Ординатура / Офтальмология / Учебные материалы / Retinal Vascular Disease Joussen Springer
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4 Hematopoietic Stem Cells in Vascular Development and Ocular Neovascularization |
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Fig. 4.6. Retinal neovascularization. The top panel (original image ×4) shows a composite image of a neural retina from a chimeric mouse that had branch retinal vein occlusion. The bottom image (original image ×100) shows a close-up from the same tissue. The red fluorescence shows blood vessels while the green fluorescence shows HSC-derived cells. The colocalization between the two channels is especially obvious in the lower panel, indicating that HSCs do differentiate into blood vessels
dilated and branch retinal vessel occlusion is performed by laser photocoagulation. An argon green laser system (HGM Corporation, Salt Lake City, UT) is used for photocoagulation with the aid of a 78diopter lens; the laser is applied to selected venous sites next to the optic nerve. Venous occlusion (approximately 50 – 100 burns) is accomplished using laser parameters of 1-s duration, 50 μm spot
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Fig. 4.7. Iris neovascularization after injection of various growth factors and laser photocoagulation. The top panel shows a low magnification (original ×10) while the bottom panel shows a high magnification (original ×40). Both show rhoda- mine-dextran in the red channel and gfp+ cells in the green channel. Note the large number of gfp+ cells in both images, especially in the bottom panel, where they can be seen incorporating with the resident vasculature
size, and 50 – 100 mW intensity. Three weeks after laser injury, mice are killed and their eyes are enucleated. The eyes may then be examined immunohistochemically (flat-mounts and cryosections) using epifluorescence and confocal microscopy for the presence of gfp+ cells and their colocalization with endothelial cells in the neural retina (Fig. 4.6) [51].
4.9.2 Iris Neovascularization
Chimeric mice are also used to induce iris neovascularization. A cocktail of growth factors including VEGF, IGF-1, and FGF2 are injected intravitreally at the time of laser. An argon green laser is used with a 78-diopter lens at an intensity of 150 mW. Multiple burns are applied to completely occlude the branch
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Mountain View, CA) coupled to a slit lamp set to a 100 μm spot focused on the retinal pigment epithelium (RPE) is used to deliver a 250 mW pulse for 0.1 s. Three burns are applied approximately one disk diameter from the optic nerve in three quadrants. This laser application results in a bubble, indicative of Bruch’s membrane rupture, more than 95 % of the time. Neovascularization of the choriocapillaris occurs within this lesion and peaks at 2 – 3 weeks after injury, at which time the animals are put to death and the eyes removed and fixed. For flatmount examination the anterior segment is dissected and discarded, and the neural retina is removed. Figure 4.8 depicts a typical flat-mounted posterior cup that was reacted with rhodamine-conjugated Ricinus communis agglutinin I (Vector Laboratories, Burlingame, VT) to delineate vasculature, and shows colocalization of gfp+ HSC-derived cells with the CNV [119].
Fig. 4.8. Choroidal neovascularization. The top panel shows a CNV lesion from a chimeric mouse in which Bruch’s membrane was ruptured. The bottom image shows a close-up from the same tissue. The red fluorescence shows blood vessels while the green fluorescence shows HSC-derived cells. The colocalization between the two channels is especially obvious in the lower panel
vessels to stimulate compensatory neovascularization in the iris. Peak neovascularization is seen 4 weeks after the laser injury (Fig. 4.7).
4.9.3 Choroidal Neovascularization
To induce choroidal neovascularization (CNV), stably reconstituted mice are anesthetized and their eyes dilated and subjected to laser injury in a manner similar to Ryan’s [113]. A 532 nm laser (Iridex,
4.10 Conclusion
Ocular neovascular diseases such as ROP, PDR, and ARMD affect all age groups and cause vision impairment for millions in developed nations. It was believed that aberrant neovascularization as in these diseases was from the migration, proliferation, and organization of resident endothelial cells. However, it is now known that HSCs participate in postnatal neovascularization along with resident cells, signifying that neovascularization consists of both angiogenesis and vasculogenesis.
