tmp921F TMP

From bloodjournal.hematologylibrary.org by guest on May 11, 2014. For personal use only.
Prepublished online March 25, 2011;

doi:10.1182/blood-2010-12-326017
Lysophosphatidic acid suppresses endothelial cell CD36 expression and

promotes angiogenesis via a PKD-1 dependent signaling pathway
Bin Ren, James Hale, Sowmya Srikanthan and Roy L. Silverstein
Information about reproducing this article in parts or in its entirety may be found online at:
http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml
Advance online articles have been peer reviewed and accepted for publication but have not yet
appeared in the paper journal (edited, typeset versions may be posted when available prior to
final publication). Advance online articles are citable and establish publication priority; they are
indexed by PubMed from initial publication. Citations to Advance online articles must include
digital object identifier (DOIs) and date of initial publication.
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of
Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.
Blood First Edition Paper, prepublished online March 25, 2011; DOI 10.1182/blood-2010-12-326017
Lysophosphatidic Acid Suppresses Endothelial Cell CD36 Expression and Promotes

Angiogenesis via a PKD-1 Dependent Signaling Pathway
Bin Ren1, James Hale1,2, Sowmya Srikanthan1,3, Roy L. Silverstein1,3
Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland,
OH 44195;
Department of Biology, Geology and Environmental Science, Cleveland State
University, Cleveland, OH 44115; 3Department of Molecular Medicine, Cleveland Clinic Lerner

College of Medicine, Case Western Reserve University, Cleveland, OH 44195, USA.
Running Title: LPA suppresses endothelial CD36 via PKD-1

Scientific Category: Vascular Biology
Corresponding author:
Roy L. Silverstein, MD
Chair, Department of Cell Biology
Lerner Research Institute, NC10
Cleveland Clinic Foundation
9500 Euclid Ave
Cleveland, OH 44195
Tel: 216-444-5220
Fax: 216-444-9404
Email: silverr2@ccf.org
1
Copyright 2011 American Society of Hematology
Abstract
In pathological settings including retinal ischemia and malignant tumors, robust angiogenesis
occurs despite the presence in the micro-environment of anti-angiogenic proteins containing
thrombospondin structural homology (TSR) domains. We hypothesized that anti-angiogenesis
mediated by TSR containing proteins could be blunted by localized down-regulation of their
cognate receptor on microvascular endothelial cells (MVEC), CD36. Through screening a panel
of EC agonists we found that lysophosphatidic acid (LPA) dramatically down-regulated CD36
surface expression on primary MVEC. LPA is a lipid signaling mediator known to have proangiogenic activity, but the mechanisms are largely unknown. We observed that LPA caused
CD36 down-regulation in a dose- and time-dependent manner and was long lasting. Downregulation occurred at the transcriptional level via a signaling pathway involving specific LPA
receptors and protein kinase D. LPA induced MVEC CD36 repression significantly attenuated
in vitro anti-angiogenic responses to thrombospondin-1, including blockade of migration, tube
formation, and VEFGR-2 signaling in response to FGF-2. In vivo relevance was demonstrated
by showing that LPA abrogated thrombospondin-1 mediated inhibition of neo-vascularization of
matrigel plugs implanted in mice. Our data thus indicate that the pro-angiogenic mechanism of
LPA may in part be via switching off the anti-angiogenic switch mediated by TSR proteins and
CD36.
Introduction
Angiogenesis or the growth of new blood vessels from existing microvasculature is

essential for organ growth and tissue repair. Under normal conditions, angiogenesis is tightly
regulated by a dynamic balance between pro- and anti-angiogenic signaling pathways. Loss of
balance between these pathways can occur as a consequence of many diseases and can lead to
either inadequate or excess angiogenesis. The latter contributes to tumor progression, diabetic
retinopathy, macular degeneration, and rheumatoid arthritis.1, 2 We have been interested in an
endogenous anti-angiogenic pathway triggered by proteins containing a conserved domain first
identified in the platelet and matrix glycoprotein thrombospondin-1 (TSP-1).3, 4 This domain,
called the TSP type 1 repeat or TSR, is also found in TSP-2, 5 vasculostatin, 6, 7 and other antiangiogenic proteins and has been shown to exert its activity by binding to a specific receptor,
CD36, expressed on microvascular endothelial cells (MVEC).8 The anti-angiogenic activities of
TSP-1 and -2 and vasculostatin are absent or significantly reduced in CD36 knockout mice.4-6
CD36 is a widely expressed cell surface glycoprotein with two major classes of ligand in
addition to TSR-containing proteins. 9, 10 On adipocytes, myocytes, specialized neurosensory
cells and gut epithelium, CD36 functions as a transporter and/or sensor of free fatty acids. On
phagocytic cells and platelets CD36 functions in the innate immune response as a scavenger
receptor, facilitating binding and internalization of numerous endogenous and exogenous danger
signals, including oxidized LDL. In these contexts CD36 has been shown to play a role in
chronic inflammation, atherosclerosis, arterial thrombosis, and insulin resistance.11-13
The mechanisms by which CD36 inhibits angiogenesis are based on its ability to
transduce signals in MVEC that turn off pro-angiogenic responses and turn on anti3
angiogenic responses in newly formed microvasculature. TSR-CD36 interactions on MVEC

inhibit cell migration and tube formation and induce apoptosis by recruiting and activating
specific SRC-family and MAP kinases, including Fyn, p38 and JNK, directly activating
caspases, and inducing expression of endogenous pro-apoptotic receptors, such as TNFR, Fas
and TRAIL receptors DR4 and DR5, and suppressing AKT activation in response to
VEGF. 3, 4, 8, 14-16
CD36 expression on monocyte/macrophages and striated muscle cells is highly regulated

and has been extensively studied. Monocyte expression is influenced by cytokines such as IL-4
and M-CSF, nuclear hormone receptors such as PPAR and LXR, lipids and lipoproteins, and
statin and anti-HIV drugs, while muscle cell expression is influenced by insulin and energy
demands.9, 17, 18 In contrast, although CD36 is broadly and constitutively expressed in
microvascular beds there is surprisingly little known regarding regulation of its expression on
MVEC. Mwaikambo et al19 recently reported that retinal MVEC CD36 expression was upregulated by hypoxia via the hypoxia-inducible factor-1 transcription factor, suggesting that upregulation of a natural anti-angiogenic pathway may accompany up-regulation of hypoxia-driven
pro-angiogenic pathways, perhaps to provide a brake to prevent excess neo-vascularization.
In many pathological settings, such as retinal ischemia and malignant tumors, robust
angiogenesis occurs despite the abundant presence of TSR-containing proteins in the microenvironment. We thus hypothesized that one mechanism by which TSR-mediated antiangiogenesis could be blunted would be via localized down-regulation of the receptor CD36 on
MVEC. In this manuscript we report that the biologically active extracellular lipid signaling
molecule lysophosphatidic acid (LPA) dramatically down-regulated CD36 transcription and

expression in primary human dermal MVEC. The down-regulation was long-lasting and
mediated by a signaling pathway involving specific G-protein coupled LPA receptors and protein
kinase D-1, a ser/thr kinase also known as protein kinase C (PKC), which induced
transcriptional repression of the CD36 gene. LPA treatment of MVEC in vitro abrogated TSP-1mediated anti-angiogenic activities, including FGF-2 induced cell migration, branching
morphogenesis, and VEGF receptor signaling. The in vivo relevance of these discoveries was
demonstrated by showing that LPA blunted TSP-1 anti-angiogenesis in mouse matrigel assays
and that this was associated with loss of neovascular CD36 expression.
LPA is an important regulator of vascular and inflammatory cells. It is produced by

