Sumathi Shanmugam

Effects of ischemic preconditioning and bevacizumab on apoptosis and vascular permeability following retinal ischemia-reperfusion injury

PURPOSE Using transient ischemia followed by reperfusion (IR) to model ischemic retinal disease, this study compares the effects of ischemic preconditioning (IPC) and therapies targeting vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF)-α on retinal apoptosis, vascular permeability, and mRNA expression. METHODS Rats were subjected to 30 or 45 minutes of retinal ischemia followed by reperfusion for up to 48 hours. Neurodegeneration was quantified by caspase-3 (DEVDase) activity and by measuring nucleosomal DNA content (cell death ELISA). Vascular leakage was quantified by the Evans Blue dye method. A set of IR-responsive mRNAs was identified by whole-genome microarray and confirmed by RT-PCR analyses. VEGF protein was measured by Western blot analysis. IPC was accomplished with 10 minutes of ischemia 24 hours before IR. VEGF and TNFα signaling was inhibited by intravitreal injection of bevacizumab or etanercept, respectively. RESULTS IR caused significant retinal cell apoptosis and vascular permeability after 4 and 48 hours. Whereas IR decreased VegfA mRNA, VEGF protein was significantly increased. IPC effectively inhibited neurodegeneration, bevacizumab effectively inhibited vascular permeability, and etanercept failed to affect either outcome. IPC significantly altered the IR responses of 15 of 33 IR-responsive mRNAs, whereas bevacizumab had no significant effect on these mRNAs. CONCLUSIONS IR provides an acute model of ischemic retinopathy that includes neurodegeneration and VEGF-dependent vascular permeability and is amenable to rapid drug therapy testing. The distinct effects of IPC and bevacizumab demonstrate that the apoptotic and vascular responses to IR may be separated and that therapeutics targeting each pathologic endpoint may be warranted in treating ischemic retinal diseases.

Ischemia–Reperfusion Injury Induces Occludin Phosphorylation/Ubiquitination and Retinal Vascular Permeability in a VEGFR-2-Dependent Manner

Retinal ischemia–reperfusion (IR) induces neurodegenaration as well as blood–retinal barrier (BRB) breakdown causing vascular permeability. Whereas the neuronal death has been extensively studied, the molecular mechanisms related to BRB breakdown in IR injury remain poorly understood. In this study, we investigated the early changes in tight junctional (TJ) proteins in response to IR injury. Ischemia–reperfusion injury was induced in male rat retinas by increasing the intraocular pressure for 45 minutes followed by natural reperfusion. The results demonstrate that IR injury induced occludin Ser490 phosphorylation and ubiquitination within 15 minutes of reperfusion with subsequent vascular permeability. Immunohistochemical analysis revealed a rapid increase in occludin Ser490 phosphorylation and loss of Zonula occludens-1 (ZO-1) protein, particularly in arterioles. Ischemia–reperfusion injury also rapidly induced the activation and phosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) at tyrosine 1175. Blocking vascular endothelial growth factor (VEGF) function by intravitreal injection of bevacizumab prevented VEGFR-2 activation, occludin phosphorylation, and vascular permeability. These studies suggest a novel mechanism of occludin Ser490 phosphorylation and ubiquitination downstream of VEGFR2 activation associated with early IR-induced vascular permeability.

Effect of IL-1β on Survival and Energy Metabolism of R28 and RGC-5 Retinal Neurons

PURPOSE Interleukin-(IL)1beta expression is increased in the retina during a variety of diseases involving the death of retinal neurons and contributes to neurodegenerative processes through an unknown mechanism. This study was conducted to examine the effects of IL-1beta on the metabolism and viability of RGC-5 and R28 retinal neuronal cells. METHODS Cellular reductive capacity was evaluated using WST-1 tetrazolium salt. Mitochondrial transmembrane potential was determined by JC-1 fluorescence. Cellular ATP levels were measured with a luciferase assay. Caspase-3/7 activation was detected with a DEVDase activity assay. Cell death and lysis was evaluated by measuring release of lactate dehydrogenase (LDH). Glycolysis was assessed by measuring glucose disappearance and lactate appearance in cell culture medium. Cellular respiration was followed polarographically. RESULTS IL-1beta treatment caused a pronounced decrease in cellular reductive potential. IL-1beta caused depletion of intracellular ATP, loss of mitochondrial transmembrane potential, caspase-3/7 activation, and LDH release. IL-1beta treatment increased rates of glucose utilization and lactate production. The cells were partially protected from IL-1beta toxicity by ample ambient glucose. However, glucose did not block the ability of IL-1beta to cause a decline in mitochondrial transmembrane potential or ATP depletion. IL-1beta decreased oxygen consumption of the R28 cells by nearly half, but did not lower cytochrome c oxidase activity. CONCLUSIONS The present results suggest that IL-1beta inhibits mitochondrial energy metabolism of these retinal neuronlike cells.

Vitreous Cytokine Expression and a Murine Model Suggest a Key Role of Microglia in the Inflammatory Response to Retinal Detachment

Purpose Retinal detachment (RD) separates the retina from the underlying retinal pigment epithelium, resulting in a gradual degeneration of photoreceptor (PR) cells. It is known that RD also results in an inflammatory response, but its contribution to PR degeneration is unknown. In this study we examine the inflammatory responses to RD in patient vitreous and validate a mouse experimental RD as a model of this phenomenon. Methods Multiplex bead arrays were used to examine cytokine levels in vitreous samples from 24 patients with macula-off rhegmatogenous retinal detachment (RRD) undergoing reattachment surgery and from 10 control patients undergoing vitrectomy for vitreous opacities or epiretinal membrane. Activation of the innate immune response was then examined in a mouse model of RD. Results Twenty-eight factors were significantly increased in vitreous from RD patients versus controls. Notable were the cytokines MCP-1 (CCL2), IP-10 (CXCL10), fractalkine (CX3CL1), GRO (CXCL1), MDC (CCL22), IL-6, and IL-8, which all exhibited relatively high concentrations and several-fold increases in the vitreous of RD patients. Concentrations of various analytes correlated with a range of clinical variables such as duration of detachment and visual acuity. Retinal detachment in the mouse resulted in cytokine mRNA expression changes consistent with human RD vitreous results, as well as microglial proliferation and migration toward the outer retina. Conclusions The findings suggest that an inflammatory response involving microglia is a component of the reaction to retinal detachment that may impact visual acuity after surgical repair and that mouse experimental RD can serve as a model to study this effect.

Erratum to: CX3CR1 deficiency accelerates the development of retinopathy in a rodent model of type 1 diabetes (Journal of Molecular Medicine, (2016), 94, 11, (1255-1265), 10.1007/s00109-016-1433-0)

The original version of this article unfortunately contains a mistake in Fig. 1a; the wrong figure panels were used. The correct Fig. 1a is shown in this paper.