Acta Physiol 2016

PAFAH1B1 and the lncRNA NONHSAT073641 maintain an

angiogenic phenotype in human endothelial cells
I. Josipovic,1,2 C. Fork,1,2 J. Preussner,3 K-K. Prior,1,2 D. Iloska,4 A. E. Vasconez,1,2 S. Labocha,5
C. Angioni,5 D. Thomas,5 N. Ferreir

os,5 M. Looso,3 S. S. Pullamsetti,4,6 G. Geisslinger,5
7
1,2
D. Steinhilber, R. P. Brandes and M. S. Leisegang1,2
1
2
3
4

Institute for Cardiovascular Physiology, Goethe-University, Frankfurt, Germany

German Center for Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt, Germany
Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany
Department of Lung Development and Remodeling, German Center for Lung Research (DZL), Max-Planck-Institute for Heart
and Lung Research, Bad Nauheim, Germany
5 Pharmazentrum Frankfurt, Institute of Clinical Pharmacology, Goethe-University, Frankfurt, Germany
6 Department of Internal Medicine, Universities of Giessen and Marburg Lung Center (UGMLC), German Center for Lung Research
(DZL), Justus-Liebig University, Giessen, Germany
7 Institute of Pharmaceutical Chemistry/ZAFES, Goethe-University, Frankfurt, Germany

Received 29 March 2016,

revision requested 25 April 2016,
revision received 16 April 2016,
accepted 25 April 2016
Correspondence: M. S. Leisegang,
PhD, Institut fur Kardiovaskulare
Physiologie, Fachbereich Medizin
der Goethe-Universitat,
Theodor-Stern Kai 7, 60590
Frankfurt am Main, Germany.
Email: Leisegang@vrc.uni-frankfurt.de

Abstract
Aim: Platelet-activating factor acetyl hydrolase 1B1 (PAFAH1B1, also known
as Lis1) is a protein essentially involved in neurogenesis and mostly studied in
the nervous system. As we observed a significant expression of PAFAH1B1 in
the vascular system, we hypothesized that PAFAH1B1 is important during
angiogenesis of endothelial cells as well as in human vascular diseases.
Method: The functional relevance of the protein in endothelial cell angiogenic function, its downstream targets and the influence of NONHSAT073641, a long non-coding RNA (lncRNA) with 92% similarity to
PAFAH1B1, were studied by knockdown and overexpression in human
umbilical vein endothelial cells (HUVEC).
Results: Knockdown of PAFAH1B1 led to impaired tube formation of
HUVEC and decreased sprouting in the spheroid assay. Accordingly, the
overexpression of PAFAH1B1 increased tube number, sprout length and
sprout number. LncRNA NONHSAT073641 behaved similarly. Microarray analysis after PAFAH1B1 knockdown and its overexpression indicated
that the protein maintains Matrix Gla Protein (MGP) expression. Chromatin immunoprecipitation experiments revealed that PAFAH1B1 is
required for active histone marks and proper binding of RNA Polymerase
II to the transcriptional start site of MGP. MGP itself was required for
endothelial angiogenic capacity and knockdown of both, PAFAH1B1 and
MGP, reduced migration. In vascular samples of patients with chronic
thromboembolic pulmonary hypertension (CTEPH), PAFAH1B1 and MGP
were upregulated. The function of PAFAH1B1 required the presence of
the intact protein as overexpression of NONHSAT073641, which was
highly upregulated during CTEPH, did not affect PAFAH1B1 target genes.
Conclusion: PAFAH1B1 and NONHSAT073641 are important for
endothelial angiogenic function.
Keywords angiogenesis, HETE, lncRNA, Matrix Gla Protein,
PAFAH1B1, pulmonary hypertension.

2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12700

PAFAH1B1 and endothelial cell function

I Josipovic et al.

Platelet-activating factor acetyl hydrolase 1b1

(PAFAH1B1, also known as Lissencephaly1) is the
regulatory subunit of the platelet-activating factor
acetyl hydrolase IB, an enzyme consisting of
PAFAH1B1, PAFAH1B2 and PAFAH1B3 (Hattori
et al. 1994). The complex catalyses the removal of an
acetyl group from platelet-activating factor (PAF) and
thus inactivates this signalling lipid (Farr et al. 1980,
Hanahan 1986, Stafforini et al. 1987). PAFAH1B1
was also the first identified gene involved in neuronal
migration diseases (Reiner et al. 1993) and its deletion
results in lissencephaly (Reiner & Sapir 2013).
PAFAH1B1 is an essential gene as homozygous
knockout mice die early in embryogenesis. Moreover,
mice with one inactive allele still exhibit disorganized
cortical, hippocampal and olfactory bulb areas due to
neuronal migration defects (Hirotsune et al. 1998). As
an underlying mechanism, it was proposed that the
isolated PAFAH1B1, that is the protein not in complex with the other interaction partners of the
PAFAH1B complex, binds to dynein and thereby
interacts with microtubules (Wynshaw-Boris & Gambello 2001). This process is facilitated by Ndel1,
which blocks the binding of PAFAH1B1 to the other
members of the PAFAH-1B enzyme complex (Tarricone et al. 2004).
Angiogenesis is a complex process involving
endothelial sprouting, migration and proliferation
(Carmeliet 2000). An important signalling protein in
this process is vascular endothelial growth factor-A
(VEGF-A), which not only increases the endothelial
angiogenic function in culture but also induces angiogenesis and hyperpermeability in vivo (Senger et al.
1983, Dvorak et al. 1995). VEGF-A is also important
for neuronal development and required for neurogenesis (Rosenstein et al. 2010). Indeed, brain development and neurogenesis are tightly connected to
angiogenesis and other proteins coordinating both
processes are Eph receptors and ephrin proteins (Egea
& Klein 2007, Kuijper et al. 2007) or Id1-Id3 (Lyden
et al. 1999). Inhibition of PAFAH1B1 led to PAF
accumulation and enhanced adherence of tumour cells
to the endothelium (Kispert et al. 2014). By inhibition
of PAF receptor, tumorigenesis could be inhibited and
microvessel density decreased in a colitis-associated
cancer model (Sun et al. 2015).
Thus, it appears plausible that the protein, due to
its role in microtubule function, also influences key
angiogenic functions like endothelial sprouting or network formation. On this basis, we hypothesized that
PAFAH1B1 is important in angiogenesis and angiogenesis-associated vascular diseases. These aspects
were studied in cultured human umbilical vein
endothelial cells and patient samples.

Acta Physiol 2016

Material and methods

Material
The following antibodies were used: Anti-Lis1
(A300-409A, Bethyl, Montgomery, TX, USA), AntiGAPDH (PA1-16777, ThermoFisher), Anti-b-Actin
(4970, Cell Signaling, Davers, MA, USA), AntiH3K4me3 (C15410003, Diagenode, Seraing, Belgium), Anti-PolII (C15100055, Diagenode), Anti-MGP
(A-11) (sc-271906, Santa Cruz). Human recombinant
VEGF-A 165 was obtained from R&D (293-VE).

Cell culture
Pooled HUVEC were purchased from Lonza (#CC2519, Lot No.186864; 191772; 192485; 76524;
76921, 7F3111, Walkersville, MD, USA) and PELOBiotech (#PB-CH-190-8013, Lot No. QC-18P13F11,
Planegg, Germany). Immortalized human microvascular endothelial cells (HMEC-1) (#98247) were
obtained from the CDC (Atlanta, GA, USA), human
aortic endothelial cells (HAoEC) were from PeloBiotech (304K-05a), and human coronary artery endothelial cells (HCAEC) were obtained also from
PeloBiotech (PB-CH-182-2011). HUVEC, HMEC-1,
HAoEC and HCAEC were cultured in a humidified
atmosphere of 5% CO2 at 37 C on fibronectincoated (#356009, Corning Incorporated, Tewksbury,
MA, USA) dishes. Endothelial growth medium (EGM)
consisting of endothelial basal medium (EBM) supplemented with human recombinant epidermal growth
factor (EGF), EndoCGS-Heparin (PeloBiotech, Planegg, Germany, 8% fetal calf serum (FCS) (#S0113,
Biochrom, Berlin, Germany), penicillin (50 U mL
1)
and streptomycin (50 lg mL
1) (#15140-122, Gibco
Lifetechnologies (Carlsbad, CA, USA) was used to
expand cells. For each experiment, at least three different batches of HUVEC passage 3 were used.
Human embryonic kidney (HEK) 293 cells were purchased from ATCC (Manassas, VA, USA) and cultured
in a humidified atmosphere of 5% CO2 at 37 C in Dulbeccos modified Eagles medium (DMEM), high glucose, GlutaMAX from Gibco, Lifetechnologies
(Carlsbad, CA, USA), supplemented with 8% FCS, penicillin (50 U mL
1) and streptomycin (50 lg mL
1).
Human aortic smooth muscle cells (HAoSMC)
(#354-05a) were purchased from PeloBiotech (Planegg,
Germany) and cultured in a humidified atmosphere of
5% CO2 at 37 C in smooth muscle cell medium (#PBMH-200-2190) supplemented with 8% FCS, penicillin
(50 U mL
1), streptomycin (50 lg mL
1), EGF, FGF,
glutamine and insulin from singlequots (PeloBiotech,
Planegg, Germany).

