Continuing Education

Diabetic retinopathy (DR) is the main cause of blindness among the working age group in the UK and most other developed countries.

The overall prevalence of DR varies across different populations, but it is the major blinding ocular complication of diabetes. The increasing number of individuals with diabetes worldwide suggests that DR and diabetic maculopathy (DM) will continue to be major contributors to vision loss and associated functional impairment for years to come.

DR is potentially a visually devastating complication of chronic hyperglycaemia and other associated systemic abnormalities. Advanced stages of DR are characterised by the growth of abnormal retinal blood vessels secondary to ischemia. These blood vessels grow in an attempt to supply oxygenated blood to the hypoxic retina. At any time during the progression of DR, patients can also develop DM, which involves retinal thickening in the macular area. DM occurs after breakdown of the blood-retinal barrier due to leakage of dilated hyperpermeable capillaries and microaneurysms.

Numerous large, prospective randomised clinical trials have delineated the current standard prevention and treatment protocols, including intensive glycaemic and blood pressure control, and laser photocoagulation for neovascularisation and clinically significant macula oedema.

The exact mechanisms by which elevated glucose initiates the vascular disruption in retinopathy remain poorly defined, and, not surprisingly, several pathways have been implicated. The vascular disruptions of DR and DM are characterised by abnormal vascular flow, disruptions in permeability, and/or closure or non-perfusion of capillaries.

Microvascular leakage and microvascular occlusion are the two main pathological processes responsible for development of sight-threatening diabetic retinopathy. The structural abnormalities that develop within the retinal capillary wall that lead to these processes include:

Increased retinal vascular permeability results in haemorrhages, exudates and retinal oedema (Figure 1).

DEVELOPMENT OF MICROANGIOPATHIC CHANGES IN DIABETIC RETINOPATHY

The mechanism of development of microangiopathy is not fully understood, but relates to a combination of changes in ultrastructural, biochemical and haemostatic processes. These include capillary basement membrane thickening (CBMT), non-enzymatic glycosylation, possibly increased free radical activity, increased flux through the polyol pathway and haemostatic abnormalities. The central feature is hyperglycaemia, which is directly linked to the above changes, and ultimately causes tissue ischaemia.

Many studies have demonstrated that chronic hyperglycaemia, as well as hyperlipidaemia and hypertension, contribute to the pathogenesis of DR.

Endothelial cells are responsible for maintaining the blood-retinal barrier, and damage to them results in increased vascular permeability. The histological hallmark of microangiopathy is CBMT. Thickening of the capillary basement membrane and increased deposition of extracellular matrix components may contribute to the development of abnormal retinal haemodynamics, including abnormal auto-regulation of retinal blood flow. This is the mechanism whereby a vessel wall alters tension to maintain as constant a pressure within as needed.

The exact mechanism of thickening and leakiness of the basement membrane appears to involve several biochemical mechanisms and is still not fully understood. The major structural elements involved in CBMT are type IV collagen, heparin sulphate – an important proteoglycan – together with laminin and fibronectin. Heparin sulphate, produced by the endothelial cells, is highly negatively charged and creates a regular lattice structure of anionic sites that hinder the filtration of negatively charged proteins, such as albumin. In diabetes, there appears to be impaired synthesis of proteoglycans and an increase in hydroxylysine and its glycosidally linked disaccharide units. Such alterations lead to abnormal packing of the peptide chains which produces excessive leakiness of the membrane. This in part explains the development of, for example, micro-albuminuria seen in diabetics.

There is also evidence that retinal leukostasis may play a significant role in the pathogenesis of DR. Leukocytes (white blood cells) possess large cell volumes, high cytoplasmic rigidity, a natural tendency to adhere to the vascular endothelium, and a capacity to generate toxic superoxide radicals and proteolytic enzymes. In diabetes, there is increased retinal leukostasis, which affects retinal endothelial function, retinal perfusion, angiogenesis, and vascular permeability. In particular, leukocytes in diabetes are less deformable, a higher proportion are activated, and they may be involved in capillary nonperfusion, endothelial cell damage, and vascular leakage in the retinal microcirculation. Capillary occlusions, capillary drop-out or degeneration associated with leukocytes in the diabetic retina is now thought to be common.

Carbohydrates interact with protein side chains in a non-enzymatic fashion to form Amadori products, which may subsequently form advanced glycosylation end-products (AGEs), particularly where there is a high glucose concentration. These products are formed non-enzymatically via a series of intermediate steps. An example is the production of glycosylated haemoglobin (as measured by the HbA1c blood test). Such products then undergo a series of changes resulting in AGE.

These are resistant to degradation and continue to accumulate indefinitely on long-lived proteins, and may therefore be responsible for the production of CBMT. AGEs may affect such functions as enzyme activity, binding of regulatory molecules, and susceptibility of proteins to proteolysis.

