Missense variants in ANO4 cause sporadic encephalopathic or familial epilepsy with evidence for a dominant-negative effect.

Anoctamins are a family of Ca2+-activated proteins that may act as ion channels and/or phospholipid scramblases with limited understanding of function and disease association. Here, we identified five de novo and two inherited missense variants in ANO4 (alias TMEM16D) as a cause of fever-sensitive developmental and epileptic or epileptic encephalopathy (DEE/EE) and generalized epilepsy with febrile seizures plus (GEFS+) or temporal lobe epilepsy. In silico modeling of the ANO4 structure predicted that all identified variants lead to destabilization of the ANO4 structure. Four variants are localized close to the Ca2+ binding sites of ANO4, suggesting impaired protein function. Variant mapping to the protein topology suggests a preliminary genotype-phenotype correlation. Moreover, the observation of a heterozygous ANO4 deletion in a healthy individual suggests a dysfunctional protein as disease mechanism rather than haploinsufficiency. To test this hypothesis, we examined mutant ANO4 functional properties in a heterologous expression system by patch-clamp recordings, immunocytochemistry, and surface expression of annexin A5 as a measure of phosphatidylserine scramblase activity. All ANO4 variants showed severe loss of ion channel function and DEE/EE associated variants presented mild loss of surface expression due to impaired plasma membrane trafficking. Increased levels of Ca2+-independent annexin A5 at the cell surface suggested an increased apoptosis rate in DEE-mutant expressing cells, but no changes in Ca2+-dependent scramblase activity were observed. Co-transfection with ANO4 wild-type suggested a dominant-negative effect. In summary, we expand the genetic base for both encephalopathic sporadic and inherited fever-sensitive epilepsies and link germline variants in ANO4 to a hereditary disease.

Splenic monocytes drive pathogenic subretinal inflammation in age-related macular degeneration.

Age-related macular degeneration (AMD) is invariably associated with the chronic accumulation of activated mononuclear phagocytes in the subretinal space. The mononuclear phagocytes are composed of microglial cells but also of monocyte-derived cells, which promote photoreceptor degeneration and choroidal neovascularization. Infiltrating blood monocytes can originate directly from bone marrow, but also from a splenic reservoir, where bone marrow monocytes develop into angiotensin II receptor (ATR1)+ splenic monocytes. The involvement of splenic monocytes in neurodegenerative diseases such as AMD is not well understood. Using acute inflammatory and well-phenotyped AMD models, we demonstrate that angiotensin II mobilizes ATR1+ splenic monocytes, which we show are defined by a transcriptional signature using single-cell RNA sequencing and differ functionally from bone marrow monocytes. Splenic monocytes participate in the chorio-retinal infiltration and their inhibition by ATR1 antagonist and splenectomy reduces the subretinal mononuclear phagocyte accumulation and pathological choroidal neovascularization formation. In aged AMD-risk ApoE2-expressing mice, a chronic AMD model, ATR1 antagonist and splenectomy also inhibit the chronic retinal inflammation and associated cone degeneration that characterizes these mice. Our observation of elevated levels of plasma angiotensin II in AMD patients, suggests that similar events take place in clinical disease and argue for the therapeutic potential of ATR1 antagonists to inhibit splenic monocytes for the treatment of blinding AMD.

FoxP3 expression by retinal pigment epithelial cells: transcription factor with potential relevance for the pathology of age-related macular degeneration.

BACKGROUND: Forkhead-Box-Protein P3 (FoxP3) is a transcription factor and marker of regulatory T cells, converting naive T cells into Tregs that can downregulate the effector function of other T cells. We previously detected the expression of FoxP3 in retinal pigment epithelial (RPE) cells, forming the outer blood-retina barrier of the immune privileged eye. METHODS: We investigated the expression, subcellular localization, and phosphorylation of FoxP3 in RPE cells in vivo and in vitro after treatment with various stressors including age, retinal laser burn, autoimmune inflammation, exposure to cigarette smoke, in addition of IL-1ß and mechanical cell monolayer destruction. Eye tissue from humans, mouse models of retinal degeneration and rats, and ARPE-19, a human RPE cell line for in vitro experiments, underwent immunohistochemical, immunofluorescence staining, and PCR or immunoblot analysis to determine the intracellular localization and phosphorylation of FoxP3. Cytokine expression of stressed cultured RPE cells was investigated by multiplex bead analysis. Depletion of the FoxP3 gene was performed with CRISPR/Cas9 editing. RESULTS: RPE in vivo displayed increased nuclear FoxP3-expression with increases in age and inflammation, long-term exposure of mice to cigarette smoke, or after laser burn injury. The human RPE cell line ARPE-19 constitutively expressed nuclear FoxP3 under non-confluent culture conditions, representing a regulatory phenotype under chronic stress. Confluently grown cells expressed cytosolic FoxP3 that was translocated to the nucleus after treatment with IL-1ß to imitate activated macrophages or after mechanical destruction of the monolayer. Moreover, with depletion of FoxP3, but not of a control gene, by CRISPR/Cas9 gene editing decreased stress resistance of RPE cells. CONCLUSION: Our data suggest that FoxP3 is upregulated by age and under cellular stress and might be important for RPE function.