2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12700

Acta Physiol 2016

Human foreskin fibroblasts were purchased from Gibco

(Lifetechnologies, Carlsbad, CA, USA) and cultured in
DMEM/F12 (#11039-021) supplemented with 10% FCS,
penicillin (50 U mL
1) and streptomycin (50 lg mL
1) in
a humidified atmosphere of 5% CO2 at 37 C.
The human monocytic cell line THP-1 was obtained
from ATCC (LGC Promochem, Wesel, Germany) and
cultured in RPMI medium containing stable glutamine, 8% FCS and 1% penicillin/streptomycin (PAA
Laboratories, C
olbe, Germany) in a humidified atmosphere of 5% CO2 at 37 C.

Human lung samples and patient characteristics

Human explanted lung tissues from subjects with chronic
thromboembolic pulmonary hypertension (CTEPH,
n = 4), pulmonary veno-occlusive disease (PVOD, n = 2)
or control donors (n = 4) were obtained during lung transplantation. Samples of donor lung tissue were taken from
lungs that had not been transplanted. All lungs were
reviewed for pathology. The ethics committee of the
University Hospital Giessen (Giessen, Germany) approved
the study protocols. The study protocol for tissue donation
was approved by the ethics committee (Ethik Kommission
am Fachbereich Humanmedizin der Justus Liebig Universitat Giessen) of the University Hospital Giessen (Giessen,
Germany) in accordance with national law and with Good
Clinical Practice/International Conference on Harmonisation guidelines. Written informed consent was obtained
from each individual patient or the patients next of kin
(AZ 31/93).

Illumina BeadChip Array

Three batches of HUVEC were transfected with control or PAFAH1B1-locked nucleic acids (LNA)-GapmeRs (Exiqon) for 48 h, and RNA was isolated
subsequently with the miRNeasy Mini Kit (Qiagen).
Next, an Illumina HumanHT12-v4 Expression BeadChip Array was performed (ServiceXS). Raw data
were processed to produce a data intensity table by
extracting all average microarray signals per gene
symbol and sample. Next, a log2 data transformation
was carried out to variance-stabilize the signals and
the robust spline normalization (Lin et al. 2008)
implemented in the lumi package (Du et al. 2008) to
normalize variance-stabilized data. For the detection
of differentially expressed genes, limma was used
(Ritchie et al. 2015) to fit a linear model for each gene
and a series of normalized array data. We next calculated moderated t-statistics, F-statistics and log-odds
of differential expression by empirical Bayes moderation of the standard errors towards a common value
(Ritchie et al. 2015) and limited the results to only
those genes with an adjusted P-value lower than 0.05.

I Josipovic et al.

PAFAH1B1 and endothelial cell function

RNA isolation, Reverse transcription and quantitative

real-time PCR (qRT-PCR)
Total RNA isolation was performed with the RNA Mini
Kit (Bio&Sell, Feucht, Germany). For reverse transcription, SuperScript III Reverse Transcriptase (Thermo
Fisher, Schwerte, Germany) and oligo(dT)23 together
with random hexamer primers (Sigma, St. Louis, MI,
USA) were used. CDNA amplification was measured
with qRT-PCR in a Mx3000P cycler (Stratagene, Agilent,
Santa Clara, CA, USA). Eva Green Master Mix and
ROX as reference dye (Bio&Sell) were used. Relative
expression of target genes was normalized to b-Actin and
analysed by the deltadelta Ct method with the MxPro
qPCR software (Stratagene).
List of primer used for qRT-PCR
Name
b-Actin

Forward Primer (50 -30 ) Reverse Primer (50 -30 )

AAA GAC CTG TAC

GCC AAC AC
PAFAH1B1 GGC CAT GAC CAC
AAT GTT TC
NONHSAT- AGA AAT AAA GTA
073641
GGC TGG TG
MGP
TCC GAG AAC GCT
CTA AGC CT
COX2
CCA GCA CTT CAC
GCA TCA GT
Endothelin- TGC CAC CTG GAC
1
ATC ATT TG
VCAM
TGT CAT CAT CTC
TTG TAC ATG
TGG
ALOX5
TGG ACA AGC CCT
TCT ACA AC

GTC ATA CTC CTG

CTT GCT GAT
CAG TAG CCA GTT
TGC ACT TC
CTG GCA GAA AAT
ACC CAT TC
GCA AAG TCT GTA
GTC ATC ACA GG
ACG CTG TCT AGC
CAG AGT TTC AC
TTC ACG GTC TGT
TGC CTT TG
GTT TTC TCT
TCC TTG AAC
ATC AAG
GTA CTT GCG CTT
CTC GAT TC

Protein isolation and western analyses

Cells were washed in Hanks solution (Applichem, Darmstadt, Germany). After lysis in Triton X-100 lysis buffer
[20 mM TRIS/Cl pH 7.5, 150 mM NaCl, 10 mM NaPPi,
20 mM NaF, 1% Triton, 2 mM orthovanadate (OV),
10 nM okadaic acid, protein-inhibitor mix (PIM),
40 lg mL
1phenylmethanesulfonyl fluoride (PMSF)],
cells were centrifuged for 10 min at 16 000 g and the
protein concentration of the cell extract was determined
by the Bradford assay. Afterwards, the extract was
boiled in Laemmli buffer. Proteins were separated by
SDS-PAGE, and gels were blotted onto a nitrocellulose
membrane. Membranes were blocked in Rotiblock solution prior to first antibody application. Infrared-fluorescent-dye-conjugated
secondary
antibodies
were
purchased from Licor (Bad Homburg, Germany), and
the signals were detected with an infrared-based laser

2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12700

PAFAH1B1 and endothelial cell function

I Josipovic et al.

scanning detection system (Odyssey Classic, Licor, Bad

Homburg, Germany).

Knockdown and overexpression procedures

For LNA-GapmeR (Exiqon, Vedbaek, Denmark) treatment, cells (70-80% confluent) were transfected with
Lipofectamin RNAiMAX (Invitrogen) transfection
reagent according to manufacturers protocol. All LNA
transfections were performed for 48 h. The LNA-GapmeRs were designed with the Exiqon LNA probe designer
and contained the following sequences: PAFAH1B1 #1:
50 -TTTGATTCTAATTCC-30 , PAFAH1B1 #2: 50 -TGG
AAAATGACTCGAG-30 ,
MGP:
50 -GCGCTTCCT
0
0
GAAGTAG-3 , CTL A: 5 -AACACGTCTATACGC-30 .
For expression studies, the NEON electroporation system (Invitrogen) was used with the following plasmids:
pcDNA3.1+-PAFAH1B1, pcDNA3.1+-PAFAH1B1_R22and pcDNA3.1+-NONHSAT073641. As a negative control, empty pcDNA3.1+ or pEGFP-C1 was used.

