Long-term plasticity of inhibitory synapses in the hippocampus and spatial learning depends on matrix metalloproteinase 3.

Learning and memory are known to depend on synaptic plasticity. Whereas the involvement of plastic changes at excitatory synapses is well established, plasticity mechanisms at inhibitory synapses only start to be discovered. Extracellular proteolysis is known to be a key factor in glutamatergic plasticity but nothing is known about its role at GABAergic synapses. We reveal that pharmacological inhibition of MMP3 activity or genetic knockout of the Mmp3 gene abolishes induction of postsynaptic iLTP. Moreover, the application of exogenous active MMP3 mimics major iLTP manifestations: increased mIPSCs amplitude, enlargement of synaptic gephyrin clusters, and a decrease in the diffusion coefficient of synaptic GABAA receptors that favors their entrapment within the synapse. Finally, we found that MMP3 deficient mice show faster spatial learning in Morris water maze and enhanced contextual fear conditioning. We conclude that MMP3 plays a key role in iLTP mechanisms and in the behaviors that presumably in part depend on GABAergic plasticity.

IL-33trap-mediated IL-33 neutralization does not exacerbate choroidal neovascularization, but fails to protect against retinal degeneration in a dry age-related macular degeneration model.

The progressive and sight-threatening disease, age-related macular degeneration (AMD), is a growing public health concern due to ageing demographics, with the highest unmet medical need for the advanced stage of dry AMD, geographic atrophy. The pathogenesis underlying AMD is driven by a complex interplay of genetic and environmental factors. There is ample evidence that inflammation is strongly involved in AMD development. Interleukin-33 (IL-33) has been proposed to be critically involved in retinal degeneration, but a protective role in eye pathophysiology was also demonstrated. The current study investigated the therapeutic potential of IL-33trap, a novel IL-33-neutralizing biologic, in dry AMD/geographic atrophy and, based on controversial data regarding the protective versus detrimental functions of IL-33 in neovascularization, evaluated the risk of progression to wet AMD by IL-33 neutralization. Repeated intravitreal (IVT) injections of IL-33trap in the mouse laser-induced choroidal neovascularization model did not exacerbate neovascularization or leakage, while it significantly inhibited inflammatory cell infiltration in the retinal pigment epithelium and choroid. On the contrary, IVT treatment with IL-33trap significantly induced retinal inflammation and could not prevent retinopathy induction in the mouse sodium iodate (NaIO3) model. Overall, these data suggest a complex and dichotomous role of IL-33 in eye pathology and indicate that IL-33 neutralization is not able to prevent onset and progression of dry AMD pathogenesis.

Single-cell transcriptome analysis of the Akimba mouse retina reveals cell-type-specific insights into the pathobiology of diabetic retinopathy.

AIMS/HYPOTHESIS: Diabetic retinopathy is a common complication of diabetes and a leading cause of visual impairment and blindness. Despite recent advances, our understanding of its pathophysiology remains incomplete. The aim of this study was to provide deeper insight into the complex network of molecular and cellular changes that underlie diabetic retinopathy by systematically mapping the transcriptional changes that occur in the different cellular compartments of the degenerating diabetic mouse retina. METHODS: Single-cell RNA sequencing was performed on retinal tissue from 12-week-old wild-type and Akimba (Ins2Akita×Vegfa+/-) mice, which are known to replicate features of clinical diabetic retinopathy. This resulted in transcriptome data for 9474 retinal cells, which could be annotated to eight distinct retinal cell types. Using STRING analysis, we studied differentially expressed gene networks in neuronal, glial and immune cell compartments to create a comprehensive view on the pathological changes that occur in the Akimba retina. Using subclustering analysis, we further characterised macroglial and inflammatory cell subpopulations. Prominent findings were confirmed at the protein level using immunohistochemistry, western blotting and ELISA. RESULTS: At 12 weeks, the Akimba retina was found to display degeneration of rod photoreceptors and presence of inflammatory cells, identified by subclustering analysis as monocyte, macrophage and microglial populations. Analysis of differentially expressed genes in the rod, cone, bipolar cell and macroglial compartments indicated changes in cell metabolism and ribosomal gene expression, gliosis, activation of immune system pathways and redox and metal ion dyshomeostasis. Experiments at the protein level supported a metabolic shift from glycolysis to oxidative phosphorylation (glyceraldehyde 3-phosphate dehydrogenase), activation of microglia/macrophages (isolectin-B4), metal ion and oxidative stress response (metallothionein and haem oxygenase-1) and reactive macroglia (glial fibrillary acidic protein and S100) in the Akimba retina, compared with wild-type mice. Our single-cell approach also indicates macroglial subpopulations with distinct fibrotic, inflammatory and gliotic profiles. CONCLUSIONS/INTERPRETATION: Our study identifies molecular pathways underlying inflammatory, metabolic and oxidative stress-mediated changes in the Akimba mouse model of diabetic retinopathy and distinguishes distinct functional subtypes of inflammatory and macroglial cells. DATA AVAILABILITY: RNA-seq data have been deposited in the ArrayExpress database at EMBL-EBI ( www.ebi.ac.uk/arrayexpress ) under accession number E-MTAB-9061. Graphical abstract.

