CD34 marks angiogenic tip cells in human vascular endothelial cell cultures

The functional shift of quiescent endothelial cells into tip cells that migrate and stalk cells that proliferate is a key event during sprouting angiogenesis. We previously showed that the sialomucin CD34 is expressed in a small subset of cultured endothelial cells and that these cells extend filopodia: a hallmark of tip cells in vivo. In the present study, we characterized endothelial cells expressing CD34 in endothelial monolayers in vitro. We found that CD34-positive human umbilical vein endothelial cells show low proliferation activity and increased mRNA expression of all known tip cell markers, as compared to CD34-negative cells. Genome-wide mRNA profiling analysis of CD34-positive endothelial cells demonstrated enrichment for biological functions related to angiogenesis and migration, whereas CD34-negative cells were enriched for functions related to proliferation. In addition, we found an increase or decrease of CD34-positive cells in vitro upon exposure to stimuli that enhance or limit the number of tip cells in vivo, respectively. Our findings suggest cells with virtually all known properties of tip cells are present in vascular endothelial cell cultures and that they can be isolated based on expression of CD34. This novel strategy may open alternative avenues for future studies of molecular processes and functions in tip cells in angiogenesis

Differential TGF-beta Signaling in Retinal Vascular Cells: A Role in Diabetic Retinopathy?

PURPOSE. An early hallmark of preclinical diabetic retinopathy is thickening of the capillary basal lamina (BL). TGF-beta, a multipotent cytokine acting through its receptors ALK5 and -1, has been postulated to be involved in this phenomenon. In light of this possible role, TGF-beta signaling and its downstream molecular effects were characterized in cultured vascular endothelial cells and pericytes of the retina. METHODS. Bovine retinal endothelial cells and pericytes were stimulated with TGF-beta 1 in the presence or absence of SD-208, a specific inhibitor of the TGF-beta type I receptor ALK5, or ALK5 small interfering (si) RNA. TGF-beta-signaling pathways were characterized by analysis of phosphorylated Smad2 or -1/5/8 proteins and TGF-beta target genes (PAI-1, fibronectin, CTGF, Smad7, and Id1) and protein (fibronectin). RESULTS. ALK5 was expressed in both cell types, whereas ALK1 was exclusively expressed in endothelial cells. In endothelial cells, TGF-beta induced Smad2 phosphorylation at high concentrations, which was efficiently blocked by ALK5 inhibition. In contrast, in pericytes, Smad2 phosphorylation was rapidly induced at low concentrations of TGF-beta. The ALK1-Smad1/5/8 pathway was activated by TGF-beta in endothelial cells only. TGF-beta caused ALK5-mediated upregulation of PAI-1, Smad7, and fibronectin and in pericytes at lower TGF-beta concentrations than in endothelial cells. CTGF mRNA expression was induced only in pericytes. Fibronectin protein was confirmed to be regulated by TGF-beta in both cell types. CONCLUSIONS. TGF-beta signaling in retinal endothelial cells and pericytes show that these cells, and in particular the pericytes, have the essential characteristics to allow for a role of TGF-beta in BL thickening in preclinical diabetic retinopathy. (Invest Ophthalmol Vis Sci. 2010; 51:1857-1865) DOI: 10.1167/iovs.09418

Altered expression of genes related to blood-retina barrier disruption in streptozotocin-induced diabetes

