Significance Inducing the growth of new blood vessels by specific factors is an attractive strategy to restore blood flow in ischemic tissues. Vascular endothelial growth factor (VEGF) is the master regulator of angiogenesis, yet clinical trials of VEGF gene delivery failed. Major challenges include the need to control the tissue distribution of factor dose and the duration of expression. Here, we developed a highly tunable fibrin-based platform to precisely control the dose and duration of VEGF protein delivery in tissues. Optimized delivery of fibrin-bound VEGF ensured normal, stable, and functional angiogenesis and improved perfusion of ischemic tissues, without genetic modification and with limited duration of VEGF delivery. These findings suggest a strategy to improve both safety and efficacy of therapeutic angiogenesis.

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.

Phase I clinical trials applying autologous progenitor cells to treat heart failure have yielded promising results; however, improvement in function is modest, indicating a need to enhance cardiac stem cell reparative capacity. Notch signaling plays a crucial role in cardiac development, guiding cell fate decisions that underlie myocyte and vessel differentiation. The Notch pathway is retained in the adult cardiac stem cell niche, where level and duration of Notch signal influence proliferation and differentiation of cardiac progenitors. In this study, Notch signaling promotes growth, survival and differentiation of cardiac progenitor cells into smooth muscle lineages in vitro. Cardiac progenitor cells expressing tamoxifen-regulated intracellular Notch1 (CPCeK) are significantly larger and proliferate more slowly than control cells, exhibit elevated mTORC1 and Akt signaling, and are resistant to oxidative stress. Vascular smooth muscle and cardiomyocyte markers increase in CPCeK and are augmented further upon ligand-mediated induction of Notch signal. Paracrine signals indicative of growth, survival and differentiation increase with Notch activity, while markers of senescence are decreased. Adoptive transfer of CPCeK into infarcted mouse myocardium enhances preservation of cardiac function and reduces infarct size relative to hearts receiving control cells. Greater capillary density and proportion of vascular smooth muscle tissue in CPCeK-treated hearts indicate improved vascularization. Finally, we report a previously undescribed signaling mechanism whereby Notch activation stimulates CPC growth, survival and differentiation via mTORC1 and paracrine factor expression. Taken together, these findings suggest that regulated Notch activation potentiates the reparative capacity of CPCs in the treatment of cardiac disease.