EphrinB2/EphB4 signaling regulates non-sprouting angiogenesis by VEGF.

Vascular endothelial growth factor (VEGF) is the master regulator of angiogenesis, whose best-understood mechanism is sprouting. However, therapeutic VEGF delivery to ischemic muscle induces angiogenesis by the alternative process of intussusception, or vascular splitting, whose molecular regulation is essentially unknown. Here, we identify ephrinB2/EphB4 signaling as a key regulator of intussusceptive angiogenesis and its outcome under therapeutically relevant conditions. EphB4 signaling fine-tunes the degree of endothelial proliferation induced by specific VEGF doses during the initial stage of circumferential enlargement of vessels, thereby limiting their size and subsequently enabling successful splitting into normal capillary networks. Mechanistically, EphB4 neither inhibits VEGF-R2 activation by VEGF nor its internalization, but it modulates VEGF-R2 downstream signaling through phospho-ERK1/2. In vivo inhibitor experiments show that ERK1/2 activity is required for EphB4 regulation of VEGF-induced intussusceptive angiogenesis. Lastly, after clinically relevant VEGF gene delivery with adenoviral vectors, pharmacological stimulation of EphB4 normalizes dysfunctional vascular growth in both normoxic and ischemic muscle. These results identify EphB4 as a druggable target to modulate the outcome of VEGF gene delivery and support further investigation of its therapeutic potential.

VEGF dose regulates vascular stabilization through Semaphorin3A and the Neuropilin-1+ monocyte/TGF-ß1 paracrine axis.

VEGF is widely investigated for therapeutic angiogenesis, but while short-term delivery is desirable for safety, it is insufficient for new vessel persistence, jeopardizing efficacy. Here, we investigated whether and how VEGF dose regulates nascent vessel stabilization, to identify novel therapeutic targets. Monoclonal populations of transduced myoblasts were used to homogeneously express specific VEGF doses in SCID mouse muscles. VEGF was abrogated after 10 and 17 days by Aflibercept treatment. Vascular stabilization was fastest with low VEGF, but delayed or prevented by higher doses, without affecting pericyte coverage. Rather, VEGF dose-dependently inhibited endothelial Semaphorin3A expression, thereby impairing recruitment of Neuropilin-1-expressing monocytes (NEM), TGF-ß1 production and endothelial SMAD2/3 activation. TGF-ß1 further initiated a feedback loop stimulating endothelial Semaphorin3A expression, thereby amplifying the stabilizing signals. Blocking experiments showed that NEM recruitment required endogenous Semaphorin3A and that TGF-ß1 was necessary to start the Semaphorin3A/NEM axis. Conversely, Semaphorin3A treatment promoted NEM recruitment and vessel stabilization despite high VEGF doses or transient adenoviral delivery. Therefore, VEGF inhibits the endothelial Semaphorin3A/NEM/TGF-ß1 paracrine axis and Semaphorin3A treatment accelerates stabilization of VEGF-induced angiogenesis.

Therapeutic angiogenesis due to balanced single-vector delivery of VEGF and PDGF-BB.

Therapeutic angiogenesis by delivery of vascular growth factors is an attractive strategy for treating debilitating occlusive vascular diseases, yet clinical trials have thus far failed to show efficacy. As a result, limb amputation remains a common outcome for muscle ischemia due to severe atherosclerotic disease, with an overall incidence of 100 per million people in the United States per year. A challenge has been that the angiogenic master regulator vascular endothelial growth factor (VEGF) induces dysfunctional vessels, if expressed outside of a narrow dosage window. We tested the hypothesis that codelivery of platelet-derived growth factor-BB (PDGF-BB), which recruits pericytes, could induce normal angiogenesis in skeletal muscle irrespective of VEGF levels. Coexpression of VEGF and PDGF-BB encoded by separate vectors in different cells or in the same cells only partially corrected aberrant angiogenesis. In marked contrast, coexpression of both factors in every cell at a fixed relative level via a single bicistronic vector led to robust, uniformly normal angiogenesis, even when VEGF expression was high and heterogeneous. Notably, in an ischemic hindlimb model, single-vector expression led to efficient growth of collateral arteries, revascularization, increased blood flow, and reduced tissue damage. Furthermore, these results were confirmed in a clinically applicable gene therapy approach by adenoviral-mediated delivery of the bicistronic vector. We conclude that coordinated expression of VEGF and PDGF-BB via a single vector constitutes a novel strategy for harnessing the potency of VEGF to induce safe and efficacious angiogenesis.

To sprout or to split? VEGF, Notch and vascular morphogenesis.

Therapeutic angiogenesis is an attractive strategy to treat patients suffering from peripheral or coronary artery disease. VEGF (vascular endothelial growth factor-A) is the fundamental factor controlling vascular growth in both development and postnatal life. The interplay between the VEGF and Notch signalling pathway has been recently found to regulate the morphogenic events leading to the growth of new vessels by sprouting. Angiogenesis can also take place by an alternative process, i.e. intussusception or vascular splitting. However, little is known about its role in therapeutic angiogenesis and its molecular regulation. In the present article, we briefly review how VEGF dose determines the induction of normal or aberrant angiogenesis and the molecular regulation of sprouting angiogenesis by Notch signalling, and compare this process with intussusception.

Taming of the wild vessel: promoting vessel stabilization for safe therapeutic angiogenesis.

VEGF (vascular endothelial growth factor) is the master regulator of blood vessel growth. However, it displayed substantial limitations when delivered as a single gene to restore blood flow in ischaemic conditions. Indeed, uncontrolled VEGF expression can easily induce aberrant vascular structures, and short-term expression leads to unstable vessels. Targeting the second stage of the angiogenic process, i.e. vascular maturation, is an attractive strategy to induce stable and functional vessels for therapeutic angiogenesis. The present review discusses the limitations of VEGF-based gene therapy, briefly summarizes the current knowledge of the molecular and cellular regulation of vascular maturation, and describes recent pre-clinical evidence on how the maturation stage could be targeted to achieve therapeutic angiogenesis.