EGF-like-domain-7 is required for VEGF-induced Akt/ERK activation and vascular tube formation in an ex vivo angiogenesis assay.

EGFL7 is a secreted angiogenic factor, which in contrast to the well-known secreted angiogenic molecules VEGF and FGF-2, is almost exclusively expressed by endothelial cells and may act in an autocrine fashion. Prior studies have shown EGFL7 to mediate its angiogenic effects by interfering with the Notch pathway and/or via the intronic miR126. Less is known about its effects on VEGF signaling. We wanted to investigate the role of epidermal growth factor-like domain 7 (EGFL7) in VEGF-driven angiogenesis using an ex vivo Matrigel-embedded mouse eye cup assay and siRNA mediated knockdown of EGFL7 by siRNA. Our results suggested that VEGF-induced vascular tube formation was significantly impaired after siRNA downregulation of EGFL7. In addition, knockdown of EGFL7 suppressed VEGF upregulation of phospho-Akt and phospho-Erk(1/2) in endothelial cells, but did not alter VEGFR phosphorylation and neuropilin-1 protein expression or miR126 expression. Thus, in conclusion, EGFL7 is required for VEGF upregulation of the Akt/Erk (1/2) pathway during angiogenesis, and may represent a new therapeutic target in diseases of pathological neovascularization.

AMP-dependent kinase inhibits oxidative stress-induced caveolin-1 phosphorylation and endocytosis by suppressing the dissociation between c-Abl and Prdx1 proteins in endothelial cells.

Caveolin-1 is the primary structural component of endothelial caveolae that is essential for transcellular trafficking of albumin and is also a critical scaffolding protein that regulates the activity of signaling molecules in caveolae. Phosphorylation of caveolin-1 plays a fundamental role in the mechanism of oxidant-induced vascular hyper permeability. However, the regulatory mechanism of caveolin-1 phosphorylation remains unclear. Here we identify a previously unexpected role for AMPK in inhibition of caveolin-1 phosphorylation under oxidative stress. A pharmacological activator of AMPK, 5-amino-4-imidazole carboxamide riboside (AICAR), inhibited oxidative stress-induced phosphorylation of both caveolin-1 and c-Abl, which is the major kinase of caveolin-1, and endocytosis of albumin in human umbilical vein endothelial cell. These effects were abolished by treatment with two specific inhibitors of AICAR, dipyridamole, and 5-iodotubericidin. Consistently, knockdown of the catalytic AMPKα subunit by siRNA abolished the inhibitory effect of AICAR on oxidant-induced phosphorylation of both caveolin-1 and c-Abl. Pretreatment with specific c-Abl inhibitor, imatinib mesylate, and knock down of c-Abl significantly decreased the caveolin-1 phosphorylation after H2O2 exposure and abolished the inhibitory effect of AICAR on the caveolin-1 phosphorylation. Interestingly, knockdown of Prdx-1, an antioxidant enzyme associated with c-Abl, increased phosphorylation of both caveolin-1 and c-Abl and abolished the inhibitory effect of AICAR on the caveolin-1 phosphorylation. Furthermore, co-immunoprecipitation experiment showed that AICAR suppressed the oxidant-induced dissociation between c-Abl and Prdx1. Overall, our results suggest that activation of AMPK inhibits oxidative stress-induced caveolin-1 phosphorylation and endocytosis, and this effect is mediated in part by stabilizing the interaction between c-Abl and Prdx-1.

Aminoimidazole carboxamide ribonucleotide (AICAR) inhibits the growth of retinoblastoma in vivo by decreasing angiogenesis and inducing apoptosis.

5-Aminoimidazole-4-carboxamide-1-ß-4-ribofuranoside (AICAR), an analog of AMP is widely used as an activator of AMP-kinase (AMPK), a protein that regulates the responses of the cell to energy change. Recently, we showed that AICAR-induced AMPK activation inhibits the growth of retinoblastoma cells in vitro by decreasing cyclins and by inducing apoptosis and S-phase arrest. In this study, we investigated the effects of AMPK activator AICAR on the growth of retinoblastoma in vivo. Intraperitoneal injection of AICAR resulted in 48% growth inhibition of Y79 retinoblastoma cell tumors in mice. Tumors isolated from mice treated with AICAR had decreased expression of Ki67 and increased apoptotic cells (TUNEL positive) compared with the control. In addition, AICAR treatment suppressed significantly tumor vessel density and macrophage infiltration. We also showed that AICAR administration resulted in AMPK activation and mTOR pathway inhibition. Paradoxically observed down-regulation of p21, which indicates that p21 may have a novel function of an oncogene in retinoblastoma tumor. Our results indicate that AICAR treatment inhibited the growth of retinoblastoma tumor in vivo via AMPK/mTORC1 pathway and by apoptogenic, anti-proliferative, anti-angiogenesis mechanism. AICAR is a promising novel non-chemotherapeutic drug that may be effective as an adjuvant in treating Retinoblastoma.

