Variation in Glycocalyx Structure in Diabetes

Diabetic Retinopathy and Diabetic Nephropathy cause blindness and kidney failure associated by leaky blood vessels. The proposed research is focused on the endothelial glycocalyx. This is a specialised layer that covers blood vessel walls and acts as a molecular filter so the correct molecules transverse blood vessel walls. In normal physiology this filter is a layer of fibres spaced regularly at 20nm apart. The systemic disruption of this layer is well established in diabetes but what the disruption is, and what mechanism causes it, is totally unknown.
My overarching aim is to determine the basis of the filtration functionality by understanding the glycocalyx fibre organisational structure and how this is changed in diabetic model systems. The proposal will use state-of-the-art three dimensional, structural, and analytical electron microscopy techniques to determine structure whilst systematically altering the constituent components and functionality (permeability) of the glycocalyx. This will be achieved as follows:

Aim 1): Determine the glycocalyx macro structure by selective stain and stain-free imaging.
I will determine the components and location of the glycocalyx fibres using in vitro culture of human cells as an easily manipulated test environment. Fibres will be imaged using predominantly metal-based optical histology stains that are observable using world-class electron microscopy. To unpick the glycocalyx complexity and identify and locate specific organisational components I will target known constituents of the fibres (glycocsaminoglycans) using a validated shRNA approach, followed by functional and structural characterisation using image analysis quantification. Subsequently, I will use a combination stains and modern stain free techniques to determine the fibre structure chemically and any parts that were previously impossible to observe. The outcome from this aim will be a detailed understanding of the major structural components of the glycocalyx that I can then go on to study in the disease state.

Aim 2): Determine the structural aspect of glycocalyx dysfunction in diabetic retinopathy and nephropathy.
I will again take advantage of the controlled conditions of in vitro cell culture from human retinal and renal endothelial cell lines to image and probe the structure of the glycocalyx built by cells (eye or kidney) in normal or hyperglycaemic environments. Recognising that in vitro culture can produce artefacts, findings will be verified by conducting a targeted set of imaging studies on biopsy material.

This project will, for the first time, link structural changes in the glycocalyx to the early stages of blindness and kidney disease in diabetes. Improved structural knowledge will allow us to hypothesise and test the underlying mechanisms behind glycocalyx failure. This will pave the way for future work , providing novel druggable targets, with an aim of alleviating disease by fixing, altering or circumnavigating the biochemical mechanism.

My broad hypothesis is "Diabetic induced hyperglycaemia causes alterations to the glycocalyx marcostructure resulting in the observed increase in permeability" of the vasculature. To test this a system must be used that can determine the macrostucture, the structure that determines the permeability. Electron microscopy is a system is able to determine the glycocalyx fibre parameters of permeability (Depth, Diameter, spacing, lattice and overall coverage) in physiological cases. I propose to use glycocalyx generated by cultured endothelial cells (human choroid, human retinal, human glomerular and mouse bEND5) to observe the structure under a variety of electron microscopy stains (Alcian Blue, Ruthenium Red, Lathanum and Dysprosium) that target differing elements. The permeability will be inferred form an electrical cell impendence system. Moreover fenestrations, the primary hydraulic route if present, will be induced by VEGF-A or latrunculin A. These systems will be compared to cells grown in normal or hyperglycaemic conditions.
A key component of the glycocalyx that has been implicated in hyperglycaemic loss of function is the glycosaminoglycan heparan sulphate (HS). The role of HS will be uncovered using shRNA for components of the HS biosynthetic pathway together with characterisation of the resulting shifts in sulphation patterning and HS distribution - linking these to function in the in vitro model. The positioning of elemental components will then be detected using altered staining and cryo-analytical electron microscopy. Following validation of the relationship between cryo-analytical EM/altered staining and HS composition in the in vitro setting, the protocols will move to physiological models of diabetic retinopathy and nephropathy where the permeability of the vasculature and the quantitative glycocalyx structure are compared to the structure with specific components missing to compare phenology via quantification of the permeability parameters.

Diabetic complications cause early blindness and kidney failure. The research will determine a mechanism for the leaky blood vessels that are systematic with the complications enabling a new area of focused research to prevent the debilitating and life threatening complications. This will be achieved by high impact and technical publications initiating an area of mechanistic research.
The fundamental aspect of this work will benefit the greater cardiovascular community due to understanding of permeability being a point of focus in, amongst other things, cancer, sepsis and perioperative fluid management. Clinical research, particularly on the eyes and systemic glycocalyx is available with non-invasive human methodologies. The understanding of the significance of this data taken is limited by structural information. Therefore as soon as any structural information is publicised the clinical researchers can include the context in a medical environment.


Polysaccharide imaging is in its infancy. This project will bring it up to date with modern capabilities at a world leading level. It is expected that the novel electron microscopy will make strides in medical research impact. The showcasing of the work at selected conferences in fields other than diabetes is important to create a synergy between related fields. Laboratory exchange visits are a likely outcome.

On a national level workshop seminars will be held at the University of Nottingham to bring together clinicians and basic scientists across the glycocalyx field in order to focus and create efficiency in future research. The protocols developed and shared by the electron microscopy community will enable regional centres of excellence with specific niches, as recommended by BioImaging UK.

The future legacy of the project will also depend in the development of student scientists that touch upon this project in the group and the laboratory. To achieve a full interdisciplinary impact this will include physical scientists.

The social impact of research must also be considered and to do this a meeting with a diabetic patient group in Nottingham will be planned.