Ординатура / Офтальмология / Английские материалы / Retinal and Choroidal Angiogenesis_Penn_2008

Figure 3-1. FITC-dextran infused mouse retinae at P12. Normoxia-raised mice (A) exhibit normal retinal vascular development. 5 days at 75% O2 (B) induces vaso-attenuation and atrophy of the central retinal bed, as well as substantial vascular leakage.

Since the advent and widespread use of the mouse model of retinopathy, extensive research has been conducted on the susceptibility of various inbred strains of mice to pathological retinal neovascularization. In order to evaluate genetic heterogeneity in angiogenic susceptibility, D’Amato and colleagues24 implanted a pellet containing an angiogenic protein, basic fibroblast growth factor (bFGF), into the corneas of 25 strains of mice. Normally, the blood vessels of the limbus do not grow into the avascular cornea. Strain differences in angiogenic response were observed by analyzing the growth of blood vessels into the cornea upon angiogenic stimulation. A 10-fold range of responsiveness was observed, with 129/SvImJ mice eliciting the most potent angiogenic response, while the commonly used C57BL/6J mice fell near the middle of the response profile. Subsequent studies revealed that vascular endothelial growth factor (VEGF) elicited a response profile that correlated closely with the bFGF response.25

Following D’Amato’s report, Hinton and colleagues analyzed strainrelated differences in retinal angiogenesis using the mouse OIR model.26 They demonstrated that the angiogenic response of the retina paralleled the results of the corneal assay. Analyzing retinal angiogenesis in mice with different genetic backgrounds has allowed for the identification of various proand anti-angiogenic factors (to be discussed in detail later) potentially involved in the pathogenesis of ROP.

Figure 3-2. P19 hematoxylin and eosin (H&E) stained retinal cross sections. In contrast to normoxia-raised mice (A), mice that have been exposed to hyperoxia (B) demonstrate a substantial number of retinal cell nuclei that penetrate the inner limiting membrane. These nuclei allow for retinal neovascularization to be quantified.

2.1.4Disadvantages of the mouse model

Unfortunately, the retinal vascular pathology observed in mouse OIR is the opposite of that observed in human ROP. In the human condition, the central retina is vascularized, and the peripheral retina is avascular. In contrast, the mouse exhibits a central area of avascularity, and the peripheral retina is vascularized. These differences in patterning cannot be ascribed to any obvious differences between these two species. Claxton and Fruttiger27 studied the retinal vascular patterning in mice that had been exposed to hyperoxia. They hypothesized that because the retinal arteries and the hyaloidal blood supply pass through the optical nerve head, it is reasonable to suspect that the proximal retina has a relatively high oxygen tension. VEGF is a survival factor for endothelial cells and is downregulated in response to high oxygen tension, explaining the pruning of peri-arterial capillaries around the optic nerve head as well as around the central retinal arteries in the mouse. Hyperoxic exposure further increases the retinal oxygen tension, expanding regions of decreased retinal VEGF, inducing endothelial cell apoptosis and vascular atrophy, and resulting in the expansion of capillary-free zones within the retina.

In addition, and in contrast to human ROP, the mouse OIR model does not lead to retinal detachment. The lens occupies 40% of the mouse eye,

resulting in less tractional force on the retina compared to that observed in humans.

2.1.5Advantages of the mouse model

The mouse model of ROP is the most commonly used model in studies of retinal angiogenesis. Mice reliably produce large litters, are relatively inexpensive to purchase and maintain, and consistently produce a neovascular response. The mouse model of ROP has provided much of what is currently known about the pathogenesis of retinopathy of prematurity, its progression, and potential means by which to prevent and/or ameliorate it. Importantly, the ability to manipulate the mouse genome has facilitated our understanding of the various genetic contributions and their interactions in producing the angiogenic phenotype.

2.2Rat

2.2.1Rat vascular development

As in the human, the retinal vasculature of the rat appears to derive from the adventitia of the hyaloid artery. Vascularization of a superficial network of arteries and veins occurs first, followed by the angiogenic vascularization of a deeper capillary network. In the human, retinal vascularization is usually complete at the time of birth. This process does not complete until postnatal day 15 (P15) in the rat. For this reason, the retinal vasculature of the newborn rat pup resembles that of a preterm infant and a newborn mouse— incomplete, largely avascular, and susceptible to OIR.

