2-Chlorofatty Aldehyde Elicits Endothelial Cell Activation

Article abstract of DOI:10.3389/fphys.2020.00460

Endothelial activation and dysfunction are hallmarks of inflammation. Neutrophil-vascular endothelium interactions have significant effects on vascular wall physiology and pathology. Myeloperoxidase (MPO)-derived products released from activated neutrophils can mediate the inflammatory response and contribute to endothelial dysfunction. 2-Chlorofatty aldehyde (2-ClFALD) is the direct oxidation product of MPO-derived hypochlorous acid (HOCl) targeting plasmalogen phospholipids. The role of 2-ClFALD in endothelial dysfunction is poorly understood and may be dependent on the vascular bed. This study compared the role of 2-ClFALD in eliciting endothelial dysfunction in human coronary artery endothelial cells (HCAEC), human lung microvascular endothelial cells (HLMVEC), and human kidney endothelial cells (HKEC). Profound increases in selectin surface expression as well as ICAM-1 and VCAM-1 surface expression were observed in HCAEC and HLMVEC. The surface expression of these adherence molecules resulted in robust adherence of neutrophils and platelets to 2-ClFALD treated endothelial cells. In contrast to HCAEC and HLMVEC, 2-ClFALD-treated HKEC had substantially reduced adherence molecule surface expression with no resulting increase in platelet adherence. 2-ClFALD-treated HKEC did have an increase in neutrophil adherence. All three endothelial cell lines treated with 2-ClFALD displayed a time-dependent loss of barrier function. Further studies revealed 2-ClHDyA localizes to ER and Golgi when using a synthetic alkyne analog of 2-ClFALD in HCAEC and HLMVEC. These findings indicate 2-ClFALDs promote endothelial cell dysfunction with disparate degrees of responsiveness depending on the vascular bed of origin.

Full text of DOI:10.3389/fphys.2020.00460

ORIGINAL RESEARCH
published: 08 May 2020
doi: 10.3389/fphys.2020.00460
2-Chlorofatty Aldehyde Elicits
Endothelial Cell Activation
Jane McHowat1,2, Shubha Shakya2,3 and David A. Ford2,3
*
1 Department of Pathology, Saint Louis University School of Medicine, St. Louis, MO, United States, 2 Center
for Cardiovascular Research, Saint Louis University School of Medicine, St. Louis, MO, United States, 3 Edward A. Doisy
Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO, United States
Endothelial activation and dysfunction are hallmarks of inflammation. Neutrophil-
vascular endothelium interactions have significant effects on vascular wall physiology
and pathology. Myeloperoxidase (MPO)-derived products released from activated
neutrophils can mediate the inflammatory response and contribute to endothelial
dysfunction. 2-Chlorofatty aldehyde (2-ClFALD) is the direct oxidation product of
MPO-derived hypochlorous acid (HOCl) targeting plasmalogen phospholipids. The
role of 2-ClFALD in endothelial dysfunction is poorly understood and may be
dependent on the vascular bed. This study compared the role of 2-ClFALD in eliciting
endothelial dysfunction in human coronary artery endothelial cells (HCAEC), human lung
microvascular endothelial cells (HLMVEC), and human kidney endothelial cells (HKEC).
Profound increases in selectin surface expression as well as ICAM-1 and VCAM-1
surface expression were observed in HCAEC and HLMVEC. The surface expression
of these adherence molecules resulted in robust adherence of neutrophils and platelets
to 2-ClFALD treated endothelial cells. In contrast to HCAEC and HLMVEC, 2-ClFALD-
treated HKEC had substantially reduced adherence molecule surface expression with no
resulting increase in platelet adherence. 2-ClFALD-treated HKEC did have an increase
in neutrophil adherence. All three endothelial cell lines treated with 2-ClFALD displayed
a time-dependent loss of barrier function. Further studies revealed 2-ClHDyA localizes
to ER and Golgi when using a synthetic alkyne analog of 2-ClFALD in HCAEC and
HLMVEC. These findings indicate 2-ClFALDs promote endothelial cell dysfunction with
disparate degrees of responsiveness depending on the vascular bed of origin.
Edited by:
David H. Wasserman,
Vanderbilt University, United States
Reviewed by:
Nathan C. Winn,
Vanderbilt University, United States
Ian Williams,
Stanford University School of
Medicine, United States
*Correspondence:
David A. Ford
david.ford@health.slu.edu
Keywords: endothelial cells, fatty acids, inflammation, lipid mediators, plasmalogens, vascular biology
Specialty section:
This article was submitted to
Lipid and Fatty Acid Research,
a section of the journal
INTRODUCTION
Frontiers in Physiology
Changes at the blood-endothelial interface can elicit profound changes in organ physiology.
