The mouse retinal pigment epithelium mounts an innate immune defense response following retinal detachment.

The retinal pigment epithelium (RPE) maintains photoreceptor viability and function, completes the visual cycle, and forms the outer blood-retinal barrier (oBRB). Loss of RPE function gives rise to several monogenic retinal dystrophies and contributes to age-related macular degeneration. Retinal detachment (RD) causes separation of the neurosensory retina from the underlying RPE, disrupting the functional and metabolic relationships between these layers. Although the retinal response to RD is highly studied, little is known about how the RPE responds to loss of this interaction. RNA sequencing (RNA-Seq) was used to compare normal and detached RPE in the C57BL6/J mouse. The naïve mouse RPE transcriptome was compared to previously published RPE signature gene lists and from the union of these 14 genes (Bmp4, Crim1, Degs1, Gja1, Itgav, Mfap3l, Pdpn, Ptgds, Rbp1, Rnf13, Rpe65, Slc4a2, Sulf1 and Ttr) representing a core signature gene set applicable across rodent and human RPE was derived. Gene ontology enrichment analysis (GOEA) of the mouse RPE transcriptome identified expected RPE features and functions, such as pigmentation, phagocytosis, lysosomal and proteasomal degradation of proteins, and barrier function. Differentially expressed genes (DEG) at 1 and 7 days post retinal detachment (dprd) were defined as mRNA with a significant (padj≤0.05) fold change (FC) of 0.67 ≥ FC ≥ 1.5 in detached versus naïve RPE. The RPE transcriptome exhibited dramatic changes at 1 dprd, with 2297 DEG identified. The KEGG pathways and biological process GO groups related to innate immune responses were significantly enriched. Lipocalin 2 (Lcn2) and several chemokines were upregulated, while numerous genes related to RPE functions, such as pigment synthesis, visual cycle, phagocytosis, and tight junctions were downregulated at 1 dprd. The response was largely transient, with only 18 significant DEG identified at 7 dprd, including upregulation of complement gene C4b. Validation studies confirmed RNA-Seq results. Thus, the RPE quickly downregulates cell-specific functions and mounts an innate immune defense response following RD. Our data demonstrate that the RPE contributes to the inflammatory response to RD and may play a role in attraction of immune cells to the subretinal space.

Delineating effects of angiopoietin-2 inhibition on vascular permeability and inflammation in models of retinal neovascularization and ischemia/reperfusion.

Introduction: Clinical trials demonstrated that co-targeting angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF-A) with faricimab controls anatomic outcomes and maintains vision improvements, with strong durability, through 2 years in patients with neovascular age-related macular degeneration and diabetic macular edema. The mechanism(s) underlying these findings is incompletely understood and the specific role that Ang-2 inhibition plays requires further investigation. Methods: We examined the effects of single and dual Ang-2/VEGF-A inhibition in diseased vasculatures of JR5558 mice with spontaneous choroidal neovascularization (CNV) and in mice with retinal ischemia/reperfusion (I/R) injuries. Results: In JR5558 mice, Ang-2, VEGF-A, and dual Ang-2/VEGF-A inhibition reduced CNV area after 1 week; only dual Ang-2/VEGF-A inhibition decreased neovascular leakage. Only Ang-2 and dual Ang-2/VEGF-A inhibition maintained reductions after 5 weeks. Dual Ang-2/VEGF-A inhibition reduced macrophage/microglia accumulation around lesions after 1 week. Both Ang-2 and dual Ang-2/VEGF-A inhibition reduced macrophage/microglia accumulation around lesions after 5 weeks. In the retinal I/R injury model, dual Ang-2/VEGF-A inhibition was statistically significantly more effective than Ang-2 or VEGF-A inhibition alone in preventing retinal vascular leakage and neurodegeneration. Discussion: These data highlight the role of Ang-2 in dual Ang-2/VEGF-A inhibition and indicate that dual inhibition has complementary anti-inflammatory and neuroprotective effects, suggesting a mechanism for the durability and efficacy of faricimab in clinical trials.

