PEDF: Raising Both Hopes and Questions in Controlling Angiogenesis

Commentary PEDF: Raising both hopes and questions in controlling angiogenesis Gerald J. Chader* The Foundation Fighting Blindness, 11350 McCormick Road, Hunt Valley, MD 21031 epending on timing and venue, new abnormal blood vessel growth. Although found in most tissues, normal cell types Dblood vessel growth can be either this gives hope for ultimately controlling (neuronal and nonneuronal), and tumors, beneficial or pathological. Vigorous vessel neovascularization, it also raises interest- as surveyed by Western and Northern blot growth is absolutely necessary for tissue, ing questions about PEDF’s biological analysis (8). DNA sequence analysis shows organ, and limb development as was activities. that PEDF belongs to the serine protease graphically demonstrated a few decades In 1989, a novel neurotrophic activity inhibitor (SERPIN) gene family (5, 8) ago with thalidomide, now known to be a was first described by Tombran-Tink and which includes a large number of proteins compound with antiangiogenic proper- Johnson (3) in conditioned medium from of widely divergent function, such as ties. As one ages though, the need for cultured fetal retinal pigment epithelial ovalbumin, alpha 1–antitrypsin, angio- angiogenesis decreases or stops entirely (RPE) cells. In a test system using cul- tensinogen, and GDN͞PN-1, another ser- except for specialized cases such as wound tured, primitive retinoblastoma cells, ex- pin with neurotrophic properties. Inter- repair. In fact, neovascularization in the tensive neuronal-like processes were in- estingly, PEDF does not seem to exhibit adult is usually associated with some form duced after exposure to the RPE antiprotease activity with the neurotro- of pathology—tumor growth, for example, conditioned medium (Fig. 1). This factor, phic active site residing at the other end of or diabetes. New work by Bouck and co- PEDF, has now been purified (4) and the peptide chain from the antiprotease workers (1), as reported in PNAS, now cloned from both human (5) and mouse active center (9, 10) seen in the classical gives evidence that a natural protein, pig- (6). The gene is active at 17 weeks in serpins. The noninhibitory nature of the ment epithelium-derived factor (PEDF), human fetal RPE cells (7), making it a PEDF molecule has been well studied by is a potent inhibitor of abnormal blood candidate as a factor involved in early Becerra and coworkers (11). vessel growth in a murine model of ocular neuronal development. During the time when the PEDF pro- neovascularization. This, together with Subsequent work has demonstrated that tein and gene were being isolated, Cristo- previous data from the same group (2), PEDF is mainly a secreted glycoprotein of falo and coworkers (12) described a pro- gives compelling evidence that PEDF may about 50 kDa that is not uniquely synthe- tein in WI-38 fibroblast cells, a prime be pivotal in controlling both normal and sized by fetal RPE cells. PEDF mRNA is model system for studying aging and senescence, whose expression was (i) re- stricted to the Go stage of the cell cycle in young cells and (ii) not expressed in older, senescent cells. This protein, called EPC-1, was found to be identical to PEDF. Subsequent work (13, 14) rein- forces the notion that PEDF is intimately associated with both the cell cycle and senescence. The expression of PEDF mRNA and the secreted protein are much more highly (100-fold) expressed in Go (growth arrest) of early-passaged cells than in rapidly proliferating young cells or in later-passaged (senescent) cells. Palm- ieri and coworkers (15) have found similar results in PEDF down-regulation in proliferating and in senescent human endo- metrial stromal fibroblasts. With the finding that PEDF was neurotrophic in cultured retinoblastoma tumor cells, it was of interest to determine whether PEDF had an effect on normal neurons. For this purpose, the effect(s) of both native and recombinant PEDF on normal cerebellar granule cells (CGCs) in primary culture was examined. Tanawaki Fig. 1. Effect of PEDF on retinoblastoma morphology. (A) Proliferating, undifferentiated Y-79 retinoblastoma cells in culture. (B) Nonproliferating cells treated with PEDF demonstrating neuronal-like See companion article on page 2593. morphology. [Reproduced with permission from ref. 27 (Copyright 1994, Academic Press).] *E-mail: [email protected]. 