Ординатура / Офтальмология / Английские материалы / Retinal and Choroidal Angiogenesis_Penn_2008

identification of tetradecanoyl-13-phorbol acetateand mitogen-inducible sequences in Swiss 3T3 cells, reported the upregulaion of COX DNA sequences. These data, along with the results of other studies, pointed to the existence of constitutive and inducible forms of COX referred to as COX-1 and COX-2, respectively.14

4.PROSTANOID RECEPTORS

Bioassays performed in various tissues suggested that activity profiles of the prostanoids overlap to some degree but possess sufficient differences to allow distinction.15 These studies led researchers to propose the existence of multiple types of prostanoid receptors with cell-specific expression profiles that could perhaps explain the variety of actions and the sometimes opposing effects elicited by the prostanoids. Additional investigations linked prostanoid activities with the activation of intracellular second messenger systems such as phosphatidylinositol (PI) turnover, Ca2+ mobilization and changes in cAMP levels.15 These studies allowed functional correlation of cell or tissue binding activities to bioactivities or the activation of second messenger systems and led Coleman et al. to propose the existence of the prostanoid receptors and to classify them. Specific putative receptors for TX, PGI, PGE, PGF, and PGD were named TP, IP, EP, FP, and DP receptors, respectively. The EP receptor classification was further broken down into four subtypes, namely EP1, EP2, EP3, and EP4.15-17 Hirata et al. cloned the human TXA2 receptor in 1991,18 and homology-based screening of cDNA libraries from several species with probes based on this sequence were performed. All of the prostanoid receptors classified by previous pharmacological and biochemical studies were identified.15 These functional and genetic analyses have classified the prostanoid receptors into a subfamily of G-protein-coupled receptors with seven transmembrane domains belonging to the superfamily of the rhodopsin-type receptors.

5.INHIBITION OF COX VIA NON-STEROIDAL ANTI-INFLAMMATORY DRUGS

In 1971, J. R. Vane and colleagues discovered that non-steroidal antiinflammatory drugs (NSAIDs) are potent inhibitors of COX.9 These seminal findings provided a powerful pharmacological tool to investigate the physiology and pathophysiology of COX-dependent processes. COX inhibition studies revealed that COX products are the mediators of pain, fever and inflammation. However, it is important to recognize that

COX-dependent processes are often integrated with other signaling cascades to produce a physiological outcome. For example, intradermal injection of a histamine-PGE2 mixture causes greater pain than if either of the compounds is administered alone. Furthermore, PGE2 will augment the effect of histamine at doses that produce no effect when administered alone.9

6.COX-2-SELECTIVE INHIBITION

The discovery and characterization of the two COX isoforms (COX-1 and COX-2) led to the hypothesis that selective inhibition of COX-2 would alleviate pain and inflammation without the adverse side effects associated with COX-1 inhibition (e.g. gastrointestinal damage). The results of large clinical trials testing the COX-2 selective inhibitors, the coxibs, have shown this hypothesis to be true in a general sense.19 However, in the recent Adenomatous Polyp Prevention on Viox (APPOVe) study,20-22 the COX-2 selective inhibitor, rofecoxib, was linked to an increased risk of myocardial infarction, resulting in its withdrawal from the market. Other COX-2

selective inhibitors may also have a detrimental effect on the cardiovascular

system.20,23,24

7.ANGIOGENESIS

Angiogenesis, the formation of new capillaries from existing blood vessels, occurs in reproduction, growth and development, and wound healing.25-30 In normal physiological processes, angiogenesis is tightly regulated. However, in various pathologies such as arthritis, tumor growth and retinopathies, dysregulated and persistent angiogenesis occurs.30-32

Endothelial cells and pericytes are two prominent cell types found in microvessels, capillaries, and collecting venules. These cells are induced by angiogenic stimuli to proliferate and differentiate, ultimately leading to a capillary network.25-28,33 Endothelial cells within microvessels normally remain quiescent for several years under physiological conditions (except in female reproductive organs), maintained by an intricate balance of proand anti-angiogenic stimuli.26,27 In certain disease states, the balance is tipped in

favor of angiogenesis, and the resting phenotype is converted to an angiogenic phenotype leading to the formation of new microvessels. 25-27,31,34-37

