p38α MAPK promotes angiogenesis · p38α MAPK promotes angiogenesis 1 LY2228820 Dimesylate, a Selective Inhibitor of p38 Mitogen-Activated Protein Kinase, Reduces Angiogenic Endothelial

p38α MAPK promotes angiogenesis

1

LY2228820 Dimesylate, a Selective Inhibitor of p38 Mitogen-Activated Protein Kinase, Reduces Angiogenic

Endothelial Cord Formation In Vitro and In Vivo

Courtney Tate1, Wayne Blosser

1, Lisa Wyss

2, Glenn Evans

1, Qi Xue

3, Yong Pan

3, and Louis Stancato

1

1Eli Lilly and Company, Oncology, Lilly Corporate Center, Indianapolis, IN, 46285

2Discovery Research, Advanced Testing Laboratories, Cincinnati, OH, 45242

3ImClone Systems, New York, NY 10016

Running title: p38α MAPK promotes angiogenesis

To whom correspondence should be addressed: Louis Stancato, Oncology Research, Eli Lilly and Company,

Lilly Corporate Center, Indianapolis, IN, USA, Tel: (317) 655-6910; Fax: (317) 276-1414; E-mail:

[email protected]

Keywords: p38 MAPK, angiogenesis, endothelial cord formation

Background: Angiogenesis is a critical process for

tumor growth and survival.

Results: LY2228820 dimesylate, a p38 MAPK

specific inhibitor, or shRNA knockdown of p38α,

MK2, or HSP27 reduced angiogenic cord formation.

Conclusion: p38 MAPK modulated soluble factors

released from stromal and tumor cells and reduced

their downstream signaling in endothelial cells.

Significance: Antiangiogenic activity of LY2228820

dimesylate may lead to anti-tumor growth effects.

SUMMARY

LY2228820 dimesylate is a highly selective

small molecule inhibitor of p38α and p38β mitogen

activated protein kinases (MAPKs) that is

currently under clinical investigation for human

malignancies. p38 MAPK is implicated in a wide

range of biological processes, in particular those

that support tumorigenesis. One such process,

angiogenesis, is required for tumor growth and

metastasis, and many new cancer therapies are

therefore directed against the tumor vasculature.

Using an in vitro co-culture endothelial cord

formation assay, a surrogate of angiogenesis, we

investigated the role of p38 MAPK in growth

factor and tumor-driven angiogenesis using

LY2228820 dimesylate treatment and by shRNA

gene knockdown. p38 MAPK was activated in

endothelial cells upon growth factor stimulation

with inhibition by LY2228820 dimesylate

treatment causing a significant decrease in VEGF,

bFGF, EGF, and IL-6 induced endothelial cord

formation and an even more dramatic decrease in

tumor-driven cord formation. In addition to

involvement in downstream cytokine signaling,

p38 MAPK was important for VEGF, bFGF, EGF,

IL-6 and other proangiogenic cytokine secretion in

stromal and tumor cells. LY2228820 dimesylate

results were substantiated using p38α MAPK

specific shRNA and shRNA against the

downstream p38 MAPK effectors MAPKAPK-2

and HSP27. Using in vivo models of functional

neoangiogenesis, LY2228820 dimesylate treatment

reduced hemoglobin content in a plug assay and

decreased VEGF-A stimulated vascularization in a

mouse ear model. Thus, p38α MAPK is implicated

in tumor angiogenesis through direct tumoral

effects and through reduction of proangiogenic

cytokine secretion via the microenvironment.

The p38 mitogen-activated protein kinases

(MAPKs) are strongly activated by stress and

inflammatory cytokines leading to modulation of

many cellular functions including proliferation,

differentiation, and survival (1). Four different p38

MAPK isoforms have been identified, p38α, β, γ, and

δ which may have both overlapping and specific

functions (1-2). p38α MAPK (p38α) and p38β MAPK

(p38β) are ubiquitously expressed, while p38γ MAPK

and p38δ MAPK demonstrate specific tissue

expression. p38α, the most abundant isoform, is

present in most cells and is exclusively critical for

mouse development (3-5). Upstream p38 MAPK

kinases (MKKs), such as MKK3 and MKK6, can

differentially regulate p38 isoforms, as evidenced by

http://www.jbc.org/cgi/doi/10.1074/jbc.M112.425553The latest version is at JBC Papers in Press. Published on January 18, 2013 as Manuscript M112.425553

Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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the inability of MKK3 to effectively activate p38β (6).

A major substrate for p38 MAPK is MAPK-activated

protein kinase-2 (MAPKAPK-2; MK2), a serine

threonine kinase that directly phosphorylates the

ubiquitously expressed heat shock protein 27

(HSP27). HSP27 is activated in response to osmotic

stress, reactive oxygen species, and inflammatory

cytokines, and may play a role in cell migration,

apoptosis, and actin cytoskeleton organization (7).

Previous reports indicate a role for p38

MAPK in a wide range of biological processes, in

particular tumor cell proliferation in vitro and in vivo

(8-9). Angiogenesis is required for tumor growth and

metastasis; therefore, many new potential cancer

therapies are directed against the tumor vasculature. Angiogenesis is the formation of vascular tubes

composed of an inner lining of endothelial cells and,

as they mature, vessels acquire a coating of

perivascular cells (referred to as pericytes, smooth

muscle cells, or mural cells) that envelop the surface

of the vascular tube and are critical for the

development and maintenance of the vasculature (10-

11). Angiogenesis is stimulated by a variety of

soluble factors, including vascular endothelial growth

factor (VEGF), basic fibroblast growth factor (bFGF),

endothelial growth factor (EGF), and interleukin 6

(IL-6) (12-13). Endothelial cells and pericytes

communicate via cytokine signaling, and pericytes

play a role in maintaining the integrity of endothelial

cells by serving as support structures (14). In addition

to vascular stabilization, pericytes are important for

modulation of endothelial cell migration,

proliferation, and survival (11,15). Previous findings

suggest a role for p38 MAPK in modulating tumor

angiogenesis in tumor cells and/or host endothelial

cells (7,16-20), but this potential role is not well

defined.

We investigated the role of p38 MAPK in

individual cytokine and tumor-driven angiogenesis

through pharmacological inhibition of p38 MAPK

using LY2228820 dimesylate treatment and by

shRNA gene knockdown. LY2228820 dimesylate

(Fig. 1A) is a potent small molecule ATP-competitive

inhibitor of p38 MAPK that is highly selective for the

p38α and p38β isoforms and is currently under

clinical investigation for human malignancies. We

report that the p38 MAPK pathway is activated in

endothelial cells in response to VEGF, bFGF, EGF,

and IL-6 stimulation and is involved in individual

cytokine-driven and tumor-driven cord formation.

