Mid-term surgical success after transscleral ab interno glaucoma gel stent implantation.

PURPOSE: To investigate the surgical success and efficacy of XEN45 implantation (XEN45 µm, AbbVie Inc., USA) with and without combined cataract surgery up to the first 5 years. METHODS: In a prospective observational monocentric trial, 192 eyes of 157 patients with open-angle glaucoma received either XEN45 implants only (solo surgery group) or combined surgery/cataract surgeries (combined surgery group). Surgical success (qualified and full success; IOP-limit: ≤12, 15, 18, 21 mmHg), time to secondary IOP-lowering procedure, IOP and number of IOP-lowering medications were analysed for 1, 2, 3, 4 and 5 years. RESULTS: Compared to baseline, IOP (24.1 ± 8.1 to 12.6 ± 2.8 mmHg, -48%, p < 0.001) and the number of IOP-lowering medications (3.0 ± 1.0 to 1.5 ± 1.2, -50%, p < 0.001) decreased significantly at 5 years. Although no differences between IOP and the number of IOP-lowering medication courses between the groups were detected at 5 years (p > 0.11), the combined procedure (63%, 37%) showed better success rates compared to the solo procedure (36%, 13%) in the definition IOP ≤18 and ≤12 mmHg (p = 0.035, 0.028). Solo XEN45 procedures had a higher rate of secondary IOP-lowering procedures compared to combined XEN45 cataract procedures (hazard ratio: 2.02, 95%CI: 1.03-3.97, p = 0.04). Twenty per cent of the eyes, including both procedures, required a secondary IOP-lowering procedure within 5 years. CONCLUSIONS: The XEN45 implant is effective in lowering IOP and the number of IOP-lowering medications in patients with open-angle glaucoma in the mid-term. Comparing XEN45 implant results with the results of trabeculectomy available in current literature, we speculate that there might be a higher surgical success rate without medications in favour of trabeculectomy.

Inhibition of CysLTR1 reduces the levels of aggregated proteins in retinal pigment epithelial cells.

The endosomal-lysosomal system (ELS), which carries out cellular processes such as cellular waste degradation via autophagy, is essential for cell homeostasis. ELS inefficiency leads to augmented levels of damaged organelles and intracellular deposits. Consequently, the modulation of autophagic flux has been recognized as target to remove damaging cell waste. Recently, we showed that cysteinyl leukotriene receptor 1 (CysLTR1) antagonist application increases the autophagic flux in the retinal pigment epithelial cell line ARPE-19. Consequently, we investigated the effect of CysLTR1 inhibition-driven autophagy induction on aggregated proteins in ARPE-19 cells using flow cytometry analysis. A subset of ARPE-19 cells expressed CysLTR1 on the surface (SE+); these cells showed increased levels of autophagosomes, late endosomes/lysosomes, aggregated proteins, and autophagy as well as decreased reactive oxygen species (ROS) formation. Furthermore, CysLTR1 inhibition for 24 h using the antagonist zafirlukast decreased the quantities of autophagosomes, late endosomes/lysosomes, aggregated proteins and ROS in CysLTR1 SE- and SE+ cells. We concluded that high levels of plasma membrane-localized CysLTR1 indicate an increased amount of aggregated protein, which raises the rate of autophagic flux. Furthermore, CysLTR1 antagonist application potentially mimics the physiological conditions observed in CysLTR1 SE+ cells and can be considered as strategy to dampen cellular aging.

Distribution of the cysteinyl leukotriene system components in the human, rat and mouse eye.

