Vegfc lineage tracing identifies progenitor cells that change cell fate after musculoskeletal injury

Aberrant repair after musculoskeletal injury is one of the most costly and common clinical scenarios in health care. While advances in our understanding of the key cells and pathways during the repair process have improved, there remains a gap in knowledge of the key progenitor cells and their interaction with the surrounding microenvironment. Heterotopic ossification (HO) is a pathological process caused by aberrant cell-fate determination resulting in the formation of bone in non-skeletal tissues after musculoskeletal injury. While the involvement of blood vessels in musculoskeletal repair is well-documented, the role of lymphatic vessels and the cells responsible for their ingrowth in musculoskeletal tissues during both homeostasis and after injury remains poorly understood. In this study, we employed a mouse model of traumatic HO to investigate lymphangiogenesis during musculoskeletal injury and repair and to uncover the key progenitor cells responsible to stimulate lymphangiogenesis and undergo aberrant cell fate differentiation. Our findings demonstrated that musculoskeletal injury triggered lymphangiogenesis at the injury site, including invasion of lymphatic vessels into the tendon proper. This increased lymphangiogenesis was driven by elevated levels of active Vascular Endothelial Growth Factor C (VEGF-C) one-week post-injury. Through single-cell transcriptomic analyses, we identified mesenchymal progenitor cells (MPCs) as a source of Vegfc following injury as well as the source of enzymes responsible for activating VEGF-C. These Vegfc-expressing MPCs underwent osteochondral differentiation and actively contributed to the formation of HO following injury. Notably, Vegfc haploinsufficiency resulted in a near 50% reduction in lymphangiogenesis and HO formation. Collectively, these findings suggest that MPC expression of VEGF-C and its activating enzymes in response to soft tissue trauma play a critical role in stimulating pathologic lymphatic vessel growth. Furthermore, Vegfc expressing MPCs undergo aberrant osteochondral differentiation responsible for HO. Targeting Vegfc expression and Vegfc-expressing progenitors therapeutically could potentially mitigate pathologic lymphangiogenesis, preventing aberrant tissue repair and subsequent HO formation.