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Angiopoietin 1 and Vascular Endothelial Growth Factor Modulate Human Glomerular Endothelial Cell Barrier Properties

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Angiopoietin 1 and Vascular Endothelial Growth Factor Modulate Human Glomerular Endothelial Cell Barrier Properties J Am Soc Nephrol 15: 566–574, 2004 Angiopoietin 1 and Vascular Endothelial Growth Factor Modulate Human Glomerular Endothelial Cell Barrier Properties SIMON C. SATCHELL, KAREN L. ANDERSON, and PETER W. MATHIESON Academic Renal Unit, University of Bristol, Southmead Hospital, Bristol, United Kingdom Abstract. Normal glomerular filtration depends on the com- porous supports were investigated by measurement of transen- bined properties of the three layers of glomerular capillary dothelial electrical resistance (TEER) and passage of labeled wall: glomerular endothelial cells (GEnC), basement mem- albumin. Responses to a cAMP analogue and thrombin were brane, and podocytes. Podocytes produce endothelial factors, examined before those to ang1 and VEGF. Results confirmed including angiopoietin 1 (ang1), and vascular endothelial the endothelial origin of GEnC and their expression of Tie2 growth factor (VEGF), whereas GEnC express their respective and VEGFR2. GEnC formed monolayers with a mean TEER receptors Tie2 and VEGFR2 in vivo. As ang1 acts to maintain of 30 to 40 ⍀/cm2. The cAMP analogue and thrombin in- the endothelium in other vascular beds, regulating some ac- creased and decreased TEER by 34.4 and 14.8 ⍀/cm2, respec- tions of VEGF, these observations suggest a mechanism tively, with corresponding effects on protein passage. Ang1 whereby podocytes may direct the unique properties of the increased TEER by 11.4 ⍀/cm2 and reduced protein passage glomerular endothelium. This interaction was investigated by by 45.2%, whereas VEGF reduced TEER by 12.5 ⍀/cm2 but studies on the barrier properties of human GEnC in vitro. had no effect on protein passage. Both ang1 and VEGF mod- GEnC were examined for expression of endothelium-specific ulate GEnC barrier properties, consistent with potential in vivo markers by immunofluorescence and Western blotting and for roles; ang1 stabilizing the endothelium and resisting angiogen- typical responses to TNF-␣ by a cell-based immunoassay. esis while VEGF induces the high permeability to water, Expression of angiopoietin and VEGF receptors was examined characteristic of the glomerular endothelium. similarly. Barrier properties of GEnC monolayers cultured on Angiopoietins (ang) and vascular endothelial growth factors antagonist of the actions of ang1 on the Tie2 receptor, and in (VEGF) form two groups of growth factors that are of partic- the adult its expression is limited to tissues in which there is ular importance in directing endothelial cell (EnC) behavior. active remodeling (7). VEGF-A (often referred to simply as These molecules are widely expressed and have essential and VEGF) is the most widely expressed member of the VEGF coordinated actions in physiologic regulation of vascular de- family and is present in a variety of biologically active iso- velopment, maturation, and permeability (1). They are also key forms arising by differential splicing, VEGF165 being most players in pathologic angiogenesis and alterations in perme- abundant (8). Like ang1, VEGF is expressed by mural cells ability (2) and as such have been implicated in major diseases, (including vascular smooth muscle cells [VSMC] and peri- including cancer, diabetic retinopathy, and arteriosclerosis. cytes), which are well positioned to act as paracrine regulators Ang1 acts via the endothelial Tie2 receptor and in the adult of endothelial behavior. VEGF has powerful angiogenic and continued expression of ang1 in periendothelial cells (3), and permeability-inducing effects acting through endothelial the phosphorylation of Tie2, even in quiescent vessels, sug- VEGF receptors (VEGFR1 to 3) (8). gests a role in vascular maintenance (4). Ang1 is unique in its We and others have shown that ang1 and VEGF are pro- ability to reduce EnC permeability (5) and seems to regulate duced by podocytes in the adult renal glomerulus, whereas the some of the actions of VEGF, stabilizing the endothelium and adjacent glomerular EnC (GEnC) express the cognate receptors resisting permeability increases, inflammation, and angiogen- Tie2 and VEGFR1 and 2 (9–12). This suggests a relationship, esis in the mature endothelium (5, 6). Ang2 is a competitive analogous to that seen between mural cells and EnC in other vessels, whereby podocytes may influence endothelial pheno- type and hence their contribution to the glomerular filtration Received September 18, 2003. Accepted November 29, 2003. barrier. Evidence in support of such an interaction is accumu- Correspondence to Dr. Simon Satchell, Academic Renal Unit, University of lating. Both experimental administration of neutralizing anti- Bristol, Southmead