Single-cell transcriptomic profiling provides insights into retinal endothelial barrier properties.

Purpose: To better characterize retinal endothelial barrier properties through analysis of individual transcriptomes of primary bovine retinal microvascular endothelial cells (RMECs). Methods: Individual RMECs were captured on the Fluidigm C1 system, cDNA libraries were prepared using a Nextera XT kit, and sequencing was performed on a NextSeq system (Illumina). Data analysis was performed using R packages Scater, SC3, and Seurat, and the browser application Automated Single-cell Analysis Pipeline (ASAP). Alternative splicing events in single cells were quantified with Outrigger. Cytoscape was used for network analyses. Results: Application of a single-cell RNA sequencing (scRNA-seq) analysis workflow showed that RMECs form a relatively homogeneous population in culture, with the main differences related to proliferation status. Expression of markers from along the arteriovenous tree suggested that most cells originated from capillaries. Average gene expression levels across all cells were used to develop an in silico model of the inner blood-retina barrier incorporating junctional proteins not previously reported within the retinal vasculature. Correlation of barrier gene expression among individual cells revealed a subgroup of genes highly correlated with PECAM-1 at the center of the correlation network. Numerous alternative splicing events involving exons within microvascular barrier genes were observed, and in many cases, individual cells expressed one isoform exclusively. Conclusions: We optimized a workflow for single-cell transcriptomics in primary RMECs. The results provide fundamental insights into the genes involved in formation of the retinal-microvascular barrier.

Ion channels and myogenic activity in retinal arterioles.

Retinal pressure autoregulation is an important mechanism that protects the retina by stabilizing retinal blood flow during changes in arterial or intraocular pressure. Similar to other vascular beds, retinal pressure autoregulation is thought to be mediated largely through the myogenic response of small arteries and arterioles which constrict when transmural pressure increases or dilate when it decreases. Over recent years, we and others have investigated the signaling pathways underlying the myogenic response in retinal arterioles, with particular emphasis on the involvement of different ion channels expressed in the smooth muscle layer of these vessels. Here, we review and extend previous work on the expression and spatial distribution of the plasma membrane and sarcoplasmic reticulum ion channels present in retinal vascular smooth muscle cells (VSMCs) and discuss their contribution to pressure-induced myogenic tone in retinal arterioles. This includes new data demonstrating that several key players and modulators of the myogenic response show distinctively heterogeneous expression along the length of the retinal arteriolar network, suggesting differences in myogenic signaling between larger and smaller pre-capillary arterioles. Our immunohistochemical investigations have also highlighted the presence of actin-containing microstructures called myobridges that connect the retinal VSMCs to one another. Although further work is still needed, studies to date investigating myogenic mechanisms in the retina have contributed to a better understanding of how blood flow is regulated in this tissue. They also provide a basis to direct future research into retinal diseases where blood flow changes contribute to the pathology.

Müller glial dysfunction during diabetic retinopathy in rats is reduced by the acrolein-scavenging drug, 2-hydrazino-4,6-dimethylpyrimidine.

AIMS/HYPOTHESIS: Recent studies suggest that abnormal function in Müller glial cells plays an important role in the pathogenesis of diabetic retinopathy. This is associated with the selective accumulation of the acrolein-derived advanced lipoxidation end-product, Nε-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine), on Müller cell proteins. The aim of the current study was to identify more efficacious acrolein-scavenging drugs and determine the effects of the most potent on Müller cell FDP-lysine accumulation and neuroretinal dysfunction during diabetes. METHODS: An ELISA-based in vitro assay was optimised to compare the acrolein-scavenging abilities of a range of drugs. This identified 2-hydrazino-4,6-dimethylpyrimidine (2-HDP) as a new and potent acrolein scavenger. The ability of this agent to modify the development of diabetic retinopathy was tested in vivo. Male Sprague Dawley rats were divided into three groups: (1) non-diabetic; (2) streptozotocin-induced diabetic; and (3) diabetic treated with 2-HDP in their drinking water for the duration of diabetes. Liquid chromatography high-resolution mass spectrometry was used to detect 2-HDP reaction products in the retina. Immunohistochemistry, real-time quantitative (q)RT-PCR and electroretinography were used to assess retinal changes 3 months after diabetes induction. RESULTS: 2-HDP was the most potent of six acrolein-scavenging agents tested in vitro (p < 0.05). In vivo, administration of 2-HDP reduced Müller cell accumulation of FDP-lysine at 3 months in rats rendered diabetic with streptozotocin (p < 0.001). A 2-HDP adduct was identified in the retinas of diabetic animals treated with this compound. 2-HDP supplementation was associated with reduced Müller cell gliosis (p < 0.05), reduced expression of the oxidative stress marker haem oxygenase-1 (p < 0.001) and partial normalisation of inwardly rectifying K+ channel 4.1 (Kir4.1) expression (p < 0.001 for staining in perivascular regions and the innermost region of the ganglion cell layer). Diabetes-induced retinal expression of inflammatory markers, inflammatory signalling compounds and activation of retinal microglial cells were all reduced in 2-HDP-treated animals. Retinal neurophysiological defects in diabetic animals, as indicated by changes in the electroretinogram 7 weeks after induction of diabetes, were also reduced by 2-HDP (p < 0.05-0.01 for b-wave amplitudes at flash intensities from -10 to +10 dB; p < 0.01 for time to peak of summed oscillatory potentials at +10 dB). CONCLUSIONS/INTERPRETATION: These findings support the hypothesis that Müller cell accumulation of FDP-lysine plays an important role in the development of diabetic retinopathy. Our results also suggest that 2-HDP may have therapeutic potential for delaying or treating this sight-threatening complication.