Tightening the retinal glia limitans attenuates neuroinflammation after optic nerve injury.

Increasing evidence suggests that functional impairments at the level of the neurovascular unit (NVU) underlie many neurodegenerative and neuroinflammatory diseases. While being part of the NVU, astrocytes have been largely overlooked in this context and only recently, tightening of the glia limitans has been put forward as an important neuroprotective response to limit these injurious processes. In this study, using the retina as a central nervous system (CNS) model organ, we investigated the structure and function of the glia limitans, and reveal that the blood-retina barrier and glia limitans function as a coordinated double barrier to limit infiltration of leukocytes and immune molecules. We provide in vitro and in vivo evidence for a protective response at the NVU upon CNS injury, which evokes inflammation-induced glia limitans tightening. Matrix metalloproteinase-3 (MMP-3) was found to be a crucial regulator of this process, thereby revealing its beneficial and immunomodulatory role in the CNS. in vivo experiments in which MMP-3 activity was deleted via genetic and pharmacological approaches, combined with a comprehensive study of tight junction molecules, glial end feet markers, myeloid cell infiltration, cytokine expression and neurodegeneration, show that MMP-3 attenuates neuroinflammation and neurodegeneration by tightening the glia limitans, thereby pointing to a prominent role of MMP-3 in preserving the integrity of the NVU upon injury. Finally, we gathered promising evidence to suggest that IL1b, which is also regulated by MMP-3, is at least one of the molecular messengers that induces glia limitans tightening in the injured CNS.

The retinal tyrosine kinome of diabetic Akimba mice highlights potential for specific Src family kinase inhibition in retinal vascular disease.

Although anti-VEGF therapies have radically changed clinical practice, there is still an urgent demand for novel, integrative approaches for sight-threatening retinal vascular diseases. As we hypothesize that protein tyrosine kinases are key signaling mediators in retinal vascular disease, we performed a comprehensive activity-based tyrosine kinome profiling on retinal tissue of 12-week-old Akimba mice, a translational model displaying hallmarks of early and advanced diabetic retinopathy. Western blotting was used to confirm retinal tyrosine kinase activity in Akimba mice. HUVEC tube formation and murine organotypic choroidal sprouting assays were applied to compare tyrosine kinase inhibitors with different specificity profiles. HUVEC toxicity and proliferation were evaluated using the CellTox™ Green Cytotoxicity and PrestoBlue™ Assays. Our results indicate a shift of the Akimba retinal tyrosine kinome towards a hyperactive state. Functional network analysis of significantly hyperphosphorylated peptides and upstream kinase prediction revealed a central role for Src-FAK family kinases. Western blotting confirmed hyperactivity of this signaling node in the retina of Akimba mice. We demonstrated that not only Src but also FAK family kinase inhibitors with different selectivity profiles were able to suppress angiogenesis in vitro and ex vivo. In the latter model, the novel selective Src family kinase inhibitor eCF506 was able to achieve potent reduction of angiogenesis, comparable to the less specific inhibitor Dasatinib. None of the tested compounds demonstrated acute endothelial cell toxicity. Overall, the collected findings provide the first comprehensive overview of retinal tyrosine kinome changes in the Akimba model of diabetic retinopathy and for the first time highlight Src family kinase inhibition using highly specific inhibitors as an attractive therapeutic intervention for retinal vascular pathology.