Hypoxia-Inducible Factor-1α in Rods Is Neuroprotective Following Retinal Detachment.

Purpose: Following retinal detachment (RD) photoreceptors (PRs) sustain hypoxic stress and eventually die. Hypoxia-inducible factor-1α (HIF-1α) plays a central role in cellular adaptation to hypoxia. The purpose of this study is to determine the necessity of HIF-1α on PR cell survival after RD. Methods: Experimental RD was created in mice by injection of hyaluronic acid (1%) into the subretinal space. Mice with conditional HIF-1α knockout in rods (denoted as HIF-1αΔrod) were used. HIF-1α expression in retinas was measured real-time polymerase chain reaction (RT-PCR) and Western blotting. PR cell death after RD was evaluated using TUNEL assay. Optical coherence tomography (OCT) and histology were used to evaluate retinal layer thicknesses and PR cell densities. A hypoxia signaling pathway PCR array was used to examine the expression of HIF-1α target genes after RD. Results: HIF-1α protein levels were significantly increased after RD, and depletion of HIF-1α in rods blunted this increase. A compensatory increase of HIF-2α protein was observed in HIF-1αΔrod mice. Conditional knockout (cKO) of HIF-1α in rods did not lead to any morphologic change in attached retinas but resulted in significantly increased PR cell loss after RD. HIF-1α cKO in rods altered the responses to retinal detachment for 25 out of 83 HIF-1α target genes that were highly enriched for genes involved in glycolysis. Conclusions: Rod-derived HIF-1α plays a key role in the PR response to RD, mediating the transcriptional activity of a battery of genes to promote PR cell survival.

mTORC1 regulates high levels of protein synthesis in retinal ganglion cells of adult mice.

Mechanistic target of rapamycin (mTOR) and mTOR complex 1 (mTORC1), linchpins of the nutrient sensing and protein synthesis pathways, are present at relatively high levels in the ganglion cell layer (GCL) and retinal ganglion cells (RGCs) of rodent and human retinas. However, the role of mTORCs in the control of protein synthesis in RGC is unknown. Here, we applied the SUrface SEnsing of Translation (SUnSET) method of nascent protein labeling to localize and quantify protein synthesis in the retinas of adult mice. We also used intravitreal injection of an adeno-associated virus 2 vector encoding Cre recombinase in the eyes of mtor- or rptor-floxed mice to conditionally knockout either both mTORCs or only mTORC1, respectively, in cells within the GCL. A novel vector encoding an inactive Cre mutant (CreΔC) served as control. We found that retinal protein synthesis was highest in the GCL, particularly in RGC. Negation of both complexes or only mTORC1 significantly reduced protein synthesis in RGC. In addition, loss of mTORC1 function caused a significant reduction in the pan-RGC marker, RNA-binding protein with multiple splicing, with little decrease of the total number of cells in the RGC layer, even at 25 weeks after adeno-associated virus-Cre injection. These findings reveal that mTORC1 signaling is necessary for maintaining the high rate of protein synthesis in RGCs of adult rodents, but it may not be essential to maintain RGC viability. These findings may also be relevant to understanding the pathophysiology of RGC disorders, including glaucoma, diabetic retinopathy, and optic neuropathies.

Conditional Knock out of High-Mobility Group Box 1 (HMGB1) in Rods Reduces Autophagy Activation after Retinal Detachment.

