Hydrogen sulfide as a novel guardian of the retinal vascular endothelial glycocalyx; new therapeutic opportunities.

Type 2 diabetes has a significant personal impact on the sufferer since it is often associated with life-changing complications. One of the most feared complications is retinopathy, which can result in blindness due to damage to the small blood vessels in the eye. This not only severely affects the quality of life of the sufferer and their family, but also impacts on wider society through the financial burden of both healthcare and benefits.

One of the first steps in the development of diabetic retinopathy is blockage of the small blood vessels in the eye due to white blood cells in the blood sticking to the endothelial cells lining the vessel walls. This stops blood flowing through the small vessels and triggers a series of biological events that, if not treated effectively, may cause blindness.

The attachment of white blood cells is regulated by "sticky" proteins called adhesion molecules on the endothelial cells. These are usually embedded within a protective layer on the endothelial cell surface called the glycocalyx. In health, the glycocalyx extends out from these cells like a "furry" layer, protecting them from damage and is so thick that the adhesion molecules are hidden within it, preventing white cell adhesion.

In people with diabetes the glycocalyx in the small vessels in the eye is reduced in thickness. This may expose the adhesion molecules, leading to increased white blood cell adhesion resulting in vessel blockage. Thus, therapies to reduce loss of the glycocalyx in diabetes could prevent progression of retinopathy by preventing white cells attaching to the vessel walls.

We think that a gas called hydrogen sulfide may help maintain the glycocalyx in health. Our laboratories are world leading in the field of hydrogen sulfide biology and we have developed, and are patenting, a range of novel donors that can release hydrogen sulfide at physiological rates. We have shown that blood hydrogen sulfide levels are significantly lowered in individuals with type 2 diabetes, and that these reduced levels are associated with abnormal small vessel function. We have also shown that in retinal endothelial cells in the laboratory our donors can reverse the reduction in glycocalyx caused by high glucose (used to mimic diabetes). Thus, we think that reduced hydrogen sulfide in the blood of people with diabetes is linked to glycocalyx loss which in turn exposes endothelial adhesion molecules and leads to increased adhesion of white cells to vessel walls and ultimately vessel blockage. From this evidence we believe that our hydrogen sulfide donors could be used to treat retinopathy in type 2 diabetes.

We will investigate this further using a range of techniques. Firstly, using retinal endothelial cells in culture we will determine the mechanism by which hydrogen sulfide prevents glycocalyx loss; does it inhibit the degradation or increase synthesis of the components of the glycocalyx or both? We will complement these studies by examining whether our donors can prevent adhesion molecule exposure in high glucose conditions. Next, using an animal model, we will examine whether administration of hydrogen sulfide donors into the eye can prevent small blood vessel changes induced by diabetes. Finally we will bring our studies back to humans and examine whether the levels of blood hydrogen sulfide and glycocalyx components are related to the degree of severity of diabetic retinopathy in patients with diabetes. Since during glycocalyx degradation its components are shed into the blood, measuring these will tell us how much glycocalyx has been lost.

We have extensive experience in the biology of hydrogen sulfide and the glycocalyx and our combined experience will ensure that this project provides reliable results that will inform clinicians, the pharmaceutical industry, scientists and most importantly patients on the therapeutic potential of hydrogen sulfide donors in treating diabetic retinopathy.

We will examine whether our novel slow release hydrogen sulfide (H2S) donors can be used to treat diabetic retinopathy.

The early stages of diabetic retinopathy are characterised by enhanced leukostasis in the retinal microvessels, resulting in vessel blockage. Reduction in thickness of the endothelial surface layer i.e. glycocalyx, in diabetes may exacerbate this by exposing endothelial adhesion molecules. Thus, interventions to prevent/reverse glycocalyx damage caused by diabetic insult could offer a novel treatment option for diabetic retinopathy. Our previous data suggest that therapeutic administration of the gaseous transmitter H2S may fulfil this role; we showed a significant loss of plasma H2S in diabetes which was associated with microvascular dysfunction and importantly have shown that the H2S donor GYY1437 prevents hyperglycaemia-associated glycocalyx loss.

