Expression patterns of endothelial permeability pathways in the development of the blood-retinal barrier in mice.

Insight into the molecular and cellular processes in blood-retinal barrier (BRB) development, including the contribution of paracellular and transcellular pathways, is still incomplete but may help to understand the inverse process of BRB loss in pathologic eye conditions. In this comprehensive observational study, we describe in detail the formation of the BRB at the molecular level in physiologic conditions, using mice from postnatal day (P)3 to P25. Our data indicate that immature blood vessels already have tight junctions at P5, before the formation of a functional BRB. Expression of the endothelial cell-specific protein plasmalemma vesicle-associated protein (PLVAP), which is known to be involved in transcellular transport and associated with BRB permeability, decreased during development and was absent when a functional barrier was formed. Moreover, we show that PLVAP deficiency causes a transient delay in retinal vascular development and changes in mRNA expression levels of endothelial permeability pathway proteins.-Van der Wijk, A.-E., Wisniewska-Kruk, J., Vogels, I. M. C., van Veen, H. A., Ip, W. F., van der Wel, N. N., van Noorden, C. J. F., Schlingemann, R. O., Klaassen, I. Expression patterns of endothelial permeability pathways in the development of the blood-retinal barrier in mice.

Endothelial ß-Catenin Signaling Supports Postnatal Brain and Retinal Angiogenesis by Promoting Sprouting, Tip Cell Formation, and VEGFR (Vascular Endothelial Growth Factor Receptor) 2 Expression.

OBJECTIVE: Activation of endothelial ß-catenin signaling by neural cell-derived Norrin or Wnt ligands is vital for the vascularization of the retina and brain. Mutations in members of the Norrin/ß-catenin pathway contribute to inherited blinding disorders because of defective vascular development and dysfunctional blood-retina barrier. Despite a vital role for endothelial ß-catenin signaling in central nervous system health and disease, its contribution to central nervous system angiogenesis and its interactions with downstream signaling cascades remains incompletely understood. Approach and Results: Here, using genetically modified mouse models, we show that impaired endothelial ß-catenin signaling caused hypovascularization of the postnatal retina and brain because of deficient endothelial cell proliferation and sprouting. Mosaic genetic analysis demonstrated that endothelial ß-catenin promotes but is not required for tip cell formation. In addition, pharmacological treatment revealed that angiogenesis under conditions of inhibited Notch signaling depends upon endothelial ß-catenin. Importantly, impaired endothelial ß-catenin signaling abrogated the expression of the VEGFR (vascular endothelial growth factor receptor)-2 and VEGFR3 in brain microvessels but not in the lung endothelium. CONCLUSIONS: Our study identifies molecular crosstalk between the Wnt/ß-catenin and the Notch and VEGF-A signaling pathways and strongly suggest that endothelial ß-catenin signaling supports central nervous system angiogenesis by promoting endothelial cell sprouting, tip cell formation, and VEGF-A/VEGFR2 signaling.

Disruption of the Extracellular Matrix Progressively Impairs Central Nervous System Vascular Maturation Downstream of ß-Catenin Signaling.

Objective- The Wnt/ß-catenin pathway orchestrates development of the blood-brain barrier, but the downstream mechanisms involved at different developmental windows and in different central nervous system (CNS) tissues have remained elusive. Approach and Results- Here, we create a new mouse model allowing spatiotemporal investigations of Wnt/ß-catenin signaling by induced overexpression of Axin1, an inhibitor of ß-catenin signaling, specifically in endothelial cells ( Axin1 iEC- OE). AOE (Axin1 overexpression) in Axin1 iEC- OE mice at stages following the initial vascular invasion of the CNS did not impair angiogenesis but led to premature vascular regression followed by progressive dilation and inhibition of vascular maturation resulting in forebrain-specific hemorrhage 4 days post-AOE. Analysis of the temporal Wnt/ß-catenin driven CNS vascular development in zebrafish also suggested that Axin1 iEC- OE led to CNS vascular regression and impaired maturation but not inhibition of ongoing angiogenesis within the CNS. Transcriptomic profiling of isolated, ß-catenin signaling-deficient endothelial cells during early blood-brain barrier-development (E11.5) revealed ECM (extracellular matrix) proteins as one of the most severely deregulated clusters. Among the 20 genes constituting the forebrain endothelial cell-specific response signature, 8 ( Adamtsl2, Apod, Ctsw, Htra3, Pglyrp1, Spock2, Ttyh2, and Wfdc1) encoded bona fide ECM proteins. This specific ß-catenin-responsive ECM signature was also repressed in Axin1 iEC- OE and endothelial cell-specific ß-catenin-knockout mice ( Ctnnb1-KOiEC) during initial blood-brain barrier maturation (E14.5), consistent with an important role of Wnt/ß-catenin signaling in orchestrating the development of the forebrain vascular ECM. Conclusions- These results suggest a novel mechanism of establishing a CNS endothelium-specific ECM signature downstream of Wnt-ß-catenin that impact spatiotemporally on blood-brain barrier differentiation during forebrain vessel development. Visual Overview- An online visual overview is available for this article.