The potent small molecule integrin antagonist THR-687 is a promising next-generation therapy for retinal vascular disorders.

Integrins are associated with various eye diseases such as diabetic retinopathy (DR) and wet age-related macular degeneration (AMD) and implicated in main pathologic disease hallmarks like neovascularization, inflammation, fibrosis and vascular leakage. Targeting integrins has the potential to attenuate these vision-threatening processes, independent of anti-vascular endothelial growth factor (VEGF) responsiveness. The current investigation characterized THR-687 as a novel pan RGD (arginylglycylaspartic acid) integrin receptor antagonist able to compete for binding with the natural ligand with nanomolar potency (e.g. αvß3 (IC50 of 4.4 ±â€¯2.7 nM), αvß5 (IC50 of 1.3 ±â€¯0.5 nM) and α5ß1 (IC50 of 6.8 ±â€¯3.2 nM)). THR-687 prevented the migration of human umbilical vein endothelial cells (HUVECs) into a cell-free area (IC50 of 258 ±â€¯113 nM) as well as vessel sprouting in an ex vivo mouse choroidal explant model (IC50 of 236 ±â€¯173 nM), and was able to induce the regression of pre-existing vascular sprouts. Moreover, combined intravitreal and intraperitoneal administration of THR-687 potently inhibited VEGF-induced leakage in the mouse retina. In addition, THR-687 injected intravitreally at 3 different dose levels (0.45 mg, 2.25 mg or 4.5 mg/eye) potently inhibited neovascularization-induced leakage in the cynomolgus laser-induced choroidal neovascularization (CNV) model. These data suggest that THR-687 is a promising drug candidate for the treatment of vision-threatening retinal vascular eye diseases such as DR and wet AMD.

Mechanisms of NMDA Receptor- and Voltage-Gated L-Type Calcium Channel-Dependent Hippocampal LTP Critically Rely on Proteolysis That Is Mediated by Distinct Metalloproteinases.

Long-term potentiation (LTP) is widely perceived as a memory substrate and in the hippocampal CA3-CA1 pathway, distinct forms of LTP depend on NMDA receptors (nmdaLTP) or L-type voltage-gated calcium channels (vdccLTP). LTP is also known to be effectively regulated by extracellular proteolysis that is mediated by various enzymes. Herein, we investigated whether in mice hippocampal slices these distinct forms of LTP are specifically regulated by different metalloproteinases (MMPs). We found that MMP-3 inhibition or knock-out impaired late-phase LTP in the CA3-CA1 pathway. Interestingly, late-phase LTP was also decreased by MMP-9 blockade. When both MMP-3 and MMP-9 were inhibited, both early- and late-phase LTP was impaired. Using immunoblotting, in situ zymography, and immunofluorescence, we found that LTP induction was associated with an increase in MMP-3 expression and activity in CA1 stratum radiatum. MMP-3 inhibition and knock-out prevented the induction of vdccLTP, with no effect on nmdaLTP. L-type channel-dependent LTP is known to be impaired by hyaluronic acid digestion. We found that slice treatment with hyaluronidase occluded the effect of MMP-3 blockade on LTP, further confirming a critical role for MMP-3 in this form of LTP. In contrast to the CA3-CA1 pathway, LTP in the mossy fiber-CA3 projection did not depend on MMP-3, indicating the pathway specificity of the actions of MMPs. Overall, our study indicates that the activation of perisynaptic MMP-3 supports L-type channel-dependent LTP in the CA1 region, whereas nmdaLTP depends solely on MMP-9. SIGNIFICANCE STATEMENT: Various types of long-term potentiation (LTP) are correlated with distinct phases of memory formation and retrieval, but the underlying molecular signaling pathways remain poorly understood. Extracellular proteases have emerged as key players in neuroplasticity phenomena. The present study found that L-type calcium channel-dependent LTP in the CA3-CA1 hippocampal projection is critically regulated by the activity of matrix metalloprotease 3 (MMP-3), in contrast to NMDAR-dependent LTP regulated by MMP-9. Moreover, the induction of LTP was associated with an increase in MMP-3 expression and activity. Finally, we found that the digestion of hyaluronan, a principal extracellular matrix component, disrupted the MMP-3-dependent component of LTP. These results indicate that distinct MMPs might act as molecular switches for specific types of LTP.

