Ординатура / Офтальмология / Английские материалы / Retinal and Choroidal Angiogenesis_Penn_2008

against glutamate toxicity36 and motor neurons from chronic glutamatemediated neurodegeneration.37 It also promotes the survival and neuriteoutgrowth of developing avian and murine spinal motor neurons.38 In addition to these neurotrophic activities, PEDF also exhibits gliastatic activity, having direct effects on metabolism and proliferation of microglia from newborn rat brain.39 Thus, PEDF is a multipotent neurotrophic factor that may play a protective role in the retina and CNS in vivo and could be used as a therapeutic agent for the treatment of diseases characterized by neuronal and retinal degeneration.

4.PEDF, AN ANTI-ANGIOGENIC FACTOR

PEDF has been described as the most potent anti-angiogenic factor for the retina1. It inhibits endothelial cell migration activated by pro-angiogenic factors (e.g., fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) or interleukin-8), suppresses VEGF-induced retinal microvascular endothelial cell proliferation,40 and causes apoptosis of activated endothelial cells.41 It inhibits aberrant blood vessel growth in models of retinal neovascularization, such as the murine model of ischemiainduced retinopathy42,43 and the transgenic mice with increased expression of VEGF in photoreceptors.42 It prevents choroidal neovascularization upon laser-induced rupture of Bruch’s membrane42 and also the infiltration of new vessels into the cornea, a process that can be induced with exogenous angiogenic stimulators.1 Consequently, PEDF in the interphotoreceptor matrix (IPM) is not only a protector of photoreceptor cells but also a barrier for vessel intrusion from the choroid into the neural retina. By preventing the infiltration of vessels into the vitreous and cornea, this interesting extracellular factor allows external light to reach the retina, where the main chemical reactions of the vision process occur.

Because neovascularization also occurs in tumor growth, PEDF has been tested also in cancer models. PEDF can inhibit the formation of new vessels in tumors, such as melanomas,44,45 in hepatocellular carcinoma,46,47 in syngeneic murine models of thoracic malignancies,48 and in prostate tumors.49 In addition, it can also act as a tumor stabilizer or suppressor as shown for prostatic and pancreatic growth, Wilm’s tumors, and neuroblastomas, by inducing both tumor cell and endothelial cell apoptosis. Finally, kidney microvascular density, prostate size, and

angiogenesis increase in PEDF knockout mice compared with wildtype.49,50 Thus, overexpression of PEDF in animal models has also proven

beneficial in preventing pathological angiogenesis, and could be exploited

as a therapeutic means for the treatment of diseases characterized by neovascularization.

5.PEDF, A NATURAL COMPONENT OF THE EYE

There is extensive evidence for the expression and production of PEDF in the native eye. From the moment of its discovery, PEDF was associated with the retina. It was identified as a protein secreted by human fetal RPE

cells in culture, hence its name.26 RPE cells from several mammalian species express high levels of PEDF transcripts and protein.12,28,51-53 The

polarized RPE cells release the mature protein, preferably from their apical side, to be deposited in the IPM,12 where it is an essential component for

neurotrophic activity.15 PEDF is highly concentrated in the IPM (Tables 2 and 3).12,15 Although it represents less than 1% of the total soluble protein,

PEDF can be purified from saline lavages of the bovine IPM by ammonium sulfate fractionation followed by S-sepharose ion-exchange column chromatography.15

Expression of PEDF is not exclusive to the RPE. The protein is also found in vitreous,54 aqueous humor,55 cornea and choroid, as well as in non-ocular tissues. Its distribution correlates with the avascularity of the outer retinal layers, vitreous and cornea.56 Retinal glial (Müller) cells in culture also secrete PEDF,57 and photoreceptors, inner nuclear layer cells, and ganglion cells in adult human retina contain PEDF mRNA,58 indicative of their potential contribution to PEDF deposition in the IPM. Similarly, cultures of cells from the ciliary epithelium, trabecular meshwork, and anterior segments release PEDF to the media and effluents,55,59 suggesting contributions to PEDF accumulation in vitreous and aqueous. In the

vitreous, PEDF accumulates at concentrations of 20-30 nM, and in the aqueous at lower concentrations (3-6 nM).54,12 Given that the volume of the

