Spatial and temporal recruitment of the neurovascular unit during development of the mouse blood-retinal barrier.

The inner blood-retinal barrier (BRB) is made up by the neurovascular unit, consisting of endothelial cells, pericytes and glial cells. The BRB maintains homeostasis of the neural retina, but in pathological eye conditions the neurovascular unit is often disrupted, causing BRB loss. Here, we investigated in detail temporal and spatial recruitment of the neurovascular unit in the neonatal mouse retina from postnatal day (P)3 to P25 employing immunohistochemical staining of vascular endothelium (isolectin B4), pericytes (α-SMA and NG2) and astrocytes (GFAP). In addition, we investigated gene expression of polarized astrocytic end-feet markers aquaporin-4 and laminin α2 chain with qPCR. We observed GFAP-positive cells migrating ahead of the retinal vasculature during the first postnatal week, suggesting that the retinal vasculature follows an astrocytic meshwork. From P9 onwards, astrocytes acquired a mature phenotype, with a more stellate shape and increased expression of aquaporin-4. NG2-positive cells and tip cells co-localized at P5 and invaded the retina together as a vascular sprouting front. In summary, these data suggest that recruitment of the cell types of the neurovascular unit is a prerequisite for proper retinal vascularization and BRB formation

The role of glycolysis and mitochondrial respiration in the formation and functioning of endothelial tip cells during angiogenesis.

During sprouting angiogenesis, an individual endothelial tip cell grows out from a pre-existing vascular network and guides following and proliferating stalk cells to form a new vessel. Metabolic pathways such as glycolysis and mitochondrial respiration as the major sources of adenosine 5'-triphosphate (ATP) for energy production are differentially activated in these types of endothelial cells (ECs) during angiogenesis. Therefore, we studied energy metabolism during angiogenesis in more detail in tip cell and non-tip cell human umbilical vein ECs. Small interfering RNA was used to inhibit transcription of glycolytic enzymes PFKFB3 or LDHA and mitochondrial enzyme PDHA1 to test whether inhibition of these specific pathways affects tip cell differentiation and sprouting angiogenesis in vitro and in vivo. We show that glycolysis is essential for tip cell differentiation, whereas both glycolysis and mitochondrial respiration occur during proliferation of non-tip cells and in sprouting angiogenesis in vitro and in vivo. Finally, we demonstrate that inhibition of mitochondrial respiration causes adaptation of EC metabolism by increasing glycolysis and vice versa. In conclusion, our studies show a complex but flexible role of the different metabolic pathways to produce ATP in the regulation of tip cell and non-tip cell differentiation and functioning during sprouting angiogenesis

Endothelial tip cells in vitro are less glycolytic and have a more flexible response to metabolic stress than non-tip cells.

Formation of new blood vessels by differentiated endothelial tip cells, stalk cells, and phalanx cells during angiogenesis is an energy-demanding process. How these specialized endothelial cell phenotypes generate their energy, and whether there are differences between these phenotypes, is unknown. This may be key to understand their functions, as (1) metabolic pathways are essentially involved in the regulation of angiogenesis, and (2) a metabolic switch has been associated with angiogenic endothelial cell differentiation. With the use of Seahorse flux analyses, we studied metabolic pathways in tip cell and non-tip cell human umbilical vein endothelial cell populations. Our study shows that both tip cells and non-tip cells use glycolysis as well as mitochondrial respiration for energy production. However, glycolysis is significantly lower in tip cells than in non-tip cells. Additionally, tip cells have a higher capacity to respond to metabolic stress. Finally, in non-tip cells, blocking of mitochondrial respiration inhibits endothelial cell proliferation. In conclusion, our data demonstrate that tip cells are less glycolytic than non-tip cells and that both endothelial cell phenotypes can adapt their metabolism depending on microenvironmental circumstances. Our results suggest that a balanced involvement of metabolic pathways is necessary for both endothelial cell phenotypes for proper functioning during angiogenesis

Glucocorticoids exert differential effects on the endothelium in an in vitro model of the blood-retinal barrier.