Angiogenesis consists of a cascade of carefully orchestrated events. Initially, there is the production of angiogenic growth factors such as vascular endothelial cell growth factor (VEGF) and basic fibroblast growth factor (bFGF) that may occur in response to tissue injury or ischemia. Extracellular

proteinases degrade the microvessel basement membrane and remodel the extracellular matrix to allow migration of endothelial cells into the extravascular space. Endothelial cell proliferation and differentiation, resulting in tube formation, with subsequent anastomoses of the adjacent

tubes, leads to a microvasculature that is stabilized by the attachment of supportive cells (e.g., pericytes).25-28,33,34

8.OCULAR DISEASE AND ANGIOGENESIS

Retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR) and age-related macular degeneration (ARMD) are vasoproliferative disorders that can lead to blindness in affected individuals. ROP occurs in premature infants, with PDR and ARMD primarily affecting working age individuals and the elderly, respectively.38-40 Pathological angiogenesis, common to each of these conditions and referred to as ocular neovascularization (NV), causes vascular permeability leading to retinal edema, the development of fragile vessels, and abnormal pre-retinal fibrovascular structures commonly referred to as neovascular tufts. These conditions predispose the affected individual to hemorrhage, tractional retinal detachment, and vision loss.41 Laser photocoagulation procedures are performed to treat ocular neovascular conditions; however, these procedures are plagued with undesirable side effects and do not target the underlying pro-angiogenic stimuli.42-46

9.MECHANISMS OF OCULAR ANGIOGENESIS

Ischemia is common to retinal neovascular conditions and leads to retinal hypoxia that initiates the angiogenic cascade.47,48 In 1948, Michaelson proposed a link between retinal ischemia and retinal angiogenesis in terms of a diffusible pro-angiogenic factor that is synthesized and released in response to hypoxia. Since then, several pro-angiogenic factors have been identified including: fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), the angiopoietins, platelet-derived growth factor (PDGF), and tumor necrosis factor (TNF). 49,50

Several lines of evidence suggest that VEGF is the principal mediator of retinal angiogenesis.51 VEGF is a homodimeric glycoprotein that induces vasopermeability and angiogenic behaviors.52-55 There are five main homodimeric VEGF isoforms. The corresponding monomers have 121, 145, 165, 189 and 206 amino acids resulting from alternative splicing of a single VEGF transcript.56 VEGF165 and VEGF121 are diffusible isoforms, whereas

the VEGF145, VEGF189, and VEGF206 isoforms are bound to the heparincontaining proteoglycans of the extracellular matrix. VEGF receptor-1

(VEGFR1 or Flt-1) and VEGF receptor-2 (VEGFR2, KDR, or Flk-1) are two high-affinity plasma membrane receptors that bind VEGF and mediate its biological signals. They each have an extracellular VEGF-binding domain consisting of seven immunoglobulin–like domains, a transmembrane domain, and a cytoplasmic tyrosine kinase sequence interrupted by a kinase insert domain. Microvascular endothelial cells co-express these receptors, as do other endothelial cell types.57

Hypoxia induces VEGF synthesis in retinal cell types including endothelial cells, pericytes, retinal pigmented epithelial cells (RPE), Müller cells, and ganglion cells.58-62 Müller cells have been shown to be the principal source of VEGF in animal models of neovascular disease.60-62 The hypoxia-inducible transcription factor (HIF)-1, accumulates in response to hypoxia and stimulates transcription of the VEGF gene from a binding site at -975 in the human VEGF promoter.63-66 VEGF is also post-transcriptionally regulated by hypoxia.63,66

The observation that increased expression of VEGF correlates with retinal NV identifies VEGF as a major inducer of the angiogenic program.