Acta Physiol 2016

(BB)-2A-Puro (PX459) v2.0 was a gift from Feng

Zhang (Addgene plasmid # 62988) and used as cloning backbone. The CRISPR/Cas9 procedure in
HEK293 was carried out after (Ran et al. 2013). The
following oligonucleotides were used for annealing:
For pSpCas9(BB)-2A-Puro-Exon4-PAFAH1B1 50 -CAC
CGGCATATTTTTCTGGCGGACG-30 and 50 -AAAC
CGTCCGCCAGAAAAATATGCC-30 and for pSpCas
9(BB)-2A-Intron4-PAFAH1B1 50 -CACCGCATCAGA
ACCATGCACGGTA-30 and 50 -AAACTACCGTGCA
TGGTTCTGATGC-30 . Plasmids were purified and
sequenced. After cloning of the gRNAs into
PX459 V2.0, two PX459 V2.0 plasmids, one targeting
the PAFAH1B1 gene inside Exon4, one inside the
Intron between Exon4 and Exon5, were used for
transfection with the NEON electroporation system
(Invitrogen). Afterwards, positive cells were selected
using puromycin (1 lg mL
1) for five days. The
empty pSpCas9(BB)-2A-Puro was used as negative
control. After 5 days, without clonal expansion, RNA
was isolated and qRT-PCR was performed.

Matrigel assay (Tube formation)

Matrigel assay was performed similar to the study of
Fork et al. (2015). Briefly, 8x103 HUVECs in EBM +
1% FCS were seeded onto polymerized growth factor
reduced Matrigel (BD) for 4 h and subsequently fixed
with paraformaldehyde (4%). Images were taken on
an Axiovert135 microscope (Zeiss, Oberkochen, Germany) and number of branching points as well as
length quantification was carried out with the AxioVision software (Zeiss).

Spheroid sprouting assay

Spheroid sprouting assays in HUVEC were generated
as described in (Korff & Augustin 1998), and images
were generated with an Axiovert135 microscope
(Zeiss). The cumulative sprout number was quantified
by analysis of ten spheroids per condition with the
AxioVision software (Zeiss).

Scratch-wound assay
Scratch-wound assays were performed similar to the
study of Gu et al. (2016). Briefly, 2 9 105 HUVEC
were cultured in endothelial basal medium (EBM)
containing FCS (2%) in 24-well plates. Migration was
monitored by live cell imaging with a Zeiss TIRF System LASOS77, and the distance of migration was calculated using ImageJ software.

CRISPR/Cas9
Guide RNAs were developed with the web interface of
CRISPR design (http://crispr.mit.edu/). The pSpCas9
4

LC-MS/MS analysis
The LC-MS/MS system consisted of a 5500 QTrap mass
spectrometer (Sciex, Darmstadt, Germany), equipped
with a Turbo-V-source operating in negative electrospray
mode, an Agilent 1200 binary HPLC pump and degasser
(Agilent, Waldbronn, Germany) and an HTC Pal
autosampler (Chromtech, Idstein, Germany) fitted with a
25-lL LEAP syringe (Hamilton, Bonaduz, Switzerland).
High-purity nitrogen for the mass spectrometer was produced by a NGM 22-LCMS nitrogen generator (cmc
Instruments, Eschborn, Germany). Acetonitrile and water
for chromatography were of LC-MS grade and were
obtained from Carl Roth (Karlsruhe, Germany).
Data Acquisition was done using Analyst Software
v1.6, and quantification was performed with MultiQuant
Software V3.0 (Sciex, Darmstadt, Germany) employing
the internal standard method (isotope dilution mass spectrometry). Ratios of analyte peak area and internal standard area (y-axis) were plotted against concentration (xaxis), and calibration curves were calculated by least
square regression with 1/x2 weighting for the arachidonic
acid metabolites and by linear regression with 1/x weighting for PAF.

Determination of 5(S)-HETE, 12(S)-HETE and 15(S)HETE

The arachidonic acid metabolites 5(S)-HETE (5Shydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid), 12(S)HETE (12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic
acid) and 15(S)-HETE (15S-hydroxy-5Z,8Z,11Z,13Eeicosatetraenoic acid) were purchased from Cayman

2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12700

Acta Physiol 2016

I Josipovic et al.

PAFAH1B1 and endothelial cell function

Chemical (Ann Arbor, MI, USA). Stock solutions with a

concentration of 10,000 ng mL
1of all analytes were
prepared in methanol. Working solutions were obtained
by mixing the three stock solutions and further dilution
with methanol to a concentration range of 0.25
2,500 ng mL
1. 5(S)-HETE-d8 (5S- hydroxy-6E,8Z,
11Z,14Z-eicosatetraenoic-5,6,8,9,11,12,14,15-d8 acid),
12(S)-HETE-d8 (12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic-5,6,8,9,11,12,14,15- d8-acid), 15(S)-HETE-d8,
(15S-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic-5,6,8,9,
11,12,14,15-d8 acid) were used as internal standards.
Sample extraction was performed using liquidliquid extraction. Cell pellets or supernatants were
gently mixed with 200 lL PBS, 20 lL of the internal
standard mix (5(S)-HETE-d8, 12(S)-HETE-d8 and 15
(S)-HETE-d8, 25 ng mL
1each) and 600 lL ethyl
acetate. This mixture was allowed to grind with a
swing mill at 30 Hz for 3 min (Retsch, Haan, Germany) using five zirconium oxide grinding balls (
3 mm). This procedure was performed twice. The
organic phase was removed at a temperature of
45 C under a gentle stream of nitrogen. The residues
were reconstituted with 50 lL of methanol/water/
0.1% BHT (50 : 50 : 0.1, v/v/v), centrifuged for
2 min at 10 000 g and transferred to glass vials prior
to injection into the LC-MS/MS system. For the chromatographic separation, Gemini NX C18 column and
precolumn were used (150 mm 9 2 mm I.D., 5 lm
particle size and 110 
A pore size from Phenomenex,
Aschaffenburg, Germany). A linear gradient was
employed at a flow rate of 0.5 ml min
1 mobile
phase with a total run time of 17 min. Mobile phase
A consisted of water/formic acid (100:0.1, v/v), and
B, of acetonitrile/formic acid (100:0.1, v/v). The gradient started with 90% A and changed from 90% to
10% A within 10 min and this was held for 11 min.
Within 2 min, the mobile phase shifted back to 90%
A and was held for 5 min to equilibrate the column
for the next sample. The injection volume was 20 lL.
Retention time of 5(S)-HETE, 12(S)-HETE and 15(S)HETE was 10.29 min, 10.20 min and 10.02 min
respectively.

For sample preparation, cell pellets were suspended in 50 lL PBS. These suspensions or 50 lL of
the cell culture supernatant samples were mixed with
40 lL internal standard, containing Lyso-PAF C16d4 (1000 ng mL
1), and 250 lL cold methanol for
protein precipitation. After vortexing and centrifugation at 20,000 g for 5 min, the supernatant was
transferred to a new tube and evaporated at 45 C
under a slight stream of nitrogen. The residues were
reconstituted in 50 lL methanol, centrifuged for
1 min at 20,000 g and transferred to glass vials prior
to injection into the LC-MS/MS system. For chromatographic separation, a Mercury Luna C18 column with the appropriate guard column was used
(20 9 2.1 mm, 5 l, Phenomenex, Aschaffenburg,
Germany). Mobile phase A was composed of water
with 0.2% formic acid and 50 mM ammonium formate, while mobile phase B was acetonitrile with
0.2% formic acid. The gradient started with 40% B
for 0.5 min was linearly increased to 100% B within
2 min and held for 1.5 min. Fraction B was
decreased to 40% again within 1 min, and the column was re-equilibrated for 2.5 min. Overall run
time was 7.5 min, and the injection volume was
10 lL. Measurements were taken and quantifications
were performed according to the arachidonic acid
metabolites.

Determination of C16 Lyso-PAF

PAFAH1B1 is important for VEGF-A-induced sprouting

and the formation of capillary-like structures

C16 Lyso-PAF (1-O-hexadecyl-2-hydroxy-sn-glycero3-phosphocholine) was obtained from Avanti Polar

Lipids (Alabaster, AL, USA). Stock solutions with a
concentration of 10,000 ng mL
1were prepared in
methanol, mixed and further diluted with methanol to
working solutions in the concentration range of 1
600 ng mL
1. The internal standards C16 Lyso-PAFd4
(1-O-hexadecyl-(7,7,8,8-d4)-sn-glyceryl-3-phosphorylcholine) were purchased from Cayman Chemical
(Ann Arbor, MI, USA).

