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Effects of fibulin-5 on attachment, adhesion, and proliferation of primary human endothelial cells
BBRC Biochemical and Biophysical Research Communications 348 (2006) 1024–1033 www.elsevier.com/locate/ybbrc
Effects of fibulin-5 on attachment, adhesion, and proliferation of primary human endothelial cells M. Preis a,e,1, T. Cohen a,b,1, Y. Sarnatzki a,b, Y. Ben Yosef a,b, J. Schneiderman b,c, Z. Gluzman a,b, B. Koren a,b, B.S. Lewis a,b,e, Y. Shaul b,d, M.Y. Flugelman a,b,e,* a
Department of Cardiovascular Medicine, Lady Davis Carmel Medical Center, 7 Michal Street, Haifa 34632, Israel b MultiGene Vascular Systems (MGVS) Ltd., Technion IIT, Haifa, Israel c Vascular Surgery, Sheeba Medical Center, Tel Hasomer, Technion IIT, Haifa, Israel d Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Technion IIT, Haifa, Israel e Bruce Rappaport Faculty of Medicine, Technion IIT, Haifa, Israel Received 14 July 2006 Available online 1 August 2006
Abstract Background: Fibulin-5 is a novel extracellular protein that is thought to act as a bridging peptide between elastin fibers and cell surface integrins in blood vessel wall. Fibulin-5 binding to endothelial cell (EC) surface integrins may effect cell proliferation and cell attachment to extracellular matrix (ECM) or to artificial surfaces. In this paper, we describe the effects of fibulin-5 on attachment, adhesion, and proliferation of primary human EC. After demonstrating that fibulin-5 over-expression inhibited EC proliferation, we tested the hypothesis that co-expression of fibulin-5 and VEGF165 will lead to unique EC phenotype that will exhibit increased adherence properties and retain its proliferation capacity. Methods and results: Fibulin-5 and VEGF165 gene transfer to primary human saphenous vein endothelial cells was accomplished using retroviral vectors encoding the two genes. Transgene expression was verified using immunohistochemistry, Western blotting, and ELISA. Fibulin 5 over-expression tended to improve immediate EC attachment (30 min after seeding) and improved significantly adhesion (>40%) under shear stress tested 24 h after EC seeding. The effects of fibulin-5 and VEGF165 on EC proliferation in the presence or absence of basic FGF were also tested. EC expressing fibulin-5 had reduced proliferation while VEGF165 co-expression ameliorated this effect. Conclusion: Fibulin-5 improved EC attachment to artificial surfaces. Dual transfer of fibulin-5 and VEGF165 resulted in EC phenotype with increased adhesion and improved proliferation. This unique EC phenotype can be useful for tissue engineering on endovascular prostheses. 2006 Elsevier Inc. All rights reserved. Keywords: Fibulin-5; Endothelial cells; Adhesion
Endothelial cells (ECs) have a pivotal role in vascular development, regulation of homeostasis, inflammation, vascular tone, and angiogenesis. Fibulin-5 is an extracellular matrix protein also known as UP50 (Urine Protein 50), EVEC (embryonic vascular EGF-like repeat-containing protein) [1], and DANCE (developmental arteries and neu*
1
Corresponding author. Fax: +972 4 8250936. E-mail address: [email protected] (M.Y. Flugelman). Both the authors contributed equally to this work.
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.07.156
ral crest epidermal growth factor-like) [2]. Currently, there are five known members in the fibulin family. The fibulins are secreted glycoproteins, associated with the formation and stabilization of extracellular matrix (ECM) proteins and elastic fibers, and are particularly abundant in tissues rich in elastic fibers [3]. Fibulin-5 contains six EGF-like repeats, all containing consensus sequences for Ca2+-binding (CB) EGF-like repeats. The first CB-EGF-like repeat of fibulin-5 also contains a RGD sequence, which mediates interaction with cell surface integrins. Nakamura et al.
M. Preis et al. / Biochemical and Biophysical Research Communications 348 (2006) 1024–1033
demonstrated that fibulin-5 may increase cell attachment to tissue culture plates through RGD-integrin-dependent binding [2]. The binding of fibulin-5 to cellular surface was mediated by cellular integrins including avb3 and avb5, which have significant role in EC biology and angiogenesis [4]. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and is a heparin-binding homodimeric glycoprotein of 45 kDa. In vitro, VEGF stimulates EC proliferation, migration, ECM degradation, and tube formation of endothelial cells [5,6]. VEGF was demonstrated to be a key survival factor for endothelial cells, both in vitro and in vivo [7]. The biological effects of VEGF are mediated by two tyrosine kinase receptors, VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1, KDR) [8,9]. In this work, we tested the hypothesis that fibulin-5 alone will increase EC adhesion and retention on artificial surface following exposure to shear stress. After finding that increased adhesion was associated with decreased EC proliferation, we tested whether co-expression of VEGF165 will reverse the inhibitory effect of fibulin-5 on proliferation without affecting the improved adhesion. To test the hypothesis, we investigated the effect of fibulin-5 alone or in combination with VEGF165 on human EC immediate attachment within 30 min after seeding, adherence to artificial surfaces 24 h after seeding followed by exposure to shear stress, and proliferation after fibulin 5 and VEGF165 gene transfer. For gene transfer we used pseudo-typed retroviral vectors encoding fibulin-5, VEGF165, and Green fluorescent protein (GFP) as control. By co-expression of fibulin 5 and VEGF165 we generated unique phenotype of EC with increased adhesion and improved proliferation. Although we focused in this study on the biology of fibulin 5 and VEGF in EC, the findings of the study may have practical implications for tissue engineering of artificial surfaces covered with autologous EC. Methods EC isolation, expansion, and characterization Human venous derived endothelial cells were used for the experiments. Endothelial cells were isolated using an enzymatically based method from a 5 to 10 cm vein segment and were cultured on tissue culture plate as previously described [10]. Endothelial cells were characterized by typical ‘‘cobblestone’’ morphology and by immunostaining for CD-31, and von-Willebrand Factor (vWF). For CD31 staining, cells were seeded on 4-well tissue culture slides (Nunc, USA) for 24 h and then fixed with 4% paraformaldehyde (PFA). Following antigen retrieval, slides were incubated with CAS block (Zymed, USA) and then immunostained with mouse anti-human CD-31 antibodies (DAKO, Denmark). The secondary antibody was biotin-conjugated goat antimouse (Chemicon, USA). Bound antibody detection was performed with horseradish peroxidase-conjugated streptavidin (Chemicon, USA). Staining for vWF was performed using similar procedure. EC at passage 6–7 were used in all experiments. Retroviral vector production For the current experiments, we produced 3 retroviral vectors encoding: Green fluorescent protein (GFP); VEGF165-IRES-GFP; fibulin-
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5-IRES-GFP. Human cDNA of VEGF165 and fibulin-5 was used for vector construction. For the vectors encoding VEGF and fibulin 5 we used bicistronic expression cassettes in which the GFP expression was controlled by IRES element. The 600 bp BamH1 fragment of human VEGF165 was cut from pUC18-VEGF165 (GenBank Accession No. AB021221) including the signal sequence for secretion (a kind gift of Dr. J. Abraham, Scios Nova, Mountain View, CA). Construction of pLXSN-VEGF165-IRES-GFP, a bicistronic plasmid co-expressing VEGF165 and the GFP genes, was done in two steps as follows: First, a 600 bp BamHI fragment of VEGF165 was inserted into the BamHI site of pIRES2-GFP (#6029-1 Clontech, USA). Second, a 2.0 kb EcoRI–MunI fragment containing VEGF165 (600 bp), IRES element, and GFP genes, was excised from pVEGF165-IRES-GFP plasmid and inserted into the EcoRI restriction site at the multiple cloning site (MCS) of pLXSN. For retroviral vector production, 10 lg of plasmids was transfected into 293FLYA packaging cells using Lipofectamine (Gibco-BRL, USA). After 48 h the supernatant from confluent cultures of viral producer cells was collected, filtered (0.45 lm), and added to 293FLYGALV packaging cells (a kind gift from F-L Cosset, Lyon, France) [11,12]. The transduced cells were grown under G418 selection (400 lg/ml; Calbiochem, USA) and individual colonies were collected and screened for VEGF165 expression by Western blot analysis of conditioned medium samples. Generation of pseudo-typed retroviral vectors encoding fibulin-5-GFP was performed in a similar way. Cell transduction 4 · 105 ECs were seeded on 60 mm fibronectin-coated plates (4.5 lg/ ml) 24 h prior to transduction. Transduction was initiated by incubation with DEAE-dextran (1 mg/ml; Sigma, USA). The cells were then washed 3 times with PBS and exposed to the retroviral vectors in transduction medium for 4 h. The media containing the retroviruses were then replaced with growth medium. EC were cultured in the presence of G418 (0.5 mg/ml, Gibco, USA). Transgene expression was monitored by GFP expression under florescent microscope, and Western blot, ELISA, and immunohistochemistry (IHC) for the relevant transgene. Sequential (‘‘dual’’) cell transduction Sequential ‘‘Dual’’ transduction of EC was achieved using a two-step process: (1) initial transduction with the retroviral vector encoding fibulin5, followed by selection with G418 (0.5 mg/ml) until >80% of the EC population was expressing fibulin-5 as detected by GFP expression by the cells; (2) transduction of this fibulin-5 expressing EC population with VEGF165 encoding retroviral vector. ECs were cultured in the presence of G418 and trans-gene expression was monitored by IHC for VEGF165 and fibulin-5. VEGF165 expression was also confirmed by ELISA and Western blotting. Since VEGF165 gene transfer was performed after fibulin-5 gene transfer VEGF165 encoding vector transduction rate was at the level of 10– 30% of cells. Western blot Western blot was used for verification of transgene expression in the transduced cells. Conditioned medium from the transduced cells was collected and was separated by 8–10% SDS–PAGE gels. For fibulin-5 the proteins were electro-transferred to nitrocellulose paper which was blocked with 10% low-fat milk and incubated at a 1:500 dilution with rabbit polyclonal antibodies directed against a synthetic peptide derived from human fibulin-5 sequence (custom made, Sigma Israel). Horseradish peroxidase-labeled goat anti-rabbit antibodies were used to visualize bound antibody using the ECL detection system. VEGF165 detection was performed using goat anti-human-VEGF antibody (Santa-Cruz, USA). Horseradish peroxidase-labeled donkey anti-goat antibodies were used to visualize bound antibody using the ECL detection system.
