Phosphomimetic Thrombospondin-1 Modulates Integrin β1-FAK Signaling and Vascular Cell Functions

Thrombospondin-1 (TSP1) is a multifunctional glycoprotein that plays a crucial role in angiogenesis and vascular remodeling. Ser93 of TSP1 has recently been identified as a novel phosphorylation site, influencing angiogenic properties; however, the underlying signaling mechanism remains unclear. Here, we investigated the functional impact of Ser93 phosphorylation using phosphomimetic (TSP1^S93D) and phosphonull (TSP1^S93A) mutants. Endothelial cell (EC) migration was analyzed using scratch assay and electric cell-substrate impedance sensing. Activation of key pathways (Akt, p38, ERK, and FAK) was analyzed by immunoblotting. TSP1 secretion was quantified by ELISA. Downstream effects on smooth muscle cells were examined by Western blot using conditioned media of endothelial cells. Expression of TSP1^S93D significantly impaired endothelial migration and wound closure, associated with reduced phosphorylation of FAK and paxillin. Upstream of FAK signaling, TSP1^S93D showed enhanced binding to integrin β1 and promoted its clustering. In contrast, TSP1^S93D stimulated smooth muscle cell proliferation, migration, cytoskeletal remodeling, and phenotypic switching toward a synthetic, pro-inflammatory state characterized by elevated marker protein expression. Together, these findings demonstrate that the impaired angiogenic properties induced by TSP1^S93D result from the modulation of integrin β1-FAK pathways in ECs, suppressing endothelial motility while promoting smooth muscle activation, suggesting a role in early vascular remodeling and inflammation.