Outgrowth Endothelial Cells: Characterization and Their Potential for Reversing Ischemic Retinopathy

Purpose. Endothelial progenitor cells (EPCs) have potential for promoting vascular repair and revascularization of ischemic retina. However, the highly heterogeneous nature of these cells causes confusion when assessing their biological functions. The purpose of this study was to provide a comprehensive comparison between the two main EPC subtypes, early EPCs (eEPCs) and outgrowth endothelial cells (OECs), and to establish the potential of OECs as a novel cell therapy for ischemic retinopathy.

Methods. Two types of human blood-derived EPCs were isolated and compared using immunophenotyping and multiple in vitro functional assays to assess interaction with retinal capillary endothelial cells and angiogenic activity. OECs were delivered intravitreally in a mouse model of ischemic retinopathy, and flat mounted retinas were examined using confocal microscopy.

Results. These data indicate that eEPCs are hematopoietic cells with minimal proliferative capacity that lack tube-forming capacity. By contrast, OECs are committed to an endothelial lineage and have significant proliferative and de novo tubulogenic potential. Furthermore, only OECs are able to closely interact with endothelial cells through adherens and tight junctions and to integrate into retinal vascular networks in vitro. The authors subsequently chose OECs to test a novel cell therapy approach for ischemic retinopathy. Using a murine model of retinal ischemia, they demonstrated that OECs directly incorporate into the resident vasculature, significantly decreasing avascular areas, concomitantly increasing normovascular areas, and preventing pathologic preretinal neovascularization.

Conclusions. As a distinct EPC population, OECs have potential as therapeutic cells to vascularize the ischemic retina.

Arteriolar involvement in the microvascular lesions of diabetic retinopathy: Implications for pathogenesis

Diabetic retinopathy (DR) is the most widespread complication of diabetes mellitus and a major cause of blindness in the working population of developed countries. The clinicopathology of the diabetic retina has been extensively studied, although the relative contribution of the various biochemical and molecular sequelae of hyperglycemia remains ill defined. Many neural and microvascular abnormalities occur in the retina of short-term diabetic animals but it remains uncertain how closely these acute changes relate to chronic human disease. It is important to determine the relationship between alterations observed within the first weeks or months in short-term aminal models, and human disease, where clinically manifest retinopathy occurs only after durations of diabetes measured in years. This review is focused on the retinal microvasculature, although it should be appreciated that pathological changes in this system often occur in parallel with abnormalities in the neural parenchyma that may be derivative or even causal. Nevertheless, it is useful to reevaluate the microvascular lesions that are manifest in the retina during diabetes in humans and long-term animal models, since in addition to providing useful clues to the pathogenic basis of DR as a disease entity, it is in the deterrence of such changes that the efficacy of any novel treatment regimes will be measured. In particular, an emphasis will be placed on the relatively unappreciated role of arteriolar dysfunction in the clinical manifestations and pathology of this disease.

Retinopathy is reduced during experimental diabetes in a mouse model of outer retinal degeneration

PURPOSE. Diabetic patients who also have retinitis pigmentosa (RP) appear to have fewer and less severe retinal microvascular lesions. Diabetic retinopathy may be linked to increased inner retinal hypoxia, with the possibility that this is exacerbated by oxygen usage during the dark-adaptation response. Therefore, patients with RP with depleted rod photoreceptors may encounter proportionately less retinal hypoxia, and, when diabetes is also present, there may be fewer retinopathic lesions. This hypothesis was tested in rhodopsin knockout mice (Rho(-/-)) as an RP model in which the diabetic milieu is superimposed. The study was designed to investigate whether degeneration of the outer retina has any impact on hypoxia, to examine diabetes-related retinal gene expression responses, and to assess lesions of diabetic retinopathy.

Chloroquine causes lysosomal dysfunction in neural retina and RPE: Implications for retinopathy

Chronic use of chloroquine has been shown to induce numerous pathophysiological defects in the retina. This drug has the ability to alter pH of intracellular compartments and lysosomal function of the retinal pigment epithelium (RPE) and retinal neurons may constitute the basis of chloroquine retinopathy. The aim of the current study was to investigate pathogenic alterations in retinal cells continuously exposed to chloroquine using appropriate in vivo and in vitro models.