Inhibition of p38 MAPK in tumor cells also led to

decreased secretion of VEGF, bFGF, EGF, IL-6, IL-8,

and other proangiogenic factors. Small molecule

inhibitor results were substantiated by shRNA

knockdown of p38α and downstream p38 MAPK

effectors MK2 and HSP27 but not p38β.

Consequently, p38α plays a role in endothelial cell

angiogenesis along with stromal and tumor cell

cytokine secretion. LY2228820 dimesylate treatment

yielded antiangiogenic effects in vivo via decreased

hemoglobin content in a Matrigel™ plug assay, a

measure of functional neoangiogenesis, and decreased

VEGF-A stimulated vascularization in a mouse ear

model. p38α and its downstream effectors, MK2 and

HSP27, are therefore implicated in tumor

angiogenesis, and p38α plays an integral role in key

proangiogenic cytokine secretion.

EXPERIMENTAL PROCEDURES

Cell Culture—U-87-MG, MDA-MB-231, SK-OV-3,

A-2780, NCI-H1650 and PC-3 cells were grown

according to American Type Culture Collection

(ATCC, Manassas, VA) guidelines. LXFA-629 non-

small-cell lung adenocarcinoma cells (Oncotest,

Freiburg, Germany) were maintained in RPMI 1640

medium supplemented with 10% heat-inactivated

FBS and 1% glutamine (all from Invitrogen, Carlsbad,

CA). All cells were grown and treated in uncoated

tissue-culture treated flasks in a humidified

atmosphere at 37°C and 5% CO2.

shRNA Knockdown—U-87-MG and MDA-MB-231

cells were transduced (multiplicity of infection 9)

with MISSION® shRNA lentiviral transduction

particles (Sigma-Alrich, St. Louis, MO; non-target

control: SCH202V; p38α: NM_001315; p38β:

NM_002751), selected with 5 µg/ml puromycin and

screened for protein knockdown by Western blot

analysis as described below. ADSC/ECFC co-

cultures were transduced following ECFC plating in

cord formation as described below with 30 ul of

MISSION® shRNA lentiviral transduction particles

(Sigma-Alrich; non-target control: SCH202V; p38α:

NM_001315; p38β: NM_002751; MK2:

NM_032960; HSP27: NM_001540) for 72 hours prior

to analysis for Western blot, cord formation, cytokine

secretion, or phosphoprotein immunoassay as

described below.

In vitro Cord Formation Assay—Adipose derived

stem cells (ADSCs, Zen-Bio, Research Triangle Park,

NC) were plated at 75,000 cells per well into 96-well

HTS Transwell® (Corning, Lowell, MA) receiver

plates (tumor-driven) or 50,000 cells per well (growth

factor-driven) into 96-well black poly-D lysine coated

plates, and tumor cells were plated at 25,000 cells per


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well in 96-well HTS Transwell® (Corning) plates in

co-culture media [MCDB-131 media (Invitrogen)

supplemented with L-ascorbic acid 2-phosphate,

dexamethasone, tobramycin, insulin (all from Sigma-

Aldrich), and cell prime r-transferrin AF (Millipore,

Ballerica, MA)] for 24 h. ADSC media was removed

and 6,000 (tumor-driven) or 5,000 (growth factor-

driven) human endothelial colony forming cells

(ECFCs; Lonza, Basel, Switzerland) per well were

over seeded. Treatment with 10 ng/ml VEGF, bFGF,

EGF, or 100 ng/ml IL-6 (all from Invitrogen) and

DMSO or 1 µM LY2228820 dimesylate treatment

occurred 4 h following ECFC plating and continued

for 96 h. Cells were directly fixed for 10 min with

3.7% formaldehyde (Sigma Aldrich) followed by ice-

cold 70% ethanol for 20 min at 25°C. Cells were

rinsed once with PBS, blocked for 30 min with 1%

BSA, and immunostained for 1 h with antiserum

directed against CD31 (R&D Systems, Minneapolis,

MN) diluted to 1 μg/ml in 1% BSA. Cells were

washed 3 times with PBS and incubated for 1 h with 5

μg/ml donkey α-sheep-Alexa-488 (Invitrogen), α-

Smooth Muscle Actin Cy3 conjugate (1:200, Sigma-

Aldrich), and 200 ng/ml Hoechst 33342 (Invitrogen)

in 1% BSA, washed with PBS, then imaged using the

cord formation algorithm on the Cellomics®

ArrayScan® VTI at an image magnification of 5X

(Thermo Fisher Scientific, Pittsburgh, PA). For

assessment of proliferation and apoptosis, cells were

plated, treated, and fixed for cord formation as

mentioned above then immunostained with Ki67

(1:100, Millipore), 5 μg/ml goat α-rabbit-Alexa-647

(Invitrogen), and 200 ng/ml Hoechst 33342

(Invitrogen) or the In Situ Cell Death Detection Kit

(Roche, Indianapolis, IN) according to the

manufacturer’s recommendations, then imaged using

the target activation algorithm on the Cellomics®

ArrayScan® VTI at an image magnification of 20X

(Thermo Fisher Scientific, Pittsburgh, PA). Cell

motility was analyzed using the Cellomics® Cell

Motility Kit (Thermo Fisher Scientific) by plating 500

ADSC or ECFC cells on prepared blue fluorescent

microsphere plates according to the manufacturer’s

recommendations. Treatment with DMSO or 1 µM

LY2228820 dimesylate and 10 ng/ml VEGF, bFGF,

EGF, or 100 ng/ml IL-6 occurred 24 h following cell

plating and continued for 18 h. Cells were then fixed,

stained, and imaged using the cell motility algorithm

on the Cellomics® ArrayScan

® VTI at an image

magnification of 20X (Thermo Fisher Scientific)

according to the manufacturer’s recommendations.

Western Blot—Whole-cell protein extracts were

isolated by cell lysis with 1% sodium dodecyl sulfate

(SDS) and brief sonication, and protein concentration

was quantified using the Bradford method. Thirty

micrograms of protein were subjected to

electrophoresis on 4 to 20% pre-cast Tris-glycine

gradient gels (Invitrogen), transferred to nitrocellulose

(Invitrogen), blocked with 5% blotting grade blocker

(Biorad, Hercules, CA) in Tris-buffered saline

containing 0.1% tween (TBST), probed with primary

antiserum, washed with TBST, and incubated with an

appropriate horseradish peroxidise-labeled secondary

antibody. Membranes were washed with TBST, and

signal was detected by ECL (Thermo Fisher

Scientific). Antiserum directed against p38α, p38β,

total HSP27 (all from Cell Signaling Technology,

Danvers, MA); phospho-p38 MAPK

(Thr180/Tyr182), phospho-HSP27 (S15), phospho-

MAPKAPK2 (Thr334), total MAPKAPK2 (all from

Epitomics, Burlingame, CA), and β-actin (Santa-Cruz

Biotechnology, Santa Cruz, CA) were diluted with

5% blotting grade blocker (Biorad) in TBST. Densitometry was performed using Image J analysis

software (NIH) as per the request of the Image J

developers.

