Retinoblastoma from human stem cell-derived retinal organoids.

Retinoblastoma is a childhood cancer of the developing retina that initiates with biallelic inactivation of the RB1 gene. Children with germline mutations in RB1 have a high likelihood of developing retinoblastoma and other malignancies later in life. Genetically engineered mouse models of retinoblastoma share some similarities with human retinoblastoma but there are differences in their cellular differentiation. To develop a laboratory model of human retinoblastoma formation, we make induced pluripotent stem cells (iPSCs) from 15 participants with germline RB1 mutations. Each of the stem cell lines is validated, characterized and then differentiated into retina using a 3-dimensional organoid culture system. After 45 days in culture, the retinal organoids are dissociated and injected into the vitreous of eyes of immunocompromised mice to support retinoblastoma tumor growth. Retinoblastomas formed from retinal organoids made from patient-derived iPSCs have molecular, cellular and genomic features indistinguishable from human retinoblastomas. This model of human cancer based on patient-derived iPSCs with germline cancer predisposing mutations provides valuable insights into the cellular origins of this debilitating childhood disease as well as the mechanism of tumorigenesis following RB1 gene inactivation.

Nucleome Dynamics during Retinal Development.

More than 8,000 genes are turned on or off as progenitor cells produce the 7 classes of retinal cell types during development. Thousands of enhancers are also active in the developing retinae, many having features of cell- and developmental stage-specific activity. We studied dynamic changes in the 3D chromatin landscape important for precisely orchestrated changes in gene expression during retinal development by ultra-deep in situ Hi-C analysis on murine retinae. We identified developmental-stage-specific changes in chromatin compartments and enhancer-promoter interactions. We developed a machine learning-based algorithm to map euchromatin and heterochromatin domains genome-wide and overlaid it with chromatin compartments identified by Hi-C. Single-cell ATAC-seq and RNA-seq were integrated with our Hi-C and previous ChIP-seq data to identify cell- and developmental-stage-specific super-enhancers (SEs). We identified a bipolar neuron-specific core regulatory circuit SE upstream of Vsx2, whose deletion in mice led to the loss of bipolar neurons.

Retinal Cell Type DNA Methylation and Histone Modifications Predict Reprogramming Efficiency and Retinogenesis in 3D Organoid Cultures.

Diverse cell types can be reprogrammed into pluripotent stem cells by ectopic expression of Oct4 (Pou5f1), Klf4, Sox3, and Myc. Many of these induced pluripotent stem cells (iPSCs) retain memory, in terms of DNA methylation and histone modifications (epigenetic memory), of their cellular origins, and this may bias subsequent differentiation. Neurons are difficult to reprogram, and there has not been a systematic side-by-side characterization of reprogramming efficiency or epigenetic memory across different neuronal subtypes. Here, we compare reprogramming efficiency of five different retinal cell types at two different stages of development. Retinal differentiation from each iPSC line was measured using a quantitative standardized scoring system called STEM-RET and compared to the epigenetic memory. Neurons with the lowest reprogramming efficiency produced iPSC lines with the best retinal differentiation and were more likely to retain epigenetic memory of their cellular origins. In addition, we identified biomarkers of iPSCs that are predictive of retinal differentiation.

Inhibition of MMP-2 and MMP-9 decreases cellular migration, and angiogenesis in in vitro models of retinoblastoma.

