Growth hormone promotes the survival of retinal cells in vivo.

Growth hormone (GH) is synthesized and present in the developing chick retina, where it may have local actions in retinal cell differentiation similar to those of conventional growth factors. We have previously shown that retinal GH has neuroprotective effects in retinal ganglion cells. In this paper, we extend our earlier functional studies by examining the in vivo effects of a GH siRNA (NR-cGH-1) after microinjection into the eye cup of the developing chick embryo in ovo. We show that intra-vitreous cGH siRNA lowers both GH mRNA and insulin-like growth factor-1 (IGF-1) mRNA levels in the retina in vivo, and concomitantly elevates the numbers of apoptotic cells in the retina. These effects are apparent 6h after treatment, and persist for at least 24h. The apoptotic cells induced by GH withdrawal were primarily located close to the optic fissure of the developing eye, and were distributed in clusters, suggesting that there are sub-populations of retinal cells that are particularly susceptible to apoptotic stimuli. These results support our view that a GH/IGF-1 axis in retinal cells regulates retinal cell survival in vivo.

Growth hormone expression and neuroprotective activity in a quail neural retina cell line.

We have previously shown that growth hormone (GH) is produced within cells of the chick embryo retina where it appears to act as an autocrine/paracrine anti-apoptotic factor in the regulation of programmed cell death during retinal development. These investigations were carried out on cultured chick embryo retinal ganglion cells (RGCs) as well as on the chick embryo retina in ovo, using GH protein knock-down by immunoneutralization. We have now investigated the putative neuroprotective actions of GH using a quail embryo neural retina cell line (QNR/D) treated with GH siRNA to silence the local synthesis of GH. We now show that knock-down of GH by gene silencing in cells of this cultured embryonic neural retina cell line, using NR-cGH-1 siRNA, correlates with the increased appearance in the cultures of cells with apoptotic nuclear morphology. This result is consistent with our previous results using protein knock-down by immunoneutralization. We thus validate, using different technology and a different culture system, our contention that GH, produced locally by cells of the neural retina acts in an autocrine or paracrine manner to regulate cell survival in the retina.

Endogenous growth hormone in human retinal ganglion cells correlates with cell survival.

PURPOSE: Locally synthesized growth hormone (GH) may act as a survival factor in several tissues. Experimental studies with chick retinal ganglion cells (RGCs) suggest that GH, synthesized within the developing retina, may have autocrine or paracrine roles in the regulation of the waves of cell death characteristic of RGC differentiation. There is also evidence that GH may have a similar neuroprotective function in the rat retina, however, there is no information concerning such a role in the human retina. In this paper we extended our earlier work by determining whether the local expression of retinal GH correlates with RGC apoptosis in human retinas. METHODS: In the absence of experimental approaches to survival factor function in the human retina, we have used a correlative immunocytochemical technique to determine how the expression of GH relates to cell death in RGCs. We used sections of human retinas, taken postmortem, that we double-labeled for GH and apoptotic cell death using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). RESULTS: We found that approximately 35% of cells in the ganglion cell layer (GCL) were both GH positive and GH receptor (GHR) positive, and that GH colocalized with GHR in these cells. However, none of the apoptotic cells in the GCL were GH immunoreactive. Labeling of sections with the RGC marker, synuclein, indicated that at least 95% of the cells in the GCL were RGCs, leading us to conclude that the majority of the cells that we have examined in the GCL are RGCs. CONCLUSIONS: The results are consistent with the earlier proposal, based on in vivo and in vitro experimental chick embryo studies, that GH promotes survival in adult human RGCs.

Growth hormone is present in the human retina and vitreous fluid.

Growth factors have been found in vitreous fluid, in which they regulate retinal function and provide markers of ocular dysfunction. Since growth hormone (GH) has recently been discovered in the eyes of rodents and embryonic chicks and found to be neuroprotective for retinal ganglion cells, the possible presence of GH in the human retina and vitreous fluid has been assessed. GH-immunoreactivity in the retina and vitreous fluid of cadavers and in the vitreous fluid of patients with ocular dysfunction was determined by Western blotting. GH-immunoreactivity, identical in size (22kDa) to recombinant pituitary GH was found in proteins extracted from the retina and in the vitreous fluid of patients with ocular disease (proliferative diabetic retinopathy, epiretinal membrane and vascular hemorrhage) and individuals with no history of ocular disease. GH-immunoreactivity was also detected in large, discrete cells in the retinal ganglion cell layer, in which GH staining was mainly within the nuclear compartment. The novel presence of GH in the human retina and vitreous fluid suggests GH may have roles in visual function and be involved in the pathogenesis of ocular disease.

