Robert N. Mames

Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization.

Adults maintain a reservoir of hematopoietic stem cells that can enter the circulation to reach organs in need of regeneration. We developed a novel model of retinal neovascularization in adult mice to examine the role of hematopoietic stem cells in revascularizing ischemic retinas. Adult mice were durably engrafted with hematopoietic stem cells isolated from transgenic mice expressing green fluorescent protein. We performed serial long-term transplants, to ensure activity arose from self-renewing stem cells, and single hematopoietic stem-cell transplants to show clonality. After durable hematopoietic engraftment was established, retinal ischemia was induced to promote neovascularization. Our results indicate that self-renewing adult hematopoietic stem cells have functional hemangioblast activity, that is, they can clonally differentiate into all hematopoietic cell lineages as well as endothelial cells that revascularize adult retina. We also show that recruitment of endothelial precursors to sites of ischemic injury has a significant role in neovascularization.

SDF-1 is both necessary and sufficient to promote proliferative retinopathy

Diabetic retinopathy is the leading cause of blindness in working-age adults. It is caused by oxygen starvation in the retina inducing aberrant formation of blood vessels that destroy retinal architecture. In humans, vitreal stromal cell-derived factor-1 (SDF-1) concentration increases as proliferative diabetic retinopathy progresses. Treatment of patients with triamcinolone decreases SDF-1 levels in the vitreous, with marked disease improvement. SDF-1 induces human retinal endothelial cells to increase expression of VCAM-1, a receptor for very late antigen-4 found on many hematopoietic progenitors, and reduce tight cellular junctions by reducing occludin expression. Both changes would serve to recruit hematopoietic and endothelial progenitor cells along an SDF-1 gradient. We have shown, using a murine model of proliferative adult retinopathy, that the majority of new vessels formed in response to oxygen starvation originate from hematopoietic stem cell-derived endothelial progenitor cells. We now show that the levels of SDF-1 found in patients with proliferative retinopathy induce retinopathy in our murine model. Intravitreal injection of blocking antibodies to SDF-1 prevented retinal neovascularization in our murine model, even in the presence of exogenous VEGF. Together, these data demonstrate that SDF-1 plays a major role in proliferative retinopathy and may be an ideal target for the prevention of proliferative retinopathy.

Insulin-like growth factor I acts as an angiogenic agent in rabbit cornea and retina: comparative studies with basic fibroblast growth factor

SummaryThe release of growth factors from ischaemic retina has been hypothesized as the central stimulus for retinal neovascularization in proliferative diabetic retinopathy. Two of the growth factors implicated are insulin-like growth factor-I and basic fibroblast growth factor. We examined the effect of insulin-like growth factor-I on in vivo neovascularization using the established angiogenic model of the rabbit cornea (n=30), and also compared the effects of insulin-like growth factor-I and basic fibroblast growth factor using two new in vivo systems. Either supraphysiologic concentrations of each growth factor (600 μg) were injected intravitreally into pigmented rabbits (n=21) or porous polyfluorotetraethylene chambers filled with an emulsion containing collagen and growth factor (500 ng) were placed on the retina surface (n=8). Our results demonstrate that when insulin-like growth factor-I was implanted together with a slow release carrier into the pocket of the normally avascular cornea, insulin-like growth factor-I (10 μg/pellet) induced angiogenesis in all rabbits. This degree of angiogenesis was comparable to that previously shown for basic fibroblast growth factor. For the intravitreal studies, the fibrotic component was greater in the basic fibroblast growth factor injected eyes, whereas the vascular component was accentuated in the eyes injected with insulin-like growth factor-I. Light and electron microscopy demonstrated areas of vascular proliferation in both groups. Porous polyfluorotetraethylene chamber studies with insulin-like growth factor-I and basic fibroblast growth factor demonstrated vascular proliferation in the vicinity of the chamber similar to the intravitreal injected eyes, but to a lesser degree than the injected eyes. Our experiments overall support the angiogenic potential of both insulin-like growth factor-I and basic fibroblast growth factor and support distinct but complimentary roles for each growth factor in the pathogenesis of proliferative diabetic retinopathy.

The role of growth factors in the pathogenesis of diabetic retinopathy.