Amyloid ß Oligomers Disrupt Blood-CSF Barrier Integrity by Activating Matrix Metalloproteinases.

The blood-CSF barrier (BCSFB) consists of a monolayer of choroid plexus epithelial (CPE) cells that maintain CNS homeostasis by producing CSF and restricting the passage of undesirable molecules and pathogens into the brain. Alzheimer's disease is the most common progressive neurodegenerative disorder and is characterized by the presence of amyloid ß (Aß) plaques and neurofibrillary tangles in the brain. Recent research shows that Alzheimer's disease is associated with morphological changes in CPE cells and compromised production of CSF. Here, we studied the direct effects of Aß on the functionality of the BCSFB. Intracerebroventricular injection of Aß1-42 oligomers into the cerebral ventricles of mice, a validated Alzheimer's disease model, caused induction of a cascade of detrimental events, including increased inflammatory gene expression in CPE cells and increased levels of proinflammatory cytokines and chemokines in the CSF. It also rapidly affected CPE cell morphology and tight junction protein levels. These changes were associated with loss of BCSFB integrity, as shown by an increase in BCSFB leakage. Aß1-42 oligomers also increased matrix metalloproteinase (MMP) gene expression in the CPE and its activity in CSF. Interestingly, BCSFB disruption induced by Aß1-42 oligomers did not occur in the presence of a broad-spectrum MMP inhibitor or in MMP3-deficient mice. These data provide evidence that MMPs are essential for the BCSFB leakage induced by Aß1-42 oligomers. Our results reveal that Alzheimer's disease-associated soluble Aß1-42 oligomers induce BCSFB dysfunction and suggest MMPs as a possible therapeutic target. SIGNIFICANCE STATEMENT: No treatments are yet available to cure Alzheimer's disease; however, soluble Aß oligomers are believed to play a crucial role in the neuroinflammation that is observed in this disease. Here, we studied the effect of Aß oligomers on the often neglected barrier between blood and brain, called the blood-CSF barrier (BCSFB). This BCSFB is formed by the choroid plexus epithelial cells and is important in maintaining brain homeostasis. We observed Aß oligomer-induced changes in morphology and loss of BCSFB integrity that might play a role in Alzheimer's disease progression. Strikingly, both inhibition of matrix metalloproteinase (MMP) activity and MMP3 deficiency could protect against the detrimental effects of Aß oligomer. Clearly, our results suggest that MMP inhibition might have therapeutic potential.

MMP-3 Deficiency Alleviates Endotoxin-Induced Acute Inflammation in the Posterior Eye Segment.

Matrix metalloproteinase-3 (MMP-3) is known to mediate neuroinflammatory processes by activating microglia, disrupting blood-central nervous system barriers and supporting neutrophil influx into the brain. In addition, the posterior part of the eye, more specifically the retina, the retinal pigment epithelium (RPE) and the blood-retinal barrier, is affected upon neuroinflammation, but a role for MMP-3 during ocular inflammation remains elusive. We investigated whether MMP-3 contributes to acute inflammation in the eye using the endotoxin-induced uveitis (EIU) model. Systemic administration of lipopolysaccharide induced an increase in MMP-3 mRNA and protein expression level in the posterior part of the eye. MMP-3 deficiency or knockdown suppressed retinal leukocyte adhesion and leukocyte infiltration into the vitreous cavity in mice subjected to EIU. Moreover, retinal and RPE mRNA levels of intercellular adhesion molecule 1 (Icam1), interleukin 6 (Il6), cytokine-inducible nitrogen oxide synthase (Nos2) and tumor necrosis factor α (Tnfα), which are key molecules involved in EIU, were clearly reduced in MMP-3 deficient mice. In addition, loss of MMP-3 repressed the upregulation of the chemokines monocyte chemoattractant protein (MCP)-1 and (C-X-C motif) ligand 1 (CXCL1). These findings suggest a contribution of MMP-3 during EIU, and its potential use as a therapeutic drug target in reducing ocular inflammation.

