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Bone regeneration is a complex process requiring highly orchestrated interactions between different cells and signals to form new mineralized tissue. Blood vessels serve as a structural template, around which bone development takes place, and also bring together the key elements for bone homeostasis into the osteogenic microenvironment, including minerals, growth factors and osteogenic progenitor cells. Vascular endothelial growth factor (VEGF) is the master regulator of vascular growth and it is required for effective coupling of angiogenesis and osteogenesis during both skeletal development and postnatal bone repair. Here, we will review the current state of knowledge on the molecular cross-talk between angiogenesis and osteogenesis. In particular, we will focus on the role of VEGF in coupling these two processes and how VEGF dose can control the outcome, addressing in particular: (1) the direct influence of VEGF on osteogenic differentiation of mesenchymal progenitors; (2) the angiocrine functions of endothelium to regulate osteoprogenitors; (3) the role of immune cells, e.g., myeloid cells and osteoclast precursors, recruited by VEGF to the osteogenic microenvironment. Finally, we will discuss emerging strategies, based on the current biological understanding, to ensure rapid vascularization and efficient bone formation in regenerative medicine.

Use of long-term constitutive expression of VEGF for therapeutic angiogenesis may be limited by the growth of abnormal blood vessels and hemangiomas. We investigated the relationship between VEGF dosage and the morphology and function of newly formed blood vessels by implanting retrovirally transduced myoblasts that constitutively express VEGF164 into muscles of adult mice. Reducing VEGF dosage by decreasing the total number of VEGF myoblasts implanted did not prevent vascular abnormalities. However, when clonal populations of myoblasts homogeneously expressing different levels of VEGF were implanted, a threshold between normal and aberrant angiogenesis was found. Clonal myoblasts that expressed low to medium levels of VEGF induced growth of stable, pericyte-coated capillaries of uniform size that were not leaky and became VEGF independent, as shown by treatment with the potent VEGF blocker VEGF-TrapR1R2. In contrast, clones that expressed high levels of VEGF induced hemangiomas. Remarkably, when different clonal populations were mixed, even a small proportion of cells with high production of VEGF was sufficient to cause hemangioma growth. These results show for the first time to our knowledge that the key determinant of whether VEGF-induced angiogenesis is normal or aberrant is the microenvironmental amount of growth factor secreted, rather than the overall dose. Long-term continuous delivery of VEGF, when maintained below a threshold microenvironmental level, can lead to normal angiogenesis without other exogenous growth factors

Highlights d Endothelial loss of pfkfb3 impairs ischemic muscle revascularization and regeneration d EC-derived lactate instructs MCT1-dependent macrophage functional polarization d Lactate-polarized macrophages promote muscle revascularization and regeneration d Restoring lactate levels improves macrophage polarization and recovery from ischemia

The critical role of vascular endothelial growth factor (VEGF) expression levels in developmental angiogenesis is well established. Nonetheless, the effects of different local (microenvironmental) VEGF concentrations in ischemia have not been studied in the adult organism, and VEGF delivery to patients has been disappointing. Here, we demonstrate the existence of both lower and upper threshold levels of microenvironmental VEGF concentrations for the induction of therapeutic vessel growth in ischemia. In the ischemic hind limb, implantation of myoblasts transduced to express VEGF164 at different levels per cell increased blood flow only moderately, and vascular leakage and aberrant preangiomatous vessels were always induced. When the same total dose was uniformly distributed by implanting a monoclonal population derived from a single VEGF-expressing myoblast, blood flow was fully restored to nonischemic levels, collateral growth was induced, and ischemic damage was prevented. Hemangiomas were avoided and only normal, pericyte-covered vessels were induced persisting over 15 mo. Surprisingly, clones uniformly expressing either lower or higher VEGF levels failed to provide any functional benefit. A biphasic effect of VEGF dose on vessel number and diameter was found. Blood flow was only improved if vessels were increased both in size and in number. Microenvironmental VEGF concentrations determine efficacy and safety in a therapeutic setting.

Bone marrow stromal cells (BMSCs) can be easily isolated from adult marrow and contain a population of pluripotent progenitors that can give rise to different mesenchymal lineages both in vitro and in vivo. These properties make BMSCs an attractive target for cell-based therapeutic strategies for a variety of disorders. However, because of their low frequency in vivo, to obtain a sufficient number of cells for tissue engineering a step of extensive in vitro expansion is required, which could significantly alter BMSC properties. Therefore, effective therapeutic use of BMSCs requires the design of appropriate approaches for in vitro cell expansion. In this study we have investigated the biological effects of in vitro expansion on BMSC proliferative ability and on their spontaneous differentiation. Telomerase activity and telomere shortening kinetics were evaluated together with variations in osteogenic, chondrogenic, and adipogenic gene expression throughout the BMSC life span. In culture BMSCs never displayed telomerase activity and during in vitro expansion telomere length decreased. Furthermore, gene expression patterns spontaneously varied during expansion, indicating a progressive commitment of the population toward the osteogenic lineage. In conclusion, BMSCs in culture undergo progressive replicative aging and osteogenic differentiation. These observations are relevant to their successful use in clinics and should be considered when designing novel therapeutic strategies.