Gut-liver axis calibrates intestinal stem cell fitness.

The gut and liver are recognized to mutually communicate through the biliary tract, portal vein, and systemic circulation. However, it remains unclear how this gut-liver axis regulates intestinal physiology. Through hepatectomy and transcriptomic and proteomic profiling, we identified pigment epithelium-derived factor (PEDF), a liver-derived soluble Wnt inhibitor, which restrains intestinal stem cell (ISC) hyperproliferation to maintain gut homeostasis by suppressing the Wnt/ß-catenin signaling pathway. Furthermore, we found that microbial danger signals resulting from intestinal inflammation can be sensed by the liver, leading to the repression of PEDF production through peroxisome proliferator-activated receptor-α (PPARα). This repression liberates ISC proliferation to accelerate tissue repair in the gut. Additionally, treating mice with fenofibrate, a clinical PPARα agonist used for hypolipidemia, enhances colitis susceptibility due to PEDF activity. Therefore, we have identified a distinct role for PEDF in calibrating ISC expansion for intestinal homeostasis through reciprocal interactions between the gut and liver.

Primary Retinal Cell Cultures as a Model to Study Retina Biology.

Since its inception, primary retinal cultures have been an in vitro tool for modeling the in vivo environment of the retina for biological studies on development and disease. They offer simple and controlled experimental approaches when compared to in vivo models. In this review we highlight the strengths and weaknesses of primary retinal culture models, and the features of dispersed retinal cell cultures.

Ablation of pigment epithelium-derived factor receptor (PEDF-R/Pnpla2) causes photoreceptor degeneration.

Photoreceptor cells express the patatin-like phospholipase domain-containing 2 (PNPLA2) gene that codes for pigment epithelium-derived factor receptor (PEDF-R) (also known as ATGL). PEDF-R exhibits phospholipase activity that mediates the neurotrophic action of its ligand PEDF. Because phospholipids are the most abundant lipid class in the retina, we investigated the role of PEDF-R in photoreceptors by generating CRISPR Pnpla2 knock-out mouse lines in a retinal degeneration-free background. Pnpla2-/- mice had undetectable retinal Pnpla2 gene expression and PEDF-R protein levels as assayed by RT-PCR and immunofluorescence, respectively. The photoreceptors of mice deficient in PEDF-R had deformities as examined by histology and transmission electron microscopy. Pnpla2 knockdown diminished the PLA2 enzymatic activity of PEDF-R in the retina. Lipidomic analyses revealed the accumulation of lysophosphatidyl choline-DHA and lysophosphatidyl ethanolamine-DHA in PEDF-R-deficient retinas, suggesting a possible causal link to photoreceptor dysfunction. Loss of PEDF-R decreased levels of rhodopsin, opsin, PKCα, and synaptophysin relative to controls. Pnpla2-/- photoreceptors had surface-exposed phosphatidylserine, and their nuclei were TUNEL positive and condensed, revealing an apoptotic onset. Paralleling its structural defects, PEDF-R deficiency compromised photoreceptor function in vivo as indicated by the attenuation of photoreceptor a- and b-waves in Pnpla2-/- and Pnpla2+/- mice relative to controls as determined by electroretinography. In conclusion, ablation of PEDF-R in mice caused alteration in phospholipid composition associated with malformation and malperformance of photoreceptors. These findings identify PEDF-R as an important component for photoreceptor structure and function, highlighting its role in phospholipid metabolism for retinal survival and its consequences.

Pigment epithelium-derived factor is an interleukin-6 antagonist in the RPE: Insight of structure-function relationships.

Retinal and choroidal inflammatory lesions increase the levels of the pro-inflammatory cytokine interleukin-6 (IL-6). Pigment epithelium-derived factor (PEDF) has anti-inflammatory properties, but it is not known if it can prevent the production of IL-6 by the retinal pigment epithelium. To investigate the anti-inflammatory effects of PEDF in the RPE, we used human ARPE-19 cells stimulated with human recombinant tumor necrosis factor-alpha (TNF-α) to induce overexpression of the IL6 gene. We found that the viability of ARPE-19 cells decreased by 22% with TNF-α at 10 ng/ml, being drastically decreased at ≥50 ng/ml. TNF-α at 5-100 ng/ml elevated the production and secretion of IL-6 protein, as measured by ELISA. To challenge the TNF-α-mediated stimulation of IL-6, we used recombinant human PEDF protein. PEDF at 100 nM recovered the TNF-α-mediated loss of cell viability and repressed IL-6 gene expression as determined by RT-PCR. PEDF at 10-100 nM attenuated the IL-6 protein secretion in a dose dependent fashion (IC50 = 65 nM), being abolished with 100 nM PEDF. To map the region that confers the IL-6 blocking effect to the PEDF polypeptide, we used chemically synthesized peptides designed from its biologically active domains, pro-death 34-mer, and pro-survival 44-mer and 17-mer (H105A), to challenge the IL-6 overproduction. The pro-survival peptides recovered the TNF-α-mediated cell viability loss, and inhibited IL-6 secretion, while the 34-mer did not have an effect, suggesting a role for the pro-survival domain in blocking TNF-α-mediated cell death and IL-6 stimulation. Our findings position PEDF as a novel antagonistic agent of IL-6 production in RPE cells, underscoring its use for the management of retinal disease-related inflammation.