Signaling mechanisms mediating local GH action in the neural retina of the chick embryo.

Growth hormone (GH) is found in the retina and vitreous of the chick embryo, where it appears to act as a growth and differentiation factor, having neuroprotective effects on retinal ganglion cells (RGCs). Here, we review the molecular mechanisms of the anti-apoptotic effect of GH in chick RGCs. GH treatment of RGCs reduces Akt levels, while raising Akt-phos levels, consistent with a role for Akt signaling pathways in the GH neuroprotective action. The induction of apoptosis by immunoneutralization with GH antiserum is accompanied by an increase in caspase-3 and caspase-9 activation, and also PARP-1 cleavage. Calpain activation also appears to be a major caspase-independent pathway to PARP-1 cleavage and apoptosis in these cells, supporting the view that caspase and calpain inhibitors are major neuroprotective agents for RGCs, and that pathways that activate both caspases and calpains are important for the anti-apoptotic actions of GH in these cells. These pathways involve the activation of cytosolic tyrosine kinases (Trks) and extracellular-signal-related kinases (ERKs). Occupation of the GH receptor by GH involves downstream intracellular Trk pathways. The Akt and Trk pathways appear to converge on the activation of cAMP response element binding protein (CREB), which is able to initiate transcription of pro- or anti-apoptotic genes. These results indicate that the action of GH in the neuroprotection of embryonic RGCs involves pathways common to with other neurotrophins, and that GH can be considered to be a growth and differentiation factor in the development of the embryonic retina. We have also investigated the relationship between the overlapping anti-apoptotic effects of GH and insulin-like growth factor-1 (IGF-1), two functionally closely related factors. We find that simultaneous immunoneutralization of GH and IGF-1 does not increase the level of apoptosis in the cultures above that achieved by immunoneutralization of GH alone. We therefore conclude that the neuroprotective actions of GH in the developing retina are likely mediated in large part through the action of IGF-1.

Growth hormone-mediated survival of embryonic retinal ganglion cells: signaling mechanisms.

Growth hormone (GH) is found in the developing eye, where it is synthesized by retinal ganglion cells (RGCs). In this location, GH variants appear to have an autocrine or paracrine anti-apoptotic neuroprotective role, and may contribute to the regulation of the developmental waves of apoptosis that characterize RGC differentiation. Here, we investigate the intracellular signaling pathways that are activated by GH as a neuroprotective agent in cultured chick embryo RGCs. We show that GH treatment reduces the cleavage of caspase-9, and that an inhibitor of caspase-9 cleavage can abrogate the pro-apoptotic effect of GH immunoneutralization. These findings complement previous results implicating caspase-3 in GH action on these cells. We had also previously shown that Akt pathways are involved in the neuroprotection of RGCs by GH. We now extend those findings to show that these pathways involve the activation of cytosolic tyrosine kinases (Trks) and extracellular-signal-related kinases (ERKs). Therefore, although the GH receptor, unlike other neurotrophin receptors, is not itself a receptor tyrosine kinase (receptor Trk), occupation of the receptor by GH involves downstream intracellular Trk pathways. Finally, we show that the Akt and Trk pathways converge on the activation of cAMP response element binding protein (CREB) which is able to initiate transcription of pro- or anti-apoptotic genes. These results indicate that the action of GH in the neuroprotection of embryonic RGCs involves pathways that are common to other neurotrophins, and that GH can be considered to be an authentic growth and differentiation factor in the development of the embryonic retina.

Retinal ganglion cell survival in development: mechanisms of retinal growth hormone action.