2122–2124 ͉ PNAS ͉ February 27, 2001 ͉ vol. 98 ͉ no. 5 www.pnas.org͞cgi͞doi͞10.1073͞pnas.061024098 Downloaded by guest on September 27, 2021 and coworkers (16) found that PEDF did not induce a more neuronal phenotype in the CGCs, but did have a marked effect on CGC survival. Usually, most of the cells are dead by 2–3 weeks in culture. How- ever, with PEDF added to the medium, the cells live much longer. It was also established that PEDF did not enhance mitotic activity of the CGCs and thus appeared to have an effect on increased survival instead of increased replication. In more detailed experiments, Araki and coworkers (17) found that cell death of the CGCs was by ‘‘natural apoptosis’’ and that, at least in immature CGCs, PEDF was a potent inhibitor of natural and induced apoptosis. The effects of PEDF are not limited to brain neurons. Houenou and coworkers (18), demonstrated that PEDF effectively promotes both the dif- ferentiation and survival of developing spinal motor neurons. Similarly, Bilek and coworkers (19) found that, in an organo- typic culture model system for motor neuron degeneration, PEDF spared the spinal chord’s gross morphological appearance and total number of neurons. Along with chronic, long-term neurotrophic effect, PEDF is also quite effective in protecting neurons against rapid neurotoxic effects such as glutamate toxicity (20). The pro- Fig. 2. In situ angiogenesis in PEDF-treated retinas. Low-power (Left) and high-power (Right) views of tective effect is seen with only a very short retinas of mice exposed to hyperoxia and subsequently treated with vehicle (a–d), or with PEDF at 11.2 preincubation with PEDF (30 min) and at (e and f) or 22.4 (g and h) ␮g͞day. a very low dose (0.47 nM). The level of protection is inversely correlated with the intracellular calcium level after glutamate Diabetes is certainly a disease with CNS into the vitreal surface were counted. As COMMENTARY poisoning. DeCoster and coworkers (21) involvement and with neovascular pathol- expected, PEDF was found to fully inhibit noted a protective effect of PEDF on ogy as well. Nowhere is this more ‘‘visible’’ extrusion of the endothelial cells into the hippocampal neurons, albeit of a smaller than in the eye, where the neural retina is vitreous cavity. The low dose seen to be nature, whereas Cao and coworkers (22) normally protected by an antiangiogenic effective in these studies is within the same found that rat retinal neurons were pro- shield or environment but, with breaching low dose range (i.e., nM) of PEDF needed tected against hydrogen peroxide-induced of the shield, is devastated by neovascular to effect CGC neuroprotection as cited apoptosis. It is thus clear that PEDF and growth as in diabetic retinopathy or reti- above. The case for PEDF as a (if not the) its recombinant peptides can protect neu- nopathy of prematurity. The article by major antiangiogenic factor in the eye is rons in both long-term and more acute Bouck and coworkers (1) now gives im- greatly strengthened if one considers pre- situations. Protection is seen in both the portant information about aberrant blood vious work from Bouck’s group. Dawson central nervous system (CNS) and the vessel proliferation in the eye and the and coworkers (2) demonstrated that spine making PEDF of potential use possible role of PEDF in controlling this PEDF is probably responsible for the nor- against diseases of the CNS (brain, retina) process. By using a well known murine mal avascularity of several of the ocular and of peripheral neurons as well. model of ischemia-induced retinopathy components. Also, loss of PEDF plays a If these characteristics were not suffi- (24), they demonstrate that the neovascu- ‘‘permissive role’’ in ischemia-induced ciently interesting for a protein, Sugita lar process in the eye can be controlled by neovascular growth. and coworkers (23) noted another striking administering relatively low doses of How does PEDF prevent the retinal property of PEDF. CGC cultures contain PEDF. Interestingly, treatment with endothelial cells from responding to isch- contaminating glial elements that, as the PEDF is i.p. as opposed to intraocular. emic signals that induce neovasculariza- neurons die, proliferate and ultimately The latter is the more usual route used in tion? Bouck and coworkers (1) suggest dominate the culture dish. In the presence getting agents to the retina, which is nor- that it may be through an apoptotic mech- of PEDF, however, the growth of astroglia mally protected behind a blood-retina bar- anism. Experimentally, cultured human is considerably slowed. Microglia change rier much as in the brain. Retinal neovas- endothelial cells were subjected to mild morphologically and become more meta- cularization with tufts extending into the stress and the numbers of TUNEL- bolically active in the presence of PEDF. vitreous body were seen in all ischemia- positive cells were monitored. Addition of PEDF also completely blocks GMCSF- induced animals (Fig. 2 a–d; originally PEDF to the cultures markedly increased stimulated cell division of microglia. This appeared in ref. 1), but new vessel growth the number of positive cells. Similarly, ‘‘gliastatic’’ effect of PEDF, along with was markedly reduced or eliminated in apoptotic endothelial cells were more the neuron-survival character, could be PEDF-treated animals (Fig. 2 e–h). To common in retinas of hyperoxic animals of benefit in the overall management corroborate these findings, endothelial treated with PEDF than in those not of a number of diseases with CNS cell nuclei extending beyond the internal receiving PEDF.

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