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Immunohistochemistry (IHC) for transgene expression IHC was used for identification of transgene expression in the transduced cells. Cells were prepared as described above. For VEGF165 staining slides were incubated with CAS block (Zymed), and then immunostained with goat anti-human VEGF antibodies (R&D). Biotin-conjugated donkey anti-goat Ab (Chemicon) or Rhodamin-conjugated secondary antibodies were used. For fibulin-5 staining, slides were incubated with mixture of CAS block (Zymed) and goat serum (Zymed) and then immunostained with fibulin-5 rabbit anti-human antibody in blocking solution supplemented with 2% Tween 20. Envision-rabbit peroxidase conjugate (Dako) or FITC-conjugated secondary antibody were used. ELISA Human VEGF immunoassay kit was used according to manufacturer’s instructions (R&D, USA) for VEGF165 levels. Fibulin-5 protein level was measured using an ELISA that was developed in our laboratory. Standards and samples were distributed into a non-antibody-coated 96-well plate. Rabbit anti-human fibulin-5 antibody was added and was bound to fibulin-5 present in the well. Secondary antibody was Envision-rabbit peroxidase conjugate (Dako). TMB/E solution (Chemicon, USA) was used as a substrate for color reaction. The plate was read using ELISA reader at 450/540 nm wave length. Proliferation assays Human saphenous vein endothelial cells (2 · 104 cells/well) were seeded in 24-well culture dishes pre-coated with fibronectin (20 lg/ml). EC were harvested with trypsin (Biological industries, Israel) and counted
using a cell counter (Coulter, USA) 4 h after seeding, and at 48, 96, and 120 h following seeding. Each experiment was performed in triplicate and was repeated in three different primary human saphenous vein endothelial cells. Proliferation rate was tested in non-transduced EC (naive), retrovirally transduced EC over-expressing GFP, fibulin-5, VEGF165, and retrovirally transduced EC co-expressing fibulin-5 and VEGF165. The assay was performed with and without supplementation of bFGF (2 ng/mL) and in the presence of 20% fetal calf sera (HyClone Perbio Science co. USA) supplemented to growth media. Early attachment to ECM This experiment was design to test whether fibulin-5 incorporated in ECM and supplemented to culture media have an effect on immediate EC attachment to ECM. ECM was generated in 48-well plates as previously described by Schneider et al. [13] We produce ECM by naive EC, EC transduced to express fibulin 5-GFP or GFP alone. We seeded 2 · 104 EC in each well. The cells seeded on ECM generated by fibulin-5-GFP transduced EC were re-suspended and incubated with conditioned medium from fibulin-5 transduced cells. The cells seeded on ECM generated by GFP transduced cells were re-suspended and incubated with conditioned media collected from GFP expressing cells, and the control cells were seeded on ECM generated by naive EC and were re-suspended and incubated with naive EC conditioned media. The cells in three groups were incubated for 30 min and then washed with PBS. The number of cells remaining attached to the dishes after washing was quantified using XTT colorimetric assay according to manufacturer’s instruction (Biological Industries, Israel) 4 h following exposure to the reagent. Presence of fibulin in ECM was confirmed by immunohistochemistry and in conditioned media by ELISA (data not shown).
Fig. 1. In vitro automated flow apparatus. Pulsatile flow was generated using a Harvard pump (Harvard apparatus #1421, USA) that was connected to the tubing and container system, positioned in an incubator of 37 C and 5% CO2. The tubing system consisted of stainless steel (#316 Hamlet, Israel) and Teflon tubes (6 mm). A physiological pulse wave was achieved with the pulse wave tank (Tank A). Controlling pulse wave amplitude and flow increment were performed using valve A. Adjustment and control of flow through the grafts was achieved using valves B and C. Adjustment and control of the pressure in the system was achieved using valve D. Fluid equilibrium with the incubator atmosphere (37 C, 5% CO2) was achieved through compensation tank B which also served as a fluid reservoir. The system was monitored online by both pressure (#742 Mennen Medical, Israel) and flow (Doppler flow monitor #T106 Transonic Systems Inc. USA) monitors. Grafts used in the experiments were 5 cm long, 6 mm diameter; thick wall ePTFE grafts (GORE, USA).
M. Preis et al. / Biochemical and Biophysical Research Communications 348 (2006) 1024–1033 Endothelial cell adhesion under continuous shear stress In this experiment we tested whether fibulin-5 expression by EC improves attachment to tissue culture plates under continuous shear stress. Cells were seeded 24–48 h before exposure to shear stress. Unlike previous experiment in which cells were tested for immediate attachment (30 min) to ECM enriched with fibulin, in this experiment we tested EC adherence to tissue culture dishes pre-coated with fibronectin 24–48 h after seeding. Testing cell retention under continuous (24 h) shear stress was achieved by horizontal shaking of the cell culture dishes on a standard laboratory linear shaker. Endothelial cells were counted prior to exposure to continuous shear stress and following exposure. The assay was performed with naive EC, EC over-expressing GFP, and EC over-expressing fibulin-5. EC adhesion under continuous pulsatile flow In order to test the effect of fibulin-5 over-expression on EC adhesion under physiological flow conditions, we established an automated in vitro pulsatile flow apparatus. The cells were seeded on ePTFE grafts (Gore, USA) and were subjected to 2 h of pulsatile flow with shear stress similar to human femoral artery shear stress. In this set of experiments, ePTFE grafts (6 mm, Gore co., USA) were pre-incubated with fibronectin (Sigma, USA) 24 h prior to seeding. EC seeding was performed using a rotation device to allow homogeneous, gravity independent graft seeding. The vascular grafts were filled with an EC suspension, which contained 4.5 · 105 cells/cm2 graft surface area in EC growth medium. The grafts with cell suspension were rotated for 48 h in a 5% CO2, 37 C sterile environment. This procedure yielded ePTFE grafts with luminal coverage by confluent EC monolayer. Flow apparatus. In order to test the effect of fibulin-5 over-expression on endothelial cell retention, we established an in vitro automated flow apparatus (Fig. 1). Flow and pressure were monitored and analyzed
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continuously by a data acquisition system (Lab view software), which presented the calculated shear forces generated in the grafts. The flow conditions were set to simulate the flow in the femoral artery: pressure of 120/80 mmHg, flow of 300–350 ml/min, and shear stress of 7–10 dyn/cm2. Tested grafts. ePTFE (6 mm; GORE Co. USA) was used, an average of 5 cm long grafts were seeded as described above with human venous EC. Grafts were seeded with naive endothelial cells (non-transduced) (n = 6), GFP transduced EC (n = 6), fibulin-5-GFP transduced EC (n = 11), and EC transduced with fibulin-5 and subsequently with VEGF165 (n = 8). Evaluation of graft seeding. At the end of seeding procedure seeded grafts edge (5 mm segment) were sliced from the seeded graft and were cut open and fixed in 2.5% Glutaraldehyde. The luminal aspects of the fixed graft segments were used for evaluation of EC coverage of the graft material. After fixation we stained EC nuclei with DAPI containing VECTRA-SHIELD mounting solution (VECTOR, USA). Number of DAPI stained nuclei per microscopic field was used for quantitative assessment of graft coverage. Morphometric evaluation of endothelial surface coverage was performed using image analysis software (Image Pro Plus 4, Media cybernetics, USA). At least 10 randomly selected fields (100· magnification) of the graft surface were analyzed. The analysis was preformed by an operator blinded to the experimental design. Statistical methods All the data are presented as means ± SD. Comparison of multiple non-parametric groups was performed using the Kruskal–Wallis test or Bonferroni all Pairwise Comparisons Test (Statistix 8, USA). Two nonparametric groups were compared using the Mann–Whitney U test (or Wilcoxon Rank Sum Test). Two parametric groups were compared using student’s t-test. A p value <0.05 was considered significant.