Diabetic retinopathy (DR) is the most severe of several ocular complications of diabetes. The earliest clinical signs of DR are microaneurysms and haemorrhages. Later signs include dilated, tortuous irregular veins and retinal non-profusion, leading to retinal ischaemia that ultimately results in neovascularisation. Diabetic macular oedema, which involves the breakdown of the blood–retinal barrier, also occurs and is responsible for a major part of vision loss, particularly in Type 2 diabetes. The pathogenesis of DR is very complex. Many biochemical mechanisms have been proposed as explanations for the development and progression of DR. Chronic hyperglycaemia leads to oxidative injury, microthrombi formation, cell adhesion molecule activation, leukostasis and cytokine activation. Next, ischaemia-mediated overexpression of growth factors and cytokines occurs. These factors include vascular endothelial growth factor, insulin-like growth factor-1, angiopoetin-1 and -2, stromal-derived factor-1, fibroblast growth factor-2 and tumour necrosis factor. Because of the complex interplay between these factors, targeting a single growth factor will be unlikely to result in therapeutic inhibition of angiogenesis. These growth factors no doubt act in synergy to mediate the steps of angiogenesis, including protease production, endothelial cell proliferation, migration and tube formation. This review attempts to provide an overview of perspectives regarding the pathogenesis of this disease. The focus, however, is on describing the unique features of selected relevant factors and how each growth factor may act in a synergistic manner with other factors.

Insulin-Like Growth Factor Binding Protein-3 Mediates Vascular Repair by Enhancing Nitric Oxide Generation

Rationale: Insulin-like growth factor binding protein (IGFBP)-3 modulates vascular development by regulating endothelial progenitor cell (EPC) behavior, specifically stimulating EPC cell migration. This study was undertaken to investigate the mechanism of IGFBP-3 effects on EPC function and how IGFBP-3 mediates cytoprotection following vascular injury. Objective: To examine the mechanism of IGFBP-3–mediated repair following vascular injury. Methods and Results: We used 2 complementary vascular injury models: laser occlusion of retinal vessels in adult green fluorescent protein (GFP) chimeric mice and oxygen-induced retinopathy in mouse pups. Intravitreal injection of IGFBP-3–expressing plasmid into lasered GFP chimeric mice stimulated homing of EPCs, whereas reversing ischemia induced increases in macrophage infiltration. IGFBP-3 also reduced the retinal ceramide/sphingomyelin ratio that was increased following laser injury. In the OIR model, IGFBP-3 prevented cell death of resident vascular endothelial cells and EPCs, while simultaneously increasing astrocytic ensheathment of vessels. For EPCs to orchestrate repair, these cells must migrate into ischemic tissue. This migratory ability is mediated, in part, by endogenous NO generation. Thus, we asked whether the migratory effects of IGFBP-3 were attributable to stimulation of NO generation. IGFBP-3 increased endothelial NO synthase expression in human EPCs leading to NO generation. IGFBP-3 exposure also led to the redistribution of vasodilator-stimulated phosphoprotein, an NO regulated protein critical for cell migration. IGFBP-3–mediated NO generation required high-density lipoprotein receptor activation and stimulation of phosphatidylinositol 3-kinase/Akt pathway. Conclusion: These studies support consideration of IGFBP-3 as a novel agent to restore the function of injured vasculature and restore NO generation.

Central retinal and posterior ciliary artery occlusion after particle embolization of the external carotid artery system

A 15-year-old boy underwent neuroradiologic embolization of the left internal maxillary artery with polyvinyl alcohol to stop traumatic epistaxis after failure of surgical clipping and nasal packing. Selective catheterization of the external carotid artery before embolization showed a faint choroidal blush. Although the procedure provided hemostasis, embolization to the central retinal artery and ciliary arteries resulted in loss of vision. The route of the emboli to the eye was via the anastomotic network of the lacrimal artery supplied by the external carotid artery system. Neuroradiologic embolization of the external carotid artery is an effective mode of therapy for dural-cavernous fistulas when fed by the external carotid artery system. Because the blood flow to the brain and eye is predominantly supplied by the internal carotid artery, embolization of the external carotid artery is considered relatively safe. The authors document the importance of recognition of the choroidal blush during selective external carotid artery angiography as a sign of collateral blood flow to the eye. Physicians and patients need to be aware of the risk of blindness as a complication of external carotid artery embolization when this sign is present.

Endogenous Klebsiella Endophthahnitis: Report of Two Cases and Review of the Literature

Background: Endogenous endophthalmitis is a rare complication of gram-negative sepsis. The diagnosis often is delayed unless a high index of suspicion exists. Familiarity with the clinical setting in which endogenous Klebsiella endophthalmitis occurs will hasten diagnosis and may improve outcome. Methods: The authors reviewed the medical literature and describe two patients with diabetes who lost vision from endogenous Klebsiella endophthalmitis. Findings: One patient lost all vision in both eyes before the diagnosis of sepsis was clinically suspected. In the other, endophthalmitis was the only clinical sign of septicemia 8 days after hospital discharge for treatment of a Klebsiella urinary tract infection. Survey of the literature showed an increase in the number of cases of endogenous Klebsiella endophthalmitis reported over the last 12 years. Sixty-one percent of the 44 patients reported in the literature since 1981 had diabetes, 68% had suppurative liver disease, and 16% had urinary tract infection. Conclusion: An important implication of these associations is that metastatic Klebsiella endophthalmitis must be considered when vitritis occurs in patients with diabetes, particularly in those with acute liver disease or a urinary tract infection, or in any patient with vitritis and suppurative liver disease.