The cysteinyl leukotrienes (CysLTs) have important functions in the regulation of inflammation and cellular stress. Blocking the CysLT receptors (CysLTRs) with specific antagonists is beneficial against progression of retinopathies (e.g. diabetic retinopathy, wet AMD). However, the exact cellular localization of the CysLTRs and their endogenous ligands in the eye have not been elucidated in detail yet. It is also not known whether the expression patterns differ between humans and animal models. Therefore, the present study aimed to describe and compare the distribution of two important enzymes in CysLT biosynthesis, 5-lipoxygenase (5-LOX) and 5-lipoxygenase-activating protein (FLAP), and of CysLTR1 and CysLTR2 in healthy human, rat and mouse eyes. Human donor eyes (n = 10) and eyes from adult Sprague Dawley rats (n = 5) and CD1 mice (n = 8) of both sexes were collected. The eyes were fixed in 4% paraformaldehyde and cross-sections were investigated by immunofluorescence with specific antibodies against 5-LOX, FLAP (human tissue only), CysLTR1 and CysLTR2. Flat-mounts of the human choroid were prepared and processed similarly. Expression patterns were assessed and semiquantitatively evaluated using a confocal fluorescence microscope (LSM710, Zeiss). We observed so far unreported expression sites for CysLT system components in various ocular tissues. Overall, we detected expression of 5-LOX, CysLTR1 and CysLTR2 in the human, rat and mouse cornea, conjunctiva, iris, lens, ciliary body, retina and choroid. Importantly, expression profiles of CysLTR1 and CysLTR2 were highly similar between human and rodent eyes. FLAP was expressed in all human ocular tissues except the lens. Largely weak immunoreactivity of FLAP and 5-LOX was observed in a few, yet unidentified, cells of diverse ocular tissues, indicating low levels of CysLT biosynthesis in healthy eyes. CysLTR1 was predominantly detected in ocular epithelial cells, supporting the involvement of CysLTR1 in stress and immune responses. CysLTR2 was predominantly expressed in neuronal structures, suggesting neuromodulatory roles of CysLTR2 in the eye and revealing disparate functions of CysLTRs in ocular tissues. Taken together, we provide a comprehensive protein expression atlas of CysLT system components in the human and rodent eye. While the current study is purely descriptive and therefore does not allow significant functional conclusions yet, it represents an important basis for future studies in diseased ocular tissues in which distribution patterns or expression levels of the CysLT system might be altered. Furthermore, this is the first comprehensive study to elucidate expression patterns of CysLT system components in human and animal models that will help to identify and understand functions of the system as well as mechanisms of action of potential CysLTR ligands in the eye.

Pericyte-derived cells participate in optic nerve scar formation.

Introduction: Pericytes (PCs) are specialized cells located abluminal of endothelial cells on capillaries, fulfilling numerous important functions. Their potential involvement in wound healing and scar formation is achieving increasing attention since years. Thus, many studies investigated the participation of PCs following brain and spinal cord (SC) injury, however, lacking in-depth analysis of lesioned optic nerve (ON) tissue. Further, due to the lack of a unique PC marker and uniform definition of PCs, contradicting results are published. Methods: In the present study the inducible PDGFRß-P2A-CreERT2-tdTomato lineage tracing reporter mouse was used to investigate the participation and trans-differentiation of endogenous PC-derived cells in an ON crush (ONC) injury model, analyzing five different post lesion time points up to 8 weeks post lesion. Results: PC-specific labeling of the reporter was evaluated and confirmed in the unlesioned ON of the reporter mouse. After ONC, we detected PC-derived tdTomato+ cells in the lesion, whereof the majority is not associated with vascular structures. The number of PC-derived tdTomato+ cells within the lesion increased over time, accounting for 60-90% of all PDGFRß+ cells in the lesion. The presence of PDGFRß+tdTomato- cells in the ON scar suggests the existence of fibrotic cell subpopulations of different origins. Discussion: Our results clearly demonstrate the presence of non-vascular associated tdTomato+ cells in the lesion core, indicating the participation of PC-derived cells in fibrotic scar formation following ONC. Thus, these PC-derived cells represent promising target cells for therapeutic treatment strategies to modulate fibrotic scar formation to improve axonal regeneration.

Cysteinyl leukotriene receptor 1 is a potent regulator of the endosomal-lysosomal system in the ARPE-19 retinal pigment epithelial cell line.

The endosomal-lysosomal system is central for cell homeostasis and comprises the functions and dynamics of particular organelles including endosomes, lysosomes and autophagosomes. In previous studies, we found that the cysteinyl leukotriene receptor 1 (CysLTR1) regulates autophagy in the retinal pigment epithelial cell line ARPE-19 under basal cellular conditions. However, the underlying mechanism by which CysLTR1 regulates autophagy is unknown. Thus, in the present study, the effects of CysLTR1 inhibition on the endosomal-lysosomal system are analyzed in detail to identify the role of CysLTR1 in cell homeostasis and autophagy regulation. CysLTR1 inhibition in ARPE-19 cells by Zafirlukast, a CysLTR1 antagonist, depleted the lysosomal pool. Furthermore, CysLTR1 antagonization reduced endocytic capacity and internalization of epidermal growth factor and decreased levels of the transferrin receptor, CD71. Serum starvation abolished the effect of Zafirlukast on the autophagic flux, which identifies the endocytic regulation of serum components by CysLTR1 as an important autophagy-modulating mechanism. The role of CysLTR1 in inflammation and cell stress has been exceedingly studied, but its involvement in the endosomal-lysosomal pathway is largely unknown. This current study provides new insights into basal activity of CysLTR1 on cellular endocytosis and the subsequent impact on downstream processes like autophagy.

