Ординатура / Офтальмология / Учебные материалы / Retinal Vascular Disease Joussen Springer

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Fig. 1.39. Trypsin digest autoradiogram of experimental photocoagulation burn (B) following intravitreal injection of tritiated thymidine to mark dividing cells. The involved microvasculature is acellular and represented by pale tubes of residual basement membrane. Darkly stained mononuclear and endothelial cells surrounding the burn are decorated with silver grains (arrows). Labeled monocytes (arrows) are also present in the wall of a remote venule (V)

Fig. 1.40. Trypsin digest of retinal capillaries from a rat that had received 20 Gy of X-radiation to the eye. A confluent group of capillaries show numerous darkly stained pericyte nuclei (arrows) but no surviving endothelial cells. Electron microscopy confirmed absence of endothelium in such vessels

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Fig. 1.42. Postsurgical cystoid macular edema. Incompetent capillaries pool dye, in the inner plexiform layer at the macula (M): choroidal folds (arrows) reflect hypotony

Established retinal perivasculitis is associated with focal damage to vessel walls (usually venous) and leakage of serum proteins and lipids into the extravascular space, e.g., sarcoidosis and multiple sclerosis. Following resolution of the initiating inflammatory process, veins may show residual alterations in caliber and collaterals can remain, perhaps in association with areas of capillary closure, especially if the obstruction during perivasculitis has been severe. Such retinal microvascular pathology is also common in conditions such as severe retinitis toxoplasmosis (Fig. 1.43) or herpesvirus infections, which again can lead to capillary occlusion at the site of acute inflammation. Extensive retinal phlebitis

and occlusion associated with Eales’ disease and sarcoidosis may precipitate pre-retinal neovascularization/vitreous hemorrhage.

1.2.9 Retinal-Choroidal Tumors

Pathological evaluation shows that the retinal microvasculature may play an important role in growth and metastasis of retinal and optic disk tumors. This microcirculation also reacts to localized release of vasoactive agents released from large, proximal choroidal tumors.

Optic disk tumors: these structures attract retinal vessels from superficial capillaries of the optic nerve head (Fig. 1.44). The associated retinal vessels enlarge as the metabolic requirements of the expanding tumor increase. In some circumstances, the normally “tight” vessels can become fenestrated and lose tight junction integrity, probably in response to high concentrations of VEGF in the microenvironment. The accompanying breakdown of iBRB accounts for exudates into the neural retina and vitreous.

Highly vascularized retinal tumors such as angiomata recruit their blood supply from the adjacent retinal microvasculature. As tumor oxygen requirements increase, retinal feeder vessels develop to provide the necessary nutritional support. The tumor vessels are fenestrated, irregular and incompetent, while larger hyperdynamic feeder vessels (within the neural retina) are typically competent. Some vascular tumors with “in-built shunting systems” have “arterialized” venous systems. Large vascular tumors are grossly incompetent and lipid/protein rich

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Fig. 1.45. a Large retinoblastoma posterior pole of the left eye (R). b Electron micrograph of a cluster of new vessels lying beneath the internal limiting membrane (ILM) in edematous retina adjacent to the tumor depicted in a.

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Fig. 1.46. Retained intraocular metallic foreign body left inferior fundus. Retinal vessels in the vicinity of the foreign body (FB) are grossly attenuated or absent (arrows)

1.2.10Primary Neuropile Atrophy and Degeneration

Normal function of the retinal circulation is completely dependent on intimate cell-cell communication with neural and glial elements of the retina. Neuroglial degenerative disorders can deprive the retinal microvascular component cells of trophic factors necessary for functionality and survival, e.g., NFGR, PDGF, VEGF, and BDGF. In retinitis pigmentosa, trauma, toxic retinopathy, and loss of retinal parenchyma is associated with retinal capillary cell attrition, closure of capillary beds, narrowing of supply vessels and involutional sclerosis of larger radicals (Fig. 1.46).