Matrix metalloproteinases in the mouse retina: a comparative study of expression patterns and MMP antibodies.

BACKGROUND: Matrix metalloproteinases (MMPs), a family of Zn(2+)-dependent endoproteases, have been shown to act as fine regulators of both health and disease. Limited research revealed that they are essential to maintaining ocular physiology and inordinate MMP activities have been linked to several neurodegenerative disorders of the retina, including age-related macular degeneration, proliferative diabetic retinopathy and glaucomatous optic neuropathies (GONs). Nevertheless, a clear definition of their pathology-exacerbating and/or -resolving actions is lacking, especially in the context of GONs, as most studies thus far merely focused on expression profiling in human patients. Therefore, in an initial step towards an improved understanding of MMP functions in the retina, we studied the spatial expression pattern of MMP-2, -3, -9 and MT1-MMP in the healthy mouse retina. METHODS: The spatial expression pattern of MMP-2, -3, -9 and MT1-MMP was studied in the healthy mouse retina via immunohistochemical stainings, and immunoreactivity profiles were compared to existing literature. Moreover, we considered sensitivity and specificity issues with commercially available MMP antibodies via Western blot. RESULTS: Basal expression of MMP-2,-3, -9 and MT1-MMP was found in the retina of healthy, adult mice. MMP-2 expression was seen in Müller glia, predominantly in their end feet, which is in line with available literature. MMP-3 expression was described for the first time in the retina, and was observed in vesicle-like structures along the radial fibers of Müller glia. MMP-9 expression, about which still discords exists, was seen in microglia and in a sparse subset of (apoptosing) RGCs. MT1-MMP localization was for the first time studied in adult mice and was found in RGC axons and Müller glia, mimicking the MT1-MMP expression pattern seen in rabbits and neonatal mice. Moreover, one antibody was selected for each MMP, based on its staining pattern in Western blot. CONCLUSIONS: The present MMP immunoreactivity profiles in the mouse retina and validation of MMP antibodies, can be instrumental to study MMP expression in mouse models of ocular pathologies and to compare these expression profiles to observations from clinical studies, which would be a first step in the disentanglement of the exact role MMPs in ocular/retinal diseases.

Identification of MMP-2 as a novel enhancer of cerebellar granule cell proliferation.

During the first postnatal days in the mouse, granule cells (GCs) undergo massive proliferation, which then gradually decreases. Matrix metalloproteinase-2 (MMP-2), a Zn(2+)-dependent proteolytic enzyme, is involved in a wide variety of pathological and physiological pathways. Evidence for a role of this proteinase in cell proliferation is emerging, reporting its involvement in pathological proliferation, as well as during neurogenesis and developmental proliferation of non-CNS tissues. In this study, MMP-2 protein expression was observed within the early postnatal cerebellar cortex, predominantly in Purkinje cells and within the GC proliferative zone, i.e. the superficial external granular layer (EGL). Consistently, the spatiotemporal MMP-2 mRNA and protein profiles highly correlated with the peak of GC precursor (GCP) proliferation and detailed morphometric analyses of MMP-2 deficient cerebella revealed a thinner EGL due to a decreased GCP proliferation. BrdU cumulative experiments, performed to measure the length of different cell cycle phases, further disclosed a transiently prolonged S-phase in MMP-2 deficient GCPs during early cerebellar development. In consequence, MMP-2 deficient animals displayed a transient delay in GC migration towards the IGL. In conclusion, our findings provide important evidence for a role for MMP-2 in neuronal proliferation and cell cycle kinetics in the developing CNS.

Quantification of Adeno-Associated Viral Genomes in Purified Vector Samples by Digital Droplet Polymerase Chain Reaction.