2.2.2Current rat model

Early studies by Patz, Ashton, and Gole28-30 involved exposing newborn rat pups to a constant level of extreme hyperoxia. This resulted in substantial vasoattenuation but an inconsistent vasoproliferative response. Informative though these studies were, it was not until 1993 that Penn and colleagues developed a protocol that consistently induced proliferative retinopathy in the rat.31 Penn noted that variable oxygenation is more likely to produce retinal angiogenesis than is constant hyperoxia. This is because variable oxygenation more closely mimics the fluctuating lung function and

subsequent change in arterial blood oxygen partial pressure, PaO2, of a neonatal infant in the NICU, an infant likely to develop ROP. In Penn’s 1993 study, exposing rats to 80% oxygen throughout the course of treatment did not induce any pre-retinal neovascularization following an appropriate postexposure period. However, a variable oxygen exposure (cycling between 40% and 80% oxygen every 12 hours), in combination with a post-exposure period of return to room air, induced pre-retinal neovascularization in 66% of the rats. Subsequent experiments by Penn32 led to the rat OIR model that is used today. In this model, newborn rats are cycled between 10% and 50% oxygen every 24 hours for 14 days. This oxygen profile, which more accurately reflects the fluctuating lung function and PaO2 of a preterm infant in the NICU, resulted in a high incidence (97%) of retinopathy (Figure 3). Additionally, the angiogenic pattern seen in the rat mimics the pattern of ROP seen in the human. Both exhibit a peripheral region of avascularity and develop neovascularization at the boundary of vascular and avascular retina (Figure 4). Thus, the rat provides an extremely relevant model with which to address ROP-related questions.

Figure 3-3. FITC-dextran infused rat retinae at P20. Normoxia-raised rats (A) exhibit normal retinal vascular development. The rat OIR model causes avascularity of the peripheral retina (B).

Figure 3-4. OIR-exposed rat retina at P20, stained with ADPase, an ablumenal enzyme marking the vascular endothelium. Neovascularization develops at the boundary of vascular and avascular retina, as in human ROP. Image reproduced with Permission from Investigative Ophthalmology and Visual Sciences.111

2.2.3Disadvantages of the rat model

Like the mouse model, the rat OIR model is subject to both strainand vendor-related differences in susceptibility to retinal neovascularization. Ma and colleagues33 compared the differential susceptibilities of two strains of rats, Brown Norway and Sprague Dawley, to ischemia-induced retinopathy. Using a modified constant oxygen exposure paradigm (developed by Smith and colleagues for the mouse), Ma found that at the time of removal to room air, the Brown Norway rats exhibited an avascular area approximately four times greater than that of the Sprague Dawley rats. The Brown Norway rats subsequently developed three times the amount of preretinal neovascularization. Later studies confirmed the above findings by demonstrating that Brown Norway rats exhibit an increased amount and duration of retinal vascular permeability relative to Sprague Dawley rats exposed to the same ROP paradigm.34 The difference between the two

strains is likely due to differences in retinal expression of proand antiangiogenic factors, as has been demonstrated in the mouse.26,33 These two

studies, though informative, were conducted under conditions of constant, extreme hyperoxia, instead of the more clinically relevant variable oxygen protocol. To address this issue, Holmes and colleagues used a modified protocol of cyclic hyperoxia and hypoxia. The Brown Norway strain again

demonstrated a higher incidence and severity of neovascularization than did the Sprague Dawley strain.35 Only a few Sprague Dawley rats, as opposed to all of the Brown Norway rats, developed neovascularization.

There are also vendor-related differences in susceptibility within the same strain of rat. Penn (unpublished observations) was the first to identify differences in the pathological response of a single rat strain obtained from several different vendors. Sprague Dawley rats from Charles River (Charles River Laboratories, Wilmington, MA) produced a two-fold greater area of neovascularization than those from Zivic-Miller (Zivic Laboratories, Pittsburg, PA). Sprague Dawley rats obtained from Harlan (Harlan, Indianapolis, IN) and Hilltop (Hilltop Lab Animals, Scottdale, PA) demonstrated intermediate levels of pathology compared to Charles River and Zivic-Miller rats. Similarly, Holmes and colleagues tested the OIR response of Sprague Dawley rats from Harlan and Charles River.36 Notably, the Charles River rats demonstrated a 62% greater susceptibility to and severity of oxygen-induced neovascularization. Thus, susceptibility to neovascularization depends on genetic variation, environment, and oxygen treatment paradigm.

2.2.4Advantages of the rat model

The rat is an ideal model of retinopathy due to large litter sizes (typically twice the size of mouse litters) and relatively inexpensive maintenance costs. Most importantly, unlike the mouse, the rat model consistently produces human-like patterns of vasoattenuation and vasoproliferation. For this reason, the rat is an attractive model that is often used for testing the efficacy of anti-angiogenic compounds for application in both ocular and non-ocular pathologies. It should also be noted that the rat model of ROP was the first to utilize fluoroscein angiogram imaging in order to track the progression of the disease in real time,37-39 and the first to utilize computer assisted image analysis in order to improve the speed and objectivity of pathology assessment.40