Several variants of growth hormone (GH) are found in the retina and vitreous of the chick embryo, where they appear to act as cell survival factors, having neuroprotective effects on retinal ganglion cells (RGCs). Here, we investigate the molecular mechanisms of the anti-apoptotic effect of GH in cultured RGCs. GH treatment increased Akt phosphorylation in these cells, which is an anti-apoptotic event. Whereas unphosphorylated Akt was detected in both nucleus and cytoplasm of RGCs by immunocytochemistry, the phosphorylated form of Akt (Akt-phos) was located primarily in the cytoplasm of both normal and apoptotic cells, although levels were markedly lower in the latter. It was found that GH treatment of RGCs reduced Akt levels, while concomitantly raising Akt-phos levels, consistent with a role for Akt signaling pathways in GH neuroprotective action. This was substantiated using Wortmannin, which, like GH antiserum, inhibited Akt phosphorylation and initiated apoptosis. The addition of Wortmannin to RGC cultures simultaneously with GH significantly reduced the anti-apoptotic effect of GH. The induction of apoptosis by GH antiserum was clearly accompanied by an increase in caspase-3 activation and PARP-1 cleavage, both of which were significantly reduced in the presence of the broad spectrum caspase inhibitor, Q-VD-OPh, which itself had a dramatic neuroprotective effect on cultured RGCs. Calpain activation appeared to be a major caspase-independent pathway to PARP-1 cleavage and apoptosis in these cells. Calpain inhibitor III (MDL 28170) was able to reduce PARP-1 cleavage and abrogate the apoptogenic effect of GH antiserum. The results support the view that caspase and calpain inhibitors are major neuroprotective agents for RGCs, and that pathways that activate both caspases and calpains are important for the anti-apoptotic actions of GH in these cells.

Retinal growth hormone is an anti-apoptotic factor in embryonic retinal ganglion cell differentiation.

Cells of the neural retina in the chick embryo undergo several waves of apoptosis during development, including peaks at approximately embryonic day (ED) 7 and 12. Prominent among the cells involved in these phases of cell death are the retinal ganglion cells (RGCs). We have previously shown that growth hormone (GH) is expressed in the neural retina, and particularly, in the RGCs. Here we study the ability of GH to rescue retinal cells from apoptosis, both in vitro and in vivo. When retinas from embryos at ED 6-8 are explanted on collagen gels, the application of recombinant GH, at 10(-6)m, significantly reduced the incidence of apoptotic cells in the cultures as judged by terminal deoxynucleotide transferase-mediated dUTP-biotin nick end labelling (TUNEL). GH was delivered to neural retinas in ovo, by microinjection into the eye cup at ED 2. When these embryos were examined at ED 6-8, no reduction in cell death was observed below the normal low control levels. However, when antibodies to GH were microinjected, the incidence of cell death increased significantly at ED 6, providing evidence that in vivo immunoneutralization of endogenous GH results in triggering of apoptotic signaling pathways. Evidence that RGCs are a particular target of this neuroprotective effect of GH was provided by examination of cultures enriched for RGCs by immunopanning. In serum-free culture, RGCs, identified by anti-Islet 1 immunolabelling, were found to be susceptible to the effect of GH immunoneutralization, which approximately quadrupled the incidence of apoptosis in the cultures. We propose that GH is a naturally occurring autocrine and/or paracrine neuroprotective agent in the developing retina which is involved in the regulation of retinal cell numbers during early embryogenesis.

Opticin binds retinal growth hormone in the embryonic vitreous.

PURPOSE: Opticin is a small leucine-rich repeat proteoglycan that is abundant in several ocular tissues, including the vitreous. Like other proteoglycans, opticin may have the ability to bind and regulate the release of growth factors. Previous work has shown that isoforms of growth hormone (GH) are present in the embryonic retina, where they may act as a growth factor. The current study was conducted to investigate the possibility that opticin binds retinal GH in the vitreous of the chick embryo. METHODS: The vitreous and retina of embryonic day-8 chicks were examined for the presence of opticin and GH, by Western immunoblot analysis, coimmunoprecipitation, and immunocytochemistry. RESULTS: Opticin associated with GH in the embryonic vitreous to produce a 60- to 62-kDa complex. Opticin and GH were also colocalized in the retina in retinal ganglion cells. CONCLUSIONS: The binding of retinal GH by opticin in the vitreous suggests that GH, secreted by the retinal ganglion cells, may be sequestered and concentrated in the vitreous and could act there as a paracrine differentiation factor in ocular development. During development, opticin could therefore regulate growth factor-like actions of retinal GH, both in the vitreous and the retinal ganglion cells. The physiological roles of GH in this location remain to be determined, but may include the regulation of cell proliferation and cell death.

Retroviral overexpression of bcl-2 in the embryonic chick lens influences denucleation in differentiating lens fiber cells.