BACKGROUND: Retinoblastoma (Rb) is the most common primary intraocular tumor in children. Local treatment of the intraocular disease is usually effective if diagnosed early; however advanced Rb can metastasize through routes that involve invasion of the choroid, sclera and optic nerve or more broadly via the ocular vasculature. Metastatic Rb patients have very high mortality rates. While current therapy for Rb is directed toward blocking tumor cell division and tumor growth, there are no specific treatments targeted to block Rb metastasis. Two such targets are matrix metalloproteinases-2 and -9 (MMP-2, -9), which degrade extracellular matrix as a prerequisite for cellular invasion and have been shown to be involved in other types of cancer metastasis. Cancer Clinical Trials with an anti-MMP-9 therapeutic antibody were recently initiated, prompting us to investigate the role of MMP-2, -9 in Rb metastasis. METHODS: We compare MMP-2, -9 activity in two well-studied Rb cell lines: Y79, which exhibits high metastatic potential and Weri-1, which has low metastatic potential. The effects of inhibitors of MMP-2 (ARP100) and MMP-9 (AG-L-66085) on migration, angiogenesis, and production of immunomodulatory cytokines were determined in both cell lines using qPCR, and ELISA. Cellular migration and potential for invasion were evaluated by the classic wound-healing assay and a Boyden Chamber assay. RESULTS: Our results showed that both inhibitors had differential effects on the two cell lines, significantly reducing migration in the metastatic Y79 cell line and greatly affecting the viability of Weri-1 cells. The MMP-9 inhibitor (MMP9I) AG-L-66085, diminished the Y79 angiogenic response. In Weri-1 cells, VEGF was significantly reduced and cell viability was decreased by both MMP-2 and MMP-9 inhibitors. Furthermore, inhibition of MMP-2 significantly reduced secretion of TGF-ß1 in both Rb models. CONCLUSIONS: Collectively, our data indicates MMP-2 and MMP-9 drive metastatic pathways, including migration, viability and secretion of angiogenic factors in Rb cells. These two subtypes of matrix metalloproteinases represent new potential candidates for targeted anti-metastatic therapy for Rb.

The Dynamic Epigenetic Landscape of the Retina During Development, Reprogramming, and Tumorigenesis.

In the developing retina, multipotent neural progenitors undergo unidirectional differentiation in a precise spatiotemporal order. Here we profile the epigenetic and transcriptional changes that occur during retinogenesis in mice and humans. Although some progenitor genes and cell cycle genes were epigenetically silenced during retinogenesis, the most dramatic change was derepression of cell-type-specific differentiation programs. We identified developmental-stage-specific super-enhancers and showed that most epigenetic changes are conserved in humans and mice. To determine how the epigenome changes during tumorigenesis and reprogramming, we performed integrated epigenetic analysis of murine and human retinoblastomas and induced pluripotent stem cells (iPSCs) derived from murine rod photoreceptors. The retinoblastoma epigenome mapped to the developmental stage when retinal progenitors switch from neurogenic to terminal patterns of cell division. The epigenome of retinoblastomas was more similar to that of the normal retina than that of retina-derived iPSCs, and we identified retina-specific epigenetic memory.

Brg1 coordinates multiple processes during retinogenesis and is a tumor suppressor in retinoblastoma.

Retinal development requires precise temporal and spatial coordination of cell cycle exit, cell fate specification, cell migration and differentiation. When this process is disrupted, retinoblastoma, a developmental tumor of the retina, can form. Epigenetic modulators are central to precisely coordinating developmental events, and many epigenetic processes have been implicated in cancer. Studying epigenetic mechanisms in development is challenging because they often regulate multiple cellular processes; therefore, elucidating the primary molecular mechanisms involved can be difficult. Here we explore the role of Brg1 (Smarca4) in retinal development and retinoblastoma in mice using molecular and cellular approaches. Brg1 was found to regulate retinal size by controlling cell cycle length, cell cycle exit and cell survival during development. Brg1 was not required for cell fate specification but was required for photoreceptor differentiation and cell adhesion/polarity programs that contribute to proper retinal lamination during development. The combination of defective cell differentiation and lamination led to retinal degeneration in Brg1-deficient retinae. Despite the hypocellularity, premature cell cycle exit, increased cell death and extended cell cycle length, retinal progenitor cells persisted in Brg1-deficient retinae, making them more susceptible to retinoblastoma. ChIP-Seq analysis suggests that Brg1 might regulate gene expression through multiple mechanisms.

Quantification of Retinogenesis in 3D Cultures Reveals Epigenetic Memory and Higher Efficiency in iPSCs Derived from Rod Photoreceptors.

Cell-based therapies to treat retinal degeneration are now being tested in clinical trials. However, it is not known whether the source of stem cells is important for the production of differentiated cells suitable for transplantation. To test this, we generated induced pluripotent stem cells (iPSCs) from murine rod photoreceptors (r-iPSCs) and scored their ability to make retinae by using a standardized quantitative protocol called STEM-RET. We discovered that r-iPSCs more efficiently produced differentiated retinae than did embryonic stem cells (ESCs) or fibroblast-derived iPSCs (f-iPSCs). Retinae derived from f-iPSCs had fewer amacrine cells and other inner nuclear layer cells. Integrated epigenetic analysis showed that DNA methylation contributes to the defects in f-iPSC retinogenesis and that rod-specific CTCF insulator protein-binding sites may promote r-iPSC retinogenesis. Together, our data suggest that the source of stem cells is important for producing retinal neurons in three-dimensional (3D) organ cultures.

