5 research outputs found
Molecular Orientation of Tropoelastin is Determined by Surface Hydrophobicity
Tropoelastin is the precursor of the extracellular protein
elastin
and is utilized in tissue engineering and implant technology by adapting
the interface presented by surface-bound tropoelastin. The preferred
orientation of the surface bound protein is relevant to biointerface
interactions, as the C-terminus of tropoelastin is known to be a binding
target for cells. Using recombinant human tropoelastin we monitored
the binding of tropoelastin on hydrophilic silica and on silica made
hydrophobic by depositing a self-assembled monolayer of octadecyl
trichlorosilane. The layered organization of deposited tropoelastin
was probed using neutron and X-ray reflectometry under aqueous and
dried conditions. In a wet environment, tropoelastin retained a solution-like
structure when adsorbed on silica but adopted a brush-like structure
when on hydrophobized silica. The orientation of the surface-bound
tropoelastin was investigated using cell binding assays and it was
found that the C-terminus of tropoelastin faced the bulk solvent when
bound to the hydrophobic surface, but a mixture of orientations was
adopted when tropoelastin was bound to the hydrophilic surface. Drying
the tropoelastin-coated surfaces irreversibly altered these protein
structures for both hydrophilic and hydrophobic surfaces
Mechanism of EPC attachment to rhTE.
<p>(A) EPCs attached to 40 μg/ml rhTE in the presence of α-lactose, β-lactose, heparan sulfate, or EDTA. (B) Attachment of EPCs to 40 μg/ml rhTE in the presence of Ca<sup>2+</sup>, Mg<sup>2+</sup>, or Mn<sup>2+</sup>. (C) and (D) Inhibition of EPC attachment and spreading on 40 μg/ml rhTE using antibodies to integrins α<sub>2</sub>β<sub>1</sub>, α<sub>5</sub>β<sub>1</sub>, and α<sub>v</sub>β<sub>3</sub>. Error bars represent S.E.M. of triplicate measurements.</p
Schematic diagram showing full-length rhTE and constructs N18, N25 and N10.
<p>Key domain features are indicated.</p
Mechanism of EPC attachment to truncated tropoelastin constructs.
<p>(A) and (B) EPCs attached to 40 μg/ml N25 and N18 respectively, in the presence of α-lactose, β-lactose, heparan sulfate or EDTA. (C) and (D) Attachment of EPCs to 40 μg/ml N25 and N18, respectively, in the presence of Ca<sup>2+</sup>, Mg<sup>2+</sup>, or Mn<sup>2+</sup>. (E) and (F), Inhibition of EPC spreading on 40 μg/ml N25 and N18 respectively, using antibody that inhibits binding to integrin α<sub>v</sub>β<sub>3</sub>. Error bars represent S.E.M. of triplicate measurements.</p
OEC characterization by flow cytometry.
<p>A) Stained cells are shown as blue histograms, while unstained controls are shown in black. The percentage of positive cells is shown in the top right of each graph. The OECs are CD34/31/54/VEGFR2 positive and CD45/14 negative. B) Representative images of the binding of isothiocyanate-Ulex europaeus agglutinin I lectin binding (ULEX), uptake of acetylated low density lipoprotein (AcLDL) and staining for CD31 by EPCs (bottom row of panel) but not by fibroblasts (top row of panel). Together, these results are indicative of a positive endothelial cell phenotype.</p