Soon after fertilization, stem/precursor cells are in the blood islands of the yolk sac. Upon further development, the cells then reside in the AGM, the blood, the fetal liver, and eventually the bone marrow. After activation, bone marrow HSCs proliferate and differentiate into progenitor cells that are found in the peripheral blood [59]. The progenitor cells can then differentiate further if needed. Also, in order to be considered a true stem cell, the HSCs must yield undifferentiated progeny with identical characteristics. HSCs are now known to have hemangioblast activity, differentiating into all of the components of the vascular system. It has been shown that HSCs also have the potential to differentiate into several other types of tissue, such as liver, muscle and neurons, demonstrating their plasticity.
In addition to plasticity, stem cells are also characterized by their self-renewal capacity. Although stem cells are considered an endless source for tissue or organ regeneration, the extent has not yet been quantified and is thus debatable. Most researchers would consider the most reliable indicator of selfrenewal capacity to be the long-term multilineage repopulating activity, detectable by transplantation
4 Hematopoietic Stem Cells in Vascular Development and Ocular Neovascularization
experiments. The capacity of HSCs to self-renew is quite heterogeneous. This translates to differences in proliferation capacity and multilineage differentiation capacity. This may provide an explanation as to why bone marrow cells cannot be serially transplanted in mice indefinitely. Homing could also influence the repopulating ability of HSCs. In some systems, stem cell plasticity has been explained by cell fusion. Donor-derived cells fused with differentiated cells in resident tissue, leading to cells with double the normal number of chromosomes in their nuclei. However, fusion is not thought to account for all issues of (trans)differentiation, especially in the eye.
As well as being plastic and self-renewing, stem cells are also thought to occupy niches. The qualities of the niche are thought to promote stem cell quiescence, maintenance or expansion. Interaction of HSCs with their niche is thought to be necessary for adult hematopoiesis in the bone marrow. HSCs must maintain a balance between quiescence and selfrenewal in the stem cell niche as well as maintain long-term hematopoiesis. Cell engraftment after transplantation depends upon the physical availability of niche space, as well as homing signals. The spatial requirement is usually met through either chemical or radiological bone marrow ablation. Some of the same signals that induce HSC mobilization from the bone marrow are also involved in repopulating the bone marrow niche.
Stem cells must leave their bone marrow niche to respond to injury/ischemia. Based upon stimuli (such as SDF-1) outside of the bone marrow, the HSCs can be mobilized and released into the circulation. They can then home to where they are needed, often an ischemic area, and then differentiate at some point between their origin and their destination. This homing often involves the binding of cell surface ligands and receptors, either of which can be on the HSCs.
In order to examine intricacies of HSCs, they often must be removed and purified from the entire bone marrow population. Characterizing HSCs involves identifying specific cell populations with antibodies conjugated to either magnetic particles or fluorochromes, and then sorting the cells thus identified using MACS or FACS. HSCs and their progeny can be identified immunologically through the presence or lack of certain cell surface markers, the composition of which changes during differentiation. Sca-1 and CD117 are most often used to distinguish HSCs from other cell types. The presence of these two markers yields a population of cells that is 95 % HSCs. Isolated cell lines have been used to study many aspects of stem cell behavior, especially characterizing the growth factors responsible for cell maintenance and
differentiation in vitro. In addition stromal cell lines have been reported to support the maintenance of HSCs in culture.
We used murine HSCs as a tool to study stem cell maintenance, migration and differentiation. Since a hallmark of PDR and wet ARMD is ocular neovascularization, we mimicked this by laser injury. In both models, gfp chimeric mice provided a technique to distinguish resident vasculature from the donorderived contribution to the neovascularization. Our studies that use HSCs for transplantation support that these stem cells significantly contribute to neovasculature; these can then be used for examining the mechanisms underlying stem cell homing, differentiation, and involvement in ocular neovascularization.
