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

Core Messages

Hypoxic signaling is mediated via the hypoxiainducible factor (HIF) pathway including HIF-1 subunits that contain binding sites to hypoxiaresponse elements

HIF-1 regulates target genes including angiogenic mediators, glucose transporters, cytokines, apoptotic genes, regulators of erythropoiesis and genes involved in DNA repair

von Hippel-Lindau disease (VHL) regulates vascular endothelial growth factor (VEGF) through HIF-1 dependent and independent processes Hypoxia, hyperglycemia and reactive oxygen intermediates, growth factors and inflammatory mediators upregulate VEGF

7.1 Introduction

Oxygen cannot passively diffuse for more than a radius of 100 μm around capillaries. As a result, adequate O2 supply to each cell depends on effective regulation of the integrity and function of the vascular network. On the other hand, high O2 tissue levels would result in reactive oxygen species (ROS) generation and cellular damage. Therefore, an optimal O2 concentration is needed to avoid hypoxia or ROSmediated cellular injury. The retinal tissue is very active metabolically and, therefore, exquisitely dependent on adequate O2 supply for its function [4]. The delivery of oxygen to the retina is dependent not only on systemic blood pressure, hemoglobin content and integrity of local vasculature, but on the level of intraocular pressure and local autoregulatory mechanisms as well. Hypoxia and its sequelae are implicated in the pathogenesis of most retinal diseases, especially those that involve pathologic neovascularization. This is due to the potent stimulation of production of vascular endothelial growth factor (VEGF), mediated by the hypoxia-inducible factor (HIF)-1 pathway, in response to hypoxia.

7.2The HIF Pathway and Its Role in Hypoxia Signaling

Essentials

The HIF family includes HIF-1 and less well characterized proteins such as HIF-2, HIF- 3, ARNT2, and ARNT3

The HIF-1 complex consists of two subunits: HIF-1 and HIF-1

HIF-1 subunits contain binding sites to hyp- oxia-response elements through which they regulate their target genes and coactivators such as CREB, CBP and p300

HIF-1 is constitutively expressed in normoxic cells whereas HIF-1 is tightly regulated by hypoxia both at the transcriptional level and the post-transcriptional level

Under normoxic conditions HIF-1 is hydroxylated, bound to von Hippel-Lindau disease (VHL) protein, which targets it for destruction by the proteasome. Hydroxylation is regulated by factors such as FIH-1- p300 and CBP and Fe chelating agents such as CoCl. During hypoxia hydroxylation is inhibited secondary to the inhibition of hydroxylases

HIF-2 may play a more important role in chronic hypoxia because it is not degraded HIF-1 can be induced in an oxygen-inde- pendent manner by cytokines, growth fac-

122

7 I

1 protein expression can be regulated by hypoxia in at least two ways:

Transcriptional level: The mRNA for HIF-1 is upregulated in hypoxic/ischemic tissues [10]. This effect requires hours to occur.

Post-transcriptional level: The HIF-1 protein is stabilized in hypoxic cells. This effect occurs within minutes. Jewell et al. [51] demonstrated that the HIF-1 protein accumulates in the nucleus within 2 min of anoxic/hypoxic exposure. HIF-1 has a half-life of 1 – 5 min in normoxic conditions [131, 140]. HIF-1 protein expression is negatively regulated in normoxic cells by hydroxylation of two conserved proline residues (Pro-402 and Pro-564). This reaction is O2-dependent and the O2 concentration is rate limiting for enzymatic activity, thus providing a mechanism for the direct regulation of HIF-1 protein levels by O2. This hydroxylation promotes HIF-1 binding to the von Hippel Lindau protein (VLH) and, through that, to the E3 ubi- quitin-protein ligase complex, which ubiquitinates HIF-1 and targets it for destruction by the proteasome [46, 58, 107]. This is the main mechanism of HIF-1 inactivation in normoxia and it is the inhibition of this pathway in O2-deprived cells that leads to the rapid accumulation of HIF- 1. The HIF-1 prolyl hydroxylases contain Fe(II) at the active site, which can be chelated by desferrioxamine (DFX) or replaced by Co(II), thus inactivating the enzyme. This explains the observation that cell treatment with CoCl2 or desferrioxamine mimics hypoxia in activating the HIF- 1 pathway and stimulating VEGF expression. Missense mutations and/or deletions of the HIF- 1 regions involved in this degradation pathway result in stabilization of the protein and overexpression even under normoxic conditions [123].