Prediction of Functional Consequences of Missense Mutations in ANO4 Gene.

The anoctamin (TMEM16) family of transmembrane protein consists of ten members in vertebrates, which act as Ca2+-dependent ion channels and/or Ca2+-dependent scramblases. ANO4 which is primarily expressed in the CNS and certain endocrine glands, has been associated with various neuronal disorders. Therefore, we focused our study on prioritizing missense mutations that are assumed to alter the structure and stability of ANO4 protein. We employed a wide array of evolution and structure based in silico prediction methods to identify potentially deleterious missense mutations in the ANO4 gene. Identified pathogenic mutations were then mapped to the modeled human ANO4 structure and the effects of missense mutations were studied on the atomic level using molecular dynamics simulations. Our data show that the G80A and A500T mutations significantly alter the stability of the mutant proteins, thus providing new perspective on the role of missense mutations in ANO4 gene. Results obtained in this study may help to identify disease associated mutations which affect ANO4 protein structure and function and might facilitate future functional characterization of ANO4.

Anti-Inflammatory Role of Netrin-4 in Diabetic Retinopathy.

Diabetic retinopathy is characterized by dysfunction of the retinal vascular network, combined with a persistent low-grade inflammation that leads to vision-threatening complications. Netrin-4 (NTN4) is a laminin-related secreted protein and guidance cue molecule present in the vascular basal membrane and highly expressed in the retina. A number of studies inferred that the angiogenic abilities of NTN4 could contribute to stabilize vascular networks and modulate inflammation. Analyzing human specimens, we show that NTN4 and netrin receptors are upregulated in the diabetic retina. We further evaluated a knock-out model for NTN4 undergoing experimental diabetes induced by streptozotocin. We investigated retina function and immune cells in vivo and demonstrated that NTN4 provides a protective milieu against inflammation in the diabetic retina and prevents cytokine production.

Effects of TNFα receptor TNF-Rp55- or TNF-Rp75- deficiency on corneal neovascularization and lymphangiogenesis in the mouse.

Tumor necrosis factor (TNF)α is an inflammatory cytokine likely to be involved in the process of corneal inflammation and neovascularization. In the present study we evaluate the role of the two receptors, TNF-receptor (TNF-R)p55 and TNF-Rp75, in the mouse model of suture-induced corneal neovascularization and lymphangiogenesis. Corneal neovascularization and lymphangiogenesis were induced by three 11-0 intrastromal corneal sutures in wild-type (WT) C57BL/6J mice and TNF-Rp55-deficient (TNF-Rp55d) and TNF-Rp75-deficient (TNF-Rp75d) mice. The mRNA expression of VEGF-A, VEGF-C, Lyve-1 and TNFα and its receptors was quantified by qPCR. The area covered with blood- or lymphatic vessels, respectively, was analyzed by immunohistochemistry of corneal flatmounts. Expression and localization of TNFα and its receptors was assessed by immunohistochemistry of sagittal sections and Western Blot. Both receptors are expressed in the murine cornea and are not differentially regulated by the genetic alteration. Both TNF-Rp55d and TNF-Rp75d mice showed a decrease in vascularized area compared to wild-type mice 14 days after suture treatment. After 21 days there were no differences detectable between the groups. The number of VEGF-A-expressing macrophages did not differ when comparing WT to TNF-Rp55d and TNF-Rp75d. The mRNA expression of lymphangiogenic markers VEGF-C or LYVE-1 does not increase after suture in all 3 groups and lymphangiogenesis showed a delayed effect only for TNF-Rp75d. TNFα mRNA and protein expression increased after suture treatment but showed no difference between the three groups. In the suture-induced mouse model, TNFα and its ligands TNF-Rp55 and TNF-Rp75 do not play a significant role in the pathogenesis of neovascularisation and lymphangiogenesis.