activated platelets and leukocytes and plasma levels are dramatically increased during vascular
injury. 20 Our data are consistent with studies showing that LPA regulates endothelial cell
behavior and angiogenesis21 and that autotaxin, a phospholipase that generates LPA from
lysophosphatidylcholine, is angiogenic and essential for vascular development in mice.22
Despite the prominent role of LPA signaling in endothelial cells the mechanisms by which LPA
mediates angiogenesis remain incompletely understood. Our study reveals a novel mechanism
by which LPA modulates angiogenesis through interrupting an anti-angiogenic pathway and
suggests that the LPA-PKD-1-CD36 signaling axis could be a useful target for therapeutic
intervention.
Methods
Reagents. 1-Palmitoyl lysophosphatidic acid (LPA) and 1-Oleoyl LPA, dioctanoylglycerol
pyrophosphate (DGPP) were purchased from Avanti Polar-Lipids Inc. (Alabaster, AL); Ki16425,
and antibodies to LPA1 and LPA3 were from Cayman Chemical (Ann Arbor, MI);
Thrombospondin-1 was purchased from Calbiochem (Darmstadt, Germany) or isolated from
human platelets as previously described3, 4; FGF-2 and Matrigel were from BD Biosciences
(Woburn, MA), Bradykinin and the CellLytic cell lysis/extraction reagent were from Sigma
(St. Louis, MO); the PKD inhibitor CID 755673 was from TOCRIS bioscience (Bristol, UK), GLISA RhoA Activation Assay Biochem Kit and the cell permeable Rho inhibitor C3
Transferase were from Cytoskeleton (Denver, CO), SYBR green was from Applied Biosystems
(Foster City, CA), reverse transcriptase AMV was from Roche Applied Science (Mannheim,
Germany), RNeasy Mini Kit was from Qiagen (Valencia, CA), and PKD-1 siRNA plasmids were
from Addgene (Cambridge, MA). Antibodies to PKD/PKC, phospho-PKD (Ser916), phosphoPKD (Ser744/748) (which recognizes human PKD-1 phosphorylation site Ser738/742), phospho-Akt
(Ser473), Akt, phospho-VEGFR2 (Tyr1175), VEGFR2, and VE-Cadherin were from Cell
Signaling Technology (Danvers, MA). Antibody to -actin and PE- or FITC-conjugated antiCD36 were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Monoclonal antibody to
mouse CD36 (clone CRF D-2712) was purchased from (Hycult Biotechnology (Plymouth
Meeting, PA) and CD36 antibody (ab78054) was from Abcam (Cambridge, MA). AlexaFluor
568 Phalloidin was from Molecular Probes, Invitrogen (Carlsbad, CA). VECTASHIELD
Mounting Medium with DAPI was purchased from Vector Laboratories, Inc. (Burlingame, CA).
Endothelial cell culture. Primary human dermal MVEC were purchased from Lonza
(Walkersville, MD) and were maintained in Microvascular Endothelial Cell Basal Medium-2
(EBM-2) obtained from Lonza supplemented with 5% fetal bovine serum (FBS), growth factors,
cytokines, supplements, and 200 units/mL penicillin, 100 units/mL streptomycin, and 250 g/mL
amphotericin. Early passage cells (passages 4-8) were grown to 90% confluence and the
medium was replaced with EBM-2 containing 1% FBS overnight prior to the studies and then
replaced again with fresh EBM-2/1% FBS for experiments. Mouse cardiac MVEC were isolated
as previously described23 and maintained as above.
Flow cytometry. Endothelial cells exposed to various stimuli or vehicle controls were retrieved
from culture dishes by trypsinization, washed with PBS and then incubated with FITCconjugated anti-CD36 monoclonal IgG or isotype-matched control IgG. Surface CD36
expression was determined by fluorescence-activated flow cytometry using a FACSCalibur
system (BD Biosciences, Woburn, MA) and the data were analyzed with FlowJo software (Tree
Star, Inc., Ashland, OR).
Real-time quantitative reverse transcriptase (RT)-PCR. RNA was isolated from MVEC using
the RNeasy Mini Kit (QIAGEN, MO) and then subjected to real-time RT-PCR using an iCycle
iQ Multicolor Real time PCR detection system (Bio-Rad Laboratories, Hercules, CA). CD36,
GAPDH and PPIA RT2 qPCR Primers were from SABiosciences, (Frederick, MD). GAPDH or
cyclophilin A (PPIA) transcripts were amplified in a separate tube to normalize variances in
input RNA. Relative Ct values were used to compare changes of mRNA expression.
Immunoprecipitation and immunoblot assays. Immunoprecipitations and immunoblots were