Moreover, HIF-1 transcriptional activity can be inhibited by:

Inactivation of the transactivating capacity of HIF-1 via hydroxylation of its carboxy-terminal domain at Asn-803 by factor inhibiting HIF-1 (FIH-1). This enzymatic reaction is also O2-de- pendent and prevents the interaction of HIF-1 with the coactivators CBP and p300 [33, 52, 57, 101]. This provides an additional O2-dependent, VHL-independent, mechanism for inhibition of HIF-1 activity.

Degradation of HIF-1 mRNA, which occurs in prolonged hypoxia. This is an autoregulatory mechanism that prevents prolonged activation of the HIF-1 pathway. HIF-2 mRNA is resistant to this mechanism, suggesting that in chronic hyp-

7.2.2 Downstream Targets of HIF

In hypoxic cells, overall protein synthesis is suppressed. However, a specific set of transcripts is upregulated. HIF-1 binds to promoter/enhancer elements and plays a pivotal role in stimulating expression of genes involved in energy metabolism, angiogenesis, cell proliferation and apoptosis (for a detailed list, see [110] and [109]). Prominent targets are genes for:

Mediators of angiogenesis and vascular physiology, such as vascular endothelial growth factor (VEGF), VEGF receptor FLT-1, adrenomedullin, endothelin- 1, heme oxygenase 1, nitric oxide synthase 2, and plasminogen activator inhibitor 1. HIF-1 increases VEGF mRNA levels via stimulation of transcription by binding to a hypoxia response element located

1 kb 5-prime to the transcriptional start site of VEGF. Also, hypoxia, via HIF-1, may increase the stability of VEGF mRNA [71, 72, 75].

Glucose transporters (glucose transporter 1, glucose transporter 3) and glycolytic enzymes [adenylate kinase 3, aldolase A, aldolase C, enolase 1 (ENO1), glucose transporter 1, glucose transporter 3, glyceraldehyde-3-phosphate dehydrogenase, hexokinase 1, hexokinase 2, insulin-like growth factor 2 (IGF-2), lactate dehydrogenase A, phosphoglycerate kinase 1, pyruvate kinase M].

Cytokines and intracellular mediators of cell proliferation and apoptosis: Bcl2/adenovirus EIB 19-kDa-interacting protein 3 (BNIP3) is a cell death factor that is a member of the Bcl-2 proapoptotic family recently shown to induce necrosis rather than apoptosis. The BNIP3 promoter contains a functional HIF-1-responsive element (HRE) and is potently activated by both hypoxia and forced expression of HIF-1 [20]. BNIP3 and its homologue, Nip3-like protein X, are molecular effectors of hypoxia-mediated apoptosis [66, 117]. Moreover, HIF-1 induces CXCR4 expression [119].

Regulators of erythropoiesis: HIF-1 upregulates erythropoietin, ceruloplasmin, transferrin, and transferrin receptor mRNAs [109]. Erythropoietin is produced by the kidney in response to hypoxia and has a pivotal role in regulating erythropoiesis [37, 38]. An additional role of erythropoietin has been discovered and appears to be of particular importance for CNS and retina diseases. Preconditioning with erythropoietin protects neurons in models of ischemic and degen-

erative damage [28, 29]. Similarly, acute hypoxia 7 I in the adult mouse retina dose-dependently stimulates expression of erythropoietin, fibroblast growth factor 2 and vascular endothelial growth factor via hypoxia-inducible factor-1 (HIF-1) stabilization and protects retinal morphology and function against light-induced apoptosis by inhibiting caspase activation. The erythropoietin receptor required for erythropoietin signaling localizes to photoreceptor cells. The protective effect of hypoxic preconditioning is mimicked by systemically applied erythropoietin that crosses the blood-retinal barrier and prevents apoptosis even when given therapeutically after light insult [42]. Activation of the HIF-1 pathway and/or application of erythropoietin may, through the inhibition of apoptosis, be beneficial for the treat-

ment of different forms of retinal disease [9].

Proteins involved in DNA repair: HIF-1 inhibits MSH2 and MSH6 expression [64], suggesting that HIF-1 may be responsible for the genetic instability characteristic of cells undergoing hypoxic stress.