Phosphoprotein Immunoassays—ADSCs/ECFCs

were plated following the cord formation protocol

above or ECFCs were plated in normal growth media

(2 x 104) in 96-well tissue culture dishes for 24 h prior

to replacement of media with co-culture media for 2

h. ADSCs/ECFCs or ECFCs were then stimulated for

15 min with 10 ng/ml VEGF, bFGF, EGF, 100 ng/ml

IL-6, or conditioned media from U-87-MG or MDA-

MB-231 cells (described below) and analyzed with

the phospho-p38 (Thr180, Tyr182) or phospho-

MAPKAPK2 (Thr334) whole cell kits according to

the manufacturer’s recommendations (Meso Scale

Discovery, Rockville, MD).

Cytokine Analysis—ADSCs or ADSCs/ECFCs were

plated in co-culture media following the cord

formation protocol above. Tumor cells (2 x 105) were

plated in co-culture media in 6-well tissue culture

dishes. Following 4 h after ECFC plating or 24 h

after tumor cell plating, media was replaced and

treatments were added for 72 h prior to media

collection and cell number counts. Cell debris was

removed from conditioned media by centrifugation,

and samples were analyzed fresh or were frozen at -

20°C until analysis. Samples were analyzed with

Quantikine® Colorimetric Sandwich ELISAs (R&D


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Systems, Minneapolis, MN) according to the

manufacturer’s recommendations.

In vivo Matrigel™

Plug Angiogenesis Assay—ADSCs

(0.5 x 106) and ECFCs (2 x 10

6) were mixed with 200

µl growth factor reduced Matrigel™

(Becton,

Dickenson and Company, Bedford, MA) on ice and

subcutaneously injected into the flanks of athymic

nude female mice (Harlan, Indianapolis, IN), one

implant per animal. Mice were dosed orally three

times daily with LY2228820 dimesylate (20 and 40

mg/kg) or twice daily with sunitinib (25 mg/kg),

which were prepared internally, beginning 4 hours

prior to cell implantation. After 5 days of dosing,

implants were removed, flash frozen in liquid

nitrogen, and hemoglobin was quantified using the

QuantiChrom™ Hemoglobin Assay Kit (Bioassay,

Hayward, CA) as previously described (21).

Angiogenesis Ear Assay—Animal protocols were

approved by the Imclone System Inc. and Mispro

Biotech Services Corporation Animal Care and Use

Committee. A nonreplicating adenoviral vector

engineered to express the predominant (164 amino

acids) murine isoform of VEGF-A (Ad-VEGF-A164

;

1x108

plaque-forming units) as previously described

(22) was injected intradermally into the dorsal ears of

8 week old athymic nu/nu mice (Charles River,

Wilmington, MA) as previously described (23). Mice

(n = 5) were dosed orally with sunitinib at 40 mg/kg

daily, LY2228820 dimesylate at 30 mg/kg twice a

day, or vehicle (HEC-tween) twice a day starting one

day before injection of adenovirus VEGF-A and

harvested at day 5 after adenovirus injection. Ears

were mounted flat under a glass slide with immersion

oil and photographed using a Leica M80

photomicroscope as previously described (23). The

images of ear vasculature were quantified by Image-

Pro Analyzer 7.0 (MediaCybernetics, Bethesda, MD).

Statistical analysis—Statistical significance for data

was assessed by two-tailed Student t test with equal

variance compared to data obtained for DMSO or

non-target shRNA controls (in vitro) or vehicle

controls (in vivo). Statistical significance was

assigned to p values <0.05.

RESULTS

VEGF, bFGF, EGF, and IL-6 activate p38

MAPK signaling—LY2228820 dimesylate (Fig. 1A)

is a highly selective ATP-competitive inhibitor of

p38α and p38β that does not alter p38 MAPK

activation but reduces downstream p38 MAPK

signaling (24). The human kinome map of

LY2228820 dimesylate activity indicates the

specificity of the inhibitor for p38 MAPK compared

to that of sunitinib, a multi-targeted receptor tyrosine

kinase inhibitor with an antiangiogenic mechanism of

action (25) (Supplemental Fig. 1). Smooth muscle

cells and endothelial cells are exposed to many

growth factors and proinflammatory cytokines that

contribute to angiogenesis, and p38 MAPK is

implicated in downstream cytokine signaling (18,26);

therefore, p38 MAPK signaling was analyzed in

endothelial colony forming cells (ECFCs), a subtype

of umbilical cord blood-derived endothelial cells that

can form intrinsic in vivo vessels upon transplantation

into immunodeficient mice (27), and adipose derived

stem cells (ADSCs, which are similar to

mesenchymal stem cells), which can give rise to cells

with pericytic properties that can stabilize vascular

assembly in vitro (28). In ECFCs, there was a robust

increase in VEGF dependent phosphorylated p38

MAPK (p-p38) expression (p<0.05), while bFGF,

EGF, and IL-6 showed a modest (~25%) increase in

p-p38 by a highly sensitive immunoassay (Fig. 1C).

Importantly, ligand induced upregulation of p-p38 in

ECFCs resulted in increased expression of the

downstream effectors p-MK2 and p-HSP27 by

Western blot (Fig. 1B) and a significant increase

(p<0.05) in p-MK2 by immunoassay (Fig. 1C).

Activation of these effectors was significantly

impaired following inhibition of p38 MAPK by

LY2228820 dimesylate treatment (Fig. 1B).

Furthermore, LY2228820 dimesylate treatment

abrogated basal p38 pathway activity in ECFCs and

ADSCs (Fig. 1B).

LY2228820 dimesylate treatment reduced

VEGF, bFGF, EGF, and IL-6-driven cord

formation—Since p38 pathway activation occurs

following treatment with proangiogenic factors, a

surrogate cord formation assay to model key

morphogenic features of blood vessel formation (29)

was used to analyze the effect of LY2228820

dimesylate treatment on in vitro endothelial cord

formation. The ECFC and ADSC co-culture can

establish cord networks in vitro and functional blood

vessels in vivo (21). ADSCs, which serve as the

feeder layer for the ECFCs, can differentiate into

pericyte-like cells that express α-smooth muscle actin,

a contractile filament expressed in pericytes.