After retinal detachment (RD), the induction of autophagy protects photoreceptors (PR) from apoptotic cell death. The cytoplasmic high-mobility group box 1 (HMGB1) promotes autophagy. We previously demonstrated that the deletion of HMGB1 from rod PRs results in a more rapid death of these cells after RD. In this work, we tested the hypothesis that the lack of HMGB1 accelerates PR death after RD due to the reduced activation of protective autophagy in the retina after RD. The injection of 1% hyaluronic acid into the subretinal space was used to create acute RD in mice with a rhodopsin-Cre-mediated conditional knockout (cKO) of HMGB1 in rods (HMGB1Δrod) and littermate controls. RD sharply increased the number of apoptotic cells in the outer nuclear layer (ONL), and this number was further increased in HMGB1Δrod mouse retinas. The activation of autophagy after RD was reduced in the HMGB1Δrod mouse retinas compared to controls, as evidenced by diminished levels of autophagy regulatory proteins LC3-II, Beclin1, ATG5/12, and phospho-ATG16L1. The cKO of HMGB1 in rods increased the expression of Fas and the Bax/Bcl-2 ratio in detached retinas, promoting apoptotic cell death. In conclusion, endogenous HMGB1 facilitates autophagy activation in PR cells following RD to promote PR cell survival and reduce programmed apoptotic cell death.

Diminished retinal complex lipid synthesis and impaired fatty acid ß-oxidation associated with human diabetic retinopathy.

BACKGROUNDThis study systematically investigated circulating and retinal tissue lipid determinants of human diabetic retinopathy (DR) to identify underlying lipid alterations associated with severity of DR.METHODSRetinal tissues were retrieved from postmortem human eyes, including 19 individuals without diabetes, 20 with diabetes but without DR, and 20 with diabetes and DR, for lipidomic study. In a parallel study, serum samples from 28 American Indians with type 2 diabetes from the Gila River Indian Community, including 12 without DR, 7 with mild nonproliferative DR (NPDR), and 9 with moderate NPDR, were selected. A mass-spectrometry-based lipidomic platform was used to measure serum and tissue lipids.RESULTSIn the postmortem retinas, we found a graded decrease of long-chain acylcarnitines and longer-chain fatty acid ester of hydroxyl fatty acids, diacylglycerols, triacylglycerols, phosphatidylcholines, and ceramide(NS) in central retina from individuals with no diabetes to those with diabetes with DR. The American Indians' sera also exhibited a graded decrease in circulating long-chain acylcarnitines and a graded increase in the intermediate-length saturated and monounsaturated triacylglycerols from no DR to moderate NPDR.CONCLUSIONThese findings suggest diminished synthesis of complex lipids and impaired mitochondrial ß-oxidation of fatty acids in retinal DR, with parallel changes in circulating lipids.TRIAL REGISTRATIONClinicalTrials.gov NCT00340678.FUNDINGThis work was supported by NIH grants R24 DK082841, K08DK106523, R03DK121941, P30DK089503, P30DK081943, P30DK020572, P30 EY007003; The Thomas Beatson Foundation; and JDRF Center for Excellence (5-COE-2019-861-S-B).

Inflammatory resolution and vascular barrier restoration after retinal ischemia reperfusion injury.

BACKGROUND: Several retinal pathologies exhibit both inflammation and breakdown of the inner blood-retinal barrier (iBRB) resulting in vascular permeability, suggesting that treatments that trigger resolution of inflammation may also promote iBRB restoration. METHODS: Using the mouse retinal ischemia-reperfusion (IR) injury model, we followed the time course of neurodegeneration, inflammation, and iBRB disruption and repair to examine the relationship between resolution of inflammation and iBRB restoration and to determine if minocycline, a tetracycline derivative shown to reverse microglial activation, can hasten these processes. RESULTS: A 90-min ischemic insult followed by reperfusion in the retina induced cell apoptosis and inner retina thinning that progressed for approximately 2 weeks. IR increased vascular permeability within hours, which resolved between 3 and 4 weeks after injury. Increased vascular permeability coincided with alteration and loss of endothelial cell tight junction (TJ) protein content and disorganization of TJ protein complexes. Shunting of blood flow away from leaky vessels and dropout of leaky capillaries were eliminated as possible mechanisms for restoring the iBRB. Repletion of TJ protein contents occurred within 2 days after injury, long before restoration of the iBRB. In contrast, the eventual re-organization of TJ complexes at the cell border coincided with restoration of the barrier. A robust inflammatory response was evident a 1 day after IR and progressed to resolution over the 4-week time course. The inflammatory response included a rapid and transient infiltration of granulocytes and Ly6C+ classical inflammatory monocytes, a slow accumulation of Ly6Cneg monocyte/macrophages, and activation, proliferation, and mobilization of resident microglia. Extravasation of the majority of CD45+ leukocytes occurred from the superficial plexus. The presence of monocyte/macrophages and increased numbers of microglia were sustained until the iBRB was eventually restored. Intervention with minocycline to reverse microglial activation at 1 week after injury promoted early restoration of the iBRB coinciding with decreased expression of mRNAs for the microglial M1 markers TNF-α, IL-1ß, and Ptgs2 (Cox-2) and increased expression of secreted serine protease inhibitor Serpina3n mRNA. CONCLUSIONS: These results suggest that iBRB restoration occurs as TJ complexes are reorganized and that resolution of inflammation and restoration of the iBRB following retinal IR injury are functionally linked.

