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Vascular Endothelial Growth Factor (VEGF)-modified
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Macrophages Transdifferentiate into Endothelial-like
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Cells and Decrease Foam Cell Formation
4 Dan Yan1, *, Yujuan He 2, Jun Dai3, Lili Yang 3, Xiaoyan Wang3, Qiurong Ruan 3, *
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Department of Pathology, Medical College, Wuhan University of Science and Technology, Wuhan, Hubei, P.R. China
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Technology, Wuhan, Hubei, P.R. China
Department of Pathology, Affiliated Tianyou Hospital of Wuhan University of Science and
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Institute of Pathology of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, P.R. China
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*Corresponding Authors
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To whom correspondence should be addressed:(email:
[email protected] ).
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Running Title：endogenous VEGF overexpression on macrophages
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23 24 25 26 27 1 Use of open access articles is permitted based on the terms of the specific Creative Commons Licence under which the article is published. Archiving of non-open access articles is permitted in accordance with the Archiving Policy of Portland Press ( http://www.portlandpresspublishing.com/content/open-access-policy#Archiving).
ACCEPTED MANUSCRIPT
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Abstract
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Macrophages are largely involved in the whole process of atherosclerosis from an initiation lesion
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to an advanced lesion. Endothelial disruption is the initial step and macrophage-derived foam cells
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are the hallmark of atherosclerosis. Promotion of vascular integrity and inhibition of foam cell
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formation are two important strategies for preventing atherosclerosis. How can we inhibit even
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reverse the negative role of macrophages in atherosclerosis? This study was performed to
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investigate if overexpressing endogenous human vascular endothelial growth factor (VEGF) could
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facilitate transdifferentiation of macrophages into endothelial-like cells (ELCs) and inhibit foam
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cell formation. We demonstrated that VEGF-modified macrophages which stably overexpressed
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human VEGF165 displayed a high capability to alter their phenotype and function into ELCs in
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vitro. Exogenous VEGF could not replace endogenous VEGF to induce the transdifferentiation of
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macrophages into ELCs in vitro. We further showed that VEGF-modified macrophages
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significantly decreased cytoplasmic lipid accumulation after treatment with oxidized LDL
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(ox-LDL). Moreover, down-regulation of CD36 expression in these cells was probably one of
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mechanisms of reduction in foam cell formation. Our results provided the in vitro proof of
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VEGF-modified macrophages as atheroprotective therapeutic cells by both promotion of vascular
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repair and inhibition of foam cell formation.
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Keywords: vascular endothelial growth factor; macrophages; endothelial-like cells; foam cell
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Introduction
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Atherosclerosis is the primary cause of mortality and morbidity in cardiovascular disease, which is
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the leading cause of death in industrialized society [1,2]. It’s generally recognized that endothelial
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dysfunction or disruption is the initial process of atherosclerosis [3-5]. Endothelial progenitor cells
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(EPCs) are capable of facilitating re-endothelialization through direct differentiation into
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endothelial cells and/or via the paracrine mechanisms [6,7]. Moreover, a recent research pointes
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that endothelial to mesenchymal transition (EndMT) contributes to atherosclerotic pathobiology
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and is associated with complex plaques that may be related to clinical events [8]. Therefore,
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maintenance of the endothelial homeostasis and integrity by promoting early repair is an important
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strategy for preventing atherosclerosis [9,10].
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Monocytes recruit from peripheral blood and attach to the activated/damaged endothelium, and
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then migrate to subendothelial space and differentiate to macrophages. The uptake of ox-LDL by
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monocyte-derived macrophage induces foam cell formation, which is a hallmark of the
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development of atherosclerosis [11,12]. Inhibition of foam cell formation in early stage is another
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fascinating approach for the prevention of progression of atherosclerosis.