7.2.3Activation of HIF by Non-hypoxic Stimuli

HIF-1 can also be activated independently of O2 levels. As mentioned already, desferrioxamine and cobalt ions inhibit the prolyl hydroxylases by chelating or replacing, respectively, the Fe(II) ion in the prolyl hydroxylase catalytic site [110]. Therefore, they inhibit HIF-1 degradation, thus stabilizing its expression. This mechanism explains the stimulatory effect of desferrioxamine and cobalt ions on VEGF expression [47]. Moreover, the hydroxylation reaction requires 2-oxoglutarate as a substrate and generates succinate as a side product [110]. As a result, succinate inhibits HIF-1 alpha prolyl hydroxylases in the cytosol, leading to stabilization and activation of HIF-1. This provides a link between the tricarboxylic acid (TCA) cycle (i.e., the energy-producing metabolic pathways of the cell) and HIF-1 activation, VEGF expression and angiogenesis [108]. Moreover, this link may explain why succinate dehydrogenase (SDH) and fumarate hydratase, both enzymes of the TCA cycle, function as tumor suppressors [109].

In addition, HIF-1 can be induced in an oxygenindependent manner by various cytokines, hormones, growth factors or other stimuli (nitric oxide, increased temperature, or mechanical stress) through the PI3K-AKT-mTOR pathway [79, 82, 97, 98]. These signaling pathways may play a key role in HIF-1 activation, VEGF expression and neovascularization in the retina. For example, in the diabetic retina, IGF-I potently stimulates VEGF expression and vitreous

IGF-I levels correlate with the presence and severity of diabetic retinal neovascularization [81]. Intravitreous IGF-I injection dose-dependently increases retinal Akt, JNK, HIF-1, NF-κB and AP-1 activity, and VEGF levels and causes microangiopathy [26]. In vitro, IGF- I potently stimulates VEGF expression in RPE cells [103]. IGF-I stimulates VEGF promoter activity in vitro, mainly via HIF-1, and secondarily via NF-κB and AP-1, as demonstrated by deletional mapping of the VEGF promoter and by electric mobility shift assays (EMSA) [96]. Systemic inhibition of IGF-I signaling with a receptor neutralizing antibody, or with inhibitors of PI-3 kinase (PI-3K), c-Jun kinase (JNK) or Akt, suppressed retinal Akt, JNK, HIF-1, NF-κB and AP-1 activity, VEGF expression, as well as ICAM- 1 levels, leukostasis and blood-retinal barrier breakdown, in a diabetic animal model [96].

Insulin is another factor that can activate HIF-1. Acute intensive insulin therapy transiently worsens diabetic retinopathy and is known epidemiologically as an independent risk factor for it. Acute intensive insulin therapy markedly increases VEGF mRNA and protein levels in the retina of diabetic rats, by activating HIF-1 via a pathway that involves p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol (PI) 3-kinase, but not p42/p44 MAPK or protein kinase C [96].

7.2.4VHL and Its Role in Retinal Angiogenesis

von Hippel-Lindau disease (VHL), also known as angiomatosis retinae, is a hereditary autosomaldominant cancer syndrome predisposing to a variety of malignant and benign neoplasms (frequently retinal, cerebellar, and spinal hemangioblastoma, renal cell carcinoma, pheochromocytoma, and pancreatic islet cell tumors, endolymphatic sac tumors, and benign cysts affecting a variety of organs) which is caused by germline mutations in the VHL gene, which is located on chromosome 3p25 [76]. Hemangioblastomas tumors of the stromal cells, usually of the CNS and retina, are seen in VHL disease but also occur as sporadic non-hereditary tumors, frequently caused by somatic VHL mutations. The stromal cells have sustained the genetic damage in these tumors, as detected by tissue microdissection, in situ hybridization, and immunohistochemical studies [67, 131]. It is the activation of the HIF pathway in the stromal cells that results in upregulation in expression of VEGF mRNA and protein, which then stimulates vascular growth in a paracrine fashion [14, 39, 44].

VHL associates with elongins B and C, cullin 2 and Rbx-1 in a multiprotein complex with ubiquitin ligase activity for specific substrates such as HIF-1.

In normoxia, VHL protein recognizes the presense of hydroxylated prolines by oxygen dependent hydroxylases in the HIF-1 protein and targets it for proteasomal degradation by ubiquitylation. Therefore in normoxia the HIF-1 levels and the expression of the HIF-1 responsive genes are also low. In hypoxia, HIF-1 does not contain hydroxylated prolines because the activity of the hydroxylases is inhibited by oxygen. This inhibits the interaction with the VHL complex and the accumulation of HIF-1 and HIF- 1 responsive genes. Additionally, pVHL interacts directly with the transcription factor Sp1 and suppresses the Sp1-mediated activation of the VEGF promoter [84].

