ABSTRACT
Human endoglin is an RGD-containing transmembrane glycoprotein identified in vascular endothelial cells. Although endoglin is essential for angiogenesis and its expression is up-regulated in inflammation and at sites of leukocyte extravasation, its role in leukocyte trafficking is unknown. This function was tested in endoglin heterozygous mice (Eng(+/-)) and their wild-type siblings Eng(+/+) treated with carrageenan or LPS as inflammatory agents. Both stimuli showed that inflammation-induced leukocyte transendothelial migration to peritoneum or lungs was significantly lower in Eng(+/-) than in Eng(+/+) mice. Leukocyte transmigration through cell monolayers of endoglin transfectants was clearly enhanced in the presence of endoglin. Coating transwells with the RGD-containing extracellular domain of endoglin, enhanced leukocyte transmigration, and this increased motility was inhibited by soluble endoglin. Leukocytes stimulated with CXCL12, a chemokine involved in inflammation, strongly adhered to endoglin-coated plates and to endoglin-expressing endothelial cells. This endoglin-dependent adhesion was abolished by soluble endoglin, RGD peptides, the anti-integrin α5ß1 inhibitory antibody LIA1/2 and the chemokine receptor inhibitor AMD3100. These results demonstrate for the first time that endothelial endoglin interacts with leukocyte integrin α5ß1 via its RGD motif, and this adhesion process is stimulated by the inflammatory chemokine CXCL12, suggesting a regulatory role for endoglin in transendothelial leukocyte trafficking.
Subject(s)
Antigens, CD/metabolism , Chemotaxis, Leukocyte/physiology , Endothelial Cells/metabolism , Inflammation/metabolism , Receptors, Cell Surface/metabolism , Transendothelial and Transepithelial Migration/physiology , Animals , Cell Adhesion/physiology , Cell Migration Assays, Leukocyte , Chemokine CXCL12/metabolism , Endoglin , Flow Cytometry , Humans , Integrin alpha5beta1/metabolism , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Microscopy, Fluorescence , Transcellular Cell Migration/physiologyABSTRACT
BACKGROUND/AIMS: TGF-beta1 plays a major role in extracellular matrix (ECM) accumulation in tissue fibrosis. Connective tissue growth factor appears to play a critical role in this effect. Endoglin is a component of the transforming growth factor b (TGF-beta) receptor complex. Endoglin is upregulated by TGF-beta1, but its functional role in ECM regulation is unknown. Using rat myoblasts as a model system, we have assessed the role of endoglin on regulating CTGF expression and ECM synthesis and accumulation in the presence or absence of TGF-beta1. METHODS: L6E9 myoblast cell line was transfected with human endoglin, and collagen, fibronectin and CTGF production was assessed by Western blot and by proline incorporation to collagen proteins. RESULTS: Northern blot analysis revealed that parental rat myoblasts L6E9 do not express endogenous endoglin. Upon endoglin transfection, endoglin-expressing cells displayed a decreased CTGF expression and decreased collagen and fibronectin accumulation respect to mock transfectants. Northern blot analysis also revealed a decreased alpha2 (I) procollagen mRNA expression in endoglin transfectants. TGF-beta1 treatment induced an increase in CTGF expression and collagen synthesis and accumulation in L6E9 myoblasts. This effect was significantly lower in endoglin-transfected than in mock-transfected cells. CONCLUSION: These results demonstrate that endoglin expression negatively regulates basal and TGF-beta1-induced CTGF and collagen expression and synthesis.