1.2.11Remote Effects of Retinal Vascular Pathology

Retinal hypoxia may lead to elevated vitreous levels of angiogenic factors such as VEGF and these can have a pathogenic influence on the other ocular structures. Iris neovascularization is a clear example of this phenomenon. Vitrectomy can also cause localized vasoproliferation, especially in diabetic retinopathy where the compromised retinal circulation may develop fibrovascular responses at the site of the vitrectomy port. Complications include traction, retinal detachment, proliferative vitreoretinopathy and iris neovascularization.

Peripheral retinal vasoproliferation, as in the case of retinopathy of prematurity, can also impact on the iris, ciliary body, lens and angle anterior chamber with inflammatory and traction effects.

In summary the retinal vasculature shows a common range of responses to diverse pathological stimuli. This normally quiescent system demonstrates a remarkable ability to recover from obstructive events induced by factors ranging from radiation injury and metabolic imbalance to immune cell inundation and gross hemodynamic insult. In such situations it is physiological recanalisation and other forms of vascular remodelling that predominante. However, when this regenerative pattern is altered by chronic hypoxia/ischemia more threatening aberrations are manifest: change of endothelial phenotype, chronic neuroretinal oedema and vitreo-retinal neovascularization. In recent years advances made possible through molecular cell biology have provided us with new perspectives on the morphological changes induced by disease in the retinal vasculature. We have also gained an increased awareness of the unity of the neuro-vas- cular complex of the retina: a uniquue branch of the central nervous system imposing an equally specialised phenotype on the vasculature that serves it.

References

1.Alder VA, Su EN, et al. (1998) Overview of studies on metabolic and vascular regulatory changes in early diabetic retinopathy. Aust N Z J Ophthalmol 26(2):141 – 148

2.Alon T, Hemo I, et al. (1995) Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nat Med 1(10):1024 – 1028

3.Antonetti DA, Lieth E, et al. (1999) Molecular mechanisms of vascular permeability in diabetic retinopathy. Semin Ophthalmol 14(4):240 – 248

4.Beltramo E, Pomero F, et al. (2002) Pericyte adhesion is impaired on extracellular matrix produced by endothelial cells in high hexose concentrations. Diabetologia 45(3): 416 – 419

5.Bresnick GH, Davis MD, et al. (1977) Clinicopathologic correlations in diabetic retinopathy. II. Clinical and histologic appearances of retinal capillary microaneurysms. Arch Ophthalmol 95(7):1215 – 1220

6.Chakravarthy U, Gardiner TA (1999) Endothelium-derived agents in pericyte function/dysfunction. Prog Retin Eye Res 18(4):511 – 527

7.Chan-Ling T, Gock B, et al. (1995) The effect of oxygen on vasoformative cell division. Evidence that ’physiological hypoxia’ is the stimulus for normal retinal vasculogenesis. Invest Ophthalmol Vis Sci 36(7):1201 – 1214

8.Claxton S, Fruttiger M (2003) Role of arteries in oxygen induced vaso-obliteration. Exp Eye Res 77(3):305 – 311

9.Dery MA, Michaud MD, et al. (2005) Hypoxia-inducible factor 1: regulation by hypoxic and non-hypoxic activators. Int J Biochem Cell Biol 37(3):535 – 540

10.Frank RN, Dutta S, et al. (1987) Pericyte coverage is greater in the retinal than in the cerebral capillaries of the rat. Invest Ophthalmol Vis Sci 28(7):1086 – 1091

11.Gardiner TA, Stitt AW, et al. (1994) Selective loss of vascular smooth muscle cells in the retinal microcirculation of diabetic dogs. Br J Ophthalmol 78(1):54 – 60

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Core Messages

The retinal vasculature is highly ordered and the formation of the normal vascular plexuses involves the complex interactions of numerous cell types, including endothelial cells, glial cells, pericytes, and myeloid cells Understanding the process of normal vascular development relates to understanding pathological neovascularization

Endothelial cell guidance during retinal vascular development is mediated by an astrocytic template, filopodial extensions from the developing endothelial cells, and cell-cell adhesion

Retinal vascular development models can be used effectively to test the activity of clinical and pre-clinical compounds on neovascularization

2.1 Introduction

2.1.1 General Vascular Development

The circulatory system evolved so that nutrients and chemicals required for cellular function can be efficiently transferred from central organs to the extremities. Because of its importance to the growth and survival of other tissues, the circulatory system forms during early stages of development, and its correct development and early function is absolutely critical for survival of the embryo. During development, blood vessel formation occurs by three processes, the initial formation of vessels from yolk sacs during early embryogenesis, and by the distinct processes of vasculogenesis and angiogenesis during subsequent development [33, 35].