PEDF Deletion Induces Senescence and Defects in Phagocytosis in the RPE.

The retinal pigment epithelium (RPE) expresses the Serpinf1 gene to produce pigment epithelium-derived factor (PEDF), a retinoprotective protein that is downregulated with cell senescence, aging and retinal degenerations. We determined the expression of senescence-associated genes in the RPE of 3-month-old mice that lack the Serpinf1 gene and found that Serpinf1 deletion induced H2ax for histone H2AX protein, Cdkn1a for p21 protein, and Glb1 gene for ß-galactosidase. Senescence-associated ß-galactosidase activity increased in the Serpinf1 null RPE when compared with wild-type RPE. We evaluated the subcellular morphology of the RPE and found that ablation of Serpinf1 increased the volume of the nuclei and the nucleoli number of RPE cells, implying chromatin reorganization. Given that the RPE phagocytic function declines with aging, we assessed the expression of the Pnpla2 gene, which is required for the degradation of photoreceptor outer segments by the RPE. We found that both the Pnpla2 gene and its protein PEDF-R declined with the Serpinf1 gene ablation. Moreover, we determined the levels of phagocytosed rhodopsin and lipids in the RPE of the Serpinf1 null mice. The RPE of the Serpinf1 null mice accumulated rhodopsin and lipids compared to littermate controls, implying an association of PEDF deficiency with RPE phagocytosis dysfunction. Our findings establish PEDF loss as a cause of senescence-like changes in the RPE, highlighting PEDF as both a retinoprotective and a regulatory protein of aging-like changes associated with defective degradation of the photoreceptor outer segment in the RPE.

Pigment epithelium-derived factor engineered to increase glycosaminoglycan affinity while maintaining bioactivity.

Pigment epithelium-derived factor (PEDF) is a secreted protein that is essential in tissue homeostasis and is involved in multiple functions in the eye, such as antiangiogenesis and neuroprotection. However, short retention in the retinal microenvironment can limit its therapeutic effects. In this study, we modified the amino acid sequence of PEDF to increase its affinity for heparin and hyaluronic acid (HA), which are negatively charged extracellular matrix (ECM) molecules. HA is the major component of the vitreous humor. We selectively converted neutral or anionic residues into cationic residues to obtain engineered PEDF (ePEDF). Using in vitro binding assays, we demonstrate that ePEDF had higher affinity for heparin and HA than wild-type PEDF (wtPEDF). ePEDF exhibited antiangiogenic and retinal survival bioactivities. It inhibited endothelial cell proliferation and tube formation in vitro. In an ex vivo model mimicking retinal degeneration, ePEDF protected photoreceptors from cell death. The findings suggest that protein engineering is an approach to develop active PEDF with higher ECM affinity to potentially improve its retention in the retina microenvironment and in turn make it a more efficient therapeutic drug for retinal diseases.

Expression and production of pigment epithelium-derived factor (PEDF) and PEDF receptor variants from mammalian and bacterial cells.

Human SERPINF1 gene codes for pigment epithelium-derived factor (PEDF), a secreted glycoprotein and member of the SERPIN superfamily. To obtain large amounts of recombinant PEDF proteins, we subcloned the coding sequence of human SERPINF1 mutated versions into the pCEP4 vector and generated stably transfected HEK.Ebna cells. The cells produced and secreted recombinant PEDF proteins into the culturing media. The recombinant PEDF proteins were purified by ion-exchange column chromatography and milligram amounts of highly purified protein were recovered. PEDF has affinity for PEDF-receptor (PEDF-R), a membrane-linked lipase encoded by the PNPLA2 gene. Recombinant PEDF-R truncated versions were obtained from Escherichia coli containing expression vectors with human PNPLA2 cDNAs with 3'end deletions and by induction with isopropyl ß-d-1-thiogalactopyranoside. The bacterially derived PEDF-R proteins in insoluble inclusion bodies were solubilized with urea and purified by cation-exchange column chromatography. C-terminally truncated PEDF-R versions containing the ligand binding region retained the ability to bind PEDF. The data demonstrate that mammalian-derived recombinant PEDF and bacterially derived recombinant PEDF-R can be produced and purified in large amounts for further use in structural and biological studies.