During the course of their differentiation, embryonic lens fibers undergo loss of their cytoplasmic organelles and nuclei. The denucleation process bears similarities to the nuclear breakdown that occurs during apoptosis. This has given rise to the hypothesis that this denucleation is analogous to apoptosis, but without the plasma membrane changes characteristic of apoptotic cell death. Previous work has shown that several members of the apoptotic cascade are active during denucleation. Here, we have overexpressed the anti-apoptotic molecule bcl-2 in developing lenses of the 8-day-old chick embryo in ovo, using the replication-competent retrovirus RCAS. We find that lenses overexpressing bcl-2 show varying degrees of distortion in comparison with untreated and negative insert controls, including a more spherical shape and disorganized fiber cells. All overexpressing lenses showed significantly higher numbers of smaller nuclei in the lens core, where denucleation begins. There was no change in cell size or pattern of proliferation. These in vivo results were confirmed in vitro using lens epithelial cell cultures, which differentiate into lentoids. The lentoids in treated cultures showed the same effect on nuclear number and size. We further found that in lenses overexpressing bcl-2 there was a reduction in the activation of caspase-9 and the cleavage of the caspase substrate DFF45, and, in the lens core, a failure of the nuclear chromatin to condense. These results provide strong support for the view that embryonic lens fiber cell denucleation is analogous to the nuclear degradation that occurs during apoptosis, and that similar control pathways are involved in both these phenomena.

Expression and function of bone morphogenetic proteins in the development of the embryonic endocardial cushions.

Bone morphogenetic proteins (BMPs) are considered to be significant factors in the morphogenesis of the endocardial cushions of the developing embryonic heart. Previous studies have suggested that they are involved in the epithelial-mesenchymal transformation and migration of the cells forming the cushions, or in triggering an apoptotic cascade in a sub-population of cushion cells. We investigated the expression and function of BMP2 and BMP4 proteins in the developing heart of the chick and mouse embryos. In the chick, by immunocytochemistry, we find expression of BMP2 protein in the endocardial cushions of the outflow tract (OT) and atrio-ventricular (AV) regions at embryonic days (ED) 5-6, as well as in adjacent myocardial layers. Immunoblotting indicated that such expression persisted through ED 4-7, but peaked at ED4-5 in the OT and 5-6 in the AV cushions. This temporal sequence correlated with the peaks of apoptotic cell death found previously in the OT and AV cushions of the chick embryo. At equivalent stages in mouse, no such expression of BMP2 was found in the cushions, although expression was detected in adjacent myocardial layers. In the case of BMP4, in both chick and mouse, expression was found only in the myocardia and not in the cushions. Furthermore, BMP-specific receptors were found in the cushions, but not the myocardia, in both the AV and OT regions of the chick embryo. These results provide circumstantial evidence to support the contention that BMPs, originating from the myocardium, could be significant in the induction of apoptosis in chick embryo cushion cells, and confirms that there is species-specific variation in the expression pattern of BMP proteins, as had been predicted from previous studies of mRNA expression. Culture media conditioned by the growth of tissues from various regions of the developing heart were tested for their ability to induce apoptosis in cushion cells in culture. It was found that medium derived from the myocardia induced significant levels of cell death in the cushion cells, and that BMP4 could be detected in such media; however, retroviral over-expression of constitutively active (CA) and dominant-negative (DN) isoforms of BMP-specific receptors 1A and 1B (BMPR-1A and BMPR-1B) in cultured cells of the AV cushions did not alter levels of apoptosis or cell proliferation. Similar over-expression in cultured endocardial cells resulted in a significant change in cell shape, from endothelial to fibroblastic, with BMPR-1A CA and BMPR-1B DN, suggesting an influence of these receptors on cell transformation and/or cell migration. Taken together, these results provide support for the contention that BMP2 and BMP4 are important factors in the phenotypic transformational events involved in the morphogenesis of the chick embryo endocardial cushions, and could be involved in the induction of apoptosis in the cushion cells.

Expression of apoptosis-inducing factor during early neural differentiation in the chick embryo.

The distribution of apoptosis-inducing factor (AIF) immunoreactivity has been studied in the developing somites and nervous system of the chick embryo at embryonic day 4. AIF was found to be expressed primarily in the cytoplasm of cells of the ventral motor roots, at the points of their insertion into the neural tube. Co-localization of mitochondrial AIF immunoreactivity with the epitopes recognized by the monoclonal antibodies HNK-1 and 1E8 suggests that the AIF may be present in Schwann cell precursors as well as in nerve fibres. AIF immunoreactivity was not observed in either cell bodies in the neural tube, or in the somitic tissue surrounding the ventral roots. The results are consistent with the hypothesis that AIF may be involved in neuronal cell death during development, and that target-derived neuronal survival factors may act by controlling AIF activity.