MMP2 Sheds Glypican‐4 to Suppress Slit3‐Robo4 Signaling and Angiogenesis

ABSTRACT Axon guidance molecules, initially identified for their roles in neural development, are now recognized as crucial regulators of angiogenesis and blood vessel patterning. Among these, Slit3 promotes endothelial cell migration, proliferation, and vascular network formation by signaling through the endothelial‐specific receptor Robo4. This interaction depends on heparan sulfate (HS) as a co‐receptor for assembly of the Slit3–Robo4 complex, although the specific HS proteoglycan involved has not yet been identified. Our recent work also shows that Robo4 ectodomain shedding by ADAM10 and ADAM17 suppresses this angiogenic pathway. Here, we identify matrix metalloproteinase‐2 (MMP2) as an additional suppressor of Slit3–Robo4 signaling. MMP2 expression correlates with Slit3 in diaphragm mesenchymal and lung stromal cells and inhibits Slit3‐induced endothelial cell proliferation, migration, and angiogenesis. Furthermore, we identify glypican‐4 (GPC4) as an HS proteoglycan mediating Slit3–Robo4 co‐receptor function and show that MMP2 cleaves GPC4, thereby disrupting Slit3–Robo4 signaling. These findings establish GPC4 as a vital co‐receptor for Slit3–Robo4 signaling and reveal MMP2‐mediated GPC4 shedding as a novel mechanism to regulate angiogenic responses. This study uncovers an additional level of proteolytic regulation in Slit3–Robo4 signaling‐driven angiogenesis, broadening our understanding of how extracellular proteinases influence ligand–receptor/co‐receptor interactions.