Store-Operated Calcium Entry in Müller Glia Is Controlled by Synergistic Activation of TRPC and Orai Channels.

The endoplasmic reticulum (ER) is at the epicenter of astrocyte Ca(2+) signaling. We sought to identify the molecular mechanism underlying store-operated calcium entry that replenishes ER stores in mouse Müller cells. Store depletion, induced through blockade of sequestration transporters in Ca(2+)-free saline, induced synergistic activation of canonical transient receptor potential 1 (TRPC1) and Orai channels. Store-operated TRPC1 channels were identified by their electrophysiological properties, pharmacological blockers, and ablation of the Trpc1 gene. Ca(2+) release-activated currents (ICRAC) were identified by ion permeability, voltage dependence, and sensitivity to selective Orai antagonists Synta66 and GSK7975A. Depletion-evoked calcium influx was initiated at the Müller end-foot and apical process, triggering centrifugal propagation of Ca(2+) waves into the cell body. EM analysis of the end-foot compartment showed high-density ER cisternae that shadow retinal ganglion cell (RGC) somata and axons, protoplasmic astrocytes, vascular endothelial cells, and ER-mitochondrial contacts at the vitreal surface of the end-foot. The mouse retina expresses transcripts encoding both Stim and all known Orai genes; Müller glia predominantly express stromal interacting molecule 1 (STIM1), whereas STIM2 is mainly confined to the outer plexiform and RGC layers. Elimination of TRPC1 facilitated Müller gliosis induced by the elevation of intraocular pressure, suggesting that TRPC channels might play a neuroprotective role during mechanical stress. By characterizing the properties of store-operated signaling pathways in Müller cells, these studies expand the current knowledge about the functional roles these cells play in retinal physiology and pathology while also providing further evidence for the complexity of calcium signaling mechanisms in CNS astroglia. SIGNIFICANCE STATEMENT: Store-operated Ca(2+) signaling represents a major signaling pathway and source of cytosolic Ca(2+) in astrocytes. Here, we show that the store-operated response in Müller cells, radial glia that perform key structural, signaling, osmoregulatory, and mechanosensory functions within the retina, is mediated through synergistic activation of transient receptor potential and Orai channels. The end-foot disproportionately expresses the depletion sensor stromal interacting molecule 1, which contains an extraordinarily high density of endoplasmic reticulum cisternae that shadow neuronal, astrocytic, vascular, and axonal structures; interface with mitochondria; but also originate store-operated Ca(2+) entry-induced transcellular Ca(2+) waves that propagate glial excitation into the proximal retina. These results identify a molecular mechanism that underlies complex interactions between the plasma membrane and calcium stores, and contributes to astroglial function, regulation, and response to mechanical stress.

Mouse Models of Stargardt 3 Dominant Macular Degeneration.

Stargardt type 3 macular degeneration is dependent on a dominant defect in a single gene, ELOVL4 (elongase of very long chain fatty acids 4). The encoded enzyme, ELOVL4, is required for the synthesis of very long chain polyunsaturated fatty acids (VLC-PUFAs), a rare class of > C24 lipids. In vitro expression studies suggest that mutated ELOVL4(STGD3) proteins fold improperly, resulting in ER stress and formation of cytosolic aggresomes of wild type and mutant ELOVL4. Although a number of mouse models have been developed to determine whether photoreceptor cell loss in STGD3 results from depletion of VLC-PUFAs, aggresome-dependent cell stress or a combination of these two factors, none of these models adequately recapitulates the disease phenotype in humans. Thus, the precise molecular mechanism by which ELOVL4 mutation causes photoreceptor degeneration in mice and in human patients remains to be characterized. This mini review compares and evaluates current STGD3 mouse models and determines what conclusions can be drawn from past work.