3.MOLECULAR MECHANISMS OF ANGIOGENESIS

Angiogenesis is the growth of new capillaries from pre-existing blood vessels. Angiogenesis involves complex interactions between cells, growth factors, cytokines and extracellular matrix (ECM) components. Ischemia is a feature common to virtually all retinal vasculopathies, and this observation formed the basis of early hypotheses suggesting the presence

of hypoxia-induced retinal angiogenic factors. Beginning with ischemic insult, the angiogenic cascade involves: the hypoxia-induced expression of pro-angiogenic growth factors and cytokines; proteolytic degradation of the vascular basement membrane by endothelial cell-derived matrix metalloproteinases; proliferation and migration of invading endothelial cells to form and extend the new vasculature; and morphogenic stabilization involving the induction of vessel differentiation, matrix deposition, and mural cell recruitment. The molecular etiology of retinal angiogenesis will be discussed in the following sections.

3.1Angiogenic factors

The growth of new blood vessels can be stimulated by a number of angiogenic factors including, but not limited to, vascular endothelial cell growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), placental growth factor (PlGF), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGFβ), tumor necrosis factor (TNF), nuclear factor-kappaB (NFκB), and interleukin-8 (IL-8).11 However, experimental evidence suggests that VEGF is the most important pro-angiogenic factor in the pathogenesis of vasoproliferative retinopathies.

3.1.1Vascular endothelial growth factor

In 1954, Michaelson proposed the presence of a “vasoformative factor” produced in response to the retina’s metabolic needs. This factor was proposed to be involved in the physiological development of the retina, as well as the pathological angiogenesis that occurs, for example, in ROP.41 Thirty years later, the key vasoformative factor involved in retinal angiogenesis was discovered to be VEGF (formerly vascular permeability factor, or VPF).42,43 VEGF induces endothelial cell proliferation and tube formation in vitro and stimulates angiogenesis in vivo.44-47 VEGF binds to cell surface receptor tyrosine kinases VEGFR1/Flt-1 and VEGFR2/KDR/Flk-1, stimulating the angiogenic cascade.47 In fact, both animal models and human

patients suffering from ocular vasoproliferative disorders exhibit elevated levels of VEGF within the eye.45,48-50

Pierce and colleagues investigated the effect of hyperoxia and hypoxia on VEGF in the mouse.51 At P7, VEGF mRNA was localized just anterior to the developing vasculature. Exposing the mice to 75% oxygen for just six hours led to substantial vasoattenuation with a reduction in VEGF mRNA as measured by in situ hybridization. Claxton and Fruttiger showed a similar reduction in VEGF expression following hyperoxic exposure.27 These studies

demonstrate that exposure to hyperoxia suppresses retinal VEGF mRNA

expression and vascular growth. Conversely, VEGF is induced by hypoxia.45,52 Studies have shown that post-oxygen exposed mice show

increased levels of VEGF mRNA. This increased expression is presumably induced by the onset of retinal hypoxia resulting from both the hyperoxiainduced vasoattenuation and the relatively hypoxic room air environment.53,54 Hypoxia-induced VEGF expression is a key event promoting vasoproliferation.

Increased expression of VEGF in hypoxia appears to be mediated by the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF-1 binds to the hypoxia response element (HRE) on several hypoxia-inducible genes. The promoter sequence of the VEGF gene contains several HREs. Ozaki et al. have demonstrated a functional link between levels of HIF-1 and VEGF transcription in the development of the mouse vasculature.55,56 Post-oxygen exposed mice, whose retinas are presumably hypoxic, demonstrate substantially increased HIF-1 levels that are temporally and spatially correlated with VEGF expression and retinal vasoproliferation.

VEGF plays a prominent role in retinal vasoproliferation. For this reason, several groups have already begun to explore means by which to inhibit VEGF, as a prelude to developing human therapies. Studies have been conducted using antisense oligonucleotides, monoclonal antibodies, VEGF peptides, and chimeric proteins that inhibit VEGF binding to its receptor.57-60 These studies, conducted in the mouse and/or rat, have been effective at reducing retinal neovascularization. Inhibition of KDR and the use of

soluble Flt-1 effectively reduces the severity of retinal neovascularization in the rat.61,62 Therapies directed against VEGF are being used to treat human

patients suffering from other forms of ocular neovascularization.63,64

3.1.2Insulin-like growth factor

VEGF is not the only growth factor involved in ocular neovascularization. In 1969, research demonstrated that removing the pituitary gland had a restorative effect on proliferative diabetic retinopathy.65 This finding led to the hypothesis that insulin-like growth factor-1 (IGF1), a product of the pituitary gland, must play a role in retinal neovascularization. Using the mouse OIR model, Smith and colleagues demonstrated that exogenous IGF- 1 induced retinal neovascularization when growth hormone (GH) was inhibited, and that an IGF-1 receptor antagonist suppressed retinal neovascularization.66,67 Moreover, knockout mice lacking the vascular endothelial cell IGF-1 receptor showed a 34% reduction in oxygen-induced retinal neovascularization.68 Clearly, IGF-1 signaling plays a mediating role in the pathogenesis of ROP.