Adeno-associated virus (AAV) is a non-pathogenic virus used as a delivery vehicle to transfer therapeutic genes into patients. Accurate quantification of AAV genome copy number in vector preparations is essential for bioprocess optimization and dosage calculation in both preclinical and clinical studies of AAV-based gene therapy products. Currently, a consensus protocol for AAV viral genome titration is lacking. Here, we present a digital droplet PCR (dd_PCR) protocol for the quantification of viral genomes in purified vector samples. Samples are treated with DNase I to eliminate unpackaged contaminant DNA. DNase-treated samples are then mixed with an appropriate primer-probe set (designed according to the target AAV genome) and PCR reagents, and then loaded into a droplet generator. The prepared droplets are transferred into a PCR plate, where PCR amplification is performed and analyzed. The viral genome titer is calculated based on the concentration (copies/µL), accounting for sample dilutions. A successful measurement shows a clear separation of the positive and negative droplet clouds, has at least 10,000 accepted droplets, shows a value between 10 copies/µL and 10,000 copies/µL, and has a coefficient of variation (CV) between repeats lower than 20%. Reliable viral genome titration will aid in the development of safe and effective AAV-based gene therapy products.

Matrix metalloproteinase-3 in the central nervous system: a look on the bright side.

Matrix metalloproteinases (MMPs) are a large family of proteases involved in many cell-matrix and cell-cell signalling processes through activation, inactivation or release of extracellular matrix (ECM) and non-ECM molecules, such as growth factors and receptors. Uncontrolled MMP activities underlie the pathophysiology of many disorders. Also matrix metalloproteinase-3 (MMP-3) or stromelysin-1 contributes to several pathologies, such as cancer, asthma and rheumatoid arthritis, and has also been associated with neurodegenerative diseases like Alzheimer's disease, Parkinson's disease and multiple sclerosis. However, based on defined MMP spatiotemporal expression patterns, the identification of novel candidate molecular targets and in vitro and in vivo studies, a beneficial role for MMPs in CNS physiology and recovery is emerging. The main purpose of this review is to shed light on the recently identified roles of MMP-3 in normal brain development and in plasticity and regeneration after CNS injury and disease. As such, MMP-3 is correlated with neuronal migration and neurite outgrowth and guidance in the developing CNS and contributes to synaptic plasticity and learning in the adult CNS. Moreover, a strict spatiotemporal MMP-3 up-regulation in the injured or diseased CNS might support remyelination and neuroprotection, as well as genesis and migration of stem cells in the damaged brain.

PDGF as an Important Initiator for Neurite Outgrowth Associated with Fibrovascular Membranes in Proliferative Diabetic Retinopathy.

PURPOSE: The formation of fibrovascular membranes (FVMs) is a serious sight-threatening complication of proliferative diabetic retinopathy (PDR) that may result in retinal detachment and eventual blindness. During the formation of these membranes, neurite/process outgrowth occurs in retinal neurons and glial cells, which may both serve as a scaffold and have guiding or regulatory roles. To further understand this process, we investigated whether previously identified candidate proteins, from vitreous of PDR patients with FVMs, could induce neurite outgrowth in an experimental setting. MATERIALS AND METHODS: Retinal explants of C57BL6/N mouse pups on postnatal day 3 (P3) were cultured in poly-L-lysine- and laminin-coated dishes. Outgrowth stimulation experiments were performed with the addition of potential inducers of neurite outgrowth. Automated analysis of neurite outgrowth was performed by measuring ß-tubulin-immunopositive neurites using Image J. Expression of PDGF receptors was quantified by RT-PCR in FVMs of PDR patients. RESULTS: Platelet-derived growth factor (PDGF) induced neurite outgrowth in a concentration-dependent manner, whilst neuregulin 1 (NRG1) and connective tissue growth factor (CTGF) did not. When comparing three different PDGF dimers, treatment with PDGF-AB resulted in the highest neurite induction, followed by PDGF-AA and -BB. In addition, incubation of retinal explants with vitreous from PDR patients resulted in a significant induction of neurite outgrowth as compared to non-diabetic control vitreous from patients with macular holes, which could be prevented by addition of CP673451, a potent PDGF receptor (PDGFR) inhibitor. Abundant expression of PDGF receptors was detected in FVMs. CONCLUSION: Our findings suggest that PDGF may be involved in the retinal neurite outgrowth, which is associated with the formation of FVMs in PDR.

Targeting RGD-binding integrins as an integrative therapy for diabetic retinopathy and neovascular age-related macular degeneration.

