arg-Gly-Asp (RGD) peptides due to differences in the alphaIIb sequences contributing to the binding pocket.

Morphological and functional platelet abnormalities in Berkeley sickle cell mice.

Berkeley sickle cell mice are used as animal models of human sickle cell disease but there are no reports of platelet studies in this model. Since humans with sickle cell disease have platelet abnormalities, we studied platelet morphology and function in Berkeley mice (SS). We observed elevated mean platelet forward angle light scatter (FSC) values (an indirect measure of platelet volume) in SS compared to wild type (WT) (37+/-3.

Functional and computational studies of the ligand-associated metal binding site of beta3 integrins.

A combination of experimental and computational approaches was used to provide a structural context for the role of the beta3 integrin subunit ligand-associated metal binding site (LIMBS) in the binding of physiological ligands to beta3 integrins. Specifically, we have carried out (1) adhesion assays on cells expressing normal alphaIIbeta3, normal alphaVbeta3, or the corresponding beta3 D217A LIMBS mutants; and (2) equilibrium and nonequilibrium (steered) molecular dynamics (MD) simulations of eptifibatide in complex with either a fully hydrated normal alphaIIbeta3 integrin fragment (alphaIIb beta-propeller and the beta3 betaA (I-like), hybrid, and PSI domains) or the equivalent beta3 D217A mutant. Normal alphaIIbeta3 expressing cells adhered to immobilized fibrinogen and echistatin, whereas cells expressing the alphaIIbeta3 D217A LIMBS mutant failed to adhere to either ligand.

Application of high-throughput screening to identify a novel alphaIIb-specific small- molecule inhibitor of alphaIIbbeta3-mediated platelet interaction with fibrinogen.

Small-molecule alphaIIbbeta3 antagonists competitively block ligand binding by spanning between the D224 in alphaIIb and the MIDAS metal ion in beta3. They variably induce conformational changes in the receptor, which may have undesirable consequences. To identify alphaIIbbeta3 antagonists with novel structures, we tested 33 264 small molecules for their ability to inhibit the adhesion of washed platelets to immobilized fibrinogen at 16 muM.

Ligand density dramatically affects integrin alpha IIb beta 3-mediated platelet signaling and spreading.

The impact of ligand density on integrin-mediated cell adhesion and outside-in signaling is not well understood. Using total internal reflection fluorescent microscopy, conformation-specific antibodies, and Ca(2+) flux measurements, we found that the surface density of fibrinogen affects alpha II b beta 3-mediated platelet signaling, adhesion, and spreading. Adhesion to fibrinogen immobilized at low density leads to rapid increases in cytosolic Ca(2+) and sequential formation of filopodia and lamellipodia.

Platelets and fibrin(ogen) increase metastatic potential by impeding natural killer cell-mediated elimination of tumor cells.

To test the hypothesis that platelet activation contributes to tumor dissemination, we studied metastasis in mice lacking Galphaq, a G protein critical for platelet activation. Loss of platelet activation resulted in a profound diminution in both experimental and spontaneous metastases. Analyses of the distribution of radiolabeled tumor cells demonstrated that platelet function, like fibrinogen, significantly improved the survival of circulating tumor cells in the pulmonary vasculature.

Leukocyte engagement of fibrin(ogen) via the integrin receptor alphaMbeta2/Mac-1 is critical for host inflammatory response in vivo.

The leukocyte integrin alpha(M)beta(2)/Mac-1 appears to support the inflammatory response through multiple ligands, but local engagement of fibrin(ogen) may be particularly important for leukocyte function. To define the biological significance of fibrin(ogen)-alpha(M)beta(2) interaction in vivo, gene-targeted mice were generated in which the alpha(M)beta(2)-binding motif within the fibrinogen gamma chain (N(390)RLSIGE(396)) was converted to a series of alanine residues. Mice carrying the Fibgamma(390-396A) allele maintained normal levels of fibrinogen, retained normal clotting function, supported platelet aggregation, and never developed spontaneous hemorrhagic events.

Antibody blockade or mutation of the fibrinogen gamma-chain C-terminus is more effective in inhibiting murine arterial thrombus formation than complete absence of fibrinogen.

An elevated plasma fibrinogen level is a risk factor for thrombotic cardiovascular disease, but which of fibrinogen's functions is responsible for the increased risk is unknown. To define better the contribution of fibrinogen to large vessel thrombus formation, we studied carotid artery thrombosis in wild-type mice, mice lacking fibrinogen (fbg-/-), mice treated with 7E9 (a blocking antibody to the fibrinogen gamma-chain C-terminus), and mice expressing a mutant fibrinogen (gamma delta 5) that lacks the gamma-chain platelet-binding motif QADGV. In control mice, thrombus formation resulted in occlusion in 8 +/- 2 minutes (mean +/- SD).

Dynamics of GPIIb/IIIa-mediated platelet-platelet interactions in platelet adhesion/thrombus formation on collagen in vitro as revealed by videomicroscopy.

The conventional description of platelet interactions with collagen-coated surfaces in vitro, based on serial static measurements, is that platelets first adhere and spread to form a monolayer and then recruit additional layers of platelets. To obtain dynamic information, we studied gravity-driven platelet deposition in vitro on purified type 1 collagen by video phase-contrast microscopy at 22 degrees C. With untreated human and wild-type mouse platelets, soon after the initial adhesion of a small number of "vanguard" platelets, "follower" platelets attached to the spread-out vanguard platelets.