Fig. 2. Primary human saphenous vein endothelial cells’ identity is shown based on the following immunohistochemical staining: (a) All cells are positive for CD31staining and von-Willebrand Factor (vWF) staining, and negative for a-smooth muscle actin. (b) Transgene expression is demonstrated by immunohistochemical staining for fibulin 5 (88% of cells) and VEGF165 (29% of cells).
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Results Endothelial cell isolation and characterization Endothelial cells were isolated and characterized as described in the methods (Fig. 2a). Following retroviral vector transduction endothelial cells expressed fibulin-5 and VEGF165 as shown in Fig. 2b. Sequential viral vector transduction with VEGF165 encoding vector was also performed and co-expression was demonstrated using immunohistochemistry (Fig. 2b) and Western blotting (Fig. 3). In the ‘‘dual’’ transduced cells, fibulin-5 was expressed by more than 88% of the cells and VEGF165 was expressed by 10– 30% of the cells. When we tested transgene expression by ELISA, EC transduced to express fibulin-5 produced 0.25 ± 0.04 pg/cell/24 h of the transgene, and after VEGF gene transfer produced 0.07 ± 0.03 pg/cell/24 h VEGF. Effect of fibulin 5 on endothelial cell attachment and adherence
5.Fibulin 5 & VEGF
4.Fibulin -5
3.VEGF
2.Naive EC
1.Positive control
Early EC attachment to ECM-coated tissue culture plates was tested. Early attachment is mainly mediated by
Fibulin-5
VEGF165
Fig. 3. Western blots for transgene expression: Lane 1 is positive control for fibulin-5 (upper panel) and VEGF (lower panel). Lane 2 is of naive EC, lane 3 is of cells transduced to express VEGF, Lane 4 is of cell transduced to express fibulin-5, and Lane 5 is of cells transduced to express fibulin-5 and VEGF. Transgene expression after transduction is evident in lanes 3– 5. Both fibulin-5 and VEGF are expressed by the dually transduced EC.
cell surface integrins and ECM proteins. Four experiments were completed in triplicates. Calibration curve was performed with known number of cells in order to convert the value of XTT measurement to number of cells (data not shown). A tendency to improved short-term attachment in the presence of fibulin-5 enriched ECM and cell culture media was found (average of all experiments: 17,599 ± 1641 for naive cells, 17,319 ± 1822 for GFP transduced cells, and 19,624 ± 2281 for fibulin-5 transduced cells; p = 0.08). This experiment may imply to the mechanisms by which fibulin-5 can improve EC attachment. The effect of fibulin-5 expression on cell adhesion was examined 24–48 h after EC plating and after exposure to continuous (24 h) shear stress, achieved by horizontal shaking of the cell culture dishes on a standard laboratory linear shaker. Cells were counted prior to exposure to continuous shear stress and following exposure. The assay was performed with naive EC, EC over-expressing GFP, and EC over-expressing fibulin-5. In the fibulin-5 over-expressing EC, 75 ± 15% of the cells remained adherent to the plate, in comparison to 57 ± 16% GFP over-expressing EC and 52 ± 13% naive EC (n = 8 experiments, four experiments were performed in triplicates, and 4 in duplicates, p = 0.002) (Fig. 4). These results demonstrate a significant effect of fibulin-5 over-expression on endothelial cell adhesion under continuous shear stress. We tested EC adhesion under pulsatile flow after seeding EC on ePTFE grafts. In this experiment EC adherence was tested under physiological shear stress conditions. The use of ePTFE vascular grafts in this experiment is part of our effort to translate the observed improved EC adhesion properties for use in tissue engineering of vascular grafts that has a luminal EC monolayer. Vascular grafts made of ePTFE (6 mm diameter) were seeded with EC and exposed to pulsatile flow for 2 h. The analysis included percentage of area coverage by endothelial cells. ePTFE grafts seeded with EC over-expressing fibulin-5 or fibulin5 + VEGF165 had significantly improved retention of EC following exposure to 2 h of pulsatile flow when compared to grafts seeded with EC expressing GFP, or naive EC (Figs. 5a and b). No significant difference was found between ePTFE grafts seeded with EC over-expressing fibulin-5 and grafts seeded with EC co-expressing fibulin-5 and VEGF165. These experiments demonstrated the improved retention of EC seeded on ePTFE grafts as a result of fibulin-5 over-expression. Effects of fibulin 5 and VEGF165 on endothelial cell proliferation Proliferation experiments were performed on EC as described in Methods. Overall, fibulin-5 over-expression by human EC had inhibitory effect on EC proliferation (Fig. 6). Repeated proliferation assays using different primary EC from three different individuals demonstrated a similar effect. The inhibitory effect of fibulin-5 on cell
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Fig. 4. Endothelial cells before and after exposure to continuous shear stress. (upper part) Images of representative fields under light microscopy, before and after exposure to continuous shear stress. (lower part) Summary of the remaining attached endothelial cells, according to transgene expression.
proliferation was more pronounced in the absence of bFGF, a strong EC mitogen. In this serum concentration (20% of FCS) and absence of bFGF cell death was observed. With supplementation of bFGF to culture media no EC death was observed but proliferation of EC expressing fibulin-5 was similar to the proliferation rate of control cells and inferior to proliferation of EC expressing VEGF165. EC expressing both VEGF165 and fibulin-5 demonstrated the high proliferation rate both in the presence or absence of bFGF. These experiments demonstrated that fibulin-5 inhibit primary human venous EC proliferation. VEGF165 co-expression in cells over-expressing fibulin-5 improved cell proliferation and attenuated the inhibitory effect of fibulin-5. Similar results were obtained when we used exogenous protein supplementation in non-transduced EC (data not shown). Discussion In this study, we tested the hypothesis that fibulin-5 gene transfer will increase EC attachment and adhesion to artificial surface, and extended our research to test whether reduced EC proliferation observed after fibulin-5 gene transfer can be reversed by co-expression of VEGF165. EC attachment and adhesion was improved after fibulin-5 gene transfer as demonstrated in three different experimental settings; tendency to improved attachment was
demonstrated 30 min after cell seeding, and significantly improved adherence was observed in EC exposed to shear forces 24–48 h after seeding. Fibulin-5 contains six Ca2+-binding EGF motifs, Rao et al. demonstrated that these areas, following binding of Ca2+, can stabilize the protein structure and direct protein to extracellular protein interaction.[14] The RGD sequence on the first Ca2+-binding EGF motif of fibulin-5 can interact with cellular surface integrins [15]. Nakamura et al. also demonstrated that fibulin-5 increase HUVEC attachment to fibulin-5-coated microtiter plates. The attachment was dose dependent and was inhibited in the presence of exogenous GRDSP peptide [2]. He also identified the cellular surface integrins avb3, avb5 and a9b1 as binding surface integrins [16]. Yanagisawa et al. demonstrated that the fibulin-5 interacts with extracellular proteins and especially with tropoelastin [17]. These data suggest that fibulin-5 acts as a bridging molecule between cells and ECM protein. We tested EC attachment 30 min following cell seeding and demonstrated a tendency to improved initial EC attachment in the presence of fibulin-5. In his attachment experiments Nakamura et al. used fibulin-5 concentration of 1–16 lg/ml. In our experiments we focused on fibulin5 concentration in a more physiological context of protein production and expression. This may explain the statistically borderline effect on attachment observed in our experiments.
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a
b
Fig. 5. Improved retention of endothelial cells over-expressing fibulin-5 or co-expressing fibulin-5 and VEGF165 on ePTFE grafts (a) endothelial cells (EC) were seeded on ePTFE grafts and exposed to pulsatile flow conditions in an in vitro flow apparatus. Non-transduced-naive EC, EC over-expressing GFP, fibulin-5 or co-expressing fibulin-5 and VEGF165 were seeded on ePTFE grafts. Results are presented as mean percentage of cell retention ± SD. (b) Nucleic staining by DAPI of seeded graft sections. Non-transduced-naive EC, and EC co-expressing fibulin-5 and VEGF165 were examined following seeding and following exposure to pulsatile flow for 2 h. Although complete coverage is apparent in the two grafts after seeding, cell adherence of EC expressing the two genes is higher than naive cell adherence after exposure to flow.