Differential effects of minocycline on microvascular complications in murine models of type 1 and type 2 diabetes.

Diabetes is a global healthcare problem associated with enormous healthcare and personal costs. Despite glucose lowering agents that control glycaemia, both type 1 (T1D) and type (T2D) diabetes patients often develop microvascular complications that increase morbidity and mortality. Current interventions rely on careful glycemic control and healthy lifestyle choices, but these are ineffective at reversing or completely preventing the major microvascular complications, diabetic peripheral neuropathy (DPN), diabetic retinopathy (DR), and diabetic kidney disease (DKD). Minocycline, a tetracycline antibiotic with anti-inflammatory and anti-apoptotic properties, has been proposed as a protective agent in diabetes. However, there are no reported studies evaluating the therapeutic efficacy of minocycline in T1D and T2D models for all microvascular complications (DPN, DR, and DKD). Therefore, we performed metabolic profiling in streptozotocin-induced T1D and db/db T2D models and compared the efficacy of minocycline in preventing complications to that of insulin and pioglitazone in both models. Minocycline partially ameliorated DR and DKD in T1D and T2D animals, but was less effective than insulin or pioglitazone, and failed to improve DPN in either model. These results suggest that minocycline is unlikely to improve outcomes beyond that achieved with current available therapies in patients with T1D or T2D associated microvascular complications.

Differential Effects of Empagliflozin on Microvascular Complications in Murine Models of Type 1 and Type 2 Diabetes.

Microvascular complications account for the significant morbidity associated with diabetes. Despite tight glycemic control, disease risk remains especially in type 2 diabetes (T2D) patients and no therapy fully prevents nerve, retinal, or renal damage in type 1 diabetes (T1D) or T2D. Therefore, new antidiabetic drug classes are being evaluated for the treatment of microvascular complications. We investigated the effect of empagliflozin (EMPA), an inhibitor of the sodium/glucose cotransporter 2 (SGLT2), on diabetic neuropathy (DPN), retinopathy (DR), and kidney disease (DKD) in streptozotocin-induced T1D and db/db T2D mouse models. EMPA lowered blood glycemia in T1D and T2D models. However, it did not ameliorate any microvascular complications in the T2D model, which was unexpected, given the protective effect of SGLT2 inhibitors on DKD progression in T2D subjects. Although EMPA did not improve DKD in the T1D model, it had a potential modest effect on DR measures and favorably impacted DPN as well as systemic oxidative stress. These results support the concept that glucose-centric treatments are more effective for DPN in T1D versus T2D. This is the first study that provides an evaluation of EMPA treatment on all microvascular complications in a side-by-side comparison in T1D and T2D models.

mTORC1 and mTORC2 expression in inner retinal neurons and glial cells.