Chromatin remodelers HELLS and UHRF1 mediate the epigenetic deregulation of genes that drive retinoblastoma tumor progression.

The retinoblastoma (Rb) family of proteins are key regulators of cell cycle exit during development and their deregulation is associated with cancer. Rb is critical for normal retinal development and germline mutations lead to retinoblastoma making retinae an attractive system to study Rb family signaling. Rb coordinates proliferation and differentiation through the E2f family of transcription factors, a critical interaction for the role of Rb in retinal development and tumorigenesis. However, whether the roles of the different E2fs are interchangeable in controlling development and tumorigenesis in the retina or if they have selective functions remains unknown. In this study, we found that E2f family members play distinct roles in the development and tumorigenesis. In Rb;p107-deficient retinae, E2f1 and E2f3 inactivation rescued tumor formation but only E2f1 rescued the retinal development phenotype. This allowed the identification of key target genes for Rb/E2f family signaling contributing to tumorigenesis and those contributing to developmental defects. We found that Sox4 and Sox11 genes contribute to the developmental phenotype and Hells and Uhrf1 contribute to tumorigenesis. Using orthotopic human xenografts, we validated that upregulation of HELLS and UHRF1 is essential for the tumor phenotype. Also, these epigenetic regulators are important for the regulation of SYK.

Superselective intraophthalmic artery chemotherapy in a nonhuman primate model: histopathologic findings.

IMPORTANCE: We describe the histopathologic findings in a nonhuman primate (NHP) model of superselective intraophthalmic artery chemotherapy (SSIOAC), detailing ocular and orbital vascular adverse effects. OBJECTIVE: To further document, using comprehensive ocular and orbital histopathology, previously reported toxic effects observed with real-time ophthalmoscopy during SSIOAC in a NHP model. DESIGN: Comparative interventional case series. SETTING: Preclinical trial approved under the guidelines of the Institutional Animal Care and Utilization committee. PARTICIPANTS: Six adult male rhesus macaques (Macacca mulatta). INTERVENTIONS: The right eye of each NHP was treated with 3 cycles of SSIOAC using either melphalan (5 mg/30 mL) or carboplatin (30 mg/30 mL). Both eyes in each animal were enucleated 6 hours after the final procedure, before euthanasia and formalin perfusion of the NHP; we then performed orbital dissection of the arterial vasculature and optic nerves. MAIN OUTCOME MEASURES: Histopathologic examination of the eyes, optic nerves, and orbital vessels of the 6 treated NHPs. RESULTS: We found leukostasis with retinal arteriole occlusion in all treated eyes. Retinal endothelial cells stained positive for 2 inflammatory markers, intercellular adhesion molecule 1 and interleukin 8. Transmission electron microscopy revealed occlusion of the retinal vessels with ultrastructural changes in the endothelial cells and surrounding pericytes. Additional findings included nerve fiber layer infarcts, central retinal artery thrombosis, hypertrophy and occlusion of choroidal arteries with disruption of the internal elastic lamina, patchy choroidal inflammation, and birefringent intravascular foreign bodies. Orbital findings included ophthalmic artery and central retinal artery wall dissection, fracturing of the internal elastic lamina, intimal hyperplasia, and eyelid vessel damage. Optic nerves displayed hemorrhage, leukostasis, and foreign body crystallization. Control eyes, optic nerves, and orbital vessels were normal. CONCLUSIONS AND RELEVANCE: Histopathologic examination of our nonhuman primate model for SSIOAC revealed significant toxic effects in the ocular and orbital vasculature. These findings substantiate previous observations with real-time retinal imaging and parallel reported vascular toxic effects in children with retinoblastoma treated with SSIOAC.

Age-related susceptibility to apoptosis in human retinal pigment epithelial cells is triggered by disruption of p53-Mdm2 association.

