Lost in Translation: Neurotrophins Biology and Function in the Neurovascular Unit.

The neurovascular unit (NVU) refers to the functional building unit of the brain and the retina, where neurons, glia, and microvasculature orchestrate to meet the demand of the retina's and brain's function. Neurotrophins (NTs) are structural families of secreted proteins and are known for exerting neurotrophic effects on neuronal differentiation, survival, neurite outgrowth, synaptic formation, and plasticity. NTs include several molecules, such as nerve growth factor, brain-derived neurotrophic factor, NT-3, NT-4, and their precursors. Furthermore, NTs are involved in signaling pathways such as inflammation, apoptosis, and angiogenesis in a nonneuronal cell type. Interestingly, NTs and the precursors can bind and activate the p75 neurotrophin receptor (p75NTR) at low and high affinity. Mature NTs bind their cognate tropomyosin/tyrosine-regulated kinase receptors, crucial for maintenance and neuronal development in the brain and retina axis. Activation of p75NTR results in neuronal apoptosis and cell death, while tropomysin receptor kinase upregulation contributes to differentiation and cell growth. Recent findings indicate that modulation of NTs and their receptors contribute to neurovascular dysfunction in the NVU. Several chronic metabolic and acute ischemic diseases affect the NVU, including diabetic and ischemic retinopathy for the retina, as well as stroke, acute encephalitis, and traumatic brain injury for the brain. This work aims to review the current evidence through published literature studying the impact of NTs and their receptors, including the p75NTR receptor, on the injured and healthy brain-retina axis.

Thioredoxin interacting protein regulates age-associated neuroinflammation.

Immune system hypersensitivity is believed to contribute to mental frailty in the elderly. Solid evidence indicates NOD-like receptor pyrin domain containing-3 (NLRP3)-inflammasome activation intimately connects aging-associated chronic inflammation (inflammaging) to senile cognitive decline. Thioredoxin interacting protein (TXNIP), an inducible protein involved in oxidative stress, is essential for NLRP3 inflammasome activity. This study aims to find whether TXNIP/NLRP3 inflammasome pathway is involved in senile dementia. According to our studies on sex-matched mice, TXNIP was significantly upregulated in aged animals, paralleled by the NLRP3-inflammasome over-activity leading to enhanced caspase-1 cleavage and IL-1ß maturation, in both sexes. This was closely associated with depletion of the anti-aging and cognition enhancing protein klotho, in aged males. Txnip knockout reversed age-related NLRP3-hyperactivity and enhanced thioredoxin (TRX) levels. Further, TXNIP inhibition along with verapamil replicated TXNIP/NLRP3-inflammasome downregulation in aged animals, with FOXO-1 and mTOR upregulation. These alterations concurred with substantial improvements in both cognitive and sensorimotor abilities. Together, these findings substantiate the pivotal role of TXNIP to drive inflammaging in parallel with klotho depletion and functional decline, and delineate thioredoxin system as a potential target to decelerate senile dementia.

Modulation of p75NTR on Mesenchymal Stem Cells Increases Their Vascular Protection in Retinal Ischemia-Reperfusion Mouse Model.