performed for CD36 protein expression. For assays of PKD-1, VEGFR-2 and Akt
phosphorylation, immunoblots of MVEC lysates were probed with phospho-specific antibodies
on duplicate membranes. The membranes were re-probed with non-phospho-specific antibodies
as a loading control. Protein concentrations were assayed with a BCA kit (Pierce, Rockford, IL)
and anti--actin was used as an additional loading control for all blots. For some studies MVEC
plasma membranes were isolated prior to immunoblotting using the Qproteome Plasma
Membrane Protein Kit (Qiagen, Valencia, CA) according to manufacturers instruction.
RNA transcription assay. MVEC cultured in 10 cm dishes were digested with 0.25%
trypsin/0.1% EDTA and suspended in 5% FBS/EBM-2. The cells were centrifuged at 500g for
10 min, washed once in buffer (10 mM TrisHCl, pH 7.4/150 mM KCl/8 mM magnesium
acetate) and then lysed in the same buffer containing 0.5% Nonidet P-40. The lysates containing
intact nuclei were added onto 100 mM TrisHCl, 5 mM MgCl2 and 600 mM sucrose and
centrifuged at 500g for 10 min. The pellets (nuclei) were subsequently resuspended in 40%
glycerol, 50 mM TrisHCl, 5 mM MgCl2 and 0.1 mM EDTA and immediately stored at -80C
until use. For the nuclear run-on transcription assay, 107 nuclei were used for each reaction
based on the method of Zhang et al24 using biotin-labeled RNA and quantitative real time PCR.
Briefly, the nuclei were incubated in a reaction buffer (5 mM TrisHCl, pH 8.0, 2.5 mM MgCl2,
150 mM KCl, 2.5 mM each of ATP, GTP, CTP) and biotin-16-UTP at 30C for 45 min in a final
volume of 60L. The reaction was stopped by the addition of 1,000 units of RNase-free DNase
for 10 min at 37C. The nuclei were subsequently lysed by the addition of buffer containing 10
Mm TrisHCl, 1% SDS, and 5 mM EDTA. The reaction mixtures were treated with 20 L of
proteinase K (10 mg/ml) and RNA was extracted with TRIzol Reagent, ethanol-precipitated, and
resuspended in 50 l RNase-free H2O. The biotinylated RNA was purified by adding
streptavidin particle beads, followed by 2 h incubation at 25C on a rocking platform. Beads
were separated by centrifugation at 2,000 rpm for 5 min, and washed once with 2SSC-15%
Formamide for 10 min and twice with 2SSC for 5 min. The biotinylated RNA was used for
reverse-transcriptase cDNA synthesis and further quantitative real-time PCR.
siRNA plasmid transfection. Passage 5 cells (5 x105) were transduced with PKD-1 RNAi or
scrambled control plasmids using the Amaxa Nucleofector system (Lonza, Walkersville, MD)
specifically optimized for primary human MVEC. A GFP plasmid was used to determine
transfection efficiency. After 24 hrs the cells were switched to 1% FBS/EBM-2 overnight and
then treated with LPA or control for 24 hrs prior to detection of CD36 and PKD-1 by
immunoblot.
Cell migration assay. HMVEC plated in 8 m pore size transwell chambers (Millipore
Corporation, Billerico, MA) were placed in 24-well dishes filled with 1% FBS/EBM-2. After
the cells were exposed to LPA (5M) for 22 hrs or vehicle control, FGF-2 (50ng/ml), and TSP-1
(2 nM) in various combinations were added and 24 hrs later the filters were removed and the
cells on the upper surface removed. The filters were then fixed in 4% paraformaldehyde
(Electron Microscopy Sciences, PA) for 30-40 minutes at room temperature, mounted with
Vectashield mounting medium, and analyzed by fluorescence microscopy after staining with
DAPI to count nuclei on the lower surface. Quantification was done using NIH Image J
software.
In vitro tube formation assay. MVEC (2 x 104) were plated on 24-well culture plates coated with
growth factor-reduced Matrigel containing LPA (1 to 5M), FGF-2 (50 ng/ml) and/or TSP-1 (2
nM) in various combinations. After 24 hrs the cellular networks were stained with
AlexaFluor568 Phalloidin and visualized with a fluorescence microscope interfaced to a
computer with Q Capture Pro software (Media Cybernetics, Inc., Bethesda, MD and QImaging
Inc., Surrey, BC Canada). The total tube length and enclosure areas were quantified using NIH
Image J for 6-8 randomly chosen fields/well of double or triple wells for each experimental
condition.
In vivo angiogenesis assay. Matrigel plug assays were performed as previously described2, 23 by
intradermal injection of 8 wk old male C57/B6 mice with Matrigel pre-mixed with FGF-2, LPA,
and TSP-1 in various combinations. Mice were sacrificed 10d later and the plugs were removed,
fixed in 4% paraformaldehyde overnight at 4C and then in10% formaldehyde before embedding
in paraffin. The paraffin blocks were cut into 5m sections and stained with Massons
Trichrome. Randomly chosen sections were analyzed microscopically using NIH Image J
software to quantify vascularity; 6-8 images were obtained for each specimen. Sections were also
analyzed by immunofluorescence microscopy using antibodies to CD36 and CD31 and ALEXA
conjugated fluorescent secondary antibodies. Imaging was done with a Leica DM-RXE
microscope, interfaced to a computer with Q Capture Pro software or with a Leica confocal
microscope. An additional study using FITC-conjugated LPA (kindly provided by Dr. Thomas
10
McIntyre, Cleveland, OH) showed that intact LPA remained detectable in extracts of matrigel
plugs harvested after 1, 3, 7 and 10 days.
All procedures and manipulations of animals were approved by institutional animal care
and use committees (IACUCs) at Cleveland Clinic in accordance with the United States Public
Health Service Policy on the Humane Care and Use of Animals and the NIH Guide for the care
and use of laboratory animals (8th edition. Revised 2010).
Statistics. Quantitative data are presented as mean SD/SEM. Comparisons were done using
Students t tests (one tailed). A p value less than 0.05 was considered statistically significant.
11
Results
LPA down-regulates CD36 expression in primary MVECs
Initial studies aimed to identify pathways regulating CD36 expression on MVECs revealed
that exposure of the cells to the PKC activators PMA and/or TMA led to dramatic downregulation of surface expression over an 18-36 hrs period (Figure 1A).
We then used
fluorescence activated flow cytometry to screen a battery of endothelial cell agonists known to
signal through PKC, including thrombin, histamine, TNF-, IL-1, bradykinin, and IF-, and
found that among this group only LPA replicated the PMA effect (Figure 1A). The effect of
LPA on CD36 expression was time and dose dependent (Figure 1B), showing an average
inhibition of about 80% at a concentration of 5M by 24 hrs. The effect was long lasting as even
when LPA was withdrawn and replaced with standard complete medium, CD36 expression
remained suppressed for at least 24 hrs longer (Figure 1B). Western blot analyses of both whole
cell lysates (Figure 1B) and isolated membrane fractions (Figure 1C) confirmed that LPA
induced down-regulation of surface CD36 expression and showed that total CD36 levels were
correspondingly decreased.
Quantitative real time PCR assays demonstrated that down-regulation of primary human
MVEC surface CD36 expression by LPA was associated with nearly complete loss of CD36
steady state mRNA (Figure 1D) by ~16 hrs. Another biologically active extracellular lipid
signaling molecule, sphingosine-1-phosphate (S1P) did not alter CD36 expression,
demonstrating specificity. Nuclear run-on transcription assays and mRNA stability assays were
then employed to determine whether CD36 mRNA regulation by LPA occurs at the
transcriptional level. Figure (1E) shows that the relative levels of CD36 nuclear mRNA were
12
significantly decreased in cells exposed to LPA for 24 hrs, while RNA stability assays using
actinomycin D demonstrated no effect (data not shown), indicating transcriptional repression of
CD36 by LPA.
To determine whether mouse models could be used to study this system we assessed
CD36 expression on primary MVEC isolated from mouse tissues and found that it was also
down-regulated by LPA. Figure 1F is a western blot of mouse cardiac MVEC showing loss of
detectable CD36 expression after exposure to 1, 2 and 5 M LPA for 24 hrs.
CD36 transcriptional repression by LPA is mediated by specific G- protein coupled receptors.

LPA signaling is mediated by members of a family of plasma membrane G-protein
coupled receptors25 with varying degrees of specificity for phospholipids such as LPA, platelet
activating factor, oxidized phospholipids, lyso-PC, and S-1-P. As a first step in dissecting the
MVEC signaling pathway involved in regulating CD36 we used western blots with antibodies
specific for LPA1 and/or LPA3 to demonstrate that HMVEC in culture expressed LPA3 and to a
lesser extent LPA1 (Figure 2A).
In contrast, 2 different murine B16 melanoma cell lines
expressed mainly LPA1. Indirect immuno-fluorescence microscopy of Matrigel plugs removed

from mice 10d after implantation showed abundant expression of LPA3 (Figure 2B) in the
neovasculature. LPA3 immunofluorescence co-localized with the endothelial cell marker, VEcadherin, demonstrating that migrating EC in vivo express LPA3. We then pretreated HMVEC in
culture with a pharmacologic antagonist, Ki14625, which blocks LPA1 and LPA3 prior to
stimulating the cells with LPA (5 M) for 24 hrs. The antagonist attenuated LPA-induced down-
13
regulation of CD36 expression by ~80% (Figure 2C), strongly suggesting that LPA3 and/or LPA1
mediate CD36 repression. Ki14625 itself showed little impact on CD36 expression.
CD36 transcriptional repression by LPA receptors is mediated by PKD-1.

To define relevant signaling cascades downstream of LPA receptors we examined protein
kinase D-1 (PKD-1), which has recently been shown to mediate LPA signals in fibroblast cell
lines and to regulate angiogenesis.26, 27 Using a western blot assay with antibodies to specific
phosphorylated forms of PKD-1 we found that LPA induced a 3-5 fold increase in
phosphorylation of both Ser916 and Ser738/742 in MVEC (Figure 3A).
The Ser916
autophosphorylation site was detected as early as 10 min after LPA exposure while
phosphorylation of the activation loop sites Ser738 and Ser742 was seen after 30 min. When the
cells were pretreated with specific LPA receptor antagonists, DGPP against LPA3 or Ki14625
against both LPA1 and LPA3, we found that Ki14625 reduced LPA-induced PKD-1 active site
phosphorylation
to
baseline
levels
while
DGPP
partially diminished
LPA-induced
phosphorylation (Figure 3C).
Importantly CID 755673 a selective PKD pharmacologic inhibitor28 significantly attenuated

the effects of LPA on CD36 mRNA (Figure 3C). The inhibitor had no effect by itself. To
confirm these results, endogenous PKD-1 expression was knocked down in HMVECs with a
specific siRNA plasmid.
Western blots showed that the siRNA plasmids reduced PKD-1
expression by >80% compared to a scrambled control plasmid (Figure 3D). Figure 3E shows
that cells transfected with the PKD-1 siRNA plasmid maintained CD36 protein expression after
14
24 hrs exposure to LPA, while expression was decreased by >70% by LPA in cells transfected
with the control constructs.
Since Rho has been implicated in regulation of CD36 expression by LPA in monocytes29,
we tested the impact of inhibiting Rho family GTPases with the specific inhibitor, C3
transferase, in our system. As shown in Figure 3C, the inhibitor had no effect on LPA-induced
CD36 down-regulation in HMVEC.
LPA abrogates TSP-1 mediated anti-angiogenic activities of MVEC in vitro.