The VHL complex regulates HIF-1 protein levels not only by targeting it to ubiquitylation but also ensuring that the ubiquitylated protein is degraded by the proteasome. That is mediated through the interaction of VHL-HIF-1 protein with Tat-binding protein TBP-1, an ATPase that facilitates the recruitment of the complex to the proteasome and the ATPmediated degradation of HIF-1 during normoxia [24]. VHL complex ubiquitylates in an oxygendependent fashion a constituent of the RNA polymerase II complex, Rbp7, that regulates the transcription of VEGF in a gene and tissue specific manner. Therefore VHL is linked to VEGF in a non-HIF- dependent fashion [86].

Of course VHL’s role extends beyond the regulation of hypoxia-induced genes. Another constituent of RNA polymerase II, Rbp1 is also dependent on VHL ubiquitylation. Only the active hyperphosphorylated form of RBp1 as the one induced in response to DNA damaging factors is subject to ubiquitylation, giving rise to the hypothesis that this interaction is a safety mechanism in order to maintain efficient DNA repair and prevent cell death. VHL also interacts with the active form of PKC, preventing it from causing cytoskeletal disorganization, and cytoskeletal components such as fibronectin. It was also recently suggested that the VHL complex inhibits transcription elongation/initiation of specific genes including HIF1 by association with different elongin BC ligases [25].

It was recently reported that adenoviral-mediated transfer of the VHL gene inhibits neovascularization in a murine laser-induced multiple branch retinal vein occlusion (BRVO) model, assessed by color photographs and fluorescein angiography (FA). VEGF mRNA expression was also significantly reduced in the adVHL-treated retina and iris. In accordance with the above, there was also an observed reduction of retinal edema, neovascularization elsewhere (NVD), new vessels elsewhere (NVE) and rubeosis [2].

Essentials

VEGF is an endothelial cell mitogen that promotes the formation of new vessels. It has five different isoforms and binds to two high affinity receptors (flt-1 and flk-1)

Hypoxia, hyperglycemia, reactive oxygen intermediates, growth factors such as IGF-1 and inflammatory mediators upregulate VEGF

Signal transduction intermediates in the VEGF activated pathways include Akt, MAPK, and STAT3

VEGF, also known as vascular permeability factor, is an endothelial cell mitogen [40] that promotes the formation of new vessels. It exists in five different isoforms of 121, 145, 165, 189, and 206 amino acids, which are derived from alternatively spliced mRNAs, of which VEGF165 is the predominant molecular species. It binds two high-affinity receptors, the 180-kDa fms-like tyrosine kinase (Flt-1, also known as VEGFR1) and the 200-kDa kinase insert domaincontaining receptor (KDR), also known as fetal liver kinase (flk) or VEGFR2, but KDR transduces the signals for endothelial proliferation and chemotaxis [35, 112].

Hypoxia stimulates VEGF mRNA expression mainly via binding of HIF-1to consensus and ancillary hypoxia-response elements (HREs) in the VEGF promoter. Also, hypoxia may increase the stability of VEGF mRNA [71, 72]. Other inducers of VEGF are advanced glycation end products (AGEs), hyperglycemia, reactive oxygen intermediates, inflammatory mediators, prostaglandins, IGF-I and insulin.

VEGF participates in the pathogenesis and progression of a wide range of angiogenesis-dependent diseases, including cancer [40], certain inflammatory disorders, and diabetic retinopathy. VEGF stimulates endothelial cell proliferation and neovascularization via a MAPK-dependent pathway [85]. VEGF may also promote endothelial cell migration and vascular permeability [5]. The resulting vessel leakiness promotes interstitial edema and worsens hypoxia, further stimulating VEGF production. VEGF also provides endothelial cells with a cytoprotective, anti-apoptotic stimulus through Flk-1/KDR-mediat- ed phosphorylation/activation of Akt [41]. Since Akt can stimulate VEGF expression, the latter may be part of an autocrine loop through which VEGF stimulates its own gene expression. Another loop may involve STAT3, as VEGF can activate STAT3 signaling in retinal microvascular endothelial cells via a

VEGFR2/STAT3 complex that induces STAT3 tyro- 7 I sine phosphorylation, nuclear translocation and

stimulation of VEGF expression [136].