PURPOSE: Relatively little is known about the contribution of p53/Mdm2 pathway in apoptosis of retinal pigment epithelial (RPE) cells or its possible link to dysfunction of aging RPE or to related blinding disorders such as age-related macular degeneration (AMD). METHODS: Age-associated changes in p53 activation were evaluated in primary RPE cultures from human donor eyes of various ages. Apoptosis was evaluated by activation of caspases and DNA fragmentation. Gene-specific small interfering RNA was used to knock down expression of p53. RESULTS: We observed that the basal rate of p53-dependent apoptosis increased in an age-dependent manner in human RPE. The age-dependent increase in apoptosis was linked to alterations in several aspects of the p53 pathway. p53 phosphorylation Ser15 was increased through the stimulation of ATM-Ser1981. p53 acetylation Lys379 was increased through the inhibition of SIRT1/2. These two posttranslational modifications of p53 blocked the sequestration of p53 by Mdm2, thus resulting in an increase in free p53 and of p53 stimulation of apoptosis through increased expression of PUMA (p53 upregulated modulator of apoptosis) and activation of caspase-3. Aged RPE also had reduced expression of antiapoptotic Bcl-2, which contributed to the increase in apoptosis. Of particular interest in these studies was that pharmacologic treatments to block p53 phosphorylation, acetylation, or expression were able to protect RPE cells from apoptosis. CONCLUSIONS: Our studies suggest that aging in the RPE leads to alterations of specific checkpoints in the apoptotic pathway, which may represent important molecular targets for the treatment of RPE-related aging disorders such as AMD.

Intra-ophthalmic artery chemotherapy triggers vascular toxicity through endothelial cell inflammation and leukostasis.

Purpose. Super-selective intra-ophthalmic artery chemotherapy (SSIOAC) is an eye-targeted drug-delivery strategy to treat retinoblastoma, the most prevalent primary ocular malignancy in children. Unfortunately, recent clinical reports associate adverse vascular toxicities with SSIOAC using melphalan, the most commonly used chemotherapeutic. Methods. To explore reasons for the unexpected vascular toxicities, we examined the effects of melphalan, as well as carboplatin (another chemotherapeutic used with retinoblastoma), in vitro using primary human retinal endothelial cells, and in vivo using a non-human primate model, which allowed us to monitor the retina in real time during SSIOAC. Results. Both melphalan and carboplatin triggered human retinal endothelial cell migration, proliferation, apoptosis, and increased expression of adhesion proteins intracellullar adhesion molecule-1 [ICAM-1] and soluble chemotactic factors (IL-8). Melphalan increased monocytic adhesion to human retinal endothelial cells. Consistent with these in vitro findings, histopathology showed vessel wall endothelial cell changes, leukostasis, and vessel occlusion. Conclusions. These results reflect a direct interaction of chemotherapeutic drugs with both the vascular endothelium and monocytes. The vascular toxicity may be related to the pH, the pulsatile delivery, or the chemotherapeutic drugs used. Our long-term goal is to determine if changes in the drug of choice and/or delivery procedures will decrease vascular toxicity and lead to better eye-targeted treatment strategies.

Retinoblastoma (Rb) regulates laminar dendritic arbor reorganization in retinal horizontal neurons.

Neuronal differentiation with respect to the acquisition of synaptic competence needs to be regulated precisely during neurogenesis to ensure proper formation of circuits at the right place and time in development. This regulation is particularly important for synaptic triads among photoreceptors, horizontal cells (HCs), and bipolar cells in the retina, because HCs are among the first cell types produced during development, and bipolar cells are among the last. HCs undergo a dramatic transition from vertically oriented neurites that form columnar arbors to overlapping laminar dendritic arbors with differentiation. However, how this process is regulated and coordinated with differentiation of photoreceptors and bipolar cells remains unknown. Previous studies have suggested that the retinoblastoma (Rb) tumor suppressor gene may play a role in horizontal cell differentiation and synaptogenesis. By combining genetic mosaic analysis of individual synaptic triads with neuroanatomic analyses and multiphoton live imaging of developing HCs, we found that Rb plays a cell-autonomous role in the reorganization of horizontal cell neurites as they differentiate. Aberrant vertical processes in Rb-deficient HCs form ectopic synapses with rods in the outer nuclear layer but lack bipolar dendrites. Although previous reports indicate that photoreceptor abnormalities can trigger formation of ectopic synapses, our studies now demonstrate that defects in a postsynaptic partner contribute to the formation of ectopic photoreceptor synapses in the mammalian retina.