To show functional consequences of LPA-mediated CD36 down-regulation we
performed in vitro Boyden Chamber cell migration assays in MVEC pretreated with LPA (5M)
for 22-24 hrs. Both LPA-treated and untreated cells showed robust migration in response to
FGF-2, with no statistically significant differences between the groups (Figure 4A). However,
while co-exposure of the cells to TSP-1 suppressed FGF-2-induced migration in control cells
(p<0.05), no suppression was seen in cells pre-exposed to LPA. TSP-1 alone or LPA alone
produced no statistically significant effect on migration.
Similar results were seen with in vitro Matrigel tube formation assays (Figure 4B). Both
LPA-treated and untreated cells showed vigorous network formation in response to FGF-2, with
no statistically significant differences between the groups. Exposure of the cells to TSP-1
suppressed FGF-2-induced tube-like structure formation to baseline levels in control cells
(p<0.05), while no suppression was seen in the cells pre-exposed to LPA. The LPA effect was
15
dose-dependent, with full restoration of tube-like structure formation seen at 5 M. TSP-1 alone
or LPA alone produced no statistically significant effect.
It has previously been shown that FGF-2 induces VEGF receptor-2 (VEGFR-2)
activation and subsequent downstream phosphorylation of Akt-1 in MVEC30, and that this can be
attenuated by TSP-1. 16, 31 To show that the effects of TSP-1 on FGF-2 mediated intracellular
signaling can be blocked by LPA we assessed VEGFR-2 and Akt phosphorylation by western
blot.
Both LPA-treated and untreated cells showed increased phosphorylation of VEGR-2
(Figure 5A) and Akt ser473 (Figure 5B) in response to FGF-2. Treatment of the cells with TSP1 suppressed FGF-2-induced phosphorylation of both VEGFR-2 and Akt in control cells,
however, no suppression was seen in cells pre-exposed to LPA. Furthermore when cells were
pretreated with a selective PKD inhibitor or LPA1, 3 receptor antagonist prior to 24 hrs of LPA
exposure, TSP-1 mediated inhibition of FGF-2-induced Akt phosphorylation was maintained
(Figure 5C). These results indicate that CD36 signaling regulates crosstalk between FGF-2 and
VEGF and that LPA interrupts this regulation via PKD-1 and LPA GPCR signaling.
LPA abrogates TSP-1 mediated anti-angiogenic responses in vivo

To determine the in vivo relevance of LPA-mediated CD36 down-regulation we utilized
Matrigel plug angiogenesis assays in which combinations of FGF-2, LPA, and TSP-1 were
added to Matrigel solutions implanted subcutaneously into mice. After 10d the plugs were
removed, embedded, sectioned and stained to assess the degree of neo-vascularization and level
of CD36 expression. As reported previously by our group and others3, 4, TSP-1 alone had no
effect, but it inhibited FGF-2 induced vascular invasion and network formation by ~3 fold
16
(Figure 6A). In the presence of LPA, FGF-2 mediated invasion was not significantly changed,
but TSP-1 mediated inhibition was lost. Indirect immunofluorescence microscopy showed that
there was abundant expression of CD36 in the neovasculature in the FGF-2 containing Matrigel
plugs, while CD36 expression was markedly attenuated in the plugs containing both FGF-2 and
LPA (Figure 6B). These data suggest that LPA-induced long-lasting down-regulation of CD36
in vivo and effectively turned off the TSP-1 mediated antiangiogenic switch.
Discussion
CD36 is expressed broadly and constitutively in microvascular beds, but despite its
importance in vascular biology, there is surprisingly little known regarding regulation of its
expression. We hypothesized the existence of mechanisms to blunt the anti-angiogenic activity
of TSR-1 proteins at the receptor level via down-regulation of CD36 expression. Others have
reported immunohistochemical32 and mRNA expression profile33 data showing that CD36
expression in tumor microvasculature can vary and that expression levels correlate with degree
of angiogenesis and prognosis in human cancers, but no mechanisms have been described. To
address this issue we used pharmacologic probes to identify potential pathways by which CD36
expression could be down-regulated in early passage cultured human dermal MVEC and found
that activation of protein kinase C (PKC) with the broad spectrum activator PMA led to
substantial loss of surface CD36 protein expression. We then identified LPA as a physiologic
EC agonist that could also down-regulate CD36 in both human and murine MVEC.
Interestingly, mouse cells were modestly more sensitive to LPA than human. Down-regulation
was at the transcriptional level, as assessed by nuclear run-on experiments and a reverse
transcription-PCR, and led to loss of cell surface protein expression over an 18-24 hrs period that
17
was long lasting. The potential importance of these observations is highlighted by our studies
showing that exposure of MVEC to LPA in vitro or in vivo resulted in loss of anti-angiogenic
responses to TSP-1: i.e. in these settings MVEC maintained their capacity to migrate, invade,
undergo tube-like structure formation, and activate VEGFR-2 signals such as VEGFR-2 and Akt
phosphorylation, in response to FGF2 even in the presence of TSP-1.
LPA (1-acyl/alkyl-sn-glycerol-3-phosphate) is a bioactive phospholipid derived primarily
by the extracellular action of phospholipase D (also known as autotaxin)20 from precursor
phosphoplipids (lysophosphatidyl choline) that are produced in cell membranes34 or lipoproteins
by the action of phospholipase A1/2 (PLA1/2) and/or LCAT.34, 35 LPA can also be produced by
deacylation of phosphatidic acid by PLA1/2, acylation of glycerol 3-phosphate by
glycerophosphate acetyltransferase, or phosphorylation of monoacylglycerol (MAG) by
acylglycerol kinase (AGK).34 LPA is found in the plasma in modest concentrations (~100-500
nM) but larger amounts (>2 M) are found in plasma and tissues in the setting of an
inflammatory response, thrombosis, atherosclerosis, and in some cancers (e.g. ovarian and
breast).35-37 Autotaxin released from platelets is probably the primary source of LPA generating
activity in these settings and autotoxin expression has been associated with increased
angiogenesis. Interestingly from the perspective of CD36 biology, oxidized LDL is a
particularly rich source of both LPA precursors and LPA.
LPA signaling has been implicated in a variety of biological processes including vascular
development, neurite remodeling, inflammation, tumor progression, wound healing, and
ischemia/reperfusion injury. 20 Studies have demonstrated pro-angiogenic activity of LPA in
cancer, atherosclerosis and chronic inflammation 36-39 and LPA has been reported to directly
stimulate proliferation and migration of human umbilical and adult bovine aortic and pulmonary
18
artery endothelial cells in vitro 39, 40 and up-regulate HIF-1 and VEGF expression in tumors and
thus indirectly promote tumor angiogenesis 41 as well as to promote vascular maturation in vivo
in chicken CAM assay. 21 In our studies of microvascular EC we did not observe a significant
effect of LPA on migration. However, our data suggest that in addition to promoting angiogenic
activity in endothelial and tumor cells, LPA may also promote angiogenesis by down-regulating
an important endogenous anti-angiogenic pathway.
LPA induces biological responses via high affinity interactions with a group of G protein
coupled receptors, originally termed EDG-2, -4 and -7, but now known as LPA1, LPA2 and
LPA3. 20 Another group of receptors termed LPA4-6 related to the P2Y purinergic family have
also been identified as possible LPA receptors.35, 42 The precise role of specific LPA receptor
subtypes have not been fully characterized in the vascular system. Here we showed that human
MVEC in culture expressed abundant levels of LPA3 with less LPA1, in contrast to melanoma
cells which express mainly LPA1. We found that an LPA3 antagonist, DGPP, inhibited LPAelicited PKD-1 activation and that Ki14625, an antagonist of both LPA1 and LPA3 not only
significantly diminished LPA-elicited PKD-1 phosphorylation but also abrogated LPA-mediated
down-regulation of CD36. LPA receptors differ in their affinity for specific molecular species of
LPA; for example unsaturated LPA has specificity towards LPA3.43 Naturally occurring
unsaturated LPA has been shown to accumulate in human atherosclerotic plaques.37 It is thus
reasonable to speculate that down-regulation of MVEC CD36 by plaque LPA could contribute to
neointimal angiogenesis, which is known to promote plaque progression.
Our studies identified the calcium/calmodulin dependent ser/thr kinase PKD-1 as a
critical signaling component downstream of LPA receptors in mediating MVEC CD36
transcriptional repression. We showed that PKD pharmacologic inhibition or selective siRNA
19
knockdown of PKD-1 blocked LPA-mediated CD36 down-regulation and restored TSP-1