Role of ELOVL4 and very long-chain polyunsaturated fatty acids in mouse models of Stargardt type 3 retinal degeneration.

Stargardt type 3 (STGD3) disease is a juvenile macular dystrophy caused by mutations in the ELOVL4 (Elongation of very long chain fatty acids 4) gene. Its protein product, ELOVL4, is an elongase required for the biosynthesis of very long-chain polyunsaturated fatty acids (VLC-PUFAs). It is unclear whether photoreceptor degeneration in STGD3 is caused by loss of VLC-PUFAs or by mutated ELOVL4 protein trafficking/aggregation. We therefore generated conditional knockout (cKO) mice with Elovl4 ablated in rods or cones and compared their phenotypes to transgenic (TG) animals that express the human STGD3-causing ELOVL4(STGD3) allele. Gas chromatography-mass spectrometry was used to assess C30-C34 VLC-PUFA and N-retinylidene-N-retinylethanolamine content; electroretinography was used to measure phototransduction and outer retinal function; electron microscopy was used for retinal ultrastructure; and the optomotor tracking response was used to test scotopic and photopic visual performance. Elovl4 transcription and biosynthesis of C30-C34 VLC-PUFAs in rod cKO and TG retinas were reduced up to 98%, whereas the content of docosahexaenoic acid was diminished in TG, but not rod cKO, retinas. Despite the near-total loss of the retinal VLC-PUFA content, rod and cone cKO animals exhibited no electrophysiological or behavioral deficits, whereas the typical rod-cone dystrophic pattern was observed in TG animals. Our data suggest that photoreceptor-specific VLC-PUFA depletion is not sufficient to induce the STGD3 phenotype, because depletion alone had little effect on photoreceptor survival, phototransduction, synaptic transmission, and visual behavior.

A trans-kingdom T6SS effector induces the fragmentation of the mitochondrial network and activates innate immune receptor NLRX1 to promote infection.

Bacteria can inhibit the growth of other bacteria by injecting effectors using a type VI secretion system (T6SS). T6SS effectors can also be injected into eukaryotic cells to facilitate bacterial survival, often by targeting the cytoskeleton. Here, we show that the trans-kingdom antimicrobial T6SS effector VgrG4 from Klebsiella pneumoniae triggers the fragmentation of the mitochondrial network. VgrG4 colocalizes with the endoplasmic reticulum (ER) protein mitofusin 2. VgrG4 induces the transfer of Ca2+ from the ER to the mitochondria, activating Drp1 (a regulator of mitochondrial fission) thus leading to mitochondrial network fragmentation. Ca2+ elevation also induces the activation of the innate immunity receptor NLRX1 to produce reactive oxygen species (ROS). NLRX1-induced ROS limits NF-κB activation by modulating the degradation of the NF-κB inhibitor IκBα. The degradation of IκBα is triggered by the ubiquitin ligase SCFß-TrCP, which requires the modification of the cullin-1 subunit by NEDD8. VgrG4 abrogates the NEDDylation of cullin-1 by inactivation of Ubc12, the NEDD8-conjugating enzyme. Our work provides an example of T6SS manipulation of eukaryotic cells via alteration of the mitochondria.

The polymodal ion channel transient receptor potential vanilloid 4 modulates calcium flux, spiking rate, and apoptosis of mouse retinal ganglion cells.