Mesenchymal stem cells (MSCs) are a promising therapy to improve vascular repair, yet their role in ischemic retinopathy is not fully understood. The aim of this study is to investigate the impact of modulating the neurotrophin receptor; p75NTR on the vascular protection of MSCs in an acute model of retinal ischemia/reperfusion (I/R). Wild type (WT) and p75NTR-/- mice were subjected to I/R injury by increasing intra-ocular pressure to 120 mmHg for 45 min, followed by perfusion. Murine GFP-labeled MSCs (100,000 cells/eye) were injected intravitreally 2 days post-I/R and vascular homing was assessed 1 week later. Acellular capillaries were counted using trypsin digest 10-days post-I/R. In vitro, MSC-p75NTR was modulated either genetically using siRNA or pharmacologically using the p75NTR modulator; LM11A-31, and conditioned media were co-cultured with human retinal endothelial cells (HREs) to examine the angiogenic response. Finally, visual function in mice undergoing retinal I/R and receiving LM11A-31 was assessed by visual-clue water-maze test. I/R significantly increased the number of acellular capillaries (3.2-Fold) in WT retinas, which was partially ameliorated in p75NTR-/- retinas. GFP-MSCs were successfully incorporated and engrafted into retinal vasculature 1 week post injection and normalized the number of acellular capillaries in p75NTR-/- retinas, yet ischemic WT retinas maintained a 2-Fold increase. Silencing p75NTR on GFP-MSCs coincided with a higher number of cells homing to the ischemic WT retinal vasculature and normalized the number of acellular capillaries when compared to ischemic WT retinas receiving scrambled-GFP-MSCs. In vitro, silencing p75NTR-MSCs enhanced their secretome, as evidenced by significant increases in SDF-1, VEGF and NGF release in MSCs conditioned medium; improved paracrine angiogenic response in HREs, where HREs showed enhanced migration (1.4-Fold) and tube formation (2-Fold) compared to controls. In parallel, modulating MSCs-p75NTR using LM11A-31 resulted in a similar improvement in MSCs secretome and the enhanced paracrine angiogenic potential of HREs. Further, intervention with LM11A-31 significantly mitigated the decline in visual acuity post retinal I/R injury. In conclusion, p75NTR modulation can potentiate the therapeutic potential of MSCs to harness vascular repair in ischemic retinopathy diseases.

Thioredoxin interacting protein, a key molecular switch between oxidative stress and sterile inflammation in cellular response.

Tissue and systemic inflammation have been the main culprit behind the cellular response to multiple insults and maintaining homeostasis. Obesity is an independent disease state that has been reported as a common risk factor for multiple metabolic and microvascular diseases including nonalcoholic fatty liver disease (NAFLD), retinopathy, critical limb ischemia, and impaired angiogenesis. Sterile inflammation driven by high-fat diet, increased formation of reactive oxygen species, alteration of intracellular calcium level and associated release of inflammatory mediators, are the main common underlying forces in the pathophysiology of NAFLD, ischemic retinopathy, stroke, and aging brain. This work aims to examine the contribution of the pro-oxidative and pro-inflammatory thioredoxin interacting protein (TXNIP) to the expression and activation of NLRP3-inflammasome resulting in initiation or exacerbation of sterile inflammation in these disease states. Finally, the potential for TXNIP as a therapeutic target and whether TXNIP expression can be modulated using natural antioxidants or repurposing other drugs will be discussed.

Modulation of Mesenchymal Stem Cells for Enhanced Therapeutic Utility in Ischemic Vascular Diseases.

Mesenchymal stem cells are multipotent stem cells isolated from various tissue sources, including but not limited to bone marrow, adipose, umbilical cord, and Wharton Jelly. Although cell-mediated mechanisms have been reported, the therapeutic effect of MSCs is now recognized to be primarily mediated via paracrine effects through the secretion of bioactive molecules, known as the "secretome". The regenerative benefit of the secretome has been attributed to trophic factors and cytokines that play neuroprotective, anti-angiogenic/pro-angiogenic, anti-inflammatory, and immune-modulatory roles. The advancement of autologous MSCs therapy can be hindered when introduced back into a hostile/disease environment. Barriers include impaired endogenous MSCs function, limited post-transplantation cell viability, and altered immune-modulatory efficiency. Although secretome-based therapeutics have gained popularity, many translational hurdles, including the heterogeneity of MSCs, limited proliferation potential, and the complex nature of the secretome, have impeded the progress. This review will discuss the experimental and clinical impact of restoring the functional capabilities of MSCs prior to transplantation and the progress in secretome therapies involving extracellular vesicles. Modulation and utilization of MSCs-secretome are most likely to serve as an effective strategy for promoting their ultimate success as therapeutic modulators.

Deletion of Thioredoxin-Interacting Protein (TXNIP) Abrogates High Fat Diet-induced Retinal Leukostasis, Barrier Dysfunction and Microvascular Degeneration in a Mouse Obesity Model.