Coexpression of normally incompatible developmental pathways in retinoblastoma genesis.

It is widely believed that the molecular and cellular features of a tumor reflect its cell of origin and can thus provide clues about treatment targets. The retinoblastoma cell of origin has been debated for over a century. Here, we report that human and mouse retinoblastomas have molecular, cellular, and neurochemical features of multiple cell classes, principally amacrine/horizontal interneurons, retinal progenitor cells, and photoreceptors. Importantly, single-cell gene expression array analysis showed that these multiple cell type-specific developmental programs are coexpressed in individual retinoblastoma cells, which creates a progenitor/neuronal hybrid cell. Furthermore, neurotransmitter receptors, transporters, and biosynthetic enzymes are expressed in human retinoblastoma, and targeted disruption of these pathways reduces retinoblastoma growth in vivo and in vitro.

Inhibition of Mdm2 sensitizes human retinal pigment epithelial cells to apoptosis.

PURPOSE: Because recent studies indicate that blocking the interaction between p53 and Mdm2 results in the nongenotoxic activation of p53, the authors sought to investigate whether the inhibition of p53-Mdm2 binding activates p53 and sensitizes human retinal epithelial cells to apoptosis. METHODS: Apoptosis was evaluated by the activation of caspases and DNA fragmentation assays. The Mdm2 antagonist Nutlin-3 was used to dissociate p53 from Mdm2 and, thus, to increase p53 activity. Knockdown of p53 expression was accomplished by using p53 siRNA. RESULTS: ARPE-19 and primary RPE cells expressed high levels of the antiapoptotic proteins Bcl-2 and Bcl-xL. Exposure of these cells to camptothecin (CPT) or TNF-α/ cycloheximide (CHX) failed to induce apoptosis. In contrast, treatment with the Mdm2 antagonist Nutlin-3 in the absence of CPT or TNF-α/CHX increased apoptosis. Activation of p53 in response to Nutlin-3 also increased levels of Noxa, p53-upregulated modulator of apoptosis (PUMA), and Siva-1, decreased expression of Bcl-2 and Bcl-xL, and simultaneously increased caspases-9 and -3 activities and DNA fragmentation. Knockdown of p53 decreased the basal expression of p21Cip1 and Bcl-2, inhibited the Nutlin-3-induced upregulation of Siva-1 and PUMA expression, and consequently inhibited caspase-3 activation. CONCLUSIONS: These results indicate that the normally available pool of intracellular p53 is predominantly engaged in the regulation of cell cycle checkpoints by p21Cip1 and does not trigger apoptosis in response to DNA-damaging agents. However, the blockage of p53 binding to Mdm2 frees a pool of p53 that is sufficient, even in the absence of DNA-damaging agents, to increase the expression of proapoptotic targets and to override the resistance of RPE cells to apoptosis.

PLD1-dependent PKCgamma activation downstream to Src is essential for the development of pathologic retinal neovascularization.

Vascular endothelial growth factor (VEGF) appears to be an important mediator of pathologic retinal angiogenesis. In understanding the mechanisms of pathologic retinal neovascularization, we found that VEGF activates PLD1 in human retinal microvascular endothelial cells, and this event is dependent on Src. In addition, VEGF activates protein kinase C-gamma (PKCgamma) via Src-dependent PLD1 stimulation. Inhibition of Src, PLD1, or PKCgamma via pharmacologic, dominant negative mutant, or siRNA approaches significantly attenuated VEGF-induced human retinal microvascular endothelial cell migration, proliferation, and tube formation. Hypoxia also induced Src-PLD1-PKCgamma signaling in retina, leading to retinal neovascularization. Furthermore, siRNA-mediated down-regulation of VEGF inhibited hypoxia-induced Src-PLD1-PKCgamma activation and neovascularization. Blockade of Src-PLD1-PKCgamma signaling via the siRNA approach also suppressed hypoxia-induced retinal neovascularization. Thus, these observations demonstrate, for the first time, that Src-dependent PLD1-PKCgamma activation plays an important role in pathologic retinal angiogenesis.