Subsequent investigations further support this notion and have shown that retinal NV is suppressed by agents that bind VEGF 59,67,68 and inhibitors of

VEGF receptor tyrosine kinase activity.69,70

10.COX-MEDIATED ANGIOGENESIS

Patients who take NSAIDs on a regular basis are less prone to the development of colorectal cancer.71 Colorectal tumors express high levels of COX, suggesting that PGs may influence the growth and development of tumors; and COX inhibitors may protect against tumorigenesis.72,73 It appears that PGs may help promote tumorigenesis by stimulating angiogenesis, because a pro-angiogenic PG effect has been noted in cancer models and other systems.74-76 On the other hand, COX inhibitors block angiogenesis in several experimental systems.77-83

The prostanoid effect is likely mediated through the stimulation of proangiogenic growth factor expression.73,79 In support of this notion,

prostaglandin treatment of cells in vitro leads to increased levels of VEGF and bFGF.84,85 Furthermore, tumor viruses, such as the Epstein-Barr virus,

induce VEGF expression in a COX-2-dependent manner.86 VEGF synthesis and release is decreased in wild-type fibroblasts treated with COX-2 inhibitors and COX-2-/- mouse fibroblasts, and COX-2 overexpression upregulates several angiogenic inducers73,79 in colon carcinoma cells. The

prostanoid induction of angiogenesis may be amplified in some cases by an autocrine feedback loop. VEGF-induced COX-2 expression and activation of phospholipase A2 -mediated arachidonic acid release leads to enhanced prostaglandin synthesis and release, followed by binding of prostanoid receptors promoting enhanced VEGF expression.87,88

The influence of prostanoids on angiogenesis likely depends on the tissue, environmental and genetic background, and the mode of action (i.e. paracrine vs. autocrine). For example, TP receptor-specific agonists and antagonists have been shown to be involved in corneal and tumor angiogenesis.89,90 However, TP receptor agonists reverse angiogenesis in vitro.91

11.INHIBITION OF COX-2 RESTRICTS ANGIOGENESIS

The anti-angiogenic function of NSAIDs has largely been attributed to the inhibition of COX-2, since selective inhibition of COX-1 fails to block NV.92-95 Reduced angiogenesis by NSAIDs may result, at least in part, from decreased prostanoid production, because in some cases NSAID suppression

of angiogenesis is reversed by prostaglandins or prostanoid-receptor agonists.87,89,94 NSAIDs block the production of angiogenic factors by tumor

cells and stromal fibroblasts and also inhibit pro-angiogenic signaling pathways in endothelial cells.73,79,81 It appears that the anti-angiogenic activity of NSAIDs has COX-dependent and –independent components. COXindependent effects that may block angiogenesis have been identified and include the inhibition of transcription factors nuclear factor kB (NF-kB) and activator protein-1 (AP-1). 95 Other effects that have been reported are the inhibition of the mitogen-activated protein kinase cascade80 and the suppression of GTPases, Cdc42 and Rac, via integrin αvβ3.96 These proteins are necessary for cell spreading and migration.97 However, it is not known whether these effects are COX-dependent.

12.POTENTIAL ROLES OF COX AND THE PROSTANOIDS IN RETINAL ANGIOGENESIS

Animal models of oxygen-induced retinopathies (OIR) are crucial to understanding the pathogenesis of vasoproliferative retinopathies and have been used in studies investigating the role(s) of COX in retinal angiogenesis. A review of the development of these models is presented in Chapter 3 of this volume.

Convincing evidence exists that links tissue hypoxia to COX-2-mediated angiogenesis in tumors, suggesting the possibility that similar COXdependent mechanisms may exist for ischemic vasoproliferative retinopathies. Recent studies using animal models of OIR, choroidal NV (CNV), corneal NV and VEGF-induced vascular leakage2-5 have investigated COX-2-dependent mechanisms in ocular angiogenesis, with particular emphasis on hypoxia, the VEGF signaling cascade, and the inhibitory effects of NSAIDs.

Wilkinson-Berka et al. tested the COX-2 selective inhibitor rofecoxib in a mouse model of OIR and observed a 37% reduction in pathological retinal angiogenesis in treated mice relative to untreated controls. Rofecoxib-treated mice maintained in room air had a 45% reduction in the formation of the inner retinal vasculature compared to untreated room air mice, suggesting a potential role for COX-2 in normal development. COX-2 immunoreactivity was observed in the ganglion cell layer and the blood vessels of the room air and OIR mice. COX-2 was also localized to pre-retinal blood vessels extending into the vitreous cavity in OIR mice.