Evidence for baseline retinal pigment epithelium pathology in the Trp1-Cre mouse.

The increasing popularity of the Cre/loxP recombination system has led to the generation of numerous transgenic mouse lines in which Cre recombinase is expressed under the control of organ- or cell-specific promoters. Alterations in retinal pigment epithelium (RPE), a multifunctional cell monolayer that separates the retinal photoreceptors from the choroid, are prevalent in the pathogenesis of a number of ocular disorders, including age-related macular degeneration. To date, six transgenic mouse lines have been developed that target Cre to the RPE under the control of various gene promoters. However, multiple lines of evidence indicate that high levels of Cre expression can be toxic to mammalian cells. In this study, we report that in the Trp1-Cre mouse, a commonly used transgenic Cre strain for RPE gene function studies, Cre recombinase expression alone leads to RPE dysfunction and concomitant disorganization of RPE layer morphology, large areas of RPE atrophy, retinal photoreceptor dysfunction, and microglial cell activation in the affected areas. The phenotype described herein is similar to previously published reports of conditional gene knockouts that used the Trp1-Cre mouse, suggesting that Cre toxicity alone could account for some of the reported phenotypes and highlighting the importance of the inclusion of Cre-expressing mice as controls in conditional gene targeting studies.

Inhibitory effect of aminoimidazole carboxamide ribonucleotide (AICAR) on endotoxin-induced uveitis in rats.

PURPOSE. To investigate the anti-inflammatory effect of aminoimidazole carboxamide ribonucleotide (AICAR), an analog of adenosine monophosphate (AMP), in endotoxin-induced uveitis (EIU). METHODS. EIU was induced by subcutaneous injection of lipopolysaccharide (LPS) (200 µg) in Lewis rats. AICAR (50 mg/kg, intraperitoneally) was given 6 hours prior and at the same time as LPS injection. Clinical uveitis scores, number of anterior chamber (AC) infiltrating cells, anterior chamber protein concentration, retinal vessel leukocyte adhesion, and protein leakage were measured 24 hours later. Protein levels of C-C chemokine ligand-2 (CCL-2)/monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-α (TNF-α) and intercellular adhesion molecule-1 (ICAM-1) in aqueous humor and retina and nuclear translocation of nuclear factor-κB (NF-κB) in the retina were determined by enzyme-linked immunosorbent assay (ELISA). Both mRNA and protein levels of CD14 in peripheral blood mononuclear cells were also measured. RESULTS. AICAR treatment significantly reduced EIU clinical severity as well as inflammatory cell infiltration and protein concentration in aqueous humor. Similarly, the number of retinal vessel-adherent leukocytes and protein leakage were decreased by AICAR treatment. Protein levels of TNF-α, CCL-2/MCP-1, and ICAM-1 in aqueous humor and CCL-2/MCP-1 and ICAM-1 levels in retina were suppressed with AICAR treatment. AICAR also reduced NF-κB translocation and CD14 expression. CONCLUSIONS. AICAR reduces systemic LPS susceptibility and attenuates intraocular inflammation in a rat EIU model by limiting infiltration of leukocytes, suppressing inflammatory mediators, and inhibiting the NF-κB pathway.

AMP-activated protein kinase suppresses matrix metalloproteinase-9 expression in mouse embryonic fibroblasts.

Matrix metalloproteinase-9 (MMP-9) plays a critical role in tissue remodeling under both physiological and pathological conditions. Although MMP-9 expression is low in most cells and is tightly controlled, the mechanism of its regulation is poorly understood. We utilized mouse embryonic fibroblasts (MEFs) that were nullizygous for the catalytic α subunit of AMP-activated protein kinase (AMPK), which is a key regulator of energy homeostasis, to identify AMPK as a suppressor of MMP-9 expression. Total AMPKα deletion significantly elevated MMP-9 expression compared with wild-type (WT) MEFs, whereas single knock-out of the isoforms AMPKα1 and AMPKα2 caused minimal change in the level of MMP-9 expression. The suppressive role of AMPK on MMP-9 expression was mediated through both its activity and presence. The AMPK activators 5-amino-4-imidazole carboxamide riboside and A769662 suppressed MMP-9 expression in WT MEFs, and AMPK inhibition by the overexpression of dominant negative (DN) AMPKα elevated MMP-9 expression. However, in AMPKα(-/-) MEFs transduced with DN AMPKα, MMP-9 expression was suppressed. AMPKα(-/-) MEFs showed increased phosphorylation of IκBα, expression of IκBα mRNA, nuclear localization of nuclear factor-κB (NF-κB), and DNA-binding activity of NF-κB compared with WT. Consistently, selective NF-κB inhibitors BMS345541 and SM7368 decreased MMP-9 expression in AMPKα(-/-) MEFs. Overall, our results suggest that both AMPKα isoforms suppress MMP-9 expression and that both the activity and presence of AMPKα contribute to its function as a regulator of MMP-9 expression by inhibiting the NF-κB pathway.