Yolk Sacs. Angiogenic clusters containing hematopoietic cells (blood precursors) at the center and angioblasts (endothelial cell precursors) lining the periphery. These cells, originally differentiating from mesoderm cells, form the earliest identifiable blood vessels.

Vasculogenesis. The assembly of vessels from separate endothelial precursor cells (angioblasts) as they differentiate into mature endothelial cells.

Angiogenesis. The formation of new blood vessels from preexisting capillaries. Differentiated endothelial cells are induced to proliferate, thus facilitating the sprouting of new vessels from existing vessels.

Clinically, blood vessels of the cardiovascular system are identified mainly on the basis of the tissue in which they are found, their position relative to other vessels, the lumen size, and the pattern of branching. This system of identification is possible because of the consistency with which vascular patterns are formed from individual to individual, and even across various mammalian species.

2.1.2Basis of Clinical Identification of Blood Vessels

Essentials

The tissue that the vessels supply (i.e., retinal vasculature, brain vasculature, etc.)

Position within the vascular tree (i.e., arterial, venous, or capillary)

Lumen size Branching patterns

Historically, research investigating the vascular development has primarily focused on the creation of the basic structural unit, the endothelial lined tube. However, as numerous molecules are continuously added to the growing list of neovascular factors [6, 15, 35], the complexity of this process is becoming evident. This complexity highlights the limitations of a conceptual framework that defines vascular development solely in terms of creating the blood vessel unit. Instead, a complete analysis of the

morphogenetic events that occur beyond endothelial cell development is required to fully understand neovascular processes. These events include vessel branching, vessel guidance, recruitment of vascularassociated non-endothelial cells (mural cells), and specification of vessel identity. As a whole, these processes are critical to neovascularization of the eye, both during development and neovascular disease progression.

2.1.3Major Cellular Components of Vessel Formation

Essentials

Endothelial cells: form the vessel wall creating the lumen through which blood flows

Mural cells: perivascular cells that associate with the vessels. Critical for maturation and maintenance of the functioning vasculature (e.g., pericytes, perivascular macrophages, astroglial cells)

Hematopoietic cells: blood lineage cells that flow through the vessel lumen. In addition to their normal roles, these cells can play important roles during initiation, maturation, and regression of blood vessels.

2.2 Endothelial Cells

Endothelial cells, the cells that form the actual blood vessel wall, are generally the major cell type referred to during discussions of neovascularization. During neovascular development, these cells must undergo proliferation, migration, and final maturation to form a new mature vessel. As embryonic blood vessels become established and various tissues differentiate, endothelial cells continue to undergo tissuespecific changes, generating functionally distinct vascular beds. These differences are primarily defined by the types of junctions that form between adjacent endothelial cells in the vessel wall. For example, lymphatic vessels have a discontinuous or even partially absent basement membrane [22] and endothelial cells in the kidney are fenestrated so that waste can be cleared from the blood stream and efficiently removed from the body. The choroidal vasculature of the eye is also fenestrated. In the brain and to a lesser extent in the retina, tight junctions form connections between the endothelial cells resulting in the formation of the blood-brain and blood-reti- nal barriers. This barrier helps regulate the neural microenvironment by protecting the brain or retina from fluctuations in plasma composition, and by preventing circulating agents and small molecules

2 Retinal Vascular Development

from entering the tissue and disrupting neural function [1]. Many other differences between the vasculature from different tissues also become apparent during development, with each specific morphology evolutionarily tailored to the different vascular requirements for each tissue.

2.2.1Vascular Heterogeneity (Morphological Classification of Vessels)

Essentials

Type and complexity of interendothelial adhesions:

–Continuous. Tight junctions between adjacent endothelial cells in a vessel. Limited materials can pass through the vessel wall.