Extracellular vesicles released by human retinal pigment epithelium mediate increased polarised secretion of drusen proteins in response to AMD stressors.

Age-related macular degeneration (AMD) is a leading cause of blindness worldwide. Drusen are key contributors to the etiology of AMD and the ability to modulate drusen biogenesis could lead to therapeutic strategies to slow or halt AMD progression. The mechanisms underlying drusen biogenesis, however, remain mostly unknown. Here we demonstrate that under homeostatic conditions extracellular vesicles (EVs) secreted by retinal pigment epithelium (RPE) cells are enriched in proteins associated with mechanisms involved in AMD pathophysiology, including oxidative stress, immune response, inflammation, complement system and drusen composition. Furthermore, we provide first evidence that drusen-associated proteins are released as cargo of extracellular vesicles secreted by RPE cells in a polarised apical:basal mode. Notably, drusen-associated proteins exhibited distinctive directional secretion modes in homeostatic conditions and, differential modulation of this directional secretion in response to AMD stressors. These observations underpin the existence of a finely-tuned mechanism regulating directional apical:basal sorting and secretion of drusen-associated proteins via EVs, and its modulation in response to mechanisms involved in AMD pathophysiology. Collectively, our results strongly support an active role of RPE-derived EVs as a key source of drusen proteins and important contributors to drusen development and growth.

Delivery Systems of Retinoprotective Proteins in the Retina.

Retinoprotective proteins play important roles for retinal tissue integrity. They can directly affect the function and the survival of photoreceptors, and/or indirectly target the retinal pigment epithelium (RPE) and endothelial cells that support these tissues. Retinoprotective proteins are used in basic, translational and in clinical studies to prevent and treat human retinal degenerative disorders. In this review, we provide an overview of proteins that protect the retina and focus on pigment epithelium-derived factor (PEDF), and its effects on photoreceptors, RPE cells, and endothelial cells. We also discuss delivery systems such as pharmacologic and genetic administration of proteins to achieve photoreceptor survival and retinal tissue integrity.

Pigment epithelium-derived factor (PEDF) and derived peptides promote survival and differentiation of photoreceptors and induce neurite-outgrowth in amacrine neurons.

Pigment epithelium-derived factor (PEDF) is a cytoprotective protein for the retina. We hypothesize that this protein acts on neuronal survival and differentiation of photoreceptor cells in culture. The purpose of the present study was to evaluate the neurotrophic effects of PEDF and its fragments in an in vitro model of cultured primary retinal neurons that die spontaneously in the absence of trophic factors. We used Wistar albino rats. Cell death was assayed by immunofluorescence and flow cytometry through TUNEL assay, propidium iodide, mitotracker, and annexin V. Immunofluorescence of cells for visualizing rhodopsin, CRX, and antisyntaxin under confocal microscopy was performed. Neurite outgrowth was also quantified. Results show that PEDF protected photoreceptor precursors from apoptosis, preserved mitochondrial function and promoted polarization of opsin enhancing their developmental process, as well as induced neurite outgrowth in amacrine neurons. These effects were abolished by an inhibitor of the PEDF receptor or receptor-derived peptides that block ligand/receptor interactions. While all the activities were specifically conferred by short peptide fragments (17 amino acid residues) derived from the PEDF neurotrophic domain, no effects were triggered by peptides from the PEDF antiangiogenic region. The observed effects on retinal neurons imply a specific activation of the PEDF receptor by a small neurotrophic region of PEDF. Our findings support the neurotrophic PEDF peptides as neuronal guardians for the retina, highlighting their potential as promoters of retinal differentiation, and inhibitors of retinal cell death and its blinding consequences. Cover Image for this issue: https://doi.org/10.1111/jnc.15089.

Degradation of Photoreceptor Outer Segments by the Retinal Pigment Epithelium Requires Pigment Epithelium-Derived Factor Receptor (PEDF-R).

