Abstract 110: Thrombospondin Type-1 Domain-Containing Protein 1 (THSD1), an Intracranial Aneurysm Susceptibility Gene, Mediates Cell Adhesion in Human Umbilical Vein Endothelial Cells
Introduction: An intracranial aneurysm (IA) is a weak spot in cerebral blood vessel wall that can lead to its abnormal bulging. Previously, we reported that mutations in THSD1, encoding thrombospondin type-1 domain-containing protein 1, are associated with IA in a subset of patients. THSD1 is a transmembrane molecule with a thrombospondin type-1 repeat (TSR). Proteins with TSR domain have been implicated in a variety of processes including regulation of matrix organization, cell adhesion and migration. We have shown that in mouse brain Thsd1 is expressed in endothelial cells.
Hypothesis: THSD1 plays an important role in maintaining the integrity of the endothelium by promoting adhesion of endothelial cells to the underlying basement membrane.
Methods: Human umbilical vein endothelial cells are used to investigate the role of THSD1 in vitro. THSD1 expression was knocked-down by RNA interference. Cell adhesion assay was done on collagen I-coated plates and focal adhesion formation was visualized using immunofluorescence by paxillin and phosphorylated focal adhesion kinase (pFAK) staining. THSD1 re-expression is accomplished by transfection with a pCR3.1-THSD1-encoding plasmid.
Results: Knockdown of THSD1 caused striking change in cell morphology and size. Compared to control siRNA-treated cells that exhibited typical cobblestone morphology, THSD1 knockdown cells were narrow and elongated, and were significantly smaller (p<0.01). Cell adherence to collagen I-coated plates was also attenuated in THSD1 knockdown cells (p<0.01). Consistent with this finding is the observation that the number and size of focal adhesions, based on paxillin and pFAK staining, were significantly reduced after THSD1 knockdown (p<0.01). These defects in cell adhesion and focal adhesion formation were rescued by re-expression of wild type THSD1 (p<0.05). In contrast, initial studies indicate that expression of mutated versions of THSD1 as seen in human patients (L5F, R450*, E466G, P639L) could not restore cell adhesion and focal adhesion formation to wild type levels.
Conclusions: Our studies provide evidence for a role of THSD1 and THSD1 mutations in endothelial cell adhesion and suggest a possible mechanism underlying THSD1-mediated aneurysm disease.
Author Disclosures: X. Fang: None. D.H. Kim: None. T. Santiago-Sim: None.
This research has received full or partial funding support from the American Heart Association, SouthWest - Arkansas, Colorado, New Mexico, Oklahoma, Texas, Wyoming.
- © 2015 by American Heart Association, Inc.