Chromatin Immunoprecipitation
Preparation of cell extracts, crosslinking and isolation
of nuclei were performed with the truCHIPTM Chromatin Shearing Kit (Covaris, Woburn, USA) according
to the manufacturers protocol and as performed in
the study of Wong et al. 2014). The following primer
sequences were used: GAPDH FP: 50 - TGG TGT CAG
GTT ATG CTG GGC CAG-30 , GAPDH RP: 50 -GTG
GGA TGG GAG GGT GCT GAA CAC-30 , MGP FP:
50 - ACA GCC TTC CAC TAA CAT CC-30 and MGP
RP: 50 -TCA GGC TCT TCA TGG TTT CG-30 .

Results

Expression of PAFAH1B1 on protein and mRNA level

was readily detectable in HUVEC. The LNA-GapmeR
technique was subsequently used to determine the
function of the protein in endothelial cells. Downregulation of both mRNA and protein was successful with
two different LNA-GapmeRs (Fig. 1a,b). Despite a
small effect on diameter size of spheroids for LNA #2
(Fig. 1c,d), the knockdown of PAFAH1B1 did not
change the number of sprouts in control and VEGF-

2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12700

PAFAH1B1 and endothelial cell function

(a)

(b)

Acta Physiol 2016

(c)

Rel. RNA expression

1.5

CTL

#1

#2

1.0

CTL

LNA

#1

#2

PAFAH1B1

0.5

-Actin

0.0
#1

#2

C VEGF-A C VEGF-A C VEGF-A

CTL
#1
#2

(g)

Sprout number

5
0
C VEGF-A C VEGF-A C VEGF-A
CTL
#1
#2

LNA

*
*

0.0
LNA CTL

#1

#2

Rel. tube length

0.5

(i)
1.5

1.0

1.0

Lyso-PAF
15

0.5
0.0
LNA CTL

10

(h)
1.5

15

#1

ng/supernatant

LNA

Rel. number of
branching points

(f)
20

Cum. sprout length

[m]

(e)

120
100
80
60
40
20
0

#2

10
5

3000

LNA CTL PAFAH

1B1

1000
0

C VEGF-A C VEGF-A C VEGF-A

CTL
#1
#2

Lyso-PAF

(k)

1.0
0.5

LNA CTL PAFAH

1B1

15S HETE
0.15

1.5

0.0

*
2000

LNA

(j)
ng/total cells

(d)

ng/total cells

LNA CTL

Diameter of
spheroids [m]

I Josipovic et al.

0.10
0.05

0.00
LNA CTL PAFAH
1B1

Figure 1 Knockdown of PAFAH1B1 reduces angiogenesis in the spheroid-outgrowth and tube formation assays. (a, b) Efficiency
of LNA-GapmeR based knockdown (#1 and #2) of PAFAH1B1 in HUVEC was analysed by RT-qPCR and western analysis. CTL
was set to 1. Paired t-test was used (n = 3), *P 0.05. For loading control, b-Actin was used. (c) LNA-GapmeR-based knockdown
of PAFAH1B1 (#1 and #2) with subsequent spheroid-outgrowth assay. Representative images after VEGF-A165 treatment are
shown. (df) The diameter of spheroids, sprout number and cumulative sprout length of the spheroid-outgrowth assay were quantified by AxioVision SE64. One-way ANOVA followed by Bonferroni correction was used (n = 4), *P 0.05. (g, h) Matrigel assay.
The relative number of branching points and relative tube length were quantified after knockdown of PAFAH1B1 (#1 and #2) in
HUVEC by AxioVision SE64. Paired t-test was used for g (n = 4), and for h, unpaired t-test was used. *P 0.05. (i, j) Lyso-PAF
levels were measured with Quadrupol LC-MS/MS in supernatants as well as in cells after LNA-GapmeR-based knockdown of
PAFAH1B1 in HUVEC. Unpaired t-test, n = 3. *P 0.05. (k) 15S-HETE levels were measured in HUVEC after LNA-GapmeR
based knockdown of PAFAH1B1 with Quadrupol LC-MS/MS. Unpaired t-test was used (n = 3), *P 0.05.

A-treated samples (Fig. 1e). In contrast, sprout length

in response to VEGF-A was significantly reduced
(Fig. 1f). Tube formation assays were carried out to
determine the impact of PAFAH1B1 on the formation
of deficient cells to form capillary-like structures.
PAFAH1B1-deficient cells had a significantly reduced
number of branching points as well as a reduced tube
length (Fig. 1g,h).
To demonstrate that LNA-GapmeR knockdown of
PAFAH1B1 was effective in the alteration in endothelial cell lipid production, LC-MS/MS measurements of
Lyso-PAF were taken. Levels of Lyso-PAF were indeed
reduced in the supernatant of endothelial cells,
whereas the intracellular levels were not changed by
knockdown of PAFAH1B1 (Fig. 1i,j). Although 5HETE has been shown to modulate the biosynthesis
of PAF (Billah et al. 1985), its impact on PAF in
endothelial cells is not known. 5S-HETE and 12SHETE levels, whose relation to PAF in endothelial
6

cells is also unknown, were not affected by

PAFAH1B1 knockdown (Fig. S1a,b).
In human tracheal epithelial cells, an increase in the
pro-angiogenic diglyceride 15(S)-hydroxy-5Z,8Z,11Z,
13E-eicosatetraenoic acid (15S-HETE) was observed
after PAF stimulation (Alpert et al. (1999), Srivastava
et al. (2007), Nie et al. (2000), Zhang et al. (2005)).
Accordingly, 15S-HETE levels were reduced after
PAFAH1B1 knockdown in HUVEC (Fig. 1k).
To further support these observations, gain of function studies by plasmid-based overexpression of
PAFAH1B1 were performed. The plasmid coding for
PAFAH1B1, but not the empty vector, increased
mRNA expression by more than a hundred-fold
(Fig. 2a), and protein abundance of PAFAH1B1 was
also greatly increased (Fig. 2b). Remarkably, the
spheroid-outgrowth assay after overexpression mirrored the effect of PAFAH1B1 knockdown (Fig. 2c).
Whereas the spheroid diameter was not changed

2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12700

Acta Physiol 2016

I Josipovic et al.

(b)
*

PAFAH1B1

Diameter of
spheroids [m]

CTL

PAFAH
1B1 OE
CTL
PAFAH1B1

2
1

GAPDH

0
OE CTL PAFAH
1B1

C VEGF-A C VEGF-A

(f)
15

10

(g)
2000
1500

CTL

PAFAH1B1

1000

*
*

500
0

C VEGF-A C VEGF-A
OE

120
100
80
60
40
20
0
OE

C VEGF-A C VEGF-A
OE

CTL

PAFAH1B1

Rel. number of
branching points

(e)
Sprout number

(d)

(c)

PAFAH1B1

CTL

PAFAH1B1

(h)
2.5
2.0

1.5
1.0
0.5
0.0
OE CTL PAFAH
1B1

Rel. tube length

200
150
100

Cum. sprout length

[m]

Rel. RNA expression

(a)

PAFAH1B1 and endothelial cell function

1.5
1.0
0.5
0.0
OE CTL PAFAH
1B1

Figure 2 PAFAH1B1 overexpression increases angiogenesis in the spheroid-outgrowth and tube formation assays. (a, b) Efficiency
of overexpression of PAFAH1B1 was analysed by RT-qPCR and western analysis. CTL was set to 1. Paired t-test was used (n = 3),
*P 0.05. (c) Overexpression of PAFAH1B1 with subsequent spheroid-outgrowth assay. Representative images after VEGF-A165
treatment are shown. (df) The diameter of spheroids, sprout number and cumulative sprout length of the spheroid-outgrowth
assay were quantified by AxioVision SE64. One-way ANOVA followed by Bonferroni correction was used (n = 3), *P 0.05. (g, h)
Matrigel assay. The relative number of branching points and the relative tube length after overexpression of PAFAH1B1 were
quantified by AxioVision SE64. For g, paired t-test was used (n = 9), and for h, unpaired t-test was used, *P 0.05.