Cell adhesion under continuous shear stress was also tested. This method was described by Asada et al. as a method to test the effect of shear stress on cells seeded in tissue culture plates [18]. Over-expression of fibulin-5 improved cell retention following exposure to continuous shear stress by approximately 40% compared to naive EC or GFP transduced EC. In vitro experiments in an automated fluid circulation apparatus demonstrated the effect of fibulin-5 over-expression on endothelial cell attachment to ePTFE grafts. The flow conditions in the close circuit were set to mimic the flow conditions in the human femoral artery. The close system enabled us to maintain constant pulsatile fluid pressure and flow, while shear stress was monitored online. The fluid dynamics parameters were maintained stable throughout the experiments and enabled us to compare the different
groups. Fibulin-5 expression by endothelial cells significantly improved the adherence to ePTFE surface and increased the percentage of cell retention on the graft surface following exposure to flow. More than 40% improvement in cell retention was achieved by over-expressing fibulin-5. This can be attributed to the action of fibulin-5 as a bridging molecule that anchors the cells to the graft coating proteins. The co-expression of fibulin-5 and VEGF165 by endothelial cells had also improved retention in comparison to the control grafts seeded with naive cells (Wilcoxon rank sum test, p value = 0.04) or EC transduced with GFP (Wilcoxon rank sum test, p value = 0.01). With improved adhesion we observed that EC transduced to express fibulin-5 exhibited reduced proliferation rates. Reduced proliferation with fibulin-5 was also reported by Scheimann et al. who demonstrated that fibulin-5
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Fig. 6. Endothelial cell proliferation assay. Human saphenous vein endothelial cells (2 · 104 cells/well) were seeded in 24-well culture dishes pre-coated with fibronectin (20 lg/ml). Endothelial cells (EC) were harvested with trypsin and counted using a cell counter (Coulter, USA) 4 h after seeding, and at 48, 96, and 120 h. Each experiment was performed in triplicates. Proliferation rate was tested in non-transduced EC (naive), retrovirally transduced EC over-expressing GFP, retrovirally transduced EC over-expressing VEGF165, retrovirally transduced EC over-expressing fibulin-5, and retrovirally transduced EC co-expressing VEGF165 and fibulin-5. The assay was performed without bFGF (a) and following bFGF stimulation (b). Results are presented as average of triplicates ± SD.
significantly inhibited DNA synthesis and cell proliferation [19]. Albig et al. demonstrated that fibulin-5 inhibits EC proliferation and invasion through synthetic basement membranes [20]. After finding that increased adhesion was also associated with decreased EC proliferation we tested whether coexpression of VEGF165 will reverse the inhibitory effect of fibulin-5 on proliferation without changing the improved adhesion. We initially showed that EC could be transduced to express both fibulin-5 and VEGF165. To achieve predictable transduction rates we initially transduced the cells with pseudo-typed retroviral vector encoding fibulin-5. Subsequently, we transduced the cells with VEGF165 encoding vector. Co-transduction with the VEGF165 encoding vector reversed the inhibitory effect of fibulin-5 on EC proliferation without a significant change in adhesion properties. Co-expression of the two genes increased EC proliferation when compared to control EC or even when compared to EC expressing VEGF165 alone. Improved
proliferation was observed both with and without basic FGF. Albig et al. also investigated the opposing effects of VEGF165 and fibulin-5 on EC proliferation but did not observe synergistic effect as we observed. In a previous report we have shown that EC over-expressing VEGF165 up-regulate levels of VEGF receptor 2(KDR) [21]. Up-regulated VEGF receptors render the cells more sensitive to VEGF and consequently lead to enhanced proliferation. The synergistic effect observed with fibulin-5 and VEGF165 cannot be explained on increased number of VEGF receptors alone. Synergy may be due to the improved adhesion that is necessary for cell proliferation and the mitogenic effect of VEGF. Based on our findings, co-expression of these two genes created a unique phenotype of EC. The extracellular environment has a substantial effect on EC proliferation, migration, attachment, and survival. Failure of EC to attach to ECM results in proliferation arrest and subsequently in cell death. Both proliferation and survival are dependent on adhesion to ECM which is
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mediated by cell surface integrins [22]. ECM proteins have various effects on EC: collagens and especially collagen type I activate EC and promote basement membrane degradation, proliferation, and invasion in a similar manner to VEGF. This effect is mediated mainly through integrins avb3 and a5b1. Laminin on the contrary, promotes basement membrane formation and reduced proliferation as does TGFb, in a process mediated mainly through integrins a6b1 and a3b1 [23]. Integrins mediate not only cell adhesion and proliferation by cell–matrix and cell–cell interaction but also by multiple outside-in signals that lead to activation of downstream pathways such as tyrosine kinases and phosphatidylinositol 3-kinase [24]. The importance of using VEGF is also supported by work published by Chennazhy et al. [25] EC grown on synthetic materials lose their proliferation potential, turn apoptotic and express pro-thrombotic phenotype, but when they are grown on matrix composed of fibronectin and VEGF, the cell remain viable, proliferate, and are less thrombogenic. While av integrin activation is necessary for the angiogenic cascade, the same integrin activation may cause anti-proliferative and anti-apoptotic effect. This inhibitory effect may be caused by binding of blocking peptides to integrin that occupy the integrin active sites [26]. These studies and especially the work of Cheresh et al. may explain the findings that fibulin-5, an integrin-binding protein, when over-expressed results in inhibition of EC proliferation. Acknowledgments This work was supported in part by a grant from the Israeli Academy of Science. Based on the technology described in this paper we established a company, MGVS Ltd. (MultiGene Vascular Systems Ltd.), in our Medical Center. The following authors are either workers or consultants of MGVS Ltd., Haifa: Cohen, T., Sarnatzki, Y., Ben Yosef, Y., Schneiderman, J., Gluzman, Z., Koren, B., Lewis, B.S., Shaul, Y., Flugelman, M.Y. References [1] R.C. Kowal, J.A. Richardson, J.M. Miano, E.N. Olson, EVEC, a novel epidermal growth factor-like repeat-containing protein upregulated in embryonic and diseased adult vasculature, Circ. Res. 84 (1999) 1166–1176. [2] T. Nakamura, P. Ruiz-Lozano, V. Lindner, D. Yabe, M. Taniwaki, Y. Furukawa, K. Kobuke, K. Tashiro, Z. Lu, N.L. Andon, R. Schaub, A. Matsumori, S. Sasayama, K.R. Chien, T. Honjo, DANCE, a novel secreted RGD protein expressed in developing, atherosclerotic, and balloon-injured arteries, J. Biol. Chem. 274 (1999) 22476–22483. [3] K.S. Midwood, J.E. Schwarzbauer, Elastic fibers: building bridges between cells and their matrix, Curr. Biol. 12 (2002) R279–R281. [4] P.C. Brooks, Role of integrins in angiogenesis, Eur. J. Cancer 32A (1996) 2423–2429. [5] J. Plouet, J. Schilling, D. Gospodarowicz, Isolation and characterization of a newly identified endothelial cell mitogen produced by AtT-20 cells, EMBO J. 8 (1989) 3801–3806.