Leukocytes can initiate an inflammatory response in the vascular wall resulting in the release
of inflammatory mediators and oxidants (Szekanecz and Koch, 2004; Langer and Chavakis,
2009), which may lead to dysfunction at the blood-endothelium interface. Endothelial dysfunction
is an early event in the pathophysiological sequelae of diseases including atherosclerosis,
Received: 30 October 2019
Accepted: 16 April 2020
Published: 08 May 2020
Citation:
McHowat J, Shakya S and
Ford DA (2020) 2-Chlorofatty
Aldehyde Elicits Endothelial Cell
Activation. Front. Physiol. 11:460.
doi: 10.3389/fphys.2020.00460
Abbreviations: 2-ClFALD, 2-chlorofatty aldehyde; 2-ClHDA, 2-chlorohexadecanal; 2-ClHDyA, 2-chlorohexadec-15-ynal;
HCAEC, human coronary artery endothelial cell; HDA, hexadecanal; HDyA, hexadec-15-ynal; MPO, myeloperoxidase; PFB-
Br, pentafluorobenzyl bromide; VWF, von Willebrand factor.
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2-Chlorofatty Aldehydes Alter Endothelial Function
ischemia/reperfusion injury in the heart, and in sepsis multi- several vascular beds. Furthermore, a novel click chemistry
organ failure (Ijzerman et al., 2003; Crea et al., 2014; De approach was used to determine the subcellular localization of
Backer et al., 2014). Once activated the endothelium releases 2-ClFALD in vascular endothelial cells.
many mediators that can contribute to leukocyte adherence,
coagulation, and changes in barrier function (Ait-Oufella
et al., 2010). Through this escalating inflammation, leukocyte-
MATERIALS AND METHODS
produced reactive oxygen species and released proteolytic
enzymes propagate tissue damage and are involved in organ
Materials
failure (Rossaint and Zarbock, 2015; Santos et al., 2016).
Cell culture supplies were purchased from Sigma-Aldrich
Myeloperoxidase is the most abundant protein in the
(Vienna, Austria), Lonza (Basel, Switzerland), or Cell
neutrophil and is a key antimicrobial enzyme released by
Applications Inc. (San Diego, CA, United States). Click-It
activated neutrophils (Ysebaert et al., 2000; Klebanoff et al.,
Cell Reaction Buffer Kit was purchased from Thermo Fisher
2013). During neutrophil activation sequential NADPH oxidase
Scientific (Waltham, MA, United States; catalog no. C10269).
production of hydrogen peroxide and MPO conversion of
Rabbit polyclonal anti-calnexin (catalog no. ab22595), rabbit
hydrogen peroxide and chloride anion lead to the production
monoclonal anti- voltage-dependent anion-selective channel
of HOCl. HOCl is a potent antimicrobial agent capable of
1/porin antibody (anti-VDAC1; catalog no. ab154856), mouse
oxidizing proteins, free amino acids, carbohydrates, DNA, and
polyclonal anti-golgi matrix protein 130 (anti-GM130; catalog
lipids (Harrison and Schultz, 1976; Hazen et al., 1996; Pattison
no. ab169276), mouse monoclonal anti-Von Willebrand Factor
and Davies, 2006; Pattison et al., 2009). Plasmalogens, a major
(anti-VWF; catalog no. ab20435), goat anti-rabbit IgG H&L
(Alexa FluorꢀR 488) (catalog no: 150077), and goat anti-mouse
IgG H&L (Alexa FluorꢀR 488) (catalog no. ab150113) antibodies
were purchased from Abcam (Cambridge, United Kingdom).
Antibodies to adhesion molecules (mouse monoclonal anti-P-
selectin, sc-18834; mouse monoclonal anti-E-selectin, sc-5262;
mouse monoclonal anti-intercellular adhesion molecule-1,
ICAM-1, sc-53336; mouse monoclonal anti-vascular cell
adhesion molecule-1, VCAM-1, sc-13160) were purchased from
Santa Cruz Biotechnology (Dallas, TX, United States). All other
chemicals were purchased from Sigma-Aldrich or Thermo
Fisher Scientific.
phospholipid in many human organs, are targeted by HOCl
leading to the production of 2-ClFALDs (Dorman et al., 1976;
Chilton and Connell, 1988; Ford and Gross, 1989; Murphy et al.,
1992; Hazen et al., 1993; Portilla and Creer, 1995; McHowat
et al., 1997; Albert et al., 2001; Hsu et al., 2003). 2-ClFALDs can
enter the fatty acid-fatty alcohol cycle as an intermediate leading
to their oxidation to 2-chlorofatty acids (2-ClFAs) (Rizzo et al.,
1987; Wildsmith et al., 2006; Anbukumar et al., 2010; Brahmbhatt
et al., 2010). During inflammation, 2-ClFALDs and 2-ClFAs are
produced at the site of neutrophil infiltration and likely alter
nearby cell function (Ford, 2010).
