SIS3 attenuates traumatic heterotopic ossification by targeting TGF-β2-driven endothelial-mesenchymal transition

Heterotopic ossification (HO) is a debilitating disorder characterized by pathological ectopic bone formation with limited effective treatments. Endothelial-mesenchymal transition (EndMT) has been implicated in traumatic HO pathogenesis, but the upstream signaling cascades governing this cellular reprogramming event are poorly defined. Here, we integrated reanalyzed single‑cell RNA sequencing transcriptomic profiles with a series of in vitro and in vivo functional assays to explore the potential molecular mechanism linking EndMT to traumatic HO. Single-cell transcriptomic profiling suggests that TGF‑β2 secreted by mesenchymal cell subsets may elicit endothelial phenotypic alterations reminiscent of EndMT via the canonical TGF‑β2-Smad2/3 signaling cascade. In line with this, sustained activation of the TGF‑β2-Smad2/3 signaling cascade was detected across the development of traumatic HO. These phenotypic alterations may confer chondro‑osteogenic potential on vascular endothelial cells and promote ectopic bone formation. Both genetic ablation and antibody‑mediated neutralization of TGF‑β2 mitigated pathological ossification, likely by blunting EndMT progression. Furthermore, SIS3, a small-molecule compound preferentially suppresses Smad3 phosphorylation, substantially attenuated TGF‑β2‑induced EndMT-like phenotypic shifts and reduced ectopic bone formation under both local intratendinous and systemic administration in mice. Collectively, our study proposes a potential TGF‑β2-Smad2/3 signaling axis linked to EndMT-like phenotypic plasticity during traumatic HO, and provides preliminary mechanistic basis for further preclinical evaluation of Smad3-targeted intervention for HO prevention.