Abstract
Angiogenesis during wound healing is essential for tissue repair and also affected during cancer treatment by anti-angiogenic drugs. Here, we introduce a minimally invasive wound healing model in the mouse ear to assess angiogenesis with high spatiotemporal resolution in a longitudinal manner in vivo using two-photon microscopy in mice expressing GCaMP2 in arterial endothelial cells. The development of vascular sprouts occurred in a highly orchestrated manner within a time window of 8 days following wounding. Novel sprouts developed exclusively from the distal stump of the transsected arteries, growing towards the proximal arterial stump. This was in line with the incidence of Ca2+ transients in the arterial endothelial cells, most probably a result of VEGF stimulation, which were more numerous on the distal part. Functional analysis revealed perfusion across the wound site via arterial sprouts developed between days 6 and 8 following the incision. At day 8, proximal and distal arteries were structurally and functionally connected, though only 2/3 of all sprouts detected were actually perfused. Treatment with the FDA approved drug, sunitinib, the preclinical drug AZD4547, as well as with the combination of the two agents had significant effects on both structural and functional readouts of neo-angiogenesis. The simplicity and high reproducibility of the model makes it an attractive tool for elucidating migratory activity, phenotype and functionality of endothelial cells during angiogenesis and for evaluating specific anti-angiogenic drug interventions.
Highlights
Proper function of cells and tissues in the body depend on sufficient supply by oxygen and nutrients via blood supply, i.e. on an efficient vascular system
We have introduced a minimally invasive wound healing model in the mouse ear to assess angiogenesis with high spatiotemporal resolution in a longitudinal manner in vivo using two-photon microscopy
The high spatiotemporal resolution achieved by intra-vital microscopy provided detailed insights into the process of vessel formation
Summary
Proper function of cells and tissues in the body depend on sufficient supply by oxygen and nutrients via blood supply, i.e. on an efficient vascular system. Since many other inhibitors of angiogenesis such as sunitinib, an inhibitor of the vascular endothelial growth factor receptor 1 and 2 (VEGFR-1, VEGFR-2), fetal liver tyrosine kinase receptor 3 (FLT3), KIT (stem-cell factor receptor), and platelet derived growth factor α and β (PDGFRα, and PDGFRβ) have been approved by the regulatory authorities for treatment of various cancers[3,4]. These new anti-angiogenic drugs have shown some beneficial impact on the survival outcome, but did not fulfil the high expectations so far. We analyzed morphological and functional changes during the healing process and studied its modulation using two anti-angiogenic drugs, sunitinib and AZD4547, both individually and as a combination therapy
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