Ординатура / Офтальмология / Английские материалы / Retinal and Choroidal Angiogenesis_Penn_2008

confirm the clinical grading. Attempts to standardize this model have been published from a single laboratory and have relied upon a grading scheme employing combined clinical and histopathological endpoints. An inherent weakness of grading schemes that employ clinical grading is the difficulty in adequately masking if there are clinical signs of treatment. For instance, if there is cataract or material in the vitreous of experimental eyes that is not present in the control eyes, masking is impossible. Nevertheless, despite these limitations, this model has been employed to quantitatively describe pharmacotherapeutic effects of a variety of drugs.

Despite the complicated grading scheme and the need for detailed masked histopathological analysis, this methodology has been successfully employed in several investigations of pharmacological efficacy. The pig model was first used to demonstrate the inhibition of neovascularization achieved from a single intravitreal injection of 4 mg triamcinolone.23 Another study investigating the intravitreal effects of an antisense oligonucleotide against RAF-1 kinase used a nearly identical design, except that the control eye received an intravitreal injection of vehicle only.16 In this study, drug-treated eyes showed a reduction in NV. In both of these studies, masking of clinical exams (and photographs) was difficult due to lens opacities in the antisense oligonucleotide study and vitreous material in the triamcinolone study. Assessments relied upon masked gross and histopathological observations. A study using systemic administration of a protein kinase C beta inhibitor, ruboxistaurin (LY333531), employed two groups of 10 animals with bilateral BVO.16 Since treatment groups could not be inferred from ocular signs associated with local administration, and the treatment drug and placebo control were coded, masking was ideal in this study. The median NV score was reduced by 65% in the PKC group vs. controls. Both triamcinolone and ruboxistaurin are being evaluated in human trials.

As a model in which to study pathophysiological aspects of intraocular NV, the pig BVO model has developed a strong track record. Pournaras and colleagues have employed this model to demonstrate a reduction in preretinal oxygen tension in the area of ischemia and restoration of normal oxygen tension after scatter laser treatment.20 They have also used this model to investigate the effects of vasoactive agents on tissue oxygenation and blood flow and to characterize biochemical changes post-BVO.20 Danis and colleagues have assayed the levels of some soluble growth factors in the vitreous over the course of development of NV and demonstrated that worsening of NV can be detected with exogenous human recombinant insulin-like growth factor-1 administration.24

Advantages of the pig BVO model over some other angiogenesis models in testing pharmacotherapeutic interventions include (1) robust production of

NV, (2) ability to easily examine the eyes clinically and photographically,

(3) the relatively large eyes, and (4) a less expensive study cost vs. primate models. Moreover, small pigs are easy to handle and unlikely to injure personnel. The major disadvantages of this model include difficulty in handling the animals after several months of growth, since they gain weight at 1-2 kg per week, and the lack of a fovea. Also, the assessment of NV is semi-quantitative and difficult and has only been published from one laboratory. Masking of treatment groups is problematic if there are local signs of ocular treatment.

4.PRIMATE BVO MODELS

Laser-induced branch retinal vein occlusion was explored in macaques (rhesus and cynomolgus) by several groups decades ago.18,25 Hayreh and colleagues reliably produced iris NV in cynomolgus monkeys after occlusion of 3 of the 4 major branch veins with an argon laser.26 Several variants of the original model have emerged in order to enhance the neovascular response. A number of laboratories have employed this model of iris NV to investigate the pathophysiology of ocular angiogenesis as well as the pharmacological efficacy of potential therapeutic agents.

NV has been demonstrated by clinical examination, fluorescein angiographic documentation (upon which later quantitative assessment was developed), and histopathological documentation.7,27,28 Preretinal and optic nerve head NV is only sporadically reported; thus, this model has not been advocated as a model of posterior segment angiogenesis. Acute venous obstruction is sometimes assisted by pretreatment with intravenous fluorescein29 or use of krypton yellow laser instead of argon green.17 Acute vascular closure is accompanied by retinal edema and intraretinal hemorrhage. Reopening of retinal veins soon after occlusion is common. Retreatment with additional laser may be performed to obtain permanent obstruction. Vitreous hemorrhage may occur during treatment, complicating later clinical observation.18 Retinal edema tends to resolve quickly, as observed by clinical examination and angiography, but histopathological evidence of subtle macular edema may persist.30 The clinical course of macular edema in this model differs markedly from human patients; consequently, this model has not been utilized as a model of macular edema. Permanent vascular occlusion often results in capillary closure and venous collateral development.