Tumor necrosis factor-alpha mediates photoreceptor death in a rodent model of retinal detachment.

PURPOSE: Photoreceptor degeneration is a major cause of visual loss in various retinal diseases, including retinal detachment (RD) and neovascular AMD, but the underlying mechanisms remain elusive. In this study, the role of TNFα in RD-induced photoreceptor degeneration was investigated. METHODS: RD was induced by subretinal injection of hyaluronic acid. Photoreceptor degeneration was assessed by counting the number of apoptotic cells with TdT-dUTP terminal nick-end labeling (TUNEL) 3 days after RD and measurement of the outer nuclear layer (ONL) thickness 7 days after RD. As the target of anti-inflammatory treatment, the expression of TNFα, with or without dexamethasone (DEX) was examined in rats by real-time PCR. To understand the role of TNFα in photoreceptor degeneration, RD was induced in mice deficient in TNFα or its receptors (TNFR1, TNFR2, and TNFR1 and -2), or in wild-type (WT) mice by using a functionally blocking antibody to TNFα. CD11b(+) cells in the outer plexiform layer (OPL) and subretinal space were counted by immunohistochemistry (IHC). RESULTS: Treatment with DEX (P = 0.001) significantly suppressed RD-induced photoreceptor degeneration and the expression of TNFα. RD-induced photoreceptor degeneration was significantly suppressed with specific blockade of TNFα (P = 0.032), in mice deficient for TNFα (P < 0.001), TNFR2 (P = 0.001), or TNFR1 and -2 (P < 0.001). However, lack of TNFR1 did not protect against RD-induced photoreceptor degeneration (P = 0.060). Müller cell activation was unchanged in WT and TNFα(-/-) mice. Recruitment of CD11b(+) monocytes was significantly lower in the TNFα(-/-) mice compared to WT mice (P = 0.002). CONCLUSIONS: TNFα plays a critical role in RD-induced photoreceptor degeneration. This pathway may become an important target in the prevention of RD-induced photoreceptor degeneration.

Heat shock protein 70 (HSP70) is critical for the photoreceptor stress response after retinal detachment via modulating anti-apoptotic Akt kinase.

Photoreceptor apoptosis is a major cause of vision loss in many ocular diseases. Significant progress has been made to elucidate the molecular pathways involved in this process, yet little is known about proteins counteracting these apoptotic pathways. It is established that heat shock proteins (HSPs) function as molecular helper proteins (chaperones) by preventing protein aggregation and facilitating refolding of dysfunctional proteins, critical to the survival of all organisms. Here, we investigated the role of HSP70 on photoreceptor survival after experimental retinal detachment (RD) in mice and rats. We found that HSP70 was up-regulated after RD and associated with phosphorylated Akt, thereby preventing its dephosphorylation and further activation of cell death pathways. Administration of quercetin, which inhibits HSP70 and suppresses Akt phosphorylation significantly increased photoreceptor apoptosis. Similarly, RD-induced photoreceptor apoptosis was augmented in mice carrying hypomorphic mutations of the genes encoding HSP70. On the other hand, administration of geranylgeranylacetone, which induces an increase in HSP70 significantly decreased photoreceptor apoptosis after RD through prolonged activation of Akt pathway. Thus, HSP70 may be a favorable potential target to increase photoreceptor cell survival after RD.

Receptor interacting protein kinases mediate retinal detachment-induced photoreceptor necrosis and compensate for inhibition of apoptosis.