Plasmalemma Vesicle-Associated Protein Has a Key Role in Blood-Retinal Barrier Loss.

Loss of blood-retinal barrier (BRB) properties induced by vascular endothelial growth factor (VEGF) and other factors is an important cause of diabetic macular edema. Previously, we found that the presence of plasmalemma vesicle-associated protein (PLVAP) in retinal capillaries associates with loss of BRB properties and correlates with increased vascular permeability in diabetic macular edema. In this study, we investigated whether absence of PLVAP protects the BRB from VEGF-induced permeability. We used lentiviral-delivered shRNA or siRNA to inhibit PLVAP expression. The barrier properties of in vitro BRB models were assessed by measuring transendothelial electrical resistance, permeability of differently sized tracers, and the presence of endothelial junction complexes. The effect of VEGF on caveolae formation was studied in human retinal explants. BRB loss in vivo was studied in the mouse oxygen-induced retinopathy model. The inhibition of PLVAP expression resulted in decreased VEGF-induced BRB permeability of fluorescent tracers, both in vivo and in vitro. PLVAP inhibition attenuated transendothelial electrical resistance reduction induced by VEGF in BRB models in vitro and significantly increased transendothelial electrical resistance of the nonbarrier human umbilical vein endothelial cells. Furthermore, PLVAP knockdown prevented VEGF-induced caveolae formation in retinal explants but did not rescue VEGF-induced alterations in endothelial junction complexes. In conclusion, PLVAP is an essential cofactor in VEGF-induced BRB permeability and may become an interesting novel target for diabetic macular edema therapy.

Molecular analysis of blood-retinal barrier loss in the Akimba mouse, a model of advanced diabetic retinopathy.

The molecular mechanisms of vascular leakage in diabetic macular edema and proliferative retinopathy are poorly understood, mainly due to the lack of reliable in vivo models. The Akimba (Ins2(Akita)VEGF(+/-)) mouse model combines retinal neovascularization with hyperglycemia, and in contrast to other models, displays the majority of signs of advanced clinical diabetic retinopathy (DR). To study the molecular mechanism that underlies the breakdown of the blood-retinal barrier (BRB) in diabetic macular edema and proliferative diabetic retinopathy, we investigated the retinal vasculature of Akimba and its parental mice Kimba (trVEGF029) and Akita (Ins2(Akita)). Quantitative PCR, immunohistochemistry and fluorescein angiography were used to characterize the retinal vasculature with special reference to the inner BRB. Correlations between the degree of fluorescein leakage and retinal gene expression were tested by calculating the Spearman's correlation coefficient. Fluorescein leakage demonstrating BRB loss was observed in Kimba and Akimba, but not in Akita or wild type mice. In Kimba and Akimba mice fluorescein leakage was associated with focal angiogenesis and correlated significantly with Plvap gene expression. PLVAP is an endothelial cell-specific protein that is absent in intact blood-retinal barrier, but its expression significantly increases in pathological conditions such as DR. Furthermore, in Akimba mice BRB disruption was linked to decreased expression of endothelial junction proteins, pericyte dropout and vessel loss. Despite fluorescein leakage, no alteration in BRB protein levels or pericyte coverage was detected in retinas of Kimba mice. In summary, our data not only demonstrate that hyperglycemia sensitizes retinal vasculature to the effects of VEGF, leading to more severe microvascular changes, but also confirm an important role of PLVAP in the regulation of BRB permeability.