The chronic interaction of these products with at least one specific cell surface receptor for AGEs (AGE-specific receptor) may perpetuate a pro-inflammatory signalling process and a pro-atherosclerotic state in vascular tissues. AGE formation within the endothelial cell basement membrane deactivates endothelial derived nitric oxide, which acts on peri-vascular smooth muscle causing vasodilation. This may result in impaired blood flow. Several cells, including vascular endothelial cells, possess receptors for AGE. Binding of AGE to endothelial receptors causes changes in vascular permeability and favours thrombosis at the endothelial cell surface.

Free radicals are produced continuously during many metabolic processes and are rapidly eliminated by antioxidants such as reduced glutathione (GSH), vitamins C and E. Diabetic patients, however, have a lower concentration of GSH as well as vitamins C and E. The reduction in antioxidant reserve in diabetic patients may be due to competition for NADPH.

This is a co-factor required to recycle the oxidised free radical scavengers back to the effective form (redox cycling). NADPH is produced by the hexose monophosphate shunt and one source of competition from NADPH comes from the polyol pathway. Excess free radicals may be produced either from protein glycation or from inefficient elimination by reduced antioxidants, possibly secondary to NADPH utilisation and defective redox cycling.

The polyol pathway (Figure 3) converts hexose sugars such as glucose into sugar alcohols (polyols).

For example, glucose can be converted into sorbitol via the action of the enzyme aldose reductase. Aldose reductase is the rate-limiting enzyme for this pathway. Under normal conditions glucose is metabolised via the hexokinase pathway. In the presence of hyperglycaemia high glucose levels saturate the hexokinase pathway and glucose is then metabolised by the polyol pathway. This then has a knock-on effect for other metabolic processes. Increased aldose reductase activity and accumulation of sorbitol have been found in diabetic animal models.

As sorbitol does not easily dissolve across cell membranes, this increases cellular osmolarity, ultimately leading to cell damage. Increased polyol pathway activity also alters the redox state of the pyridine nucleotides NADPH and NAD+, thus increasing their concentrations. Since these are important factors in many enzyme catalysed reactions, many other metabolic pathways may be also affected.

Hyperglycaemia is associated with increased cellular protein kinase C activity in cultured endothelial cells, resulting from enhanced synthesis of diacylglycerol from glucose.

Protein kinase C is involved in signal transduction of responses to hormones, growth factors and neurotransmitters. It can affect growth rate, DNA synthesis, hormone receptor turnover and contraction in vascular smooth muscle cells. Protein kinase C activity may have a role in the development of microangiopathy in relation to hyperglycaemia.

In patients with early diabetic retinopathy the likelihood of micro-thrombus formation is enhanced because of an increase of factor VIII, which is produced by the endothelial cells.

Another substance called prostacyclin (PGI2), which is normally produced in endothelial cells, has a strong vasodilator effect that can reduce platelet aggregation and adherence to the cell wall. In diabetic patients, production of PGI2 is reduced. Another substance involved in thrombosis is plasminogen activator, which is in lower concentrations in diabetics. This converts plasminogen to plasmin which then promotes fibrinolysis.

Platelet function in diabetes is also abnormal. Thromboxane A2, which is released from platelets, is increased in diabetics (as with many vascular conditions). This produces significant vasoconstriction and also causes platelet aggregation. This combination of factors leads to microthrombus formation and small vessel occlusion.

There is evidence that retinal leukostasis may also play an important role in the pathogenesis of DR.

In diabetes, there is increased retinal leukostasis, which affects retinal endothelial function, retinal perfusion, angiogenesis and vascular permeability. Leukocytes possess very large cell volume, high cytoplasmic rigidity, a natural tendency to adhere to the vascular endothelium via cellular adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and beta-integrins on the leukocytes.

Other molecules also thought to be involved in this process include vascular cell adhesion molecules, fibronectin and osteopontin. Furthermore, they are thought to generate toxic superoxide radicals and proteolytic enzymes. In diabetics, leukocytes are less deformable, with a relatively high proportion in an activated state and these factors contribute to capillary non-perfusion, endothelial cell damage and vascular leakage in the retinal microcirculation.

As a result of retinal occluded capillaries, retinal ischaemia stimulates a pathologic neovascularsiation mediated by angiogenic factors, such as vascular endothelial growth factor (VEGF), which ultimately gives rise to proliferative retinopathy.

The exact pathogenesis of retinal neovascularisation is not fully understood, although there have been some significant advances in our understanding of this condition in recent years.

It is thought that systemic activation of leukocytes, particularly monocytes, leads to capillary closure through increased adhesiveness of the leukocytes to the damaged endothelial cell surface of the capillary walls.

Hyperglycaemia, with subsequent protein-kinase-C activation, and the release VEGF are important triggers to the development of retinopathy (Figure 4). VEGF is a potent pro-angiogenic and permeability factor that causes new vessel growth and vascular leakage and is 50,000 times more potent than histamine in producing vascular leakage.

It is elevated even in background diabetic retinopathy, is associated with increased vascular permeability, increases with disease severity and is detectable in the vitreous and aqueous once neovascularisation has developed. The production of VEGF is induced by hypoxia.

Further reading