Human intrinsic choroidal neurons do not alter the expression of intrinsic markers in response to pressure.

BACKGROUND: The choroid is densely innervated by all parts of the autonomic nervous system and further harbours a network of local nerve cells, the intrinsic choroidal neurons (ICN). Their function in ocular control is currently unknown. While morphological data assume a role in intraocular pressure regulation, we here test if increased pressure on isolated choroids may activate ICN. METHODS: Donor tissue was transferred into a pressurisable tissue culture chamber, and nasal and temporal choroid halves incubated for 1 or 4 hours, with pressures set to 15 or 50 mm Hg, followed by qRT-PCR expression analysis of the ICN-specific markers VIP, UCN, NOS1, UCH-L1. POL2-normalised data in the different pressure settings, incubation times and localisations were statistically analysed. RESULTS: The presence of the ICN-specific markers VIP, UCN, NOS1, UCH-L1 was confirmed using immunohistochemistry, and mRNA of all markers was detected in all experimental conditions. Marker analysis revealed no significant changes of mRNA expression levels between 15 and 50 mm Hg in the different incubation times. When comparing all samples over all experimental conditions, a significant increase of VIP and NOS1 mRNA was detected in temporal versus nasal choroids. CONCLUSION: In this functional analysis of human ICN in vitro, higher amounts of VIP and NOS1 mRNA were detected in the temporal choroid, that is, the choroidal site with ICN accumulation. Further, our data indicate that elevated pressure is apparently not able to trigger ICN responses via the investigated markers. Alternative markers and stimuli need to be investigated in upcoming studies in order to unravel ICN function.

Urocortin-positive nerve fibres and cells are present in the human choroid.

BACKGROUND: Choroidal vascular regulation is mediated by the autonomic nervous system in order to gain proper blood flow control. While the mechanisms behind this control are unknown, neuroregulatory peptides are involved in this process. To better understand choroidal function, we investigate the presence of urocortin-1 (UCN), a neuroregulatory peptide with vascular effects, in the human choroid and its possible intrinsic and extrinsic origin. METHODS: Human choroid and eye-related cranial ganglia (superior cervical ganglion- SCG, ciliary ganglion-CIL, pterygopalatine ganglion-PPG, trigeminal ganglion-TRI) were prepared for immunohistochemistry against UCN, protein-gene product 9.5 (PGP9.5), substance P (SP), tyrosine hydroxylase (TH) and vesicular acetylcholine transporter (VAChT). For documentation, confocal laser scanning microscopy was used. RESULTS: In choroidal stroma, UCN-immunoreactivity was present in nerve fibres, small cells and intrinsic choroidal neurons (ICN). Some UCN+ nerve fibres colocalised for VAChT, while others were VAChT. A similar situation was found with SP: some UCN+ nerve fibres showed colocalisation for SP, while others lacked SP. Colocalisation for UCN and TH was not observed. In eye-related cranial ganglia, only few cells in the SCG, PPG and TRI were UCN+, while many cells of the CIL displayed weak UCN immunoreactivity. CONCLUSION: UCN is part of the choroidal innervation. UCN+/VAChT+ fibres could derive from the few cells of the PPG or cells of the CIL, if these indeed supply the choroid. UCN+/SP+ fibres might originate from ICN, or the few UCN+ cells detected in the TRI. Further studies are necessary to establish UCN function in the choroid and its implication for choroidal autonomic control.

Leukotriene Signaling as a Target in α-Synucleinopathies.

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are two common types of α-synucleinopathies and represent a high unmet medical need. Despite diverging clinical manifestations, both neurodegenerative diseases share several facets of their complex pathophysiology. Apart from α-synuclein aggregation, an impairment of mitochondrial functions, defective protein clearance systems and excessive inflammatory responses are consistently observed in the brains of PD as well as DLB patients. Leukotrienes are lipid mediators of inflammatory signaling traditionally known for their role in asthma. However, recent research advances highlight a possible contribution of leukotrienes, along with their rate-limiting synthesis enzyme 5-lipoxygenase, in the pathogenesis of central nervous system disorders. This review provides an overview of in vitro as well as in vivo studies, in summary suggesting that dysregulated leukotriene signaling is involved in the pathological processes underlying PD and DLB. In addition, we discuss how the leukotriene signaling pathway could serve as a future drug target for the therapy of PD and DLB.

