FGF-2 overexpression opposes the beta amyloid toxic injuries to the vascular endothelium

Recent evidences suggest that Abeta peptides modulate endothelial cell (EC) functions. At low concentrations, Abeta1-40 enhances the pro-angiogenic activity of FGF-2, whereas deposition of excess Abeta causes EC dysfunction and cerebral amyloid angiopathy (CAA). We investigated whether FGF-2 attenuates EC dysfunction caused by pathological Abeta levels. We studied Abeta1-40 on EC survival, as well as on signals responsible of their angiogenic phenotype. At 5-50 microM Abeta1-40 reduced EC population, caused apoptosis, downregulated FGF-2 production, inhibited FGF-2 binding to heparin, and FGFR1 phosphorylation. Toxic effects were owing to lack of FGF-2 stimulation, as EC overexpressing FGF-2 displayed extraordinary resistance to Abeta1-40 injuries. The FGF-2 mechanism responsible for reversing damages, involves the downstream enhancement of Akt, a pathway independent of eNOS activation. In conclusion, we demonstrate that FGF-2 protects EC from the effects of excess Abeta1-40, suggesting that it may attenuate the consequences of Abeta deposition in pathologies as CAA.     

New in vitro model to study high glucose-dependent endothelial dysfunctions

Several thrombogenic abnormalities are associated with diabetes. Since endothelial dysfunction occurs at early stages of disease, it may reflect pathophysiological changes that are responsible for alterations in vascular structure, growth and modifications of adhesivity to platelets and leukocytes, leading to atherosclerosis and thrombosis. Predisposing factors of vascular diseases, such as diabetes, are also associated with endothelial dysfunction. Restoration or replacement of endothelium-related factors like nitric oxide impede the progression of vascular thrombogenic diseases, and prevent the action of vasoconstrictor factors such as endothelin or other prothrombotic factors such as plasminogen-activator inhibitor-1. Since high glucose concentration in blood is the hallmark of diabetes and because the vascular lesions of atherosclerosis are localized in large artheries, we have cultured endothelial cells from the human aorta. Two endothelial cell strains from the same aortic tract that show different characteristics and behavior in high glucose were isolated. Such findings reflect the importance to have well characterized and standardized cell culture systems to carry out experiments to study the glucose-dependent atherosclerotic process in vitro. Our cell strains may represent a useful in vitro model to study the complex pathophysiology of diabetes-related atherosclerosis.     

Secretion of Human Serum Albumin by Kluyveromyces lactis Overexpressing KlPDI1 and KlERO1

The control of protein conformation during translocation through the endoplasmic reticulum is often a bottleneck for heterologous protein production. The core pathway of the oxidative folding machinery includes two conserved proteins: Pdi1p and Ero1p. We increased the dosage of the genes encoding these proteins in the yeast Kluyveromyces lactis and evaluated the secretion of heterologous proteins. KlERO1, an orthologue of Saccharomyces cerevisiae ERO1, was cloned by functional complementation of the ts phenotype of an Scero1 mutant. The expression of KlERO1 was induced by treatment of the cells with dithiothreitol and by overexpression of human serum albumin (HSA), a disulfide bond-rich protein. Duplication of either PDI1 or ERO1 led to a similar increase in HSA yield. Duplication of both genes accelerated the secretion of HSA and improved cell growth rate and yield. Increasing the dosage of KlERO1 did not affect the production of human interleukin 1β, a protein that has no disulfide bridges. The results confirm that the ERO1 genes of S. cerevisiae and K. lactis are functionally similar even though portions of their coding sequence are quite different and the phenotypes of mutants overexpressing the genes differ. The marked effects of KlERO1 copy number on the expression of heterologous proteins with a high number of disulfide bridges suggests that control of KlERO1 and KlPDI1 is important for the production of high levels of heterologous proteins of this type.     

Endostatin: a promising drug for antiangiogenic therapy

Angiogenesis, the formation of new blood vessels from existing capillaries, is critical for tumors to grow beyond a few in size. Tumor cells produce one or more angiogenic factors including fibroblast growth factor and vascular endothelial growth factor. Surprisingly, antiangiogenic factors or angiogenesis inhibitors have been isolated from tumors. Some angiogenesis inhibitors, such as angiostatin, are associated with tumors while others, such as platelet-factor 4 and interferon-alpha are not. Endostatin, a C-terminal product of collagen XVIII, is a specific inhibitor of endothelial cell proliferation, migration and angiogenesis. The mechanism by which endostatin inhibits endothelial cell proliferation and migration is unknown. Endostatin was originally expressed in a prokaryotic system and, late, in a yeast system, thanks to which it is possible to obtain a sufficient quantity of the protein in a soluble and refolded form to be used in preclinical and clinical trials.     

T cell activity correlates with oligomeric peptide-major histocompatibility complex binding on T cell surface

Recognition of virally infected cells by CD8+ T cells requires differentiation between self and nonself peptide-class I major histocompatibility complexes (pMHC). Recognition of foreign pMHC by host T cells is a major factor in the rejection of transplanted organs from the same species (allotransplant) or different species (xenotransplant). AHIII12.2 is a murine T cell clone that recognizes the xenogeneic (human) class I MHC HLA-A2.1 molecule (A2) and the syngeneic murine class I MHC H-2 D(b) molecule (D(b)). Recognition of both A2 and D(b) are peptide-dependent, and the sequences of the peptides recognized have been determined. Alterations in the antigenic peptides bound to A2 cause large changes in AHIII12.2 T cell responsiveness. Crystal structures of three representative peptides (agonist, null, and antagonist) bound to A2 partially explain the changes in AHIII12.2 responsiveness. Using class I pMHC octamers, a strong correlation is seen between T cell activity and the affinity of pMHC complexes for the T cell receptor. However, contrary to previous studies, we see similar half-lives for the pMHC multimers bound to the AHIII12.2 cell surface.