2-Chlorofatty aldehyde and 2-ClFA have been linked
to inflammatory diseases, including endotoxemia and
atherosclerosis (Thukkani et al., 2003, 2005; Brahmbhatt et al.,
2010; Ford et al., 2016). Activated neutrophils and monocytes
isolated from human blood have elevated levels of 2-ClFALD,
approaching 20–90 µM (compared to undetectable levels in
inactivated neutrophils and monocytes), which represents an
approximation of the maximal concentrations at the site of
production near the leukocyte-endothelial interface (Thukkani
et al., 2002; Anbukumar et al., 2010). Other studies have
also shown neutrophil-derived HOCl can target endothelial
plasmalogens resulting in endothelial 2-ClFALD production
(Thukkani et al., 2002). Chlorinated lipids have been shown
to elicit recruitment of macrophages to the lung, disrupt the
blood-brain barrier, and induce apoptosis (Wang et al., 2014;
Ford et al., 2016; Nusshold et al., 2016). Recently 2-ClFALD
was shown to increase leukocyte rolling and adhesion in the
mesenteric microcirculation (Yu et al., 2018). Despite these
insights into the biological role of chlorinated lipids the role of
2-ClFALD and 2-ClFA elicited endothelial dysfunction remain
to be further characterized. In particular, the demonstration that
2-ClFA associates with ARDS in humans with sepsis (Meyer
et al., 2017) suggests understanding the role of 2-ClFALD, the
precursor of 2-ClFA, on tissue specific endothelium is needed.
Synthesis of 2-Chlorohexadec-15-ynal
(2-ClHDyA)
The alkyne analog of 2-ClHDA (2-ClHDyA), was synthesized
according to protocols described previously (Halland et al.,
2004; Nusshold et al., 2016). Sequentially, (1) hexadec-7-
ynol (Alfa Aesar, cat. B22113) was converted to hexadec-
15-ynol using sodium hydride (Sigma-Aldrich, cat. 452912)
and diaminopropane (Sigma-Aldrich, cat. D23602) (Nusshold
et al., 2016), hexadec-15-ynol was oxidized to HDyA in a
solution of 2-iodoxybenzoic acid (Sigma-Aldrich, cat. 661384)
in DMSO (Sigma-Aldrich, cat. D2650) (Nusshold et al., 2016),
and HDyA was chlorinated using N-chlorosuccinimide (Sigma-
Aldrich, cat. 109681) and proline (Sigma-Aldrich, cat. P0380)
for 16 h (Halland et al., 2004). Products were purified by flash
chromatography (30 g silica gel, high purity grade, pore size 60 Å,
Sigma-Aldrich, cat. 227196) (Hartman et al., 2018). 2-ClHDyA
was subsequently quantitated by GC-FID following conversion
to its dimethyl acetal derivative using heptadecanoic acid and
its methyl ester derivative as internal standard (Gross, 1985;
Albert et al., 2001).
Endothelial Cell Culture
Accordingly, the present studies were designed to elucidate Human coronary artery endothelial cells (HCAEC, Lonza, cat.
mechanisms responsible for 2-ClFALD mediated endothelial CC-2585) and human lung microvascular endothelial cells
activation and distinguish mechanisms in endothelial cells from (HLMVEC, Lonza, cat. CC-2527) were grown in EGM-2MV
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medium (Lonza, cat. CC-3202). Human renal glomerular
endothelial cells (HKEC, Sciencell, cat. 4000) were grown in
endothelial cell medium containing 5% FBS (Sciencell, cat. 1001).
Endothelial cells were used in experiments at passage 4–8. Cells
were treated with lipids in DMSO.
