Angiogenesis, the sprouting of blood vessels from preexisting vasculature is associated with both natural and pathological processes. Various angiogenesis assays involve the study of individual endothelial cells in culture conditions (1). The aortic ring assay is an angiogenesis model that is based on organ culture. In this assay, angiogenic vessels grow from a segment of the aorta (modified from (2)). Briefly, mouse thoracic aorta is excised, the fat layer and adventitia are removed, and rings approximately 1 mm in length are prepared. Individual rings are then embedded in a small solid dome of basement matrix extract (BME), cast inside individual wells of a 48-well plate. Angiogenic factors and inhibitors of angiogenesis can be directly added to the rings, and a mixed co-culture of aortic rings and other cell types can be employed for the study of paracrine angiogenic effects. Sprouting is observed by inspection under a stereomicroscope over a period of 6-12 days. Due to the large variation caused by the irregularities in the aortic segments, experimentation in 6-plicates is strongly advised. Neovessel outgrowth is monitored throughout the experiment and imaged using phase microscopy, and supernatants are collected for measurement of relevant angiogenic and anti-angiogenic factors, cell death markers and nitrite.

Pancreatic islets are a highly vascularized entity, and their transplantation into diabetic individuals requires optimal revascularization. In addition, β-cells in islets are extremely sensitive to inflammation. α-1-Antitrypsin (AAT), a circulating serine-protease inhibitor that is available for clinical use as an affinity-purified human product, has been shown to protect islets from graft failure in mouse transplantation models and to achieve readily vascularized islet grafts. AAT is known to induce vascular endothelial growth factor (VEGF) expression and release, as well as protect from proteolytic cleavage of VEGF by elastase, promote viability of endothelial cells, and enhance migration of myocytes. Our aim was to examine whether AAT enhances vasculogenesis toward islet grafts. We employed Matrigel-islet plugs as means to introduce islets in an explantable isolated compartment and examined vessel formation, vessel maturation, and inflammatory profile of explants 9 days after implantation. Also, we examined primary epithelial cell grafts that were prepared from lungs of mice that are transgenic for human AAT. In addition, aortic ring sprouting assay was performed, and HUVEC tube formation assays were studied in the presence of AAT. Our findings indicate that islet grafts exhibit mature vessels in the presence of AAT, as demonstrated by morphology, as well as expression of endothelial CD31, smooth muscle actin (SMA), and von Willebrand factor (vWF). Epithelial cells that express human AAT achieved a similar positive outcome. Aortic ring sprouting was enhanced in AAT-treated cultures and also in cultures that contained primary epithelial cells from human AAT transgenic animals in the absence of added AAT. According to the tube formation assay, HUVECs exhibited superior responses in the presence of AAT. We conclude that vasculogenesis toward islet grafts is enhanced in the presence of AAT. Together with the remarkable safety profile of AAT, the study supports its use in the relevant clinical setups.

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Alpha-1-antitrypsin (AAT) exerts anti-inflammatory and tolerogenic activities during islet allograft transplantation in diabetes mouse models. Although a serine protease inhibitor, evidence suggests that AAT possesses activities that are independent of protease inhibition. The previously reported plasmid, pEF-hAAT, contains the genomic sequence of human AAT (hAAT) and sustains circulating levels after single hydrodynamic tail-vein injection (HDI) in mice. To asses whether expression of hAAT by pEF-hAAT protects islets from acute rejection, pEF-hAAT (100 μg, n = 6) or PBS (n = 4, control) were hydrodynamically introduced to mice. Liver expression and circulating hAAT levels were determined. Supernatant of transfected Hepa1c cells exhibited anti-elastase activity. Sixteen days post HDI mice were injected streptozotocin (225 mg/kg) and grafted with 450 allogenic islets. Circulating hAAT levels (1-350 μg/ml) persisted for over 30 days. Normoglycemia was achieved and islets were accepted in 6 out of 6 recipients that expressed hAAT. All 4 control animals exhibited acute islet allograft rejection by day 12. We conclude that plasmid-derived long-term expression of as low as 1 μg/ml hAAT exhibits islet-allograft protection. Using site-directed mutagenesis of pEF-hAAT we will examine whether activities that are independent of protease inhibition partake in the protection of islet allografts.