[6] M.S. Pepper, N. Ferrara, L. Orci, R. Montesano, Potent synergism between vascular endothelial growth factor and basic fibroblast growth factor in the induction of angiogenesis in vitro, Biochem. Biophys. Res. Commun. 189 (1992) 824–831. [7] T. Alon, I. Hemo, A. Itin, J. Pe’er, J. Stone, E. Keshet, Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity, Nat. Med. 1 (1995) 1024–1028. [8] C. de Vries, J.A. Escobedo, H. Ueno, K. Houck, N. Ferrara, L.T. Williams, The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor, Science 255 (1992) 989–991. [9] T.P. Quinn, K.G. Peters, C. De Vries, N. Ferrara, L.T. Williams, Fetal liver kinase 1 is a receptor for vascular endothelial growth factor and is selectively expressed in vascular endothelium, Proc. Natl. Acad. Sci. USA 90 (1993) 7533–7537. [10] M.Y. Flugelman, R. Virmani, M.B. Leon, R.L. Bowman, D.A. Dichek, Genetically engineered endothelial cells remain adherent and viable after stent deployment and exposure to flow in vitro, Circ. Res. 70 (1992) 348–354. [11] F.L. Cosset, Y. Takeuchi, J.L. Battini, R.A. Weiss, M.K. Collins, High-titer packaging cells producing recombinant retroviruses resistant to human serum, J. Virol. 69 (1995) 7430–7436. [12] B. Koren, L. Fischer, Z. Gluzman, M. Preis, N. Abramovitz, T. Cohen, F.-L. Cosset, B.S. Lewis, M.Y. Flugelman, Efficient transduction of human endothelial cells seeded onto metallic stents using bicistronic pseudo typed retro viral vectors encoding VEGF and GFP, Cardiovascular Revascularization Medicine, (2006) in press. [13] A. Schneider, H. Schwalb, I. Vlodavsky, G. Uretzky, An improved method of endothelial seeding on small caliber prosthetic vascular grafts coated with natural extracellular matrix, Clin. Mater. 13 (1993) 51–55. [14] Z. Rao, P. Handford, M. Mayhew, V. Knott, G.G. Brownlee, D. Stuart, The structure of a Ca(2+)-binding epidermal growth factorlike domain: its role in protein–protein interactions, Cell 82 (1995) 131–141. [15] R.O. Hynes, Integrins: versatility, modulation, and signaling in cell adhesion, Cell 69 (1992) 11–25. [16] T. Nakamura, P.R. Lozano, Y. Ikeda, Y. Iwanaga, A. Hinek, S. Minamisawa, C.F. Cheng, K. Kobuke, N. Dalton, Y. Takada, K. Tashiro, J. Ross Jr., T. Honjo, K.R. Chien, Fibulin-5/DANCE is essential for elastogenesis in vivo, Nature 415 (2002) 171–175. [17] H. Yanagisawa, E.C. Davis, B.C. Starcher, T. Ouchi, M. Yanagisawa, J.A. Richardson, E.N. Olson, Fibulin-5 is an elastin-binding protein essential for elastic fibre development in vivo, Nature 415 (2002) 168– 171. [18] H. Asada, J. Paszkowiak, D. Teso, K. Alvi, A. Thorisson, J.C. Frattini, F.A. Kudo, B.E. Sumpio, A. Dardik, Sustained orbital shear stress stimulates smooth muscle cell proliferation via the extracellular signal-regulated protein kinase 1/2 pathway, J. Vasc. Surg. 42 (2005) 772–780. [19] W.P. Schiemann, G.C. Blobe, D.E. Kalume, A. Pandey, H.F. Lodish, Context-specific effects of fibulin-5 (DANCE/EVEC) on cell proliferation, motility, and invasion. Fibulin-5 is induced by transforming growth factor-beta and affects protein kinase cascades, J. Biol. Chem. 277 (2002) 27367–27377. [20] A.R. Albig, W.P. Schiemann, Fibulin-5 antagonizes vascular endothelial growth factor (VEGF) signaling and angiogenic sprouting by endothelial cells, DNA Cell Biol. 23 (2004) 367–379. [21] A. Weisz, B. Koren, T. Cohen, G. Neufeld, T. Kleinberger, B.S. Lewis, M.Y. Flugelman, Increased vascular endothelial growth factor 165 binding to kinase insert domain-containing receptor after infection of human endothelial cells by recombinant adenovirus encoding the Vegf(165) gene, Circulation 103 (2001) 1887–1892. [22] F.G. Giancotti, E. Ruoslahti, Integrin signaling, Science 285 (1999) 1028–1032. [23] G.E. Davis, D.R. Senger, Endothelial extracellular matrix: biosynthesis, remodeling, and functions during vascular morphogenesis and neovessel stabilization, Circ. Res. 97 (2005) 1093–1107.
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cultured for tissue engineering, Biomaterials 26 (2005) 5658– 5667. [26] D.A. Cheresh, D.G. Stupack, Integrin-mediated death: an explanation of the integrin-knockout phenotype? Nat. Med. 8 (2002) 193–194.
Effects of fibulin-5 on attachment, adhesion, and proliferation of primary human endothelial cells M. Preis a,e,1, T. Cohen a,b,1, Y. Sarnatzki a,b, Y. Ben Yosef a,b, J. Schneiderman b,c, Z. Gluzman a,b, B. Koren a,b, B.S. Lewis a,b,e, Y. Shaul b,d, M.Y. Flugelman a,b,e,* a
Department of Cardiovascular Medicine, Lady Davis Carmel Medical Center, 7 Michal Street, Haifa 34632, Israel b MultiGene Vascular Systems (MGVS) Ltd., Technion IIT, Haifa, Israel c Vascular Surgery, Sheeba Medical Center, Tel Hasomer, Technion IIT, Haifa, Israel d Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Technion IIT, Haifa, Israel e Bruce Rappaport Faculty of Medicine, Technion IIT, Haifa, Israel Received 14 July 2006 Available online 1 August 2006
Abstract Background: Fibulin-5 is a novel extracellular protein that is thought to act as a bridging peptide between elastin fibers and cell surface integrins in blood vessel wall. Fibulin-5 binding to endothelial cell (EC) surface integrins may effect cell proliferation and cell attachment to extracellular matrix (ECM) or to artificial surfaces. In this paper, we describe the effects of fibulin-5 on attachment, adhesion, and proliferation of primary human EC. After demonstrating that fibulin-5 over-expression inhibited EC proliferation, we tested the hypothesis that co-expression of fibulin-5 and VEGF165 will lead to unique EC phenotype that will exhibit increased adherence properties and retain its proliferation capacity. Methods and results: Fibulin-5 and VEGF165 gene transfer to primary human saphenous vein endothelial cells was accomplished using retroviral vectors encoding the two genes. Transgene expression was verified using immunohistochemistry, Western blotting, and ELISA. Fibulin 5 over-expression tended to improve immediate EC attachment (30 min after seeding) and improved significantly adhesion (>40%) under shear stress tested 24 h after EC seeding. The effects of fibulin-5 and VEGF165 on EC proliferation in the presence or absence of basic FGF were also tested. EC expressing fibulin-5 had reduced proliferation while VEGF165 co-expression ameliorated this effect. Conclusion: Fibulin-5 improved EC attachment to artificial surfaces. Dual transfer of fibulin-5 and VEGF165 resulted in EC phenotype with increased adhesion and improved proliferation. This unique EC phenotype can be useful for tissue engineering on endovascular prostheses. 2006 Elsevier Inc. All rights reserved. Keywords: Fibulin-5; Endothelial cells; Adhesion
Endothelial cells (ECs) have a pivotal role in vascular development, regulation of homeostasis, inflammation, vascular tone, and angiogenesis. Fibulin-5 is an extracellular matrix protein also known as UP50 (Urine Protein 50), EVEC (embryonic vascular EGF-like repeat-containing protein) [1], and DANCE (developmental arteries and neu*
1
Corresponding author. Fax: +972 4 8250936. E-mail address: [email protected] (M.Y. Flugelman). Both the authors contributed equally to this work.
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.07.156
ral crest epidermal growth factor-like) [2]. Currently, there are five known members in the fibulin family. The fibulins are secreted glycoproteins, associated with the formation and stabilization of extracellular matrix (ECM) proteins and elastic fibers, and are particularly abundant in tissues rich in elastic fibers [3]. Fibulin-5 contains six EGF-like repeats, all containing consensus sequences for Ca2+-binding (CB) EGF-like repeats. The first CB-EGF-like repeat of fibulin-5 also contains a RGD sequence, which mediates interaction with cell surface integrins. Nakamura et al.
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demonstrated that fibulin-5 may increase cell attachment to tissue culture plates through RGD-integrin-dependent binding [2]. The binding of fibulin-5 to cellular surface was mediated by cellular integrins including avb3 and avb5, which have significant role in EC biology and angiogenesis [4]. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and is a heparin-binding homodimeric glycoprotein of 45 kDa. In vitro, VEGF stimulates EC proliferation, migration, ECM degradation, and tube formation of endothelial cells [5,6]. VEGF was demonstrated to be a key survival factor for endothelial cells, both in vitro and in vivo [7]. The biological effects of VEGF are mediated by two tyrosine kinase receptors, VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1, KDR) [8,9]. In this work, we tested the hypothesis that fibulin-5 alone will increase EC adhesion and retention on artificial surface following exposure to shear stress. After finding that increased adhesion was associated with decreased EC proliferation, we tested whether co-expression of VEGF165 will reverse the inhibitory effect of fibulin-5 on proliferation without affecting the improved adhesion. To test the hypothesis, we investigated the effect of fibulin-5 alone or in combination with VEGF165 on human EC immediate attachment within 30 min after seeding, adherence to artificial surfaces 24 h after seeding followed by exposure to shear stress, and proliferation after fibulin 5 and VEGF165 gene transfer. For gene transfer we used pseudo-typed retroviral vectors encoding fibulin-5, VEGF165, and Green fluorescent protein (GFP) as control. By co-expression of fibulin 5 and VEGF165 we generated unique phenotype of EC with increased adhesion and improved proliferation. Although we focused in this study on the biology of fibulin 5 and VEGF in EC, the findings of the study may have practical implications for tissue engineering of artificial surfaces covered with autologous EC. Methods EC isolation, expansion, and characterization Human venous derived endothelial cells were used for the experiments. Endothelial cells were isolated using an enzymatically based method from a 5 to 10 cm vein segment and were cultured on tissue culture plate as previously described [10]. Endothelial cells were characterized by typical ‘‘cobblestone’’ morphology and by immunostaining for CD-31, and von-Willebrand Factor (vWF). For CD31 staining, cells were seeded on 4-well tissue culture slides (Nunc, USA) for 24 h and then fixed with 4% paraformaldehyde (PFA). Following antigen retrieval, slides were incubated with CAS block (Zymed, USA) and then immunostained with mouse anti-human CD-31 antibodies (DAKO, Denmark). The secondary antibody was biotin-conjugated goat antimouse (Chemicon, USA). Bound antibody detection was performed with horseradish peroxidase-conjugated streptavidin (Chemicon, USA). Staining for vWF was performed using similar procedure. EC at passage 6–7 were used in all experiments. Retroviral vector production For the current experiments, we produced 3 retroviral vectors encoding: Green fluorescent protein (GFP); VEGF165-IRES-GFP; fibulin-