Takahashi et al. tested the effects of nepafenac in three murine models of ocular NV. 98 Nepafenac, the amide derivative of the COX-1 and -2 inhibitor amfenac, easily penetrates the cornea after topical administration and is readily deaminated to amfenac in vivo. OIR or CNV was induced in mice by standard protocols, and the mice were treated with 0.1%, 0.5% nepafenac or vehicle by topical administration. Nepafenac-treated OIR mice had significantly less ischemia-induced retinal NV than the corresponding vehicle-treated controls. To investigate the effects of nepafenac on VEGF expression in mouse OIR, semiquantitative RT-PCR analysis of retinal RNA showed a nepafenac-dependent decrease in VEGF mRNA levels, providing a plausible explanation for the observed reduction in ischemiainduced retinal NV.

As previously discussed, Müller cells are a major source of VEGF in the hypoxic retina and play a key role in the pathogenesis of vasoproliferative retinopathies. COX-2 undergoes a dramatic upregulation when Müller cells are subjected to hypoxia. Furthermore, there is an approximate 3-fold increase in PGE2 synthase in hypoxic Müller cells relative to those maintained in normoxia (Penn, unpublished results). In vitro data have shown that amfenac dose-dependently inhibits hypoxia-induced VEGF production in Müller cells (Penn, unpublished results). It remains unclear if these observations are COX-dependent because COX-2-/- Müller cells showed significant hypoxia-induced VEGF expression (Penn, unpublished results). However, it has been demonstrated that PGE2 induces upregulation of VEGF and βFGF in Müller cells. Using selective inhibitors of protein kinase A, the authors inferred that EP2 and/or EP4 were responsible for

250 G. W. McCollum and J. S. Penn

VEGF induction.84 These data suggest the possibility of hypoxia-induced VEGF expression via a COX-2/PGE2 autocrine loop.

Sennlaub et al. investigated the localization of COX-2 in human retinas from non-diabetic subjects and subjects with diabetic retinopathy, and in retinas of murine and rat models of OIR.3 COX-2 immunoreactivity was localized in RPE cells, the outer segment of the photoreceptors, and to some degree the inner plexiform layer. In all diabetic patients, COX-2 immunoreactivity was also detected in the nerve fiber layer, co-localizing to a significant extent with glial fibrillary acidic protein (GFAP). This suggests that significant COX-2 expression occurs in the retinal astrocytes of these diabetic patients. Immunolocalization of COX-2 in OIR mouse retinas was similar to that found in humans. Of particular interest is that COX-2 expression was detected in astrocytes (GFAP-positive cells) of the nerve fiber layer during the normoxic period following hyperoxic exposure, which is similar to the pattern observed in retinas from humans with diabetic retinopathy. In vitro experiments were performed with primary porcine retinal astrocyte cultures exposed to hypoxia (2% oxygen) for 24 hours. They revealed an 8-fold increase in COX-2 protein levels relative to normoxic controls as measured by western blot analysis. There was a concomitant increase in PGE2 synthesis that was significantly decreased by the COX-2 selective inhibitors APHS and etodolac, and the COX-1-selective inhibitor SC-560 resulted in only a small decrease. In the same study, APHS, etodolac, or SC-560 were tested in the murine and rat models of OIR by intravitreal injection. APHS showed a dose-dependent decrease in pre-retinal NV, and SC-560 had no effect, when both were tested in the murine model. The retinal PGE2 level in these mice was reduced by 65% 24 hours after APHS treatment. Intravitreal injection of PGE2 produced a small but significant increase in pre-retinal NV. In the rat OIR model, etodolac showed a decrease in pre-retinal NV when compared to vehicle-treated controls that was reversed by intravitreal injection of PGE2. The EP2- and EP3-specific agonists, butaprost and M&B28767 respectively, were tested in etodolac treated OIR rats. Butaprost exacerbated and M&B28767 partly reversed the inhibitory effects of etodolac. EP receptor protein expression profiles were determined in the rat OIR model during the course of oxygen treatment. During hyperoxic exposure, EP1 was not detected; EP4 was slightly decreased; EP2, and to a greater extent EP3, was decreased. After 24 hours at normoxia, there was no significant change in EP4; however, there were significant increases in EP2 and EP3. These data suggest that there is a COX- 2-dependent regulatory component of retinal NV in these models that is relayed by PGE2 through the EP2 and EP3 receptors.

