Ischemic proliferative retinopathy (e.g., diabetes mellitus, retinopathy of prematurity, or retinal vein occlusion) is a major cause of blindness worldwide. Apart from neovascularization, ischemic proliferative retinopathy leads to retinal degeneration. Apoptosis has been ascribed to be the leading mechanism in ischemic retinal degeneration. We showed recently that inducible nitric oxide synthase (iNOS) is expressed in the avascular retina in proliferative retinopathy in vivo and that iNOS expression in retinal glial cells is responsible for retinal neuronal cell death in vitro. Here we show that retinal apoptosis and subsequent degeneration occur in the murine model of ischemic proliferative retinopathy. Furthermore, because NO can have beneficial or detrimental effects in the retina, we analyzed the role of iNOS on retinal apoptosis in ischemic proliferative retinopathy. Using iNOS knock-out mice and iNOS inhibitor 1400W, we demonstrate in vivo that iNOS expression induces apoptosis locally in the inner nuclear layer of the avascular retina and that protein nitration may be involved in this process. These findings are the first evidence for retinal apoptosis in an animal model of ischemic proliferative retinopathy, demonstrating that iNOS plays a crucial role not only in retinal neovascular disease but also in retinal degeneration. We show that it is an ideal target to protect the hypoxic retina from degeneration and to improve its vascularization.

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Dissociated newborn rat retinal cells were maintained in monolayer culture for periods of up to 11 d. When grown in the absence of exogenous growth factors, 1-2% of the total neuronal population expressed opsin (the photopigment that is specific for maturing photoreceptors). Addition of a single dose of 10 ng/ml basic fibroblast growth factor (bFGF) to the culture medium induced an average increase of sixfold in the numbers of neurons expressing opsin. This supplementation had little effect on the total number of differentiated neurons or of glial cells when measured at the same time points. Furthermore, another specific class of retinal neurons, the amacrine cells, showed no changes following exposure to this growth factor. Two other growth factors known to exert neurotrophic effects, epidermal and nerve growth factor, were without effect. The effect of bFGF was dose dependent, with highly significant differences being observed with as little as 100 pg/ml, and with 700 pg/ml eliciting half-maximal stimulation; maximal effects were observed at 10 ng/ml. Induction of opsin expression by low concentrations of bFGF was blocked completely by an antiserum directed specifically against bFGF, but not by preimmune serum immunoglobulins. This increase in the number of photoreceptors expressing opsin following exposure to bFGF could have been due to either increased cell survival, increased proliferation of progenitor cells, or increased differentiation of immature photoreceptors. There was no increase in overall cell survival under the experimental conditions used, and double labeling immunocytochemistry combined with autoradiographic analysis of 3H-thymidine uptake showed that proliferation of neuronal precursors was not enhanced by the addition of bFGF. In contrast to these observations, cultures established from older (postnatal day 3) retina revealed large numbers of opsin-expressing photoreceptors in all culture plates, with or without added growth factors. This reduction in the stimulatory effects of bFGF with increasing postnatal age is consistent with the period of sensitivity being limited to the cycling of neuronal precursors. It is possible that a bFGF-like molecule is secreted by neighboring cells such as the retinal pigmented epithelium, to participate in retinal development and differentiation. To our understanding, this molecule is the first protein identified to influence specifically the differentiation of photoreceptor cells.

Background-Intravitreal neovascular diseases, as in ischemic retinopathies, are a major cause of blindness. Because inflammatory mechanisms influence vitreal neovascularization and cyclooxygenase (COX)-2 promotes tumor angiogenesis, we investigated the role of COX-2 in ischemic proliferative retinopathy. Methods and Results-We describe here that COX-2 is induced in retinal astrocytes in human diabetic retinopathy, in the murine and rat model of ischemic proliferative retinopathy in vivo, and in hypoxic astrocytes in vitro. Specific COX-2 but not COX-1 inhibitors prevented intravitreal neovascularization, whereas prostaglandin E 2 , mainly via its prostaglandin E receptor 3 (EP 3 ), exacerbated neovascularization. COX-2 inhibition induced an upregulation of thrombospondin-1 and its CD36 receptor, consistent with the observed antiangiogenic effects of COX-2 inhibition; EP 3 stimulation reversed effects of COX-2 inhibitors on thrombospondin-1 and CD36. Conclusion-These findings point to an important role for COX-2 in ischemic proliferative retinopathy, as in diabetes.

