1. Identification of proteins associated with clinical and pathological features of proliferative diabetic retinopathy in vitreous and fibrovascular membranesIngeborg Klaassen, Ewout W. de Vries, Ilse M.C. Vogels, Antoine H. C. van Kampen, Machteld I. Bosscha, David H. W. Steel, Cornelis J. F. van Noorden, Sarit Y Lesnik-Oberstein, Reinier O. Schlingemann, 2017, original scientific article Abstract: Purpose
To identify the protein profiles in vitreous associated with retinal fibrosis, angiogenesis, and neurite formation in epiretinal fibrovascular membranes (FVMs) in patients with proliferative diabetic retinopathy (PDR).
Methods
Vitreous samples of 5 non-diabetic control patients with vitreous debris and 7 patients with PDR membranes were screened for 507 preselected proteins using the semi-quantitative RayBio® L-series 507 antibody array. From this array, 60 proteins were selected for a custom quantitative antibody array (Raybiotech, Human Quantibody® array), analyzing 7 control patients, 8 PDR patients with FVMs, and 5 PDR patients without FVMs. Additionally, mRNA levels of proteins of interest were measured in 10 PDR membranes and 11 idiopathic membranes and in retinal tissues and cells to identify possible sources of protein production.
Results
Of the 507 proteins screened, 21 were found to be significantly elevated in PDR patients, including neurogenic and angiogenic factors such as neuregulin 1 (NRG1), nerve growth factor receptor (NGFR), placental growth factor (PlGF) and platelet derived growth factor (PDGF). Angiopoietin-2 (Ang2) concentrations were strongly correlated to the degree of fibrosis and the presence of FVMs in patients with PDR. Protein correlation analysis showed PDGF to be extensively co-regulated with other proteins, including thrombospondin-1 and Ang2. mRNA levels of glial-derived and brain/derived neurotrophic factor (GDNF and BDNF) were elevated in PDR membranes. These results were validated in a second study of 52 vitreous samples of 32 PDR patients and 20 control patients.
Conclusions
This exploratory study reveals protein networks that potentially contribute to neurite outgrowth, angiogenesis and fibrosis in the formation of fibrovascular membranes in PDR. We identified a possible role of Ang2 in fibrosis and the formation of FVMs, and of the neurotrophic factors NRG1, PDGF and GDNF in neurite growth that occurs in all FVMs in PDR.
Published in DiRROS: 25.07.2024; Views: 105; Downloads: 124
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2. Spatial and temporal recruitment of the neurovascular unit during development of the mouse blood-retinal barrierAnne-Eva van der Wijk, Ilse M.C. Vogels, Henk A. van Veen, Cornelis J. F. van Noorden, Reinier O. Schlingemann, Ingeborg Klaassen, 2018, original scientific article Abstract: 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.
Keywords: endothelial cells, astrocytes, pericytes, retinal development
Published in DiRROS: 24.07.2024; Views: 122; Downloads: 108
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3. IGF2 and IGF1R identified as novel tip cell genes in primary microvascular endothelial cell monolayersMarchien G. Dallinga, Bahar Yetkin-Arik, Richelle P. Kayser, Ilse M.C. Vogels, Patrycja Nowak-Sliwinska, Arjan W. Griffioen, Cornelis J. F. van Noorden, Ingeborg Klaassen, Reinier O. Schlingemann, 2018, original scientific article Abstract: 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+ 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+ 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.
Keywords: Angiogenesis, tip cells, CD34, IGF2, endothelial cells, cultured cells, endothelial growth factors
Published in DiRROS: 24.07.2024; Views: 132; Downloads: 113
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4. Expression patterns of endothelial permeability pathways in the development of the blood-retinal barrier in miceAnne-Eva van der Wijk, Joanna Wisniewska-Kruk, Ilse M.C. Vogels, Henk A. van Veen, Wing Fung Ip, Nicole N. van der Wel, Cornelis J. F. van Noorden, Reinier O. Schlingemann, Ingeborg Klaassen, 2019, original scientific article Abstract: 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 Wei, 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.
Keywords: tight junctions, transcellular permeability, VEGF signaling
Published in DiRROS: 24.07.2024; Views: 119; Downloads: 94
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5. Diferential roles of eNOS in late efects ofVEGF‑A on hyperpermeability in diferent types of endothelial cellsEsmeralda K. Bosma, Shahan Darwesh, Yasmin I. Habani, Maxime Cammeraat, Paola Serrano Martinez, Mathilda E. van Breest Smallenburg, JiaY. Zheng, Ilse M.C. Vogels, Cornelis J. F. van Noorden, Reinier O. Schlingemann, Ingeborg Klaassen, 2023, original scientific article Abstract: Vascular endothelial growth factor (VEGF)-A induces endothelial hyperpermeability, but the molecular
pathways remain incompletely understood. Endothelial nitric oxide synthase (eNOS) regulates acute
efects of VEGF-A on permeability of endothelial cells (ECs), but it remains unknown whether and how
eNOS regulates late efects 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 fndings
underscore the complexity of VEGF-A-induced permeability pathways in ECs and the role of eNOS
therein, and demonstrate that diferent pathways are activated depending on the EC phenotype.
Keywords: endocytosis, RNAi, hyperpermeability
Published in DiRROS: 15.07.2024; Views: 152; Downloads: 116
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