We have shown that a high fat diet (HFD) induces the activation of retinal NOD-like receptor protein (NLRP3)-inflammasome that is associated with enhanced expression and interaction with thioredoxin-interacting protein (TXNIP). Here, the specific contribution of TXNIP and the impact of HFD on retinal leukostasis, barrier dysfunction and microvascular degeneration were investigated. Wild-type (WT) and TXNIP knockout (TKO) mice were fed with normal diet or 60% HFD for 8-18 weeks. TXNIP was overexpressed or silenced in human retinal endothelial cells (REC). At 8 weeks, HFD significantly induced retinal leukostasis and breakdown of the blood-retina barrier in WT mice, but not in TKO mice. In parallel, HFD also induced retinal expression of adhesion molecules and cleaved IL-1ß in WT mice, which were also abrogated in TKO mice. In culture, TXNIP overexpression induced NLRP3, IL-1b, and adhesion molecules expression, while TXNIP silencing inhibited them. Blocking the IL-1ß receptor significantly suppressed TXNIP-induced expression of NLRP3-inflammasome and adhesion molecules in HREC. Ex-vivo assay showed that leukocytes isolated from WT-HFD, but not from TKO-HFD, induced leukostasis and cell death. At 18 weeks, HFD triggered development of degenerated (acellular) capillaries and decreased branching density in WT but not in TKO mice. Together, HFD-induced obesity triggered early retinal leukostasis and microvascular dysfunction at least in part via TXNIP-NLRP3-inflammasome activation.

Modulating Expression of Thioredoxin Interacting Protein (TXNIP) Prevents Secondary Damage and Preserves Visual Function in a Mouse Model of Ischemia/Reperfusion.

Retinal neurodegeneration, an early characteristic of several blinding diseases, triggers glial activation, resulting in inflammation, secondary damage and visual impairment. Treatments that aim only at neuroprotection have failed clinically. Here, we examine the impact of modulating thioredoxin interacting protein (TXNIP) to the inflammatory secondary damage and visual impairment in a model of ischemia/reperfusion (IR). Wild type (WT) and TXNIP knockout (TKO) mice underwent IR injury by increasing intraocular pressure for 40 min, followed by reperfusion. An additional group of WT mice received intravitreal TXNIP-antisense oligomers (ASO, 100 µg/2 µL) 2 days post IR injury. Activation of Müller glial cells, apoptosis and expression of inflammasome markers and visual function were assessed. IR injury triggered early TXNIP mRNA expression that persisted for 14 days and was localized within activated Müller cells in WT-IR, compared to sham controls. Exposure of Müller cells to hypoxia-reoxygenation injury triggered endoplasmic reticulum (ER) stress markers and inflammasome activation in WT cells, but not from TKO cells. Secondary damage was evident by the significant increase in the number of occluded acellular capillaries and visual impairment in IR-WT mice but not in IR-TKO. Intervention with TXNIP-ASO prevented ischemia-induced glial activation and neuro-vascular degeneration, and improved visual function compared to untreated WT. Targeting TXNIP expression may offer an effective approach in the prevention of secondary damage associated with retinal neurodegenerative diseases.

Modulation of the p75 neurotrophin receptor using LM11A-31 prevents diabetes-induced retinal vascular permeability in mice via inhibition of inflammation and the RhoA kinase pathway.

