Summary Exercise is a powerful driver of physiological angiogenesis during adulthood, but the mechanisms of exercise-induced vascular expansion are poorly understood. We explored endothelial heterogeneity in skeletal muscle and identified two capillary muscle endothelial cell (mEC) populations that are characterized by differential expression of ATF3/4. Spatial mapping showed that ATF3/4 + mECs are enriched in red oxidative muscle areas while ATF3/4 low ECs lie adjacent to white glycolytic fibers. In vitro and in vivo experiments revealed that red ATF3/4 + mECs are more angiogenic when compared with white ATF3/4 low mECs. Mechanistically, ATF3/4 in mECs control genes involved in amino acid uptake and metabolism and metabolically prime red (ATF3/4 + ) mECs for angiogenesis. As a consequence, supplementation of non-essential amino acids and overexpression of ATF4 increased proliferation of white mECs. Finally, deleting Atf4 in ECs impaired exercise-induced angiogenesis. Our findings illustrate that spatial metabolic angiodiversity determines the angiogenic potential of muscle ECs.

Aims Cardiac ischaemia does not elicit an efficient angiogenic response. Indeed, lack of surgical revascularization upon myocardial infarction results in cardiomyocyte death, scarring, and loss of contractile function. Clinical trials aimed at inducing therapeutic revascularization through the delivery of pro-angiogenic molecules after cardiac ischaemia have invariably failed, suggesting that endothelial cells in the heart cannot mount an efficient angiogenic response. To understand why the heart is a poorly angiogenic environment, here we compare the angiogenic response of the cardiac and skeletal muscle using a lineage tracing approach to genetically label sprouting endothelial cells. Methods and results We observed that overexpression of the vascular endothelial growth factor in the skeletal muscle potently stimulated angiogenesis, resulting in the formation of a massive number of new capillaries and arterioles. In contrast, response to the same dose of the same factor in the heart was blunted and consisted in a modest increase in the number of new arterioles. By using Apelin-CreER mice to genetically label sprouting endothelial cells we observed that different pro-angiogenic stimuli activated Apelin expression in both muscle types to a similar extent, however, only in the skeletal muscle, these cells were able to sprout, form elongated vascular tubes activating Notch signalling, and became incorporated into arteries. In the heart, Apelin-positive cells transiently persisted and failed to give rise to new vessels. When we implanted cancer cells in different organs, the abortive angiogenic response in the heart resulted in a reduced expansion of the tumour mass. Conclusion Our genetic lineage tracing indicates that cardiac endothelial cells activate Apelin expression in response to pro-angiogenic stimuli but, different from those of the skeletal muscle, fail to proliferate and form mature and structured vessels. The poor angiogenic potential of the heart is associated with reduced tumour angiogenesis and growth of cancer cells.

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With the increased prevalence of chronic diseases, non-healing wounds place a significant burden on the health system and the quality of life of affected patients. Non-healing wounds are full-thickness skin lesions that persist for months or years. While several factors contribute to their pathogenesis, all non-healing wounds consistently demonstrate inadequate vascularization, resulting in the poor supply of oxygen, nutrients, and growth factors at the level of the lesion. Most existing therapies rely on the use of dermal substitutes, which help the re-epithelialization of the lesion by mimicking a pro-regenerative extracellular matrix. However, in most patients, this approach is not efficient, as non-healing wounds principally affect individuals afflicted with vascular disorders, such as peripheral artery disease and/or diabetes. Over the last 25 years, innovative therapies have been proposed with the aim of fostering the regenerative potential of multiple immune cell types. This can be achieved by promoting cell mobilization into the circulation, their recruitment to the wound site, modulation of their local activity, or their direct injection into the wound. In this review, we summarize preclinical and clinical studies that have explored the potential of various populations of immune cells to promote skin regeneration in non-healing wounds and critically discuss the current limitations that prevent the adoption of these therapies in the clinics.

Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): AIRC Introduction Adult mammals fail to regenerate the myocardium after ischemic injury (1). Different strategies to achieve myocardial regeneration have been considered including reactivation of progenitor cell populations, cell replacement therapies, cell reprograming and molecules able to stimulate cardiomyocyte proliferation (2). None of these approaches has been so far successful. The supply of nutrients and oxygen to the myocardium after the occlusion of a coronary artery depends on pre-existing and newly formed collateral vessels (3). The relevance of revascularization in the context of cardiac regeneration is suggested by the evidence that angiogenesis precedes cardiomyocyte proliferation in a model of neonatal heart injury (4). Our laboratory has recently demonstrated that the heart has a low angiogenetic potential (5). To what extent this is responsible for the poor regenerative capacity of the heart remains to be determined. Thus, understanding the mechanisms blocking angiogenesis in the adult heart could lead to the development of efficient strategies to promote cardiac revascularization and regeneration. Purpose Our purpose is to understand the mechanisms responsible for the low angiogenic potential of the adult mammalian heart. In particular, we designed a proteomic approach to detect differences in the composition of the perivascular extracellular matrix between the neonatal (angiogenic) with and the adult (not angiogenic) heart. Methods and Results We adopted an in vivo biotinylation strategy to label vascular extracellular proteins in vivo, followed by protein identification by mass spectrometry. A reactive derivative of biotin was systemically injected in both neonatal and adult mice (n=4) to label vascular and peri-vascular extracellular proteins. Biotinylated proteins were purified from total hearts by streptavidin-conjugated beads, eluted, and digested with trypsin. The resulting peptides were analyzed by LC-MS/MS. Among the differentially expressed proteins, we identified lumican and its receptor, integrin beta-1. This ligand-receptor pair is known to impair tube formation by endothelial cells and to interfere with both expression and activity of matrix metalloproteinases (MMPs) in vitro (6). Western blot and gene expression analysis confirmed up-regulation of lumican in the adult compared to neonatal heart, as well as a different glycosylation pattern. Lumican knock-out both ex vivo and in vivo resulted in increased vessel density. On the other hand, overexpression of lumican impaired the proliferative capacity of cardiac endothelial cells. Finally, MMP-14 activity was inhibited by adult, but not neonatal cardiac extract. Conclusions In vivo proteomic analysis identified lumican as a major contributor of the low angiogenic potential of the adult mammalian heart. These results point to lumican silencing as a promising strategy to achieve cardiac revascularization.