α(1)-Antitrypsin (AAT) is a 52-kDa circulating serine protease inhibitor. Production of AAT by the liver maintains 0.9-1.75 mg/mL circulating levels. During acute-phase responses, circulating AAT levels increase more than fourfold. In individuals with one of several inherited mutations in AAT, low circulating levels increase the risk for lung, liver and pancreatic destructive diseases, particularly emphysema. These individuals are treated with lifelong weekly infusions of human plasma-derived AAT. An increasing amount of evidence appears to suggest that AAT possesses not only the ability to inhibit serine proteases, such as elastase and proteinase-3 (PR-3), but also to exert antiinflammatory and tissue-protective effects independent of protease inhibition. AAT modifies dendritic cell maturation and promotes T regulatory cell differentiation, induces interleukin (IL)-1 receptor antagonist and IL-10 release, protects various cell types from cell death, inhibits caspases-1 and -3 activity and inhibits IL-1 production and activity. Importantly, unlike classic immunosuppressants, AAT allows undeterred isolated T-lymphocyte responses. On the basis of preclinical and clinical studies, AAT therapy for nondeficient individuals may interfere with disease progression in type 1 and type 2 diabetes, acute myocardial infarction, rheumatoid arthritis, inflammatory bowel disease, cystic fibrosis, transplant rejection, graft versus host disease and multiple sclerosis. AAT also appears to be antibacterial and an inhibitor of viral infections, such as influenza and human immunodeficiency virus (HIV), and is currently evaluated in clinical trials for type 1 diabetes, cystic fibrosis and graft versus host disease. Thus, AAT therapy appears to have advanced from replacement therapy, to a safe and potential treatment for a broad spectrum of inflammatory and immune-mediated diseases.

Antiinflammatory clinical-grade, plasma-derived human α-1 antitrypsin (hAAT) protects islets from allorejection as well as from autoimmune destruction. hAAT also interferes with disease progression in experimental autoimmune encephalomyelitis (EAE) and in collagen-induced arthritis (CIA) mouse models. hAAT increases IL-1 receptor antagonist expression in human mononuclear cells and T-regulatory (Treg) cell population size in animal models. Clinical-grade hAAT contains plasma impurities, multiple hAAT isoforms and various states of inactive hAAT. We thus wished to establish islet-protective activities and effect on Treg cells of plasmid-derived circulating hAAT in whole animals. Islet function was assessed in mice that received allogeneic islet transplants after mice were given hydrodynamic tail-vein injection with pEF-hAAT, a previously described Epstein-Barr virus (EBV) plasmid construct containing the EBV nuclear antigen 1 (EBNA1) and the family of repeat EBNA1 binding site components (designated “EF”) alongside the hAAT gene. Sera collected from hAAT-expressing mice were added to lipopolysaccharide (LPS)-stimulated macrophages to assess macrophage responsiveness. Also, maturation of peritoneal cells from hAAT-expressing mice was evaluated. hAAT-expressing mice accepted islet allografts (n = 11), whereas phosphate-buffered saline-injected animals (n = 11), as well as mice treated with truncated-hAAT-plasmid (n = 6) and untreated animals (n = 20) rapidly rejected islet allografts. In hAAT-expressing animals, local Treg cells were abundant at graft sites, and the IL-1 receptor antagonist was elevated in grafts and circulation. Sera from hAAT-expressing mice, but not control mice, inhibited macrophage responses. Finally, peritoneal cells from hAAT-expressing mice exhibited a semimature phenotype. We conclude that plasmid-derived circulating hAAT protects islet allografts from acute rejection, and human plasma impurities are unrelated to islet protection. Future studies may use this in vivo approach to examine the structure-function characteristics of the protective activities of AAT by manipulation of the hAAT plasmid.

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.