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

## TL;DR

Phosphomimetic TSP1 affects vascular cell functions by modulating integrin β1-FAK signaling, impairing endothelial migration while promoting smooth muscle cell activation.

## Contribution

The study reveals how phosphorylation of TSP1 at Ser93 modulates integrin β1-FAK signaling and vascular cell behavior.

## Key findings

- TSP1S93D impairs endothelial cell migration and wound closure via reduced FAK and paxillin phosphorylation.
- TSP1S93D enhances integrin β1 clustering and binding upstream of FAK signaling.
- TSP1S93D promotes smooth muscle cell proliferation and pro-inflammatory phenotypic changes.

## Abstract

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 (TSP1S93D) and phosphonull (TSP1S93A) 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 TSP1S93D significantly impaired endothelial migration and wound closure, associated with reduced phosphorylation of FAK and paxillin. Upstream of FAK signaling, TSP1S93D showed enhanced binding to integrin β1 and promoted its clustering. In contrast, TSP1S93D 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 TSP1S93D 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.