Epidemiological and prospective data have revealed that the stressors of the diabetic vasculature persist beyond the point when good glycaemic control had been achieved (Diabetes_Group, 2000
) and is referred to as ‘metabolic memory’. The first suggestion of this phenomenon came from a study by Engerman and Kern in the 1980s. Alloxan-induced diabetic dogs were divided into three groups according to glycaemic control: poor control for five years, good control for five years, and poor control for two-and-a-half years followed by good control for two-and-a-half years. Retinopathy developed in the latter group, despite subsequent sustained good glycaemic control having been achieved (Engerman and Kern, 1987
More recently, studies by Kowluru and coworkers have shown that hyperglycaemia-induced elevations in inflammatory mediators in retinal microvascular cells resist reversal after re-institution of normal glucose conditions (Kowluru et al., 2010
The standard parameter of glycaemic control, hemoglobin A1C is an advanced glycation endproduct (AGE). This particular cross-link formation of AGEs serves as an excellent tool to evaluate glucose control. Similar chemical changes in extracellular matrices (ECM) lead to pathological thickening of vascular basement membranes, including those of the retinal capillaries. (Bailey, 2001
; Gardiner et al., 2003
). Accumulation of AGEs has many deleterious effects on basement membrane physiology and cellular function (Boyd-White and Williams, 1996
; Monnier et al., 1986
; Wells-Knecht et al., 1995
). For example, retinal vascular endothelial cells and pericytes are co-dependent on complex, two-way communication via paracrine growth factors secreted into their shared basement to ensure continued survival of both cell types. AGE formation on this basement membrane alters retinal vascular cell function and compromises cell survival by cross-linking or otherwise inactivating vital heparin-binding growth factors and integrin-recognition domains (Paul and Bailey, 1999
Accelerated death of endothelial cells, pericytes and microvascular smooth muscle cells is known to occur in diabetic retinopathy (Mizutani et al., 1996
; Stitt et al., 1994
). This is believed due to the AGE-modification of basement membrane removing key survival factors from the matrix (Stitt et al., 2004
). The accumulation of AGEs and associated cross-links on the arginine and lysine residues of ECM is a recognized phenomenon during diabetes (Fu et al., 1994
Significantly, this hyperglycaemic damage targets endothelial cells as well as pericytes. The death of both cell types is a hallmark of diabetic retinopathy and leads to the formation of naked basement membrane tubes. These non-perfused, degenerated tubes/capillaries can be recognized histologically as ‘acellular capillaries’ in diabetic patients, dogs, rats and mice (Engerman and Kern, 1995
; Joussen et al., 2004
; Kern and Engerman, 1996a
; Mizutani et al., 1996
; Tang et al., 2003
), . In turn, acellular capillaries result in lesions that produce irreversible retinal ischemia through their inability to support blood flow. This vessel destruction leading to retinal ischemia and increased expression of angiogenic growth factors ultimately triggers retinal neovascularization. While all these histological features are well established, the mechanisms underlying diabetes-induced retinal cell injury and death are incompletely understood. Degeneration of retinal capillaries is recognized to play an important role in the pathogenesis of DR (Davis et al., 1997
), with many investigators believing that the resulting retinal ischemia stimulates pre-retinal neovascularization. Acute ischemia/reperfusion of the retina also has been recognized to cause formation of these acellular capillaries, even in non-diabetic rats and mice (Zheng et al., 2005
Endothelial progenitor cells (EPCs) play a role in maintenance of the normal vasculature () and defects in EPCs may contribute to capillary degeneration. In , a trypsin digested rat retina was stained with CD133, CD14 and lectin. EPCs are present in the vasculature in the diabetic retina and typically appear at the bifurcations of the retinal capillaries and are seen as clusters as well as single cells.
The collective evidence indicates that the loss of retinal microvascular cells, a critical early step in diabetic retinopathy, may not only be due to increased cell death but also to altered repair mechanisms. In the retina, a defect in EPCs could prevent reparation of endothelial injury early on, leading to acellular capillary lesions and retinal ischemia.
As we show in , CD34+ cells are dysfunctional in diabetics and have inability to home to areas of injury to orchestrate the repair process. In , a model of ischemia reperfusion (I/R) injury that demonstrates a key feature of diabetic retinopathy, acellular capillaries, was used to examine the function of CD34+ cells. Mouse eyes were subjected to I/R injury and CD34+ were injected into the vitreous after injury. For this study we isolated CD34+ cells from diabetic patients with vascular complications or age- and gender-matched controls. Immunofluorescence studies show that control cells but not diabetic cells home to areas of injury.
Human peripheral blood CD34+ cells from diabetic patients are dysfunctional in vascular homing and association and cannot repair damaged retinal vessels
This defect in migration is due in part to a diabetes-associated reduction in phosphorylation and intracellular redistribution of vasodilator-stimulated phosphoprotein (VASP), a critical actin motor protein required for cell migration that also controls vasodilation and platelet aggregation (Li Calzi et al., 2008
). The effect of the chemokine stromal derived factor-1(SDF-1) on VASP redistribution is markedly reduced in diabetics () whereas VASP redistributes to filopodia-like structures following SDF-1 treatment in controls. Thus, SDF-1 promotes cytoskeletal changes through site- and cell type-specific VASP phosphorylation in healthy CD34+
cell but in diabetes, blunted responses to this factor and other growth factors may lead to reduced vascular repair and tissue perfusion.
VASP redistribution is impaired in diabetic EPCs
Endothelial injury, acellular capillaries and subsequent retinal ischemia result in the compensatory release of chemokines and growth factors such as VEGF, SDF-1, and monocyte chemoattractant protein-1 (MCP-1 or CCL2) by the ischemia injured retina. Studies have already revealed correlations between the vitreous levels of SDF-1, MCP-1 and VEGF and the degree of macular edema and retinopathy in patients with DR (Butler et al., 2005
). However, many additional factors have been shown to be increased in the vitreous of diabetics including IGF-1 (Grant et al., 1986
); IL1B, IL6, IL8, CCL2, and EDN1 (Zhou et al., 2012
) and these factors likely impact also DR outcomes.
Restoration of EPC function would result in repair of areas of capillary injury (acellular capillaries). This would prevent the development of ischemia and the subsequent ‘compensatory’ retinal expression of chemokines and growth factors that accumulate in the vitreous. Central to understanding the role of EPCs in vascular repair is an understanding of how BMDC become endothelial cells.