Hospital, Bristol, BS10 5NB, UK. Phone: ϩ44-117-959- 5437; Fax: ϩ44-117-959-5438; E-mail [email protected] bodies to VEGF and podocyte-specific reduction in VEGF 1046-6673/1503-0566 production cause GEnC abnormalities, including swelling, Journal of the American Society of Nephrology vacuolation, and detachment along with proteinuria (13–15). Copyright © 2004 by the American Society of Nephrology The similar glomerular lesion of pre-eclampsia may be caused DOI: 10.1097/01.ASN.0000115397.22519.03 by reductions in circulating free VEGF (16), and use of a J Am Soc Nephrol 15: 566–574, 2004 Angiopoietin 1, VEGF, and Glomerular Endothelial Cells 567 VEGF-neutralizing antibody in clinical trials in treatment of Immunofluorescence metastatic carcinoma has been associated with proteinuria (17). Cells that were grown to confluence on collagen-coated (Autogen We investigated the nature of this interaction further by exam- Bioclear, Calne, Wilts., UK) glass cover slips were fixed in 2% ining effects of these mediators on barrier properties of human formaldehyde and permeabilized in 0.3% Triton X-100. Cells were GEnC in culture. incubated with blocking solution (5% FCS and 0.05% Tween 20 in PBS) and then with antibodies to platelet-endothelial cell adhesion Much debate has focused on which part of the glomerular molecule-1 (PECAM-1; R&D Systems, Minneapolis, MN), vascular filtration barrier is primarily responsible for restricting protein endothelial (VE)-cadherin (Santa Cruz Biochemicals, Santa Cruz, flux, but models indicate that the barrier functions as a whole, CA), and von Willebrand factor (vWF; DakoCytomation, Ely, Cams, with each part making an essential contribution (18, 19). UK). Primary antibody binding was detected using FITC-conjugated In vivo, GEnC form a continuous inner layer of the glomerular secondary antibodies (Jackson ImmunoResearch Laboratories, West capillaries. Away from the nucleus, the cytoplasm is attenuated Grove, PA). Control cells were incubated with secondary antibodies to 200 nm and is punctuated by numerous fenestrae: transcel- only. Coverslips were mounted in Vectashield aqueous mountant lular pores, 60 to 80 nm in diameter (20). Novel imaging (Vector Laboratories, Peterborough, UK) and examined using a Leitz DMRB fluorescence microscope (Leica, Solms, Germany). techniques have demonstrated a GEnC glycocalyx of 200 to Subconfluent GEnC grown on coverslips were incubated with 400 nm in thickness, which covers both fenestrae and inter- TNF-␣ (10 ng/ml; R&D Systems) in standard medium (EGM2-MV) fenestral domains (20). Whereas fenestrations are essential for or with control medium for 6 h. Cells were fixed, permeabilized, and the high hydraulic conductivity of the glomerular filtration labeled for e-selectin as above using a monoclonal antibody (R&D barrier, the glycocalyx is likely to offer significant resistance to Systems). Control cells were labeled with an irrelevant primary filtration of macromolecules (19, 21). antibody. GEnC have been little studied in comparison with other glomerular cells and EnC from other origins. This is largely Cell-Based Fluorescence Immunoassay because GEnC have been difficult to culture, and few have A cell-based fluorescence immunoassay, essentially as described published evidence of their successful isolation and propaga- previously (22), was used to quantify changes in e-selectin expression ␣ tion; fewer still have done so using human cells (22). However in response to TNF- . GEnC were seeded in 96-well plates at 10,000 ␣ EnC from different organs, types of blood vessels, and species cells/well. After 5 d, cells were treated with TNF- (0 to 10 ng/ml; R&D Systems) in standard medium. After 6 h, the cells were fixed as have different morphologies, behavior, and patterns of protein above. Cells were incubated with an antibody to e-selectin that was expression (23–25), indicating the importance of selecting the detected with a FITC-labeled secondary antibody as above. Fluores- relevant EnC type for in vitro studies. GEnC have recently cence emission at 520 nm after excitation at 490 nm was measured on become available from a commercial source. We characterized a Packard Instruments FluoroCount fluorospectro-photometer (avail- these cells to confirm their endothelial origin and investigated able through PerkinElmer Life Sciences, Boston, MA). their expression of Tie2 and VEGF receptors. We developed a system for studying the effects of ang1 and VEGF on GEnC Western Blotting permeability properties in vitro and hence to confirm the ability Cultured GEnC were lysed in Laemmli sample buffer, and solubi- of these mediators to modulate GEnC characteristics that may lized protein concentrations were determined (bicinchoninic acid as- be relevant to their barrier function in vivo. GEnC monolayers say; Pierce Chemical Co., Rockford, IL). Lysates of cultured human were grown on porous supports, and barrier
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