Title: The endothelial surface layer in diabetic microangiopathy
Funder: Medical Research Council

Abstract: Microvascular disease in type 1 diabetes mellitus (diabetic microangiopathy) causes the same phenotype (excessive leak) in diverse organs (e.g. kidney, retina), and therefore a common underlying defect has been proposed. Capillary hypertension and altered microvessel barrier properties (permeability coefficients) probably both contribute to excessive leak, but previous studies have not distinguished between these possibilities. The endothelial surface layer (ESL), a 0.5-1 m thick extracellular matrix of proteoglycans, glycosaminoglycans and adsorbed plasma proteins that - lines the luminal aspect of all endothelial cells - determines permeability coefficients in structurally diverse microvessels - is altered in individuals with type 1 diabetes and microalbuminuria. I hypothesise that altered ESL in diabetes contributes to altered microvascular permeability coefficients and increased vascular leak in diabetic microangiopathy. Aim 1: Measure permeability coefficients and ESL ultrastructure (electron microscopy) in streptozotocin-induced diabetic (and control) rats [a] before hyperglycaemia, [b] during hyperglycaemia but before albuminuria [c] during albuminuria, and [d] during frank diabetic nephropathy. I will measure microvascular permeability coefficients directly, using single microvessel cannulation studies of intact mesenteric microvessels in vivo and oncometric studies of glomerular microvessels ex vivo. This time-course work will identify whether changes in the ESL (and/or other structures) occur in tandem with altered permeability. Aim 2: Extend our current perfused microvessel assays to measure solute permeability coefficients and ESL depth simultaneously, using confocal microscopy. Mesenteric studies will be performed in the host laboratory; glomerular perfusion studies will be performed with Prof. Peti-Peterdi before translating the assay to the UK. Aim 3: Characterise the structural and molecular changes that occur in the ESL in diabetes using detailed analysis of electron micrographs, as well as lectin and antibody binding studies. I will also characterise the biochemical changes in proteoglycans using HPLC. Aim 4: Apply highly-selective enzymes and glycosaminoglycan synthesis inhibitors to reproduce diabetes-induced ESL changes, and assess whether the diabetes-induced permeability changes are reproduced. Aim 5: Assess whether restoration of the ESL, by replenishing glycosaminoglycans or applying angiopoietin-1, restores normal permeability in in diabetic microvessels. Overall, these studies will identify [1] how different permeability coefficients are altered in diabetic microangiopathy [2] whether alterations in the ESL are crucial determinants of the functional changes observed in diabetic microangiopathy, and [3] the nature of these ESL alterations. Understanding the precise, functionally important defects in diabetic microangiopathy will allow me to test whether restoring the ESL can restore normal microvascular permeability in diabetic microangiopathy, which may have therapeutic potential.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: METABOLIC AND ENDOCRINE;CARDIOVASCULAR