induced inhibition of angiogenic signals. These studies are consistent with reports that LPA can
induce PKD activation in mouse Swiss 3T3, Rat-1, and IEC6 cells. In these cells PKD activation
was shown to be PKC-dependent44 which explains our finding that direct PKC activation by
PMA also down-regulated MVEC CD36. Although there are 3 known isoforms of PKD and
both PKD-1 and -2 have been implicated in endothelial cell angiogenic responses, our siRNA
studies suggest that PKD-1 is the dominant isoform in the LPA/CD36 pathway. Unfortunately,
data on genetically engineered mouse models are limited since PKD-1 null mice are embryonic
lethal. Recently, Cantrell and colleagues45 reported data from novel models in which
catalytically inactive PKD-1 and -2 mutant alleles were knocked-in to the appropriate loci or in
which PKD-2 was deleted by a gene trap strategy. This work confirmed the role of PKD-1 in
embryogenesis, and showed that PKD-2 is not necessary for normal development, but is the
predominant isoform in lymphocytes. No studies of angiogenesis in these mice have been done.
PKD-1 transduces signals to multiple pathways, including vesicular transport,
transcriptional activation and chromatin remodeling, and it has been implicated in several
important cardiovascular processes including endothelial cell migration, proliferation, and
apoptosis, and cardiac and smooth muscle cell hypertrophy. 46 That PKD-1 can mediate
endothelial cell pro-angiogenic responses to VEGF 47, 48 as well as abrogate CD36-mediated antiangiogenic responses demonstrate that targeting PKD-1 could be a powerful way to inhibit
angiogenesis.
LPA signaling through Rho GTAPases has been implicated in down-regulation of
monocyte/macrophage CD36 by LPA. 29 While we found that LPA stimulated RhoA activity in
HMVEC (not shown), a cell permeable inhibitor which selectively inactivates RhoA, RhoB and
20
RhoC GTPase activity had no effect on LPA-mediated CD36 down-regulation (Figure 3C),
suggesting the difference of endothelial and macrophages regulatory pathways.
In summary, our studies have defined a novel pathway by which production of LPA in
angiogenic microenvironments activates a specific signaling pathway in MVEC leading to loss
of surface expression of the receptor CD36. Such receptor loss leads to loss of responsiveness to
endogenous or exogenous TSR containing anti-angiogenic proteins thereby promoting
angiogenesis. Specific targeting of the LPA-PKD-1-CD36 signaling axis could thus have
potential value in treating human diseases associated with aberrant angiogenesis or in preventing
resistance to TSR-mediated anti-angiogenic therapies.
Acknowledgement
This work was supported in part by NIH grant HL085718 to R Silverstein. We appreciate the
technical help from Drs. Angela Ting, Jinbo Liu and Thomas McIntyre.
Authorship
BR, JH and SS performed experiments and analyzed data; BR and RLS designed the studies,
analyzed the data and wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
21
References
1.
Folkman
J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med.
1995; 1(1): 2731.

2.
Ren B, Yee KO, Lawler J, Khosravi-Far R. Regulation of tumor angiogenesis by

thrombospondin-1. Biochim Biophys Acta. 2006; 1765 (2): 178-88.
3.
Simantov R, Febbraio M, Crombie R, Asch AS, Nachman RL, Silverstein RL. Histidinerich glycoprotein inhibits the antiangiogenic effect of thrombospondin-1. J Clin Invest.
2001; 107(1): 45-52.
4.
Jimnez B, Volpert OV, Crawford SE, Febbraio M, Silverstein RL, Bouck N. Signals
leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1.
Nat Med. 2000; 6 (1): 41-8.
5.
Simantov R, Febbraio M, Silverstein RL. The anti-angiogenic effect of thrombospondin2 is mediated by CD36 and inhibited by Histidine-rich Glycoprotein. Matrix Biol. 2005;
24 (1): 27-34.
6.
Kaur B, Cork SM, Sandberg EM, et al. Vasculostatin inhibits intracranial glioma growth
and negatively regulates in vivo angiogenesis through a CD36-dependent mechanism.
Cancer Res. 2009; 69 (3): 1212-20.
7.
Klenotic PA, Huang P, Palomo J, et al. Histidine-Rich Glycoprotein Modulates the AntiAngiogenic Effects of Vasculostatin. Am J Pathol. 2010; 176 (4): 2039-50.
8.
Dawson DW, Pearce SF, Zhong R, Silverstein RL, Frazier WA, Bouck NP. CD36
mediates the in vitro inhibitory effects of thrombospondin-1 on endothelial cells. J Cell
Biol. 1997; 138 (3): 707-17.
22
9.
Silverstein RL, Febbraio M. CD36, a scavenger receptor involved in immunity,

metabolism, angiogenesis, and behavior. Sci. Signal. 2009; 2 (72), re3.
10.
Febbraio M, Hajjar DP, Silverstein RL. CD36: a class B scavenger receptor involved in
angiogenesis, atherosclerosis, inflammation and lipid metabolism. J Clin Invest. 2001;
108 (6): 785-791.
11.
Febbraio M, Podrez EA, Smith JD, et al. Targeted disruption of the Class B scavenger
receptor, CD36, protects against atherosclerotic lesion development in mice. 2000; J Clin
Invest. 105 (8): 1049-1056.
12.
Podrez EA, Byzova TV, Febbraio M., et al. Platelet CD36 links hyperlipidemia, oxidant
stress and a pro-thrombotic phenotype. Nat Med. 2007; 13 (9):1086-1095.
13.
Kennedy DJ, Kuchibhotla S, Westfall KM, Silverstein RL, Morton RE, and Febbraio M.
CD36 Mediates a Pro-inflammatory Signaling Loop in Fat and Contributes to Insulin
Resistance. Cardiovasc Res. 2011; 89 (3): 604-613.
14.
Rege TA, Stewart J Jr, Dranka B., Benveniste EN, Silverstein RL, Gladson CL.
Thrombospondin-1-induced apoptosis of brain microvascular endothelial cells can be
mediated by TNF-R1. J Cell Physiol. 2009; 218 (1): 94-103.
15.
Volpert OV, Zaichuk T, Zhou W, et al. Inducer-stimulated Fas targets activated

endothelium for destruction by anti-angiogenic thrombospondin-1 and pigment
epithelium-derived factor. Nat Med. 2002; 8 (4): 349-57.
16.
Ren B, Song K, Parangi S, et al. A double hit to kill tumor and endothelial cells by
TRAIL and antiangiogenic 3TSR. Cancer Res. 2009; 69 (9): 3856-65.
23
17.
Huh HY, Pearce SF, Yesner LM, Schindler JL, Silverstein RL. Regulated expression of
CD36 during monocyte-to-macrophage differentiation: potential role of CD36 in foam
cell formation. Blood 1996; 87 (5): 20208.
18.
Feng J, Han J, Pearce SF, et al. Induction of CD36 expression by oxidized LDL and IL-4
by a common signaling pathway dependent on protein kinase C and PPAR-gamma. J
Lipid Res. 2000; 41 (5): 68896.
19.
Mwaikambo BR, Yang C, Chemtob S, Hardy P. Hypoxia up-regulates CD36 expression