AIMS/HYPOTHESIS: Breakdown of the inner blood-retinal barrier (BRB) is an early event in the pathogenesis of diabetic macular oedema, that eventually leads to vision loss. We have previously shown that diabetes causes an imbalance of nerve growth factor (NGF) isoforms resulting in accumulation of its precursor proNGF and upregulation of the p75 neurotrophin receptor (p75NTR), with consequent increases in the activation of Ras homologue gene family, member A (RhoA). We also showed that genetic deletion of p75NTR in diabetes preserved the BRB and prevented inflammatory mediators in retinas. This study aims to examine the therapeutic potential of LM11A-31, a small-molecule p75NTR modulator and proNGF antagonist, in preventing diabetes-induced BRB breakdown. The study also examined the role of p75NTR/RhoA downstream signalling in mediating cell permeability. METHODS: Male C57BL/6 J mice were rendered diabetic using streptozotocin injection. After 2 weeks of diabetes, mice received oral gavage of LM11A-31 (50 mg kg-1 day-1) or saline (NaCl 154 mmol/l) for an additional 4 weeks. BRB breakdown was assessed by extravasation of BSA-AlexaFluor-488. Direct effects of proNGF were examined in human retinal endothelial (HRE) cells in the presence or absence of LM11A-31 or the Rho kinase inhibitor Y-27632. RESULTS: Diabetes triggered BRB breakdown and caused significant increases in circulatory and retinal TNF-α and IL-1ß levels. These effects coincided with significant decreases in retinal NGF and increases in vascular endothelial growth factor and proNGF expression, as well as activation of RhoA. Interventional modulation of p75NTR activity through treatment of mouse models of diabetes with LM11A-31 significantly mitigated proNGF accumulation and preserved BRB integrity. In HRE cells, treatment with mutant proNGF (10 ng/ml) triggered increased cell permeability with marked reduction of expression of tight junction proteins, zona occludens-1 (ZO-1) and claudin-5, compared with control, independent of inflammatory mediators or cell death. Modulating p75NTR significantly inhibited proNGF-mediated RhoA activation, occludin phosphorylation (at serine 490) and cell permeability. ProNGF induced redistribution of ZO-1 in the cell wall and formation of F-actin stress fibres; these effects were mitigated by LM11A-31. CONCLUSIONS/INTERPRETATION: Targeting p75NTR signalling using LM11A-31, an orally bioavailable receptor modulator, may offer an effective, safe and non-invasive therapeutic strategy for treating macular oedema, a major cause of blindness in diabetes.

Deletion of p75NTR prevents vaso-obliteration and retinal neovascularization via activation of Trk- A receptor in ischemic retinopathy model.

Ischemic retinopathy is characterized by ischemia followed by retinal neovascularization (RNV) resulting in visual impairment. Given the role of neuron-secreted growth factors in regulating angiogenesis, we examined how genetic deletion of the neurotrophin receptor; p75NTR can overcome retinal ischemia using oxygen-induced retinopathy (OIR) mouse model. Wildtype (WT) or p75NTR-/- mice pups were subjected to hyperoxia (70% O2, p7-p12) then returned to normal air (relative hypoxia, p12-p17). Vascular alterations were assessed at p12 and p17 time-points. Deletion of p75NTR prevented hyperoxia-associated central vascular cell death (p12) and hypoxia-associated RNV and enhanced central vascular repair (p17). Decreased expression of apoptotic markers; preserved Akt survival signal decreased proNGF were also observed at p12. During hypoxia, deletion of p75NTR maintained VEGF and VEGFR2 activation and restored NGF/proNGF and BDNF/proBDNF levels. Deletion of p75NTR coincided with significant increases in expression and activation of NGF survival receptor, TrkA at basal and hyperoxic condition. Pharmacological inhibition of TrkA using compound K-252a (0.5 µg 1 µl-1/eye) resulted in 2-fold increase in pathological RNV and 1.34-fold increase in central vascular cell death in p75NTR-/- pups. In conclusion, deletion of p75NTR protected against retinal ischemia and prevented RNV, in part, through restoring neurotrophic support and activating TrkA receptor.

High Glucose-Mediated Tyrosine Nitration of PI3-Kinase: A Molecular Switch of Survival and Apoptosis in Endothelial Cells.