Apoptosis has been shown to be a significant form of cell loss in many diseases. Detachment of photoreceptors from the retinal pigment epithelium, as seen in various retinal disorders, causes photoreceptor loss and subsequent vision decline. Although caspase-dependent apoptotic pathways are activated after retinal detachment, caspase inhibition by the pan-caspase inhibitor Z-VAD fails to prevent photoreceptor death; thus, we investigated other pathways leading to cell loss. Here, we show that receptor interacting protein (RIP) kinase-mediated necrosis is a significant mode of photoreceptor cell loss in an experimental model of retinal detachment and when caspases are inhibited, RIP-mediated necrosis becomes the predominant form of death. RIP3 expression, a key activator of RIP1 kinase, increased more than 10-fold after retinal detachment. Morphological assessment of detached retinas treated with Z-VAD showed decreased apoptosis but significantly increased necrotic photoreceptor death. RIP1 kinase inhibitor necrostatin-1 or Rip3 deficiency substantially prevented those necrotic changes and reduced oxidative stress and mitochondrial release of apoptosis-inducing factor. Thus, RIP kinase-mediated programmed necrosis is a redundant mechanism of photoreceptor death in addition to apoptosis, and simultaneous inhibition of RIP kinases and caspases is essential for effective neuroprotection and may be a novel therapeutic strategy for treatment of retinal disorders.

Retinoblastoma cells are inhibited by aminoimidazole carboxamide ribonucleotide (AICAR) partially through activation of AMP-dependent kinase.

5-Aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), an analog of AMP, is widely used as an activator of AMP-kinase (AMPK), a protein that regulates the responses of the cell to energy change. We studied the effects of AICAR on the growth of retinoblastoma cell lines (Y79, WERI, and RB143). AICAR inhibited Rb cell growth, induced apoptosis and S-phase cell cycle arrest, and led to activation of AMPK. These effects were abolished by treatment with dypiridamole, an inhibitor that blocks entrance of AICAR into cells. Treatment with the adenosine kinase inhibitor 5-iodotubericidin to inhibit the conversion of AICAR to ZMP (the direct activator of AMPK) reversed most of the growth-inhibiting effects of AICAR, indicating that some of the antiproliferative effects of AICAR are mediated through AMPK activation. In addition, AICAR treatment was associated with inhibition of the mammalian target of rapamycin pathway, decreased phosphorylation of ribosomal protein-S6 and 4E-BP1, down-regulation of cyclins A and E, and decreased expression of p21. Our results indicate that AICAR-induced activation of AMPK inhibits retinoblastoma cell growth. This is one of the first descriptions of a nonchemotherapeutic drug with low toxicity that may be effective in treating Rb patients.

Induction of corneal epithelium-like cells from cynomolgus monkey embryonic stem cells and their experimental transplantation to damaged cornea.

PURPOSE: We previously reported the successful transplantation of corneal epithelium-like cells derived from mouse embryonic stem (ES) cells onto injured mouse cornea. Here, we tested whether nonhuman primate ES cells have ability to differentiate into corneal epithelial cells and whether monkey ES cell-derived corneal epithelium-like cells were applicable for the experimental transplantation to damaged cornea. METHODS: Monkey ES cells were cultivated on type IV collagen-coated dishes for various days to induce differentiation into corneal epithelium-like cells. The differentiation was evaluated by reverse transcription-polymerase chain reaction and immunostaining. The corneal epithelium-like cells were transplanted to the injured mouse cornea. Reconstitution of the corneal epithelium was evaluated by immunostaining. RESULTS: The cells cultured on type IV collagen showed cobblestone-like appearance resembling epithelial cells. They expressed messenger RNA of pax6, p63, E-cadherin, CD44, proliferating cell nuclear antigen, keratin 3, and keratin 12. Protein expressions of pax6, keratin 3/12, p63, proliferating cell nuclear antigen, E-cadherin, and CD44 were confirmed by immunostaining. When the corneal epithelium-like cells were transplanted, they adhered to the corneal stroma, leading to formation of multiple cell layers. The grafted cells were stained with anti-human nuclear protein antibody, which cross-reacted with nuclei of monkey cells but not with those of mouse cells. They retained the expressions of keratin 3/12, E-cadherin, and CD44. CONCLUSIONS: We induced corneal epithelium-like cells from monkey ES cells with moderate efficiency. The cells were successfully transplanted onto the injured mouse cornea. This is the first demonstration that nonhuman primate ES cells were induced to differentiate into corneal epithelium-like cells, which were applicable for transplantation to an animal model of corneal injury.

Transfection with pax6 gene of mouse embryonic stem cells and subsequent cell cloning induced retinal neuron progenitors, including retinal ganglion cell-like cells, in vitro.