Thus, we will examine, using cellular, animal and human studies, the mechanisms by which our H2S donors protect the glycocalyx from diabetes-associated degradation and their in vivo therapeutic potential. Using cultured retinal endothelial cells we will examine the effects of H2S on the secretion/activity of glycocalyx degrading enzymes using activity assays, zymography and immunoblotting. These studies will be complemented by studies into the effects of H2S on the synthesis of glycocalyx components using immunoblotting, chromatography and also TaqMan low density qPCR arrays to identify endothelial gene expression changes in response to H2S. The functional role of H2S-mediated glycocalyx protection will be assessed by examining the effects of the H2S donors on diabetes-associated pro-inflammatory vascular responses i.e. increased adhesion molecule exposure (in vitro) and increased leukostasis and vascular permeability in vivo using an established rat model. Finally, we will examine whether circulating H2S levels correlate with the degree of severity of diabetic retinopathy in man.

Our research has the potential to lead to the development of a new treatment for retinopathy, a potentially life-altering complication of diabetes that currently has only limited and expensive therapeutic options. Thus, this work may have a positive impact on a number of important beneficiaries expanded on below. Briefly: (1) Patients with diabetes; (2) Healthcare professionals; (3) NHS; (4) Pharmaceutical industry; (5) Wider scientific community (6) Researcher working on the project; (7) Society; (8) Public engagement; (9) Local economy.

(1) Importantly, in the longer term, this work should benefit sufferers of diabetes and their families. Blindness resulting from retinopathy is a highly feared complication and has significant impact on the quality of life and psychological welfare of sufferers and their families. Research shows that loss of sight is more feared by patients than premature death due to diabetes.
(2) Healthcare professionals including physicians, diabetes specialist nurses and ophthalmologists will benefit from increased patient satisfaction and greater patient confidence since current therapies for retinopathy are limited.
(3) The NHS would be a major beneficiary. Current therapies are expensive and limited. The cost of therapy could be significantly reduced since H2S donors are relatively cheap and easy to produce. The development of a new therapy may also alter the way diabetic retinopathy is managed; if progression of retinopathy can be prevented or slowed then routine screening of individuals with diabetes could be less frequent, reducing screening costs.
(4) Pharmaceutical industry - a widely used therapy to halt the progression of diabetic retinopathy could generate considerable income for the pharmaceutical industry and may also generate interest in the therapeutic use of slow release hydrogen sulfide donors in other common conditions.
(5) The wider scientific community in the field of diabetic retinopathy, glycocalyx biology and H2S biology will benefit. This project will improve understanding of the mechanisms involved in the development and progression of retinopathy and a greater understanding of the biological function of hydrogen sulfide and, in particular, its effects on the endothelial glycocalyx. In the short term, this project will also have an impact on the scientific community through the advancement of methods to study the expanding field of H2S biology i.e. slow release hydrogen sulfide donors. We have a track record of providing these slow release H2S donors free of charge to national and international collaborators in a diverse range of projects.
(6) The researcher undertaking the project will benefit significantly. The project involves a wide range of scientific techniques; both in vitro and in vivo and so will provide extensive training and experience and thus progress personal development and future employability.
(7) Society. There are over 1200 new cases of blindness each year due to diabetic retinopathy. This represents a significant cost to society in terms of time off work as the disease progresses, disability allowance, caring for individuals who become blind i.e. healthcare services and family if they need to give up work to become carers. Additionally, sufferers can experience depression and anxiety and the cost of treating these associated conditions would be reduced
(8) This project will have a positive impact on public engagement with science. The potential to develop a new therapy for a debilitating and feared disease will engage public interest and raise awareness of the researchers themselves, their academic institutions and the MRC.
(9) Our SRHDs may be translated in the mid-to-long term into therapeutic entities and generate patentable or commercially exploitable tools which will benefit the local economy. We currently have two patent applications on our H2S donors submitted (WPO/GB2011/00143, 30/09/10 and GB1117095.8, 20/09/11.