The retina is one of the most metabolically active tissues, yet the processes that control retinal metabolism remains poorly understood. The mTOR complex (mTORC) that drives protein and lipid biogenesis and autophagy has been studied extensively in regards to retinal development and responses to optic nerve injury but the processes that regulate homeostasis in the adult retina have not been determined. We previously demonstrated that normal adult retina has high rates of protein synthesis compared to skeletal muscle, associated with high levels of mechanistic target of rapamycin (mTOR), a kinase that forms multi-subunit complexes that sense and integrate diverse environmental cues to control cell and tissue physiology. This study was undertaken to: 1) quantify expression of mTOR complex 1 (mTORC1)- and mTORC2-specific partner proteins in normal adult rat retina, brain and liver; and 2) to localize these components in normal human, rat, and mouse retinas. Immunoblotting and immunoprecipitation studies revealed greater expression of raptor (exclusive to mTORC1) and rictor (exclusive for mTORC2) in normal rat retina relative to liver or brain, as well as the activating mTORC components, pSIN1 and pPRAS40. By contrast, liver exhibits greater amounts of the mTORC inhibitor, DEPTOR. Immunolocalization studies for all three species showed that mTOR, raptor, and rictor, as well as most other known components of mTORC1 and mTORC2, were primarily localized in the inner retina with mTORC1 primarily in retinal ganglion cells (RGCs) and mTORC2 primarily in glial cells. In addition, phosphorylated ribosomal protein S6, a direct target of the mTORC1 substrate ribosomal protein S6 kinase beta-1 (S6K1), was readily detectable in RGCs, indicating active mTORC1 signaling, and was preserved in human donor eyes. Collectively, this study demonstrates that the inner retina expresses high levels of mTORC1 and mTORC2 and possesses active mTORC1 signaling that may provide cell- and tissue-specific regulation of homeostatic activity. These findings help to define the physiology of the inner retina, which is key for understanding the pathophysiology of optic neuropathies, glaucoma and diabetic retinopathy.

All-trans-Retinaldehyde Contributes to Retinal Vascular Permeability in Ischemia Reperfusion.

Purpose: Extracellular accumulation of all-trans-retinaldehyde (atRAL), a highly reactive visual cycle intermediate, is toxic to cells of the outer retina and contributes to retinal and macular degenerations. However, the contribution of atRAL to retinal capillary function has not been studied. We hypothesized that atRAL released from the outer retina can contribute to retinal vascular permeability. We, therefore, tested the contribution of atRAL to retinal ischemia-reperfusion (IR)-induced vascular permeability. Methods: IR was induced in mice by transient increase in intraocular pressure followed by natural reperfusion. The visual cycle was ablated in the Lrat-/- mice, reduced by dark adaptation or the use of the RPE65 inhibitor and atRAL scavenger emixustat. Accumulation of FITC-BSA was used to assess vascular permeability and DNA fragmentation quantified cell death after IR. Primary bovine retinal endothelial cell (BREC) culture was used to measure the direct effects of atRAL on endothelial permeability and cell death. Results: Inhibition of the visual cycle by Lrat-/-, dark adaptation, or with emixustat, all reduced approximately half of IR induced vascular permeability at 48 hours. An increase in BREC permeability with atRAL coincided with lactate dehydrogenase (LDH) release, a measure of cell death. Both permeability and toxicity were blocked by emixustat. Conclusions: Outer retinal pathology may contribute to vascular permeability by release of atRAL, which can act directly on vascular endothelial cells to alter barrier properties and induce cell death. These studies may have implications for a variety of blinding eye diseases that include outer retinal damage and retinal vascular permeability.

Loss of High-Mobility Group Box 1 (HMGB1) Protein in Rods Accelerates Rod Photoreceptor Degeneration After Retinal Detachment.