Localization of 2-ClHDyA in HCAEC and
HLMVEC
Cells were grown to confluence on sterile coverslips and
treated with 10 µM 2-ClHDyA, or no lipid, for 30 min or
60 min. Cells were washed with PBS, fixed with formalin, and
permeabilized with 0.25% Triton X-100 for 10 min. In the
case of VDAC1, cells were permeabilized with ice cold 100%
methanol for 10 min at −20◦C. Cells were subsequently washed
with 2% (w/v) BSA in PBS and labeled with 5 µM azide-
carboxytetramethylrhodamine (azide-TAMRA) (Sigma-Aldrich;
catalog no. 760757) by using the Click-It Cell Reaction
Buffer Kit (Thermo Fisher; catalog no. C10269) following
manufacturer’s protocols. After click reaction, cells were washed
with 2% BSA in PBS. To identify subcellular localization
of 2-ClHDyA, cells were incubated with primary antibodies
against VDAC1 (1:1000), GM130 (1:142), calnexin (1:1000),
and VWF (1:100) overnight at 4◦C. Cells were washed three
times with PBS for 5 min and incubated with goat anti-
mouse IgG secondary antibody (1:500) labeled with Alexa 488
or goat anti rabbit IgG secondary antibody (1:500) for 1 h.
The coverslips were mounted onto microscope slides with a
Vectashield solution containing 40,6-diamidino-2-phenylindole
(DAPI; Vector Laboratories; catalog no. H1200).
Confocal Microscopy
A
Leica SP5 confocal microscope (Leica Microsystems,
Mannheim, Germany) with a 63 × 1.4 oil immersion objective
was used to acquire images. Alexa 488 was excited at 488 nm
and detected between 500 and 540 nm. Azide-TAMRA
fluorescence was excited at 543 nm and detected between 570
and 650 nm. DAPI fluorescence was detected between 440
and 470 nm. Alexa 488 and TAMRA fluorescence signals were
acquired simultaneously.
Adhesion Molecule Surface Expression
Human coronary artery endothelial cells, HLMVEC, and HKEC
were grown to confluence and treated with either 10 µM 2-
ClHDA or HDA for 30 min (P-selectin), 1 h (E-selectin) or
4 h (ICAM-1 and VCAM-1). Cells were not permeabilized,
but were fixed with 1% paraformaldehyde overnight and cell
surface expression of adhesion molecules measured as described
previously (Meyer et al., 2017; Hartman et al., 2018).
FIGURE 1 | Effects of 2-ClHDA on selectin, ICAM-1 and VCAM-1 surface
expression in HCAEC (A), HLMVEC (B), and HKEC (C). Cells were treated
with 10 µM HDA, 10 µM 2-ClHDA or vehicle for 0.5 (P-selectin), 1 h
(E-selectin) or 4 h (VCAM-1 and ICAM-1). Cells were incubated with primary
antibodies and then incubated with HRP-conjugated secondary antibodies.
Surface expression of selectins, ICAM-1 and VCAM-1 was measured
spectrophotometrically after addition of TMB substrate. Values are normalized
to secondary antibody alone. n = 4 for each treatment, mean ± SEM.
*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 for comparisons
with control treatment. † P < 0.05, †† P < 0.01, and †††† P < 0.0001 for
comparisons with HDA treatment.
Platelet and Neutrophil Adherence to
Endothelial Cells
Adherence of platelets to either HCAEC, HLMVEC or HKEC
was determined as previously described (Verheul et al., 2000).
Platelets were isolated from the whole blood of healthy volunteers
as previously described (Beckett et al., 2007), and as authorized
by Saint Louis University Institutional Review Board Protocol
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12369. Platelets were stained with Calcein-AM (2.5 µmol/L;
Thermo Fisher, cat. C3100MP) for 15 min at 37◦C in the dark.
Fluorescence-labeled platelets (50 × 106 cells in 500 µL) were
added to endothelial cells and incubated for 20 min at 37◦C.
Platelet adherence was determined by fluorescence measurement
(excitation at 492 nm, emission at 535 nm). Neutrophils were
prepared from whole blood of healthy volunteers as previously
described (Thukkani et al., 2002; Duerr et al., 2015), and as
authorized by Saint Louis University Institutional Review Board
Protocol 10014. 2 × 106 neutrophils were added to endothelial
cells and incubated for 20 min. Neutrophil adherence was
measured as MPO activity using 1,9-dimethyl-methylene blue,
and absorbance measured at 460 nm as described previously
(Meyer et al., 2017; Hartman et al., 2018).
Endothelial Cell Permeability
Endothelial cells were grown to confluence on Transwell
polycarbonate filters (Corning Inc., Corning, NY, United States)
mounted in a chamber insert. Resistance across cells was
monitored daily using an EVOM volt-ohmmeter (World
Precision Instruments). The lipids, 2-ClHDA or HDA, (10 µM)
were added to each well then the resistance across each well was
monitored up to 24 h.
