Alan W. Stitt

Advanced glycation: an important pathological event in diabetic and age related ocular disease

The formation of advanced glycation end products (AGEs) is a key pathophysiological event with links to a range of important human diseases. It is now clear that AGEs may act as mediators, not only of diabetic complications 12 but also of widespread age related pathology such as Alzheimer’s disease, 3 decreased skin elasticity, 45 male erectile dysfunction, 67 pulmonary fibrosis, 8 and atherosclerosis. 91 0 Since many cells and tissues of the eye are profoundly influenced by both diabetes and ageing, it is fitting that advanced glycation is now receiving considerable attention as a possible modulator in important visual disorders. An increasing number of reports confirm widespread AGE accumulation at sites of known ocular pathology and demonstrate how these products mediate crosslinking of long lived molecules in the eye. Such studies also underscore the putative pathophysiological role of advanced glycation in ocular cell dysfunction in vitro and in vivo. This article reviews some of the important eVects that advanced glycation has on ocular tissues and the role that AGEs, and their specific receptors, have in the initiation and progression of sight threatening disorders such as diabetic retinopathy, glaucoma, cataract formation, and age related macular degeneration (AMD). This review also considers pharmacological strategies to prevent or neutralise the eVects of AGEs and the recent development of potential therapies for AGE induced disease processes.

Molecular analysis of endothelial progenitor cell (EPC) subtypes reveals two distinct cell populations with different identities

BackgroundThe term endothelial progenitor cells (EPCs) is currently used to refer to cell populations which are quite dissimilar in terms of biological properties. This study provides a detailed molecular fingerprint for two EPC subtypes: early EPCs (eEPCs) and outgrowth endothelial cells (OECs).MethodsHuman blood-derived eEPCs and OECs were characterised by using genome-wide transcriptional profiling, 2D protein electrophoresis, and electron microscopy. Comparative analysis at the transcript and protein level included monocytes and mature endothelial cells as reference cell types.ResultsOur data show that eEPCs and OECs have strikingly different gene expression signatures. Many highly expressed transcripts in eEPCs are haematopoietic specific (RUNX1, WAS, LYN) with links to immunity and inflammation (TLRs, CD14, HLAs), whereas many transcripts involved in vascular development and angiogenesis-related signalling pathways (Tie2, eNOS, Ephrins) are highly expressed in OECs. Comparative analysis with monocytes and mature endothelial cells clusters eEPCs with monocytes, while OECs segment with endothelial cells. Similarly, proteomic analysis revealed that 90% of spots identified by 2-D gel analysis are common between OECs and endothelial cells while eEPCs share 77% with monocytes. In line with the expression pattern of caveolins and cadherins identified by microarray analysis, ultrastructural evaluation highlighted the presence of caveolae and adherens junctions only in OECs.ConclusionsThis study provides evidence that eEPCs are haematopoietic cells with a molecular phenotype linked to monocytes; whereas OECs exhibit commitment to the endothelial lineage. These findings indicate that OECs might be an attractive cell candidate for inducing therapeutic angiogenesis, while eEPC should be used with caution because of their monocytic nature.

Microvascular lesions of diabetic retinopathy: clues towards understanding pathogenesis?

Retinopathy is a major complication of diabetes mellitus and this condition remains a leading cause of blindness in the working population of developed countries. As diabetic retinopathy progresses a range of neuroglial and microvascular abnormalities develop although it remains unclear how these pathologies relate to each other and their net contribution to retinal damage. From a haemodynamic perspective, evidence suggests that there is an early reduction in retinal perfusion before the onset of diabetic retinopathy followed by a gradual increase in blood flow as the complication progresses. The functional reduction in retinal blood flow observed during early diabetic retinopathy may be additive or synergistic to pro-inflammatory changes, leucostasis and vaso-occlusion and thus be intimately linked to the progressive ischaemic hypoxia and increased blood flow associated with later stages of the disease. In the current review a unifying framework is presented that explains how arteriolar dysfunction and haemodynamic changes may contribute to late stage microvascular pathology and vision loss in human diabetic retinopathy.

The role of advanced glycation in the pathogenesis of diabetic retinopathy.