(Fig. 2d), increases in sprout number and length with

and without VEGF-A stimulation (Fig. 2e,f) were
observed after overexpression. Additionally, in tube
formation assays the ability to form capillary-like
structures was increased, demonstrated by an
increased number of branching points, but not by an
increase in tube length (Fig. 2g,h). This further supports not only the idea that PAFAH1B1 is required
for angiogenic function, but also that the protein
might also be of utility to increase angiogenesis.

PAFAH1B1 supports a quiescence-associated endothelial

gene expression pattern
Given that the HETE lipids are not designated substrates of the PAFAH1B complex, the changes in lipid
mediators might be a consequence of altered expression of lipid metabolizing enzymes in response to a
reduced PAFAH1B expression. The reactions of
arachidonic acid to 5S-HETE, 12S-HETE and 15SHETE are mediated by the lipid-peroxidizing enzymes
ALOX5, ALOX12, ALOX15 and ALOX15B; ALOX5
requires additionally 5-LOX activating protein
(ALOX5AP) (Maayah & El-Kadi 2016). To address
this aspect, an Illumina BeadChIP array was performed with and without knockdown of PAFAH1B1.
None of these lipid-peroxidizing enzymes were altered
significantly (Table S1). Knockdown of PAFAH1B1,
however, led to an increase in several inflammatory

RNAs, such as CCL8, TNFSF13B, TNFAIP6 and

IDO1, and to a decrease in a number of pro-angiogenic RNAs like PECAM-1, Matrix Gla protein
(MGP), MGC61598 (NRARP), EFNA1, ID3 and
ANGPT2 (Table 1).
As PAFAH1B1 misregulation led to altered endothelial cell angiogenic capacity, we focused on the most
promising candidate, MGP, as a mediator of
PAFAH1B1s actions. To validate the gene array findings, qRT-PCR experiments were performed. After
knockdown of PAFAH1B1, the expression level of MGP
was confirmed to be reduced on RNA and protein level
(Fig. 3a,b) and overexpression of PAFAH1B1 increased
MGP mRNA levels (Fig. 3c).

PAFAH1B1 and MGP mRNA expression is increased in

chronic thromboembolic pulmonary hypertension
(CTEPH)
To determine whether the interactions observed in
HUVEC are relevant for the human disease situation,
we studied samples from patients with the vascular
disorders CTEPH and PVOD. Expression levels of
both, PAFAH1B1 and MGP, were significantly
increased in CTEPH (Fig. 3d). In PVOD, MGP
expression levels in the two patients studied was not
changed, but PAFAH1B1 levels were more than double as high as in the healthy subjects (Fig. 3e). These
human data raise the possibility that the interaction of

2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12700

PAFAH1B1 and endothelial cell function

I Josipovic et al.

Table 1 Illumina Beadchip array after LNA-GapmeR based

knockdown of PAFAH1B1 (50 nM LNA, 48 h) in HUVEC
Gene name

Log FC
LNA vs. CTL

P
Value

Acc. No.

PAFAH1B1
MMRN1
PECAM1
FAM124B
MGP
MGC61598
EFNA1
ID3
ANGPT2
LXN
FAM108C1
HNRPA1P4
CLDN5
C13orf15
LAMA4
FAM174B
PPM1F
EVL
PRCP
C1orf54
ESAM
WDR54
PLOD1
CTSB
VASH1
HNRPA1L2
LOC646723
CABLES1
CHST15
HYAL2
HIST1H1C
GBP4
SAMD9L
RGS2
LITAF
FST
HERC6
CSF3
IFIH1
GCA
INDO
MIR155HG
SAMD9
CFB
LOC100128274
HIST1H2AC
LINCR
HIST2H2BE
IDO1
GBP5
GBP1
CEBPD

1.33

1.24

0.90

0.88

0.88

0.87

0.84

0.82

0.82

0.81

0.80

0.80

0.77

0.77

0.76

0.71

0.70

0.70

0.69

0.69

0.69

0.69

0.67

0.65

0.65

0.64

0.64

0.64

0.63

0.63
0.86
0.87
0.87
0.89
0.91
0.93
0.94
0.96
0.97
1.00
1.04
1.06
1.07
1.08
1.08
1.08
1.10
1.11
1.13
1.17
1.20
1.28

0.02
0.02
0.03
0.04
0.04
0.04
0.03
0.03
0.03
0.05
0.02
0.01
0.03
0.05
0.03
0.05
0.04
0.02
0.05
0.04
0.00
0.00
0.02
0.05
0.02
0.03
0.00
0.05
0.03
0.03
0.05
0.03
0.03
0.05
0.03
0.01
0.01
0.02
0.04
0.04
0.02
0.02
0.05
0.05
0.05
0.05
0.01
0.04
0.03
0.01
0.01
0.04

NM_000430.2
NM_007351.2
NM_000442.2
NM_024785.2
NM_000900.2
XM_939432.1
NM_004428.2
NM_002167.2
NM_001118888.1
NM_020169.2
XM_051862.7
XM_372050.4
NM_003277.2
NM_014059.1
NM_002290.2
NM_207446.1
NM_014634.2
NM_016337.2
NM_005040.2
NM_024579.1
NM_138961.1
NM_032118.2
NM_000302.2
NM_001908.3
NM_014909.2
NR_002944.2
NM_199187.1
NM_138375.1
NM_015892.2
NM_033158.2
NM_005319.3
NM_052941.2
NM_152703.2
NM_002923.1
NM_004862.2
NM_006350.2
NM_001013005.1
NM_000759.2
NM_022168.2
NM_012198.2
NM_002164.3
NR_001458.3
NM_017654.2
NM_001710.4
XM_001725558.1
NM_003512.3
NM_001080535.1
NM_003528.2
NM_002164.4
NM_052942.2
NM_002053.1
NM_005195.2

Table 1 (continued)

Gene name

(continued)

Acta Physiol 2016

IFIT1
TNFAIP6
OASL
HIST2H2AA3
C15orf48
RSAD2
TNFSF13B
CCL8

Log FC
LNA vs. CTL
1.30
1.33
1.33
1.42
1.72
1.74
1.80
1.97

P
Value

Acc. No.

0.02
0.02
0.04
0.05
0.05
0.02
0.04
0.02

NM_001548.2
NM_007115.2
NM_003733.2
NM_003516.2
NM_032413.2
NM_080657.3
NM_006573.3
NM_005623.2

PAFAH1B1 and MGP also occurs in humans and that

it might be relevant for disorders like CTEPH.

PAFAH1B1 maintains Pol II binding and histone

methylation state on its target genes
To directly prove that PAFAH1B1 is necessary for
MGP gene activation, chromatin immunoprecipitation
experiments for the active histone mark histone 3
lysine 4 trimethylation (H3K4me3) and for RNA
Polymerase II (Pol II) were performed. Knockdown of
PAFAH1B1 did not affect the decoration of the promoter of the housekeeping gene GAPDH (Fig. 3f,g).
In contrast, loss of PAFAH1B1 was associated with a
reduction in the active histone mark H3K4me3 on the
transcriptional start site of MGP as well as with
reduced RNA Pol II binding (Fig. 3h,i). This suggests
that PAFAH1B1 increases MGP promoter activity by
maintaining H3K4me3 levels and by facilitating
proper RNA Pol II binding.
To further link MGP expression to PAFAH1B1, we
performed knockdown and overexpression of
PAFAH1B1 and analysed MGP expression with qRTPCR. Indeed, overexpression of PAFAH1B1 after LNAs
against the gene restored MGP expression (Fig. 3j,k).

PAFAH1B1 maintains MGP expression level in HUVEC,

HMEC-1, HCAEC and HAoEC
To address whether the interaction of PAFAH1B1 and
MGP is specific to human venous endothelial cells,
additional cell types were screened. The expression of
PAFAH1B1 was similar in HUVEC, HAoEC,
HCAEC, HMEC-1, HAoSMC, fibroblasts, HEK293
and THP-1 monocytes (Fig. 4a). MGP showed the
highest expression in HUVEC compared with all other
tested cell types (Fig. 4b). Also b-Actin revealed to be
differentially expressed (Fig. S1c). Not only in
HUVEC but also in all other cell types studied
(HMEC-1, HCAEC, HAoEC, Fig. 4ce), knockdown
of PAFAH1B1 led to decreased MGP mRNA levels,
whereas in the other cell types with the exception of

2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12700

I Josipovic et al.