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5-IRES-GFP. Human cDNA of VEGF165 and fibulin-5 was used for vector construction. For the vectors encoding VEGF and fibulin 5 we used bicistronic expression cassettes in which the GFP expression was controlled by IRES element. The 600 bp BamH1 fragment of human VEGF165 was cut from pUC18-VEGF165 (GenBank Accession No. AB021221) including the signal sequence for secretion (a kind gift of Dr. J. Abraham, Scios Nova, Mountain View, CA). Construction of pLXSN-VEGF165-IRES-GFP, a bicistronic plasmid co-expressing VEGF165 and the GFP genes, was done in two steps as follows: First, a 600 bp BamHI fragment of VEGF165 was inserted into the BamHI site of pIRES2-GFP (#6029-1 Clontech, USA). Second, a 2.0 kb EcoRI–MunI fragment containing VEGF165 (600 bp), IRES element, and GFP genes, was excised from pVEGF165-IRES-GFP plasmid and inserted into the EcoRI restriction site at the multiple cloning site (MCS) of pLXSN. For retroviral vector production, 10 lg of plasmids was transfected into 293FLYA packaging cells using Lipofectamine (Gibco-BRL, USA). After 48 h the supernatant from confluent cultures of viral producer cells was collected, filtered (0.45 lm), and added to 293FLYGALV packaging cells (a kind gift from F-L Cosset, Lyon, France) [11,12]. The transduced cells were grown under G418 selection (400 lg/ml; Calbiochem, USA) and individual colonies were collected and screened for VEGF165 expression by Western blot analysis of conditioned medium samples. Generation of pseudo-typed retroviral vectors encoding fibulin-5-GFP was performed in a similar way. Cell transduction 4 · 105 ECs were seeded on 60 mm fibronectin-coated plates (4.5 lg/ ml) 24 h prior to transduction. Transduction was initiated by incubation with DEAE-dextran (1 mg/ml; Sigma, USA). The cells were then washed 3 times with PBS and exposed to the retroviral vectors in transduction medium for 4 h. The media containing the retroviruses were then replaced with growth medium. EC were cultured in the presence of G418 (0.5 mg/ml, Gibco, USA). Transgene expression was monitored by GFP expression under florescent microscope, and Western blot, ELISA, and immunohistochemistry (IHC) for the relevant transgene. Sequential (‘‘dual’’) cell transduction Sequential ‘‘Dual’’ transduction of EC was achieved using a two-step process: (1) initial transduction with the retroviral vector encoding fibulin5, followed by selection with G418 (0.5 mg/ml) until >80% of the EC population was expressing fibulin-5 as detected by GFP expression by the cells; (2) transduction of this fibulin-5 expressing EC population with VEGF165 encoding retroviral vector. ECs were cultured in the presence of G418 and trans-gene expression was monitored by IHC for VEGF165 and fibulin-5. VEGF165 expression was also confirmed by ELISA and Western blotting. Since VEGF165 gene transfer was performed after fibulin-5 gene transfer VEGF165 encoding vector transduction rate was at the level of 10– 30% of cells. Western blot Western blot was used for verification of transgene expression in the transduced cells. Conditioned medium from the transduced cells was collected and was separated by 8–10% SDS–PAGE gels. For fibulin-5 the proteins were electro-transferred to nitrocellulose paper which was blocked with 10% low-fat milk and incubated at a 1:500 dilution with rabbit polyclonal antibodies directed against a synthetic peptide derived from human fibulin-5 sequence (custom made, Sigma Israel). Horseradish peroxidase-labeled goat anti-rabbit antibodies were used to visualize bound antibody using the ECL detection system. VEGF165 detection was performed using goat anti-human-VEGF antibody (Santa-Cruz, USA). Horseradish peroxidase-labeled donkey anti-goat antibodies were used to visualize bound antibody using the ECL detection system.
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Immunohistochemistry (IHC) for transgene expression IHC was used for identification of transgene expression in the transduced cells. Cells were prepared as described above. For VEGF165 staining slides were incubated with CAS block (Zymed), and then immunostained with goat anti-human VEGF antibodies (R&D). Biotin-conjugated donkey anti-goat Ab (Chemicon) or Rhodamin-conjugated secondary antibodies were used. For fibulin-5 staining, slides were incubated with mixture of CAS block (Zymed) and goat serum (Zymed) and then immunostained with fibulin-5 rabbit anti-human antibody in blocking solution supplemented with 2% Tween 20. Envision-rabbit peroxidase conjugate (Dako) or FITC-conjugated secondary antibody were used. ELISA Human VEGF immunoassay kit was used according to manufacturer’s instructions (R&D, USA) for VEGF165 levels. Fibulin-5 protein level was measured using an ELISA that was developed in our laboratory. Standards and samples were distributed into a non-antibody-coated 96-well plate. Rabbit anti-human fibulin-5 antibody was added and was bound to fibulin-5 present in the well. Secondary antibody was Envision-rabbit peroxidase conjugate (Dako). TMB/E solution (Chemicon, USA) was used as a substrate for color reaction. The plate was read using ELISA reader at 450/540 nm wave length. Proliferation assays Human saphenous vein endothelial cells (2 · 104 cells/well) were seeded in 24-well culture dishes pre-coated with fibronectin (20 lg/ml). EC were harvested with trypsin (Biological industries, Israel) and counted
using a cell counter (Coulter, USA) 4 h after seeding, and at 48, 96, and 120 h following seeding. Each experiment was performed in triplicate and was repeated in three different primary human saphenous vein endothelial cells. Proliferation rate was tested in non-transduced EC (naive), retrovirally transduced EC over-expressing GFP, fibulin-5, VEGF165, and retrovirally transduced EC co-expressing fibulin-5 and VEGF165. The assay was performed with and without supplementation of bFGF (2 ng/mL) and in the presence of 20% fetal calf sera (HyClone Perbio Science co. USA) supplemented to growth media. Early attachment to ECM This experiment was design to test whether fibulin-5 incorporated in ECM and supplemented to culture media have an effect on immediate EC attachment to ECM. ECM was generated in 48-well plates as previously described by Schneider et al. [13] We produce ECM by naive EC, EC transduced to express fibulin 5-GFP or GFP alone. We seeded 2 · 104 EC in each well. The cells seeded on ECM generated by fibulin-5-GFP transduced EC were re-suspended and incubated with conditioned medium from fibulin-5 transduced cells. The cells seeded on ECM generated by GFP transduced cells were re-suspended and incubated with conditioned media collected from GFP expressing cells, and the control cells were seeded on ECM generated by naive EC and were re-suspended and incubated with naive EC conditioned media. The cells in three groups were incubated for 30 min and then washed with PBS. The number of cells remaining attached to the dishes after washing was quantified using XTT colorimetric assay according to manufacturer’s instruction (Biological Industries, Israel) 4 h following exposure to the reagent. Presence of fibulin in ECM was confirmed by immunohistochemistry and in conditioned media by ELISA (data not shown).
Fig. 1. In vitro automated flow apparatus. Pulsatile flow was generated using a Harvard pump (Harvard apparatus #1421, USA) that was connected to the tubing and container system, positioned in an incubator of 37 C and 5% CO2. The tubing system consisted of stainless steel (#316 Hamlet, Israel) and Teflon tubes (6 mm). A physiological pulse wave was achieved with the pulse wave tank (Tank A). Controlling pulse wave amplitude and flow increment were performed using valve A. Adjustment and control of flow through the grafts was achieved using valves B and C. Adjustment and control of the pressure in the system was achieved using valve D. Fluid equilibrium with the incubator atmosphere (37 C, 5% CO2) was achieved through compensation tank B which also served as a fluid reservoir. The system was monitored online by both pressure (#742 Mennen Medical, Israel) and flow (Doppler flow monitor #T106 Transonic Systems Inc. USA) monitors. Grafts used in the experiments were 5 cm long, 6 mm diameter; thick wall ePTFE grafts (GORE, USA).