Retinopathy is one of the commonest microvascular complications of diabetes and is still the prevailing cause of registerable blindness in the working population of developed countries. The clinicopathology of microvascular lesions and the dysregulation of an array of biochemical pathways in the diabetic retina have been extensively studied, although the relative contribution of various biochemical sequelae of hyperglycaemia remains ill- defined. There is little doubt that the pathogenesis of this diabetic complication is highly complex and there is a pressing need to establish new therapeutic regimens that can effectively prevent or retard the initiation and progression of retinal microvascular cell dysfunction and death which is characteristic of the vasodegenerative stages of diabetic retinopathy. Among the several pathogenic mechanisms that may contribute to diabetic retinopathy are the formation and accumulation of advanced glycation endproducts (AGEs). AGEs can form on the amino groups of proteins, lipids, and DNA through a number of complex pathways, including nonenzymatic glycation by glucose and reaction with metabolic intermediates and reactive dicarbonyl intermediates. These reactions not only modify the structure and function of proteins, but also cause intramolecular and intermolecular cross-link formation. AGEs are known to accumulate in the diabetic retina where they may have important effects on retinal vascular cell function in vitro and in vivo. Evidence now points toward a pathogenic role for advanced glycation in the initiation and progression of diabetic retinopathy. This review will examine the basis of AGE-related pathology in the diabetic retina at cellular and molecular levels. It will also outline how recent strategies to inhibit AGE formation or limit their pathogenic influence during chronic diabetes may have an important role to play in the treatment of retinopathy.

Inhibition of Tumor Necrosis Factor-α Improves Physiological Angiogenesis and Reduces Pathological Neovascularization in Ischemic Retinopathy

The present study was undertaken to test whether inhibition of the proangiogenic inflammatory cytokine tumor necrosis factor (TNF)-alpha can modulate retinal hypoxia and preretinal neovascularization in a murine model of oxygen-induced retinopathy (OIR). OIR was produced in TNF-alpha-/- and wild-type (WT) control C57B6 neonatal mice by exposure to 75% oxygen between postnatal days 7 and 12 (P7 to P12). Half of each WT litter was treated with the cytokine inhibitor semapimod (formerly known as CNI-1493) (5 mg/kg) by daily intraperitoneal injection from the time of reintroduction to room air at P12 until P17. The extent of preretinal neovascularization and intraretinal revascularization was quantified by image analysis of retinal flat-mounts and retinal hypoxia correlated with vascularization by immunofluorescent localization of the hypoxia-sensitive drug pimonidazole (hypoxyprobe, HP). HP adducts were also characterized by Western analysis and quantified by competitive enzyme-linked immunosorbent assay. TNF-alpha-/- and WT mice showed a similar sensitivity to hyperoxia-induced retinal ischemia at P12. At P13 some delay in early reperfusion was evident in TNF-alpha-/- and WT mice treated with semapimod. However, at P17 both these groups had significantly better vascular recovery with less ischemic/hypoxic retina and preretinal neovascularization compared to untreated retinopathy in WT mice. Immunohistochemistry showed deposition of HP in the avascular inner retina but not in areas underlying preretinal neovascularization, indicating that such aberrant vasculature can reduce retinal hypoxia. Inhibition of TNF-alpha significantly improves vascular recovery within ischemic tissue and reduces pathological neovascularization in OIR. HP provides a useful tool for mapping and quantifying tissue hypoxia in experimental ischemic retinopathy.

Endothelial Progenitors as Tools to Study Vascular Disease

Endothelial progenitor cells (EPCs) have great clinical value because they can be used as diagnostic biomarkers and as a cellular therapy for promoting vascular repair of ischaemic tissues. However, EPCs also have an additional research value in vascular disease modelling to interrogate human disease mechanisms. The term EPC is used to describe a diverse variety of cells, and we have identified a specific EPC subtype called outgrowth endothelial cell (OEC) as the best candidate for vascular disease modelling because of its high-proliferative potential and unambiguous endothelial commitment. OECs are isolated from human blood and can be exposed to pathologic conditions (forward approach) or be isolated from patients (reverse approach) in order to study vascular human disease. The use of OECs for modelling vascular disease will contribute greatly to improving our understanding of endothelial pathogenesis, which will potentially lead to the discovery of novel therapeutic strategies for vascular diseases.

Histological and ultrastructural investigation of retinal microaneurysm development in diabetic patients.

BACKGROUND--Although microaneurysms are a clinicopathological hallmark of diabetic retinopathy, there have been few ultrastructural studies of these important lesions. As a result, knowledge of the mechanisms involved in the pathogenesis of microaneurysms remains fragmentary. This study provides histological and ultrastructural evidence of various stages in microaneurysm formation within the retinal vasculature. METHODS--The eyes of three type II diabetic patients, obtained within 24 hours of death, were studied by the trypsin digest technique. Eyes from two further type II diabetics were fixed in 2.5% glutaraldehyde within 12 hours of death and processed for electron microscopy. RESULTS--In the trypsin digest preparations, small saccular and fusiform microaneurysms were observed in the peripheral retinal. In the central retina, the microaneurysms ranged in morphology from thin walled, cellular forms to dense, acellular, hyalinised forms. Ultrastructurally, four distinct groups of microaneurysm were observed. Type I showed an extensive accumulation of polymorphonuclear cells into the lumen. The endothelium remained intact, although pericytes were invariably absent. Type II microaneurysms were typified by large numbers of red blood cells (RBCs) in the lumen. Endothelial cells and pericytes were completely absent. The type III microaneurysm was also non-perfused and contained aggregates of irregularly shaped RBC profiles and RBC breakdown products. Recanalisation by new vessels into the occluded lumen was observed in one microaneurysm. Type IV microaneurysms were almost or completely sclerosed, with extensive fibrosis and lipid infiltration into the lumen and basement membrane wall. CONCLUSION--This investigation describes several distinctive stages in the formation of microaneurysms during diabetic retinopathy. With reference to the pathogenesis of retinal microaneurysms, the interaction of various cell types is discussed and the significance of vascular cell death and localised hypertensive events highlighted.