Diabetes and hyperglycemia are associated with increased retinal oxidative and nitrative stress and vascular cell death. Paradoxically, high glucose stimulates expression of survival and angiogenic growth factors. Therefore, we examined the hypothesis that high glucose-mediated tyrosine nitration causes inhibition of the survival protein PI3-kinase, and in particular, its regulatory p85 subunit in retinal endothelial cell (EC) cultures. Retinal EC were cultured in high glucose (HG, 25 mM) for 3 days or peroxynitrite (PN, 100 µM) overnight in the presence or absence of a peroxynitrite decomposition catalyst (FeTPPs, 2.5 µM), or the selective nitration inhibitor epicatechin (100 µM). Apoptosis of ECs was assessed using TUNEL assay and caspase-3 activity. Immunoprecipitation and Western blot were used to assess protein expression and tyrosine nitration of p85 subunit and its interaction with the p110 subunit. HG or PN accelerated apoptosis of retinal ECs compared to normal glucose (NG, 5 mM) controls. HG- or PN-treated cells also showed significant increases in tyrosine nitration on the p85 subunit of PI3-kinase that inhibited its association with the catalytic p110 subunit and impaired PI3-kinase/Akt kinase activity. Decomposing peroxynitrite or blocking tyrosine nitration of p85 restored the activity of PI3-kinase, and prevented apoptosis and activation of p38 MAPK. Inhibiting p38 MAPK or overexpression of the constitutively activated Myr-Akt construct prevented HG- or peroxynitrite-mediated apoptosis. In conclusion, HG impairs pro-survival signals and causes accelerated EC apoptosis, at least in part via tyrosine nitration and inhibition of PI3-kinase. Inhibitors of nitration can be used in adjuvant therapy to delay diabetic retinopathy and microvascular complication.

Deletion of Thioredoxin-interacting protein ameliorates high fat diet-induced non-alcoholic steatohepatitis through modulation of Toll-like receptor 2-NLRP3-inflammasome axis: Histological and immunohistochemical study.

Endemic prevalence of obesity is associated with alarming increases in non-alcoholic steatohepatitis (NASH) with limited available therapeutics. Toll-like receptor2 (TLR2) and Nod-like receptor protein 3 (NLRP3) Inflammasome are implicated in hepatic steatosis, inflammation and fibrosis; the histological landmark stages of NASH. TXNIP, a member of α-arrestin family activates NLRP3 in response to various danger stimuli. The aim of current work was to investigate the effect of TXNIP genetic deletion on histological manifestations of high fat diet-induced steatohepatitis and activation of TLR2-NLRP3-inflammasome axis. Wild-type mice (WT) and TXNIP knock out (TKO) littermates were randomized to normal diet (WT-ND and TKO-ND) or high fat diet (HFD, 60% fat) (WT-HFD and TKO-HFD). After 8-weeks, liver samples from all groups were evaluated by histological, immunohistochemical and western blot analysis. HFD resulted in significant induction of micro and macrovesicular hepatic steatosis, that was associated with increased inflammatory immune cell infiltration in WT-HFD compared with WT-ND and TKO-ND controls, but not in TKO-HFD group. In parallel, WT-HFD group showed significant fibrosis and α-SMA expression; a marker of pro-fibrotic stellate-cell activation, in areas surrounding the central vein and portal circulation, versus all other groups. Western blot revealed increased activation of TLR2-NLRP3 inflammasome pathway and downstream IL-1ß and TNFα in WT-HFD group, but not in TKO-HFD group. IL-1ß expression coincided within the same areas of steatosis, inflammatory cell infiltration, collagen deposition and α-SMA expression in WT-HFD mice, that was significantly reduced in TKO-HFD mice. In conclusion, TXNIP deletion ameliorates the HFD-induced steatosis, inflammatory and fibrotic response via modulation of TLR2-NLRP3 inflammasome axis. Targeting TXNIP-TLR2-NLRP3 pathway may provide potential therapeutic modalities for NASH treatment.

Inducible overexpression of endothelial proNGF as a mouse model to study microvascular dysfunction.