Effects of empagliflozin and target-organ damage in a novel rodent model of heart failure induced by combined hypertension and diabetes.

Type 2 diabetes mellitus and hypertension are two major risk factors leading to heart failure and cardiovascular damage. Lowering blood sugar by the sodium-glucose co-transporter 2 inhibitor empagliflozin provides cardiac protection. We established a new rat model that develops both inducible diabetes and genetic hypertension and investigated the effect of empagliflozin treatment to test the hypothesis if empagliflozin will be protective in a heart failure model which is not based on a primary vascular event. The transgenic Tet29 rat model for inducible diabetes was crossed with the mRen27 hypertensive rat to create a novel model for heart failure with two stressors. The diabetic, hypertensive heart failure rat (mRen27/tetO-shIR) were treated with empagliflozin (10 mg/kg/d) or vehicle for 4 weeks. Cardiovascular alterations were monitored by advanced speckle tracking echocardiography, gene expression analysis and immunohistological staining. The novel model with increased blood pressure und higher blood sugar levels had a reduced survival compared to controls. The rats develop heart failure with reduced ejection fraction. Empagliflozin lowered blood sugar levels compared to vehicle treated animals (182.3 ± 10.4 mg/dl vs. 359.4 ± 35.8 mg/dl) but not blood pressure (135.7 ± 10.3 mmHg vs. 128.2 ± 3.8 mmHg). The cardiac function was improved in all three global strains (global longitudinal strain - 8.5 ± 0.5% vs. - 5.5 ± 0.6%, global radial strain 20.4 ± 2.7% vs. 8.8 ± 1.1%, global circumferential strain - 11.0 ± 0.7% vs. - 7.6 ± 0.8%) and by increased ejection fraction (42.8 ± 4.0% vs. 28.2 ± 3.0%). In addition, infiltration of macrophages was decreased by treatment (22.4 ± 1.7 vs. 32.3 ± 2.3 per field of view), despite mortality was not improved. Empagliflozin showed beneficial effects on cardiovascular dysfunction. In this novel rat model of combined hypertension and diabetes, the improvement in systolic and diastolic function was not secondary to a reduction in left ventricular mass or through modulation of the afterload, since blood pressure was not changed. The mRen27/tetO-shIR strain should provide utility in separating blood sugar from blood pressure-related treatment effects.

Inhibition of Ca2+ channel surface expression by mutant bestrophin-1 in RPE cells.

The BEST1 gene product bestrophin-1, a Ca2+ -dependent anion channel, interacts with CaV 1.3 Ca2+ channels in the retinal pigment epithelium (RPE). BEST1 mutations lead to Best vitelliform macular dystrophy. A common functional defect of these mutations is reduced trafficking of bestrophin-1 into the plasma membrane. We hypothesized that this defect affects the interaction partner CaV 1.3 channel affecting Ca2+ signaling and altered RPE function. Thus, we investigated the protein interaction between CaV 1.3 channels and bestrophin-1 by immunoprecipitation, CaV 1.3 activity in the presence of mutant bestrophin-1 and intracellular trafficking of the interaction partners in confluent RPE monolayers. We selected four BEST1 mutations, each representing one mutational hotspot of the disease: T6P, F80L, R218C, and F305S. Heterologously expressed L-type channels and mutant bestrophin-1 showed reduced interaction, reduced CaV 1.3 channel activity, and changes in surface expression. Transfection of polarized RPE (porcine primary cells, iPSC-RPE) that endogenously express CaV 1.3 and wild-type bestrophin-1, with mutant bestrophin-1 confirmed reduction of CaV 1.3 surface expression. For the four selected BEST1 mutations, presence of mutant bestrophin-1 led to reduced CaV 1.3 activity by modulating pore-function or decreasing surface expression. Reduced CaV 1.3 activity might open new ways to understand symptoms of Best vitelliform macular dystrophy such as reduced electro-oculogram, lipofuscin accumulation, and vision impairment.

Correction to: Lack of netrin-4 alters vascular remodeling in the retina.

The article "Lack of netrin-4 alters vascular remodeling in the retina".

Control of the retinal local RAS by the RPE: An interface to systemic RAS activity.

