The endothelial surface layer in diabetic microangiopathy
Lead Research Organisation:
University of Bristol
Department Name: Physiology and Pharmacology
Abstract
Small blood vessel (microvessel) changes in diabetes cause excessive leakiness, resulting in kidney, eye and nerve disease. Identifying leak-inducing changes in microvessel structures may lead to new treatments. Changes in the carpet-like layer of sugar-coated proteins that forms the innermost lining of all blood vessel walls (endothelial surface layer:ESL) may cause this leak, since the ESL regulates leakiness, and is thinner in diabetes. I will induce diabetes (type 1 diabetes) in rats, and examine kidney and gut microvessels (much current ESL knowledge comes from gut microvessels, because extremely precise measurements of microvessel structure and function can be made).
I will use state-of-the-art technology to measure [1]microvessel leakiness [2]ESL depth and [3]ESL composition in the same blood vessel, and compare changes with/without diabetes, and between kidney and gut microvessels. I intend to identify the precise ESL changes in diabetes, show that these are crucial in microvessel leakiness, and then use specific experimental agents to reverse the ESL changes and thereby restore normal microvessel function: this could help develop new treatments. The work will also allow me to establish highly informative, unique techniques for future investigations into why individuals with leaky kidney vessels are prone to other diseases e.g. heart disease.
I will use state-of-the-art technology to measure [1]microvessel leakiness [2]ESL depth and [3]ESL composition in the same blood vessel, and compare changes with/without diabetes, and between kidney and gut microvessels. I intend to identify the precise ESL changes in diabetes, show that these are crucial in microvessel leakiness, and then use specific experimental agents to reverse the ESL changes and thereby restore normal microvessel function: this could help develop new treatments. The work will also allow me to establish highly informative, unique techniques for future investigations into why individuals with leaky kidney vessels are prone to other diseases e.g. heart disease.
Technical Summary
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.
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.