Endosomal disentanglement of a transducible artificial transcription factor targeting endothelin receptor A.

Cell-penetrating peptides (CPPs) hold great promise for intracellular delivery of therapeutic proteins. However, endosomal entrapment of transduced cargo is a major bottleneck hampering their successful application. While developing a transducible zinc finger protein-based artificial transcription factor targeting the expression of endothelin receptor A, we identified interaction between the CPP and the endosomal membrane or endosomal entanglement as a main culprit for endosomal entrapment. To achieve endosomal disentanglement, we utilized endosome-resident proteases to sever the artificial transcription factor from its CPP upon arrival inside the endosome. Using this approach, we greatly enhanced the correct subcellular localization of the disentangled artificial transcription factor, significantly increasing its biological activity and distribution in vivo. With rational engineering of proteolytic sensitivity, we propose a new design principle for transducible therapeutic proteins, helping CPPs attain their full potential as delivery vectors for therapeutic proteins.

Characterization of the Two Inducible Cre Recombinase-Based Mouse Models NG2-CreER™ and PDGFRb-P2A-CreERT2 for Pericyte Labeling in the Retina.

PURPOSE: Pericytes (PCs), located abluminal of endothelial cells on capillaries, are essential for vascular development and stability. They display a heterogeneous morphology depending on organ localization, differentiation state, and function. Consequently, PCs show a diverse gene expression profile, impeding the usage of a unique PC marker and therefore the distinct identification of PCs. Inducible reporter mouse models represent an important tool for investigating the fate of PCs under physiological and pathophysiological conditions. PC-specific expression efficiency of the fluorescence reporter tdTomato following tamoxifen induction was analyzed and compared in two inducible Cre recombinase-expressing mouse models under control of the NG2 and PDGFRb promotor. METHODS: The NG2-CreER™-tdTomato and the PDGFRb-P2A-CreERT2-tdTomato mice were treated with tamoxifen at three defining time points of retinal vascular development: post-natal days (P)5, P10/11/12, and P48/49/50/51. TdTomato reporter induction efficiency was determined by analyzing retinal whole mounts utilizing confocal microscopy, using the antibodies Anti-neural/glial antigen 2 (PCs), Anti-Collagen IV (basement membrane), and Anti-Glutamine Synthetase (Müller glial cells). RESULTS: Tamoxifen induction at the three different time points resulted in PC-specific expression of tdTomato in both reporter models. In the NG2-CreER™-tdTomato mouse, the induction efficiency ranged from 21.9 to 35.5%. In the PDGFRb-P2A-CreERT2-tdTomato mouse, an induction efficiency between 78.9 and 94.1% was achieved. TdTomato expression in the retina was restricted to PCs and vascular smooth muscle cells in the NG2-CreER™-tdTomato mouse, however, in the PDGFRb-P2A-CreERT2-tdTomato mouse, tdTomato was also expressed in Müller glial cells. CONCLUSION: Both reporter mouse models represent promising tools for fate-mapping studies of PCs. While the NG2-CreER™-tdTomato mouse reveals very specific labeling of PCs in the retina, its induction efficiency is lower compared to the PDGFRb-P2A-CreERT2-tdTomato mouse. Although the latter revealed a high percentage of tdTomato-positive PCs in the retina, additional labeling of Müller cells potentially hampers analysis of reporter-positive PCs.

Chronobiological activity of cysteinyl leukotriene receptor 1 during basal and induced autophagy in the ARPE-19 retinal pigment epithelial cell line.

