Atherosclerosis and Ischaemic Disease
Hepatic lipase inactivation decreases atherosclerosis in insulin
resistance by reducing LIGHT/Lymphotoxin β-Receptor pathway
Irene Andrés-Blasco*,1; Ángela Vinué*,1; Andrea Herrero-Cervera1; Sergio Martínez-Hervás1,2,3; Laura Nuñez1; Laura Piqueras1;
Juan F. Ascaso1,2,3; María Jesús Sanz1,4; Deborah Jane Burks3,5; Herminia González-Navarro1,3
1Institute of Health Research-INCLIVA, Valencia, Spain; 2Endocrinology and Nutrition Department Clinic Hospital and Department of Medicine, University of Valencia, Spain;
3CIBERDEM, Madrid, Spain; 4Department of Farmacology, University of Valencia, Spain; 5Centro de Investigación Príncipe Felipe, Valencia, Spain
Summary
Coexistence of insulin resistance (IR) and metabolic syndrome (MetS)
increases the risk of cardiovascular disease (CVD). Genetic studies in
diabetes have linked Hepatic Lipase (HL) to an enhanced risk of CVD
while others indicate a role of HL in inflammatory cells. Thus, we ex-
plored the role of HL on atherosclerosis and inflammation in a mouse
model of MetS/IR, (apoE-/-Irs2+/- mice) and in patients with MetS and
IR. HL-deficiency in apoE-/-Irs2+/- mice reduced atheroma size, plaque
vulnerability, leukocyte infiltration and macrophage proliferation.
Compared with apoE-/-Irs2+/-HL+/+ mice, MCP1, TNFα and IL6 plasma
levels, pro-inflammatory Ly6Chi monocytes and activated(CD69+)-T
lymphocytes were also decreased in apoE-/-Irs2+/-HL-/- mice. The LIGHT
(Tumour necrosis factor ligand superfamily member 14, TNFSF14)/
Lymphotoxin β-Receptor(LTβ-R) pathway, which is involved in T-cell
and macrophage activation, was diminished in plasma and in apoE-/-
Irs2+/-HL-/- mouse atheromas. Treatment of apoE-/-Irs2+/-HL-/- mice with
LIGHT increased the number of Ly6Chi-monocytes and lesion size.
Acutely LIGHT-treated apoE-/- mice displayed enhanced proliferating
Correspondence to:
Herminia González-Navarro
Institute of Health Research-INCLIVA
Avda. Menéndez Pelayo, 4, 46010, Valencia, Spain
Tel.: +34 96 386 44 03, Fax: +34 96 398 78 60
E-mail: gonzaleh@uv.es
* These authors contributed equally to this work.
Introduction
Type 2 diabetes mellitus (T2DM) and metabolic syndrome (MetS)
are major risk factors for developing cardiovascular disease (CVD)
(1). Their incidence is increasing at an alarming rate (1) and des-
pite the use of statin therapy in T2DM/MetS patients, acute CVD
events still occur, indicating the medical need to reduce the resid-
ual CV risk (2). Insulin resistance (IR), is a main metabolic alter-
ation resulting from the dyslipidaemia and abdominal obesity that
partly defines MetS and is thought to be one of the linking factors
between T2DM/MetS and CVD (3).
© Schattauer 2016
1
Ly6Chi-monocytes and increased activation of the mitogen-activated
protein kinase p38, suggesting that LIGHT/LT-R axis might promote
atherogenesis by increasing proinflammatory monocytes and prolifer-
ation. Notably, MetS-IR subjects with increased atherosclerosis dis-
played up-regulation of the LIGHT/LTβ-R axis, enhanced inflammatory
monocytes and augmented HL mRNA expression in circulating leuko-
cytes. Thus, HL-deficiency decreases atherosclerosis in MetS/IR states
by reducing inflammation and macrophage proliferation which are
partly attributed to reduced LIGHT/LTβ-R pathway. These studies
identify the LIGHT/LTβ-R axis as a main pathway in atherosclerosis
and suggest that its inactivation might ameliorate inflammation and
macrophage proliferation associated with atherosclerosis burden in
MetS/IR.
Keywords
Atherosclerosis, inflammation, insulin resistance, hepatic lipase,
macrophage
Financial support:
This study was supported by grants from the Carlos III Health Institute (CP10/00555
and PI13/00834 to HG-N) from the Spanish Ministry of Economy and Competitiveness
(SAF2011–23777 to MJS), from the European Regional Development Fund (FEDER)
and Generalitat Valenciana (GVACOMP2014–006 and PROMETEO II/2013/014 to
MJS). H. G.-N. is a ‘Miguel Servet’ investigator (CP10/00555). I.A-B. and A. V. received
salary support from Proyecto Paula. This work was supported by CIBERDEM.