and function via hypoxia-inducible factor-1- and phosphatidylinositol 3-kinase-dependent
mechanisms. J Biol Chem. 2009; 284 (39): 26695-707.
20.
Morris AJ, Panchatcharam M, Cheng HY, et al. Regulation of blood and vascular cell
function by bioactive lysophospholipids. J Thromb Haemost. 2009; 7 (S1):38-43.
21.
Rivera-Lopez CM, Tucker AL, Lynch KR. Lysophosphotidic acid (LPA) and
angiogenesis. Angiogenesis. 2008; 11 (3): 301-10.
22.
Nam SW, Clair T, Kim YS, et al. Autotaxin (NPP2), a metastasis-enhancing motogen, is
an angiogenic factor. Cancer Res. 2001; 61 (18): 69386944.
23.
Ren B, Deng Y, Mukhopadhyay A, et al. ERK1/2-Akt1 crosstalk regulates arteriogenesis

in mice and zebrafish. J Clin Invest. 2010; 120 (4): 1217-28.
24.
Zhang MX, Ou H, Shen YH, et al. Regulation of endothelial nitric oxide synthase by
small RNA. Proc Natl Acad Sci U S A. 2005; 102 (47): 16967-72.
25.
Tigyi, G., and A. Parrill. 2003. Molecular mechanisms of lysophosphatidic acid action.
Prog. Lipid Res. 42 (6): 498526.
24
26.
Taylor CJ, Motamed K, Lilly B. Protein kinase C and downstream signaling pathways in
a three-dimensional model of phorbol ester-induced angiogenesis. Angiogenesis. 2006; 9
(2): 39-51.
27.
Ha CH, Wang W, Jhun BS, et al. Protein kinase D-dependent phosphorylation and
nuclear export of histone deacetylase 5 mediates vascular endothelial growth factorinduced gene expression and angiogenesis. J Biol Chem. 2008; 283 (21): 14590-9.
28.
Sharlow ER, Giridhar KV, LaValle CR, et al. Potent and Selective Disruption of Protein
Kinase D Functionality by a Benzoxoloazepinolone. J Biol Chem. 2008; 283 (48): 3351626.
29.
Ruiz-Velasco N, Domnguez A, Vega MA. Statins upregulate CD36 expression in human

monocytes, an effect strengthened when combined with PPAR-gamma ligands Putative
contribution of Rho GTPases in statin-induced CD36 expression. Biochem Pharmacol.
2004; 67 (2): 303-13.
30.
Murakami M. Simons M. Fibroblast growth factor regulation of neovascularization. Curr

Opin Hematol. 2008; 15 (3): 215220.
31.
Zhang X, Kazerounian S, Duquette M, et al. Thrombospondin-1 modulates vascular

endothelial growth factor activity at the receptor level. FASEB J. 2009; 23 (10): 3368-76.
32.
Tsuchida T, Kijima H, Tokunaga T, et al. Expression of the thrombospondin-1 receptor

CD36 is correlated with decreased stromal vascularization in colon cancer. Int. J. Oncol
1999; 14 (1): 47-51.
33.
Chen CN, Lin JJ, Chen JJ, et al. Gene expression profile predicts patient survival of
gastric cancer after surgical resection. J Clin Oncol. 2005; 23 (29): 7286-95.
25
34.
Aoki J. Mechanisms of lysophosphatidic acid production. Semin Cell Dev Biol. 2004; 15
(5): 477-89.
35.
Liu S, Umezu-Goto M, Murph M, et al. Expression of autotaxin and lysophosphatidic

acid receptors increases mammary tumorigenesis, invasion, and metastases. Cancer Cell.
2009 2; 15 (6): 539-50.
36.
Mills BG, Moolenaar WH. The emerging role of lysophosphatidic acid in cancer. Nat
Rev Cancer. 2003; 3 (8): 582-91.
37.
Siess W, Zangl KJ, Essler M, et al. Lysophosphatidic acid mediates the rapid activation
of platelets and endothelial cells by mildly oxidized low density lipoprotein and
accumulates in human atherosclerotic lesions. Proc Natl Acad Sci U S A. 1999; 96 (12):
6931-6.
38.
Zhang C, Baker DL, Yasuda S, et al. Lysophosphatidic acid induces neointima formation
through PPARgamma activation. J Exp Med. 2004; 199 (6): 763-74.
39.
Lee H, Goetzl EJ, An S. Lysophosphatidic acid and sphingosine 1-phosphate stimulate

endothelial cell wound healing. Am J Physiol Cell Physiol. 2000; 278 (3): C612C618.
40.
English D, Kovala AT, Welch Z, et al. Induction of endothelial cell chemotaxis by

sphingosine 1-phosphate and stabilization of endothelial monolayerbarrier function by
lysophosphatidic acid, potential mediators of hematopoietic angiogenesis. J Hematother
Stem Cell Res. 1999; 8 (6): 627634.
41.
Hu X., Mendoza F. J., Sun J., Banerji V., Johnston J. B., Gibson S. B.
Lysophosphatidic acid (LPA) induces the expression of VEGF leading to protection
against apoptosis in B-cell derived malignancies. Cell. Signal. 2008; 20 (6): 11981208.
26
42.
An S, Bleu T, Hallmark OG, Goetzl EJ. Characterization of a novel subtype of human G

protein coupled receptor for lysophosphatidic acid. J. Biol. Chem. 1998; 273 (14): 7906
7910.
43.
Bandoh K, Aoki J, Hosono H, et al. Molecular cloning and characterization of a novel

human G-protein-coupled receptor, EDG7, for lysophosphatidic acid. J. Biol. Chem.
1999; 274 (39): 2777685.
44.
Chiu T, Rozengurt E. PKD in intestinal epithelial cells: rapid activation by phorbol esters,
LPA, and angiotensin through PKC. Am J Physiol Cell Physiol. 2001; 280 (4): C929-42.
45.
Matthews SA, Navarro MN, Sinclair LV, Emslie E, Feijoo-Carnero C, Cantrell DA.
Unique functions for protein kinase D1 and protein kinase D2 in mammalian cells.
Biochem J. 2010; 432 (1): 153-63.
46.
Rozengurt E, Rey O, Waldron RT. Protein kinase D signaling. J. Biol. Chem. 2005; 280
(14), 13205-8.
47.
Qin L, Zeng H, Zhao D. Requirement of protein kinase D tyrosine phosphorylation for