Sustained increase in intraocular pressure represents a major risk factor for eye disease, yet the cellular mechanisms of pressure transduction in the posterior eye are essentially unknown. Here we show that the mouse retina expresses mRNA and protein for the polymodal transient receptor potential vanilloid 4 (TRPV4) cation channel known to mediate osmotransduction and mechanotransduction. TRPV4 antibodies labeled perikarya, axons, and dendrites of retinal ganglion cells (RGCs) and intensely immunostained the optic nerve head. Müller glial cells, but not retinal astrocytes or microglia, also expressed TRPV4 immunoreactivity. The selective TRPV4 agonists 4α-PDD and GSK1016790A elevated [Ca2+]i in dissociated RGCs in a dose-dependent manner, whereas the TRPV1 agonist capsaicin had no effect on [Ca2+](RGC). Exposure to hypotonic stimulation evoked robust increases in [Ca2+](RGC). RGC responses to TRPV4-selective agonists and hypotonic stimulation were absent in Ca2+ -free saline and were antagonized by the nonselective TRP channel antagonists Ruthenium Red and gadolinium, but were unaffected by the TRPV1 antagonist capsazepine. TRPV4-selective agonists increased the spiking frequency recorded from intact retinas recorded with multielectrode arrays. Sustained exposure to TRPV4 agonists evoked dose-dependent apoptosis of RGCs. Our results demonstrate functional TRPV4 expression in RGCs and suggest that its activation mediates response to membrane stretch leading to elevated [Ca2+]i and augmented excitability. Excessive Ca2+ influx through TRPV4 predisposes RGCs to activation of Ca2+ -dependent proapoptotic signaling pathways, indicating that TRPV4 is a component of the response mechanism to pathological elevations of intraocular pressure.

Store-operated channels regulate intracellular calcium in mammalian rods.

Exposure to daylight closes cyclic nucleotide-gated (CNG) and voltage-operated Ca(2+) -permeable channels in mammalian rods. The consequent lowering of the cytosolic calcium concentration ([Ca(2+)](i)), if protracted, can contribute to light-induced damage and apoptosis in these cells. We here report that mouse rods are protected against prolonged lowering of [Ca(2+)](i) by store-operated Ca(2+) entry (SOCE). Ca(2+) stores were depleted in Ca(2+)-free saline supplemented with the endoplasmic reticulum (ER) sequestration blocker cyclopiazonic acid. Store depletion elicited [Ca(2+)](i) signals that exceeded baseline [Ca(2+)](i) by 5.9 ± 0.7-fold and were antagonized by an inhibitory cocktail containing 2-APB, SKF 96365 and Gd(3+). Cation influx through SOCE channels was sufficient to elicit a secondary activation of L-type voltage-operated Ca2+ entry. We also found that TRPC1, the type 1 canonical mammalian homologue of the Drosophila photoreceptor TRP channel, is predominantly expressed within the outer nuclear layer of the retina. Rod loss in Pde6b(rdl) (rd1), Chx10/Kip1(-/-rdl) and Elovl4(TG2) dystrophic models was associated with ∼70% reduction in Trpc1 mRNA content whereas Trpc1 mRNA levels in rodless cone-full Nrl(-/-) retinas were decreased by ∼50%. Genetic ablation of TRPC1 channels, however, had no effect on SOCE, the sensitivity of the rod phototransduction cascade or synaptic transmission at rod and cone synapses. Thus, we localized two new mechanisms, SOCE and TRPC1, to mammalian rods and characterized the contribution of SOCE to Ca(2+) homeostasis. By preventing the cytosolic [Ca(2+)](i) from dropping too low under sustained saturating light conditions, these signalling pathways may protect Ca(2+)-dependent mechanisms within the ER and the cytosol without affecting normal rod function.

Dynamic Navigation for Dental Implant Placement.

In modern implantology, the application of surgical navigation systems is becoming increasingly important. In addition to static surgical navigation methods, a guide-independent dynamic navigation implant placement procedure is becoming more widespread. The procedure is based on computer-guided dental implant placement utilizing optical control. This work aims to demonstrate the technical steps of a new dynamic computer-aided implant surgery (DCAIS) system (design, calibration, surgery) and check the accuracy of the results. Based on cone-beam computed tomography (CBCT) scans, the exact positions of implants are determined with dedicated software. The first step of the operation is the calibration of the navigation system, which can be performed in two ways: 1) based on CBCT images taken with a marker or 2) based on CBCT images without markers. Implants are inserted with the aid of real-time navigation according to the preoperative plans. The accuracy of the interventions can be evaluated based on postoperative CBCT images. The preoperative images containing the planned positions of the implants and postoperative CBCT images were compared based on the angulation (degree), platform, and apical deviation (mm) of the implants. To evaluate the data, we calculated the standard deviation (SD), mean, and standard error of the mean (SEM) of deviations within planned and performed implant positions. Differences between the two calibration methods were compared based on this data. Based on the interventions performed so far, the use of DCAIS allows for high-precision implant placement. A calibration system that does not require labeled CBCT recording allows for surgical intervention with similar accuracy as a system that uses labeling. The accuracy of the intervention can be improved by training.

