Visualizing Endothelial Cells And Pericytes During Cutaneous Wound Angiogenesis In Living Adult Zebrafish.
chikage noishiki, M.D.1, Shinya Yuge, Ph.D.1, Koji Ando, Ph.D.2, Yuki Wakayama, Ph.D.2, Naoki Mochizuki, M.D., Ph.D2, Rei Ogawa, M.D., Ph.D., F.A.C.S.1, Shigetomo Fukuhara, Ph.D.1.
1nippon medical school, tokyo, Japan, 2National Cerebral and Cardiovascular Center Research Institute, Osaka, Japan.
PURPOSE: Cutaneous wound healing is a complex and dynamic process by which skin tissue repairs itself after injury. Angiogenesis, the growth of new blood vessels from pre-existing vessels, is critical for wound healing. Here, we aimed to elucidate how endothelial cells (ECs) and pericytes (PCs) establish neovascular networks during cutaneous wound angiogenesis.
METHODS: To this end, we set up a fluorescence-based live-imaging system for adult zebrafish and visualized ECs and PCs during cutaneous wound healing in living animals.
RESULTS: In normal skin tissues, ECs and PCs remained dormant to maintain quiescent vessels, whereas cutaneous injury immediately induced angiogenesis through VEGF signaling pathway. At 2 days post-injury (dpi), the injured vessels elongated, and some uninjured vessels became tortuous and began to sprout new branches. Then, vessel sprouting, elongation, bifurcation, and anastomosis were progressively induced to form the tortuous and disorganized vascular networks at 6 dpi. Thereafter, tortuosity of the blood vessels gradually decreased through regression of excessive vessels, thereby leading to formation of well-organized vessel networks at 42 dpi. These findings indicate that cutaneous wounding immediately induces angiogenesis to form dense and disorganized vascular networks within a few weeks, and subsequently those vessels become normalized through regression of excess vessels by 1−2 months. To investigate how ECs and PCs establish neovessels during cutaneous angiogenesis, we injured a single capillary in the skin and monitored subsequent vessel repair (Figure 1). Injured vessels immediately elongated upon injury and anastomosed with each other at 2−3 dpi. Subsequently, the ECs and PCs in the repaired vessels further increased, thereby forming the PC-covered tortuous vessels at 7 dpi. Thereafter, they became gradually normalized by decreasing the number of ECs and PCs for several months. Similarly, the ECs and PCs in the adjacent uninjured vessels increased to form the PC-covered tortuous vessels, which were subsequently normalized through decreasing the number of ECs and PCs. These findings reveal that both ECs and PCs increase to establish the PC-covered tortuous vessels at the early stage of cutaneous angiogenesis and decrease for their normalization at the late stage. This result conflicts with the current concept that induction of angiogenesis induces PC detachment from the vessel wall to facilitate EC sprouting. Therefore, we analyzed the relationship between the site of EC sprouting and the position of PCs, and found that ECs sprouted from the vessels regardless of the position of PCs. These findings indicate that detachment of PCs from the vessel wall is not essential for EC sprouting during cutaneous angiogenesis.
CONCLUSION: We show how ECs and PCs establish neovascular networks during cutaneous angiogenesis. Importantly, we found that PCs proliferate and migrate to cover the tortuous vessels upon induction of angiogenesis, revealing an unexpected role of PCs in cutaneous wound angiogenesis. Therefore, this live-imaging system for adult zebrafish will be a valuable contribution to advance the field of wound healing research.
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