(c)

CTL

CTL
LNA

#1

#2

LNA
MGP

Actin

(d)
80
60
40
2.0
1.5
1.0
0.5
0.0

PAF MGP
AH1B1

4
2
0

GAPDH TSS
0.8
0.6
0.4
0.2
0.0

LNA

H3K4me3

30
10
0

(k)

*
*

0.5

+
+

(e)

CTEPH
3
2

*
*

1
0

Donor
CTL

PAF MGP
AH1B1

PVOD

2
Donor
CTL

1
0
PAF MGP
AH1B1

MGP TSS
0.15
0.10

0.05
0.00

LNA

Pol II

H3K4me3

1.5

20

LNA

MGP

2.0

0.0
LNA PAFAH1B1
OE PAFAH1B1

40

Pol II

1.0

CTL
LNA

(i)

MGP TSS
50

Rel. RNA expression

Rel. RNA expression

(j)

(h)
% Input recovery

% Input recovery

(g)

GAPDH TSS
8

LNA

PAF MGP
AH1B1

% Input recovery

% Input recovery

(f)

Rel. RNA expression

(b)
1.2
1.0
0.8
0.6
0.4
0.2
0.0

Rel. RNA expression

Rel. RNA expression

(a)

PAFAH1B1 and endothelial cell function

Rel. RNA expression

Acta Physiol 2016

PAFAH1B1

*
2

0
LNA PAFAH1B1
OE PAFAH1B1

+
+

Figure 3 PAFAH1B1 regulates MGP expression. (a) LNA-GapmeR-based knockdown of PAFAH1B1 in HUVEC with subsequent qRT-PCR for PAFAH1B1 and MGP. Unpaired t-test was used (n = 6), *P 0.05. (b) Western analyses with antibodies
against MGP and b-actin after LNA-GapmeR-based knockdown of PAFAH1B1 in HUVEC. (c) Overexpression of PAFAH1B1
in HUVEC and qRT-PCR for PAFAH1B1 and MGP. Unpaired t-test was used (n = 3), *P 0.05. (d) qRT-PCR analysis of
PAFAH1B1 and MGP in human CTEPH lungs. Paired t-test was used (n = 4), *P 0.05. (e) qRT-PCR analysis of PAFAH1B1
and MGP in human PVOD lungs (n = 2). (fi) Chromatin immunoprecipitation after CTL- or PAFAH1B1 LNA-GapmeR-based
knockdown in HUVEC and qPCR with primers for GAPDH (f, g) and MGP (h, i) transcriptional start sites. H3K4me3, RNA
Polymerase II and IgG antibodies were used. All samples were normalized to Input and IgG. Unpaired t-test was used (n = 3),
*P 0.05. (j, k) Overexpression after 24 h of LNA-GapmeR-based knockdown of PAFAH1B1 in HUVEC and qRT-PCR of
MGP (j) or PAFAH1B1 (k). CTL was set to 1. Paired t-test was used (n = 3), *P 0.05. Dashed line indicates Control.

fibroblasts, the PAFAH1B1 reduction was not accompanied by a reduction of MGP (Fig. 4fh).
To exclude that the lack of effect of PAFAH1B1 knockdown on MGP in non-endothelial cells was a consequence
of insufficient knockdown, we performed CRISPR/Cas9 of
PAFAH1B1 Exon4/Intron4 in HEK293 to knock out
PAFAH1B1. Seven days after the transfection with Cas9
and the guide RNAs, meaning 5 days of puromycin selection, the expression levels of PAFAH1B1 were already
reduced and this effect was indeed accompanied by reduced
MGP expression (Fig. 4i). This suggests that functional
PAFAH1B1 is also required to control MGP expression in
cells other than endothelial cells, such as HEK293 cells.

MGP is a pro-angiogenic factor in HUVEC

To further support our model that the pro-angiogenic properties of PAFAH1B1 are mediated partially

by MGP, we performed LNA-GapmeR-based knockdown of MGP (Fig. 5a). This approach not only
reduced MGP to nearly 50%, but also decreased the
diameter of the spheroids in control and VEGF-Atreated samples (Fig. 5b,c). Whereas the number of
sprouts was only decreased in the VEGF-A condition
after MGP knockdown (Fig. 5d), the sprout length
was decreased in the control and VEGF-A-treated
samples after MGP knockdown (Fig. 5e). In tube
formation experiments, the number of branching
points as well as the tube length was reduced after
MGP knockdown (Fig. 5f,g). This decreased the ability to form capillary-like structures as well as the
spheroid data suggests that MGP is a pro-angiogenic
molecule. To determine whether MGP is also relevant for angiogenic migration, we performed a
scratch-wound assay. The migration speed of
HUVEC was indeed drastically reduced by

2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12700

(e)

HCAEC
CTL
0.057 LNA

1.0
0.8
0.6
0.4
0.2

0.0

HAoEC
1.0
0.058

0.8
0.6
0.4
0.2

5
10
15
1

0.0

(f)
CTL
LNA

CTL
LNA

1.0
0.5
0.0
PAF MGP
AH1B1

(g)

Fibroblasts
1.5

PAF MGP
AH1B1

PAF MGP
AH1B1

(i)

Rel. RNA expression

Rel. RNA expression

Rel. RNA expression

(c)

MGP

Rel. RNA expression

Rel. RNA expression

[log2]

(d)

Rel. RNA expression

1-HUVEC
2-HAoEC
3-HCAEC
4-HMEC-1
5-HAoSMC
6-Fibroblast
7-HEK293
8-THP1

Acta Physiol 2016

(b)

PAFAH1B1

Rel. RNA expression

Rel. RNA expression

[log2]

(a)

I Josipovic et al.

0.5

0.0
PAF MGP
AH1B1

0.8
0.6

CTL
LNA

CTL
LNA

0.055

0.4
0.2
0.0
PAF MGP
AH1B1

(h)

HAoSMC
1.5
1.0

HMEC-1
1.0

Rel. RNA expression

PAFAH1B1 and endothelial cell function

THP-1
1.5
CTL
LNA

1.0
0.5

0.0
PAF MGP
AH1B1

HEK293
1.0

0.8
0.6
0.4

CTL
CRISPR

0.2
0.0
PAF MGP
AH1B1

Figure 4 PAFAH1B1 affects MGP expression in different cell types and is upregulated in CTEPH. (a, b) Expression analysis of
PAFAH1B1 (a) and MGP (b) with qRT-PCR in HUVEC (1), HAoEC (2), HCAEC (3), HMEC-1 (4), HAoSMC (5), Fibroblasts
(6), HEK293 (7) and THP-1 cells (8). HUVEC was set to 1. n = 6. (ch) LNA-GapmeR-based knockdown of PAFAH1B1 in
HMEC-1 (c), HCAEC (d), HAoEC (e), fibroblasts (f), HAoSMC (g) and THP-1 cells (h) and analysis of PAFAH1B1 and MGP
expression with qRT-PCR. Unpaired t-test, n = 4. *P 0.05. (i) CRISPR/Cas9 of Exon4/Intron4 of PAFAH1B1 in HEK293
cells. Five days after puromycin treatment, RNA of the mixed population (CRISPR positive and negative) was harvested and
PAFAH1B1 and MGP expression levels were analysed with qRT-PCR. As a negative control (CTL CRISPR), empty vector was
used. Unpaired t-test, n = 4. *P 0.05. Dashed line indicates negative control.

knockdown of MGP. Knockdown of PAFAH1B1 had

similar, yet less pronounced effects (Fig. 5h,i). This
favours a model where a PAFAH1B1-dependent
decrease in tube length and branching points is mediated by MGP.