Formation of vascular networks by ECFCs can be

visualized by cluster of differentiation 31 (CD31)

immunostaining (30), accompanied by ADSC

migration and increased density near the cords along

with increased α-smooth muscle actin expression.


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LY2228820 dimesylate treatment significantly

reduced (p<0.05) basal, VEGF, bFGF, EGF, and IL-6

driven cord formation along with α-smooth muscle

actin expression (Fig. 1D). In addition, cords that

were established without stimulation (basal) or with

VEGF, bFGF, EGF, and IL-6 for 4 days prior to

LY2228820 dimesylate treatment showed significant

regression (p<0.05) in both cord formation and α-

smooth muscle actin expression (Supplemental Fig.

2). Cell viability of the ADSC/ECFC co-culture was

not altered by LY2228820 dimesylate treatment

which ensured that the observed effects on cord

formation were not due to cell toxicity (data not

shown).

Endothelial cells require cytokine stimulation

for survival, proliferation, and migration, all of which

are essential for angiogenesis, and the paracrine

signaling between endothelial cells and pericytes is

important for cord formation. In addition,

endothelial cells secrete factors, partly to attract

pericytes to envelop the vessel wall and promote

vessel maturation (31); therefore, a role for p38

MAPK on ADSC/ECFC co-culture, ADSC, and

ECFC cytokine secretion was analyzed. The

ADSC/ECFC co-culture along with ADSCs alone

showed a significant reduction (p<0.05) in VEGF,

bFGF, EGF, and IL-6 secretion upon LY2228820

dimesylate treatment (Fig. 1E). ECFCs secreted

undetectable to minimal amounts (<8 pg/ml) of

VEGF, bFGF, EGF, and IL-6 (data not shown).

However, the amount of VEGF, bFGF, EGF, and IL-6

was slightly enhanced (~10-30%) in the ADSC/ECFC

co-cultures compared to ADSCs alone, indicating that

cell contact between ADSCs and ECFCs may be

important for cytokine secretion. These data indicate

that p38 MAPK is involved both in downstream

cytokine signaling and cytokine release from stromal

cells. Statistically significant decreases (p<0.05) in

ECFC proliferation, assessed by expression of the

proliferation marker Ki67, ECFC motility, or

increases in ECFC apoptosis, assessed by TUNEL

analysis, were not observed with LY2228820

dimesylate treatment upon VEGF, bFGF, EGF, or IL-

6 stimulation (Supplemental Fig. 3).

Knockdown of p38α, MK2, or HSP27 reduced

VEGF, bFGF, EGF, IL-6-driven cord formation—

The use of a p38α/β selective inhibitor such as

LY2228820 dimesylate is critical in establishing a

role for p38 MAPK function in angiogenesis but does

not distinguish between the functions of p38α and

p38β. To further investigate a role for the p38 MAPK

pathway in angiogenesis, shRNA gene knockdown of

p38α and p38β in ADSC/ECFC co-cultures were

generated. Knockdown of p38α but not p38β in

ADSCs/ECFCs was effective in blocking p38 MAPK

signaling, as evidenced by reduced levels of both p-

p38, p-MK2, and p-HSP27 by Western blot (Fig. 2B)

and by immunoassay (Fig. 2D) which led to a

significant reduction (p<0.05) in VEGF, bFGF, EGF,

and IL-6 (knockdown of p38β also significantly

reduced IL-6 driven-cord formation but to a lesser

extent than p38α) driven cord formation (Fig. 2A).

These results are similar to those of LY2228820

dimesylate treatment, which strengthens the notion

that compound effects are specific to inhibition of

p38α. To further support a role for p38 MAPK and

the p38 MAPK pathway in cord formation,

knockdown of downstream signaling effectors MK2

and HSP27 in the ADSC/ECFC co-culture also

significantly reduced (p<0.05) VEGF, bFGF, EGF,

and IL-6 driven cord formation (Fig. 2A).

Knockdown of p38α, MK2, or HSP27 but not p38β

also significantly reduced (p<0.05) cytokine secretion

of VEGF, bFGF, and IL-6 from ADSC/ECFC co-

cultures (Fig. 2C). Secretion of EGF from

ADSC/ECFC co-cultures was significantly reduced

(p<0.05) with p38α knockdown and was only slightly

reduced with MK2 or HSP27 knockdown (Fig. 2C).

Knockdown of p38α significantly inhibited (p<0.05)

activation of p-p38 and p-MK2 following VEGF

stimulation (Fig. 2D). Knockdown of MK2 also

significantly reduced (p<0.05) basal and VEGF

induced p-MK2 expression (Fig. 2D). In contrast,

knockdown of p38β, MK2, or HSP27 did not alter

VEGF induced activation of p-p38 by immunoassay

analysis (Fig. 2D). This indicates that p38α and not

p38β is the main mediator of VEGF, EGF, bFGF, and

IL-6 cytokine secretion and downstream signaling

through MK2 and HSP27 in our proangiogenic co-

culture system. As observed with LY2228820

dimesylate treatment, cell viability of ADSC/ECFC

co-cultures was not altered upon shRNA treatment;

therefore, the anti-cord forming effects did not stem

from a cytotoxic event (data not shown). Similar

results were obtained with two additional shRNA

clones targeting different mRNA regions of p38α,

p38β, MK2 and HSP27, indicating that the effects

observed are likely due to reduced expression of the

intended target genes (data not shown).


tumor-driven cord formation—To more closely

represent tumor angiogenesis, instead of individual

cytokines stimulating cord formation, LY2228820

dimesylate effects on tumor-conditioned media-driven


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and tumor cell-driven cord formation were analyzed.

Conditioned media from commonly used, well

characterised U-87-MG glioblastoma and MDA-MB-

231 breast cancer cells increased protein expression of

p-p38, p-MK2, and p-HSP27 by Western blot and

significantly increased (p<0.05) p-p38 and p-MK2 by

immunoassay (Supplemental Fig. 4A-B). This

indicates that cytokines secreted from U-87-MG and

MDA-MB-231 tumor cells activate p38 MAPK

signaling. LY2228820 dimesylate treatment

significantly reduced (p<0.05) U-87-MG and MDA-

MB-231 tumor-conditioned media-driven cord

formation, indicating a function for p38 MAPK in

stromal cells downstream of tumor-secreted cytokines

(Supplemental Fig. 4C). LY2228820 dimesylate also

significantly reduced (p<0.05) tumor-driven cord

formation and smooth muscle actin expression from a

range of tumor histologies, including U-87-MG,

MDA-MB-231, ovarian (A-2780, SK-OV-3), lung

(LXFA-629, NCI-H1650), and prostate (PC-3) (Fig.