Autophagy is an important cellular mechanism for maintaining cellular homeostasis, and its impairment correlates highly with age and age-related diseases. Retinal pigment epithelial (RPE) cells of the eye represent a crucial model for studying autophagy, as RPE functions and integrity are highly dependent on an efficient autophagic process. Cysteinyl leukotriene receptor 1 (CysLTR1) acts in immunoregulation and cellular stress responses and is a potential regulator of basal and adaptive autophagy. As basal autophagy is a dynamic process, the aim of this study was to define the role of CysLTR1 in autophagy regulation in a chronobiologic context using the ARPE-19 human RPE cell line. Effects of CysLTR1 inhibition on basal autophagic activity were analyzed at inactive/low and high lysosomal degradation activity with the antagonists zafirlukast (ZTK) and montelukast (MTK) at a dosage of 100 nM for 3 hours. Abundances of the autophagy markers LC3-II and SQSTM1 and LC3B particles were analyzed in the absence and presence of lysosomal inhibitors using western blot analysis and immunofluorescence microscopy. CysLTR1 antagonization revealed a biphasic effect of CysLTR1 on autophagosome formation and lysosomal degradation that depended on the autophagic activity of cells at treatment initiation. ZTK and MTK affected lysosomal degradation, but only ZTK regulated autophagosome formation. In addition, dexamethasone treatment and serum shock induced autophagy, which was repressed by CysLTR1 antagonization. As a newly identified autophagy modulator, CysLTR1 appears to be a key player in the chronobiological regulation of basal autophagy and adaptive autophagy in RPE cells.

Inhibition of the cysteinyl leukotriene pathways increases survival of RGCs and reduces microglial activation in ocular hypertension.

Glaucoma is the second leading cause of blindness worldwide. This multifactorial, neurodegenerative group of diseases is characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons, leading to irreversible visual impairment and blindness. There is a huge unmet and urging need for the development of new and translatable strategies and treatment options to prevent this progressive loss of RGC. Accumulating evidence points towards a critical role of neuroinflammation, in particular microglial cells, in the pathogenesis of glaucoma. Leukotrienes are mediators of neuroinflammation and are involved in many neurodegenerative diseases. Therefore, we tested the leukotriene receptors CysLT1R/GPR17-selective antagonist Montelukast (MTK) for its efficacy to modulate the reactive state of microglia in order to ameliorate RGCs loss in experimental glaucoma. Ocular hypertension (OHT) was induced unilaterally by injection of 8 µm magnetic microbead (MB) into the anterior chamber of female Brown Norway rats. The contralateral, untreated eye served as control. Successful induction of OHT was verified by daily IOP measurement using a TonoLab rebound tonometer. Simultaneously to OHT induction, one group received daily MTK treatment and the control group vehicle solution by oral gavage. Animals were sacrificed 13-15 days after MB injection. Retina and optic nerves (ON) of OHT and contralateral eyes were analyzed by immunofluorescence with specific markers for RGCs (Brn3a), microglial cells/macrophages (Iba1 and CD68), and cysteinyl leukotriene pathway receptors (CysLT1R and GPR17). Protein labeling was documented by confocal microscopy and analyzed with ImageJ plugins. Further, mRNA expression of genes of the inflammatory and leukotriene pathway was analyzed in retinal tissue. MTK treatment resulted in a short-term IOP reduction at day 2, which dissipated by day 5 of OHT induction in MTK treated animals. Furthermore, MTK treatment resulted in a decreased activation of Iba1+ microglial cells in the retina and ON, and in a significantly increased RGC survival in OHT eyes. Within the retina, GPR17 and CysLT1R expression was demonstrated in single RCGs and in microglial cells respectively. Further, increased mRNA expression of pro-inflammatory genes was detected in OHT induced retinas. In the ON, OHT induction increased the number of GPR17+ cells, showing a trend of reduction following MTK treatment. This study shows for the first time a significantly increased RGC survival in an acute OHT model following treatment with the leukotriene receptor antagonist MTK. These results strongly suggest a neuroprotective effect of MTK and a potential new therapeutic strategy for glaucoma treatment.

Cysteinyl leukotriene receptor 1 modulates autophagic activity in retinal pigment epithelial cells.

The retinal pigment epithelium (RPE), which is among the tissues in the body that are exposed to the highest levels of phagocytosis and oxidative stress, is dependent on autophagy function. Impaired autophagy and continuous cellular stress are associated with various disorders, such as dry age-related macular degeneration (AMD), a disease for which effective therapies are lacking. Cysteinyl leukotriene receptor (CysLTR) 1 is a potential modulator of autophagy; thus, the aim of this study was to investigate the role of CysLTR1 in autophagy regulation in the RPE cell line ARPE-19. The polarized ARPE-19 monolayer exhibited expression of CysLTR1, which was colocalized with ß-tubulin III. In ARPE-19 cells, autophagic activity was rhythmically regulated and was increased upon CysLTR1 inhibition by Zafirlukast (ZK) treatment. H2O2 affected the proautophagic regulatory effect of ZK treatment depending on whether it was applied simultaneously with or prior to ZK treatment. Furthermore, mRNA levels of genes related to the leukotriene system, autophagy and the unfolded protein response were positively correlated. As CysLTR1 is involved in autophagy regulation under basal and oxidative stress conditions, a dysfunctional leukotriene system could negatively affect RPE functions. Therefore, CysLTR1 is a potential target for new treatment approaches for neurodegenerative disorders, such as AMD.