Cherubism in three siblings. / A cherubismus elofordulása három testvérben.

Összefoglaló. A cherubismus ritka, autoszomális dominánsan öröklodo megbetegedés. A fibroossealis elváltozások csoportjába tartozik. Jellemzoje az állcsontok szimmetrikus duzzanata, a típusos radiológiai elváltozások és az SH3BP2-gén mutációja. Szövettanilag nem különül el az óriássejtes granulomától. A csontelváltozások és a fibroticus szövet felszaporodása pubertás elott kezdodik, ezután stagnálás vagy visszafejlodés következik be. A magyar orvosi irodalomban a szerzok elsoként tárgyalják három testvér kórtörténete alapján a cherubismust. A diagnózist a hasonló klinikai tünetek, a típusos kórlefolyás, a szinte azonos radiológiai kép, a szövettan és a genetikai elváltozások biztosítják. A testvérek és az anya csíravonalában kimutatott azonos mutáció akkor is megfelel egy dominánsan öröklodo szindrómának (például cherubismusnak), ha a betegség az anyában klinikailag nem manifesztálódott, de genetikailag igen. A szerzok összefoglalják a kórkép kezelési lehetoségeit: a sebészi (excochleatio, ,,decountouring", esetleg reszekció) és a gyógyszeres (biszfoszfonát, kalcitonin, szteroid stb.) terápiát. Egyezik a véleményük azokéval, akik azon az állásponton vannak, hogy a beavatkozásokkal várni kell, és meg kell figyelni a betegeket a várható regresszió miatt. Saját eseteikben csak a növekvo tumorrész excochleatióját végezték, foleg kozmetikai okok és a szövettan biztosítása érdekében. Orv Hetil. 2022; 163(11): 446-452. Summary. Cherubism is a rare autosomal, dominant bone disorder, characterised by symmetrical expansion of the jaws along the typical radiological and genetic (SH3BP2 mutation) features. It belongs to the heterogenous group of fibro-osseous lesions. Its histology is the same as that of giant-cell granuloma. The bone lesions and fibrous tissue expansion increase before puberty and regress thereafter. For the first time in Hungarian medical literature, the authors discuss the condition of cherubism in the case of three siblings. The diagnosis of these three siblings is supported by the clinical, radiological, microscopic and genetic data. In all three, the bone lesions and fibrous tissue expansion increased before puberty and stabilized thereafter. The radiological results and the molecular findings were nearly identical. The identical mutation shown in the germ lines of the three siblings and the mother correspond to a dominantly inherited syndrome (e.g., cherubism) even if the condition did not manifest in the mother. The authors summarize the treatment options of the disease: surgical (excochleation, decountouring, in rare case resection) and drug (bisphosphonate, calcitonin, steroid, etc.) therapy. They agree with those who are of the opinion that interventions should wait and the patients should be observed ("wait and see") for the expected regression. In their own cases, only excochleation of the growing tumor was performed, mainly for cosmetic reasons and to secure the tissue. Orv Hetil. 2022; 163(11): 446-452.

Loss of TRPV2-mediated blood flow autoregulation recapitulates diabetic retinopathy in rats.

Loss of retinal blood flow autoregulation is an early feature of diabetes that precedes the development of clinically recognizable diabetic retinopathy (DR). Retinal blood flow autoregulation is mediated by the myogenic response of the retinal arterial vessels, a process that is initiated by the stretch­dependent activation of TRPV2 channels on the retinal vascular smooth muscle cells (VSMCs). Here, we show that the impaired myogenic reaction of retinal arterioles from diabetic animals is associated with a complete loss of stretch­dependent TRPV2 current activity on the retinal VSMCs. This effect could be attributed, in part, to TRPV2 channel downregulation, a phenomenon that was also evident in human retinal VSMCs from diabetic donors. We also demonstrate that TRPV2 heterozygous rats, a nondiabetic model of impaired myogenic reactivity and blood flow autoregulation in the retina, develop a range of microvascular, glial, and neuronal lesions resembling those observed in DR, including neovascular complexes. No overt kidney pathology was observed in these animals. Our data suggest that TRPV2 dysfunction underlies the loss of retinal blood flow autoregulation in diabetes and provide strong support for the hypothesis that autoregulatory deficits are involved in the pathogenesis of DR.

Intracellular pH modulates inner segment calcium homeostasis in vertebrate photoreceptors.