Advanced glycation end products and diabetic complications.

Diabetic complications are major cause of morbidity and mortality in patients with diabetes. While the precise pathogenic mechanism(s) underlying conditions such as diabetic retinopathy, diabetic nephropathy and increased risk of atherosclerosis remain ill-defined, it is clear that hyperglycaemia is a primary factor that initiates and promotes complications. Formation of advanced glycation end products (AGEs) correlate with glycaemic control, and these reactive adducts form on DNA, lipids and proteins where they represent pathophysiological modifications that precipitate dysfunction at a cellular and molecular level. Many of these adducts form rapidly during diabetes and promote progression of a raft of diabetes-related complications. Recent evidence also suggests an important interaction with other pathogenic mechanisms activated within the diabetic milieu. This review outlines the nature of AGE formation in biological systems and highlights accumulative evidence that implicates these adducts in diabetic complications. As more therapeutic agents are developed to inhibit AGE formation or limit their pathogenic influence during chronic diabetes, it is becoming clear that these anti-AGE strategies have an important role to play in the treatment of diabetic patients.

Nitric oxide synthase activity and expression in retinal capillary endothelial cells and pericytes

The purpose of this study was to examine nitric oxide synthase (NOS) expression in the retinal vasculature in vivo and to study nitric oxide (NO) synthesis in vitro in retinal microvascular endothelial cells and pericytes. Immunoreactivity was examined using a polyclonal antibody raised against porcine cerebellar nitric oxide synthase on frozen sections cut from postmortem human retina and trypsin digests of rat retinal vasculature. The synthesis of nitrite, a stable end product from the interaction of NO with molecular oxygen, was measured in culture supernatants of retinal microvascular cells under basal and stimulated conditions. Expression of constitutive NOS (cNOS) in these cells was examined using the polymerase chain reaction (PCR). Strong NOS immunoreactivity was seen in the endothelium of choroidal and retinal vessels. Nitrite synthesis was documented in supernatants from cultured microvascular endothelial cells which increased significantly following exposure to A23187 and cytokines. Nitrite synthesis by pericytes was not detectable under basal conditions or following stimulation with A23187. Bacterial lipopolysaccharide (LPS), a potent inducer of NOS, caused an increase in nitrite concentrations in pericyte supernatants 24 h after stimulation suggesting the presence of inducible NOS (iNOS). PCR amplification confirmed the presence of the cNOS gene in endothelial cells but not in pericytes. Retinal vascular endothelial cells express significant amounts of NOS constitutively in vivo and in vitro which is activated by Ca++. Also, endothelial cells can be stimulated to synthesize iNOS by cytokines. Retinal pericytes too show iNOS activity following exposure to bacterial LPS. These results suggest that the nitric oxide synthase/nitric oxide pathway may be involved in the regulation of microcirculatory haemodynamics in the retina.

AGEs and Diabetic Retinopathy

The pathogenesis of diabetic retinopathy is multifactorial, and a range of hyperglycemia-linked pathways have been implicated in the initiation and progression of this condition. All cells in the retina are affected by the diabetic milieu, and in view of such disease and tissue complexity, it is unlikely that any single process is solely responsible for retinal pathophysiology. Nevertheless, establishing causal mechanisms remains an important research goal. This review concentrates on the formation of advanced glycation end products (AGEs) and the role they play in diabetic retinopathy. Perspective is provided on advanced glycation in the retina, the impact that this process has on retinal cell function, and how it relates to other pathogenic pathways. Emphasis is also placed the modulatory role of the receptor for AGEs (RAGE) and how its activation could evoke retinal inflammatory disease. Further research is needed to achieve a clear understanding of the cellular and molecular processes that underpin diabetic retinopathys initiation and progression. Such advances in basic mechanisms may lead to effective treatments that can prevent progression of retinopathy from the point of the diagnosis of diabetes to sight-threatening proliferative diabetic retinopathy (PDR) and diabetic macular edema (DME).