Work in this research group is aimed at elucidating the mechanism of action of pigment epithelium-derived factor (PEDF), an extracellular glycoprotein that acts in neuronal differentiation and survival in cells derived from the retina and CNS, and in angiogenesis inhibiting neovascularization in the cornea, retina and choroid. PEDF is a member of the serpin, but it has no known inhibitory activity against serine proteases. The first step in the biological events of PEDF's neurotrophic activity is the binding to receptors on the surface of target cells, e.g. retinoblastoma, cerebellar granule cells and photoreceptors. It has been shown before that PEDF protects motor neurons from chronic glutamate-mediated neurodegeneration. In collaboration with Drs. Ralph Kuncl and Masako Bilak, we studied the mechanisms for motor neuron protection by PEDF. A small peptide from human PEDF containing the receptor binding site and termed 44-mer protected motor neurons from chronic glutamate-mediated degeneration, as has been shown for the full length PEDF. 125I-PEDF and fluoresceinated-PEDF bound specifically and high affinity to receptors in spinal cord motor neurons in organotypic and cells cultures. We quantified PEDF in cerebrospinal fluid samples from ALS patients with amyotrophic lateral sclerosis and found that elevated levels of PEDF in the CSF may be an autoprotective reaction in ALS. PEDF is identified in tissues rich in collagen, e.g., cornea, vitreous, bone, cartilage. We showed that recombinant human PEDF formed complexes with collagens from the bovine cornea and vitreous. We examined the direct binding of PEDF to collagen I and found that interactions were ionic in nature and occurred when PEDF and collagen I were both in solution, when either one was immobilized, or even when collagen I was denatured under reducing conditions. 125I-PEDF bound to immobilized collagen I in a saturable fashion. Compared to neurotrophic PEDF-derived peptides, ovalbumin and angiogenic inhibitors, only full-length PEDF competed efficiently with 125I-PEDF for the binding to immobilized collagen I. We analyzed the collagen-binding region using controlled proteolysis and chemically modified PEDF. Cleavage of the serpin exposed loop did not prevent binding to collagen I. Conjugation of lysines with fluorescein increased the collagen binding affinity. However, treatment of PEDF with EDC abolished it, implicating the PEDF aspartic and/or glutamic acid residues in its interaction with collagen I. A negatively charged region on the surface of the PEDF molecule is rich in acidic residues available to interact directly with positively charged areas of collagen. This represented the first collagen-binding site described for a serpin, which in PEDF, is distinct from its heparin-binding region, neurotrophic active site and its serpin exposed loop. We investigated the distribution of PEDF in the retinal pigment epithelium-interphotoreceptor matrix region of the retina. Secretion of neurotrophically active PEDF was preferred towards the apical side of RPE in culture than the basal side. In situ, PEDF was found polarized preferentially to the apical side, i.e. next to the photoreceptors in the interphotoreceptor matrix where PEDF is identified as a soluble component. In collaboration with the laboratory of Dr JX Ma we compared the susceptibilities of Sprague Dawley (SD) and Brown Norway (BN) rats with ischemia-induced retinal neovascularization by comparing the ratio of angiogenic to antoangiogenic factor, such as VEGF to PEDF. The hyperoxia-treated BN rats showed a significant reduction in retinal PEDF, but they showed a substantial increase of VEGF at both the protein and RNA levels, resulting in an increased VEGF-to-PEDF ratio. Hyperoxia-treated SD rats showed changes in PEDF and VEGF levels that were less in magnitude and of shorter duration than in BN rats. In age-matched normal BN and SD rats, however, there was no detectable difference in the basal VEGF-to-PEDF ratio between the strains.
Showing the most recent 10 out of 18 publications