Cell Viability Assay
The metabolic activity of HLMVEC was examined
using
the
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-
tetrazolium bromide (MTT) assay as previously described
FIGURE 2 | Effects of 2-ClHDA on Neutrophil (A,C,E) and Platelet (B,D,F) Adherence to HCAEC (A,B), HLMVEC (C,D) and HKEC (E,F). Cells were treated with
10 µM HDA, 10 µM 2-ClHDA or vehicle for 4 h. Subsequently endothelial cells were incubated with either freshly isolated human neutrophils or platelets for 20 min
to assess adherence. n = 4 for each treatment, mean ± SEM. * P < 0.05, and **** P < 0.0001 for comparisons with control treatment. †††† P < 0.0001 for
comparisons with HDA treatment.
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(Hartman et al., 2018) to test changes in metabolic activity Interestingly although 2-ClHDA mediated neutrophil adherence
elicited by either no lipid, 2-ClHDA or 2-ClHDyA (10 µM).
to HCAEC and HLMVEC was ∼4-fold greater than that
elicited by HDA, significant adherence of neutrophils in
response to HDA was detected in comparison to vehicle
treatments. Although both platelet and neutrophil adherence
to HKEC was significantly increased in response to 2-
ClHDA treatments (Figures 2E,F), the response in HKEC
was weaker compared to that observed in HCAEC and
HLMVEC. Additionally HDA did not elicit neutrophil or platelet
adhesion to HKEC.
Statistical Analyses
ANOVA with the Tukey’s post hoc test was used for comparisons
between three groups. All data are presented as mean ± SEM
unless otherwise noted.
RESULTS
Effect of 2-ClHDA on Surface Expression
of Adhesion Molecules as Well as
Neutrophil and Platelet Adherence
Previous studies have shown 2-ClHDA increases leukocyte
rolling and adhesion in the mesenteric microcirculation (Yu
et al., 2018). To better understand mechanisms responsible for
in vivo leukocyte rolling and adhesion we examined the role
of 2-ClHDA as a mediator of both adhesion molecule surface
expression as well as adhesion of neutrophils and platelets
in isolated endothelial cells. Furthermore these studies were
performed with human endothelial cells from several vascular
beds including HCAEC, HLMVEC and HKEC. Data shown
in Figure 1A highlight the responsiveness of HCAEC to 2-
ClHDA treatment. Significant increases in surface expression of
P-selectin, E-selectin, ICAM-1, and VCAM-1 in response to 2-
ClHDA compared to treatments with either vehicle (control)
or HDA. The differential response to 2-ClHDA compared to
HDA reveals the importance of the α-carbon chlorination of
the aldehyde. In comparison to HCAEC, HLMVEC (Figure 1B)
had a weaker response to 2-ClHDA treatment, but did show
significant increases in E-selectin, ICAM-1, and VCAM-1 when
compared to control conditions. Again, HDA did not cause
surface expression of these molecules in HLMVEC. In a separate
study, cell surface expression of adhesion molecules in response
to 2-ClHDA was compared to LPS incubation (50 ng/ml) in
HLMVEC. Similar, but smaller, increases were observed in
response to 2-ClHDA when compared to LPS for P-selectin
(0.170 ± 0.005 2-ClHDA vs. 0.188 ± 0.005 LPS), E-selectin
(0.135 ± 0.003 2-ClHDA vs. 0.258 ± 0.008 LPS), ICAM-
1 (0.189 ± 0.006 2-ClHDA vs. 0.258 ± 0.006 LPS), and
VCAM-1 (0.219 ± 0.007 2-ClHDA vs. 0.253 ± 0.006 LPS).
Surface expression of the selectins and ICAM-1 did not
significantly increase in HKEC in comparison to control
conditions (Figure 1C).
Selectin adhesion molecules mediate the adherence of
leukocytes to the endothelium (Etzioni, 1996; Peyvandi et al.,
2011). Because these molecules are increased following 2-
ClHDA treatment, the adherence of neutrophils and platelets
to endothelium after 2-ClHDA treatment was examined. In
concert with elevated surface expression of adhesion molecules,
both neutrophil and platelet adhesion to HCAEC and HLMVEC
in response to 2-ClHDA was robustly increased in both
HCAEC and HLMVEC in comparison to treatments with
vehicle as well as in response to HDA (Figures 2A–D).
FIGURE 3 | Permeability Barrier of HCAEC (A), HLMVEC (B), and HKEC (C).