vitreous is larger than that of the IPM and aqueous, purification of PEDF from vitreous yields the highest amounts of pure PEDF protein per eye. The concentrations of PEDF have been measured in the monkey and the cow (Tables 2 and 3). In human, the concentrations of PEDF in vitreous and aqueous were obtained from patients with macular hole and cataract surgery with no angiogenic eye diseases. Three different research groups

have reported values for the vitreal PEDF similar to those in bovine and monkey,4,6,60 while a fourth has reported values ten-fold higher.3 In

aqueous, PEDF concentrations were reported 3-20-fold higher than in bovine and monkey.5,7,61

aThe estimated volumes of IPM, vitreous and aqueous were 0.26 mL, 10 mL and 0.15 mL, respectively.

aThe estimated volumes of IPM, vitreous and aqueous were 0.063 mL, 1.2 mL and 0.2 mL, respectively.

Outside the eye, PEDF is also secreted by a variety of cells and deposited

in extracellular matrices, such as cerebral spinal fluid, human blood serum, and bone marrow matrix.17,62,63 The molecular interactions that govern the

deposition of PEDF in extracellular matrices are represented by interactions with glycosaminoglycans and collagens (see 8.2.4.).

6.PEDF IN DEVELOPMENT, AGING AND DISEASE

PEDF expression patterns in the human eye suggest that modulation of this protein over time may play a role in the development of normal neural and

vascular retina. RPE of the fetal human eye (7.4-21.5 weeks of gestation) expresses PEDF mRNA and protein.51,58 The protein is also detected in

photoreceptors and inner retinal cell types in developing human eyes,58 demonstrating its potential for action in vivo during early retinal development. Using a mouse model, Behling et al.64 detected PEDF expression in cells of the retina by late in gestation (E 18.5) and in a variety of ocular cell types over the next two weeks, with an evolution of expression pattern coinciding with the period during which retinal vasculature develops. In the choroid, PEDF was not detected until development was close to completion. Then it remained high throughout the life of the animal, suggesting that it may play an anti-angiogenic role in this tissue. PEDF protein was detected in the RPE at all time points.

Given that PEDF mRNA expression decreases with passage number of cultured RPE cells and that this is accompanied by loss of their proliferative potential and phenotype,51 it has been proposed that these changes are typical

of senescence and that PEDF expression in the human eye also decreases with age. Although there are no reports yet on age-related changes in PEDF expression in the eye, these have been reported in the skin.65 PEDF, also called EPC-1, is detected primarily in the dermal layer of the skin, and its expression declines with increasing donor age. This decline is statistically significant between young (less than 31 years old) and middle-aged (between 30 and 60 years old) donors, becoming less dramatic at older ages. An age-related decline in the retina could lead to lower PEDF-mediated neurotrophic and anti-angiogenic activities and an increase of ocular pathologies with age. It is worth noting here that several ocular neurodegenerative diseases are targeted at a young age.

Downregulation of PEDF correlates with several ocular neovascular and neurodegenerative diseases, like diabetic retinopathy (DR), age-related macular degeneration (AMD), glaucoma and retinitis pigmentosa. It has been shown that the vitreous of patients with choroidal neovascularization (CNV) due to AMD had lower PEDF levels and lacked the vitreal antiangiogenic activity of age-matched controls.3 PEDF levels in the aqueous

humor of eyes with retinitis pigmentosa and advanced glaucoma were significantly lower than those in eyes with cataract alone.61,66 Comparison

between levels of PEDF in vitreous of eyes with DR and idiopathic macular hole, between proliferative and nonproliferative DR, and between active and inactive DR showed lower vitreal concentration of PEDF with higher levels of vascular endothelial growth factor (VEGF) in each case.2,5-8 In experimental animal models for ocular neovascularization, the levels of PEDF were inversely correlated with formation of CNV by laser-mediated rupturing of Bruch’s membrane.67 These observations suggest that loss of PEDF creates a permissive environment for angiogenesis and neuropathy in patients that may contribute to progression of ocular neovascular and neurodegenerative diseases.