Impaired maturation of nerve growth factor precursor (proNGF) and its accumulation has been reported in several neurodegenerative diseases, myocardial infarction and diabetes. To elucidate the direct impact of proNGF accumulation identified the need to create a transgenic model that can express fully mutated cleavage-resistant proNGF. Using Cre-Lox technology, we developed an inducible endothelial-specific proNGF transgenic mouse (proNGFLoxp) that overexpresses GFP-conjugated cleavage-resistant proNGF123 when crossed with VE-cadherin-CreERT2 (Cre). Expression of proNGF, inflammatory mediators, NGF and VEGF was evaluated by PCR, Western blot and immunohistochemistry. EC-proNGF overexpression was confirmed using colocalization of anti-proNGF within retinal vasculature. EC-proNGF did not cause retinal neurotoxicity or marked glial activation at 4-weeks. Microvascular preparation from Cre-proNGF mice showed significant imbalance of proNGF/NGF ratio, enhanced expression of TNF-α and p75NTR, and tendency to impair TrkA phosphorylation compared to controls. EC-proNGF overexpression triggered mRNA expression of p75NTR and inflammatory mediators in both retina and renal cortex compared to controls. EC-proNGF expression induced vascular permeability including breakdown of BRB and albuminuria in the kidney without affecting VEGF level at 4-weeks. Histopathological changes were assessed after 8-weeks and the results showed that EC-proNGF triggered formation of occluded (acellular) capillaries, hall mark of retinal ischemia. EC-proNGF resulted in glomerular enlargement and kidney fibrosis, hall mark of renal dysfunction. We have successfully created an inducible mouse model that can dissect the contribution of autocrine direct action of cleavage-resistant proNGF on systemic microvascular abnormalities in both retina and kidney, major targets for microvascular complication.

Implication of the neurotrophin receptor p75NTR in vascular diseases: beyond the eye.

INTRODUCTION: The p75 neurotrophin receptor (p75NTR) is a member of TNF-α receptor superfamily that bind all neurotrophins, mainly regulating their pro-apoptotic actions. Ischemia is a common pathology in different cardiovascular diseases affecting multiple organs, however the contribution of p75NTR remains not fully addressed. The aim of this work is to review the current evidence through published literature studying the impact of p75NTR receptor in ischemic vascular diseases. AREAS COVERED: In the eye, several ischemic ocular diseases are associated with enhanced p75NTR expression. Ischemic retinopathy including diabetic retinopathy, retinopathy of prematurity and retinal vein occlusion are characterized initially by ischemia followed by excessive neovascularization. Beyond the eye, cerebral ischemia, myocardial infarction and critical limb ischemia are ischemic cardiovascular diseases that are characterized by altered expression of neurotrophins and p75NTR expression. We surveyed both clinical and experimental studies that examined the impact of p75NTR receptor in ischemic diseases of eye, heart, brain and peripheral limbs. EXPERT COMMENTARY: p75NTR receptor is a major player in multiple ischemic vascular diseases affecting the eye, brain, heart and peripheral limbs with significant increases in its expression accompanying neuro-vascular injury. This has been addressed in the current review along with the beneficial vascular outcomes of p75NTR inhibition.

Correction: Diabetes-Induced Superoxide Anion and Breakdown of the Blood-Retinal Barrier: Role of the VEGF/uPAR Pathway.

[This corrects the article DOI: 10.1371/journal.pone.0071868.].

Deletion of TXNIP Mitigates High-Fat Diet-Impaired Angiogenesis and Prevents Inflammation in a Mouse Model of Critical Limb Ischemia.