To probe for potential COX-2-dependent mechanisms of angiogenesis, the effect of COX-2 inhibition and EP3 stimulation on retinal pro-angiogenic

VEGF and VEGFR2 and anti-angiogenic Thrombospondin-1 (TSP-1) and its receptor (CD36) was investigated during the post-hyperoxic period in the rat OIR model. Exposure to normoxia for 24 hours after the hyperoxic insult resulted in increased TSP-1 protein expression. The COX-2 inhibitor etodolac induced a substantial increase in TSP-1 and CD36, and addition of M&B28767 reversed this effect, suggesting that EP3 stimulation inhibits the production of anti-angiogenic factors. This explains at least in part the antiangiogenic effect of COX-2 inhibition. VEGF expression was only marginally affected by etodolac and M&B28767, while VEGFR2 expression was not changed. According to these data, the influence of COX-2 on retinal NV could not be explained by modulation of VEGF protein levels.

NSAIDs have been shown to inhibit endothelial cell angiogenic behaviors such as proliferation and tube formation, and hypoxia-induced VEGF expression in Müller cells. The role of COX remains unclear, because NSAIDs have non-specific activities that may contribute to the antiangiogenic effect observed in cultured cells and in animal models of neovascular disease.97,99 For example, amfenac inhibits the phosphorylation of Erk in human retinal microvascular endothelial cells (HRMEC), which is a major downstream signaling intermediate of VEGFR2 involved in cell proliferation (Penn, unpublished results). As a result, studies performed with NSAIDs must be interpreted with caution. To assess the role of COX in retinal angiogenesis, and, at the same time, avoid the complications associated with the non-specific effects of pharmacological COX-inhibitors, Cryan et al. investigated the effects of either COX-1 or COX-2 gene deletion in the mouse model of OIR.20 Histological analysis of retinas from wild-type, COX-1-/- and COX-2-/- mice raised in room air showed no differences in the development of the retinal vasculature. Pre-retinal NV, retinal vascular/avascular areas, and perfused retinal areas were measured in COX- 1-/-, COX-2-/-and wild type OIR mice. Interestingly, there was essentially no difference in pre-retinal NV for the COX-1-/- strain and a non-significant trend toward less pre-retinal NV for the COX-2-/- strain compared to the wild-type. Isolectin B4-staining of retinal vasculature, a technique that does not distinguish between perfused and nonperfused vessels, revealed similar percentages of capillary-free zones among these strains. As measured by fluorescein angiography, perfused retinal areas were reduced in COX-2-/- mice compared to the other two strains. Immunohistochemical analysis showed increased fibrin deposits and thrombocyte staining in the retinas of COX-2-/- mice, suggesting that COX-2 protects against vascular obstruction (thrombosis). The authors postulate that the absence or reduction of COX-2- derived PGI leaves the pro-thrombosis effects of COX-1-derived TX from platelets unbalanced, because PGI inhibits platelet activation and TX is a potent platelet activator. A substantial neovascular response occurred in the

COX-2-/- mice. However, is there any evidence for a COX-2-dependent component? In rodent models of OIR, the size of the nonperfused retinal area frequently correlates with the severity of pre-retinal NV. Explaining this correlation, a commonly accepted hypothesis states that the level of proangiogenic stimulus depends on the level of tissue hypoxia, which is proportional to the size of the nonperfused retinal area. Although the nonperfused retinal area of the COX-2-/- mice was higher than the other two strains, there was a comparable neovascular response. Based on this observation, the authors speculate that a COX-2-dependent component of the neovascular response exists; however, the primary finding of increased retinal thrombosis in the COX-2-/- mice complicates the evaluation of COX- 2-dependent retinal pro-angiogenic mechanisms.

The studies outlined above leave large gaps in our understanding of the COX-dependent mechanisms involved in ocular angiogenesis. No attempt has yet been made to examine systematically which PGs are important in neovascular eye pathology or to discern the COX-dependent and/or COXindependent effects of NSAIDs in animal models of proliferative retinopathy or the angiogenic endothelial cell behaviors. Thus, little in the way of mechanistic information has been uncovered. These questions are left to future studies.

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