Neuronal metabolic and electrical activity is associated with shifts in intracellular pH (pH(i)) proton activity and state-dependent changes in activation of signaling pathways in the plasma membrane, cytosol, and intracellular compartments. We investigated interactions between two intracellular messenger ions, protons and calcium (Ca²(+)), in salamander photoreceptor inner segments loaded with Ca²(+) and pH indicator dyes. Resting cytosolic pH in rods and cones in HEPES-based saline was acidified by ∼0.4 pH units with respect to pH of the superfusing saline (pH = 7.6), indicating that dissociated inner segments experience continuous acid loading. Cytosolic alkalinization with ammonium chloride (NH4Cl) depolarized photoreceptors and stimulated Ca²(+) release from internal stores, yet paradoxically also evoked dose-dependent, reversible decreases in [Ca²(+)](i). Alkalinization-evoked [Ca²(+)](i) decreases were independent of voltage-operated and store-operated Ca²(+) entry, plasma membrane Ca²(+) extrusion, and Ca²(+) sequestration into internal stores. The [Ca²(+)](i)-suppressive effects of alkalinization were antagonized by the fast Ca²(+) buffer BAPTA, suggesting that pH(i) directly regulates Ca²(+) binding to internal anionic sites. In summary, this data suggest that endogenously produced protons continually modulate the membrane potential, release from Ca²(+) stores, and intracellular Ca²(+) buffering in rod and cone inner segments.

Calcium signals in the nucleus accumbens: activation of astrocytes by ATP and succinate.

BACKGROUND: Accumulating evidence suggests that glial signalling is activated by different brain functions. However, knowledge regarding molecular mechanisms of activation or their relation to neuronal activity is limited. The purpose of the present study is to identify the characteristics of ATP-evoked glial signalling in the brain reward area, the nucleus accumbens (NAc), and thereby to explore the action of citric acid cycle intermediate succinate (SUC). RESULTS: We described the burst-like propagation of Ca2+ transients evoked by ATP in acute NAc slices from rat brain. Co-localization of the ATP-evoked Ca2+ signalling with immunoreactivities of the astroglia-specific gap junction forming channel protein connexin43 (Cx43) and the glial fibrillary acidic protein (GFAP) indicated that the responsive cells were a subpopulation of Cx43 and GFAP immunoreactive astrocytes. The ATP-evoked Ca2+ transients were present under the blockade of neuronal activity, but were inhibited by Ca2+ store depletion and antagonism of the G protein coupled purinergic P2Y1 receptor subtype-specific antagonist MRS2179. Similarly, Ca2+ transients evoked by the P2Y1 receptor subtype-specific agonist 2-(Methylthio)adenosine 5'-diphosphate were also blocked by MRS2179. These characteristics implied that intercellular Ca2+ signalling originated from the release of Ca2+ from internal stores, triggered by the activation of P2Y1 receptors. Inhibition by the gap junction blockers carbenoxolone and flufenamic acid and by an antibody raised against the gating-associated segment of Cx43 suggested that intercellular Ca2+ signalling proceeded through gap junctions. We demonstrated for the first time that extracellular SUC also evoked Ca2+ transients (EC50 = 50-60 µM) in about 15% of the ATP-responsive NAc astrocytes. By contrast to glial cells, electrophysiologically identified NAc neurons surrounded by ATP-responsive astrocytes were not activated simultaneously. CONCLUSIONS: We concluded, therefore, that ATP- and SUC-sensitive Ca2+ transients appear to represent a signalling layer independent of NAc neurons. This previously unrecognised glial action of SUC, a major cellular energy metabolite, may play a role in linking metabolism to Ca2+ signalling in astrocytic networks under physiological and pathological conditions such as exercise and metabolic diseases.

COMP-Ang1: Therapeutic potential of an engineered Angiopoietin-1 variant.

The Angiopoietin-1/2 system is an opportune target for therapeutic intervention in a wide range of vascular pathologies, particularly through its association with endothelium. The complex multi-domain structure of native human Angiopoietin-1 has hindered its widespread applicability as a therapeutic agent, prompting the search for alternative approaches to mimicking the Ang1:Tie2 signalling axis; a system with highly complex patterns of regulation involving multiple structurally similar molecules. An engineered variant, Cartilage Oligomeric Matrix Protein - Angiopoietin-1 (COMP-Ang1), has been demonstrated to overcome the limitations of the native molecule and activate the Tie2 pathway with several fold greater potency than Ang1, both in vitro and in vivo. The therapeutic efficacy of COMP-Ang1, at both the vascular and systemic levels, is evident from multiple studies. Beneficial impacts on skeletal muscle regeneration, wound healing and angiogenesis have been reported alongside renoprotective, anti-hypertensive and anti-inflammatory effects. COMP-Ang1 has also demonstrated synergy with other compounds to heighten bone repair, has been leveraged for potential use as a co-therapeutic for enhanced targeted cancer treatment, and has received considerable attention as an anti-leakage agent for microvascular diseases like diabetic retinopathy. This review examines the vascular Angiopoietin:Tie2 signalling mechanism, evaluates the potential therapeutic merits of engineered COMP-Ang1 in both vascular and systemic contexts, and addresses the inherent translational challenges in moving this potential therapeutic from bench-to-bedside.