Examples: retina, heart, lung, brain, skeletal muscle

–Fenestrated. Fewer tight junctions between adjacent endothelial cells. Vessels are more permeable to cells and larger molecules. Examples: choroidal plexus, intestinal villi, endocrine and exocrine glands

–Discontinuous. Incomplete membrane wall. These types of vessels are often found in “filtration tissues.” Examples: hepatic sinusoids in the liver

Size of vessels (arteries or veins with large lumens vs. smaller capillaries)

Shape of individual vessels (straight vs. tortuous)

Patterning of the vascular plexus (hexagonal patterning, planar vs. three-dimensional, etc.) Presence or absence of diaphragms or valves to prevent misdirection of blood flow [8]

2.3 Mural Cells

Once a new vessel has formed, whether by vasculogenesis or angiogenesis, it must undergo maturation to become a stable, functional vessel. This involves mural cell recruitment and, ultimately, remodeling of the vascular bed. Perivascular mural cells are closely associated with vessels and lie just external to the endothelial cells. Just as vessel morphology is tissue specific, the type of associated mural cells, their morphology, and their function, also vary depending on the location of the associated vessel. For example, in arteries and larger vessels, smooth muscle cells form a multilayered sheath around the vessel wall. Smaller arterioles and venules also recruit smooth muscle cells, but mainly as a continuous

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sheet around the vessels. Unlike larger arteries, these smaller vessels contain a basement membrane layer between the endothelial cells and the associated smooth muscle cells. In general, pericytes mainly associate with, and extend long processes along, capillaries and postcapillary venules [9, 20]. The type of associated mural cells also differs by tissue. For example, astrocytes remain closely associated with the brain and retinal vasculature while Schwann glial cells are associated with the peripheral vasculature during development [27]. Appropriate recruitment of the various mural cells is important for stabilization and maturation of new vessels during developmental neovascularization. Vessels lacking mural cells have been found to be more susceptible to apoptosis and degeneration during vascular remodeling processes than vessels that have appropriately associated mural cells [3]. Mural cells are also important for maintaining vascular quiescence after vascular development is complete [28]. In fact, the loss of mural cell association has been correlated with a number of neovascular ocular diseases including diabetic retinopathy.

2.4 Vascular Patterning

The appropriate organization and development of vascular plexuses is critical for proper tissue function. The loss of these guidance mechanisms can lead to abnormal vascularization and a variety of pathological conditions. This is particularly true for the development of the retina, a highly organized neuronal tissue with perfectly layered neuronal cells and synaptic plexuses. The retina consists of many parallel, anatomically equipotent microcircuits derived from millions of neuronal cells. Each of the approximately 55 distinct cell types (for mammalian species) have different, important functions [26] and must be organized in such a way that visual information can be passed from the photoreceptors in the back of the retina, through various neurons in the inner nuclear layer, to the ganglion cells in the front of the retina, and eventually on to the brain. Thus, the neurons and their glial helper cells must be organized perfectly during development so that functional synapses can form between appropriate neurons within the correct retinal regions (Fig. 2.1).

Similarly, the retinal vasculature must also develop appropriately and must localize to specific retinal layers. Incorrect vascular organization can lead to the disruption of retinal function. In most higher order mammals, three planar vascular plexuses are formed during development; the superficial plexus forms within the ganglion cell layer at the front of the retina, and the deep and intermediate vascular plex-

uses form at the outer and inner edges of the inner nuclear layer respectively (Fig. 2.1). If any of the three vascular layers fail to form, neuronal degeneration can occur due to hypoxic stress. If the vasculature forms but the layers are disorganized, or if vessels become located within regions of the retina that normally remain avascular, such as the photoreceptor layer or the macula, these vessels can disrupt the neuronal synapses, again leading to the disruption of visual function [41]. For proof of the importance of proper vessel formation in the retina, one needs only to look at the devastating diseases that lead to a loss of vision because of abnormal neovascularization [5, 23]. Many of the ocular diseases that can cause a catastrophic loss of vision due to complications from abnormal neovascularization are reviewed extensively in this book.