VEGF-A165-induced angiogenesis through its interaction and regulation of
phospholipase Cgamma phosphorylation. J Biol Chem. 2006; 281 (43): 32550-8.
48.
Wong C, Jin ZG. Protein kinase C-dependent protein kinase D activation modulates ERK
signal pathway and endothelial cell proliferation by vascular endothelial growth factor. J
Biol Chem. 2005; 280 (39): 3326233269.
27
Figure Legends
Figure 1. LPA down-regulates CD36 expression in human and mouse microvascular

endothelial cells. (A) Representative flow cytometry histogram (left) of human MVEC after
exposure in complete media to LPA (50 M; green), PMA (100 ng/ml; blue), or vehicle control
(red) for 36 hrs. Cells were analyzed with FITC-conjugated anti-CD36 IgG. An isotype
matched non-immune IgG was used as a control (brown). On the right is a bar graph showing
means SD of CD36 median fluorescence intensity (MFI) for triplicate experiments (p<0.05).
(B) Time course (left) and dose response (right) of CD36 down-regulation in MVEC exposed to
LPA. Time course was done using 5 M LPA and dose response was assessed at 24 hrs. In both
cases cells were grown first to 90% confluence and then incubated overnight in basic media
containing 1% FBS prior to adding LPA. Insert shows a representative blot. Bar graph for the
time course shows means SEM of band densities (normalized to -actin control) for 3
independent experiments. The 48 hrs bar on the time course indicates relative expression 48 hrs
after initiating LPA exposure; in this experiment the LPA-containing media was removed after
24 hrs and replaced with complete medium for an additional 24 hrs. The dose response (right
panel) was determined by flow cytometry as in panel A. Mean median fluorescence intensities
relative to the isotype control were obtained for each treatment and the fluorescence intensity
from cells not exposed to LPA was set as 100. (C) Membrane fractions of human MVEC
exposed to LPA were obtained by differential centrifugation and then analyzed by western blot
for CD36 expression as in panel B (D) Quantitative real time PCR for CD36 mRNA from
HMVEC exposed to LPA (5 M) or S1P (1 M) for timed periods from 8-24 hrs. Data are
expressed relative to a control transcript, GAPDH and the mean value for untreated samples was
28
set to 1 and bar graph shows mean SEM of 3 independent experiments. (E) CD36 mRNA
transcription (nuclear run-on) assays performed on nuclei isolated from human MVEC treated
with LPA (5 M) or vehicle control for 24 hrs. Nuclei were incubated with ATP, CTP, GTP,
and biotin-16-UPT (2.5 mM) at 30C for 45 min and the biotinylated transcripts then purified
and used for cDNA synthesis and real-time PCR. Data are presented as relative mRNA levels of
CD36 compared to GAPDH and are mean SEM from n=3. (F) Mouse cardiac MVEC CD36
protein expression detected by western blot of whole cell lysates 24 hrs after exposure to LPA
(1-5 M). Blots were performed as in panel B using anti-CD36 antibody and then stripped and
re-probed with anti--actin as a loading control. Gel is representative of 3 separate experiments
and numbers below show the mean band density SD compared to the control lane.
Figure 2. LPA receptors LPA1 and LPA3 regulate CD36 MVEC expression. (A) Lysates
prepared from confluent cultures of human umbilical vein (HUVEC), HMVEC and 2 strains of
murine B16 melanoma cells were analyzed by western blot using an antibody that recognizes
LPA1 (upper band) and LPA3 (lower band). Blots were stripped and re-probed with anti--actin
as a loading control. (B) Matrigel containing FGF-2 was injected subcutaneously into mice and
after 10 days the plugs were removed, sectioned and analyzed by indirect immunofluorescence
microscopy using antibodies to LPA3 (green) or VE-cadherin (red). A representative merged
image is shown in the third panel. The far right image shows a control with secondary antibody
alone. Scale bar = 100 m. (C) Human MVEC were pretreated with the LPA1, 3 antagonist
Ki14625 (2 M) or vehicle control followed by LPA (5 M) for 24 hrs. Quantitative real time
PCR of CD36 mRNA was then done as in Figure 1D. Data are expressed relative to a control
29
transcript, cyclophilin A (PPIA) and the mean value for untreated samples was set to 1 and bar
graph shows mean SEM of 3 independent experiments.
Figure 3. PKD-1 mediates LPA induced down-regulation of CD36. (A) Human MVECs
were exposed to LPA (5 M) or vehicle control for 30 min and then cell lysates prepared and
analyzed by western blot using antibodies specific for PKD-1 phosphorylated at Ser916 or
Ser738/742. Blots were stripped and re-probed with an antibody to total PKD-1 as a loading
control. Bar graph shows relative phosphorylation levels (mean SEM) from 3 independent
experiments. The blot is a representative image of a time course of Ser916 phosphorylation.
Relative band densities are shown below the blot. (B) MVEC were treated with the LPA3
antagonist DGPP (2-20 M) or LPA1,3 antagonist Ki14625 (1-2 M) for 30 min followed by
LPA (5 M) for 30 min. Western blots for phosphoPKD-1 (Ser738/742) were performed as in
panel A. Relative band densities are shown above the blot. (C) MVEC were pretreated with a
selective PKD inhibitor CID 755673 (25 M) or C3 transferase (C3*, 5 g/ml) for 30 min
followed by LPA (5 M) for 24 hrs. Quantitative real time PCR for CD36 mRNA was then
performed as in Figure 1D, and relative CD36 expression was normalized with PPIA. (D)
Lysates from MVEC transfected with pmaxGFP, scrambled RNAi plasmids or pSUPER PKD-1
RNAi were analyzed by western blot using an antibody to PKD-1. Blots were stripped and reprobed with anti--actin as loading control. (E) MVEC transfected with RNAi plasmids as in
panel E were treated with LPA (5 M) for 24 hrs and analyzed by western blot for CD36
expression. Blots were stripped and re-probed with anti--actin as loading control.
30
Figure 4. LPA abrogates TSP-1 mediated inhibition of migration and tube-like structure
formation in vitro. (A) MVEC treated with LPA (5 M) or vehicle control for 22 hrs were
subjected migration assays using a modified Boydens chamber assay in which cells were
exposed to FGF-2 (50 ng/ml), TSP-1 (2 nM), or their combination. Migration was assessed
microscopically after 24 hrs by staining nuclei with DAPI (NS = not significant; ** p< 0.01). (B)
MVEC were exposed to 5 M (left) or 1-5 M of LPA (right) and then cultured for 24 hrs in
Matrigel containing FGF-2 (50 ng/ml), TSP-1 (2 nM), or their combination. Cells were imaged
with fluorescence microscope interfaced to a computer with Q capture software and extent of
tube (cord)-like structure quantified microscopically using Image J software. Representative
images are shown in top left panel (scale bar = 50 m); bar graphs show mean SEM (**p<
0.01; *p<0.05).
Figure 5. LPA abrogates TSP-1 mediated inhibition of angiogenic signaling via LPA1, 3 and
PKD-1. MVEC were exposed to LPA (5 M) 24 hrs and then treated with FGF-2 (50 ng/ml)
with or without TSP-1 (2 nM) for 30 min. Cell lysates were subjected to western blot using
antibodies to phospho-VEGFR-2Tyr1175 (A) or phospho-Akt (B). (C) Cells were pretreated
with CID 755673 (25 M) or Ki14625 (2 M) for 30 min followed by LPA for 24 hrs and then
analyzed as in panel B. Blots were stripped and re-probed with antibodies to total VEGFR-2 or
Akt as loading controls. Blots are representative of two independent experiments.
Figure 6. LPA abrogates TSP-1 mediated anti-angiogenesis in vivo. Matrigel solutions

containing FGF-2 (100 ng/ml) alone or in combinations with TSP-1 (400 ng/ml) and/or LPA (5
M) were implanted subcutaneously into C57Bl6 mice. After 10d the plugs were removed,
31
sectioned, and stained with Massons Trichrome to measure neovascularization (A) or antibodies
to CD36 (green) and CD31 (red) to assess relative expression levels (B). Vascular invasion was
quantified from digital images using Image J software. Representative images are shown in (A)
top panel (scale bar =50 m) . Bar graph shows mean SEM (NS = non-significant; *p< 0.01).
Arrows in panel B indicate new blood vessels with weak CD36 expression.
32
Figure 1
A
Cell Number
Median Fluorescence
Intensity
50
40
30
*
*
20
10
0
ISOTYPE
Control
LPA
PMA
* p < 0.05 vs. control
Relative Fluorescence Intensity
LPA (5 M)
18
24
48
(hr)
Relative CD36 Expression
CD36
Relative Density
-actin
1.2
1
0.8
0.6
0.4
0.2
0
18
Time (hr)
24
48
Dose response
120
100
80
60
40
20
0
-20
CTL
2
LPA (M)
Figure 1
LPA (5 M)
C
24
CD36
Relative Expression to GAPDH