Disruption of the blood-retina barrier (BRB) is an early phenomenon in preclinical diabetic retinopathy (PCDR). Two vascular permeability pathways may be affected, the paracellular pathway involving endothelial cell tight junctions, and the endothelial transcellular pathway mediated by endocytotic vesicles (caveolae). The relative contribution of both pathways to vascular permeability in PCDR is unknown. We compared transcription levels in entire rat retina of genes related to these pathways between control conditions and after 6 and 12 weeks of streptozotocin-induced diabetes, as well as in bovine retinal endothelial cells (BRECs) exposed to VEGF and bovine retinal pericytes (BRPCs), using real-time quantitative RT-PCR. To confirm endothelial-specificity, immunohistochemical staining was performed in rat retina, and mRNA transcript levels were compared between BRECs and BRPCs. mRNA and protein of most paracellular transport-related genes were specifically expressed by retinal endothelial cells, whereas vesicle transport-related mRNA and proteins were present in various retinal cell types, including endothelial cells. Expression of selected endothelial cell tight junction genes and particularly that of occludin and claudin-5 was reduced in the diabetic retina and in BRECs after exposure to VEGF. Expression of 6 out of 11 vesicular transport-related genes was upregulated after induction of diabetes. Of these, only plasmalemma vesicle-associated protein (PV-1) was exclusively expressed in BRECs and not in BRPCs. PV-1 transcription was markedly induced in diabetic retina and by VEGF in BRECs. Caveolin-1 immunostaining was primarily found in the retinal vasculature, and its mRNA levels in BRECs were highly abundant and VEGF-inducible. Whereas the endothelial tight junction genes occludin and claudin-5 showed a transient downregulation, we observed long-term upregulation in diabetic retina and VEGF-induced expression in BRECs of the vesicular transport-related genes caveolin-1 and PV-1. The altered gene expression profiles observed in this study suggest a transient induction of the paracellular pathway and prolonged involvement of transcellular endothelial transport mechanisms in the increased permeability of retinal capillaries in PCDR. (C) 2009 Elsevier Ltd. All rights reserve

TNF alpha-Induced Disruption of the Blood-Retinal Barrier In Vitro Is Regulated by Intracellular 3',5'-Cyclic Adenosine Monophosphate Levels

PURPOSE. Proinflammatory cytokines such as tumor necrosis factor ( TNFa) may have a causative role in blood-retinal barrier (BRB) disruption, which is an essential step in the development of diabetic macular edema. The purpose of our study was to determine whether TNFa increases permeability in an in vitro model of the BRB and to explore the mechanisms involved. METHODS. Primary bovine retinal endothelial cells (BRECs) were grown on Transwell inserts and cells were stimulated with TNF alpha or a combination of TNF alpha, IL1 beta, and VEGF. Molecular barrier integrity of the BRB was determined by gene and protein expression of BRB-specific components, and barrier function was assessed using permeability assays. RESULTS. TNFa reduced the expression of tight and adherens junctions in BRECs. Permeability for a 376 Da molecular tracer was increased after TNF alpha stimulation, but not for larger tracers. We found that 3',5'-cyclic adenosine monophosphate (cAMP) stabilized the barrier properties of BRECs, and that TNF alpha significantly decreased intracellular cAMP levels. When BRECs were preincubated with a membrane-permeable cAMP analog, the effects of TNF alpha on claudin-5 expression and permeability were mitigated. The effects of TNF alpha on barrier function in BRECs were largely independent of the small Rho guanosine triphosphate (GTP) ases RhoA and Rac1, which is in contrast to TNF alpha effects on the nonbarrier endothelium. The combination of TNF alpha, IL1 beta, and VEGF increased permeability for a 70 kDa-FITC tracer, also mediated by cAMP. CONCLUSIONS. TNFa alone, or in combination with IL1b and VEGF, induces permeability of the BRB in vitro for differently sized molecular tracers mediated by cAMP, but independently of Rho/ Rac signalin

Association of Circulating Markers With Outcome Parameters in the Bevacizumab and Ranibizumab in Diabetic Macular Edema Trial

PURPOSE. The purpose of this study was to evaluate selected candidate biomarkers as potential markers for patients with diabetic macular edema (DME) who receive antivascular endothelial growth factor (VEGF) therapy METHODS. Selected biomarkers included blood levels of messenger RNA (mRNA) of retinoschisin, RPE65, rhodopsin, and endothelial progenitor cell markers CD34 and CD133. Blood samples were obtained from 89 patients with DME according to the study protocol of the Bevacizumab and Ranibizumab in Diabetic Macular Edema (BRDME) study. During each monthly visit, patients underwent optical coherence tomography scanning and visual acuity was measured. Anti-VEGF injections were administered at fixed monthly intervals over 6 months. Analyses of covariance using simplified and linear mixed models were used to examine the correlations between candidate markers and changes in visual acuity and central subfield thickness. RESULTS. Plasma mRNA levels of retinoschisin were negatively associated with visual acuity, and plasma mRNA levels of rhodopsin were positively associated with visual acuity in patients with DME (P <0.01 and P <0.05, respectively). In addition, changes in central subfield thickness between baseline and months 1, 2, and 3 during anti-VEGF treatment were associated with mRNA levels of retinoschisin, rhodopsin, and the ratio of retinoschisin-torhodopsin (P <0.01, all). CONCLUSIONS. This prospective, multicenter study found that circulating mRNA levels of retinoschisin and rhodopsin are associated with visual acuity and changes in central subfield thickness during anti-VEGF therapy in patients with DME