Pathological progression-related changes have also been reported outside the eye, such as in some prostate cancers. Studies with PEDF gene ablated mice have shown that a developmental deficiency in PEDF can cause profound changes in the size and cellularity of the prostate and pancreas.49 In both a rat model and in human tumors, the proliferation index and vascular count inversely correlated with PEDF mRNA levels, suggesting that loss of PEDF expression could be associated with the progression toward a metastatic phenotype in prostate cancer.68 In neurodegenerative diseases, elevated PEDF protein levels have been detected in the cerebrospinal fluid of patients affected by amyotrophic lateral sclerosis compared with patients with other neurodegenerative diseases,62 suggesting an autoprotective reaction in amyotrophic lateral sclerosis. Similar suggestions have been made from PEDF increases in

rodent retinas after panretinal photocoagulation53,60 and in penetrating ocular injury, which suppressed retinal neovascularization.69 These increases might represent an attempt by the tissue to limit neuronal damage and/or to counterbalance the neovascularization stimulus.

Although the lines of evidence for the association of changes in PEDF levels with development, age and pathology are increasing, little is known about the molecular basis for these changes (see 8.2.5.).

7.MECHANISMS OF ACTION

7.1Structure-function relationships

Segmentation of the PEDF polypeptide by chemical proteolysis and recombinant DNA technology provided much of the information accumulated to date on structure-function. PEDF cleaved at its serpin exposed loop retains neurotrophic23 and anti-angiogenic properties.70 More

importantly, even when PEDF is truncated from the C-terminal end (e.g., bacterially expressed BH (Asp44-Pro418), BP (Asp44-Pro267), BX (Asp44-

Leu228) and BA (Asp44-Thr121)), it retains its neuronal differentiating and

survival activities in retinoblastoma cells and cerebellar granule cell neurons and motor neurons.23,33-35,38,71 Furthermore, synthetic peptides designed from the smallest BA region, 34-mer (Asp44-Asn77) and 44-mer (Val78-Thr121),

exhibit anti-angiogenic and neurotrophic activities, respectively.70,72-74 These observations demonstrate that two distinct regions on the PEDF primary structure, namely the aforementioned 34-mer and 44-mer, confer antiangiogenic and neurotrophic properties to the PEDF polypeptide, respectively. Since both are separated from the homologous serpin reactive site in the primary as well as in the tertiary structure of PEDF, inhibition of serine proteases is not a mechanism of action for PEDF, and its overall native conformation is not required for its biological activities. These findings provided an example of the separation of inhibitory and other activities in a serpin.

7.2Search for a receptor

Given that serine protease inhibition cannot explain the biological activities of PEDF, investigations were directed toward the hypothesis that PEDF’s neurotrophic activity could be mediated by interactions with cell surfaces. Focusing on human retinoblastoma cells, bovine retina, rat cerebellar granule

cells, and mouse motor neurons, we prospected for PEDF receptors and found evidence for (1) a saturable, specific, and high-affinity class of receptors on the surface of all cells, with characteristics of an ~80-kDa

plasma membrane protein; and (2) the concurrence of receptor-binding and neurotrophic activity in the same region of PEDF (the 44-mer).72,73,75

Examination of the expression of PEDF receptors in native retinas provided evidence for PEDF binding to cell-surface receptors distributed discretely in the neural retina of adult steers, i.e., inner segments of photoreceptor and ganglion cell layer.75 These receptor sites correlate with PEDF effects on the survival and morphogenesis of photoreceptor cells in vivo, retinal ganglion cells in vivo, and retina cells in culture, in agreement with their functionality. The binding parameters for the ligand-receptor interactions from different species provide support for PEDF receptor homologs (Table 4). Thus, we established that the first step in the biological activity of PEDF is the binding to receptors on the surface of target cells, a significant advance in the elucidation of PEDF’s mechanisms of action. Moreover, the data are of particular importance because they offer an anatomical basis for studies to assess PEDF’s neurotrophic effects on the adult retina.