7.4Retinal Hypoxia and Retinopathy of Prematurity

Essentials

Retinopathy of prematurity (ROP) is a disease of abnormal vasculogenesis in premature babies of very low weight

The prevalent pathogenesis for ROP features an early occurring insult (hyperoxia, sepsis, etc.) that results in arrest of vasculogenesis that subsequently resumes without forward progress resulting in a ridge formation with neovascularization and sequelae such as hemorrhage and retinal detachment

The pathophysiology of ROP involves the hyperoxia-induced vascular growth arrest (Phase I) and the hypoxia induced vascular growth (Phase II)

Phase I involves the downregulation of angiogenic factors (VEGF, IGF-I) and the upregulation of anti-angiogenic factors [pigment epithelium-derived factor (PEDF), endothelin-1 and platelet-activating factor (PAF)]. Free radical intermediates generated from oxygen species or nitric oxide oxidants overwhelm the scavenging cellular mechanisms and result in microvascular dysfunction and apoptosis

Phase II involves the HIF-1- and HLF-medi- ated upregulation of VEGF

IGF-1 is downregulated in ROP and the duration for which its levels are low correlates with the severity of the disease

7.4.1 Introduction

Retinopathy of prematurity is a disease of delayed or abnormal retinal vascular growth in premature babies especially of very low birth weight. Normally, the retina vasculogenesis begins in the 16th week of gestation as waves of mesenchymal spindle cells originating from the optic nerve lead shunts that are later covered by endothelium forming lumens. The retina is normally completely vascularized at full term (40 weeks of gestation) [6]. However, in the premature neonate, the retina remains incompletely vascularized at the time of birth and, consequently, very susceptible to toxic insults. Several theories exist on the pathogenesis of ROP, featuring an early occurring insult (high oxygen concentration, vita-

min E deficiency, sepsis, intraventricular hemorrhage) to the premature vessels growing to the nasal and temporal retinal periphery, resulting in the temporary arrest of vasculogenesis. After that initial injury the vessel growth resumes but the vessels start growing without forward progress forming a vascular ridge that can be of sizeable proportions. This vascular ridge can regress or progress into the vitreous cavity, resulting in hemorrhage and retina detachment [87]. Unchecked neovascularization can lead to local scarring, contracture, and detachment of the retina, with or without intraocular hemorrhage. Extensive retinal detachment in premature infants typically causes severe visual impairment, even with successful surgical reattachment of the retina [93]. Although low birth weight, low gestational age, and use of supplemental oxygen are the most important predisposing factors for ROP, it has been suggested that genetic factors may confer additional susceptibility [135].

7.4.2Pathophysiology of Retinopathy of Prematurity

The pathophysiology of ROP can be separated into two distinct phases [116]: Phase I involves the hype- roxia-induced vascular growth arrest and Phase II involves the relative hypoxia-induced vascular growth. Multiple factors and signal transduction pathways operate in each phase, whereas it seems that single exposure to hyperoxia does not result in the pathologic sequela of ROP but a combination of hyperoxia and hyperbarism is necessary. Hyperbarism can impede further the retinal and choroidal flow by inducing vasoconstriction in the choriocapillaris [21].

Phase I is characterized by the oxidative damage, the downregulation of angiogenic factors such as VEGF and IGF-I [69], the upregulation of anti-angio- genic factors such as PEDF [27] and modulating factors such as endothelin-1 and PAF [8]. Upon exposure to high oxygen concentrations (hyperoxia) the retinal tissue acts like an oxygen sink that is especially susceptible to oxidative damage. That is due to its high content of fatty acids with labile double bonds and a constant exposure to light that by photoexcitation can induce free radical formation and peroxidation reaction products acting on these double bonds. Oxygen radicals, peroxynitrite, hydroxyl radicals and lipid peroxidation products that are formed overwhelm the endogenous antioxidant mechanisms and cause cellular damage by reacting with membrane lipids, nucleic acids and metal containing compounds. That leads to microvascular dysfunction and selective death of the vascular endothelium that is more susceptible to damage than the pericytes

HYPEROXIA

Vascular insult

Vascular growth arrest

(Phase I)

HYPOXIA

↑ HIF-1α, HLF

↑ VEGF

Vascular growth

(Phase II)