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. (C) 2011 Elsevier Ltd. All rights reserve

Effect of VEGF-A on expression of profibrotic growth factor and extracellular matrix genes in the retina

PURPOSE. Vascular endothelial growth factor-A ( VEGF) causes increased vascular permeability and leukocyte adhesion in preclinical diabetic retinopathy ( PCDR). Another hallmark of PCDR is thickening of the capillary basement membrane ( BM). Recently, VEGF has been shown to induce expression of pro-fibrotic genes such as transforming growth factor ( TGF)-beta 1 and connective tissue growth factor ( CTGF or CCN2) in cultured endothelial cells. Moreover, neutralization of VEGF prevented BM thickening in diabetic mice in vivo. The authors hypothesize that VEGF directly contributes to BM thickening in the diabetic retina by inducing expression of profibrotic growth factors and extracellular matrix (ECM) components. METHODS. Transcription and protein levels of ECM-related genes were evaluated in the rat retina after intravitreal VEGF injection by real-time quantitative PCR, Western blot analysis, and immunohistochemistry. In addition, expression profiles of the same genes in response to VEGF stimulation were investigated in bovine retinal vascular cells in vitro. RESULTS. Intravitreal VEGF injection induced retinal transcription of CYR61 (CCN1), CTGF, TGF-beta 1, tissue inhibitor of metalloproteases ( TIMP)-1 and fibronectin, and protein expression of CYR61, CTGF, TGF-beta 1 and fibronectin. In bovine retinal endothelial cells and pericytes stimulated by VEGF in vitro, gene expression profiles were similar to those in the intact retina in vivo. CONCLUSIONS. VEGF induces profibrotic growth factors and extracellular matrix genes in the retina in vivo, as well as in cultured retinal vascular cells in vitro. The current findings have relevance for understanding the pathogenesis of preclinical DR, where early upregulation of VEGF may cause BM thickening by induction of ECM-related gene

Involvement of the ubiquitin-proteasome system in the expression of extracellular matrix genes in retinal pigment epithelial cells

Emerging evidence suggests that dysfunction of the ubiquitin-proteasome system is involved in the pathogenesis of numerous senile degenerative diseases including retinal disorders. The aim of this study was to assess whether there is a link between proteasome regulation and retinal pigment epithelium (RPE)-mediated expression of extracellular matrix genes. For this purpose, human retinal pigment epithelial cells (ARPE-19) were treated with different concentrations of transforming growth factor-β (TGFβ), connective tissue growth factor (CTGF), interferon-γ (IFNγ) and the irreversible proteasome inhibitor epoxomicin. First, cytotoxicity and proliferation assays were carried out. The expression of proteasome-related genes and proteins was assessed and proteasome activity was determined. Then, expression of fibrosis-associated factors fibronectin (FN), fibronectin EDA domain (FN EDA), metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinases-1 (TIMP-1) and peroxisome proliferator-associated receptor-γ (PPARγ) was assessed. The proteasome inhibitor epoxomicin strongly arrested cell cycle progression and down-regulated TGFβ gene expression, which in turn was shown to induce expression of pro-fibrogenic genes in ARPE-19 cells. Furthermore, epoxomicin induced a directional shift in the balance between MMP-2 and TIMP-1 and was associated with down-regulation of transcription of extracellular matrix genes FN and FN-EDA and up-regulation of the anti-fibrogenic factor PPARγ. In addition, both CTGF and TGFβ were shown to affect expression of proteasome-associated mRNA and protein levels. Our results suggest a link between proteasome activity and pro-fibrogenic mechanisms in the RPE, which could imply a role for proteasome-modulating agents in the treatment of retinal disorders characterized by RPE-mediated fibrogenic response

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