3.2ECM breakdown

Binding of growth factor to its receptor leads to the degradation of the vascular basement membrane, permitting the extravasation and subsequent proliferation of endothelial cells. Matrix metalloproteinases (MMPs) are responsible for the proteolytic degradation of the vascular basement membrane, making proliferative neovascularization possible. Das and colleagues showed increased MMP-2 and MMP-9 mRNA expression in mice with retinal neovascularization. The increased mRNA expression correlated with protein level and proteolytic activity, as measured by zymographic analysis.69 Administering a nonselective MMP inhibitor to OIR-exposed mice leads to a reduction in MMP2 and MMP9 activity and a 72% reduction in retinal neovascularization.69

3.3Cellular adhesion

Much of what is known about endothelial cell attachment and migration in pathological retinal neovascularization comes from studies investigating the mechanisms of normal retinal development in the rodent. Several classes of endothelial cell adhesion molecules bind to the ECM and initiate a number of endothelial cell-specific responses. The integrins are an example of such adhesion molecules, and studies have shown that integrins αvβ3 and αvβ5 are specifically involved in retinal neovascularization. A murine model of ischemia-induced retinopathy demonstrated an upregulation of integrin αvβ3 in endothelial cells undergoing neovascularization.70 Administering a cyclic RGD peptide that inhibits integrin αv activity effectively reduces hypoxiainduced retinal neovascularization by more than 70% in the mouse.71 Peptide antagonists of integrins αvβ3 and αvβ5 also reduce retinal neovascularization in the mouse model.72

3.4Blood vessel remodeling

Following migration, adhesion, and proliferation, endothelial cells undergo several maturation processes that serve to stabilize the newly formed blood vessels. The endothelial cell surface receptor Tie2 and its ligands, the angiopoietins (Ang1 and Ang2), are involved in blood vessel maturation. Ang1 binds with high affinity to the Tie2 receptor, stimulating receptor phosphorylation that leads to downstream signaling events involved in vascular development.73 Ang1 stabilizes newly formed blood vessels by promoting an interaction between the endothelial cells and a network of support cells. Ang2 also binds to Tie2 with high affinity. However, Ang2 does not stimulate receptor phosphorylation.74 For this reason, Ang2 is

considered to be a competitive inhibitor of Ang1. As such, Ang2 has been shown to cooperate with VEGF to prevent stasis of the newly formed vasculature. Unstable vessels are more likely to respond to a VEGF signal with sprouting.75-78

Consistent with its role in vessel stabilization, over-expression of Ang1 in the mouse retina has been shown to decrease VEGF-induced leakiness of the vasculature. In addition, over-expressing Ang1 inhibits the initiation and progression of retinal neovascularization in the mouse OIR model.79,80 On the other hand, Ang2 mRNA expression is increased in rat pups exposed to a model of retinopathy in which animals are raised in 80% oxygen for the first 11 days of life and then removed to room air for 7 days.81 Mice lacking Ang2 exhibit abnormal vascular development, and Ang2-null mice are protected from retinal neovascularization upon oxygen exposure. These observations indicate a critical role for Ang2 in the development of the retinal vasculature and in neovascularization.82, 83

3.5Additional proteins involved in blood vessel growth

3.5.1Ephrins and EphRs

Recently, much attention has focused on the A and B classes of the Ephrin receptor tyrosine kinases (Eph RTKs) present on the surface of endothelial cells and their ligands, the ephrins. Ephrin-B2 is expressed in endothelial cells and has been detected in retinal endothelial cells isolated from patients suffering from either ROP or proliferative diabetic retinopathy.84 Reverse signaling through ephrin-B2 stimulates retinal endothelial cell proliferation and migration.85 Intravitreal injections of soluble ephrin-B2 or Eph-B4 are able to reduce the severity of retinal neovascularization in the mouse OIR model.86 Together, these data suggest that class B Eph RTKs and ephrin B ligands play a role in retinal neovascularization.

Class A Eph RTKs and their ephrin A ligands also have a role in ocular neovascularization. Experiments performed with a soluble Eph-A2 chimeric protein suggest that the interaction between Eph-A2 and ephrin-A1 is required for maximal VEGF-induced neovascularization in a mouse corneal angiogenesis assay. The chimeric protein inhibited VEGF-induced endothelial cell survival, migration, sprouting, and corneal angiogenesis,87 suggesting a functional link between VEGF and the EphA RTKs. Cheng et al.88 sought to determine the effect of soluble Eph-A2 on retinal neovascularization using the rat model. Soluble Eph-A2 significantly lowered the severity of retinal neovascularization by 50%, hypothetically through competitive binding to available ephrin ligands. In addition, the