Purpose: Retinal detachment (RD) disrupts the nutritional support and oxygen delivery to photoreceptors (PRs), ultimately causing cell death. High-mobility group box 1 (HMGB1) can serve as an extracellular alarmin when released from stressed cells. PRs release HMGB1 after RD. The purpose of this study was to investigate the relationship between HMGB1 and PR survival after RD. Methods: Acute RD was created by injection of hyaluronic acid (1%) into the subretinal space in C57BL/6 mice and mice with a rhodopsin-Cre-mediated conditional knockout (cKO) of HMGB1 in rods (HMGB1ΔRod). Immunofluorescence (IF) in retinal sections was used to localize HMGB1, rhodopsin, and Iba-1 proteins. Optical coherence tomography and electroretinography were used to quantify retinal thickness and function, respectively. The morphology of the retina was assessed by hematoxylin and eosin. Results: HMGB1 protein was localized to the nuclei of all retinal neurons, including PRs, with cones staining more intensely than rods. HMGB1 protein was also found in the inner and outer segments of cones but not rods. Creation of RD caused a dramatic increase of HMGB1 protein IF in rods. cKO of HMGB1 in rods did not affect retinal structure or function. However, after RD, loss of rods and reduction in the thickness of the outer nuclear layer were significantly increased in the HMGB1ΔRod retinas as compared to the control. Interestingly, depletion of HMGB1 in rods did not affect the activation and mobilization of microglia/macrophages normally seen after RD. Conclusions: Increased HMGB1 expression in stressed rods may represent an intrinsic mechanism regulating their survival after RD.

New insights into the mechanisms of diabetic complications: role of lipids and lipid metabolism.

Diabetes adversely affects multiple organs, including the kidney, eye and nerve, leading to diabetic kidney disease, diabetic retinopathy and diabetic neuropathy, respectively. In both type 1 and type 2 diabetes, tissue damage is organ specific and is secondary to a combination of multiple metabolic insults. Hyperglycaemia, dyslipidaemia and hypertension combine with the duration and type of diabetes to define the distinct pathophysiology underlying diabetic kidney disease, diabetic retinopathy and diabetic neuropathy. Only recently have the commonalities and differences in the metabolic basis of these tissue-specific complications, particularly those involving local and systemic lipids, been systematically examined. This review focuses on recent progress made using preclinical models and human-based approaches towards understanding how bioenergetics and metabolomic profiles contribute to diabetic kidney disease, diabetic retinopathy and diabetic neuropathy. This new understanding of the biology of complication-prone tissues highlights the need for organ-specific interventions in the treatment of diabetic complications.

Mitochondrial uncoupling has no effect on microvascular complications in type 2 diabetes.

Diabetic peripheral neuropathy (DPN), diabetic kidney disease (DKD), and diabetic retinopathy (DR) contribute to significant morbidity and mortality in diabetes patients. The incidence of these complications is increasing with the diabetes epidemic, and current therapies minimally impact their pathogenesis in type 2 diabetes (T2D). Improved mechanistic understanding of each of the diabetic complications is needed in order to develop disease-modifying treatments for patients. We recently identified fundamental differences in mitochondrial responses of peripheral nerve, kidney, and retinal tissues to T2D in BKS-db/db mice. However, whether these mitochondrial adaptations are the cause or consequence of tissue dysfunction remains unclear. In the current study BKS-db/db mice were treated with the mitochondrial uncoupler, niclosamide ethanolamine (NEN), to determine the effects of mitochondrial uncoupling therapy on T2D, and the pathogenesis of DPN, DKD and DR. Here we report that NEN treatment from 6-24 wk of age had little effect on the development of T2D and diabetic complications. Our data suggest that globally targeting mitochondria with an uncoupling agent is unlikely to provide therapeutic benefit for DPN, DKD, or DR in T2D. These data also highlight the need for further insights into the role of tissue-specific metabolic reprogramming in the pathogenesis of diabetic complications.

Inhibition of Atypical Protein Kinase C Reduces Inflammation-Induced Retinal Vascular Permeability.