Glutamate-induced internalization of Ca(v)1.3 L-type Ca(2+) channels protects retinal neurons against excitotoxicity.

Glutamate-induced rise in the intracellular Ca(2+) level is thought to be a major cause of excitotoxic cell death, but the mechanisms that control the Ca(2+) overload are poorly understood. Using immunocytochemistry, electrophysiology and Ca(2+) imaging, we show that activation of ionotropic glutamate receptors induces a selective internalization of Ca(v)1.3 L-type Ca(2+) channels in salamander retinal neurons. The effect of glutamate on Ca(v)1.3 internalization was blocked in Ca(2+)-free external solution, or by strong buffering of internal Ca(2+) with BAPTA. Downregulation of L-type Ca(2+) channel activity in retinal ganglion cells by glutamate was suppressed by inhibitors of dynamin-dependent endocytosis. Stabilization of F-actin by jasplakinolide significantly reduced the ability of glutamate to induce internalization suggesting it is mediated by Ca(2+)-dependent reorganization of actin cytoskeleton. We showed that the Ca(v)1.3 is the primary L-type Ca(2+) channel contributing to kainate-induced excitotoxic death of amacrine and ganglion cells. Block of Ca(v)1.3 internalization by either dynamin inhibition or F-actin stabilization increased vulnerability of retinal amacrine and ganglion cells to kainate-induced excitotoxicity. Our data show for the first time that Ca(v)1.3 L-type Ca(2+) channels are subject to rapid glutamate-induced internalization, which may serve as a negative feedback mechanism protecting retinal neurons against glutamate-induced excitotoxicity.

Do calcium channel blockers rescue dying photoreceptors in the Pde6b ( rd1 ) mouse?

Retinitis pigmentosa (RP) is a genetically heterogeneous set of blinding diseases that affects more than a million people worldwide. In humans, ~5-8% of recessive and dominant RP cases are caused by nonsense mutations in the Pde6b gene coding for the ss-subunit of the rod photoreceptor cGMP phosphodiesterase 6 (PDE6-ss). The study of the disease has been greatly aided by the Pde6b ( rd1 ) (rd1) mouse model of RP carrying a null PDE6ss allele. Degenerating rd1 rods were found to experience a pathological increase in intracellular calcium concentration ('Ca overload') when they enter the apoptotic process at postnatal day 10. A 1999 study suggested that the Ca(2+) channel antagonist D-cis diltiazem delays the kinetics of rd1 rod degeneration, conferring partial rescue of scotopic vision. Subsequent reports were mixed: whereas several studies failed to replicate the original results, others appeared to confirm the neuroprotective effects of Ca(2+) channel antagonists such as diltiazem, nilvadipine and verapamil. We discuss the discrepancies between the results of different groups and suggest plausible causes for the discordant results. We also discuss potential involvement of recently identified Ca(2+)-dependent mechanisms that include protective calcium ATPase mechanisms, ryanodine and IP3 calcium stores, and store operated channels in Pde6b ( rd1 ) neurodegeneration.

VEGF-B Is an Autocrine Gliotrophic Factor for Müller Cells under Pathologic Conditions.