Cells were grown to confluence on Transwell polycarbonate filters mounted in
a chamber insert. Resistance across the cells was monitored daily using an
EVOM volt-ohmmeter. Once the resistance remained constant for 3
consecutive days, experiments were performed. 10 µM lipids were added to
each well then the resistance across each well was monitored at 0.5, 1, 2, 4,
8, 12, 18, and 24 h. n = 6 for each treatment, mean ± SEM. * P < 0.05,
** P < 0.01, *** P < 0.001, **** P < 0.0001 for comparisons with control
treatment. † P < 0.05, †† P < 0.01 and †††† P < 0.0001 for comparisons with
HDA treatment.
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FIGURE 4 | Subcellular localization of 2-ClHDyA in HLMVEC at 30 min. Cells were grown to confluence on sterile coverslips and treated with 10 µM 2-ClHDyA for
30 min. Cells were fixed with formalin, permeabilized with Triton X-100, except for VDAC1 when cells were permeabilized with ice cold 100% methanol and clicked
with azide TAMRA (red). Cells were incubated with primary antibodies against GM130 (Golgi), VWF, calnexin (ER), and VDAC1 (mitochondria), then labeled with Alexa
488 labeled secondary antibodies (green). Cells were mounted in DAPI-containing solution (blue) and imaged with a Leica SP5 microscope. All fluorescence was
taken simultaneously.
albeit this loss was not as great as that elicited by 2-ClHDA
Endothelial Cell Monolayer Permeability
(Figure 3A). Changes in electrical resistance in response to 2-
in Response to 2-ClHDA
ClHDA were similar to those observed when HLMVEC were
incubated with LPS (50 ng/ml) for up to 24 h (data not shown).
Similar to conditions comparing adhesion molecule surface
expression and neutrophil adhesion to endothelial cells, the
impact of 2-ClHDA on endothelial permeability barrier function
Subcellular Localization of 2-ClHDyA
was determined. HCAEC, HLMVEC, and HKEC all significantly
lost barrier function, measured as changes in electrical resistance, Since HLMVEC and HCAEC gave robust responses to 2-ClHDA,
in response to 2-ClHDA treatments in comparison to vehicle additional studies were performed to examine the subcellular
control treatment (Figures 3A–C). Comparing the response distribution of 2-ClHDA in these cells. For these studies the click-
of the three endothelial cell lines to 2-ClHDA indicated chemistry analog of 2-ClHDA, 2-ClHDyA, was used. Following
HCAEC > HLMVEC > HKEC. Additionally, HCAEC was either a 30 min or 60 min incubation of 2-ClHDyA with
uniquely susceptible to HDA-mediated loss of barrier function endothelial cells, cells were fixed and then click reactions were
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FIGURE 5 | Subcellular localization of 2-ClHDyA in HLMVEC at 60 min. Cells were grown to confluence on sterile coverslips and treated with 10 µM 2-ClHDyA for
60 min. Cells were fixed with formalin, permeabilized with Triton X-100, except for VDAC1 when cells were permeabilized with ice cold 100% methanol and clicked
with azide TAMRA (red). Cells were incubated with primary antibodies against GM130 (Golgi), VWF, calnexin (ER), and VDAC1 (mitochondria), then labeled with Alexa
488 labeled secondary antibodies (green). Cells were mounted in DAPI-containing solution (blue) and imaged with a Leica SP5 microscope. All fluorescence was
taken simultaneously.
performed using an azide with TAMRA reporter. Colocalization with ER, Golgi and mitochondria were observed in HCAEC
analyses with known organelle markers showed that 2-ClHDyA (Figures 7, 8). 2-ClHDyA did not colocalize with the Weibel-
localized with the ER, Golgi and mitochondria as indicated Palade bodies (detected with VWF in HCAEC) or VWF storage
by calnexin, GM130 and VDAC1 at both 30 and 60 min in granules in HLMVEC.