Purpose: To examine the contribution of pigment epithelium-derived factor receptor (PEDF-R) to the phagocytosis process. Previously, we identified PEDF-R, the protein encoded by the PNPLA2 gene, as a phospholipase A2 in the retinal pigment epithelium (RPE). During phagocytosis, RPE cells ingest abundant phospholipids and protein in the form of photoreceptor outer segment (POS) tips, which are then hydrolyzed. The role of PEDF-R in RPE phagocytosis is not known. Methods: Mice in which PNPLA2 was conditionally knocked out (cKO) in the RPE were generated. Mouse RPE/choroid explants were cultured. Human ARPE-19 cells were transfected with siPNPLA2 silencing duplexes. POSs were isolated from bovine retinas. The phospholipase A2 inhibitor bromoenol lactone was used. Transmission electron microscopy, immunofluorescence, lipid labeling, pulse-chase experiments, western blots, and free fatty acid and ß-hydroxybutyrate assays were performed. Results: The RPE of the cKO mice accumulated lipids, as well as more abundant and larger rhodopsin particles, compared to littermate controls. Upon POS exposure, RPE explants from cKO mice released less ß-hydroxybutyrate compared to controls. After POS ingestion during phagocytosis, rhodopsin degradation was stalled both in cells treated with bromoenol lactone and in PNPLA2-knocked-down cells relative to their corresponding controls. Phospholipase A2 inhibition lowered ß-hydroxybutyrate release from phagocytic RPE cells. PNPLA2 knockdown also resulted in a decline in fatty acids and ß-hydroxybutyrate release from phagocytic RPE cells. Conclusions: PEDF-R downregulation delayed POS digestion during phagocytosis. The findings imply that the efficiency of RPE phagocytosis depends on PEDF-R, thus identifying a novel contribution of this protein to POS degradation in the RPE.

Pigment Epithelium-Derived Factor (PEDF) Fragments Prevent Mouse Cone Photoreceptor Cell Loss Induced by Focal Phototoxicity In Vivo.

Here, we evaluated the effects of PEDF (pigment epithelium-derived factor) and PEDF peptides on cone-photoreceptor cell damage in a mouse model of focal LED-induced phototoxicity (LIP) in vivo. Swiss mice were dark-adapted overnight, anesthetized, and their left eyes were exposed to a blue LED placed over the cornea. Immediately after, intravitreal injection of PEDF, PEDF-peptide fragments 17-mer, 17-mer[H105A] or 17-mer[R99A] (all at 10 pmol) were administered into the left eye of each animal. BDNF (92 pmol) and bFGF (27 pmol) injections were positive controls, and vehicle negative control. After 7 days, LIP resulted in a consistent circular lesion located in the supratemporal quadrant and the number of S-cones were counted within an area centered on the lesion. Retinas treated with effectors had significantly greater S-cone numbers (PEDF (60%), 17-mer (56%), 17-mer [H105A] (57%), BDNF (64%) or bFGF (60%)) relative to their corresponding vehicle groups (≈42%). The 17-mer[R99A] with no PEDF receptor binding and no neurotrophic activity, PEDF combined with a molar excess of the PEDF receptor blocker P1 peptide, or with a PEDF-R enzymatic inhibitor had undetectable effects in S-cone survival. The findings demonstrated that the cone survival effects were mediated via interactions between the 17-mer region of the PEDF molecule and its PEDF-R receptor.

Cell-based assays to identify novel retinoprotective agents.

Degeneration of the retina can lead ultimately to devastating irreversible vision loss, such as in inherited retinitis pigmentosa and age-related macular degeneration. Currently there is no cure to prevent retinal degeneration. Quantitative cell-based assays can be used to test potential drugs that prevent the death of retinal cells. Here, we describe in detail three semi-automated cell-based protocols to identify retinoprotective factors with two retinal cell lines, rat R28 cells and mouse 661W cells. In these protocols, cells are induced to undergo death by photo-oxidation stress, growth factor depletion or cytotoxicity with sodium iodate. Pigment epithelium-derived factor, an established neurotrophic factor for retinal cells, was used as a positive control. We discuss how these protocols will prove useful in high-throughput quantitative screening to identify novel therapeutics for retinal disorders.

PEDF deficiency increases the susceptibility of rd10 mice to retinal degeneration.