Immunohistochemical Characterization of Neurotransmitters in the Episcleral Circulation in Rats.

Purpose: Episcleral venous pressure (EVP) greatly influences steady-state IOP and recent evidence suggests a neuronal influence on EVP. Yet little is known about the innervation of the episcleral circulation and, more specifically, the neurotransmitters involved. We identify possible neurotransmitter candidates in the episcleral circulation of rats. Methods: Eight immersion-fixated rat eyes taken from four animals were cut into serial sections, followed by standard immunohistochemistry. Antibodies against choline acetyltransferase, dopamine-ß-hydroxylase, synaptophysine, PGP 9.5, VIP, neuronal nitric oxide synthase (nNOS), substance P, CGRP, and galanin were used. Additionally, colocalization experiments with smooth muscle actin and neurofilament (200 kDa) were performed. Results: In all specimens, the episcleral vessels showed immunoreactivity for smooth muscle actin and were reached by neurofilament (200 kDa)-positive structures. Furthermore, these structures colocalized with immunoreactivity for PGP 9.5, synaptophysine, choline acetyl transferase (ChAT), dopamine-ß-hydroxylase, VIP, CGRP, nNOS, substance P and galanin. Conclusions: These findings indicate that there is neuronal input to the episcleral circulation. ChAT and VIP as well as dopamine-ß-hydroxylase suggest parasympathetic and sympathetic innervation. Further studies are needed on whether the positively-stained structures are of functional significance for the regulation of the episcleral venous pressure and thereby IOP.

Pericytes in the Retina.

Pericytes (PCs) are specialized cells located abluminal of endothelial cells (ECs) on capillaries, embedded within the same basement membrane. They are essential regulators of vascular development, remodeling, and blood-retina-barrier (BRB) tightness and are therefore important components to maintain tissue homeostasis. The perivascular localization and expression of contractile proteins suggest that PCs participate in capillary blood flow regulation and neurovascular coupling. Due to their ability to differentiate into various cell types in vitro, they are regarded as potential cells for tissue repair and therapeutic approaches in regenerative medicine. Altered function or loss of PCs is associated with a multitude of CNS diseases, including diabetic retinopathy (DR). In this chapter, we will provide a short overview of retinal vascular development, the origin of PCs, and focus on PCs in retinopathy of prematurity (ROP) and in the diabetic retina. Further, animal models to study the fate of PCs and the potential role of (retinal) PCs in regeneration and wound healing will be discussed.

Low-dose calcipotriol can elicit wound closure, anti-microbial, and anti-neoplastic effects in epidermolysis bullosa keratinocytes.

Recessive dystrophic epidermolysis bullosa (RDEB) patients suffer from chronic and repeatedly infected wounds predisposing them to the development of aggressive and life-threatening skin cancer in these areas. Vitamin D3 is an often neglected but critical factor for wound healing. Intact skin possesses the entire enzymatic machinery required to produce active 1-alpha,25-dihydroxyvitamin D3 (calcitriol), underscoring its significance to proper skin function. Injury enhances calcitriol production, inducing the expression of calcitriol target genes including the antimicrobial peptide cathelicidin (hCAP18), an essential component of the innate immune system and an important wound healing factor. We found significantly reduced hCAP18 expression in a subset of RDEB keratinocytes which could be restored by calcipotriol treatment. Reduced scratch closure in RDEB cell monolayers was enhanced up to 2-fold by calcipotriol treatment, and the secretome of calcipotriol-treated cells additionally showed increased antimicrobial activity. Calcipotriol exhibited anti-neoplastic effects, suppressing the clonogenicity and proliferation of RDEB tumor cells. The combined wound healing, anti-microbial, and anti-neoplastic effects indicate that calcipotriol may represent a vital therapeutic option for RDEB patients which we could demonstrate in a single-patient observation study.