Inhibition of platelet-derived growth factor promotes pericyte loss and angiogenesis in ischemic retinopathy

We investigated whether inhibition of platelet-derived growth factor (PDGF) receptor tyrosine kinase activity would affect pericyte viability, vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor-2 (VEGFR-2) expression and angiogenesis in a model of retinopathy of prematurity (ROP). ROP was induced in Sprague Dawley rats by exposure to 80% oxygen from postnatal (P) days 0 to 11 (with 3 hours/day in room air), and then room air from P12-18 (angiogenesis period). Shams were neonatal rats in room air from P0-18. STI571, a potent inhibitor of PDGF receptor tyrosine kinase, was administered from P12-18 at 50 or 100 mg/kg/day intraperitoneal (i.p.). Electron microscopy revealed that pericytes in the inner retina of both sham and ROP rats appeared normal; however STI571 induced a selective pericyte and vascular smooth muscle degeneration. Immunolabeling for caspase-3 and a-smooth muscle cell actin in consecutive paraffin sections of retinas confirmed that these degenerating cells were apoptotic pericytes. In all groups, VEGF and VEGFR-2 gene expression was located in ganglion cells, the inner nuclear layer, and retinal pigment epithelium. ROP was associated with an increase in both VEGF and VEGFR-2 gene expression and blood vessel profiles in the inner retina compared to sham rats. STI571 at both doses increased VEGF and VEGFR-2 mRNA and exacerbated angiogenesis in ROP rats, and in sham rats at 100 mg/kg/day. In conclusion, PDGF is required for pericyte viability and the subsequent prevention of VEGF/VEGFR-2 overexpression and angiogenesis in ROP.

The AGE inhibitor pyridoxamine inhibits development of retinopathy in experimental diabetes

We examined the ability of pyridoxamine (PM), an inhibitor of formation of advanced glycation end products (AGEs) and lipoxidation end products (ALEs), to protect against diabetes-induced retinal vascular lesions. The effects of PM were compared with the antioxidants vitamin E (VE) and R-alpha-lipoic acid (LA) in streptozotocin-induced diabetic rats. Animals were given either PM (1 g/l drinking water), VE (2,000 IU/kg diet), or LA (0.05%/kg diet). After 29 weeks of diabetes, retinas were examined for pathogenic changes, alterations in extracellular matrix (ECM) gene expression, and accumulation of the immunoreactive AGE/ALE N-epsilon-(carboxymethyl)lysine (CML). Acellular capillaries were increased more than threefold, accompanied by significant upregulation of laminin immunoreactivity in the retinal microvasculature. Diabetes also increased mRNA expression for fibronectin (2-fold), collagen IV (1.6-fold), and laminin beta chain (2.6-fold) in untreated diabetic rats compared with nondiabetic rats. PM treatment protected against capillary drop-out and limited laminin protein upregulation and ECM mRNA expression and the increase in CML in the retinal vasculature. VE and LA failed to protect against retinal capillary closure and had inconsistent effects on diabetes-related upregulation of ECM mRNAs. These results indicate that the AGE/ALE inhibitor PM protected against a range of pathological changes in the diabetic retina and may be useful for treating diabetic retinopathy.

Müller glial dysfunction during diabetic retinopathy in rats is linked to accumulation of advanced glycation end-products and advanced lipoxidation end-products.

Aims/hypothesis: The impact of AGEs and advanced lipoxidation end-products (ALEs) on neuronal and Müller glial dysfunction in the diabetic retina is not well understood. We therefore sought to identify dysfunction of the retinal Müller glia during diabetes and to determine whether inhibition of AGEs/ALEs can prevent it.

Methods: Sprague-Dawley rats were divided into three groups: (1) non-diabetic; (2) untreated streptozotocin-induced diabetic; and (3) diabetic treated with the AGE/ALE inhibitor pyridoxamine for the duration of diabetes. Rats were killed and their retinas were evaluated for neuroglial pathology. Results: AGEs and ALEs accumulated at higher levels in diabetic retinas than in controls (p<0.001). AGE/ALE immunoreactivity was significantly diminished by pyridoxamine treatment of diabetic rats. Diabetes was also associated with the up-regulation of the oxidative stress marker haemoxygenase-1 and the induction of glial fibrillary acidic protein production in Müller glia (p<0.001). Pyridoxamine treatment of diabetic rats had a significant beneficial effect on both variables (p<0.001). Diabetes also significantly altered the normal localisation of the potassium inwardly rectifying channel Kir4.1 and the water channel aquaporin 4 to the Müller glia end-feet interacting with retinal capillaries. These abnormalities were prevented by pyridoxamine treatment.