(Fold Change)
Membrane Fraction of HMVEC
1.5
1
0.5
0
CTL
16
LPA (5 M)
24 (hr)
S1P (24hr)
Relative mRNA Level
E
1.5
LPA (M)
1
CTL
CD36
0.5
-actin
0
CTL
LPA
Density
0.370.14
0.210.18
0.200.17
Figure 2
HMVECs
HUVECs
B16-F1 B16-F10
LPA1
LPA3
-actin
B
VE-cadherin
Relative Expression to
PPIA (Fold Change)
LPA3
LPA3/VE-cadherin
2
1.5
1
0.5
0
CTL
Ki14625 (2 M)
LPA (5 M)
Ki + LPA
Negative CTL
Figure 3
LPA 5 (M)
6
Control
LPA
CTL
10
30
60
(min)
3.5
3.7
1.8
P-PKD-1(Ser916)
2
Total-PKD-1
Relative Density
Ser738/742
Ser916
Phospho-PKD-1
B
Relative Density
4.3
5.8
3.1
Relative Density
P-PKD-1(Ser738/742)
P-PKD-1(Ser738/742)
Total-PKD-1
Total-PKD-1
2.1
1.7
0.6
LPA (M)
LPA (M)
DGPP (M)
20
Ki14625 (M)
Relative Phosphorylation
Figure 3
C
GFP
Relative Expression
PKD-1
100
80
-actin
60
40
20
0
CTL
LPA
CID+LPA
C3 *+LPA
E
RNAi CTL
Vehicle
CD36
-actin
LPA
PKD-1 RNAi
Vehicle
LPA
Scrambled PKD-1 RNAi
CD36 mRNA
120
**
15
10
5
(Fluorescence Unit)
No LPA
LPA
NS
+
+
+
TSP-1
NS
** p < 0.01
+
+
+
+
+
FGF-2
**
20
Relative Migration
Figure 4
Figure 4
No LPA
With LPA
TSP-1
FGF-2
FGF-2+TSP-1
Relative Tube Formation

(Fluorescence Unit)
NS
45
(Fluorescence Unit)
Vehicle
Relative Branching
40
30
20
10
**
**
30
15
LPA (M)
TSP-1
FGF-2
*p < 0.05
0
FGF-2
TSP-1
+
-
No LPA
+
+
+
-
LPA
+
+
** p < 0.01
Figure 5
No LPA
LPA
P-VEGFR-2
VEGFR-2
FGF-2 (ng/ml)
50
50
50
50
TSP-1 (nM)
C
No LPA
LPA
LPA
P-Akt
P-Akt
Akt
Akt
FGF-2 (ng/ml)
TSP-1 (nM)
50
-
50
2
50
-
50
FGF-2 (ng/ml)
50
50
50
TSP-1 (nM)
CID 755673 (M)
25
Ki14625 (M)
Figure 6
FGF-2
FGF-2+TSP-1
FGF-2+LPA+TSP-1
FGF-2+LPA
Relative Vascular Invasion

(Mean Gray Value)
FGF-2+LPA
FGF-2 CTL
CD36
NS
40
35
30
25
20
15
10
5
0
LPA CTL
CD31
FGF-2
FGF-2+TSP-1
FGF-2+LPA
FGF-2+LPA+TSP-1
*p < 0.01

tmp921F TMP

Загружено:

Сведения о документе

Исходное описание:

Оригинальное название

Авторское право

Доступные форматы

Поделиться этим документом

Поделиться или встроить документ

Параметры публикации

Этот документ был вам полезен?

Это неприемлемый материал?

Авторское право:

Доступные форматы

tmp921F TMP

Загружено:

Авторское право:

Доступные форматы

From bloodjournal.hematologylibrary.org by guest on May 11, 2014. For personal use only.

Prepublished online March 25, 2011;

Lysophosphatidic acid suppresses endothelial cell CD36 expression and

Lysophosphatidic Acid Suppresses Endothelial Cell CD36 Expression and Promotes

Bin Ren1, James Hale1,2, Sowmya Srikanthan1,3, Roy L. Silverstein1,3

Department of Biology, Geology and Environmental Science, Cleveland State

University, Cleveland, OH 44115; 3Department of Molecular Medicine, Cleveland Clinic Lerner

Running Title: LPA suppresses endothelial CD36 via PKD-1

Angiogenesis or the growth of new blood vessels from existing microvasculature is

angiogenic responses in newly formed microvasculature. TSR-CD36 interactions on MVEC

CD36 expression on monocyte/macrophages and striated muscle cells is highly regulated

molecule lysophosphatidic acid (LPA) dramatically down-regulated CD36 transcription and

LPA is an important regulator of vascular and inflammatory cells. It is produced by

Immunoprecipitation and immunoblot assays. Immunoprecipitations and immunoblots were

CD36 transcriptional repression by LPA is mediated by specific G- protein coupled receptors.

In contrast, 2 different murine B16 melanoma cell lines

expressed mainly LPA1. Indirect immuno-fluorescence microscopy of Matrigel plugs removed

CD36 transcriptional repression by LPA receptors is mediated by PKD-1.

phosphorylation (Figure 3C).

Importantly CID 755673 a selective PKD pharmacologic inhibitor28 significantly attenuated

Western blots showed that the siRNA plasmids reduced PKD-1

LPA abrogates TSP-1 mediated anti-angiogenic activities of MVEC in vitro.

Both LPA-treated and untreated cells showed increased phosphorylation of VEGR-2

LPA abrogates TSP-1 mediated anti-angiogenic responses in vivo

knockdown of PKD-1 blocked LPA-mediated CD36 down-regulation and restored TSP-1

Conflict-of-interest disclosure: The authors declare no competing financial interests.

J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med.

1995; 1(1): 2731.

Ren B, Yee KO, Lawler J, Khosravi-Far R. Regulation of tumor angiogenesis by

Silverstein RL, Febbraio M. CD36, a scavenger receptor involved in immunity,

Volpert OV, Zaichuk T, Zhou W, et al. Inducer-stimulated Fas targets activated

Mwaikambo BR, Yang C, Chemtob S, Hardy P. Hypoxia up-regulates CD36 expression

Ren B, Deng Y, Mukhopadhyay A, et al. ERK1/2-Akt1 crosstalk regulates arteriogenesis

Ruiz-Velasco N, Domnguez A, Vega MA. Statins upregulate CD36 expression in human

Murakami M. Simons M. Fibroblast growth factor regulation of neovascularization. Curr

Zhang X, Kazerounian S, Duquette M, et al. Thrombospondin-1 modulates vascular

Tsuchida T, Kijima H, Tokunaga T, et al. Expression of the thrombospondin-1 receptor

Liu S, Umezu-Goto M, Murph M, et al. Expression of autotaxin and lysophosphatidic

Lee H, Goetzl EJ, An S. Lysophosphatidic acid and sphingosine 1-phosphate stimulate

English D, Kovala AT, Welch Z, et al. Induction of endothelial cell chemotaxis by

An S, Bleu T, Hallmark OG, Goetzl EJ. Characterization of a novel subtype of human G

Bandoh K, Aoki J, Hosono H, et al. Molecular cloning and characterization of a novel

Qin L, Zeng H, Zhao D. Requirement of protein kinase D tyrosine phosphorylation for

Figure 1. LPA down-regulates CD36 expression in human and mouse microvascular

Figure 6. LPA abrogates TSP-1 mediated anti-angiogenesis in vivo. Matrigel solutions

* p < 0.05 vs. control

Relative Fluorescence Intensity

Relative Expression to GAPDH

Membrane Fraction of HMVEC

Relative mRNA Level

Scrambled PKD-1 RNAi

Relative Tube Formation

CID 755673 (M)

Relative Vascular Invasion

Вам также может понравиться