Table 17-4. Physicochemical Parameters of PEDF Binding

7.3Signal transduction pathways

Evidence on the downstream events that occur upon PEDF binding to cell surface receptors is beginning to be obtained. One line of evidence for signaling comes from studies performed with systems other than the retina.33 In cerebellar granule cell neurons, the neuroprotective effect of PEDF against glutamate toxicity involves alterations in the intracellular homeostasis of calcium.34 PEDF could interact and modulate the membrane Na+/Ca++ exchanger, or increase the expression of intracellular proteins like calbindin or parvalbumin, which are implicated in sequestering calcium. The survival effect of PEDF on granule cell neurons involves the nuclear

factor-κΒ (NF-κΒ) pathway, which is known to play a role in the protection of neuronal cells in several models of apoptotic death induction.76 A member of the IκΒ inhibitory proteins normally sequesters NF-κΒ in the cytoplasmic compartment. When cells are exposed to activators of NF-κΒ, these IκΒ proteins are phosphorylated, ubiquitinated, and degraded by proteosomes, and then NF-κΒ is released and translocates to the nucleus, where it binds to responsive elements on the DNA. PEDF treatments decrease IκΒα and IκΒβ proteins in a time-dependent fashion. Furthermore, pretreatment of cells with an inhibitor of either IκΒ phosphorylation (BAY-11-7082) or of the proteosome activity on IκΒ (ALLN) inhibits NF-κΒ activation by PEDF. The downstream events following NF-κΒ activation have also been investigated. In immature cerebellar granule cells, but not in mature ones, the PEDF is able to induce anti-apoptotic effectors, like Bcl-2, Bcl-x and Mn-SOD. PEDF is also able to induce other neurotrophic factors like nerve growth factor, brain-derived neurotrophic factor, and glial cell-derived neurotrophic factor.77 Thus, PEDF promotes neuronal survival through activation of NF-κΒ, which in turn induces expression of anti-apoptotic and/or neurotrophic factor genes. Although PEDF enhances expression of other neurotrophic factors or chemokines, it does not exert its neuroprotective effect on cerebellar granule cell neurons through their production.77

In contrast to the neuronal survival effects, the anti-angiogenic effects of PEDF have been associated with induction of endothelial cell apoptosis.41,43

Several pro-angiogenic stimuli are able to induce elevated membrane levels of CD95/Fas, a receptor related to apoptotic cell death, whereas antiangiogenic factors like PEDF are able to increase the expression of endothelial Fas ligand (FasL). When expressed simultaneously, these two molecules are able to induce apoptosis in endothelial cells, and therefore to inhibit angiogenesis.78 This provides an example of cooperation between proand anti-angiogenic factors in the inhibition of angiogenesis and is one explanation for the ability of angiogenesis inhibitors to select remodeling capillaries for destruction.

Induction of angiogenesis by vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) requires activation of the nuclear factor of activated T cells (NFAT).79 In a study by Zaichuk et al.,41 such activation is blocked by PEDF. A variety of upstream regulatory molecules are used in NFATc2 regulation by PEDF. A dramatic increase in phosphoJNK levels and JNK-dependent substrate phosphorylation by PEDF is observed exclusively in activated endothelial cells. SP600125, a generic JNK inhibitor, reduces NFATc2 phosphorylation and restores nuclear localization in affected endothelial cells. The same compound has no effect on VEGFor bFGF-dependent angiogenesis, but destroys the response to PEDF both in

vitro and in vivo, pointing to JNK kinases and their downstream targets as critical elements of PEDF angioinhibitory signaling. Expression of a novel NFAT target, caspase-8 inhibitor cellular Fas-associated death domain-like interleukin 1α-converting enzyme inhibitory protein (c-FLIP), which mediates resistance to apoptotic signaling, is coregulated by VEGF and PEDF. The VEGF-dependent increase of NFATc2 binding to the c-FLIP promoter in vivo is attenuated by PEDF. Thus PEDF may counter-regulate VEGF or bFGF in angiogenesis via cFLIP controlled by NFAT and its upstream JNK.

It is thought that in the neural and vascular retina, PEDF triggers signaling events similar to those described above to exert survival and antiangiogenic effects, respectively.

8.REGULATION OF PEDF

Given that aberrant regulation of PEDF may be conducive to neovascularization and/or neuronal cell death, it is of interest to define the mechanisms that govern its regulation and turnover to better exploit this interesting protein as a relevant therapeutic factor for the eye. Alterations in the expression, structure, and localization of PEDF can be exploited to modulate its efficacy. A scheme summarizing this section is shown in Figure 2.