The Contribution of Adult Hematopoietic Stem Cells to Retinal Neovascularization

Retinal neovascular diseases such as diabetic retinopathy and retinopathy of prematurity are among the leading causes of vision impairment throughout the world. Retinal neovascularization is thought to occur in response to a hypoxic insult, which leads to changes in the existing microvasculature such as pericyte death and subsequent endothelial cell proliferation.1.2 Compensatory neovascularization then results in the formation of aberrant and pathologic capillaries. An important question whose answer would have broad implications for potential therapeutic strategies is the origin of the cells responsible for compensatory neovascularization.

Novel Protective Properties of IGFBP-3 Result in Enhanced Pericyte Ensheathment, Reduced Microglial Activation, Increased Microglial Apoptosis, and Neuronal Protection after Ischemic Retinal Injury

This study was conducted to determine the perivascular cell responses to increased endothelial cell expression of insulin-like growth factor binding protein-3 (IGFBP-3) in mouse retina. The contribution of bone marrow cells in the IGFBP-3-mediated response was examined using green fluorescent protein-positive (GFP(+)) adult chimeric mice subjected to laser-induced retinal vessel occlusion injury. Intravitreal injection of an endothelial-specific IGFBP-3-expressing plasmid resulted in increased differentiation of GFP(+) hematopoietic stem cells (HSCs) into pericytes and astrocytes as determined by immunohistochemical analysis. Administration of IGFBP-3 plasmid to mouse pups that underwent the oxygen-induced retinopathy model resulted in increased pericyte ensheathment and reduced pericyte apoptosis in the developing retina. Increased IGFBP-3 expression reduced the number of activated microglial cells and decreased apoptosis of neuronal cells in the oxygen-induced retinopathy model. In summary, IGFBP-3 increased differentiation of GFP(+) HSCs into pericytes and astrocytes while increasing vascular ensheathment of pericytes and decreasing apoptosis of pericytes and retinal neurons. All of these cytoprotective effects exhibited by IGFBP-3 overexpression can result in a more stable retinal vascular bed. Thus, endothelial expression of IGFBP-3 may represent a physiologic response to injury and may represent a therapeutic strategy for the treatment of ischemic vascular eye diseases, such as diabetic retinopathy and retinopathy of prematurity.

Free Insulin-like Growth Factor Binding Protein-3 (IGFBP-3) Reduces Retinal Vascular Permeability in Association with a Reduction of Acid Sphingomyelinase (ASMase)

PURPOSE To examine the effect of free insulin-like growth factor (IGF) binding protein-3 (IGFBP-3), independent of the effect of insulin-like growth factors, in modulating retinal vascular permeability. METHODS We assessed the ability of a form of IGFBP-3 that does not bind IGF-1 (IGFBP-3NB), to regulate the blood retinal barrier (BRB) using two distinct experimental mouse models, laser-induced retinal vessel injury and vascular endothelial growth factor (VEGF)-induced retinal vascular permeability. Additionally, in vitro studies were conducted. In the animal models, BRB permeability was quantified by intravenous injection of fluorescein labeled serum albumin followed by digital confocal image analysis of retinal flat-mounts. Claudin-5 and vascular endothelial-cadherin (VE-cadherin) localization at interendothelial junctions was studied by immunofluorescence. In vitro changes in transendothelial electrical resistance (TEER) and flux of fluorescent dextran in bovine retinal endothelial monolayers (BREC) were measured after IGFBP-3NB treatment. Acid (ASMase) and neutral (NSMase) sphingomyelinase mRNA levels and activity were measured in mouse retinas. RESULTS Four days postinjury, laser-injured mouse retinas injected with IGFBP-3NB plasmid demonstrated reduced vascular permeability compared with retinas of laser-injured mouse retinas injected with control plasmid. IGFBP-3NB administration resulted in a significant decrease in laser injury-associated increases in ASMase and NSMase mRNA and activity when compared with laser alone treated mice. In vivo, intravitreal injection of IGFBP-3NB reduced vascular leakage associated with intravitreal VEGF injection. IGFBP-3NB partially restored VEGF-induced in vivo permeability and dissociation of claudin-5 and VE-cadherin at junctional complexes. When IGFBP-3NB was applied basally to bovine retinal endothelial cells (BREC) in vitro, TEER increased and macromolecular flux decreased. CONCLUSIONS Intravitreal administration of IGFBP-3NB preserves junctional integrity in the presence of VEGF or laser injury by reducing BRB permeability in part by modulating sphingomyelinase levels.