HLMVEC (Figures 4, 5). Additional control experiments showed
azide-TAMRA specifically reacts with HLMVEC treated with
2-ClHDyA (Figure 6A, lower panel) and was undetectable in
cells that were not treated with 2-ClHDyA (Figure 6A, upper
DISCUSSION
panel). Additional test also showed 2-ClHDyA and 2-ClHDA Much of the biological role of chlorinated lipids on endothelial
both elicited barrier dysfunction similar to LPS in HLMVEC activation have focused on the longer lived chlorinated lipid,
(Figure 6B) and did not alter cell metabolic activity as determined 2-ClFA, while the mechanisms underlying biological effects of
by MTT assay (Figure 6C). A similar distribution of 2-ClHDyA the 2-ClFA precursor, 2-ClFALD, remain to be explored. Using
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FIGURE 6 | 2-ClHDyA in HLMVEC: Specificity of TAMRA-Azide Click Reaction and Comparisons to 2-ClHDA Changes in Barrier Function and MTT Activity. (A) Cells
were grown to confluence on sterile coverslips and treated with 10 µM 2-ClHDyA (bottom panel) or no lipid (top panel) for 60 min. Indicated GM130 (Golgi) staining
and DAPI staining as well as confocal microscopy were performed as described in Figure 5. (B) Cells were grown to confluence on Transwell polycarbonate filters
mounted in a chamber insert. Resistance across the cells was monitored daily using an EVOM volt-ohmmeter. Once the resistance remained constant for 3
consecutive days, experiments were performed. Either 10 µM lipids or LPS (50 ng/ml) were added to each well then the resistance across each well was monitored
for indicated times. n = 6 for each treatment, mean ± SEM. In some cases the SEM is within the symbol. * P < 0.05, ** P < 0.01, **** P < 0.0001 for comparisons
with control treatment for each of the lipids or LPS as indicated by color of symbols. (C) HLMVEC cells were treated with either 2-ClHDA or 2-ClHDyA (10 µM) for
indicated time point at 37◦C. Metabolic activity of HLMVEC was measured using an MTT assay following manufacturer’s protocol. MTT reduction is expressed as
percent of control MTT reduction (vehicle only, designated as 100%). n = 4 for each treatment, mean ± SEM. SEM is within the symbol of mean for majority of data
points.
intravital microscopy in the rat we recently showed 2-ClFALD
Human coronary artery endothelial cells were very sensitive
elicits leukocyte rolling and adhesion, as well as permeability to activation by the 2-ClFALD, 2-ClHDA at a concentration
changes, in the mesenteric microcirculation. Others have also (10 µM), which is below maximal concentrations estimated at
shown 2-ClFALD causes blood brain barrier dysfunction, but did the leukocyte-endothelial interface (20–90 µM) (Thukkani et al.,
not examine leukocyte adhesion (Nusshold et al., 2016). These 2002; Anbukumar et al., 2010). Robust surface expression of
studies also showed 2-ClFALD associated with Golgi, ER and P-selectin, E-selectin, ICAM-1, and VCAM-1 were observed.
mitochondria in an immortalized human brain endothelial cell Neutrophil and platelet adhesion to HCAEC accompanied the
line. To enhance our understanding of the biological role of 2- surface expression of adhesion molecules in response to 2-
ClFALD we now show 2-ClFALD elicits endothelial activation in ClHDA. Interestingly there was robust adhesion of neutrophils
primary human endothelial cells from three disparate vascular and platelets to 2-ClHDA treated HLMVEC and HKEC in the
beds (i.e., coronary artery, lung microvascular, and kidney) absence of robust increases of surface expression of adhesion
resulting in adhesion molecule surface expression, neutrophil and molecules. Disparate responses to agonists by endothelial cells
platelet adherence, and permeability barrier dysfunction. These originating from different vascular beds have previously been
findings are significant since they provide important information observed (Scott and Patel, 2013), and thus these results most
on tissue beds associated with multi-organ failure during sepsis, likely represent differences in selectin density and mobilization
and in particular plasma 2-ClFA levels associate with ARDS and in response to 2-ClHDA in these cell systems. It can also be
30-day mortality in human sepsis (Meyer et al., 2017).
speculated that at least for neutrophils, adherence may involve
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FIGURE 7 | Subcellular localization of 2-ClHDyA in HCAEC at 30 min. Cells were grown to confluence on sterile coverslips and treated with 10 µM 2-ClHDyA for
30 min. Cells were fixed with formalin, permeabilized with Triton X-100, except for VDAC1 when cells were permeabilized with ice cold 100% methanol and clicked
with azide TAMRA (red). Cells were incubated with primary antibodies against GM130 (Golgi), VWF, calnexin (ER), and VDAC1 (mitochondria), then labeled with Alexa
488 labeled secondary antibodies (green). Cells were mounted in DAPI-containing solution (blue) and imaged with a Leica SP5 microscope. All fluorescence was
taken simultaneously.
neutrophil netosis, which has been shown to be accelerated by caused P-selectin surface expression and neutrophil adhesion.
the 2-ClHDA oxidation product, 2-chlorofatty acid (Palladino However, unlike the click analog of 2-ClFA, 2-ClHDyA did
et al., 2018). Further investigations need to determine if there are not localize to the HCAEC Weibel-Palade bodies. 2-ClHDyA
novel protein ligands and mechanisms that may be responsible localized to the ER, Golgi and mitochondria of both HCAEC
for neutrophils and platelet adherence to 2-ClHDA treated and HLMVEC. Thus the mechanism responsible for 2-ClHDA
endothelial cells.
