Regulation of VEGF-induced intussusceptive angiogenesis by Notch4 signaling

Introduction. Vascular endothelial growth factor-A (VEGF) is the master regulator of vascular growth and a key target for therapeutic angiogenesis. However, VEGF causes either normal or aberrant angiogenesis depending on its dose in the microenvironment around each producing cell in vivo. We previously found that therapeutic doses of VEGF in skeletal muscle do not induce angiogenesis by the well-studied mechanism of sprouting, but rather through intussusception, whose regulation is essentially unknown. Notch signaling is critical in sprouting angiogenesis and endothelium expresses both Notch1 and Notch4. We previously found that Notch1 activation and Dll4 expression are lost during the transition between normal or aberrant angiogenesis by VEGF. Here we investigated whether and how Notch4 and Jag1 regulate the process of splitting angiogenesis by increasing VEGF doses, in the therapeutic target tissue of skeletal muscle.
Methods. VEGF doses inducing either normal or aberrant angiogenesis were delivered in hindlimb skeletal muscles of (a) SCID mice, by implanting well-characterized monoclonal populations of transduced myoblasts, or (b) Notch4-WT (N4+/+) and Notch4-KO (N4-/-) mice, by injecting either (i) fibrin matrices decorated with an engineered, cross-linkable version of VEGF protein or (ii) a therapeutically relevant adenoviral vector. In SCID mice, Notch signaling was globally inhibited by co-expressing a secreted form of the ligand Dll4 (sDll4). In N4+/+, Jag1 interaction with Notch receptors was specifically prevented by systemically delivering a Jag1-neutralizing antibody.
Results. In SCID mice, global Notch inhibition by sDll4 did not impair normal angiogenesis, but completely prevented aberrant vascular growth, converting it into morphologically normal and functionally perfused microvascular networks. Similar results were observed when only Notch4 signaling was genetically abrogated in N4-/- mice, without interfering with Notch1 signaling. Angiogenic normalization was not dependent on the mode of VEGF delivery, as it could be observed with both controlled VEGF protein delivery within fibrin matrices and uncontrolled expression by a clinically relevant adenoviral gene therapy vector. Jag1 blockade failed to prevent aberrant structures, but also increased Dll4 expression, thus suggesting the involvement of a compensatory mechanism which could result in Notch4 activation despite the absence of Jag1-mediated stimulus. The lack of Notch4 signaling did not affect the total amount of proliferating endothelial cells, but it significantly reduced their speed of proliferation, leading to a more moderate degree of circumferential enlargement and therefore enabling successful splitting into normal vascular structures. Gene set enrichment analysis revealed a downregulation of sets connected with cell proliferation and metabolism in N4-/- mice, including Myc target gene sets, which points to a potential crosstalk between Notch4 and Myc signaling pathways.
Conclusion. Notch4 signaling determines the switch between normal and aberrant angiogenesis by modulating the speed of endothelial proliferation by VEGF and therefore emerging as a relevant target to ensure therapeutic angiogenesis. The role of Jag1 and Dll4 in the process is currently being investigated.