Integrins are a class of transmembrane receptors that are involved in a wide range of biological functions. Dysregulation of integrins has been implicated in many pathological processes and consequently, they are attractive therapeutic targets. In the ophthalmology arena, there is extensive evidence suggesting that integrins play an important role in diabetic retinopathy (DR), age-related macular degeneration (AMD), glaucoma, dry eye disease and retinal vein occlusion. For example, there is extensive evidence that arginyl-glycyl-aspartic acid (Arg-Gly-Asp; RGD)-binding integrins are involved in key disease hallmarks of DR and neovascular AMD (nvAMD), specifically inflammation, vascular leakage, angiogenesis and fibrosis. Based on such evidence, drugs that engage integrin-linked pathways have received attention for their potential to block all these vision-threatening pathways. This review focuses on the pathophysiological role that RGD-binding integrins can have in complex multifactorial retinal disorders like DR, diabetic macular edema (DME) and nvAMD, which are leading causes of blindness in developed countries. Special emphasis will be given on how RGD-binding integrins can modulate the intricate molecular pathways and regulate the underlying pathological mechanisms. For instance, the interplay between integrins and key molecular players such as growth factors, cytokines and enzymes will be summarized. In addition, recent clinical advances linked to targeting RGD-binding integrins in the context of DME and nvAMD will be discussed alongside future potential for limiting progression of these diseases.

Reevaluation of the role of VEGF-B suggests a restricted role in the revascularization of the ischemic myocardium.

OBJECTIVE: The endogenous role of the VEGF family member vascular endothelial growth factor-B (VEGF-B) in pathological angiogenesis remains unclear. METHODS AND RESULTS: We studied the role of VEGF-B in various models of pathological angiogenesis using mice lacking VEGF-B (VEGF-B(-/-)) or overexpressing VEGF-B(167). After occlusion of the left coronary artery, VEGF-B deficiency impaired vessel growth in the ischemic myocardium whereas, in wild-type mice, VEGF-B(167) overexpression enhanced revascularization of the infarct and ischemic border zone. By contrast, VEGF-B deficiency did not affect vessel growth in the wounded skin, hypoxic lung, ischemic retina, or ischemic limb. Moreover, VEGF-B(167) overexpression failed to enhance vascular growth in the skin or ischemic limb. CONCLUSIONS: VEGF-B appears to have a relatively restricted angiogenic activity in the ischemic heart. These insights might offer novel therapeutic opportunities.

Longitudinal In Vivo Characterization of the Streptozotocin-Induced Diabetic Mouse Model: Focus on Early Inner Retinal Responses.

Purpose: The goal of this study was to perform an extensive temporal characterization of the early pathologic processes in the streptozotocin (STZ)-induced diabetic retinopathy (DR) mouse model, beyond the vascular phenotype, and to investigate the potential of clinically relevant compounds in attenuating these processes. Methods: Visual acuity and contrast sensitivity (CS) were studied in the mouse STZ model until 24 weeks postdiabetes onset. ERG, spectral domain optical coherence tomography (SD-OCT), leukostasis, and immunohistochemistry were applied to investigate neurodegeneration, inflammation, and gliosis during early-, mid- and late-phase diabetes. Aflibercept or triamcinolone acetonide (TAAC) was administered to investigate their efficacy on the aforementioned processes. Results: Visual acuity and CS loss started at 4 and 18 weeks postdiabetes onset, respectively, and progressively declined over time. ERG amplitudes were diminished and OP latencies increased after 6 weeks, whereas SD-OCT revealed retinal thinning from 4 weeks postdiabetes. Immunohistochemical analyses linked these findings to retinal ganglion and cholinergic amacrine cell loss at 4 and 8 weeks postdiabetes onset, respectively, which was further decreased after aflibercept administration. The number of adherent leukocytes was augmented after 2 weeks, whereas increased micro- and macroglia reactivity was present from 4 weeks postdiabetes. Aflibercept or TAAC showed improved efficacy on inflammation and gliosis. Conclusions: STZ-induced diabetic mice developed early pathologic DR hallmarks, from which inflammation seemed the initial trigger, leading to further development of functional and morphologic retinal changes. These findings indicate that the mouse STZ model is suitable to study novel integrative non-vascular therapies to treat early DR.