BACKGROUND: Previous work demonstrated that high-fat diet (HFD) triggered thioredoxin-interacting protein (TXNIP) and that silencing TXNIP prevents diabetes-impaired vascular recovery. Here, we examine the impact of genetic deletion of TXNIP on HFD-impaired vascular recovery using hind limb ischemia model. METHODS: Wild type mice (WT, C57Bl/6) and TXNIP knockout mice (TKO) were fed either normal chow diet (WT-ND and TKO-ND) or 60% high-fat diet (WT-HFD and TKO-HFD). After four weeks of HFD, unilateral hind limb ischemia was performed and blood flow was measured using Laser doppler scanner at baseline and then weekly for an additional three weeks. Vascular density, nitrative stress, infiltration of CD68+ macrophages, and expression of inflammasome, vascular endothelial growth factor (VEGF), VEGF receptor-2 were examined by slot blot, Western blot and immunohistochemistry. RESULTS: By week 8, HFD caused similar increases in weight, cholesterol and triglycerides in both WT and TKO. At week 4 and week 8, HFD significantly impaired glucose tolerance in WT and to a lesser extent in TKO. HFD significantly impaired blood flow and vascular density (CD31 labeled) in skeletal muscle of WT mice compared to ND but not in TKO. HFD and ischemia significantly induced tyrosine nitration, and systemic IL-1ß and infiltration of CD68+ cells in skeletal muscle from WT but not from TKO. HFD significantly increased cleaved-caspase-1 and IL-1 ß compared to ND. Under both ND, ischemia tended to increase VEGF expression and increased VEGFR2 activation in WT only but not TKO. CONCLUSION: Similar to prior observation in diabetes, HFD-induced obesity can compromise vascular recovery in response to ischemic insult. The mechanism involves increased TXNIP-NLRP3 (nucleotide-binding oligomerization domain-like receptor protein 3) inflammasome activation, nitrative stress and impaired VEGFR2 activation. Deletion of TXNIP restored blood flow, reduced nitrative stress and blunted inflammasome-mediated inflammation; however, it did not impact VEGF/VEGFR2 in HFD. Targeting TXNIP-NLRP3 inflammasome can provide potential therapeutic target in obesity-induced vascular complication.

High fat diet dysregulates microRNA-17-5p and triggers retinal inflammation: Role of endoplasmic-reticulum-stress.

AIM: To elucidate how high diet-induced endoplasmic reticulum-stress upregulates thioredoxin interacting protein expression in Müller cells leading to retinal inflammation. METHODS: Male C57Bl/J mice were fed either normal diet or 60% high fat diet for 4-8 wk. During the 4 wk study, mice received phenyl-butyric acid (PBA); endoplasmic reticulum-stress inhibitor; for 2 wk. Insulin resistance was assessed by oral glucose tolerance. Effects of palmitate-bovine serum albumin (BSA) (400 µmol/L) were examined in retinal Müller glial cell line and primary Müller cells isolated from wild type and thioredoxin interacting protein knock-out mice. Expression of thioredoxin interacting protein, endoplasmic reticulum-stress markers, miR-17-5p mRNA, as well as nucleotide-binding oligomerization domain-like receptor protein (NLRP3) and IL1ß protein was determined. RESULTS: High fat diet for 8 wk induced obesity and insulin resistance evident by increases in body weight and impaired glucose tolerance. By performing quantitative real-time polymerase chain reaction, we found that high fat diet triggered the expression of retinal endoplasmic reticulum-stress markers (P < 0.05). These effects were associated with increased thioredoxin interacting protein and decreased miR-17-5p expression, which were restored by inhibiting endoplasmic reticulum-stress with PBA (P < 0.05). In vitro, palmitate-BSA triggered endoplasmic reticulum-stress markers, which was accompanied with reduced miR-17-5p and induced thioredoxin interacting protein mRNA in retinal Müller glial cell line (P < 0.05). Palmitate upregulated NLRP3 and IL1ß expression in primary Müller cells isolated from wild type. However, using primary Müller cells isolated from thioredoxin interacting protein knock-out mice abolished palmitate-mediated increase in NLRP3 and IL1ß. CONCLUSION: Our work suggests that targeting endoplasmic reticulum-stress or thioredoxin interacting protein are potential therapeutic strategies for early intervention of obesity-induced retinal inflammation.

Deletion of the Neurotrophin Receptor p75NTR Prevents Diabetes-Induced Retinal Acellular Capillaries in Streptozotocin-Induced Mouse Diabetic Model.