Glucocorticoids (GCs) are used as treatment in diabetic macular oedema, a condition caused by blood-retinal barrier (BRB) disruption. The proposed mechanisms by which GCs reduce macular oedema are indirect anti-inflammatory effects and inhibition of VEGF production, but direct effects on the BRB endothelium may be equally important. Here, we investigated direct effects of GCs on the endothelium to understand the specific pathways of GC action, to enable development of novel therapeutics lacking the adverse side-effects of the presently used GCs. Primary bovine retinal endothelial cells (BRECs) were grown on Transwell inserts and treated with hydrocortisone (HC), dexamethasone (Dex) or triamcinolone acetonide (TA). Molecular barrier integrity of the BRB was determined by mRNA and protein expression, and barrier function was assessed using permeability assays. In addition, we investigated whether TA was able to prevent barrier disruption after stimulation with VEGF or cytokines. Treatment of BRECs with GCs resulted in upregulation of tight junction mRNA (claudin-5, occludin, ZO-1) and protein (claudin-5 and ZO-1). In functional assays, only TA strengthened the barrier function by reducing endothelial permeability. Moreover, TA was able to prevent cytokine-induced permeability in human retinal endothelial cells and VEGF-induced expression of plasmalemma vesicle-associated protein (PLVAP), a key player in VEGF-induced retinal vascular leakage. Glucocorticoids have differential effects in an experimental in vitro BRB model. TA is the most potent in improving barrier function, both at the molecular and functional levels, and TA prevents VEGF-induced expression of PLVAP

IGF2 and IGF1R identified as novel tip cell genes in primary microvascular endothelial cell monolayers.

Tip cells, the leading cells of angiogenic sprouts, were identified in cultures of human umbilical vein endothelial cells (HUVECs) by using CD34 as a marker. Here, we show that tip cells are also present in primary human microvascular endothelial cells (hMVECs), a more relevant endothelial cell type for angiogenesis. By means of flow cytometry, immunocytochemistry, and qPCR, it is shown that endothelial cell cultures contain a dynamic population of CD34 <sup>+</sup> cells with many hallmarks of tip cells, including filopodia-like extensions, elevated mRNA levels of known tip cell genes, and responsiveness to stimulation with VEGF and inhibition by DLL4. Furthermore, we demonstrate that our in vitro tip cell model can be exploited to investigate cellular and molecular mechanisms in tip cells and to discover novel targets for anti-angiogenesis therapy in patients. Small interfering RNA (siRNA) was used to knockdown gene expression of the known tip cell genes angiopoietin 2 (ANGPT2) and tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE1), which resulted in similar effects on tip cells and sprouting as compared to inhibition of tip cells in vivo. Finally, we identified two novel tip cell-specific genes in CD34 <sup>+</sup> tip cells in vitro: insulin-like growth factor 2 (IGF2) and IGF-1-receptor (IGF1R). Knockdown of these genes resulted in a significant decrease in the fraction of tip cells and in the extent of sprouting in vitro and in vivo. In conclusion, this study shows that by using our in vitro tip cell model, two novel essential tip cells genes are identified

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

Differential roles of eNOS in late effects of VEGF-A on hyperpermeability in different types of endothelial cells.

Vascular endothelial growth factor (VEGF)-A induces endothelial hyperpermeability, but the molecular pathways remain incompletely understood. Endothelial nitric oxide synthase (eNOS) regulates acute effects of VEGF-A on permeability of endothelial cells (ECs), but it remains unknown whether and how eNOS regulates late effects of VEGF-A-induced hyperpermeability. Here we show that VEGF-A induces hyperpermeability via eNOS-dependent and eNOS-independent mechanisms at 2 days after VEGF-A stimulation. Silencing of expression of the eNOS gene (NOS3) reduced VEGF-A-induced permeability for dextran (70 kDa) and 766 Da-tracer in human dermal microvascular ECs (HDMVECs), but not in human retinal microvascular ECs (HRECs) and human umbilical vein ECs (HUVECs). However, silencing of NOS3 expression in HRECs increased permeability to dextran, BSA and 766 Da-tracer in the absence of VEGF-A stimulation, suggesting a barrier-protective function of eNOS. We also investigated how silencing of NOS3 expression regulates the expression of permeability-related transcripts, and found that NOS3 silencing downregulates the expression of PLVAP, a molecule associated with trans-endothelial transport via caveolae, in HDMVECs and HUVECs, but not in HRECs. Our findings underscore the complexity of VEGF-A-induced permeability pathways in ECs and the role of eNOS therein, and demonstrate that different pathways are activated depending on the EC phenotype