M. Preis et al. / Biochemical and Biophysical Research Communications 348 (2006) 1024–1033 Endothelial cell adhesion under continuous shear stress In this experiment we tested whether fibulin-5 expression by EC improves attachment to tissue culture plates under continuous shear stress. Cells were seeded 24–48 h before exposure to shear stress. Unlike previous experiment in which cells were tested for immediate attachment (30 min) to ECM enriched with fibulin, in this experiment we tested EC adherence to tissue culture dishes pre-coated with fibronectin 24–48 h after seeding. Testing cell retention under continuous (24 h) shear stress was achieved by horizontal shaking of the cell culture dishes on a standard laboratory linear shaker. Endothelial cells were counted prior to exposure to continuous shear stress and following exposure. The assay was performed with naive EC, EC over-expressing GFP, and EC over-expressing fibulin-5. EC adhesion under continuous pulsatile flow In order to test the effect of fibulin-5 over-expression on EC adhesion under physiological flow conditions, we established an automated in vitro pulsatile flow apparatus. The cells were seeded on ePTFE grafts (Gore, USA) and were subjected to 2 h of pulsatile flow with shear stress similar to human femoral artery shear stress. In this set of experiments, ePTFE grafts (6 mm, Gore co., USA) were pre-incubated with fibronectin (Sigma, USA) 24 h prior to seeding. EC seeding was performed using a rotation device to allow homogeneous, gravity independent graft seeding. The vascular grafts were filled with an EC suspension, which contained 4.5 · 105 cells/cm2 graft surface area in EC growth medium. The grafts with cell suspension were rotated for 48 h in a 5% CO2, 37 C sterile environment. This procedure yielded ePTFE grafts with luminal coverage by confluent EC monolayer. Flow apparatus. In order to test the effect of fibulin-5 over-expression on endothelial cell retention, we established an in vitro automated flow apparatus (Fig. 1). Flow and pressure were monitored and analyzed
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continuously by a data acquisition system (Lab view software), which presented the calculated shear forces generated in the grafts. The flow conditions were set to simulate the flow in the femoral artery: pressure of 120/80 mmHg, flow of 300–350 ml/min, and shear stress of 7–10 dyn/cm2. Tested grafts. ePTFE (6 mm; GORE Co. USA) was used, an average of 5 cm long grafts were seeded as described above with human venous EC. Grafts were seeded with naive endothelial cells (non-transduced) (n = 6), GFP transduced EC (n = 6), fibulin-5-GFP transduced EC (n = 11), and EC transduced with fibulin-5 and subsequently with VEGF165 (n = 8). Evaluation of graft seeding. At the end of seeding procedure seeded grafts edge (5 mm segment) were sliced from the seeded graft and were cut open and fixed in 2.5% Glutaraldehyde. The luminal aspects of the fixed graft segments were used for evaluation of EC coverage of the graft material. After fixation we stained EC nuclei with DAPI containing VECTRA-SHIELD mounting solution (VECTOR, USA). Number of DAPI stained nuclei per microscopic field was used for quantitative assessment of graft coverage. Morphometric evaluation of endothelial surface coverage was performed using image analysis software (Image Pro Plus 4, Media cybernetics, USA). At least 10 randomly selected fields (100· magnification) of the graft surface were analyzed. The analysis was preformed by an operator blinded to the experimental design. Statistical methods All the data are presented as means ± SD. Comparison of multiple non-parametric groups was performed using the Kruskal–Wallis test or Bonferroni all Pairwise Comparisons Test (Statistix 8, USA). Two nonparametric groups were compared using the Mann–Whitney U test (or Wilcoxon Rank Sum Test). Two parametric groups were compared using student’s t-test. A p value <0.05 was considered significant.
Fig. 2. Primary human saphenous vein endothelial cells’ identity is shown based on the following immunohistochemical staining: (a) All cells are positive for CD31staining and von-Willebrand Factor (vWF) staining, and negative for a-smooth muscle actin. (b) Transgene expression is demonstrated by immunohistochemical staining for fibulin 5 (88% of cells) and VEGF165 (29% of cells).
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Results Endothelial cell isolation and characterization Endothelial cells were isolated and characterized as described in the methods (Fig. 2a). Following retroviral vector transduction endothelial cells expressed fibulin-5 and VEGF165 as shown in Fig. 2b. Sequential viral vector transduction with VEGF165 encoding vector was also performed and co-expression was demonstrated using immunohistochemistry (Fig. 2b) and Western blotting (Fig. 3). In the ‘‘dual’’ transduced cells, fibulin-5 was expressed by more than 88% of the cells and VEGF165 was expressed by 10– 30% of the cells. When we tested transgene expression by ELISA, EC transduced to express fibulin-5 produced 0.25 ± 0.04 pg/cell/24 h of the transgene, and after VEGF gene transfer produced 0.07 ± 0.03 pg/cell/24 h VEGF. Effect of fibulin 5 on endothelial cell attachment and adherence
5.Fibulin 5 & VEGF
4.Fibulin -5
3.VEGF
2.Naive EC
1.Positive control
Early EC attachment to ECM-coated tissue culture plates was tested. Early attachment is mainly mediated by
Fibulin-5
VEGF165
Fig. 3. Western blots for transgene expression: Lane 1 is positive control for fibulin-5 (upper panel) and VEGF (lower panel). Lane 2 is of naive EC, lane 3 is of cells transduced to express VEGF, Lane 4 is of cell transduced to express fibulin-5, and Lane 5 is of cells transduced to express fibulin-5 and VEGF. Transgene expression after transduction is evident in lanes 3– 5. Both fibulin-5 and VEGF are expressed by the dually transduced EC.
cell surface integrins and ECM proteins. Four experiments were completed in triplicates. Calibration curve was performed with known number of cells in order to convert the value of XTT measurement to number of cells (data not shown). A tendency to improved short-term attachment in the presence of fibulin-5 enriched ECM and cell culture media was found (average of all experiments: 17,599 ± 1641 for naive cells, 17,319 ± 1822 for GFP transduced cells, and 19,624 ± 2281 for fibulin-5 transduced cells; p = 0.08). This experiment may imply to the mechanisms by which fibulin-5 can improve EC attachment. The effect of fibulin-5 expression on cell adhesion was examined 24–48 h after EC plating and after exposure to continuous (24 h) shear stress, achieved by horizontal shaking of the cell culture dishes on a standard laboratory linear shaker. Cells were counted prior to exposure to continuous shear stress and following exposure. The assay was performed with naive EC, EC over-expressing GFP, and EC over-expressing fibulin-5. In the fibulin-5 over-expressing EC, 75 ± 15% of the cells remained adherent to the plate, in comparison to 57 ± 16% GFP over-expressing EC and 52 ± 13% naive EC (n = 8 experiments, four experiments were performed in triplicates, and 4 in duplicates, p = 0.002) (Fig. 4). These results demonstrate a significant effect of fibulin-5 over-expression on endothelial cell adhesion under continuous shear stress. We tested EC adhesion under pulsatile flow after seeding EC on ePTFE grafts. In this experiment EC adherence was tested under physiological shear stress conditions. The use of ePTFE vascular grafts in this experiment is part of our effort to translate the observed improved EC adhesion properties for use in tissue engineering of vascular grafts that has a luminal EC monolayer. Vascular grafts made of ePTFE (6 mm diameter) were seeded with EC and exposed to pulsatile flow for 2 h. The analysis included percentage of area coverage by endothelial cells. ePTFE grafts seeded with EC over-expressing fibulin-5 or fibulin5 + VEGF165 had significantly improved retention of EC following exposure to 2 h of pulsatile flow when compared to grafts seeded with EC expressing GFP, or naive EC (Figs. 5a and b). No significant difference was found between ePTFE grafts seeded with EC over-expressing fibulin-5 and grafts seeded with EC co-expressing fibulin-5 and VEGF165. These experiments demonstrated the improved retention of EC seeded on ePTFE grafts as a result of fibulin-5 over-expression. Effects of fibulin 5 and VEGF165 on endothelial cell proliferation Proliferation experiments were performed on EC as described in Methods. Overall, fibulin-5 over-expression by human EC had inhibitory effect on EC proliferation (Fig. 6). Repeated proliferation assays using different primary EC from three different individuals demonstrated a similar effect. The inhibitory effect of fibulin-5 on cell
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Fig. 4. Endothelial cells before and after exposure to continuous shear stress. (upper part) Images of representative fields under light microscopy, before and after exposure to continuous shear stress. (lower part) Summary of the remaining attached endothelial cells, according to transgene expression.
proliferation was more pronounced in the absence of bFGF, a strong EC mitogen. In this serum concentration (20% of FCS) and absence of bFGF cell death was observed. With supplementation of bFGF to culture media no EC death was observed but proliferation of EC expressing fibulin-5 was similar to the proliferation rate of control cells and inferior to proliferation of EC expressing VEGF165. EC expressing both VEGF165 and fibulin-5 demonstrated the high proliferation rate both in the presence or absence of bFGF. These experiments demonstrated that fibulin-5 inhibit primary human venous EC proliferation. VEGF165 co-expression in cells over-expressing fibulin-5 improved cell proliferation and attenuated the inhibitory effect of fibulin-5. Similar results were obtained when we used exogenous protein supplementation in non-transduced EC (data not shown). Discussion In this study, we tested the hypothesis that fibulin-5 gene transfer will increase EC attachment and adhesion to artificial surface, and extended our research to test whether reduced EC proliferation observed after fibulin-5 gene transfer can be reversed by co-expression of VEGF165. EC attachment and adhesion was improved after fibulin-5 gene transfer as demonstrated in three different experimental settings; tendency to improved attachment was
demonstrated 30 min after cell seeding, and significantly improved adherence was observed in EC exposed to shear forces 24–48 h after seeding. Fibulin-5 contains six Ca2+-binding EGF motifs, Rao et al. demonstrated that these areas, following binding of Ca2+, can stabilize the protein structure and direct protein to extracellular protein interaction.[14] The RGD sequence on the first Ca2+-binding EGF motif of fibulin-5 can interact with cellular surface integrins [15]. Nakamura et al. also demonstrated that fibulin-5 increase HUVEC attachment to fibulin-5-coated microtiter plates. The attachment was dose dependent and was inhibited in the presence of exogenous GRDSP peptide [2]. He also identified the cellular surface integrins avb3, avb5 and a9b1 as binding surface integrins [16]. Yanagisawa et al. demonstrated that the fibulin-5 interacts with extracellular proteins and especially with tropoelastin [17]. These data suggest that fibulin-5 acts as a bridging molecule between cells and ECM protein. We tested EC attachment 30 min following cell seeding and demonstrated a tendency to improved initial EC attachment in the presence of fibulin-5. In his attachment experiments Nakamura et al. used fibulin-5 concentration of 1–16 lg/ml. In our experiments we focused on fibulin5 concentration in a more physiological context of protein production and expression. This may explain the statistically borderline effect on attachment observed in our experiments.