8.1Regulation of expression of the PEDF gene

Retinoids, in particular all trans-retinoic acids, are potent regulators of cell proliferation.80 They regulate gene transcription by binding to and activating two classes of nuclear transcription factors: the RA receptors (RARs) and the retinoid X receptors (RXRs). It has been shown that the PEDF promoter has a functional retinoic acid responsive region.81 PEDF expression can be increased in Y-79 cells, ARPE19, an immortalized cell line derived from RPE cells, and endothelial cells by retinoids. PEDF, in turn, can upregulate the expression of specific RARs and RXRs.

Dexamethasone, a synthetic glucocorticoid used especially as an antiinflammatory and anti-allergy agent, also induces PEDF mRNA expression in both mouse Muller glial cells and C6 rat glioma cells,81 as well as in human trabecular meshwork from human eyes.82 PEDF protein levels also increased significantly in dexamethasone-treated trabecular meshwork cells over non-treated controls,59 implying a direct effect of dexamethasone on upregulating PEDF expression.

Collagen

GAG

MMPs

Receptor

DEX, retinoids

DNARNA

PEDF mRNA

Figure 17-2. Production and regulation of PEDF. Synthesis of PEDF mRNA is induced by retinoids and dexamethasone (DEX). The PEDF mRNA codes for a precursor PEDF protein that is glycosylated, and its N-terminal signal peptide is removed upon secretion from the cell. In the extracellular matrix (ECM), the mature PEDF is a highly protease-resistant protein that binds to collagens and glycosaminoglycans (GAG), ECM components. However, matrix metalloproteinases (MMPs) can degrade the PEDF protein, resulting in inactivation of its anti-angiogenic and neurotrophic activities. PEDF has affinity for a cell-surface receptor, and this interaction seems necessary for its biological activities. The PEDF ligand-receptor interactions can be modulated by GAGs.

In contrast, leptin, a circulating hormone secreted mainly from adipose tissues involved in the control of body weight, downregulates PEDF gene expression, while it upregulates the VEGF gene in bovine cultured retinal pericytes.83 Interestingly, like VEGF, leptin acts as an angiogenic factor and is found elevated in vitreous from patients with angiogenic eye diseases, supporting a control for a pro-/anti-angiogenic balance.

8.2Post-translational regulation of PEDF

8.2.1Post-translational modifications

Glycosylation, N-terminus modifications or unfolding of the PEDF protein do not affect its biological activities, as demonstrated by the activity of bacterially-derived PEDF.71 However, different phosphorylation sites can convert PEDF from a neurotrophic to an anti-angiogenic factor. For example, prevention of phosphorylation at Ser24 and Ser114 of human PEDF abolished its neurotrophic activity but enhanced its anti-angiogenic activity, while phosphorylation at Ser227 decreased the PEDF anti-angiogenic activity.13

8.2.2Cell polarization

Because polarization is constantly under the control of regulating factors coming from the choroid and the retina or of other extracellular stimuli (e.g., extracellular molecules of oxygen), the mechanisms that control cell polarization may be important for regulating PEDF. Defects in or loss of cell polarity may determine changes in PEDF secretion and play a role in the pathogenesis of retinal degeneration, and choroidal and retinal neovascularization. Although PEDF is released preferentially from apical membranes,12 little is known about control of intracellular PEDF trafficking and release in the polarized RPE cells.

8.2.3Interactions with glycosaminoglycans

Ligand-receptor interactions can regulate the activity of PEDF. A study on the effects of glycosaminoglycans on the ligand-receptor interactions of PEDF shows that heparan sulfate (HS) is a cofactor that can modulate these interactions.84 PEDF has binding affinity for heparan sulfate and heparin,16 and the PEDF-heparin/HS complex has a higher affinity for receptors. Interaction with cofactors may induce a conformational change in PEDF that accelerates the ligand-receptor interactions. The receptor may also form a complex with heparin/HS to facilitate interactions with the ligand. These observations offer interesting possibilities for regulation of the activity of PEDF. The ratio and amount of production of heparin and HS by cells bearing PEDF receptors may be just as important for controlling the activity of PEDF as modulation of the rate of expression of PEDF receptors. Different types of glycosaminoglycans may differentially modulate ligand-receptor interactions by increasing or even decreasing their affinities.