An Antagonistic Axon-Dendrite Interplay Enables Efficient Neuronal Repair in the Adult Zebrafish Central Nervous System.

Neural insults and neurodegenerative diseases typically result in permanent functional deficits, making the identification of novel pro-regenerative molecules and mechanisms a primary research topic. Nowadays, neuroregenerative research largely focuses on improving axonal regrowth, leaving the regenerative properties of dendrites largely unstudied. Moreover, whereas developmental studies indicate a strict temporal separation of axogenesis and dendritogenesis and thus suggest a potential interdependency of axonal and dendritic outgrowth, a possible axon-dendrite interaction during regeneration remains unexplored. To unravel the inherent dendritic response of vertebrate neurons undergoing successful axonal regeneration, regeneration-competent adult zebrafish of either sex, subjected to optic nerve crush (ONC), were used. A longitudinal study in which retinal ganglion cell (RGC) dendritic remodeling and axonal regrowth were assessed side-by-side after ONC, revealed that-as during development-RGC axogenesis precedes dendritogenesis during central nervous system (CNS) repair. Moreover, dendrites majorly shrank before the start of axonal regrowth and were only triggered to regrow upon RGC target contact initiation, altogether suggestive for a counteractive interplay between axons and dendrites after neuronal injury. Strikingly, both retinal mechanistic target of rapamycin (mTOR) and broad-spectrum matrix metalloproteinase (MMP) inhibition after ONC consecutively inhibited RGC synapto-dendritic deterioration and axonal regrowth, thus invigorating an antagonistic interplay wherein mature dendrites restrain axonal regrowth. Altogether, this work launches dendritic shrinkage as a prerequisite for efficient axonal regrowth of adult vertebrate neurons, and indicates that molecular/mechanistic analysis of dendritic responses after damage might represent a powerful target-discovery platform for neural repair.

Complementary research models and methods to study axonal regeneration in the vertebrate retinofugal system.

Due to the lack of axonal regeneration, age-related deterioration in the central nervous system (CNS) poses a significant burden on the wellbeing of a growing number of elderly. To overcome this regenerative failure and to improve the patient's life quality, the search for novel regenerative treatment strategies requires valuable (animal) models and techniques. As an extension of the CNS, the retinofugal system, consisting of retinal ganglion cells that send their axons along the optic nerve to the visual brain areas, has importantly contributed to the current knowledge on mechanisms underlying the restricted regenerative capacities and to the development of novel strategies to enhance axonal regeneration. It provides an extensively used research tool, not only in amniote vertebrates including rodents, but also in anamniote vertebrates, such as zebrafish. Indeed, the latter show robust regeneration capacities, thereby providing insights into the factors that contribute to axonal regrowth and proper guidance, complementing studies in mammals. This review provides an integrative and critical overview of the classical and state-of-the-art models and methods that have been employed in the retinofugal system to advance our knowledge on the signaling pathways underlying the restricted versus robust axonal regeneration in rodents and zebrafish, respectively. In vitro, ex vivo and in vivo models and techniques to improve the visualization and analysis of regenerating axons are summarized. As such, the retinofugal system is presented as a valuable model to further facilitate research on axonal regeneration and to open novel therapeutic avenues for CNS pathologies.

Matrix Metalloproteinases During Axonal Regeneration, a Multifactorial Role from Start to Finish.

By proteolytic cleavage, matrix metalloproteinases (MMPs) not only remodel the extracellular matrix (ECM) but they also modify the structure and activity of other proteinases, growth factors, signaling molecules, cell surface receptors, etc. Their vast substrate repertoire adds a complex extra dimension of biological control and turns MMPs into important regulatory nodes in the protease web. In the central nervous system (CNS), the detrimental impact of elevated MMP activities has been well-described for traumatic injuries and many neurodegenerative diseases. Nonetheless, there is ample proof corroborating MMPs as fine regulators of CNS physiology, and well-balanced MMP activity is instrumental to development, plasticity, and repair. In this manuscript, we review the emerging evidence for MMPs as beneficial modulators of axonal regeneration in the mammalian CNS. By exploring the multifactorial causes underlying the inability of mature axons to regenerate, and describing how MMPs can help to overcome these hurdles, we emphasize the benign actions of these Janus-faced proteases.