As many other organs, the retina has a local renin-angiotensin-system (RAS). All main elements of the RAS are active in the retina: renin, angiotensinogen, angiotensin-converting enzymes. The functional role of the intraretinal RAS is not fully understood. So far, histological and functional analysis point to a regulation of ganglion cell activity and maybe also of bipolar cell activity, but it is not clear how RAS contributes to retinal signal processing. In contrast to local RAS in other organs, the retinal RAS is clearly separated from the systemic RAS. The angiotensin-2 (AngII)/AngI ratio in the retina is different to that in the plasma. However, it appears that the retinal pigment epithelium (RPE), that forms the outer blood/retina barrier, is a major regulator of the retinal RAS by producing renin. Interestingly, comparable to the kidney, the renin production in the RPE is under control of the angiotensin-2 receptor type-1 (AT1). AT1 localizes to the basolateral membrane of the RPE and faces the blood side of the blood/retina barrier. Increases in systemic AngII reduce renin production in the RPE and therefore decrease the intraretinal RAS activity. The relevance of the local RAS for retinal function remains unclear. Nevertheless, it is of fundamental significance to understand the pathology of systemically induced retinal diseases such as hypertension or diabetes.

Systemic ß adrenergic stimulation/ sympathetic nerve system stimulation influences intraocular RAS through cAMP in the RPE.

Several lines of evidence support the existence of a renin-angiotensin system (RAS) in the retina that is separated from the blood stream by the retinal pigment epithelium (RPE). Under physiological conditions, increased activity of intraretinal RAS regulates neuronal activity of the retina but patho-physiologically participates in retinal degeneration such as hypertensive or diabetic retinopathy. Interestingly, the RPE appears to be a modulator of intraretinal RAS in response to changes in systemic RAS. As increased systemic RAS activity is associated with increased sympathetic tonus, we investigated whether systemic ß-adrenergic stimulation of the RPE also modulates renin expression in the RPE. In vivo, the mouse RPE expresses the ß-adrenergic receptor subtypes 1 and 2. Staining of retina sagittal sections showed tyrosine hydroxylase positive nerve endings in the choroid indicating adrenaline/noradrenaline production sites in close proximity to the RPE. Systemic infusion of isoproterenol increased renin expression in the RPE but not in the retina. This increase was sensitive to concomitant systemic application of the angiotensin-2 receptor-type-1 blocker losartan. In vitro analysis of renin gene expression using polarized porcine RPE showed that the activity of the renin promoter can be increased by cAMP stimulation (IBMX/forskolin) but was not influenced by angiotensin-2. Thus, with the identification of the ß-adrenergic system we added a new regulator of the retinal RAS with relevance for retinal function and pathology. Furthermore, it appears that the RPE is not only a close interaction partner of the photoreceptors but also a regulator or retinal activity in general.

Lack of netrin-4 alters vascular remodeling in the retina.

PURPOSE: Netrin-4 (NTN4) is a protein that plays an important role in the regulation of angiogenesis in the pathological retina. Some evidences show that it can also have a role in inflammation and vascular stability. We will explore these questions in vivo in the mature mouse retina. METHODS: We created a NTN4 knockout that expresses EGFP in mononuclear phagocytes (CSFR1-positive cells) to track inflammation in vivo in the retina by scanning laser ophthalmoscopy (SLO). Fundus angiography permitted to study blood vessels. Retinal function was assessed with electroretinography (ERG). RESULTS: Lack of NTN4 leads to an increased amount of amoeboid mononuclear phagocytes in the adult retina, and blood vessels displayed increased tortuosity when compared with the wildtype. Inner retina function also seemed affected in NTN4 null. Lack of NTN4 resulted in a higher persistence of hyaloid artery and spontaneous leakage in the adult retina. No differences were found regarding vessel bifurcation, vessel width, or vein/artery ratio. CONCLUSIONS: These in vivo data show for the first time that lack of NTN4 induces changes in the retinal vascular phenotype in a non-pathological scenario. This evidence widens the role of NTN4 as a guidance cue in vascular remodeling.

Anoctamin-4 is a bona fide Ca2+-dependent non-selective cation channel.

Changes in cell function occur by specific patterns of intracellular Ca2+, activating Ca2+-sensitive proteins. The anoctamin (TMEM16) protein family has Ca2+-dependent ion channel activity, which provides transmembrane ion transport, and/or Ca2+-dependent phosphatidyl-scramblase activity. Using amino acid sequence analysis combined with measurements of ion channel function, we clarified the so far unknown Ano4 function as Ca2+-dependent, non-selective monovalent cation channel; heterologous Ano4 expression in HEK293 cells elicits Ca2+ activated conductance with weak selectivity of K+ > Na+ > Li+. Endogenously expressed Ca2+-dependent cation channels in the retinal pigment epithelium were identified as Ano4 by KO mouse-derived primary RPE cells and siRNA against Ano4. Exchanging a negatively charged amino acid in the putative pore region (AA702-855) into a positive one (E775K) turns Ano4-elicited currents into Cl- currents evidencing its importance for ion selectivity. The molecular identification of Ano4 as a Ca2+-activated cation channel advances the understanding of its role in Ca2+ signaling.

