VEGF induces normal or aberrant angiogenesis depending on its dose in the microenvironment around each producing cell in vivo. This transition depends on the balance between VEGF-induced endothelial stimulation and PDGF-BB-mediated pericyte recruitment, and co-expression of PDGF-BB normalizes aberrant angiogenesis despite high VEGF doses. We recently found that VEGF over-expression induces angiogenesis in skeletal muscle through an initial circumferential vascular enlargement followed by longitudinal splitting, rather than sprouting. Here we investigated the cellular mechanism by which PDGF-BB co-expression normalizes VEGF-induced aberrant angiogenesis. Monoclonal populations of transduced myoblasts, expressing similarly high levels of VEGF alone or with PDGF-BB, were implanted in mouse skeletal muscles. PDGF-BB co-expression did not promote sprouting and angiogenesis that occurred through vascular enlargement and splitting. However, enlargements were significantly smaller in diameter, due to a significant reduction in endothelial proliferation, and retained pericytes, which were otherwise lost with high VEGF alone. A time-course of histological analyses and repetitive intravital imaging showed that PDGF-BB co-expression anticipated the initiation of vascular enlargement and markedly accelerated the splitting process. Interestingly, quantification during in vivo imaging suggested that a global reduction in shear stress favored the initiation of transluminal pillar formation during VEGF-induced splitting angiogenesis. Quantification of target gene expression showed that VEGF-R2 signaling output was significantly reduced by PDGF-BB co-expression compared to VEGF alone. In conclusion, PDGF-BB co-expression prevents VEGF-induced aberrant angiogenesis by modulating VEGF-R2 signaling and endothelial proliferation, thereby limiting the degree of circumferential enlargement and enabling efficient completion of vascular splitting into normal capillary networks despite high VEGF doses.

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.

Despite major advances in medical, catheter-based or surgical treatment, cardiovascular diseases such as peripheral artery disease and coronary artery disease still cause significant morbidity and mortality. Furthermore, many patients do not qualify for catheter-based treatment or bypass surgery because of advanced disease or surgical risk. There is therefore an urgent need for novel treatment strategies. Therapeutic angiogenesis aims to restore blood flow to ischaemic tissue by stimulating the growth of new blood vessels through the local delivery of angiogenic factors, and may thus be an attractive treatment alternative for these patients. Angiogenesis is a complex process and the growth of normal, stable and functional vasculature depends on the coordinated interplay of different cell types and growth factors. Vascular endothelial growth factor-A (VEGF) is the fundamental regulator of vascular growth and the key target of therapeutic angiogenesis approaches. However, first-generation clinical trials of VEGF gene therapy have been disappointing, and a clear clinical benefit has yet to be established. In particular, VEGF delivery (a) appears to have a very limited therapeutic window in vivo: low doses are safe but mostly inefficient, whereas higher doses become rapidly unsafe; and (b) requires a sustained expression in vivo of at least about four weeks to achieve stable vessels that persist after cessation of the angiogenic stimulus. Here we will review the current understanding of how VEGF induces the growth of normal or pathological blood vessels, what limitations for the controlled induction of safe and efficient angiogenesis are intrinsically linked to the biological properties of VEGF, and how this knowledge can guide the design of more effective strategies for therapeutic angiogenesis.

Vascularization is a critical step in the restoration of cellular homeostasis. Several strategies including localized growth factor delivery, endothelial progenitor cells, genetically engineered cells, gene therapy, and prevascularized implants have been explored to promote revascularization. But, long‐term stabilization of newly induced vessels remains a challenge. It has been shown that fibroblasts and mesenchymal stem cells can stabilize newly induced vessels. However, whether an injected biomaterial alone can serve as an instructive environment for angiogenesis remains to be elucidated. It is reported here that appropriate vascular branching, and long‐term stabilization can be promoted simply by implanting a hydrogel with stiffness matching that of fibrin clot. A unique subpopulation of circulating CD11b+myeloid and CD11b+/CD115+monocytes that express the stretch activated cation channel Piezo‐1, which is enriched prominently in the clot‐like hydrogel, is identified. These findings offer evidence for a mechanobiology paradigm in angiogenesis involving an interplay between mechanosensitive circulating cells and mechanics of tissue microenvironment.

Non-healing ulcers are a serious complication of diabetes mellitus and a major unmet medical need. A major cause for the lack of healing is the impairment of spontaneous vascularization in the skin, despite mostly normal blood flow in deeper large vessels. Therefore, pro-angiogenic treatments are needed to increase therapeutic perfusion by recruiting new arterial connections (therapeutic arteriogenesis). Vascular endothelial growth factor (VEGF) is the master regulator of angiogenesis in physiology and disease, but exploitation of its therapeutic potential requires careful control of its dose distribution in tissue. Co-delivery of platelet derived growth factor-BB (PDGF-BB) has been shown to expand the therapeutic window of VEGF and also improve associated arteriogenesis. We used a highly controlled protein delivery system, based on a clinically applicable fibrin-based platform, to investigate the angiogenic and arteriogenic potential of engineered versions (TG-) of VEGF and PDGF-BB proteins in the skin of diabetic and obese db/db mice. Intradermal delivery of therapeutically relevant doses of TG-VEGF and TG-PDGF-BB induced robust growth of new microvascular networks with similar efficacy as in normal littermate control mice. Further, TG-PDGF-BB prevented the formation of aberrant vascular enlargements by high TG-VEGF levels. As fibrin was degraded after the first week, the induced angiogenesis mostly regressed by 4 weeks, but it promoted effective arteriogenesis in the dermal layer. Therefore, controlled co-delivery of TG-VEGF and TG-PDGF-BB recombinant proteins is effective to induce angiogenesis and arteriogenesis in diabetic mouse skin and should be further investigated to promote diabetic wound healing.

Chronic wounds in type-2 diabetic patients present areas of severe local skin ischemia despite mostly normal blood flow in deeper large arteries. Therefore, restoration of blood perfusion requires the opening of arterial connections from the deep vessels to the superficial skin layer, that is, arteriogenesis. Arteriogenesis is regulated differently from microvascular angiogenesis and is optimally stimulated by high doses of Vascular Endothelial Growth Factor-A (VEGF) together with Platelet-Derived Growth Factor-BB (PDGF-BB). Here we found that fibrin hydrogels decorated with engineered versions of VEGF and PDGF-BB proteins, to ensure protection from degradation and controlled delivery, efficiently accelerated wound closure in diabetic and obese db/db mice, promoting robust microvascular growth and a marked increase in feeding arterioles. Notably, targeting the arteriogenic factors to the intact arterio-venous networks in the dermis around the wound was more effective than the routine treatment of the inflamed wound bed. This approach is readily translatable to a clinical setting.

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