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Cells of the monocyte-macrophage lineage are characterized by considerable diversity and
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plasticity [13]. Monocytes are not only the precursors of lipid-laden foam cell macrophages, but
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also display high developmental plasticity to differentiate under appropriate stimulation into
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different cell types, including endothelial lineage cells [14]. Can we take the advantage of the
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potential developmental relationship between monocytes/macrophages and endothelial lineage
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cells to alter some properties of macrophages to inhibit even reverse the negative role of
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macrophages in atherosclerosis? 3
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In our previous research, we transiently transfected mouse primary macrophages with human
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vascular endothelial growth factor 165 (hVEGF165) plasmid and found that they could
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transdifferentiate into ELCs and incorporated in the newly formed vessel [15,16]. It indicated that
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the macrophages overexpressing VEGF might become a promising role like EPCs for cell-based
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therapy. However, the primary macrophages transiently transfected with hVEGF165 only express
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the target protein for a few days. To further investigate the effect of stable endogenous VEGF on
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macrophages, we already have successfully established hVEGF165-ZsGreen1-RAW264.7 cells - a
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mouse macrophage cell line stably overexpressing human VEGF165 by lentiviral vector [17].
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Then the aims of the present study were to (1) identify the phenotype and function of
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hVEGF165-ZsGreen1-RAW264.7 cells to determine whether they transdifferentiate into ELCs; (2)
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investigate the capability of hVEGF165-ZsGreen1-RAW264.7 cells to become foam cells and the
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underlying mechanism.
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Materials and Methods
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Cell culture
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RAW 264.7 macrophages stably overexpressing human VEGF named as hVEGF165-
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ZsGreen1-RAW264.7 in this paper was the already established cell line in our laboratory[17].
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Briefly,
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pLVX-IRES-ZsGreen1 to construct recombinant pLVX-hVEGF165-IRES-ZsGreen1. RAW264.7
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cells were infected with this recombined lentiviral vector containing human VEGF165 and
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ZsGreen1 then purified by fluorescence-activated cell sorting (FACS) twice according to green
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fluorescence protein ZsGreen1. Then we obtained the mouse macrophage cell line stably
human
VEGF165
was
subcloned
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into
the
lentiviral
expression
vector
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overexpressing human VEGF165 after verification by fluorescent microscopy, PCR, Western Blot
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and ELISA. hVEGF165- ZsGreen1-RAW264.7 cells were cultured in Dulbecco’s modified Eagle’s
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medium (DMEM; Invitrogen Life Technologies, Carlsbad, CA, USA) high glucose containing
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20% (v/v) fetal bovine serum (FBS; Lonza, Walkersville, MD, USA) and 10 ng/ml basic fibroblast
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growth factor (bFGF; Invitrogen). RAW 264.7 macrophages only transfected with ZsGreen1
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named as ZsGreen1-RAW264.7 and untransfected RAW264.7 (ATCC, Manassas,VA, USA) were
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both as controls. There is another group of untransfected RAW264.7 with treatment of exogenous
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recombinant human VEGF165 protein (PeproTech Inc, Rocky Hill, NJ, USA) in 50ng/ml for 48 h
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named as VEGF-treated-RAW264.7.
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qRT-PCR
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To detect the level of some endothelial marker genes, mRNA expression of vascular endothelial
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growth factor receptor-2 fetal liver kinase 1 (FLK-1), von Willebrand factor (vWF), endothelial
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NO synthase (eNOS), vascular endothelial-cadherin (VE-cadherin) and Tie-2 were evaluated by
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qRT-PCR using the Thunderbird SYBR qPCR Mix (TOYOBO, Japan) with gene specific primers
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on a 7500 Fast real-time PCR system (Applied Biosystems, Alameda, CA, USA). The expression
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of CD36 mRNA in ox-LDL-induced hVEGF165-ZsGreen1-RAW264.7 cells was also detected as
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above. Specific fragments were amplified and β-actin was also amplified to serve as internal
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standard. The results were normalized to β-actin and presented as fold difference relative to
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RAW267.4 control. All primer sequences are shown in Supplementary Table S1. All experiments
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were repeated three times, and the representative data are shown.