Epithelial-Mesenchymal Transdifferentiation in Pediatric Lens Epithelial Cells.

Purpose: Posterior capsule opacification (PCO) is a complication after cataract surgery, particularly in children. Epithelial-mesenchymal transition (EMT) of lens epithelial cells, mediated by transforming growth factor beta (TGFß), contributes to PCO. However, its pathogenesis in children is poorly understood. We correlated cell growth in culture with patient characteristics, studied gene expression of pediatric lens epithelial cells (pLEC), and examined the effects of TGFß-2 on these cells in vitro. Methods: Clinical characteristics of children with cataracts correlated with growth behavior of pLEC in vitro. mRNA expression of epithelial (αB-crystallin, connexin-43) and mesenchymal (αV-integrin, α-smooth muscle actin, collagen-Iα2, fibronectin-1) markers was quantified in pLEC and in cell line HLE-B3 in the presence and absence of TGFß-2. Results: Fifty-four anterior lens capsules from 40 children aged 1 to 180 months were obtained. Cell outgrowth occurred in 44% of the capsules from patients ≤ 12 months and in 33% of capsules from children aged 13 to 60 months, but in only 6% of capsules from children over 60 months. TGFß-2 significantly upregulated expression of αB-crystallin (HLE-B3), αV-integrin (HLE-B3), collagen-Iα2, and fibronectin-1 (in pLEC and HLE-B3 cells). Conclusions: Patient characteristics correlated with growth behavior of pLEC in vitro, paralleling a higher clinical incidence of PCO in younger children. Gene expression profiles of pLEC and HLE-B3 suggest that upregulation of αV-integrin, collagen-Iα2, and fibronectin-1 are involved in EMT.

Correction: Individual and temporal variability of the retina after chronic bilateral common carotid artery occlusion (BCCAO).

[This corrects the article DOI: 10.1371/journal.pone.0193961.].

Individual and temporal variability of the retina after chronic bilateral common carotid artery occlusion (BCCAO).

Animal models of disease are an indispensable element in our quest to understand pathophysiology and develop novel therapies. Ex vivo studies have severe limitations, in particular their inability to study individual disease progression over time. In this respect, non-invasive in vivo technologies offer multiple advantages. We here used bilateral common carotid artery occlusion (BCCAO) in mice, an established model for ischemic retinopathy, and performed a multimodal in vivo and ex vivo follow-up. We used scanning laser ophthalmoscopy (SLO), ocular coherence tomography (OCT) and electroretinography (ERG) over 6 weeks followed by ex vivo analyses. BCCAO leads to vascular remodeling with thickening of veins starting at 4 weeks, loss of photoreceptor synapses with concomitant reduced b-waves in the ERG and thinning of the retina. Mononuclear phagocytes showed fluctuation of activity over time. There was large inter-individual variation in the severity of neuronal degeneration and cellular inflammatory responses. Ex vivo analysis confirmed these variable features of vascular remodeling, neurodegeneration and inflammation. In summary, we conclude that multimodal follow-up and subgroup analysis of retinal changes in BCCAO further calls into question the use of ex vivo studies with distinct single end-points. We propose that our approach can foster the understanding of retinal disease as well as the clinical translation of emerging therapeutic strategies.

Nitric oxide-sensitive guanylyl cyclase stimulation improves experimental heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) can arise from cardiac and vascular remodeling processes following long-lasting hypertension. Efficacy of common HF therapeutics is unsatisfactory in HFpEF. Evidence suggests that stimulators of the nitric oxide-sensitive soluble guanylyl cyclase (NOsGC) could be of use here. We aimed to characterize the complex cardiovascular effects of NOsGC stimulation using NO-independent stimulator BAY 41-8543 in a double-transgenic rat (dTGR) model of HFpEF. We show a drastically improved survival rate of treated dTGR. We observed less cardiac fibrosis, macrophage infiltration, and gap junction remodeling in treated dTGR. Microarray analysis revealed that treatment of dTGR corrected the dysregulateion of cardiac genes associated with fibrosis, inflammation, apoptosis, oxidative stress, and ion channel function toward an expression profile similar to healthy controls. Treatment reduced systemic blood pressure levels and improved endothelium-dependent vasorelaxation of resistance vessels. Further comprehensive in vivo phenotyping showed an improved diastolic cardiac function, improved hemodynamics, and less susceptibility to ventricular arrhythmias. Short-term BAY 41-8543 application in isolated untreated transgenic hearts with structural remodeling significantly reduced the occurrence of ventricular arrhythmias, suggesting a direct nongenomic role of NOsGC stimulation on excitation. Thus, NOsGC stimulation was highly effective in improving several HFpEF facets in this animal model, underscoring its potential value for patients.