Changes in permeability of retinal blood vessels contribute to macular edema and the pathophysiology of numerous ocular diseases, including diabetic retinopathy, retinal vein occlusions, and macular degeneration. Vascular endothelial growth factor (VEGF) induces retinal permeability and macular thickening in these diseases. However, inflammatory agents, such as tumor necrosis factor-α (TNF-α), also may drive vascular permeability, specifically in patients unresponsive to anti-VEGF therapy. Recent evidence suggests VEGF and TNF-α induce permeability through distinct mechanisms; however, both require the activation of atypical protein kinase C (aPKC). We provide evidence, using genetic mouse models and therapeutic intervention with small molecules, that inhibition of aPKC prevented or reduced vascular permeability in animal models of retinal inflammation. Expression of a kinase-dead aPKC transgene, driven by a vascular and hematopoietic restricted promoter, reduced retinal vascular permeability in an ischemia-reperfusion model of retinal injury. This effect was recapitulated with a small-molecule inhibitor of aPKC. Expression of the kinase-dead aPKC transgene dramatically reduced the expression of inflammatory factors and blocked the attraction of inflammatory monocytes and granulocytes after ischemic injury. Coinjection of VEGF with TNF-α was sufficient to induce permeability, edema, and retinal inflammation, and treatment with an aPKC inhibitor prevented VEGF/TNF-α-induced permeability. These data suggest that aPKC contributes to inflammation-driven retinal vascular pathology and may be an attractive target for therapeutic intervention.

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.

Developmental and light regulation of tumor suppressor protein PP2A in the retina.

Protein phosphatases are a group of universal enzymes that are responsible for the dephosphorylation of various proteins and enzymes in cells. Cellular signal transduction events are largely governed by the phosphorylation of key proteins. The length of cellular response depends on the activation of protein phosphatase that dephosphorylates the phosphate groups to halt a biological response, and fine-tune the defined cellular outcome. Dysregulation of these phosphatase(s) results in various disease phenotypes. The retina is a post-mitotic tissue, and oncogenic tyrosine and serine/ threonine kinase activities are important for retinal neuron survival. Aberrant activation of protein phosphatase(s) may have a negative effect on retinal neurons. In the current study, we characterized tumor suppressor protein phosphatase 2 (PP2A), a major serine/ threonine kinase with a broad substrate specificity. Our data suggest that PP2A is developmentally regulated in the retina, localized predominantly in the inner retina, and expressed in photoreceptor inner segments. Our findings indicate that PKCα and mTOR may serve as PP2A substrates. We found that light regulates PP2A activity. Our studies also suggest that rhodopsin regulates PP2A and its substrate(s) dephosphorylation. PP2A substrate phosphorylation is increased in mice lacking the A-subunit of PP2A. However, there is no accompanying effect on retina structure and function. Together, our findings suggest that controlling the activity of PP2A in the retina may be neuroprotective.

Shared and distinct lipid-lipid interactions in plasma and affected tissues in a diabetic mouse model.

Lipids are ubiquitous metabolites with diverse functions; abnormalities in lipid metabolism appear to be related to complications from multiple diseases, including type 2 diabetes. Through technological advances, the entire lipidome has been characterized and researchers now need computational approaches to better understand lipid network perturbations in different diseases. Using a mouse model of type 2 diabetes with microvascular complications, we examined lipid levels in plasma and in renal, neural, and retinal tissues to identify shared and distinct lipid abnormalities. We used correlation analysis to construct interaction networks in each tissue, to associate changes in lipids with changes in enzymes of lipid metabolism, and to identify overlap of coregulated lipid subclasses between plasma and each tissue to define subclasses of plasma lipids to use as surrogates of tissue lipid metabolism. Lipid metabolism alterations were mostly tissue specific in the kidney, nerve, and retina; no lipid changes correlated between the plasma and all three tissue types. However, alterations in diacylglycerol and in lipids containing arachidonic acid, an inflammatory mediator, were shared among the tissue types, and the highly saturated cholesterol esters were similarly coregulated between plasma and each tissue type in the diabetic mouse. Our results identified several patterns of altered lipid metabolism that may help to identify pathogenic alterations in different tissues and could be used as biomarkers in future research into diabetic microvascular tissue damage.