Vascular Endothelial Factor-A (VEGF) is the master regulator of angiogenesis, but its therapeutic modes of action are still elusive. In the therapeutic target tissue of skeletal muscle, VEGF does not induce vascular sprouting, but rather intussusceptive angiogenesis, which is yet poorly understood. Intussusceptive morphogenesis entails: 1) circumferential enlargement; 2) formation and fusion of intraluminal endothelial pillars; 3) longitudinal splitting into new vessels. Here we sought to resolve the transient states of endothelium underlying intussusceptive angiogenesis by VEGF delivery in skeletal muscle.

Endothelial cells (ECs) were isolated from tibialis anterior limb muscles of C57/Bl6 mice 3, 4, 5 and 7 days after delivery of a therapeutically relevant VEGF dose, and sequenced. These time-points comprise all stages of intussusception. Protein expression was verified by immunofluorescent confocal microscopy.

A community specifically induced by VEGF was identified and sub-clustering revealed 5 individual functional communities. Based on expression signatures, enrichment of biological processes, temporal behaviour and pseudotime trajectories, the 5 communities were identified as: 1) Angiogenic Capillary; 2) Proliferation; 3) Migratory Vein; 4) Interferon; and 5) Endothelial-to-Mesenchymal Transition (EndoMT). The Interferon community appeared only during the resolution phase sfter splitting, while the others co-existed during the enlargement/pillar stages. The EndoMT signature appeared distinctive for intussusception, in comparison with a pure sprouting model in the retina. Transcriptionally, the EndoMT signature identified no positive event in retina sprouting databases. Conversely, previously published “Tip Cell” signatures from multiple databases failed to identify a Tip Cell community in the intussusception dataset, but correctly identified the Proliferation community. Multiplex immunostaining identified Endo-MT cells co-expressing activated TGF-b signaling (pSMAD), Zeb2 transcription factor and fibronectin-1 during the enlargement/pillar stages, with resolution after splitting, whereas these were absent in the retina sprouting front.

The angiogenic mechanisms of sprouting and intussusception are transcriptionally distinct and EndoMT is a biological process uniquely associated with intussusceptive angiogenesis induced by VEGF delivery in skeletal muscle.