Fig. 7.2. Pathophysiology of phases I and II of retinopathy of prematurity. Phase I is characterized by vascular growth arrest that results from the hyperoxia-induced vascular insult through reactive oxygen species (ROS) and the downregulation of protective growth factors such as IGF. The resultant hyperoxia results in rebound increase of transcription factors such as HIF- 1 and through them of angiogenic factors such as VEGF. This is a compensatory mechanism that promotes vascular growth in phase II

and the smooth muscle cells [19]. The importance of reactive oxygen intermediates in the pathogenesis of ROP is evident with administration of antioxidants to murine models and subsequent prevention of retinopathy. In parallel, protein levels of “free radical scavenger enzymes” such as superoxide dismutase (SOD) decrease upon exposure to constant hyperoxia whereas delivery of SOD via long-circulating liposomes reduces the oxygen-induced vaso-obliter- ation [90]. Among free radical donors, NO seems to play an important role in the hyperoxia-induced vasoattenuation of the early phases of ROP. NO derived oxidants such as peroxynitrite are generated in various models of ischemia and their levels are found to correlate with exposure to high concentrations of inspired oxygen in human infants who develop bronchopulmonary dysplasia. Peroxynitritemediated cellular injury includes the modification of important cellular proteins, the impairment of endothelial cell proliferation and migration and VEGF-

induced phosphorylation of the KDR receptor. Additionally it seems that NO and VEGF are reciprocally I 7 regulated [127]; NO donors inhibit VEGF expression

in vivo whereas activation of VEGF receptors such as KDR activates endothelial NO synthetase (eNOS) that upregulates NO and inhibits VEGF expression via a negative feedback inhibition loop involving the AP-1 binding to the VEGF promoter. In agreement with the above the hyperoxia-induced VEGF downregulation in eNOS knock-out animals is less pronounced, contributing to the improved oxygen tolerance of their retinal capillaries in murine models of ROP. The same protective effect can be elicited with pharmacological inhibition of the NOS activity [19].

The oxidative stress can be augmented by the upregulation of PAF during this period. PAF seems to increase the generation of oxygen radicals and to also exert a direct and selective cytotoxic effect on the neurovascular retinal endothelial cells, inducing a form of death that seems to be intermediate between apoptosis and necrosis [68]. This cytotoxicity is independent of platelet aggregation and involves thromboxane TXA2, which along with PAF is a strong stimulant of calcium mobilization, an important element of both necrotic and apoptotic cell death processes. The PAF and TXA2 increase in cellular calcium results in the activation of specific phospholipases and proteases, the disruption of mitochondrial permeability transition pores and the arrest in ATP production that leads to energy starvation and sustains the cytotoxic cycle. The role of PAF in retinal vaso-obliteration has been demonstrated with the protective effect of PAF receptor blockers in murine models of ROP [8].

During Phase I, hyperoxia induces a downregulation in various VEGF isoforms and the VEGF receptors flk-1 and flt-1. Although the signal transduction pathway of the hyperoxia-induced VEGF downregulation is not fully elucidated, it seems to involve the kinase c-abl and to be influenced by the administration of dexamethazone and COX-2 inhibitors [92]. VEGF regression during the period of hyperoxia correlates well with the apoptosis of the vascular endothelium and the regression of newly formed capillaries. Parallel to the VEGF downregulation, PEDF, an anti-angiogenic factor, is upregulated during the period of hyperoxia [103, 114]. PEDF is likely to contribute to the regulation of blood vessel growth in the eye by creating a permissive environment for angiogenesis when oxygen is limiting (as it is in tumors and in retinopathies) and an inhibitory environment when oxygen concentrations are normal or high [114]. Given its high potency and the broad range of angiogenic inducers against which it can act, PEDF may prove to be a useful therapeutic for pathologic ocular neovascularization as well as for retinoblasto-

mas, where its dual activities of inducing cell differ- 7 I entiation and inhibiting angiogenesis may be partic-

ularly effective [7].

Vasoconstriction during the hyperoxic phase can also be caused by the increased expression of endo- thelin-1 that is supposed to be secreted by the endothelial cells. The role of endothelin-1 is controversial and the signal transduction pathway involved is only partially known and involves the angiotensin-con- verting enzyme (ACE) [137]. ACE inhibition blocks hyperoxic-induced ET-1 secretion from retinal capillary endothelial cells and exerts a protective role in animal models of ROP [113].