3A). LY2228820 dimesylate treatment inhibited

downstream p38 MAPK signaling (p-MK2, p-HSP27)

in U-87-MG and MDA-MB-231 cells (Fig. 3B) along

with each tumor cell line analyzed (data not shown).

In tumor-driven cord formation, LY2228820

dimesylate effects on the tumor cells and downstream

cytokine signaling could not be separated because all

three cell lines (tumor, ADSC, and ECFC) were

concurrently treated with LY2228820 dimesylate,

necessitating pre-treatment of U-87-MG or MDA-

MB-231 tumor cells with LY2228820 dimesylate

prior to cell plating. Pre-treatment of tumor cells with

LY2228820 dimesylate significantly reduced

(p<0.05) cord formation (Supplemental Fig. 5) and

secretion of VEGF, bFGF, EGF, and IL-6 from U-87-

MG (Fig. 3C), along with A-2780, SK-OV-3, and PC-

3 tumor cell lines (data not shown), and VEGF,

bFGF, and IL-6 from MDA-MB-231 tumor cells (Fig.

3C; basal EGF is below the limit of detection in

MDA-MB-231 cells). LY2228820 dimesylate

treatment also reduced secretion of IL-8 and other

proangiogenic cytokines (angiogenin, HGF, PlGF,

PDGF-AA) secreted from U-87-MG, MDA-MB-231,

SK-OV-3, and A-2780 tumor cells (manuscript in

preparation and data not shown). Pre-treatment of

tumor cells with LY2228820 dimesylate and addition

of compound into the cord formation assay led to the

greatest inhibition of cord formation (Supplemental

Fig. 5), further supporting the notion that LY2228820

dimesylate treatment has a direct effect on tumor cell

cytokine secretion and cytokine signaling in

ADSC/ECFC cells, especially since cell viability of

tumor cells and ADSC/ECFC co-cultures were

unchanged (data not shown).

Knockdown of p38α in tumor cells reduced

tumor-driven cord formation—To further investigate

the role of p38α and p38β in tumor cell cytokine

secretion and tumor-induced cord formation, stable

knockdown of p38α or p38β was assessed in U-87-

MG and MDA-MB-231 cells. It is important to note

that complete protein knockdown was not achieved

for either isoform which may have affected results on

cord formation. Knockdown of p38α but not p38β in

both cell lines reduced expression of p-p38, p-MK2,

and p-HSP27 (Fig. 4B, Supplemental Fig. 6B), and

significantly reduced (p<0.05) tumor-driven cord

formation (Fig. 4A, Supplemental Fig. 6A) along with

VEGF, bFGF, EGF (in U-87-MG, undetectable in

MDA-MB-231), and IL-6 secretion (Fig. 4C,

Supplemental Fig. 6C). Similar results were obtained

with additional U-87-MG and MDA-MB-231 stable

shRNA knockdown lines which confirmed the

importance of p38α in controlling tumor cell cytokine

secretion and cord formation.

Cord formation rescue with addition of

VEGF, bFGF, EGF, and IL-6 in conditioned media

from tumor cells with stable p38α knockdown—To

determine if the reduction in VEGF, bFGF, EGF (U-

87-MG), and IL-6 is contributing to the reduction in

cord formation observed with stable knockdown of

p38α in U-87-MG and MDA-MB-231 tumor cells, we

performed an add back experiment. Conditioned

media from control shRNA cells or p38α stable

knockdown cells for U-87-MG and MDA-MB-231

cells were collected and analyzed for VEGF, bFGF,

EGF (U-87-MG cells only, MDA-MB-231 cells had

undetectable amounts of EGF), and IL-6 cytokine

levels. Addition of 1X or 2X amounts (compared to

the control shRNA conditioned media) of the

individual deficient cytokines or a mixture of the

deficient cytokines to the p38α stable knockdown

conditioned media was used to assess cord formation.

Importantly, addition of a 1X or 2X mixture of

VEGF, bFGF, EGF (U-87-MG only), and IL-6

significantly increased (p<0.05) cord formation

compared to the U-87-MG or MDA-MB-231 p38α

knockdown tumor conditioned media (Supplemental

Fig. 7). This indicates that reduction in VEGF, bFGF

and to a lesser extent EGF and IL-6 upon p38α

knockdown in tumor cells contributes to the reduction

in cord formation observed.


hemoglobin content and ear angiogenesis in vivo—To

extend a role for the p38 MAPK pathway in


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angiogenesis in vivo, we tested LY2228820

dimesylate in a neoangiogenesis Matrigel™ plug

model consisting of ADSCs/ECFCs that form blood

vessels following co-implantation into the flank of a

nude mouse (28). Five days after implantation,

ADSC/ECFC cells formed extensive networks of

blood vessels whose functionality was assessed by

measuring hemoglobin content. LY2228820

dimesylate and sunitinib, an approved angiogenesis

inhibitor used as a positive control, were given at

clinically relevant doses and both caused a significant

reduction (p<0.05) in hemoglobin content (Fig. 5A).

To further analyze the relevant effects of p38

MAPK on angiogenesis in vivo, we tested

LY2228820 dimesylate in an ear angiogenesis model

consisting of intradermal injection of Ad-VEGF-A164

into nude mouse ears that induces a robust angiogenic

response (32). Similar to the Matrigel™ plug assay,

both LY2228820 dimesylate and sunitinib treatment

caused a significant reduction (p<0.05) in ear

vascularity (Fig. 5B) which indicates that LY2228820

dimesylate treatment impaired neoangiogenesis in

vivo. All in vitro and in vivo experiments were

confirmed with a second generation p38α and p38β

specific ATP-competitive inhibitor, further supporting

that the results obtained in these studies are due to

specific inhibition of p38 MAPK signaling (data not

shown).

DISCUSSION

Our work examined the role of p38 MAPK

signaling on cord formation in vitro and

neoangiogenesis in vivo, and our data implicate p38

MAPK both in the release of cytokines from tumor

and stromal cells and in mediating their downstream

effects on endothelial cells. Previous studies using

p38 MAPK inhibitors were limited by an inability to

distinguish tumor versus stromal cell effects and

delineate p38 MAPK isoform specific effects. Our

data support a positive role for p38 MAPK activation

in angiogenesis, similar to what was reported by

Jackson et al. where small molecule inhibitors of p38

MAPK kinase reduced angiogenesis in an

inflammatory angiogenesis model (33). Others have

also reported a role for p38 MAPK in pro-

inflammatory angiogenesis in vivo in a prostate cancer

rat model (16), in shear stress-mediated angiogenesis

(34), in human coronary artery endothelial cell tube

formation (17), and in vitro in basal and tumor

conditioned media capillary-like structure formation

in a co-culture system (35). Further strengthening a

potential role for p38 MAPK in angiogenesis, lack of

the p38 MAPK upstream activator, MKK3 causes

deficiency in both primary and placental blood vessel

development (36).