mediated mobilization of Weibel-Palade bodies and other
We previously used the click-chemistry analog of 2-ClFA granules containing adherence molecules remain to be resolved.
to show it selectively associates with the Weibel-Palade bodies In this respect it also should be noted that the HLMVEC
of HCAEC and subsequently elicits the cascade of events contain P-selectin and VWF in separate storage granules (Ochoa
including P-selectin surface expression and neutrophil adhesion et al., 2010; Wu et al., 2014). Future studies need to be
(Hartman et al., 2018). The association of 2-ClFA to the Weibel- performed to determine the mechanism responsible for 2-
Palade body suggested the localization may have a key role ClHDA as well as 2-ClFA elicited endothelial activation. These
in mobilizing these storage granules with subsequent surface studies using confocal microscopy highlight that there may
expression of P-selectin. Similar to 2-ClFA, 2-ClHDA also be unique mechanisms responsible for 2-ClHDA and 2-ClFA
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2-Chlorofatty Aldehydes Alter Endothelial Function
FIGURE 8 | Subcellular localization of 2-ClHDyA in HCAEC at 60 min. Cells were grown to confluence on sterile coverslips and treated with 10 µM 2-ClHDyA for
60 min. Cells were fixed with formalin, permeabilized with Triton X-100, except for VDAC1 when cells were permeabilized with ice cold 100% methanol and clicked
with azide TAMRA (red). Cells were incubated with primary antibodies against GM130 (Golgi), VWF, calnexin (ER), and VDAC1 (mitochondria), then labeled with Alexa
488 labeled secondary antibodies (green). Cells were mounted in DAPI-containing solution (blue) and imaged with a Leica SP5 microscope. All fluorescence was
taken simultaneously.
mediated endothelial activation. Additionally the relationship of and platelet adherence and loss of permeability barrier function.
chlorinated lipids and their association with adhesion molecule There are modest differences in the responses to 2-ClFALD by
receptors needs to be examined.
endothelial cells from three origins including human coronary
2-Chlorohexadecanal caused permeability barrier dysfunction arteries, human lung, and human kidney. In conjunction with
in HCAEC, HLMVEC, and HKEC. HDA did have some modest our previous studies using intravital microscopy (Yu et al., 2018),
effects in causing endothelial permeability barrier dysfunction, these data suggest this is a key mechanism through which 2-
but not to the degree observed with 2-ClHDA treatment. The ClFALD can elicit in vivo endothelial dysfunction leading to
mechanism responsible for permeability barrier dysfunction may microcirculatory collapse. Several key observations were also
involve adheren dysfunction with intercellular gap formation.
made indicating there are additional key gaps that need to be
These studies demonstrate that the initial product of HOCl examined in the future including the precise mechanism that
targeting plasmalogens, 2-ClFALD, activates endothelial cells 2-ClFALD elicits adhesion molecule surface expression and the
resulting in adhesion molecule surface expression, neutrophil mechanisms responsible for platelet and neutrophil adherence to
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2-ClFALD treated endothelial cells in the absence of a response
with adhesion molecule surface expression (e.g., in HKEC).
AUTHOR CONTRIBUTIONS
DF was responsible for oversight of all aspects of studies
and manuscript preparation. JM performed endothelial
cell functional assays and manuscript preparation. SS
performed endothelial cell sub cellular localization studies and
manuscript preparation.
DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the
article/supplementary material.
FUNDING
This study was supported (in part) by research funding
from the National Institutes of Health R01 GM-
ETHICS STATEMENT
The studies involving human participants were reviewed 115553 and R01 GM129508 to DF. The content is
and approved by the Saint Louis University IRB. The solely the responsibility of the authors and does not
patients/participants provided their written informed consent necessarily represent the official views of the National
to participate in this study.
Institutes of Health.
Ford, D. A., Honavar, J., Albert, C. J., Duerr, M. A., Oh, J.-Y., Doran, S., et al.
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Gross, R. W. (1985). Identification of plasmalogen as the major phospholipid
constituent of cardiac sarcoplasmic reticulum. Biochemistry 24, 1662–1668.
doi: 10.1021/bi00328a014
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potential conflict of interest.
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Thukkani, A. K., Albert, C. J., Wildsmith, K. R., Messner, M. C., Martinson, B. D.,
Hsu, F. F., et al. (2003). Myeloperoxidase-derived reactive chlorinating species
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