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a
b
Fig. 5. Improved retention of endothelial cells over-expressing fibulin-5 or co-expressing fibulin-5 and VEGF165 on ePTFE grafts (a) endothelial cells (EC) were seeded on ePTFE grafts and exposed to pulsatile flow conditions in an in vitro flow apparatus. Non-transduced-naive EC, EC over-expressing GFP, fibulin-5 or co-expressing fibulin-5 and VEGF165 were seeded on ePTFE grafts. Results are presented as mean percentage of cell retention ± SD. (b) Nucleic staining by DAPI of seeded graft sections. Non-transduced-naive EC, and EC co-expressing fibulin-5 and VEGF165 were examined following seeding and following exposure to pulsatile flow for 2 h. Although complete coverage is apparent in the two grafts after seeding, cell adherence of EC expressing the two genes is higher than naive cell adherence after exposure to flow.
Cell adhesion under continuous shear stress was also tested. This method was described by Asada et al. as a method to test the effect of shear stress on cells seeded in tissue culture plates [18]. Over-expression of fibulin-5 improved cell retention following exposure to continuous shear stress by approximately 40% compared to naive EC or GFP transduced EC. In vitro experiments in an automated fluid circulation apparatus demonstrated the effect of fibulin-5 over-expression on endothelial cell attachment to ePTFE grafts. The flow conditions in the close circuit were set to mimic the flow conditions in the human femoral artery. The close system enabled us to maintain constant pulsatile fluid pressure and flow, while shear stress was monitored online. The fluid dynamics parameters were maintained stable throughout the experiments and enabled us to compare the different
groups. Fibulin-5 expression by endothelial cells significantly improved the adherence to ePTFE surface and increased the percentage of cell retention on the graft surface following exposure to flow. More than 40% improvement in cell retention was achieved by over-expressing fibulin-5. This can be attributed to the action of fibulin-5 as a bridging molecule that anchors the cells to the graft coating proteins. The co-expression of fibulin-5 and VEGF165 by endothelial cells had also improved retention in comparison to the control grafts seeded with naive cells (Wilcoxon rank sum test, p value = 0.04) or EC transduced with GFP (Wilcoxon rank sum test, p value = 0.01). With improved adhesion we observed that EC transduced to express fibulin-5 exhibited reduced proliferation rates. Reduced proliferation with fibulin-5 was also reported by Scheimann et al. who demonstrated that fibulin-5
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Fig. 6. Endothelial cell proliferation assay. Human saphenous vein endothelial cells (2 · 104 cells/well) were seeded in 24-well culture dishes pre-coated with fibronectin (20 lg/ml). Endothelial cells (EC) were harvested with trypsin and counted using a cell counter (Coulter, USA) 4 h after seeding, and at 48, 96, and 120 h. Each experiment was performed in triplicates. Proliferation rate was tested in non-transduced EC (naive), retrovirally transduced EC over-expressing GFP, retrovirally transduced EC over-expressing VEGF165, retrovirally transduced EC over-expressing fibulin-5, and retrovirally transduced EC co-expressing VEGF165 and fibulin-5. The assay was performed without bFGF (a) and following bFGF stimulation (b). Results are presented as average of triplicates ± SD.
significantly inhibited DNA synthesis and cell proliferation [19]. Albig et al. demonstrated that fibulin-5 inhibits EC proliferation and invasion through synthetic basement membranes [20]. After finding that increased adhesion was also associated with decreased EC proliferation we tested whether coexpression of VEGF165 will reverse the inhibitory effect of fibulin-5 on proliferation without changing the improved adhesion. We initially showed that EC could be transduced to express both fibulin-5 and VEGF165. To achieve predictable transduction rates we initially transduced the cells with pseudo-typed retroviral vector encoding fibulin-5. Subsequently, we transduced the cells with VEGF165 encoding vector. Co-transduction with the VEGF165 encoding vector reversed the inhibitory effect of fibulin-5 on EC proliferation without a significant change in adhesion properties. Co-expression of the two genes increased EC proliferation when compared to control EC or even when compared to EC expressing VEGF165 alone. Improved
proliferation was observed both with and without basic FGF. Albig et al. also investigated the opposing effects of VEGF165 and fibulin-5 on EC proliferation but did not observe synergistic effect as we observed. In a previous report we have shown that EC over-expressing VEGF165 up-regulate levels of VEGF receptor 2(KDR) [21]. Up-regulated VEGF receptors render the cells more sensitive to VEGF and consequently lead to enhanced proliferation. The synergistic effect observed with fibulin-5 and VEGF165 cannot be explained on increased number of VEGF receptors alone. Synergy may be due to the improved adhesion that is necessary for cell proliferation and the mitogenic effect of VEGF. Based on our findings, co-expression of these two genes created a unique phenotype of EC. The extracellular environment has a substantial effect on EC proliferation, migration, attachment, and survival. Failure of EC to attach to ECM results in proliferation arrest and subsequently in cell death. Both proliferation and survival are dependent on adhesion to ECM which is
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mediated by cell surface integrins [22]. ECM proteins have various effects on EC: collagens and especially collagen type I activate EC and promote basement membrane degradation, proliferation, and invasion in a similar manner to VEGF. This effect is mediated mainly through integrins avb3 and a5b1. Laminin on the contrary, promotes basement membrane formation and reduced proliferation as does TGFb, in a process mediated mainly through integrins a6b1 and a3b1 [23]. Integrins mediate not only cell adhesion and proliferation by cell–matrix and cell–cell interaction but also by multiple outside-in signals that lead to activation of downstream pathways such as tyrosine kinases and phosphatidylinositol 3-kinase [24]. The importance of using VEGF is also supported by work published by Chennazhy et al. [25] EC grown on synthetic materials lose their proliferation potential, turn apoptotic and express pro-thrombotic phenotype, but when they are grown on matrix composed of fibronectin and VEGF, the cell remain viable, proliferate, and are less thrombogenic. While av integrin activation is necessary for the angiogenic cascade, the same integrin activation may cause anti-proliferative and anti-apoptotic effect. This inhibitory effect may be caused by binding of blocking peptides to integrin that occupy the integrin active sites [26]. These studies and especially the work of Cheresh et al. may explain the findings that fibulin-5, an integrin-binding protein, when over-expressed results in inhibition of EC proliferation. Acknowledgments This work was supported in part by a grant from the Israeli Academy of Science. Based on the technology described in this paper we established a company, MGVS Ltd. (MultiGene Vascular Systems Ltd.), in our Medical Center. The following authors are either workers or consultants of MGVS Ltd., Haifa: Cohen, T., Sarnatzki, Y., Ben Yosef, Y., Schneiderman, J., Gluzman, Z., Koren, B., Lewis, B.S., Shaul, Y., Flugelman, M.Y. References [1] R.C. Kowal, J.A. Richardson, J.M. Miano, E.N. Olson, EVEC, a novel epidermal growth factor-like repeat-containing protein upregulated in embryonic and diseased adult vasculature, Circ. Res. 84 (1999) 1166–1176. [2] T. Nakamura, P. Ruiz-Lozano, V. Lindner, D. Yabe, M. Taniwaki, Y. Furukawa, K. Kobuke, K. Tashiro, Z. Lu, N.L. Andon, R. Schaub, A. Matsumori, S. Sasayama, K.R. Chien, T. Honjo, DANCE, a novel secreted RGD protein expressed in developing, atherosclerotic, and balloon-injured arteries, J. Biol. Chem. 274 (1999) 22476–22483. [3] K.S. Midwood, J.E. Schwarzbauer, Elastic fibers: building bridges between cells and their matrix, Curr. Biol. 12 (2002) R279–R281. [4] P.C. Brooks, Role of integrins in angiogenesis, Eur. J. Cancer 32A (1996) 2423–2429. [5] J. Plouet, J. Schilling, D. Gospodarowicz, Isolation and characterization of a newly identified endothelial cell mitogen produced by AtT-20 cells, EMBO J. 8 (1989) 3801–3806.
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