In this report, VEGF, bFGF, EGF, and IL-6

were shown to activate the p38 MAPK pathway in

endothelial cells. LY2228820 dimesylate treatment

reduced VEGF, bFGF, EGF, and IL-6 driven cord

formation and α-smooth muscle actin expression

(pericyte marker) along with the secretion of those

soluble factors from ADSCs and a proangiogenic

ADSC/ECFC co-culture. Our results are consistent

with previous findings that p38 MAPK mediates both

signaling downstream of cytokine receptors and

cytokine release (33-34,37). Thus, p38 MAPK-

dependent production of VEGF, bFGF, EGF, and IL-

6 may be critical for angiogenesis. VEGF was

reported to induce activation of p38 MAPK and

HSP27 in endothelial cells through a p38 MAPK

dependent pathway (7,18,26). p38 MAPK signaling

was also shown to be responsible for activation of

HSP27 and mediate smooth muscle cell migration

upon PDGF, IL-1β, and TGFβ stimulation (38). In

endothelial cells, p38 MAPK was also activated by

bFGF, and inhibition of p38 MAPK abrogated bFGF-

mediated tube formation of MSS31 stromal cells and

endothelial cell migration (39). Therefore, p38

MAPK is activated by many proangiogenic factors

and likely mediates endothelial cell function.

p38 MAPK signaling has also been implicated

in the regulation of endothelial and mural cell

migration (37-38) and recruitment during

angiogenesis (11). Inhibition of p38 MAPK led to

reduced HSP27 phosphorylation, actin reorganization,

and endothelial cell migration (18), which was

dependent on MK2 following VEGF stimulation (26).

Furthermore, overexpression of MKK6, an upstream

activator of p38 MAPK kinase enhanced HSP27

phosphorylation and migration in human umbilical

vein endothelial cells (HUVECs) (40), while siRNA-

mediated MK2 knockdown in MSS31 spleen

endothelial cells inhibited VEGF-induced cell

migration (26). It remains to be determined whether

p38 MAPK mediates ECFC or ADSC migration in

our cord formation system, but we did not observe

significant reduction in migration with cytokine

stimulated ECFCs alone or ADSCs alone upon

LY2228820 dimesylate treatment.

In addition to reducing individual cytokine-

induced cord formation, LY2228820 dimesylate

treatment displayed a more pronounced reduction in

tumor-conditioned media-driven and tumor cell-

driven cord formation. The fact that p38 MAPK was


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shown to be activated downstream of many

proangiogenic cytokine receptors suggests that

LY2228820 dimesylate treatment may have an

additive effect when a multitude of cytokines are

being released from tumor cells and/or that

LY2228820 dimesylate treatment has an effect on

tumor cell cytokine secretion and signaling

downstream of cytokine receptors in stromal cells.

Previous reports also indicate that p38 MAPK can

regulate cytokine secretion from tumor cells,

including VEGF secretion in malignant gliomas (41)

and other tumor cells (19). Pre-treatment of tumor

cells with LY2228820 dimesylate, which does not

affect tumor cell viability, reduced cord formation

which suggests that LY2228820 dimesylate treatment

alters cytokine secretion from tumor cells. Indeed,

LY2228820 dimesylate treatment significantly

reduced secretion of VEGF, bFGF, EGF, IL-6, and

other proangiogenic (IL-8, angiogenin) cytokines

from a variety of tumor cell lines. While IL-8 has

been shown to play an important role in tumor

growth, angiogenesis, and metastasis (42) it was not a

potent inducer of cord formation in our in vitro

system (data not shown). Pre-treatment of tumor cells

with LY2228820 dimesylate along with addition of

LY2228820 dimesylate into the cord formation

system led to a greater inhibition of cord formation

than just pre-treatment of tumor cells with

LY2228820 dimesylate or addition of LY2228820

dimesylate into the cord formation assay. This

suggests that LY2228820 dimesylate treatment

reduced tumor-driven cord formation in part by

decreasing cytokine secretion from tumor and/or

stromal cells and by affecting the stromal cells

response to cytokine stimulation.

In addition to small molecule inhibition of

p38 MAPK, protein knockdown of p38α in stromal or

tumor cells reduced cord formation. Protein

knockdown of p38β only reduced IL-6 driven cord

formation in stromal cells, indicating a role for both

p38α and p38β in IL-6 mediated cord formation.

Targeted inactivation of the mouse p38α gene results

in embryonic death due to a placental defect (3-5) and

angiogenesis was abnormal in the yolk sac and the

embryo itself, resulting in immature networks of

vessels (4). In contrast, single knockout of p38β,

p38γ and p38δ, or both p38γ and δ result in viable,

healthy mice (43-44). In addition, MK2 activity is

abolished in p38α knock out mice (3), while p38β

knock out mice exhibit normal MK2 activity (43),

which highlights diverse downstream pathway

activation among p38 MAPK isoforms. One study

using SB203580, a small molecule which completely

inhibits p38α and only partially inhibits p38β suggests

that p38α is the principal isoform controlling

proliferation and migration of endothelial cells (40).

Similarly, our results indicate that downstream p38

MAPK signaling through MK2 and HSP27 is

mediated by p38α and not p38β in stromal and tumor

cells which suggests independent, non-redundant

functions between the p38 MAPK isoforms.

In our experiments, knockdown of p38α but

not p38β reduced secretion of VEGF, bFGF, EGF,

and IL-6 production from stromal and tumor cells

implicating p38α as a critical mediator of

proangiogenic cytokine secretion. Importantly,

addition of the reduced VEGF, bFGF, EGF (U-87-

MG only), and IL-6 in stable U-87-MG or MDA-MB-

231 p38α knockdown tumor cells was able to rescue

cord formation indicating that these cytokines appear

to be main players involved in p38 MAPK signaling

and cord formation on our co-culture system.

