Constantine S. Mitsiades

Suppression of Fas-FasL-induced endothelial cell apoptosis prevents diabetic blood–retinal barrier breakdown in a model of streptozotocin-induced diabetes

Diabetic macular edema, resulting from increased microvascular permeability, is the most prevalent cause of vision loss in diabetes. The mechanisms underlying this complication remain poorly understood. In the current study, diabetic vascular permeability (blood‐retinal barrier breakdown) is demonstrated to result from a leukocyte‐mediated Fas‐FasL‐dependent apoptosis of the retinal vasculature. Following the onset of streptozotocin‐induced diabetes, FasL expression was increased in rat neutrophils (P<0.005) and was accompanied by a simultaneous increase in Fas expression in the retinal vasculature. Static adhesion assays demonstrated that neutrophils from diabetic, but not control, rats induced endothelial cell apoptosis in vitro (P<0.005). The latter was inhibited via an antibody‐based FasL blockade (P<0.005). In vivo, the inhibition of FasL potently reduced retinal vascular endothelial cell injury, apoptosis, and blood‐retinal barrier breakdown (P<0.0001) but did not diminish leukocyte adhesion to the diabetic retinal vasculature. Taken together, these data are the first to identify leukocyte‐mediated FasFasL‐dependent retinal endothelial cell apoptosis as a major cause of blood‐retinal barrier breakdown in early diabetes. These data imply that the targeting of the Fas‐FasL pathway may prove beneficial in the treatment of diabetic retinopathy.

Insulin-Like Growth Factor-I Plays a Pathogenetic Role in Diabetic Retinopathy

Diabetic retinopathy is a leading cause of blindness in the Western world. Aberrant intercellular adhesion molecule-1 expression and leukocyte adhesion have been implicated in its pathogenesis, raising the possibility of an underlying chronic inflammatory mechanism. In the current study, the role of insulin-like growth factor (IGF)-I in these processes was investigated. We found that systemic inhibition of IGF-I signaling with a receptor-neutralizing antibody, or with inhibitors of PI-3 kinase (PI-3K), c-Jun kinase (JNK), or Akt, suppressed retinal Akt, JNK, HIF-1alpha, nuclear factor (NF)-kappaB, and AP-1 activity, vascular endothelial growth factor (VEGF) expression, as well as intercellular adhesion molecule-1 levels, leukostasis, and blood-retinal barrier breakdown, in a relevant animal model. Intravitreous administration of IGF-I increased retinal Akt, JNK, HIF-1alpha, NF-kappaB, and AP-1 activity, and VEGF levels. IGF-I stimulated VEGF promoter activity in vitro, mainly via HIF-1alpha, and secondarily via NF-kappaB and AP-1. In conclusion, IGF-I participates in the pathophysiology of diabetic retinopathy by inducing retinal VEGF expression via PI-3K/Akt, HIF-1alpha, NF-kappaB, and secondarily, JNK/AP-1 activation. Taken together, these in vitro and in vivo signaling studies thus identify potential targets for pharmacological intervention to preserve vision in patients with diabetes.

Constitutive Nuclear Factor-κB Activity Is Crucial for Human Retinoblastoma Cell Viability

Retinoblastoma (Rb) is the most common intraocular malignancy of childhood. Although systemic and intrathecal chemotherapy with local and cranial radiotherapy have improved overall survival, the prognosis for patients with central nervous system involvement is still poor. We investigated the role of the transcription factor nuclear factor (NF)-kappaB, which promotes cell survival in several other models, in the pathophysiology of Rb. The human Rb cell lines Y79 and WERI-Rb1 were treated with the cell permeable peptide SN50, that specifically inhibits the transcriptional activity of NF-kappaB by blocking its translocation into the nucleus. We found that NF-kappaB inhibition up-regulated Bax; down-regulated the anti-apoptotic proteins Bcl-2, A1, and cIAP-2; and induced loss of the mitochondrial transmembrane potential and caspase-independent, calpain-dependent apoptosis in Rb cells. Inhibition of the p38 kinase sensitized cells to SN50-induced cell death, whereas insulin-like growth factor-1 activated NF-kappaB and attenuated the proapoptotic effect of SN50. Finally, NF-kappaB inhibition sensitized Rb cells to doxorubicin. In conclusion, inhibition of NF-kappaB activity in Rb cells leads to loss of mitochondrial transmembrane potential and caspase-independent, calpain-dependent apoptosis. Therapeutic strategies targeting NF-kappaB could be beneficial in the clinical management of Rb, either alone or in combination with conventional chemotherapy.