Purpose: Müller glia are important in retinal health and disease and are a major source of retinal VEGF-A. Of the different VEGF family members, the role of VEGF-A in retinal health and disease has been studied extensively. The potential contribution of other VEGF family members to retinal pathophysiology, however, remains poorly defined. This study aimed to understand the role of VEGF-B in Müller cell pathophysiology. Methods: The expression of different VEGFs and their receptors in human MIO-M1 and mouse QMMuC-1 Müller cell lines and primary murine Müller cells was examined by RT-PCR, ELISA, and Western blot. The effect of recombinant VEGF-B or VEGF-B neutralization on Müller cell viability and survival under normal, hypoxic, and oxidative (4-hydroxynonenal [4-HNE]) conditions was evaluated by Alamar Blue, Yo-Pro uptake, and immunocytochemistry. The expression of glial fibrillary acidic protein, aquaporin-4, inward rectifying K+ channel subtype 4.1, glutamine synthetase, and transient receptor potential vanilloid 4 under different treatment conditions was examined by RT-PCR, immunocytochemistry, and Western blot. Transient receptor potential vanilloid 4 channel activity was assessed using a Fura-2-based calcium assay. Results: VEGF-B was expressed in Müller cells at the highest levels compared with other members of the VEGF family. VEGF-B neutralization did not affect Müller cell viability or functionality under normal conditions, but enhanced hypoxia- or 4-HNE-induced Müller cell death and decreased inward rectifying K+ channel subtype 4.1 and aquaporin-4 expression. Recombinant VEGF-B restored Müller cell glutamine synthetase expression under hypoxic conditions and protected Müller cells from 4-HNE-induced damage by normalizing transient receptor potential vanilloid 4 channel expression and activity. Conclusions: Autocrine production of VEGF-B protects Müller cells under pathologic conditions.

The Role of Lipoxidation in the Pathogenesis of Diabetic Retinopathy.

Lipids can undergo modification as a result of interaction with reactive oxygen species (ROS). For example, lipid peroxidation results in the production of a wide variety of highly reactive aldehyde species which can drive a range of disease-relevant responses in cells and tissues. Such lipid aldehydes react with nucleophilic groups on macromolecules including phospholipids, nucleic acids, and proteins which, in turn, leads to the formation of reversible or irreversible adducts known as advanced lipoxidation end products (ALEs). In the setting of diabetes, lipid peroxidation and ALE formation has been implicated in the pathogenesis of macro- and microvascular complications. As the most common diabetic complication, retinopathy is one of the leading causes of vision loss and blindness worldwide. Herein, we discuss diabetic retinopathy (DR) as a disease entity and review the current knowledge and experimental data supporting a role for lipid peroxidation and ALE formation in the onset and development of this condition. Potential therapeutic approaches to prevent lipid peroxidation and lipoxidation reactions in the diabetic retina are also considered, including the use of antioxidants, lipid aldehyde scavenging agents and pharmacological and gene therapy approaches for boosting endogenous aldehyde detoxification systems. It is concluded that further research in this area could lead to new strategies to halt the progression of DR before irreversible retinal damage and sight-threatening complications occur.

COMP-Ang1 Stabilizes Hyperglycemic Disruption of Blood-Retinal Barrier Phenotype in Human Retinal Microvascular Endothelial Cells.

Purpose: Current treatments for diabetic retinopathy (DR) have considerable limitations, underpinning the need for new therapeutic options. In this article, the ability of an engineered angiopoietin-1 variant (COMP-Ang1) to ameliorate the injurious effects of hyperglycemia on barrier integrity in a human retinal microvascular endothelial cell (HRMvEC) model is comprehensively investigated. Methods: Confluent HRMvECs were treated (0-72 hours) with d-glucose (5 or 30 mM) in the absence and presence of COMP-Ang1 (10-200 ng/mL). l-glucose (30 mM) was used as osmotic control. Posttreatment, intact cell monolayers were monitored for permeability to FITC-dextran 40 kDa. Cells were also harvested for analysis of interendothelial junction targets by RT-qPCR and Western blotting. The impact of receptor tyrosine kinase Tie2 gene silencing on COMP-Ang1 efficacy was also evaluated. Results: Treatment with 30 mM d-glucose (but not l-glucose) demonstrated a time-dependent elevation in the mean rate of FITC-dextran diffusion across intact HRMvEC monolayers, in parallel with significant reductions in mRNA/protein levels of occludin, claudin-5, ZO-1, and VE-Cadherin. These effects were all attenuated by COMP-Ang1 in a concentration-dependent fashion, with 200 ng/mL recovering barrier function by ∼88%, and recovering reduced interendothelial junction protein levels by more than 50%. Finally, Tie2 knockdown by small interfering RNA silencing blocked the ability of COMP-Ang1 to mitigate against hyperglycemia-induced permeabilization of HRMvECs and depletion of junctional expression levels. Conclusions: In summary, this article presents a reproducible in vitro cell study that quantifies the concentration-dependent efficacy of COMP-Ang1 to mitigate the injurious effects of hyperglycemic challenge on HRMvEC barrier properties via Tie2-mediated signaling.