Additional studies indicate that p38α is the

predominant isoform involved in cytokine production

in vivo following lipopolysaccharide (LPS)

stimulation (43), and gene transfer of p38α and the

upstream activator MKK3 significantly increased

expression of bFGF and PDGF-A in the normal heart

(45), further supporting the concept of independent,

non-redundant functions of p38 MAPK isoforms. In

addition to p38α, knockdown of the p38 MAPK

pathway proteins MK2 or HSP27 in stromal cells also

reduced cord formation along with VEGF, bFGF, and

IL-6 secretion, a result substantiated in MK2 knock

out mice where reduced angiogenesis in wound

healing is in concert with reduced expression of

several cytokines (GM-CSF, VEGF, IFNγ, MCP1,

TNF, IL-6, and IL-1β) compared to wild-type mice

(46). In addition, targeted deletion of MK2 in

macrophages led to decreased production of LPS-

induced tumor necrosis factor (TNF), IL-6, and other

cytokines (47). Taken together, these results reveal a

role for the p38 MAPK pathway components MK2

and HSP27 in cytokine production and angiogenesis.

In addition to endothelial cells, pericytes play

an important role in endothelial cell function and

vascular formation (10-11). Pericytes have been

associated mainly with stabilization of blood vessels

but also can sense angiogenic stimuli, guide sprouting

tubes, elicit endothelial survival, and exhibit

macrophage-like activities (31). Several molecules

regulate pericyte contractile tone and function as

paracrine signals that reveal an interaction between

endothelial cells and pericytes in the regulation of


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blood flow (31). Tumors usually contain a small

number of functional pericytes important for vessel

stability, function, and endothelial cell survival (31).

Highlighting the importance of pericyte vessel

stability function, glioblastomas frequently contain

vessels that are not covered by pericytes, and these

vessels are more dependent on VEGF as an

endothelial cell survival factor (48). Furthermore,

blocking PDGFR signaling resulted in detachment of

pericytes from tumor vessels and restriced tumor

growth (49). Inhibition of p38 MAPK signaling with

LY2228820 dimesylate treatment or knockdown of

p38α significantly reduced α-smooth muscle actin

expression from perictye-like cells in our cord

formation assay. This suggests that p38 MAPK may

play an important role in paracrine signaling between

endothelial cells and perictyes but it remains unknown

whether p38 MAPK signaling is important for

pericyte function or recruitment in vivo. Tumor

vessels without pericytes appear more vulnerable and

may be more responsive to anti-endothelial cell drugs

(31). Multiple human renal tumor models treated

with a combination of LY2228820 dimesylate and

sunitinib show potentiation of sunitinib activity

(unpublished data), which may be due in part to

decreased numbers of pericytes interacting with the

vessels, causing the endothelial cells to be more

susceptible to the antiangiogenic therapy.

MAPK kinases (MKK) are crucial enzymes

involved in several biological pathways that control

cell differentiation, proliferation, and survival (50). In

response to extracellular stimuli, MKKs become

activated and phosphorylate MAPKs, including

extracellular signal-regulated protein kinase (ERK), c-

Jun-NH2 kinase (JNK), and p38 MAPK. Others

report important roles for the other MAPK signaling

pathways (ERK, JNK) in tumor angiogenesis (13,51).

A variety of small molecule inhibitors that target

MEK, ERK, and JNK were observed to be

antiangiogenic in our in vitro co-culture system (data

not shown), further evidence indicating the

importance of MAPK signaling in angiogenesis. Our

results indicate a positive role for p38 MAPK

signaling, in particular p38α MAPK, MK2, and

HSP27 in angiogenesis and p38α MAPK in tumor and

stromal cell cytokine release.

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FOOTNOTES

1To whom correspondence should be addressed: Louis Stancato, Oncology Research, Eli Lilly and Company,

Lilly Corporate Center, Indianapolis, IN, USA, Tel: (317) 655-6910; Fax: (317) 276-1414; E-mail:

[email protected] 1Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN, 46285

2Discovery Research, Advanced Testing Laboratories, Cincinnati, OH, 45242

3ImClone Systems, New York, NY 10016

4The abbreviations used are: VEGF, vascular endothelial growth factor; bFGF, fibroblast growth factor; EGF,

endothelial growth factor; MAPK, mitogen-activated protein kinase; MK2, MAP kinase-activated protein

kinase 2; HSP27, heat shock protein 27; CD31, cluster of differentiation 31; SMA, α-smooth muscle actin 5Authors affiliated with Eli Lilly have Eli Lilly shares received via 401(k) and bonus plans.

ACKNOWLEDGEMENTS—We thank Mark Uhlik, Michelle Swearingen, and Simon Chen for in vitro cord

formation assay development, D’Arcy Brewer for in vivo matrigel™

plug assistance, and Susan Pratt for helpful

discussions.


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FIGURE LEGENDS

FIGURE 1. LY2228820 dimesylate treatment reduced VEGF, bFGF, EGF, and IL-6 driven cord formation. A.

Chemical structure of LY2228820 dimesylate (LY). B. Whole cell protein extracts were isolated from ECFCs or

ADSCs following pre-treatment with DMSO (-) or 1 µM LY (+) for 30 minutes prior to addition of 10 ng/ml

VEGF, bFGF, EGF, or 100 ng/ml IL-6, then the extracts were subjected to Western blot analysis using

antiserum directed against phospho-p38 MAPK (p-p38), p38α, p38β, phospho-MAPKAPK2 (p-MK2), total

MK2, phospho-HSP27 (p-HSP27), total HSP27, and β-actin as a loading control. C. Whole cell protein extracts

were isolated from ECFCs following 15 minute PBS (basal), 10 ng/ml VEGF, bFGF, EGF, or 100 ng/ml IL-6

treatment, then extracts were subjected to p-p38 and p-MK2 analysis by phosphoprotein immunoassay. Graphs

represent mean ± standard error from three independent experiments, and asterisks denote statistically significant

(*, p<0.05) differences compared to basal controls. D. The ADSC/ECFC co-cultures were treated with DMSO

or 1 µM LY simultaneously with PBS (basal), 10 ng/ml VEGF, bFGF, EGF, or 100 ng/ml IL-6 for 96 hours

prior to immunohistochemistry for CD31 (green), α-smooth muscle actin (red), and Hoechst 33342 to stain all

nuclei (blue). Representative images (5X magnification) are shown, graphs represent mean ± standard error

after basal cord formation data was subtracted from VEGF, bFGF, EGF, and IL-6 data from three independent

experiments, and asterisks denote statistically significant (*, p<0.05) differences compared to DMSO controls.

E. Conditioned media was collected from the ADSC/ECFC co-cultures or ADSCs alone treated with DMSO or 1

µM LY for 72 hours and subjected to ELISA analysis for VEGF, bFGF, EGF, and IL-6. Graphs represent mean

± standard error from three independent experiments, and asterisks denote statistically significant (*, p<0.05)

differences compared to DMSO controls.