The SERPINF1 gene encodes pigment epithelium-derived factor (PEDF), a member of the serpin superfamily with neurotrophic and antiangiogenic properties in the retina. We hypothesized that absence of PEDF would lead to increased stress-associated retinal degeneration in Serpinf1 null mice. Accordingly, using a Serpinf1 null mouse model, we investigated the impact of PEDF absence on retinal morphology, and susceptibility to induced and inherited retinal degeneration. We studied the pattern of Serpinf1 expression in the mouse retina layers. PEDF protein was detected by western blotting. Transmission electron microscopy was performed on mouse retina. Serpinf1 null mice and wild type littermates were injected with NaIO3 (30 mg/kg body weight) intraperitonially. At post-injection day 1, 3, 4, 6 and 8 mice were euthanized, and eyes were enucleated. Serpinf1 null and rd10 double mutant mice were generated and their eyes enucleated at different time points from post-natal day 15 to post-natal day 28. Enucleated eyes were processed for hematoxylin and eosin staining and histopathological evaluations. We found that Serpinf1 was expressed in the retinal pigment epithelium, in the inner nuclear layer and in the ganglion cell layer, but undetectable in the outer nuclear layer of wild type mice. Plasma PEDF protein levels were undetectable in Serpinf1 null animals. RPE atrophy and retinal thinning were observed in NaIO3-treated wild type mice that progressed with time post-injection. NaIO3-treated Serpinf1 null mice showed comparatively better retinal morphology than wild type mice at day 4 post-injection. However, the absence of PEDF in Serpinf1 null x rd10 mice increased the susceptibility to retinal degeneration relative to that of rd10 mice. We concluded that histopathological evaluation of retinas lacking PEDF showed that removal of the Serpinf1 gene may activate PEDF-independent compensatory mechanisms to protect the retina against oxidative stress, while it increases the susceptibility to degenerate the retina in inherited retinal degeneration models.

Role of the PNPLA2 Gene in the Regulation of Oxidative Stress Damage of RPE.

Oxidative stress-mediated injury of the retinal pigment epithelium (RPE) can precede progressive retinal degeneration and ultimately lead to blindness (e.g., age-related macular degeneration (AMD)). The RPE expresses the PNPLA2 gene and produces its protein product PEDF-R that exhibits lipase activity. We have shown that transient PNPLA2 overexpression decreases dead-cell proteolytic activity and that synthetic peptides derived from a central region of PEDF-R efficiently protect ARPE-19 and pig primary RPE cells from oxidative stress. This study aims to evaluate the effect of loss of PNPLA2 in RPE cells undergoing oxidative stress. Loss of PNPLA2 conferred increased resistance to cells when subjected to oxidative stress.

Signaling Mechanisms Involved in PEDF-Mediated Retinoprotection.

Pigment epithelium-derived factor (PEDF) is involved in signal transduction cascades necessary for protection of the retina. The interaction between PEDF and retinal cells elicits neuroprotective effects in vitro and in vivo. The direct substrates and signaling mechanisms involved in the survival response derived from such interaction are beginning to be revealed. It is of interest to elucidate cell death pathways that are crucial for the retinoprotective response of PEDF for the identification of targets that interfere with retina degeneration with potential therapeutic value. Here we review the molecular pathways triggered by PEDF that are involved in retinal survival activity.

PEDF peptides promote photoreceptor survival in rd10 retina models.

The purpose of the study is to evaluate the protective properties of PEDF peptide fragments on rd10 mouse models of retinal degeneration ex vivo. Human recombinant PEDF and synthetic peptides were used. Rd10 retinal explants as well as wild-type retinal explants treated with zaprinast to mimic the rd10 photoreceptor cell death were employed. PEDF protein was intravitreally administered into rd10 mice. Outer nuclear layer thickness measurements in retinal sections, TUNEL labeling in retinal explants, western blots and immunofluorescence with retinal samples were performed. PEDF protein levels in the RPE of rd10 mice decreased with age (P15 - P25). Levels of PEDF receptor PEDF-R declined in the photoreceptor inner segments from rd10 relative to wild-type mice at P25. PEDF administration increased the outer nuclear layer thickness of rd10 retinas in vivo and decreased the number of TUNEL+ nuclei of photoreceptors in rd10 retinal explant cultures, both relative to untreated controls. Peptides containing the PEDF neurotrophic region decreased the number of TUNEL+ photoreceptors in both rd10 and zaprinast-induced cell death ex vivo models, while peptides without the neurotrophic region and/or lacking affinity for PEDF-R were ineffective in protecting photoreceptors. Thus, retinal explants are a valuable system to evaluate PEDF activity. Short peptides with the photoreceptor-protective property of PEDF may prove useful for the development of therapeutic agents for photoreceptor protection in retinal degenerations.