Changes in retinoblastoma cell adhesion associated with optic nerve invasion.

In the 1970s, several human retinoblastoma cell lines were developed from cultures of primary tumors. As the human retinoblastoma cell lines were established in culture, growth properties and changes in cell adhesion were described. Those changes correlated with the ability of the human retinoblastoma cell lines to invade the optic nerve and metastasize in orthotopic xenograft studies. However, the mechanisms that underlie these changes were not determined. We used the recently developed knockout mouse models of retinoblastoma to begin to characterize the molecular, cellular, and genetic changes associated with retinoblastoma tumor progression and optic nerve invasion. Here we report the isolation and characterization of the first mouse retinoblastoma cell lines with targeted deletions of the Rb family. Our detailed analysis of these cells as they were propagated in culture from the primary tumor shows that changes in cadherin-mediated cell adhesion are associated with retinoblastoma invasion of the optic nerve prior to metastasis. In addition, the same changes in cadherin-mediated cell adhesion correlate with the invasive properties of the human retinoblastoma cell lines isolated decades ago, providing a molecular mechanism for these earlier observations. Most importantly, our studies are in agreement with genetic studies on human retinoblastomas, suggesting that changes in this pathway are involved in tumor progression.

Cells previously identified as retinal stem cells are pigmented ciliary epithelial cells.

It was previously reported that the ciliary epithelium (CE) of the mammalian eye contains a rare population of cells that could produce clonogenic self-renewing pigmented spheres in culture. Based on their ability to up-regulate genes found in retinal neurons, it was concluded that these sphere-forming cells were retinal stem cells. This conclusion raised the possibility that CE-derived retinal stem cells could help to restore vision in the millions of people worldwide who suffer from blindness associated with retinal degeneration. We report here that human and mouse CE-derived spheres are made up of proliferating pigmented ciliary epithelial cells rather than retinal stem cells. All of the cells in the CE-derived spheres, including the proliferating cells, had molecular, cellular, and morphological features of differentiated pigmented CE cells. These differentiated cells ectopically expressed nestin when exposed to growth factors and low levels of pan-neuronal markers such as beta-III-tubulin. Although the cells aberrantly expressed neuronal markers, they retained their pigmented CE cell morphology and failed to differentiate into retinal neurons in vitro or in vivo. Our results provide an example of a differentiated cell type that can form clonogenic spheres in culture, self-renew, express progenitor cell markers, and initiate neuronal differentiation that is not a stem or progenitor cell. More importantly, our findings highlight the importance of shifting the focus away from studies on CE-derived spheres for cell-based therapies to restore vision in the degenerating retina and improving techniques for using ES cells or retinal precursor cells.

A novel role for activating transcription factor-2 in 15(S)-hydroxyeicosatetraenoic acid-induced angiogenesis.

To investigate the mechanisms underlying 15(S)-HETE-induced angiogenesis, we have studied the role of the small GTPase, Rac1. We find that 15(S)-HETE activated Rac1 in human retinal microvascular endothelial cells (HRMVEC) in a time-dependent manner. Blockade of Rac1 by adenovirus-mediated expression of its dominant negative mutant suppressed HRMVEC migration as well as tube formation and Matrigel plug angiogenesis. 15(S)-HETE stimulated Src in HRMVEC in a time-dependent manner and blockade of its activation inhibited 15(S)-HETE-induced Rac1 stimulation in HRMVEC and the migration and tube formation of these cells as well as Matrigel plug angiogenesis. 15(S)-HETE stimulated JNK1 in Src-Rac1-dependent manner in HRMVEC and adenovirus-mediated expression of its dominant negative mutant suppressed the migration and tube formation of these cells and Matrigel plug angiogenesis. 15(S)-HETE activated ATF-2 in HRMVEC in Src-Rac1-JNK1-dependent manner and interference with its activation via adenovirus-mediated expression of its dominant negative mutant abrogated migration and tube formation of HRMVEC and Matrigel plug angiogenesis. In addition, 15(S)-HETE-induced MEK1 stimulation was found to be dependent on Src-Rac1 activation. Blockade of MEK1 activation inhibited 15(S)-HETE-induced JNK1 activity and ATF-2 phosphorylation. Together, these findings show that 15(S)-HETE activates ATF-2 via the Src-Rac1-MEK1-JNK1 signaling axis in HRMVEC leading to their angiogenic differentiation.