Placental growth factor and its potential role in diabetic retinopathy and other ocular neovascular diseases.

The role of vascular endothelial growth factor (VEGF), including in retinal vascular diseases, has been well studied, and pharmacological blockade of VEGF is the gold standard of treatment for neovascular age-related macular degeneration, retinal vein occlusion and diabetic macular oedema. Placental growth factor (PGF, previously known as PlGF), a homologue of VEGF, is a multifunctional peptide associated with angiogenesis-dependent pathologies in the eye and non-ocular conditions. Animal studies using genetic modification and pharmacological treatment have demonstrated a mechanistic role for PGF in pathological angiogenesis. Inhibition decreases neovascularization and microvascular abnormalities across different models, including oxygen-induced retinopathy, laser-induced choroidal neovascularization and in diabetic mice exhibiting retinopathies. High levels of PGF have been found in the vitreous of patients with diabetic retinopathy. Despite these strong animal data, the exact role of PGF in pathological angiogenesis in retinal vascular diseases remains to be defined, and the benefits of PGF-specific inhibition in humans with retinal neovascular diseases and macular oedema remain controversial. Comparative effectiveness research studies in patients with diabetic retinal disease have shown that treatment that inhibits both VEGF and PGF may provide superior outcomes in certain patients compared with treatment that inhibits only VEGF. This review summarizes current knowledge of PGF, including its relationship to VEGF and its role in pathological angiogenesis in retinal diseases, and identifies some key unanswered questions about PGF that can serve as a pathway for future basic, translational and clinical research.

Myeloid cells contribute indirectly to VEGF expression upon hypoxia via activation of Müller cells.

Anti-VEGF-directed therapies have been a milestone for treating retinal vascular diseases. Depletion of monocyte lineage cells suppresses pathological neovascularization in the oxygen-induced retinopathy mouse model. However, the question whether myeloid-derived VEGF-A expression is responsible for the pathogenesis in oxygen-induced retinopathy remained unknown. We analyzed LysMCre-driven myeloid cell-specific VEGF-A knockout mice as well as mice with complete depletion of circulating macrophages through clodronate-liposome treatment in the oxygen-induced retinopathy model by immunohistochemistry, qPCR, and flow cytometry. Furthermore, we analyzed VEGF-A mRNA expression in MIO-M1 cells alone and in co-culture with BV-2 cells in vitro. The myeloid cell-specific VEGF-A knockout did not change relative retinal VEGF-A mRNA levels, the relative avascular area or macrophage/granulocyte numbers in oxygen-induced retinopathy and under normoxic conditions. We observed an insignificantly attenuated pathology in systemically clodronate-liposome treated knockouts but evident VEGF-A expression in activated Müller cells on immunohistochemically stained sections. MIO-M1 cells had significantly higher expression levels of VEGF-A in co-culture with BV-2 cells compared to cultivating MIO-M1 cells alone. Our data show that myeloid-derived cells contribute to pathological neovascularization in oxygen-induced retinopathy through activation of VEGF-A expression in Müller cells.

Dataset on the activation of Müller cells through macrophages upon hypoxia in the retina.

The dataset presented in this article complements the article entitled "Myeloid cells contribute indirectly to VEGF expression upon hypoxia via activation of Müller cells" (C. Nürnberg, N. Kociok, C. Brockmann, T. Lischke, S. Crespo-Garcia, N. Reichhart, S. Wolf, R. Baumgrass, S.A. Eming, S. Beer-Hammer, and A.M. Joussen). This complementary dataset provides further insight into the experimental validation of the VEGFfl/fl LysMCre (here named VEGFmcko) knockout model used in the main article through genomic and quantitative Real-Time PCR in various murine tissues as well as additional flow cytometry data and immunohistochemical stainings. By providing these data, we aim to enable researcher to reproduce and critically analyze our data.