Supramolecular organization, spatial orientation and optical properties of extracellular proteins in corneas and aortas from diabetic spontaneous mice / Organização supramolecular, orientação espacial e propriedades opticas de proteinas extracelulares em conrneas e aortas de camundongos espontaneamente diabeticos

Marcela Aldrovani Rodrigues

Diabetes mellitus is a chronic disorder of the carbohydrate, lipids and protein metabolism. A characteristic feature of this disorder is the hyperglycemia due to a deficient insulin production or to metabolic defects on its response in peripheral tissues. Hyperglycemia induces non-enzymatic glycosylation of proteins, which is responsible for advanced glycosylation end-products (AGEs), and intermolecular crosslinks reactions. In this study, supramolecular organization, spatial orientation and optical properties were investigated in extracellular proteins of the cornea and of the abdominal aorta from nonobese diabetic (NOD) mice and non-diabetic mice (BALB/c). CORNEAS: Birefringence and linear dichroism were investigated in corneal stroma collagen fibers from nonobese NOD/Uni mice and BALB/c/Uni mice. The contribution of stroma proteoglycans to optical anisotropies of the cornea was also investigated in NOD and BALB/c mice. Birefringence optical retardations were measured in stroma collagen fibers of unstained whole and sectioned (8 µm) corneas. Linear dichroism and dichroic ratios were investigated in cornea sections stained with ponceau SS pH 2.5 and toluidine blue pH 4.0, using scanning microspectrophotometer and different wavelengths that were obtained with a monochromator filter ruler. Morphological analysis of the birefringences revealed that NOD and BALB/c stroma collagen fibers are intercrossed in different spatial planes and oriented in more that one direction along the corneal trajectories. NOD corneas showed higher birefringence optical retardation values than the control. Ponceau SS-complexed collagen fibers showed positive linear dichroism and dichroic ratios values that were higher for NOD corneas. After staining with toluidine blue, NOD and BALB/c corneas showed metachromatic reaction verifying the presence of anionic groups (proteoglycan glycosaminoglycans) in the stroma, and negative linear dichroism values. No significant difference was observed between NOD and BALB/c corneas stained with toluidine blue. These results demonstrate that diabetes was capable of altering the optical anisotropies of the cornea, possibly to increase the number of intermolecular crosslinks in stroma collagen fibers, making them more crystalline and aggregate than the control. However, diabetes did not affect the spatial organization of the stroma proteoglycans. Nonenzymatic glycosylation was assessed in aorta extracellular matrix from NOD/Uni mice, using nitroblue tetrazolium (NBT). Molecular and structural changes were investigated in elastic lamellae and collagen fibers of diabetic mice aortas, after staining with dansyl chloride and anilinonaphthalene sulfonate (ANS). Alterations in arterial autofluorescence and birefringence of collagen fibers were investigated in unstained aortas. Smooth muscle cells proliferation was also investigated by confocal microscopy and image analysis, after Feulgen reaction. Assessment of nonenzymatic glycosylation demonstrated glycosylation products formation in the aorta extracellular matrix from NOD mice. Elastic lamellae and collagen fibers from diabetic aortas presented less intense fluorescence after staining with dansyl chloride and ANS when compared to controls. However unstained NOD aortas showed more intense autofluorescence when compared to controls. Birefringence analysis suggests alterations in the higher molecular packing of the arterial collagen fibers in diabetic aortas. In aortas stained by Feulgen reaction no evidence of smooth muscle cells proliferation was observed in diabetic aortas

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