Molecular regulation of intussusceptive angiogenesis by ephrinB2/EphB4 signaling and its therapeutic potential

Abstract

Ischemic diseases are characterized by reduced blood flow leading to poor tissue oxygenation and tissue damage. Peripheral artery disease is a type of ischemic disease caused by occlusion of the peripheral arteries, and is a major cause of decreased mobility, functional capacity and quality of life and it increases the risks of limb amputation and/or death. Therapeutic angiogenesis is a promising strategy for treatment of ischemic diseases, as it aims to induce growth of new blood vessels, restore blood flow and ultimately improve tissue regeneration. VEGF (Vascular Endothelial Growth Factor) gene therapy, as a strategy for induction of angiogenesis, was proven safe, but failed to demonstrate clear therapeutic benefit during clinical trials. The reasons for disappointing results are low transduction efficiency and insufficient local protein production at safe vector doses. Higher VEGF doses, on the other side, lead to induction of aberrant, dysfunctional angioma-like vascular structures. Therefore, in order to take advantage of VEGF’s therapeutic potential and enable safe delivery of effective doses, we sought to identify molecular targets responsible for normalization of aberrant vascular growth. We previously found that: 1) VEGF can induce either normal and functional capillary networks or aberrant angioma-like vascular structures depending on its concentration in the microenvironment around each producing cell in vivo; and 2) VEGF induces vascular growth in skeletal muscle through the mechanism of intussusceptive angiogenesis, where both normal and aberrant vascular structures form through a process of circumferential enlargement followed by intussusceptive remodeling. The transition from normal to aberrant angiogenesis is determined by the retention or loss of pericytes during the initial stage of vascular enlargement. We have previously demonstrated that stimulation of pericytes recruitment by PDGF-BB (Platelet-Derived Growth Factor BB) co-expression can normalize aberrant vessel growth induced by high and uncontrolled VEGF levels and ensure the induction of exclusively normal and mature microvascular networks. That is why we concentrated on investigation of signaling pathways between pericytes and endothelium that could be responsible for this switch. In the first part of the Thesis, we identified ephrinB2/EphB4 signaling between pericytes and endothelium, as the one that controls the switch between normal and aberrant angiogenesis with increasing VEGF doses. Activity of ephrinB2/EphB4 determines the outcome of VEGF-induced angiogenesis by modulating VEGF downstream signaling through pERK1/2, but without directly affecting internalization or phosphorylation of VEGF-R2. The therapeutic potential of EphB4 was tested in the mouse model of limb ischemia and it was shown that activation of EphB4 signaling together with uncontrolled VEGF delivery by adenoviral vectors yields only normal and functional vascular growth, decreasing tissue necrosis and increasing tissue regeneration. In the second part of the Thesis, the crosstalk between the ephrinB2/EphB4 and Notch4 pathways was investigated. We found that aberrant angiogenesis induced by inhibition of ephrinB2/EphB4 with low VEGF can be prevented in mice with mutated Notch4 protein which is unable to signal. The absence of active Notch4 signaling reduced, but did not completely prevent, the increase in endothelial cell proliferation caused by inhibition of the ephrinB2/EphB4 pathway. These observations suggest that both ephrinB2/EphB4 and Notch4 cooperate in aberrant vessel normalization, but appear to do so via two independent molecular mechanisms. In conclusion, simultaneous activation of EphB4 and inhibition of Notch4 pathway could be a strategy for prevention of aberrant angiogenesis by high VEGF that could lead to therapeutic benefit.