Retinal Pericytes: Characterization of Vascular Development-Dependent Induction Time Points in an Inducible NG2 Reporter Mouse Model.

Purpose/aim of the study: In the retina, defects in pericytes (PCs) function/loss are associated with various complications; however, the exact pathological mechanisms are still not fully elucidated. Following the behavior of retina-resident PCs during health and disease will reveal new insights for both the understanding of pathological mechanisms and the development of new regenerative therapies for the treatment of retinopathies. The main goal of this study is to determine whether the NG2-reporter mouse (NG2CreERTM-eGFP) is a suitable model to study the fate of retina-resident PCs. MATERIAL AND METHODS: Vascular development-dependent reporter induction in retinal PCs was evaluated at different time points [(a) > P21, (b) < P21, and (c) P1 to > P21)] and additionally four different modes of application were tested. Reporter expression was evaluated by enhanced green fluorescent protein (eGFP) immunofluorescence by confocal microscopy and induction efficiency was calculated by analyzing NG2-expressing PCs in comparison to eGFP-labeled PCs in the three capillary layers. RESULTS: eGFP-positive PCs were detected in the three retinal capillary layers at all time points and administration routes tested. Multiple tamoxifen (TAM) applications in adult (> P21) NG2CreERTM-eGFP mice resulted in 3.59% eGFP-positive PCs. 2.37% eGFP-labeled PCs were detected after single intraperitoneal TAM injections at early postnatal days (P2/P5); however, just 1.61% PCs revealed reporter expression upon activation via the lactating mother (P4-P7). The highest number of eGFP-labeled PCs (7.09%) was detected following triple TAM administrations (P10-P12). The number of reporter-positive PCs doubled using homozygous animals. CONCLUSION: Despite low recombination efficiency in the used PC-specific fate mapping mouse model, changes in NG2 promoter activity of PCs during vascular development are indicated by single and multiple TAM inductions at different developmental time points. Nevertheless, these findings need further confirmation in up-coming studies by using homozygous NG2CreERTM-eGFP mice and additionally by mating the NG2CreERTM with a different reporter mouse to increase the low recombination efficiency.

Lack of Galanin Receptor 3 Alleviates Psoriasis by Altering Vascularization, Immune Cell Infiltration, and Cytokine Expression.

The neuropeptide galanin is distributed in the central and peripheral nervous systems and in non-neuronal peripheral organs, including the skin. Galanin acts via three G protein-coupled receptors which, except galanin receptor 1, are expressed in various skin structures. The galanin system has been associated with inflammatory processes of the skin and of several other organs. Psoriasis is an inflammatory skin disease with increased neovascularization, keratinocyte hyperproliferation, a proinflammatory cytokine milieu, and immune cell infiltration. In this study, we showed that galanin receptor 3 is present in endothelial cells in human and murine dermal vessels and is co-expressed with nestin in neo-vessels of psoriatic patients. Moreover, in a murine psoriasis model, we showed that C57/BL6 mice lacking galanin receptor 3 display a milder course of psoriasis upon imiquimod treatment, leading to decreased disease severity, delayed neo-vascularization, reduced infiltration of neutrophils, and significantly lower levels of proinflammatory cytokines compared with wild-type mice. In contrast, galanin receptor 2-knockout animals did not differ significantly from wild type mice at both the macroscopic and molecular levels in their inflammatory response to imiquimod treatment. Our data indicate that galanin receptor 3, but not galanin receptor 2, plays an important role in psoriasis-like skin inflammation.

Pericytes Stimulate Oligodendrocyte Progenitor Cell Differentiation during CNS Remyelination.

The role of the neurovascular niche in CNS myelin regeneration is incompletely understood. Here, we show that, upon demyelination, CNS-resident pericytes (PCs) proliferate, and parenchymal non-vessel-associated PC-like cells (PLCs) rapidly develop. During remyelination, mature oligodendrocytes were found in close proximity to PCs. In Pdgfbret/ret mice, which have reduced PC numbers, oligodendrocyte progenitor cell (OPC) differentiation was delayed, although remyelination proceeded to completion. PC-conditioned medium accelerated and enhanced OPC differentiation in vitro and increased the rate of remyelination in an ex vivo cerebellar slice model of demyelination. We identified Lama2 as a PC-derived factor that promotes OPC differentiation. Thus, the functional role of PCs is not restricted to vascular homeostasis but includes the modulation of adult CNS progenitor cells involved in regeneration.