The growth factor effect on endothelial cell dysfunction in the presence of glycated collagen and A[beta] peptide: implications for decreased angiogenesis in diabetes and Alzheimer's disease

Justin George Mathew

doi:10.17918/etd-4555

Reduced angiogenesis, the growth of new blood vessels from existing vessels, is important in disease states including diabetes and Alzheimer's disease. People with diabetes experience morbidity and mortality from unregulated micro-vascular remodeling, which may be linked to hyperglycemia. People with Alzheimer's disease experience diminished memory and motor function from a buildup of A[beta] plaques and a reduction in microvascular density. The primary focus of this research is to determine if growth factors can rescue endothelial cell angiogenic processes from altered extracellular proteins, specifically glycated collagen and A[beta] peptide. Assays were developed to discover how these altered extracellular proteins affect growth factor induced cell plasminogen system activity. The resultant data would then add additional insight into growth factor therapy for patients with diabetes and Alzheimer's disease. Endothelial cells on glycated collagen displayed decreased plasminogen system activity, which impacted angiogenic functions such as tube formation and 3D migration. FGF-2 did not fully rescue the negative effects of glycated collagen. uPA, uPAR and PAI-1 protein levels did not significantly change at short time points. However, PAI-1 bound to glycated collagen at higher levels than native collagen. Since even cells on native collagen did not show a short term response in uPA, PAI-1, or uPAR protein levels in response to FGF-2 stimulation, FGF-2 effects on vitronectin were explored. FGF-2 increased cell-associated vitronectin by 24 hours in varied endothelial cell types and vascular smooth muscle cells. xv The vitronectin appeared to be taken up on from the serum and remained on the endothelial cell membrane, as shown by immunofluorescence microscopy with and without permeabilizing cells. FGF-2 signaled intracellularly to activate the [alpha]v[beta]5 integrin, a vitronectin binding site. Blocking focal adhesion kinase phosphorylation or the [alpha]v[beta]5 integrin, or knocking down the [beta]5 integrin with siRNA, abrogated this FGF-2 induced effect. Since FGF-2 had limited influence on cells on glycated collagen, we then examined if a similar effect would be seen in the presence of A[beta]42. Endothelial cells in the presence of A[beta]42 did not alter plasminogen system activity; however specific secondary structure of the peptide attenuated growth factor induced effects. A[beta]42 oligomers were non-toxic and did not affect FGF-2 or VEGF induced cell proliferation, ERK phosphorylation, 3D migration and had limited effect on endothelial cell tube formation. Monomeric A[beta]42 impacted VEGF effects on cell proliferation and tube length, although with limited influence on plasmin activity. These data show that endothelial cell response to growth factors is dependent on the protein interactions of PAI-1 with glycated collagen, vitronectin with [alpha]v[beta]5 and A[beta]42 secondary structure. Demonstrating a new way in which growth factors impacts the plasminogen system and subsequent angiogenesis is a fundamental step in growth factor therapy. This research highlights the use of growth factory therapy in both diabetes and Alzheimer's patients where there is a reduction of angiogenic function.

The growth factor effect on endothelial cell dysfunction in the presence of glycated collagen and A[beta] peptide: implications for decreased angiogenesis in diabetes and Alzheimer's disease

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The growth factor effect on endothelial cell dysfunction in the presence of glycated collagen and A[beta] peptide: implications for decreased angiogenesis in diabetes and Alzheimer's disease

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