Blood vessel networks expand in a 2-step process that begins with vessel sprouting and is followed by vessel anastomosis. Vessel sprouting is induced by chemotactic gradients of the vascular endothelial growth factor (VEGF), which stimulates tip cell protrusion. Yet it is not known which factors promote the fusion of neighboring tip cells to add new circuits to the existing vessel network. By combining the analysis of mouse mutants defective in macrophage development or VEGF signaling with live imaging in zebrafish, we now show that macrophages promote tip cell fusion downstream of VEGF-mediated tip cell induction. Macrophages therefore play a hitherto unidentified and unexpected role as vascular fusion cells. Moreover, we show that there are striking molecular similarities between the pro-angiogenic tissue macrophages essential for vascular development and those that promote the angiogenic switch in cancer, including the expression of the cell-surface proteins TIE2 and NRP1. Our findings suggest that tissue macrophages are a target for antiangiogenic therapies, but that they could equally well be exploited to stimulate tissue vascularization in ischemic disease.

The prospect of cell-based therapy in cardiovascular regenerative medicine comes a step closer with the demonstration that a peptide can stimulate a progenitor cell population in the adult heart to act as a source of new cardiomyocytes. The stem or progenitor cells, thought to be derived from the epicardium, are activated by the small actin monomer binding protein thymosin β4, which has previously been shown to restore vascular potential to adult epicardium-derived progenitor cells after injury. The discovery of a resident source of myocardial progenitors will stimulate a search for small molecules and other factors that promote optimal progenitor activation and replacement of destroyed myocardium. A significant bottleneck in cardiovascular regenerative medicine is the identification of a viable source of stem/progenitor cells that could contribute new muscle after ischaemic heart disease and acute myocardial infarction1. A therapeutic ideal—relative to cell transplantation—would be to stimulate a resident source, thus avoiding the caveats of limited graft survival, restricted homing to the site of injury and host immune rejection. Here we demonstrate in mice that the adult heart contains a resident stem or progenitor cell population, which has the potential to contribute bona fide terminally differentiated cardiomyocytes after myocardial infarction. We reveal a novel genetic label of the activated adult progenitors via re-expression of a key embryonic epicardial gene, Wilm’s tumour 1 (Wt1), through priming by thymosin β4, a peptide previously shown to restore vascular potential to adult epicardium-derived progenitor cells2 with injury. Cumulative evidence indicates an epicardial origin of the progenitor population, and embryonic reprogramming results in the mobilization of this population and concomitant differentiation to give rise to de novo cardiomyocytes. Cell transplantation confirmed a progenitor source and chromosome painting of labelled donor cells revealed transdifferentiation to a myocyte fate in the absence of cell fusion. Derived cardiomyocytes are shown here to structurally and functionally integrate with resident muscle; as such, stimulation of this adult progenitor pool represents a significant step towards resident-cell-based therapy in human ischaemic heart disease.

The lymphatic vasculature is a blind-ended network crucial for tissue-fluid homeostasis, immune surveillance and lipid absorption from the gut. Recent evidence has proposed an entirely venous-derived mammalian lymphatic system. By contrast, here we show that cardiac lymphatic vessels in mice have a heterogeneous cellular origin, whereby formation of at least part of the cardiac lymphatic network is independent of sprouting from veins. Multiple Cre–lox-based lineage tracing revealed a potential contribution from the putative haemogenic endothelium during development, and discrete lymphatic endothelial progenitor populations were confirmed by conditional knockout of Prox1 in Tie2⁺ and Vav1⁺ compartments. In the adult heart, myocardial infarction promoted a significant lymphangiogenic response, which was augmented by treatment with VEGF-C, resulting in improved cardiac function. These data prompt the re-evaluation of a century-long debate on the origin of lymphatic vessels and suggest that lymphangiogenesis may represent a therapeutic target to promote cardiac repair following injury.

Canonical roles for macrophages in mediating the fibrotic response after a heart attack include extracellular matrix turnover and activation of cardiac fibroblasts to initiate collagen deposition. Here we reveal that macrophages directly contribute collagen to the forming post-injury scar. Unbiased transcriptomics shows an upregulation of collagens in both zebrafish and mouse macrophages following heart injury. Adoptive transfer of macrophages, from either collagen-tagged zebrafish or adult mouse GFPtpz-collagen donors, enhances scar formation via cell autonomous production of collagen. In zebrafish, the majority of tagged collagen localises proximal to the injury, within the overlying epicardial region, suggesting a possible distinction between macrophage-deposited collagen and that predominantly laid-down by myofibroblasts. Macrophage-specific targeting of col4a3bpa and cognate col4a1 in zebrafish significantly reduces scarring in cryoinjured hosts. Our findings contrast with the current model of scarring, whereby collagen deposition is exclusively attributed to myofibroblasts, and implicate macrophages as direct contributors to fibrosis during heart repair.

Myocardial infarction (MI) arising from obstruction of the coronary circulation engenders massive cardiomyocyte loss and replacement by non-contractile scar tissue, leading to pathological remodeling, dysfunction, and ultimately heart failure. This is presently a global health problem for which there is no effective cure. Following MI, the innate immune system directs the phagocytosis of dead cell debris in an effort to stimulate cell repopulation and tissue renewal. In the mammalian adult heart, however, the persistent influx of immune cells, coupled with the lack of an inherent regenerative capacity, results in cardiac fibrosis. Here, we reveal that stimulation of cardiac lymphangiogenesis with VEGF-C improves clearance of the acute inflammatory response after MI by trafficking immune cells to draining mediastinal lymph nodes (MLNs) in a process dependent on lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1). Deletion of Lyve1 in mice, preventing docking and transit of leukocytes through the lymphatic endothelium, results in exacerbation of chronic inflammation and long-term deterioration of cardiac function. Our findings support targeting of the lymphatic/immune cell axis as a therapeutic paradigm to promote immune modulation and heart repair.

During development, the axons of retinal ganglion cell (RGC) neurons must decide whether to cross or avoid the midline at the optic chiasm to project to targets on both sides of the brain. By combining genetic analyses with in vitro assays, we show that neuropilin 1 (NRP1) promotes contralateral RGC projection in mammals. Unexpectedly, the NRP1 ligand involved is not an axon guidance cue of the class 3 semaphorin family, but VEGF164, the neuropilin-binding isoform of the classical vascular growth factor VEGF-A. VEGF164 is expressed at the chiasm midline and is required for normal contralateral growth in vivo. In outgrowth and growth cone turning assays, VEGF164 acts directly on NRP1-expressing contralateral RGCs to provide growth-promoting and chemoattractive signals. These findings have identified a permissive midline signal for axons at the chiasm midline and provide in vivo evidence that VEGF-A is an essential axon guidance cue.