LncRNA NONHSAT073641 influences angiogenesis,

but not MGP expression
Given the influence of PAFAH1B1 on MGP, we speculated that the regulation of PAFAH1B1-dependent
genes is more complex and not just a consequence of
a direct or indirect action of the protein. Analysis of
the PAFAH1B1 gene revealed the presence of two
lncRNAs or pseudogenes: NONHSAT072155 (also
called NONHSAT0736411, LIS2) and NONHSAT073641 (also called NONHSAT0736412,
LIS2P, 641). Both share high similarity with the
PAFAH1B1 by cDNA sequence comparison (Fig. S2).
The lncRNA NONHSAT073641 strongly resembles a
10

copy of the cDNA of PAFAH1B1, as it has 1241 nt

(PAFAH1B1 has 1233 nt), of which 92% match to
the PAFAH1B1 cDNA nucleotides. Comparison of
PAFAH1B1 cDNA with the multiple sequence alignment tool MUSCLE (Fig. S2) showed that NONHSAT073641 comprises several nucleotide differences,
among them a critical C22T difference leading to a
premature stop codon after seven amino acids and different deletions, insertions and frame shifts, making it
incapable of coding a protein similar to PAFAH1B1,
despite the fact that the nucleotide sequence is very
similar. qRT-PCR experiments revealed NONHSAT073641 expression levels similar to VCAM, but
about 100 times less than PAFAH1B1 (Fig. 6a).
Moreover, comparison with different cell types
revealed that the expression of this lncRNA was highest in HEK293 and HMEC-1, followed by HUVEC
(Fig. 6b). This may suggest that NONHSAT073641
affects PAFAH1B1 expression. Overexpression of
NONHSAT073641,
however,
did
not
alter

2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12700

I Josipovic et al.

(b)

(c)

CTL

1.5

MGP
Diameter of
spheroids [m]

Rel. RNA expression

(a)

1.0

0.5
0.0

(d)
120
100
80
60
40
20
0

C VEGF-A
CTL

LNA CTL MGP

LNA

Distance [m]

*
*

500

140
120
100
80
60
40
20
0

C VEGF-A
CTL

C VEGF-A
MGP

1.5
1.0

0.5
0.0
LNA CTL MGP

CTL
PAFAH1B1
MGP

C VEGF-A
MGP

12
10
8
6
4
2
0
LNA

*
*

C VEGF-A
CTL

C VEGF-A
MGP

(h)
CTL

1.5
Rel. tube length

1000

LNA

(i)

(g)

(f)
*

Rel. number of
branching points

Cum. sprout length

[m]

(e)
1500

PAFAH1B1 and endothelial cell function

Sprout number

Acta Physiol 2016

1.0

0.5
0.0
LNA CTL MGP

PAFAH
1B1

MGP

8h

Figure 5 MGP promotes angiogenesis in HUVEC. (a) Efficiency of LNA-GapmeR-based knockdown of MGP in HUVEC was
analysed by RT-qPCR. CTL was set to 1. Paired t-test was used (n = 4), *P 0.05. (b) LNA-GapmeR-based knockdown of
MGP with subsequent spheroid-outgrowth assay. Representative images after VEGF-A165 treatment are shown. (ce) The diameter of spheroids, sprout number and cumulative sprout length of the spheroid-outgrowth assay were quantified by AxioVision
SE64. One-way ANOVA followed by Bonferroni correction was used (n = 4), *P 0.05. (f, g) Matrigel assay. The relative number
of branching points and relative tube length were quantified after knockdown of MGP in HUVEC by AxioVision SE64. Paired
t-test was used for f (n = 12), whereas for g unpaired t-test was used. *P 0.05. (h, i) Scratch-wound assay and statistics of
HUVEC transfected with LNA-GapmeRs against CTL, PAFAH1B1 or MGP. Representative images were taken 8 h after the
scratch (h). For statistics, 2-way ANOVA was used, n = 4. *P 0.05.

PAFAH1B1 mRNA and protein levels, ruling out conventional actions of lncRNAs like miRNA sponging
or transcriptional enhancing (Fig. 6c,d). Similarly, the
PAFAH1B1 mutant R22-, which should resemble the
lncRNA NONHSAT073641 in the way it increases
non-coding PAFAH1B1 mRNA, did not affect
PAFAH1B1 protein abundance (Fig. S1d).
Unexpectedly, NONHSAT073641 nevertheless promoted endothelial angiogenic function. Similar to
PAFAH1B1, NONHSAT073641 overexpression did
not change the diameter of endothelial spheroids
(Fig. 6e,f), but increased the number of sprouts as well
as the sprout length in the control and VEGF-A-treated samples (Fig. 6g,h). Moreover, the ability of the
cell to form capillary-like structures was increased in
response to NONHSAT073641 overexpression as
demonstrated by increased number of branching
points and relative tube length (Fig. 6i,j).
To understand whether NONHSAT073641 alters
gene expression similar to PAFAH1B1, MGP

expression was studied. Indeed, overexpression of

NONHSAT073641 resulted in a trend (P = 0.0695)
towards increased expression of MGP (Fig. 6k).
Importantly, also in the human disease samples,
expression of NONHSAT073641 was greatly
increased (Fig. 6l).

Discussion
The present study demonstrates that PAFAH1B1 as
well as the lncRNA NONHSAT073641 promotes the
angiogenic function of human endothelial cells.
PAFAH1B1 appears to mediate this effect by promoting MGP expression, but also by enhancing Lyso-PAF
and 15S-HETE production. The lncRNA NONHSAT073641 affects angiogenesis to a very similar
extent, but by a less clear mechanism. In CTEPH, all
three genes, PAFAH1B1, MGP and NONHSAT073641, were found to be significantly upregulated, suggesting that the interaction observed in

2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12700

11

 I Josipovic et al.

PAFAH1B1 and endothelial cell function

(b)
NONHSAT073641

4
3
2
1
0
1
2
3
4
5

Endo COX PAFAH 641 VCAMALOX

5
thelin 2
1B1
1

(e)

2500

2000
1500
4
3
2
1
0
OE CTL

(f)

CTL

NONHSAT073641

NONHSAT073641

PAFAH1B1

GAPDH

641 PAFAH
1B1

(g)
10

100

Sprout number

NONHSAT
CTL 073641 OE

Diameter of
spheroids [m]

(d)

Rel. RNA expression

1
0
1
2
3
4
5

(c)

Rel. RNA expression

[log2]

Rel. RNA expression

[log10]

(a)

Acta Physiol 2016

80
60
40
20
0

8
6

4
2
0
C VEGF-A

CVEGF-A

400

*
*

200
0
C VEGF-A
OE

CTL

C VEGF-A

1.5
1.0
0.5
0.0
OE

CTL

641

OE

CTL

641

1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0

MGP

OE CTL

641

Rel. RNA expression

600

2.0

CTL641

C VEGF-A
641

(l)

(k)
1.2
1.0
0.8
0.6
0.4
0.2
0.0

Rel. RNA expression

800

2.5

Rel. tube length

(j)

(i)
1000

Rel. number of
branching points

Cum. sprout length

[m]

(h)

OE

641

OE

60
40

NONHSAT073641

20
0
CTEPH PVODDONOR

641

Figure 6 LncRNA NONHSAT073641 is upregulated in CTEPH and overexpression affects angiogenesis in the spheroid-outgrowth and tube formation assays. (a) Basal expression of endothelin-1, COX2, PAFAH1B1, NONHSAT073641 (641), VCAM
and ALXO5 in HUVEC measured by qRT-PCR. n = 6. (b) Expression analysis of NONHSAT073641 with qRT-PCR in
HUVEC (1), HAoEC (2), HCAEC (3), HMEC-1 (4), HAoSMC (5), Fibroblasts (6), HEK293 (7) and THP-1 cells (8). HUVEC
was set to 1. n = 6. (c) NONHSAT073641 expression was analysed by qRT-PCR after overexpression of NONHSAT073641 or
PAFAH1B1. CTL was set to 1. For statistics, paired t-test was used (n = 3), *P 0.05. (d) PAFAH1B1 protein expression after
overexpression of NONHSAT073641 was analysed with Western blot. For loading control, GAPDH antibody was used. (e)
Overexpression of NONHSAT073641 with subsequent spheroid-outgrowth assay. Representative images after VEGF-A165
treatment were shown. (fh) The diameter of spheroids, sprout number and cumulative sprout length of the spheroid-outgrowth
assay were quantified by AxioVision SE64. One-way ANOVA followed by Bonferroni correction was used (n = 3), *P 0.05. (i, j)
The relative number of branching points and the relative tube length after overexpression of NONHSAT073641 in HUVEC
were quantified by AxioVision SE64. For i, paired t-test was used (n = 9), whereas for j, unpaired t-test was used *P 0.05. (k)
qRT-PCR analysis of MGP after overexpression of NONHSAT073641. CTL was set to 1. Paired t-test was used (n = 5),
*P 0.05. (l) qRT-PCR analysis of NONHSAT073641 in human CTEPH and PVOD lungs. Paired t-test was used (n = 4),
*P 0.05. Dashed line indicates negative control.

cultured cells also occurs in human vascular disease.

