A global approach to prevent secondary microvessel complications in diabetes

People with diabetes have high blood sugar. This can act like a toxin, causing damage to the small blood vessels all around the body and making them leakier. Leaky small blood vessels in the kidney and eye, can lead to diabetic kidney disease and diabetic eye disease (secondary complications).

All blood vessels are lined with a protective seaweed-like layer called the glycocalyx. People with diabetes have a thinner glycocalyx. People with diabetes also have more proteins in the blood (like heparanase) that can act like scissors and chop up the glycocalyx. This happens early in diabetes before secondary blood vessel complications develop, such as diabetic kidney or eye disease. We believe that damage to the glycocalyx in diabetes can lead to leakiness in all blood vessels. We have shown that repairing the glycocalyx in diabetes stops blood vessels from leaking in the kidney. We have some data to suggest this may also happen in the eye. If damage to the glycocalyx is the same in all blood vessels in diabetes, then we may be able to protect them in the same way.

Little is known about the glycocalyx in the eye and whether it is involved in the development of diabetic eye disease. We aim to understand how the glycocalyx normally prevents leakiness of small blood vessels in the eye. We then aim to find out whether diabetes affects the glycocalyx in the eye. Our main and final aim is to use a drug to stop proteins in the blood, like heparinase, from chopping up the glycocalyx. We will then find out if the small blood vessels in the eye and kidney are protected in diabetes and work better as a result. This work will be done in rats with diabetes.

If we can make the small blood vessels in the eye and kidney work better in diabetes, it is likely that all other small blood vessels will also work better. This will mean that we can use one drug to prevent all small blood vessel complications in diabetes.

Objective 1: To establish the structural changes in e-Glx at retinal endothelial cell clefts, caused by HS loss, that are associated with increased solute flux.
i. Human retinal endothelial cells: HS will be disrupted by enzymatic removal (heprainase III) or knock down (shRNA to the HS biosynthesis enzyme, Ext1). Solute permeability will be assessed across monolayers using electrical cell impedence sensing (ECIS) and labelled albumin passage. E-Glx structure and junctional cleft parameters will be assessed by electron microscopy (EM).
ii. Rat retina: E-Glx HS will be removed using an i.v. bolus of heparinase III. Retinal solute flux will be assessed by sodium fluorescein angiography (NaF). HS removal and albumin leak will be confirmed by immunofluorescence on retina. E-Glx structure and junctional cleft parameters will be quantified by EM.

Objective 2: To establish structural endothelial glycocalyx HS changes in retinal endothelial cells in diabetes
Adult male Wistar rats will receive a single dose of streptozotocin (STZ, 50mg/Kg) or vehicle. Retinal NaF will be measured at 2wk and 4wk post STZ. Urine albumin creatinine ratios (uACR) will be determined at baseline and endpoint. HS removal, retinal albumin leak, e-Glx structure and junctional cleft parameters will be confirmed as in aim 1ii. E-Glx HS immunofluorescence and heparanase activity will be carried out on healthy versus diabetic retina from human donor eyes.

Objective 3: To protect e-Glx HS and reduce microvascular permeability in experimental diabetes.
Diabetes will be induced in rats as in aim 2. A heparanase inhibitor will be administered i.p. daily for the last 2wk in cohort 1: 4wk post-STZ and cohort 2: 12wk post-STZ. E-Glx HS changes will confirmed by immunofluorescence on fixed retina and glomeruli. E-Glx structural and junctional cleft parameters in the retina and glomerulus quantified as above. Functional microvascular changes will be confirmed by NaF angiography and uACR.

Realising the objective of this programme of research will benefit the following groups:

1. Diabetic patients with and without secondary complications- The main non-academic beneficiaries will be patients with diabetes, which is an ever-growing scourge in the world (8.5% of the adult population world-wide), with major health economic implications. The likely timescale to treatment following this project is 7 years because this treatment is already being investigated clinically. We have numerous industrial links and these companies are currently actively working towards anti-proteinuric therapies. We will be ideally placed to share in the development and testing of new agents. We have clinical trial experience alongside our laboratory track record: the PIs include 2 academic clinicians who can lead translational application of our work.

2. The general public. The other non-academic beneficiaries will be the public: we are committed to public engagement and the PIs have many years of experience between them of addressing lay groups including patients, carers and the general public

3. Charities - This research programme will make conceptual changes in diabetic kidney disease and other vascular diseases in diabetes and so influence the direction of future research into avenues following the targets identified. This will allow more efficient use of scarce financial resources spent by charities on research.

4. Patient organizations - specific charities will be better able to inform patients about research that will benefit their own disease in the next 5-10 years.
5. Academic Researchers - as described under academic beneficiaries

6. Clinicians - understanding and developing treatments for patients with secondary diabetic complications, both by identifying targets and subsequent design of clinical trials, will allow clinicians to participate in the scientific advances being made as well as informing patients of new treatment options on the horizon.

7. Industry - by forming additional partnerships with industrial/pharmaceutical companies we will develop new drug/compound pipelines based on targets that we identify in this programme, thus creating commercial opportunities for a worldwide market.

8. The UK economy. Diabetes currently costs 10% of the total NHS budget with 5 million diabetic individuals in the UK. The majority of these patients will die from secondary complications. This project will target the complications associated with diabetes, in order to cut back the majority of NHS spending on this condition.