Pigment epithelium-derived factor hinders photoreceptor cell death by reducing intracellular calcium in the degenerating retina.

Calcium ions play a critical role in neuronal cell death. Pigment epithelium-derived factor (PEDF) is a promising neuroprotective protein for photoreceptor cells but the mechanisms mediating its effects against retinal degeneration are still not well characterized. We addressed this question in the rd1 degenerating mouse retina that bears a mutation in the Pde6b gene encoding one subunit of the phosphodiesterase enzyme. Loss of phosphodiesterase activity in rod photoreceptor cells increases cyclic guanosine monophosphate (cGMP) levels leading to a rise in intracellular calcium. Short-term treatments with recombinant human PEDF protein decreased intracellular calcium in photoreceptors in vivo. Taking advantage of calcium pump blockers, we defined that PEDF signaling acts on PMCA calcium pumps to lower intracellular calcium. PEDF restrained cell death pathways activated by high calcium levels and engaging calpains, BAX and AIF. The neurotrophic effects were mediated by the PEDF receptor (PEDF-R), encoded by the PNPLA2 gene. Finally, peptides containing the neurotrophic domain of PEDF targeted these same cell death pathways in vivo. The findings reveal rescue from death of degenerating photoreceptor cells by a PEDF-mediated preservation of intracellular calcium homeostasis.

Pigment Epithelium-derived Factor Protects Retinal Pigment Epithelial Cells Against Cytotoxicity "In Vitro".

Oxidative stress has been implicated in neurodegenerative diseases, such as age-related macular degeneration. Hydrogen peroxide and sodium iodate can mediate oxidative injury. Sodium iodate induces a selective retinal degeneration targeting the RPE. We describe a method of chronic sodium iodate-mediated injury on RPE cells that may serve to evaluate protective factors against oxidative stress. Cytotoxicity and cell viability curves of ARPE-19 cells with sodium iodate were generated. The antioxidant pigment epithelium-derived factor decreased sodium iodate-mediated cytotoxicity without affecting ARPE-19 cell viability. A cell culture system to evaluate protection against oxidative stress injury with PEDF is discussed.

Comparison of two neurotrophic serpins reveals a small fragment with cell survival activity.

PURPOSE: Protease nexin-1 (PN-1), a serpin encoded by the SERPINE2 gene, has serine protease inhibitory activity and neurotrophic properties in the brain. PN-1 inhibits retinal angiogenesis; however, PN-1's neurotrophic capacities in the retina have not yet been evaluated. Pigment epithelium-derived factor (PEDF) is a serpin that exhibits neurotrophic and antiangiogenic activities but lacks protease inhibitory properties. The aim of this study is to compare PN-1 and PEDF. METHODS: Sequence comparisons were performed using computer bioinformatics programs. Mouse and bovine eyes, human retina tissue, and ARPE-19 cells were used to prepare RNA and protein samples. Interphotoreceptor matrix lavage was obtained from bovine eyes. Gene expression and protein levels were evaluated with reverse-transcription PCR (RT-PCR) and western blotting, respectively. Recombinant human PN-1, a version of PN-1 referred to as PN-1[R346A] lacking serine protease inhibitory activity, and PEDF proteins were used, as well as synthetic peptides designed from PEDF and PN-1 sequences. Survival activity in serum-starved, rat-derived retinal precursor (R28) cells was assessed with terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) cell death assays. Bcl2 levels were measured with RT-PCR. RESULTS: PN-1 is analogous in primary and tertiary structure to PEDF. A region in PN-1 shares homology with the neurotrophic active region of PEDF, a 17-residue region within alpha helix C. The native human retina, ARPE-19 cells, and murine RPE and retina expressed the gene for PN-1 (SERPINE2 and Serpine2 mRNA). The retina, ARPE-19 cell lysates, and bovine interphotoreceptor matrix contained PN-1 protein. The addition of PN-1, PN-1[R346A], or the 17mer peptide of PN-1 to serum-starved retina cells decreased the number of TUNEL-positive nuclei relative to the untreated cells, such as PEDF. PN-1, PN-1[R346A], and PN-1-17mer treatments increased the Bcl2 transcript levels in serum-starved cells, as seen with PEDF. CONCLUSIONS: PN-1 and PEDF share structural and functional features, and expression patterns in the retina. These serpins' mechanisms of action as cell survival factors are independent of serine protease inhibition. We have identified PN-1 as a novel factor for the retina that may play a neuroprotective role in vivo, and small peptides as relevant candidates for preventing retinal degeneration.