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Western blot analysis
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To detect the level of some endothelial marker proteins, expression of FLK-1, vWF and eNOS
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was performed by Western Blot Analysis. Briefly, cells were lysed in RIPA buffer (Sigma-Aldrich,
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MO, USA), followed by protein quantification using Pierce BCA Protein Assay Kit (Thermo
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Fisher Scientific, Pittsburgh, PA，USA). Cell lysates containing the same amount of proteins were
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separated on SDS-polyacrylamide gel electrophoresis, followed by transferring onto the
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nitrocellulose membranes. After non-specific blocking with 5% non-fat milk in TBS containing
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0.05% Tween 20 at room temperature for 1 h, the membranes were incubated with the specific
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antibodies towards mouse FLK-1 (Invitrogen; 1:1000), vWF (Santa Cruz Biotechnology, Santa
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Cruz, CA, USA; 1:600) and eNOS (Abcam, , Cambridge, MA, USA; 1:500) at 4 °C for overnight.
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Subsequently membranes were incubated with appropriate secondary antibody, and protein bands
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were visualized using enhanced chemiluminescence (Thermo Fisher). Bands were quantified by
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densitometry and normalized to those of β-actin (Abcam; 1:1000). The expression of CD36
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protein in ox-LDL-induced hVEGF165-ZsGreen1-RAW264.7 cells was detected by the specific
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antibody (Abcam; 1:1000) as above. All experiments were repeated three times, and the
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representative data are shown.
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In vitro angiogenesis assay
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The formation of tubular-like structures was assessed using Matrigel (BD Biosciences, San Jose,
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CA USA) by in vitro angiogenesis assay. A 96-well plate was pre-coated with Matrigel and
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incubated at 37°C for 2 h prior to the addition of 2×104 cells /well suspended in 100 µl
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conditioned medium. Following additional 24 h incubations, three fields were chosen at random 6
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and the formation of tubular-like structures was observed using an inverted microscope (IX51;
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Olympus, Japan).
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ELISA for VEGF concentration
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After 72 h of culture, VEGF concentration in supernatants was measured in triplicate using the
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VEGF human ELISA kit and VEGF mouse ELISA kit (Both from Abcam), respectively. Briefly,
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VEGF standards and samples were pipetted into wells and VEGF present in a sample was bound
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to the wells by the immobilized antibody. The wells were washed and biotinylated anti-human or
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anti-mouse VEGF antibody was added. After washing away unbound biotinylated antibody,
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peroxidase (HRP)-conjugated streptavidin was pipetted to the wells. The wells were again washed,
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a TMB substrate solution was added to the wells and color developed in proportion to the amount
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of VEGF bound. The Stop Solution changed the color from blue to yellow, and the intensity of the
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color was measured at 450 nm. VEGF concentrations were calculated (in pg/ml) with the standard
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curve .
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Foam cell formation assay
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An in vitro foam cell formation assay was performed as described previously with minor
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modification [18]. Briefly, hVEGF165-ZsGreen1-RAW264.7 or ZsGreen1-RAW264.7 or
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RAW264.7 were cultured in 12-well plate in serum free medium and treated with 100 μg/ml
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ox-LDL (Yiyuan Biotechnologies, Guangzhou, China) for 24 h to induce foam cell formation. Oil
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red O powder (Sigma-Aldrich, MO, USA) was dissolved in isopropanol (0.5 %; Sigma-Aldrich).
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The stock was then diluted to 0.3 % oil red O solution with distilled H2O and filtered through a 7
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0.22-μm filter. After fixation with 4% paraformaldehyde for 1 h at room temperature, cells were
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stained with oil red-O solution to detect the lipid accumulation for 5 min. Then cells were
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observed with a microscope and those containing oil red O‐positive fat droplets were considered
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foam cells.
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Quantification of total lipid content
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Quantification of lipid accumulation in cells was measured based on a previously published
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protocol [18]. Cells stained with oil red O were treated with 1ml of 60% isopropanol for 1 h to
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redissolve the oil red O and absorbance was detected at 518 nm through a spectrophotometer.
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Statistical analysis
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Data are presented as means ± SD. The statistical significance of differences between groups was
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analyzed using one-way analysis of variance (ANOVA) with Tukey’s test post hoc. Values of
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P