OBJECTIVE: It is theoretically possible to induce various cell types, including retinal neurons, from embryonic stem cells (ESCs). pax6 regulates early events in eye development, including the generation of retinal ganglion cells (RGCs). We previously reported the successful induction of corneal epithelial cells from ESCs transfected with the pax6 gene. Here, we attempted to establish cloned RGC-like cells from ESCs transfected with the pax6 gene. METHODS: Undifferentiated mouse ESCs were transfected with pax6 cDNA by electroporation, followed by selection with G418. We conducted limiting-dilution culture of pax6-transfected cells. We expanded the cloned pax6-transfected cells, which expressed nestin and musashi-1, for further characterization in culture media containing fibronectin. The cells were characterized using RT-PCR, immunostaining, electron microscopy, renal subcapsular transplantation assay and Ca imaging. RESULTS: We obtained clonally expanding pax6-transfected cells, all of which were positive for six3, sonic hedgehog (shh), math5, brn3, thy1 and melanopsin, by using several ESCs. When transplanted into a mouse renal capsule, they differentiated into neurons with elongated axons, expressing betaIII tubulin and neurofilament middle chain, and were free from teratoma development. Electron-microscopic examination showed neurotubules and neurofilaments in the axon-like processes of the cloned pax6-transfected cells. High KCl stimulation increased free Ca influx on Ca2+ imaging. CONCLUSIONS: ESCs were applicable for the induction of retinal progenitor cells, including RGC-like cells, by transfection with the pax6 gene and subsequent limiting-dilution culture. Cloned cell lines may be useful to analyze the requirements for retinal progenitor cell differentiation, and our study suggests the clinical application of this cell type.

Experimental transplantation of corneal epithelium-like cells induced by Pax6 gene transfection of mouse embryonic stem cells.

PURPOSE: Corneal epithelial stem cells are deficient in cases of limbal disorders, leading to conjunctival epithelial ingrowth, vascularization, and eventually visual disturbance. We introduced the eye development-associated transcription factor pax6 to embryonic stem (ES) cells and tested whether pax6-transfected cells resembling purified corneal epithelial cells were applicable as a cell source for corneal transplantation. METHODS: pax6 cDNA with green fluorescence protein was electrotransfected to ES cells and the cells were cultured with G418 for 14 days. They were characterized by reverse transcription-polymerase chain reaction and immunohistochemistry. The cells were transplanted onto experimentally damaged mouse corneas. Histologic reconstitution of the corneal epithelium was assessed. RESULTS: pax6-transfected cells formed a monolayer of epithelium-like cells in vitro. They expressed cytokeratin12, a specific keratin of corneal epithelial cells, E-cadherin, and CD44, which are important adhesion molecules of corneal epithelial cells on the cell membrane. They accumulated to make a colony that gave a staining pattern of reticular configuration for cytokeratin 12, E-cadherin, and CD44. When the cells were transplanted onto damaged cornea, they have been kept alive on the cornea. CONCLUSIONS: The purified corneal epithelium-like cells derived from ES cells transfected with pax6 gene adapted to the injured cornea and were kept alive on it. These results suggested application of ES cell-derived corneal epithelial cells for treating corneal injuries.

Recent advances in corneal regeneration and possible application of embryonic stem cell-derived corneal epithelial cells.

The depletion of limbal stem cells due to various diseases leads to corneal opacification and visual loss. The unequivocal identification and isolation of limbal stem cells may be a considerable advantage because long-term, functional recovery of corneal epithelium is linked to graft constructs that retain viable stem cell populations. As specific markers of limbal stem cells, the ATP-binding cassette, sub-family G, member2 (ABCG2), a member of the multiple drug-resistance (MDR) family of membrane transporters which leads to a side population phenotype, and transcription factor p63 were proposed recently. Conventional corneal transplantation is not applicable for patients with limbal stem cells deficiency, because the conventional allograft lacks limbal stem cells. The introduction of limbal epithelial cell transplantation was a major advance in the therapeutic techniques for reconstruction of the corneal surface. Limbal epithelial cell transplantation is clinically conducted when cultured allografts as well as autografts are available; however, allografts have a risk of immunologic rejection and autografts are hardly available for patients with bilateral ocular surface disorders. Embryonic stem (ES) cells are characterized by their capacity to proliferate indefinitely and to differentiate into any cell type. We induced corneal epithelial cells from ES cells by culturing them on type IV collagen or alternatively, by introduction of the pax6 gene into ES cells. Recent advances in our study supports the possibility of their clinical use as a cell source for reconstruction of the damaged corneal surface. This review summarizes the recent advances in corneal regeneration therapies and the possible application of ES cell-derived corneal epithelial cells.