FIGURE 2. Knockdown of p38α, MK2, or HSP27 reduced VEGF, bFGF, EGF, and IL-6-driven cord

formation. A. The ADSC/ ECFC co-cultures were treated with non-targeting (control), p38α, p38β, MK2, or

HSP27 shRNA for 72 hours prior to induction of cord formation without (PBS, basal) or with 10 ng/ml VEGF,

bFGF, EGF, or 100 ng/ml IL-6 for 96 hours before immunohistochemistry for CD31 (green), α-smooth muscle

actin (red), and Hoechst 33342 to stain all nuclei (blue). Representative images (5X magnification) are shown,


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graphs represent mean ± standard error from three independent experiments after basal cord formation data was

subtracted from VEGF, bFGF, EGF, and IL-6 data, and asterisks denote statistically significant differences (*,

p<0.05) compared to non-targeting shRNA controls. B. Whole cell protein extracts were isolated from the

ADSC/ECFC co-cultures following 72 hour shRNA treatment for the indicated gene and subjected to Western

blot analysis using antiserum directed against p38α, p38β, p-p38, p-MK2, total MK2, p-HSP27, total HSP27,

and β-actin as a loading control, and protein quantification was determined with densitometry. C. Conditioned

media was collected from the ADSC/ECFC co-cultures following 72 hour shRNA treatment for the indicated

gene and subjected to ELISA analysis for VEGF, bFGF, EGF, and IL-6. Graphs represent mean ± standard

error from three independent experiments, and asterisks denote statistically significant (*, p<0.05) differences

compared to DMSO controls. D. Whole cell protein extracts were isolated from ECFCs following 72 hour

shRNA treatment for the indicated gene following by 15 minute PBS (basal) or 10 ng/ml VEGF treatment, then

extracts were subjected to p-p38 and p-MK2 analysis by phosphoprotein immunoassay. Graphs represent mean

± standard error from three independent experiments, and asterisks denote statistically significant (*,# p<0.05)

differences compared to respective shRNA control PBS (*) or shRNA control VEGF (#) treated samples.

FIGURE 3. LY2228820 dimesylate treatment reduced tumor-driven cord formation. A. ADSC/ECFC co-

cultures with permeable transwells containing media (no cells) or indicated tumor cells (U-87-MG, MDA-MB-

231, A-2780, SK-OV-3, LXFA-629, NCI-H1650, and PC-3) were treated with DMSO or 1 µM LY for 96 hours

prior to immunohistochemistry for CD31 (green), α-smooth muscle actin, (red), and Hoechst 33342 to stain all

nuclei (blue). Representative images (5X magnification) are shown, graphs represent mean ± standard error

from three independent experiments after no cells (basal) data was subtracted, and asterisks denote statistically

significant (*, p<0.05) differences compared to DMSO controls. B. Whole cell protein extracts were isolated

from the indicated tumor cells following treatment with DMSO (-) or 1 µM LY (+) for 4 hours and subjected to

Western blot analysis using antiserum directed against p-p38, p38α, p38β, p-MK2, total MK2, p-HSP27, total

HSP27, and β-actin as a loading control. C. Conditioned media was collected from U-87-MG or MDA-MB-231

tumor cells treated with DMSO or 1 µM LY for 72 hours and subjected to ELISA analysis for VEGF, bFGF,


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EGF, and IL-6. Graphs represent mean ± standard error from three independent experiments, and asterisks

denote statistically significant (*, p<0.05) differences compared to DMSO controls.

FIGURE 4. Knockdown of p38α MAPK in U-87-MG tumor cells reduced tumor-driven cord formation. A.

Stable U-87-MG shRNA cell lines for a non-targeting shRNA (control), p38α, p38β, or media (no cells) were

plated in permeable transwells with ADSC/ECFC co-cultures for 96 hours then subjected to

immunohistochemistry for CD31 (green), α-smooth muscle actin (red), and Hoechst 33342 to stain all nuclei

(blue). Representative images (5X magnification) are shown, graphs represent mean ± standard error from three

independent experiments after no cells data (basal cord formation) was subtracted, and asterisks denote

statistically significant (*, p<0.05) differences compared to the non-targeting shRNA control. B. Whole cell

protein extracts were isolated from the indicated cell lines and subjected to Western blot analysis using

antiserum directed against p38α, p38β, p-p38, p-MK2, total MK2, and β-actin as a loading control. C.

Conditioned media was collected from stable U-87-MG shRNA cell lines and subjected to ELISA analysis for

VEGF, bFGF, EGF, and IL-6. Graphs represent mean ± standard error from three independent experiments, and

asterisks denote statistically significant (*, p<0.05) differences compared to non-targeting shRNA controls.

FIGURE 5. LY2228820 dimesylate treatment reduced hemoglobin content and ear vascularization in vivo. A.

An ADSC/ECFC cell mixture was co-implanted subcutaneously into the flanks of athymic nude mice (8 mice

per treatment group). Oral dosing of mice began 4 hours prior to cell implantation and occurred three times

daily with LY (20 and 40 mg/kg) or twice daily with sunitinib (25 mg/kg). After 5 days of dosing, Matrigel®

plugs were removed and hemoglobin was quantified. Graph is representative of three independent experiments

and indicates mean ± standard error from one experiment. Asterisks denote statistically significant (*, p<0.05)

differences compared to vehicle controls. B. Mice were dosed orally with vehicle (HEC-tween), LY2228820

dimesylate at 30mg/kg twice a day, or sunitinib at 40 mg/kg daily, starting 1 day before injection of adenovirus

VEGF-A (Ad-VEGF-A164

). Ears were harvested 5 days after adenovirus injection, imaged (representative

images from two independent experiments are shown; 8X magnification), and vasculature was quantified.


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Graph is representative of two experiments and indicates mean ± standard error. Asterisks denote statistically

significant (*, p<0.05) differences compared to vehicle controls.


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StancatoCourtney Tate, Wayne Blosser, Lisa Wyss, Glenn Evans, Qi Xue, Yong Pan and Louis

Reduces Angiogenic Endothelial Cord Formation in vitro and in vivoLY2228820 dimesylate, a Selective Inhibitor of p38 Mitogen-Activated Protein Kinase,

published online January 18, 2013J. Biol. Chem.

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p38α MAPK promotes angiogenesis · p38α MAPK promotes angiogenesis 1 LY2228820 Dimesylate, a Selective Inhibitor of p38 Mitogen-Activated Protein Kinase, Reduces Angiogenic Endothelial