Abstract
Endothelial cell activation involves the elevated expression of cell adhesion molecules, chemoattractants, chemokines, and cytokines. These expression profiles may be regulated by integrin-mediated cell signaling pathways. In the current study, an alpha2beta1 integrin triple helical peptide ligand derived from type I collagen residues alpha1(I)496-507 was examined for induction of human aortic endothelial cell (HAEC) activation. In addition, a "miniextracellular matrix" composed of a mixture of the alpha1(I)496-507 ligand and a second, alpha-helical ligand incorporating the endothelial cell proliferating region of SPARC (secreted protein acidic and rich in cysteine) was studied for induction of HAEC activation. Following HAEC adhesion to alpha1(I)496-507, mRNA expression of E-selectin-1, vascular and intercellular cell adhesion molecules-1, and monocytic chemoattractant protein-1 was stimulated, whereas that of endothelin-1 was inhibited. Enzyme-linked immunosorbent assay analysis demonstrated that E-selectin-1 and monocytic chemoattractant protein-1 expression was also stimulated, whereas endothelin-1 protein expression diminished. Engagement of the alpha2beta1 integrin initiated a HAEC response similar to that of tumor necrosis factor-alpha-induced HAECs but was not sufficient to induce an inflammatory response. Addition of the SPARC119-122 region had only a slight effect on HAEC activation. Other cell-extracellular matrix interactions appear to be required to elicit an inflammatory response. The alpha2beta1 integrin specific triple helical peptide ligand described herein represents a more general in vitro model system by which gene expression and protein production profiles induced by binding to a single cellular receptor type can be quantified.
Highlights
The endothelium, under normal physiological conditions, does not bind elements in the flowing blood but instead associates with smooth muscle cells and the extracellular matrix (ECM).1 These normal cell-cell and cell-matrix interactions are maintained, in part, by integrins on the endothelial cell surface
Because type I collagen binding sites for the ␣11 and ␣21 integrins can overlap [6], the inhibition of human aortic endothelial cell (HAEC) binding to the C10-(␣1(I)496 –507) peptide-amphiphile was examined in the presence of either an anti-␣1 or an anti-␣2 integrin subunit mAb
HAECs appear to bind to the C10-(␣1(I)496 – 507) peptide-amphiphile using the ␣21 integrin
Summary
Peptide-amphiphile Sequences—The C10-(␣1(I)496 –507) peptide-amphiphile consisted of amino acid sequence (Gly-Pro-Hyp)4-Gly-Ala-ArgGly-Glu-Arg-Gly-Phe-Hyp-Gly-Glu-Arg-(Gly-Pro-Hyp)4-NH2 lipidated with a CH3-(CH2)8-CO2H (designated C10) alkyl chain. The PCRs were performed in 50 l consisting of PCR buffer (50 mM KCl and 20 mM Tris1⁄7HCl, pH 8.4), 1.5 mM MgCl2, 0.8 mM dNTPs, a 0.2 M concentration of each primer, 2.5 units of Platinum® Taq DNA polymerase (Invitrogen), and 10% of the cDNA made from the reverse transcription reactions above. BCA reagents (Pierce) were used to measure protein levels in cell lysates These assays were quantified with a SpectraMAX GeminiEM, 96-well plate spectrafluorometer using SoftMax Pro 4.3LS software. ICAM-1, VCAM-1, E-selectin-1, MCP-1, and EDN1 ELISA kits were obtained from R&D Systems These kits were used to measure protein expression in the conditioned medium and neat lysates from cells grown on surfaces coated with peptide-amphiphiles according to the manufacturer’s instructions. A SpectraMAX Pro 96-well plate spectrafluorometer was used to quantify protein levels
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