An evaluation of oligonucleotide-based therapeutic strategies for polyQ diseases.

BACKGROUND: RNA interference (RNAi) and antisense strategies provide experimental therapeutic agents for numerous diseases, including polyglutamine (polyQ) disorders caused by CAG repeat expansion. We compared the potential of different oligonucleotide-based strategies for silencing the genes responsible for several polyQ diseases, including Huntington's disease and two spinocerebellar ataxias, type 1 and type 3. The strategies included nonallele-selective gene silencing, gene replacement, allele-selective SNP targeting and CAG repeat targeting. RESULTS: Using the patient-derived cell culture models of polyQ diseases, we tested various siRNAs, and antisense reagents and assessed their silencing efficiency and allele selectivity. We showed considerable allele discrimination by several SNP targeting siRNAs based on a weak G-G or G-U pairing with normal allele and strong G-C pairing with mutant allele at the site of RISC-induced cleavage. Among the CAG repeat targeting reagents the strongest allele discrimination is achieved by miRNA-like functioning reagents that bind to their targets and inhibit their translation without substantial target cleavage. Also, morpholino analog performs well in mutant and normal allele discrimination but its efficient delivery to cells at low effective concentration still remains a challenge. CONCLUSIONS: Using three cellular models of polyQ diseases and the same experimental setup we directly compared the performance of different oligonucleotide-based treatment strategies that are currently under development. Based on the results obtained by us and others we discussed the advantages and drawbacks of these strategies considering them from several different perspectives. The strategy aimed at nonallele-selective inhibiting of causative gene expression by targeting specific sequence of the implicated gene is the easiest to implement but relevant benefits are still uncertain. The gene replacement strategy that combines the nonallele-selective gene silencing with the expression of the exogenous normal allele is a logical extension of the former and it deserves to be explored further. Both allele-selective RNAi approaches challenge cellular RNA interference machinery to show its ability to discriminate between similar sequences differing in either single base substitutions or repeated sequence length. Although both approaches perform well in allele discrimination most of our efforts are focused on repeat targeting due to its potentially higher universality.

A novel co-culture model of the blood-retinal barrier based on primary retinal endothelial cells, pericytes and astrocytes.

Loss of blood-retinal barrier (BRB) properties is an important feature in the pathology of diabetic macular edema (DME), but cellular mechanisms underlying BRB dysfunction are poorly understood. Therefore, we developed and characterized a novel in vitro BRB model, based on primary bovine retinal endothelial cells (BRECs). These cells were shown to maintain specific in vivo BRB properties by expressing high levels of the endothelial junction proteins occludin, claudin-5, VE-cadherin and ZO-1 at cell borders, and the specific pumps glucose transporter-1 (GLUT1) and efflux transporter P-glycoprotein (MDR1). To investigate the influence of pericytes and astrocytes on BRB maintenance in vitro, we compared five different co-culture BRB models, based on BRECs, bovine retinal pericytes (BRPCs) and rat glial cells. Co-cultures of BRECs with BRPCs and glial cells showed the highest trans-endothelial resistance (TEER) as well as decreased permeability of tracers after vascular endothelial growth factor (VEGF) stimulation, suggesting a major role for these cell types in maintaining barrier properties. To mimic the in vivo situation of DME, we stimulated BRECs with VEGF, which downregulated MDR1 and GLUT1 mRNA levels, transiently reduced expression levels of endothelial junctional proteins and altered their organization, increased the number of intercellular gaps in BRECs monolayers and influence the permeability of the model to differently-sized molecular tracers. Moreover, as has been shown in vivo, expression of plasmalemma vesicle-associated protein (PLVAP) was increased in endothelial cells in the presence of VEGF. This in vitro model is the first co-culture model of the BRB that mimicks in vivo VEGF-dependent changes occurring in DME.