As our knowledge of the processes that mediate neovascularization progresses, it is becoming increasingly clear that there are common mechanisms observed in developmental and pathological angiogenesis. In either circumstance, proliferation of endothelial cells must be initiated. The endothelial cells must then degrade the extracellular matrix, undergo guided migration, and ultimately mature into functional vessels. With such complex mechanisms to ensure proper regulation of neovascularization, it is not surprising that many of the factors involved in promoting vascular development are also implicated in pathological vascularization. Thus, by investigating the mechanisms that mediate vascular development, novel insights are also gained regarding potential treatments of vascular diseases. Differences between developmental and pathological angiogenesis are also important. In fact, these differences may prove to be even more useful for the development of novel anti-angiogenic treatments since factors that are solely implicated in pathological angiogenesis would be logical therapeutic targets. Disruption of their function would not affect vascular development or other important angiogenic processes such as wound healing, and thus they are likely to have few negative affects on the established vasculature.

It is reasonable to think that many vascular diseases arise from a loss of complex regulation mechanisms associated with normal developmental neovascularization. In fact, many of the problems associated with abnormal angiogenesis do not arise from the growth of new blood vessels per se, but from abnormalities in the neovessels such as leakiness and hemorrhaging. This is particularly true for the pathology of ocular diseases with associated neovascularization. For example, many of the characteristics of neovascularization that occur during retina development are mirrored in diabetic retinopathy. However, key problems begin to arise as neovessels

associated with diabetic retinopathy fail to recruit and maintain appropriate mural cell association. The apoptotic loss of pericytes surrounding the retinal vasculature occurs during early stages of diabetic retinopathy. This is likely to lead to reduced vessel stability, the onset of endothelial cell apoptosis, and subsequent retinal ischemia. New vessels that form as a result of this ischemia fail to properly recruit mural cells. Because these neovessels lack pericyte and normal basement membrane coverage, they do not mature or develop normally, leading to the observed vascular leakiness associated with retinal edema and vision loss [14]. Thus, understanding the developmental mechanisms that facilitate proper formation of mature vascular systems is important not only because of the parallels between developmental and pathological neovascularization, but also so similar mechanisms may be employed clinically to stabilize pathological vessels and treat diseases associated with abnormal, leaky vessels. To this extent, in various mouse models of retinal degeneration, bone marrow-derived cells have been used to stabilize degenerating vasculature [32]. Interestingly, stabilization of the degenerating vasculature also appears to facilitate rescue of degenerating cones (Fig. 2.2) [32]. In addition to their potential utility for the treatment of degenerative diseases, such therapeutic methods may also be useful for the treatment of neovascular eye diseases such as age related macular degeneration (ARMD) and diabetic retinopathy. In contrast to simply blocking the growth of new vessels using angiostatics, similar bone marrow-derived cells may be able to stabilize neovessels, allowing the underlying hypoxic drive to be relieved while eliminating the major clinical problems associated with neovessels [29].

2.5 Retinal Vascular Development

Along with the development of any tissue, the development of the retina requires the concomitant formation of a complex vascular system. In the human fetus, formation of the ocular vasculature occurs mainly during the second and third trimesters in utero and the retinal vasculature is one of the last vascular systems to form. During early development of the eye, the retina is initially supplied by the choroid and the hyaloidal vessels. However, due to the natural regression of the hyaloidal vasculature (Fig. 2.3), as well as the thickening of the retina due to the final development and differentiation of the neuronal layers, the retina eventually finds itself in a hypoxic environment and must begin to establish its own vascular system [36].

During development, retinal blood vessels form three vascular plexuses in a highly reproducible manner, thereby resulting in the formation of distinct vascular and avascular zones (Fig. 2.4). Mammalian retinas generally vascularize in similar fashions during development, albeit on different time scales; human retinal developmental vascularization begins during the second trimester in utero and continues, becoming complete only after birth. In most non-primate species, the retina is avascular at birth and developmental neovascularization occurs during the first few weeks postnatally. Another important distinction between primates and non-primates is the development of the macula, the highly dense region of cone photoreceptors where central vision occurs. This region, which forms in primates, remains avascular throughout development and into adulthood. The abnormal vascularization or leakage of fluid into the macular region is a major cause of devastating loss of vision.