Preconditioning protects the retinal pigment epithelium cells from oxidative stress-induced cell death.

PURPOSE: The cytotoxic effects of oxidative stress, which play an important role in ocular diseases, are well known. In this study, we investigated the effect of non-lethal doses of oxidative stress on various cell functions, namely cell viability, cell attachment and cell migration in a widely used retinal pigment epithelium (RPE) cell line (ARPE-19). METHODS: A single exposure to various concentrations of hydrogen peroxide (H(2)O(2)) was used to establish a dose response for H(2)O(2)-induced cell death. Other cellular responses, such as changes in cell attachment and migration, were monitored after exposure to increasing doses. Finally, the effects of preconditioning cells with increasing non-lethal doses of H(2)O(2), with and without a subsequent exposure to lethal doses of H(2)O(2), were determined. RESULTS: The optimum dose for inducing cell death in ARPE-19 cells was between 900 and 1000 microm H(2)O(2). Preconditioning the cells with 1, 10 and 50 microm of H(2)O(2) provided a dose-dependent protection against cell death induced by a lethal dose (900-1000 microm) of H(2)O(2). Preconditioning with higher doses caused cells to become more susceptible to the cytotoxic effects of the lethal dose. Although H(2)O(2) increased cell attachment in lower doses, it induced a dose-dependent inhibition of cell attachment to the substrate in higher doses. H(2)O(2) did not affect cell migration in sub-lethal doses. CONCLUSION: Preconditioning RPE cells with limited exposure to non-lethal oxidative stress confers significant protection against subsequent H(2)O(2)-induced cell death. It also affects cell attachment in a dose-specific manner. This finding may help in understanding the pathogenesis of diseases in which oxidative stress plays an important role and in determining the suitability of certain treatment strategies, in particular RPE transplantation in the treatment of age-related macular degeneration.

15(S)-HETE production in human retinal microvascular endothelial cells by hypoxia: Novel role for MEK1 in 15(S)-HETE induced angiogenesis.

PURPOSE: To examine for the expression of 15-lipoxygenase 1 (15-LOX1) and 15-LOX2 in human retinal microvascular endothelial cells (HRMVECs) and study the role of arachidonic acid metabolites of these enzymes in angiogenesis. METHODS: Quantitative RT-PCR and reverse-phase HPLC analyses were used to determine 15-LOX1/2 expression and their arachidonic acid metabolites in HRMVECs. The role of MEK1 in 15(S)-HETE-induced angiogenesis was studied using HRMVEC migration, tube formation, and basement membrane matrix plug angiogenesis. RESULTS: HRMVECs expressed both 15-LOX1 and 15-LOX2. Hypoxia induced the expression of 15-LOX1 and the production of its arachidonic acid metabolites 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) and 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). 15(S)-HETE stimulated HRMVEC migration and tube formation as potently as 20 ng/mL fibroblast growth factor-2 (FGF-2). In addition, 15(S)-HETE stimulated the phosphorylation of ERK1/2, JNK1, p38 MAPK, and MEK1 in a time-dependent manner in these cells. Inhibition of MEK1 by pharmacologic and dominant-negative mutant approaches attenuated 15(S)-HETE-induced phosphorylation of ERK1/2 and JNK1 but not p38 MAPK. Blockade of ERK1/2 and JNK1 activation suppressed 15(S)-HETE-induced HRMVEC migration and tube formation and basement membrane matrix plug angiogenesis. Inhibition of p38 MAPK attenuated 15(S)-HETE-induced HRMVEC migration only. Inhibition of MEK1 also blocked 15(S)-HETE-induced HRMVEC migration and tube formation and basement membrane matrix plug angiogenesis. CONCLUSIONS: These results suggest that hypoxia, through the induction of 15-LOX1 expression, leads to the production of 15(S)-HETE in HRMVECs. In addition, 15(S)-HETE, through MEK1-dependent activation of ERK1/2 and JNK1, stimulates the angiogenic differentiation of HRMVECs and basement membrane matrix plug angiogenesis.