Iris NV may be observed within the first week following BVO and tends to be maximal between one and two weeks.7 Iris NV varies in severity, ranging from subtle vessels to the most severe stage involving hyphema and

ectropion uvea. Variants of this model include performing lensectomy and vitrectomy at the time of vascular occlusion, which increases the NV response and may lead to neovascular glaucoma in a small percentage of cases.26 The increased NV is likely related to the surgical trauma in addition to the removal of the physiological barriers to the diffusion of angiogenic products into the anterior chamber. Another development has been to pass a full-thickness silk suture through the cornea to produce chronic aqueous leakage and hypotony.31 The resulting increase in NV is likely related to increased angiogenic products produced by the inflammation from the penetrating trauma and the serum products from the chronic vascular exudation produced in hypotonous globes. Given the variety of production methods utilized by different laboratories, the incidence of iris NV production ranges up to 100% in some laboratories.

Histopathologically, new vessels may be observed on the anterior surface of the iris as seen in human disease. With more profuse proliferation, fibrous tissue also develops with the vessels to produce a neovascular membrane. This membrane may eventually contract and produce ectropion in some cases.27 If the proliferation extends into the trabecular meshwork and seals off the iridocorneal angle (termed peripheral anterior synechiae), neovascular glaucoma may result.

Standardized assessments of iris NV in the primate model usually rely on fluorescein angiographic grading. Briefly, the angiographic extent of NV is categorized (sometimes with standard photographs for reference) by the density and extent of leakage from vascular abnormalities, with ectropion

uvea and/or hyphema representing the most severe endpoint (see table from Miller et al., 1993).32

Use of this model has been employed extensively to investigate pharmacotherapeutic agents31 as well as to analyze elements of the angiogenesis cascade, particularly in regard to soluble growth factors such as VEGF. Notably, intravitreal injection of exogenous VEGF produces iris NV in monkey eyes in the absence of ischemia. Moreover, elevated VEGF levels

have been documented in eyes with BVO, and inhibition of VEGF in eyes with BVO inhibits iris NV.17,31-33

Advantages of the primate BVO models include their wide acceptance and application, relatively easy production, large eyes with ease of surgical intervention and clinical evaluation, and relatively standardized assessment. Use of primates for preclinical testing is also commonly performed prior to clinical trial development. Disadvantages include the high cost of purchase and maintenance of large animals, the potential for transmission of communicable diseases and injuries, and lack of posterior segment NV, which would be needed to mimic the more common human diseases of interest.

5.MODELS OF OCULAR NEOVASCULARIZATION FROM BVO IN OTHER ANIMALS

Many groups have described angiogenesis in the setting of BVO in species other than pigs and monkeys. This effort is likely inspired by the desire to avoid the expense and complications of working with the larger animal models.

Iris NV from BVO has been produced in cats with a great deal of effort. Steffanson and colleagues produced a high proportion of iris NV in cats after surgical cautery and transaction of retinal veins followed by retinal detachment.34 Hjelmeland et al. also produced iris NV and ectropion uvea with a surgical technique employing lensectomy, vitrectomy, and retinal venous cautery and transaction.8 Preretinal or optic nerve head NV was not described in either model. Because of the technical difficulty and need for surgery to produce iris NV in cats, it is unlikely that this model will be widely employed for pharmacotherapeutic trials of angioinhibitors.

Preretinal NV after venous occlusion in rats has been described by several groups. Saito et al. demonstrated convincing preretinal NV in pigmented rats after occlusion of all retinal veins with an argon blue-green laser and intravenous fluorescein pretreatment.35 This model featured extensive exudative retinal detachment and macrophage infiltration (also noted in the pig model) and resulted in identifiable NV in 70% of animals. Other groups have described preretinal NV due to BVO using argon green

laser and Rose Bengal in rats, but based on angiographic interpretation without presenting histopathological data.11,36,37 To our knowledge, only one

group has used this methodology for pharmacotherapeutic assessment with histopathology.38 The technique is not technically difficult, and the animals are inexpensive and easily maintained. Further work with this model appears in order.