Diabetic retinopathy is characterized by early stage of retinal neuro-inflammation that triggers development of acellular capillaries and a late stage of pathological neovascularization. Due to limited treatment options, there is a pressing need to develop new therapeutics. Our group discovered that diabetes-impaired processing of the nerve growth factor precursor (proNGF) resulting in its accumulation and its receptor p75NTR. Here, we examine the protective effects of modulating p75NTR in experimental model of diabetic retinopathy. Diabetes was induced using streptozotocin in both wild type (WT) and p75NTR knockout (p75KO) mice. Retinal inflammation and microvascular dysfunction were assessed. Western blot analysis was performed to assess expression of apoptotic and inflammatory markers and levels of the neurotrophin, p75NTR and ephrin-B2. Deletion of p75NTR did not alter body weight or diabetes status compared to WT mice. In WT-mice, diabetes triggered retinal inflammation, significant decrease in pericyte count and marked increase in development of occluded (acellular) capillary formation after 24-weeks. Deletion of p75NTR prevented acellular capillary, restored pericyte count, and inhibited the retinal Ephrin-B2, activation of the stress-kinase JNK and apoptotic marker cleaved caspase-3 in the diabetic retina. Deletion of p75NTR reduced retinal inflammation, and proNGF expression. These effects coincided with increased NGF level and TrkA activation in the diabetic retina. Targeting p75NTR using genetic approach protected the retina from the impact of long-term diabetes in mediating microvascular degeneration and maintains the balance of NGF/proNGF level. Together, these results provide rationale that targeting p75NTR may offer novel and effective therapeutic strategy to combat diabetic retinopathy.

Nox4 contributes to the hypoxia-mediated regulation of actin cytoskeleton in cerebrovascular smooth muscle.

UNLABELLED: Ischemia/reperfusion and the resulting oxidative/nitrative stress impair cerebral myogenic tone via actin depolymerization. While it is known that NADPH oxidase (Nox) family is a major source of vascular oxidative stress; the extent and mechanisms by which Nox activation contributes to actin depolymerization, and equally important, the relative role of Nox isoforms in this response is not clear. AIM: To determine the role of Nox4 in hypoxia-mediated actin depolymerization and myogenic-tone impairment in cerebral vascular smooth muscle. MAIN METHODS: Control and Nox4 deficient (siRNA knock-down) human brain vascular smooth muscle cells (HBVSMC) were exposed to 30-min hypoxia/45-min reoxygenation. Nox2, Nox4, inducible and neuronal nitric oxide synthase (iNOS and nNOS) and nitrotyrosine levels as well as F:G actin were determined. Myogenic-tone was measured using pressurized arteriography in middle cerebral artery isolated from rats subjected to sham, 30-min ischemia/45-min reperfusion or ex-vivo oxygen glucose deprivation in the presence and absence of Nox inhibitors. RESULTS: Nox4 and iNOS expression were significantly upregulated following hypoxia or ischemia/reperfusion. Hypoxia augmented nitrotyrosine levels while reducing F actin. These effects were nullified by inhibiting nitration with epicatechin or pharmacological or molecular inhibition of Nox4. Ischemia/reperfusion impaired myogenic-tone, which was restored by the selective inhibition of Nox4. CONCLUSION: Nox4 activation in VSMCs contributes to actin depolymerization after hypoxia, which could be the underlying mechanism for myogenic-tone impairment following ischemia/reperfusion.

Renin-angiotensin system as a potential therapeutic target in stroke and retinopathy: experimental and clinical evidence.

As our knowledge expands, it is now clear that the renin-angiotensin (Ang) system (RAS) mediates functions other than regulating blood pressure (BP). The RAS plays a central role in the pathophysiology of different neurovascular unit disorders including stroke and retinopathy. Moreover, the beneficial actions of RAS modulation in brain and retina have been documented in experimental research, but not yet exploited clinically. The RAS is a complex system with distinct yet interconnected components. Understanding the different RAS components and their functions under brain and retinal pathological conditions is crucial to reap their benefits. The aim of the present review is to provide an experimental and clinical update on the role of RAS in the pathophysiology and treatment of stroke and retinopathy. Combining the evidence from both these disorders allows a unique opportunity to move both fields forward.