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5 Inflammation as a Stimulus for Vascular Leakage |
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A.M. Joussen, A.P. Adamis |
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Core Messages
Diabetic retinopathy shows many of the characteristics of an inflammatory disease
Diabetic retinal vascular leakage, capillary nonperfusion, and endothelial cell damage are caused, in part, by retinal leukocyte stasis in early experimental diabetes
Vascular endothelial growth factor (VEGF) plays key roles in mediating both ischemiarelated neovascularization as well as retinal leukocyte stasis
Leukocytes adhere to the retinal vasculature via intercellular adhesion molecule-1 (ICAM-1) and CD18
FasL mediated apoptosis is involved in vascular remodeling upon ischemia and diabetes These pathological processes are similar to
those underlying the leukocyte-mediated pruning of the retinal vasculature during normal development
In the past few decades, our knowledge of the mechanisms underlying retinal vasoproliferation has increased greatly (see Chapters 2, 3.1, 3.2 and 3.3). While vasoproliferation was once considered to be mainly a consequence of ischemia, current evidence also supports a contribution of inflammatory mechanisms. Inflammation is also highly related to vascular leakage in diseases that are known to result in retinal and macular edema. Recently, inflammatory mechanisms have gained interest with respect to the retinal pathology following ischemia, as well as in diseases such as diabetic retinopathy (DR) and sickle cell retinopathy (see Chapter 27.1). In this chapter, the discussion will focus on the published data relating to the inflammatory mechanisms in ischemic retinal diseases such as DR. The definition of inflammation in this setting is the involvement of any leuko- cyte-mediated pathology in the course of the disease.
We examined several lines of evidence, including correlative studies of elevated levels of inflammatory mediators in the presence of DR and the impact of anti-inflammatory agents on the disease. As a central focus, we will discuss in detail a series of preclinical studies that support a causal linkage between inflammation and two principal characteristics of the pathology associated with DR, ischemia-linked neovascularization and the breakdown of the blood-reti- nal barrier (BRB), together with the role of VEGF in mediating these events. These studies have provided
good evidence that the vascular damage that is seen in DR is mediated by processes that are very similar to those that regulate retinal vascularization during normal development.
5.1Evidence for Inflammation in the Pathogenesis of Diabetic Retinopathy
Essentials
Inflammatory mediators are upregulated in DR
Diabetic retinal pathology can be inhibited by anti-inflammatory agents
VEGF is a key mediator of inflammatory changes in the diabetic retina Angiopoietin-1 regulates vascular permeability and expression of inflammatory mediators in diabetic retinopathy
5.1.1Upregulation of Inflammatory Mediators in Diabetic Retinopathy
Both clinical and preclinical studies have associated the development of DR with elevated ocular levels of inflammatory mediators. McLeod et al. [70] reported that levels of intercellular adhesion molecule-1 (ICAM-1), an important adhesive molecule for circu-
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lating leukocytes, were elevated throughout the vasculature of diabetic patients, whereas the distribution was much more restricted in nondiabetic subjects; moreover, this elevation was accompanied by a significantly higher number of neutrophils in both the choroid and retina. Limb et al. [65] reported that levels of ICAM-1 as well as other adhesion molecules such as vascular cell adhesion molecule (VCAM-1) and E-selectin were elevated in patients with proliferative DR, while Funatsu et al. [33] have also reported elevated levels of ICAM-1 in patients with diabetic macular edema (DME). These patients also showed elevated vitreous levels of vascular endothelial growth factor (VEGF) [33], which upregulates ICAM-1 expression [107]. As discussed below, VEGF may in fact be a key factor mediating inflammatory events in the diabetic eye, and DR-correlated elevation of VEGF levels was first reported over a decade ago [1, 4]. Since then diabetes-associated elevations of VEGF in the vitreous have been reported by a number of groups, together with increases in a variety of other factors, including interleukin-6 [31], angiotensin II [32], angiopoietin 2 [109], erythropoietin [110] and stroma-derived factor-1 (SDF-1). SDF- 1 is itself a stimulator of VEGF expression [14] and an important mediator of cell migration and adhesion [60].