The characterization of DNase II and DNase I activity was undertaken to discriminate their different roles in physiological nuclear degradation during lens fiber cell differentiation. The activity of both nucleases determined in a new assay allows to discriminate DNase II from DNase I in the same extract. In fibers, both types of nuclease activities are found and appear higher than in epithelial cells. Specific polyclonal antibodies directed against these two nucleases reveal by Western blot analysis the presence of various DNase isoforms. DNase II like-nuclease, present in fibers, is represented by three major bands (60, 23, and 18 kDa), which are not detected, at least for two of them (60 and 23 kDa), in epithelial cells. DNase I like-nuclease pattern in fiber cells shows a single 32-kDa band, while several bands can be detected in epithelial cells. Immunocytochemistry studies show both nucleases present in lens cell sections. DNase II is, as usual, in cytoplasm of epithelial cells, but it appears strikingly concentrated in the nuclei of fibers. DNase I is always concentrated in nuclei of epithelial and fiber cells. DNA degradation observed in agarose gels shows that DNase II-activating medium cleaves the DNA from fiber cells more efficiently than DNase I-activating buffer. In addition, DNase II antibody is able to prevent this degradation. These results suggest a specific involvement of DNase II in nuclear degradation during lens cell differentiation.Apoptosis or programmed cell death occurs in many physiological and pathological situations where selection of cells is required (1-4). In 1980, a landmark study (5) revealed that glucocorticoids induced extensive DNA degradation in rat thymocytes in vitro at the onset of cell death. DNA cleavage occurred in a very specific pattern producing fragments of DNA that were multiples of 180 -200 base pairs. This indicated that the chromatin was cleaved at the linker DNA between nucleosomic cores. The characteristic ladder was first shown by Hewish and Burgoyne (6) To date, three different endonucleases have been involved in DNA fragmentation leading to nucleosomal appearance. Some authors, such as Peitsch et al. (8), claimed that the well characterized pancreatic deoxyribonuclease (DNase I) was constitutively expressed in cells of tissues potentially primed for apoptosis. This 30-kDa nuclease, active at neutral pH, could be responsible for DNA cleavage into oligonucleosomes during cell death. On the other hand, Barry and Eastman (9), studying apoptosis in Chinese hamster ovary cells, were unable to detect a Ca 2ϩ -Mg 2ϩ -dependent endonuclease. Instead, they identified another endonuclease, which was cation-independent, with optimal activity at pH 5. This enzyme was proposed to be DNase II (9, 10). Finally, Hughes and Cidlowski (11) showed a lower molecular weight nuclease, an 18-kDa peptide (termed NUC 18), which was activated by Ca 2ϩ and Mg 2ϩ and related to cyclophilin (12). This peptide appears as a novel enzyme whose activity correlates with apoptosis in thymocy...

The most widely recognized biochemical change associated with the majority of apoptotic systems is the degradation of genomic DNA. Among the enzymes that may participate in this cleavage, the acidic cationindependent DNase II is a likely candidate since it is activated in many apoptotic cells. To better understand its role, we purified and sequenced a DNase II extracted from porcine spleen. Protein sequencing of random peptides demonstrated that this enzyme is derived from a ubiquitous serpin, the leukocyte elastase inhibitor (LEI), by an acidic-dependent posttranslational modification or by digestion with elastase. We call this novel enzyme L-DNase II. In vitro experiments with purified recombinant LEI show that the native form has no effect on purified nuclei whereas its posttranslationally activated form induces pycnosis and DNA degradation. Antibodies directed against L-DNase II showed, in different cell lines, an increased expression and a nuclear translocation of this enzyme during apoptosis. Since the appearance of the endonuclease activity results in a loss of the anti-protease properties of LEI, the transformation from LEI to L-DNase II may act as a switch of protease and nuclease pathways, each of which is activated during apoptosis.Apoptosis, or programmed cell death, is a mechanism of cell clearance in many physiological processes such as embryogenesis, metamorphosis, and tumor regression (2). Although the signals inducing apoptosis are very different, nuclear condensation, membrane blebbing, and formation of apoptotic bodies are morphological features common to all apoptotic cells. By far the most widely recognized biochemical change is the degradation of genomic DNA. The identity of the enzymes responsible for this cleavage is the subject of considerable debate. Several endonucleases have been proposed to be responsible for DNA fragmentation. Ca 2ϩ -plus Mg 2ϩ -dependent endonucleases in thymocytes, such as NUC 18/cyclophylin A (16), DNase I (21), DNase ␥ (29), and a new 97-kDa DNase (18), are examples. Mg 2ϩ -dependent, Ca 2ϩ -independent endonucleases have been implicated in human myeloid cell line apoptosis (7,8). Barry and Eastman (1) implicated DNase II, a cation-independent acidic endonuclease, as the enzyme that degrades DNA in apoptosis associated with intracellular acidification. We have shown in our laboratory the involvement of DNase II in nuclear degradation in terminally differentiating lens fiber cells (32).To date, our knowledge of the molecular structure of DNase II is very limited. The enzymatic properties of DNase II from different tissues and animals were found to be very similar, but its physical and chemical properties showed high variability. For instance, the molecular mass of mammalian DNase II ranges between 26 and 45 kDa. The reasons for this variability remain unknown (15).To better understand the biology of DNase II, the knowledge of its protein sequence seemed to be a mandatory step. In this study, we showed that this ubiquitous L-DNase II arises from leukocyte elastase...