Development and repair of blood vessels in the zebrafish spinal cord.

The vascular system is inefficiently repaired after spinal cord injury (SCI) in mammals, resulting in secondary tissue damage and immune deregulation that contribute to the limited functional recovery. Unlike mammals, zebrafish can repair the spinal cord (SC) and restore motility, but the vascular response to injury has not been investigated. Here, we describe the zebrafish SC blood vasculature, starting in development with the initial vessel ingression in a body size-dependent manner, the acquisition of perivascular support and the establishment of ventral to dorsal blood circulation. The vascular organization grows in complexity and displays multiple barrier specializations in adulthood. After injury, vessels rapidly regrow into the lesion, preceding the glial bridge and axons. Vascular repair involves an early burst of angiogenesis that creates dysmorphic and leaky vessels. Dysfunctional vessels are later removed, as pericytes are recruited and the blood-SC barrier is re-established. This study demonstrates that zebrafish can successfully re-vascularize the spinal tissue, reinforcing the value of this organism as a regenerative model for SCI.

Low Doses of Ionizing Radiation Enhance the Angiogenic Potential of Adipocyte Conditioned Medium.

At low doses, ionizing radiation activates endothelial cells and promotes angiogenesis. However, it is still unknown if other cells may contribute to this process. In this study, the effect of low-dose ionizing radiation (LDIR) in modulating the pro-angiogenic potential of adipocytes was investigated. Adipocytes are known to secrete multiple angiogenic factors and adipokines that induce angiogenesis. In this work, a confluent monolayer of 3T3-L1 pre-adipocytes was exposed to low doses (0.1 and 0.3 Gy) and to higher doses (0.5, 0.8 and 1.0 Gy), as control. Our data show that the adipocyte-conditioned media (A-CM) from mature adipocytes differentiated from low-dose irradiated pre-adipocytes presented a higher angiogenic potential, compared to mature adipocytes differentiated from sham-irradiated control preadipocytes. The vascular endothelial growth factor (VEGF)-A levels were significantly increased in A-CM from the 0.1 Gy (P < 0.05) and 0.3 Gy (P < 0.01) experimental conditions and a significant increase was found in response to 0.3 Gy dose of radiation for VEGF-C, angiopoietin-2 (ANG-2) and hepatocyte growth factor (HGF). Moreover, 0.3 Gy dose of radiation significantly increased the expression of matrix metalloproteinase (MMP)-2 active forms. In vitro, the A-CM from the 0.1 and 0.3 Gy doses experimental conditions significantly accelerated endothelial cell migration after an in vitro wound healing assay. Importantly, in vivo, the A-CM corresponding to the 0.3 Gy experimental condition significantly induced the growth of more blood vessels towards the inoculation area in the chick embryo chorioallantoic membrane (CAM). In conclusion, this work reveals a new mechanism by which low-dose radiation might promote angiogenesis, enhancing the angiogenic potential of A-CM.

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia.

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.