CTEPH is a complex vascular disorder consisting of
major vessel remodelling and small vessel arteriopathy. It is characterized by medial hypertrophy, microthrombi formation and plexiform lesion (Lang et al.
2013). Moreover, circulating CTEPH microparticles
co-cultured with human pulmonary endothelial cells
increased TGF--induced angiogenesis, suggesting
their role in pro-angiogenic feedback of endothelial
injury (Belik et al. 2016). In spite of the hypothesis
that pulmonary capillaries cannot endure angiogenesis
(Schraufnagel 1990, Peao et al. 1994), scanning electron microscopy of microvascular casts from a patient
12

with PVOD, a rare form of pulmonary arterial hypertension, identified neovascular structures as a replacement of the damaged alveolar capillaries. This case
study reported that new, larger capillaries in fewer
amounts were not only overgrowing the smaller capillaries, but also connecting the alveoli and thus reducing the alveolar capillary interface. (Schraufnagel
et al. 1996).
Many genes have been identified to be relevant for
both, angiogenesis and neurogenesis (Senger et al.
1983, Dvorak et al. 1995, Lyden et al. 1999, Egea &
Klein 2007, Kuijper et al. 2007, Rosenstein et al.
2010). The present study suggests that also

2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12700

Acta Physiol 2016

PAFAH1B1 is such a protein and that MGP is possibly mediating this effect. MGP is an antagonist of
BMPs 2 and 4 and inhibits arterial calcification (Luo
et al. 1997, Speer et al. 2009). Mutations in the MGP
gene are the basis of the Keutel syndrome leading to
abnormal cartilage calcification, peripheral pulmonary
stenosis and midfacial hypoplasia (Munroe et al.
1999). The role of MGP in angiogenesis has been
studied extensively, but its precise role combining the
physiological and molecular data is still unclear. On
the one hand, MGP inhibits excessive branching of
pulmonary capillaries during vascular development in
mice (Yao et al. 2007). It is expressed during different
angiogenic steps (Glienke et al. 2000, Albig et al.
2007) and increased in tumour vasculature (Kuzontkoski et al. 2010). MGP increases tube formation,
migration, proliferation and the release of VEGF-A
and bFGF in bovine aortic endothelial cells (Bostrom
et al. 2004). On the other hand, MGP was described
to suppress endothelial sprouting in mouse aortic
rings, tumour growth and vascular density (Sharma &
Albig 2013).
Our data suggest that MGP is pro-angiogenic and
that PAFAH1B1 keeps the MGPs promoter histone
states active to maintain MGP transcription. The fact
that MGP acts downstream of PAFAH1B1 was indeed
observed in several endothelial cell types as well as in
HEK293. Whether the increase in pro-inflammatory
gene expression or PAF formation is important for the
pro-angiogenic roles of PAFAH1B1 or MGP remains
unclear. As reduced lyso-PAF amounts in the supernatants of endothelial cells after PAFAH1B1 knockdown were detected, the question of the molecular
function of PAFAH1B1 in endothelial cells remains.
Therefore, the participation of PAFAH1B1 in the
release of lyso-PAF as well as its enzymatic regulation
should be addressed in future studies.
The human genome contains a nearly identical copy
of the PAFAH1B1 cDNA, which represents a long
non-coding RNA or pseudogene called NONHSAT073641. Several studies demonstrate that pseudogenes can act as modulators of their protein-coding
counterparts by competing for miRNA binding. Such
competitive endogenous RNAs (ceRNAs) are therefore
RNAs that share one or more miRNA response elements (MREs) and compete with the miRNA target
sequences for miRNA binding (Poliseno et al. 2010,
Salmena et al. 2011). For example, the ceRNA
PTENP1, whose counterpart is the haploinsufficient
tumour suppressor PTEN, affects PTEN expression
levels and downstream PI3K signalling (Poliseno et al.
2010, Chen et al. 2015).
The present study does not fully answer the question of whether or not NONHSAT073641 acts as a
ceRNA for PAFAH1B1. Another possible mode of

I Josipovic et al.

PAFAH1B1 and endothelial cell function

action is that pseudogenes regulate gene expression of

their counterparts by acting as small interfering RNAs
(siRNAs) (Tam et al. 2008). Our results, however,
demonstrate
that
overexpression
of
NONHSAT073641 did not affect expression levels of
PAFAH1B1, excluding this alternative. This negative
finding is also supported by our observation that overexpression of the PAFAH1B1 mutant R22- also did
not affect expression levels of PAFAH1B1.
It has previously been reported that pseudogenes can
affect angiogenesis. For example, overexpression of the
pseudogene CYP4Z2P-30 UTR promoted network formation (Zheng et al. 2015). In the present study, we
demonstrate that the lncRNA NONHSAT073641 similarly promotes angiogenesis as PAFAH1B1 did. It
appears possible that this is also mediated by an induction of MGP. Importantly, the lncRNA was highly
upregulated in human vascular disease samples, in
which increased angiogenesis occurs. Future challenges
will be to understand the molecular relationship
between PAFAH1B1 and NONHSAT073641 as well as
the intracellular identification of the mediator between
MGP and PAFAH1B1.
In conclusion, with the present work a novel function
of PAFAH1B1, maintenance of endothelial angiogenic
capacity, was identified. PAFAH1B1 promotes the
expression of angiogenesis-associated proteins and
maintains endothelial angiogenic function.

Conflict of interest
The authors of this manuscript have no conflict of
interest in the present study.
We are grateful for excellent technical assistance of Cindy F.
H
oper, Tanja L
uneburg and Katalin Palfi. This work was
supported by the German Research Foundation (DFG SFB
1039 TP A1, TPA2 and Z1), the excellence cluster Cardiopulmonary System Ex147 ECCPS and the GoetheUniversity.

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Supporting Information
Additional Supporting Information may be found
online in the supporting information tab for this
article:
Table S1. Illumina Beadchip array after LNA-GapmeR based knockdown of PAFAH1B1 (50 nM LNA,
48 h) in HUVEC showing expression values of genes
involved in HETE-metabolism.
Figure S1. (A, B) 5S- and 12S-HETE levels were
measured in HUVEC after LNA-GapmeR based
knockdown of PAFAH1B1 with Quadrupol LC-MS/
MS. Unpaired t-test was used (n = 3), *P 0.05. (C)
Expression analysis of b-Actin with qRT-PCR in
HUVEC (1), HAoEC (2), HCAEC (3), HMEC-1 (4),
HAoSMC (5), Fibroblasts (6), HEK293 (7) and THP1 cells (8). HUVEC was set to 1. n = 6. (D) Whole
Western blot as shown in figure 6d (Dashed rectangle). PAFAH1B1 protein expression after overexpression of empty vector pcDNA3.1 + (CTL empty
vector), pEGFP-C1 (CTL GFP), pcDNA3.1 + -NONHSAT073641, pcDNA3.1 + -PAFAH1B1 R22- as well
as pcDNA3.1 + PAFAH1B1 was analysed with Western blot. For loading control, GAPDH antibody was
used. As negative control, untransfected, CTL empty
vector and CTL GFP were used. As positive control,
pcDNA3.1 + -PAFAH1B1 was used.
Figure S2. Clustal multiple sequence alignment by
MUSCLE (3.8) of PAFAH1B1 cDNA sequence,
NONHSAT073641 and NONHSAT072155 nucleotide sequences. Nucleotides marked in yellow represent
differences
between
PAFAH1B1
and
NONHSAT073641. The asterisks mark overlapping
nucleotides.

2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12700

15