Phase II of ROP is characterized by the relative hypoxia-induced proliferation of the retinal blood vessels when the infant is moved from the hyperoxic environment to normal room air. Hypoxia induces the upregulation of VEGF that leads to the retinal neovascularization and the invasion of the vitreous that characterizes clinical and experimental ROP [100, 121].The expression of VEGF in the innermost layers of retina falls during hyperoxia and increases on return to room air. Regression of retinal capillaries in neonatal rats exposed to high oxygen is preceded by a shut-off of VEGF production by nearby neuroglial cells. Intraocular injection of VEGF at the onset of experimental hyperoxia prevents apoptotic death of endothelial cells and rescues the retinal vasculature [3, 121].

The hypoxia-induced upregulation of VEGF is well studied and occurs through the activation of the transcriptional factor HIF-1 [131] and HLF (HIF-1- like factor otherwise known as EPAS) [33, 75] and its binding to multiple response elements on the VEGF promoter. Knock-out mice for HLF do not exhibit the proliferative neovascular response in an ROP murine model, highlighting the role of that transcription factor in ROP [83]. Interestingly enough these mice show reduced expression levels of erythropoietin, an HIF-regulated growth factor, and restoration of the erythropoietin levels renders these mice susceptible to retinopathy at a significant level. It is conceivable that VEGF and erythropoietin collaborate in the development of neovascularization in response to hypoxia. Recently, a dominant negative regulator of HIFs, IPAS, was isolated by Makino et al. [77] and was shown to be expressed in response to hypoxia in the retinal ganglion cell layer (GCL) and inner nuclear layer (INL), suggesting that a more complicated mechanism occurs during the neovascular response in ROP. Although knock-out mice for HIF-1 are not viable, mice deficient for HIF-1 transcriptional targets such as RTP-801 demonstrate a significant attenuation in the development of major pathologic features of this model such as retinal vaso-obliteration, retinal neovascularization and apoptosis of the INL

cells [17]. The mechanism with which RTP-801 protects against the pathological manifestation of ROP is not completely understood. Although RTP-801 loss could protect endothelial cells against hyperoxiainduced apoptosis and subsequent hypoxia-induced neovascularization, the mechanism seems more complex. In murine ROP models there always seems to be a clear correlation between the non-perfused retinal area and the neovascular response, and this relationship is perturbed in the RTP-801 knock-out mouse. An interesting alternative is that RTP-801 absence prevents the hypoxia-induced neuroretinal apoptosis exerting a direct effect rather than as a result of reduced vascular disease. RTP-801 has been shown to modulate cellular ROS levels and cell sensitivity to oxidative stress [117]. Interestingly, RTP-801 is also a transcriptional target of p53 [32], another transcriptional factor involved in the pathogenesis of oxygen-induced retinopathy [104]. p53 is known to trigger the expression of several genes involved in the cellular redox control and contribute to the mitochondrial apoptotic cascade initiated in various models of retinal ischemia. HIF-1 regulates p53 stabilization and activity via interaction with mdm2 [23]. Therefore the p53 and HIF-1 redox regulation pathways merge through the combined transcriptional regulation of RTP-801 in ROP.

On the other hand, vasculogenesis in ROP is also regulated by non-oxygen regulated factors such as IGF-I, which inhibits the retinal neovascularization when at low levels and stimulates it at high levels. IGF-I knock-out mice exhibit abnormal retinal vasculogenesis despite the presence of normal VEGF levels [116]. Low IGF-I levels in vitro prevent VEGFinduced phosphorylation of Akt, a kinase that is critical for the vascular endothelial cell survival. IGF-I levels are low in premature infants that develop ROP in comparison to the age-matched infants without the disease. The duration for which the retinal IGF levels are low correlates with the severity of the disease, making IGF-I an important player in ROP.

ROP was very prevalent in developed countries in the 1940s and 1950s, but the realization that high O2 was responsible for it led to more prudent use of O2 and a decrease in ROP. Two factors that are currently leading to an increase in ROP incidence are the improved availability of intensive care facilities for premature infants in developing countries and the increased survival of even more premature infants (very low birth weight infants) in developed countries [135]. The current therapy for prevention of the disease is generally to titrate the infant’s blood to an adequate PaO2 but to prevent systemic hyperoxia. Vitamin E supplementation may also be useful in decreasing the severity of ROP, possibly by functioning as an antioxidant [18, 60].