Tumor necrosis factor-
(TNF-
) is a proinflammatory cytokine that has also been implicated in the pathogenesis of diabetic retinopathy [64, 66, 92]; moreover, susceptibility to diabetic retinopathy has been associated with TNF-
gene polymorphism [39]. TNF-
is found in the extracellular matrix, endothelium, and vessel walls of fibrovascular tissue in proliferative diabetic retinopathy [64], and in the vitreous from eyes with this complication [29, 101]. TNF-
can stimulate VEGF expression by the retinal pigment epithelium [84] and in choroidal neovascular membranes [38], and has been implicated as an inducer of pathological angiogenesis in the retina [34].
The evidence provided by these correlative measurements of inflammatory mediators has been supplemented by other approaches. The advent of highdensity microarray technology [15, 21, 48, 100], with its capacity for simultaneous monitoring of thousands of genes, provides a unique opportunity for a high-throughput analysis of diabetic retinopathy at the molecular level. In an analysis of retinal gene expression in streptozotocin-induced diabetes in rats, numerous genes operative in inflammatory reactions were found to be upregulated [51]. Prominent among these were the genes for macrophage migration inhibitory factor (MIF), a proinflammatory lymphokine that is believed to be involved in maintaining neutrophils in the vasculature and in
facilitating their adhesion and local release of cytokines [82, 93], as well as a number of genes for adhesion molecules and apoptosis. While the findings from these approaches are purely correlative, and are not able to differentiate between potential molecular mechanisms, they nonetheless can provide important clues as to the nature of processes that may be involved in the pathogenesis of DR.
Finally, correlative studies have also been carried out examining the levels of serum factors in patients with DR [73]. These workers reported that the levels of the chemokines RANTES (Regulated on Activation, Normal T-cell Expressed and Secreted) and SDF-1
were significantly elevated in patients with at least severe nonproliferative diabetic retinopathy compared to patients with less severe diabetic retinopathy. Positive immunostaining was observed in the inner retina for RANTES and monocyte chemoattractant protein-1 (MCP-1) in patients with diabetes. In keeping with earlier findings, staining was also strongly positive throughout the diabetic retina for ICAM-1, while normal retinal tissues showed little reactivity.
5.1.2Diabetic Retinal Pathology Can Be Inhibited by Anti-inflammatory Agents
Induction of adhesion molecules on endothelial cells by proinflammatory cytokines such as TNF-
[65] and VEGF is mediated at the molecular level by the activation of a redox-sensitive transcription factor, nuclear factor (NF)-κB [108]. NF-κB upregulates ICAM-1 and various inflammatory genes such as cyclooxygenase (COX)-2 [63]. The cyclooxygenases COX-1 and COX-2 are key enzymes in the conversion of arachidonic acid to prostaglandin H2, the common precursor for all other eicosanoids. COX-1 is expressed ubiquitously and generates eicosanoids with cytoprotective function whereas COX-2 is an immediate-early gene expressed at sites of acute inflammation and generates eicosanoids with a proinflammatory role that create a positive feedback loop by further activating NF-κB and inflammatory cytokines [69, 85].
The observation that arthritic diabetic patients receiving high daily doses of aspirin exhibit reduced symptoms of diabetic retinopathy led to the hypothesis that anti-inflammatory treatment could prove beneficial [88]. Aspirin, in low doses (8 mg/day), inhibits platelet aggregation, predominantly via acetylation of COX-1 and reduction of thromboxane A2 production. In intermediate doses (2 – 4 g/day), aspirin inhibits both COX-1 and COX-2, blocking prostaglandin production, and is antipyretic [86]. In high doses (6 – 8 g/day), it is a potent anti-inflamma- tory drug suitable for the treatment of rheumatic dis-