6.SUMMARY

Ischemia-induced ocular NV due to BVO in primates is a fairly standardized, reproducible model based on clinical and angiographic grading of iris NV. This model has been employed by many investigators to study the pathogenesis of ischemia-induced NV and potential therapeutic strategies for human use. Disadvantages of the primate model include the expense and difficulty of working with primates and the relatively obscure role of iris NV in human retinal disease. Preretinal NV can be reliably produced by BVO in pigs and more closely mimics the manifestations of ischemic retinal disease observed in humans. Quantification of NV in the pig model is difficult, but

has been applied with success during pathophysiological and pharmacotherapeutic investigations. Ocular NV due to BVO in other species has been described but has not been developed into standardized models or routinely employed in research.

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MOLECULAR CHARACTERIZATION

Chapter 6

VASCULOGENESIS AND ANGIOGENESIS IN FORMATION OF THE HUMAN RETINAL VASCULATURE

Cell-Cell Interactions and Molecular Cues

Tailoi Chan-Ling

Bosch Institute, Department of Anatomy, University of Sydney, Sydney, Australia

1.INTRODUCTION

During early embryonic development, the human retina transforms from a single layer of undifferentiated neuro-epithelial cells to an organized stratified structure. Concomitant with the maturation of the neuronal elements, the retina’s vasculature develops to form an elaborate vascular tree that is well matched to the metabolic needs of the tissue. This formation of the intra-retinal vessels takes place via two distinct cellular processes under distinct molecular cues.1 Formation of the primordial superficial vessels of the central two-thirds of the human retina takes place via the process of vasculogenesis, the de novo formation of primitive vessels by differentiation from vascular precursor cells. Formation of the remaining retinal vessels takes place via angiogenesis, the process of new vessel formation by budding or intussusceptive growth from existing blood vessels. Vasculogenesis appears to take place independently of hypoxia-induced

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© Springer Science+Business Media B.V. 2008

vascular endothelial growth factor (VEGF165).1 In contrast, angiogenesis is mediated via hypoxia-induced expression of VEGF165 by retinal glia (Müller cells and astrocytes)2 and pericytes.3

Retinal vascularization in human, primate, cat, dog, and rat supports the conclusion that both vasculogenesis and angiogenesis are involved. In contrast, compelling evidence for the existence of vascular precursor cells is not available in the mouse retina, though Ash, McLeod, and Lutty4 have preliminary data suggesting the presence of ADPase+ vascular precursor cells in advance of the forming vasculature in postnatal day 3 (P3) mice. This apparent species difference between the mechanism by which retinal vessels form in humans and mice necessitates caution when extrapolating directly from mouse studies to human application. This is particularly of relevance in the development of therapies for retinopathy of prematurity (ROP), age-related macular degeneration (ARMD), and diabetic retinopathy (DR), and could in part explain the failure of novel treatments, where successful pre-clinical trials would have predicted a more positive outcome. Failure to recognize key species differences could lead to ineffective clinical trials, worsened disease, or unexpected severe adverse events. Where the mouse model reproduces specific features of the histopathology and neurobiology of the human condition, its application is warranted and highly advantageous due to the availability of genetically modified animals and experimental reagents. However, mouse models apply only in the elucidation of the role of angiogenic processes and do not mimic fully human retinal and choroidal pathogenesis. There are limitations of various animal models of ROP, ARMD, and DR, but when used appropriately, animal models of various species continue to provide a crucial tool for improving the understanding and development of neovascularizing retinopathies. The marked species differences in the mechanism of retinal vascular formation reported to date point to the necessity to undertake studies on human tissues during normal development and in disease.

1.1Three intra-retinal vascular plexuses in human retina: Superficial and deep vascular plexuses and radial peri-papillary capillaries (RPCs)

The human retina first appears as an undifferentiated neural epithelium early in embryonic development (Figure 1A). With further maturation, neuronal stratification produces an ordered multi-layered stratified structure. Concurrent with this neuronal maturation is the formation of three inherent retinal vascular plexuses (Figure 1B).

A superficial vascular plexus is located in the ganglion cell and nerve fiber layers, and a second deep plexus is located at the junction of the inner