Conclusions/interpretation: While it is established that AGE/ALE formation in the retina during diabetes is linked to microvascular dysfunction, this study suggests that these pathogenic adducts also play a role in Müller glial dysfunction.

AGEs, RAGE, and Diabetic Retinopathy

Diabetic retinopathy is a major diabetic complication with a highly complex etiology. Although there are many pathways involved, it has become established that chronic exposure of the retina to hyperglycemia gives rise to accumulation of advanced glycation end products (AGEs) that play an important role in retinopathy. In addition, the receptor for AGEs (RAGE) is ubiquitously expressed in various retinal cells and is upregulated in the retinas of diabetic patients, resulting in activation of pro-oxidant and proinflammatory signaling pathways. This AGE-RAGE axis appears to play a central role in the sustained inflammation, neurodegeneration, and retinal microvascular dysfunction occurring during diabetic retinopathy. The nature of AGE formation and RAGE signaling bring forward possibilities for therapeutic intervention. The multiple components of the AGE-RAGE axis, including signal transduction, formation of ligands, and the end-point effectors, may be promising targets for strategies to treat diabetic retinopathy.

Intervention With an Erythropoietin-Derived Peptide Protects Against Neuroglial and Vascular Degeneration During Diabetic Retinopathy.

OBJECTIVE:
Erythropoietin (EPO) may be protective for early stage diabetic retinopathy, although there are concerns that it could exacerbate retinal angiogenesis and thrombosis. A peptide based on the EPO helix-B domain (helix B-surface peptide [pHBSP]) is nonerythrogenic but retains tissue-protective properties, and this study evaluates its therapeutic potential in diabetic retinopathy.
RESEARCH DESIGN AND METHODS:
After 6 months of streptozotocin-induced diabetes, rats (n = 12) and age-matched nondiabetic controls (n = 12) were evenly split into pHBSP and scrambled peptide groups and injected daily (10 µg/kg per day) for 1 month. The retina was investigated for glial dysfunction, microglial activation, and neuronal DNA damage. The vasculature was dual stained with isolectin and collagen IV. Retinal cytokine expression was quantified using real-time RT-PCR. In parallel, oxygen-induced retinopathy (OIR) was used to evaluate the effects of pHBSP on retinal ischemia and neovascularization (1-30 µg/kg pHBSP or control peptide).
RESULTS:
pHBSP or scrambled peptide treatment did not alter hematocrit. In the diabetic retina, Müller glial expression of glial fibrillary acidic protein was increased when compared with nondiabetic controls, but pHBSP significantly reduced this stress-related response (P < 0.001). CD11b+ microglia and proinflammatory cytokines were elevated in diabetic retina responses, and some of these responses were attenuated by pHBSP (P < 0.01-0.001). pHBSP significantly reduced diabetes-linked DNA damage as determined by 8-hydroxydeoxyguanosine and transferase-mediated dUTP nick-end labeling positivity and also prevented acellular capillary formation (P < 0.05). In OIR, pHBSP had no effect on preretinal neovascularization at any dose.
CONCLUSIONS:
Treatment with an EPO-derived peptide after diabetes is fully established can significantly protect against neuroglial and vascular degenerative pathology without altering hematocrit or exacerbating neovascularization. These findings have therapeutic implications for disorders such as diabetic retinopathy.

Diabetic retinopathy: quantitative variation in capillary basement membrane thickening in arterial or venous environments

Diabetes mellitus was induced in male beagles by a single injection of an alloxan and streptozotocin cocktail and fasting blood sugar levels maintained between 15 and 20 mmol/l. Five years after induction of diabetes, three diabetic animals were sacrificed, together with sex and age-matched controls, and the retinas fixed for either transmission electron microscopy (TEM) or trypsin digestion. In TEM specimens, capillaries in close proximity to the major vessels were designated as either AE (arterial environment) or VE (venous environment) and the thickness of their basement membranes (BMs) measured using an image analyser based two dimensional morphometric analysis system. Results show that the BMs of retinal capillaries from the diabetic dogs were significantly thicker than those from control dogs. Furthermore, within the diabetic group the AE capillaries had thicker BMs than VE capillaries (p less than or equal to 0.05). The controls, however, showed no significant difference in BM thickness between AE and VE capillaries. Although many of the capillaries designated as AE or VE would actually have been derived from the opposite side of the circulation, with respect to BM thickness, they conformed to values of their specific group. The conclusion is that diabetic capillaries are more vulnerable to BM thickening in an arterial environment than in a venous environment.