Received: October 5, 2015
Accepted after major revision: April 24, 2016
Epub ahead of print: May 12, 2016
http://dx.doi.org/10.1160/TH15-10-0773
Thromb Haemost 2016; 116: ■■■
Human genetic studies have linked hepatic lipase (HL) gene
polymorphisms to progression of abdominal obesity toward
T2DM (4) and to an increased risk of CVD in T2DM subjects
(5–7). HL is a key enzyme in the lipoprotein clearance and metab-
olism that has been associated with pro- and anti-atherogenic
properties (8, 9). The net effect of HL on CVD progression in hu-
mans is highly dependent on the underlying lipoprotein pheno-
type (10). Thus, increased levels are beneficial in hypercholestero-
laemic patients but detrimental in subjects with hypertriglyceri-
daemia, central obesity and IR (7, 10, 11). A rational for this is that
HL activity, which is high in MetS/IR and obesity (12), hydrolyses
Thrombosis and Haemostasis 116.2/2016
2 Andrés-Blasco, Vinué et al. HL gene ablation decreases inflammation in insulin resistance
triglycerides in the triglyceride-rich low-density lipoprotein (LDL)
particles generating proatherogenic small dense LDL (sdLDLs). In
fact, HL gene ablation in apolipoprotein E (apoE)-deficient mice
produces large buoyant LDL, thus preventing the formation of
sdLDL and restraining atherosclerosis progression (8, 13). There-
fore, lower HL activity could have the benefit of reducing CV risk
in patients exhibiting MetS, IR, and abdominal obesity (10).
Whilst the presence of sdLDL facilitates lesion initiation and
progression, it also promotes inflammation, which is particularly
high in MetS/IR states, and favours the formation of advanced
plaques (14). Advanced plaques are characterised by a chronic de-
fective-inflammatory resolution and accumulation of inflamma-
tory cells (15, 16). These changes lead to unstable obstructive
plaques which are prone to rupture, and promote acute events
(17). Among the mechanisms underlying lesion progression in
MetS/IR is a change in macrophage phenotype that increases local
apoptosis and secretion of additional inflammatory mediators
(18). Moreover, inflammatory environments also promote macro-
phage proliferation thus facilitating cell accumulation which ex-
acerbates lesion size (19). All these events lead to a vicious cycle of
inflammation that finally affects vascular smooth muscle cell sur-
vival, thus facilitating necrotic core formation and fibrous cap
degradation which are the hallmark of acute events (20).
Many current studies are focused on therapeutic strategies tar-
geted at modulating inflammation at advanced stages of athero-
sclerosis, such as restraining the secretion of proinflammatory
mediators or the expansion of natural circulating T regulatory cells
(21, 22) which are powerful atherosclerosis inhibitors. Interest-
ingly, HL has been reported to modulate atherosclerosis progres-
sion via its expression on inflammatory cells (23–26). Thus, HL in-
activation in the myeloid lineages significantly decreased athero-
sclerosis in apoE-deficient and LCAT (lecithin cholesterol acyl-
transferase) transgenic mice, indicating that myeloid HL likely
plays a proatherogenic role (25). In contrast, atherosclerosis was
accelerated in CETP (cholesterol ester transfer protein) transgenic
mice with combined LDL-receptor and myeloid HL-deficiency
pointing to an antiatherogenic role for HL (24). In addition, others
have shown that atherosclerosis progression induced by inflam-
matory mediators or cells are accompanied by an increase in HL
expression and activity (27, 28). These studies highlight a complex
relationship between inflammation, lipid homeostasis and HL in
the development of atherosclerosis which requires further investi-
gation to be fully understood.
In the present study, we explored the effect of genetic manipu-
lation of HL in atherosclerosis development in the setting of MetS/
IR, which is characterised by high levels of inflammation. To this
end, the effect of HL deficiency was investigated in a mouse model
that develops IR, MetS, and atherosclerosis, the apoE-/-Irs2 (insu-
lin receptor substrate 2)+/- mice (29, 30). To understand the role of
HL in the inflammatory processes, and the relevance of these in
atheroma progression in MetS/IR, systemic and lesional inflam-
matory properties were also studied. Furthermore, the clinical rel-
evance of our findings was explored in human patients with MetS
and IR.
Thrombosis and Haemostasis 116.2/2016
Materials and methods
An expanded methods section can be found in the online Supple-
mentary Material (available online at www.thrombosis-online.
com).
Mice and diets
Animal care was in accordance with institutional guidelines and
following the 2010/63/EU directive from the European Parlia-
ment. HL-/- (Jackson laboratories), apoE-/- (C57BL/6J, Charles
River) and Irs2+/- mice were backcrossed at least 20 times to
C57BL/6J background and crossed to generate the experimental
and control groups. These were identified by genotyping and were
littermates (8, 29). Two-month-old apoE-/-Irs2+/-HL+/+, apoE-/-
Irs2+/-HL-/-, apoE-/-Irs2+/+HL+/+ and apoE-/-Irs2+/+HL-/- male mice
were fed an atherogenic diet for two months (10.8 % total fat,
0.75 % cholesterol, S4892-E010, Ssniff) and characterised. ApoE-/-
mice were maintained on a regular diet (2.8 % fat; Panlab).
In vivo treatment with murine recombinant LIGHT
(Tumour necrosis factor ligand superfamily member
14, TNFSF14) and bromodeoxyuridine incorporation
Four month-old regular diet-fed apoE-/- mice were treated for five
days with murine recombinant LIGHT (2 µg/kg of body weight,
[BW]/day, Peprotech) or with vehicle (saline). For LIGHT treat-
ment of apoE-/-Irs2+/-HL-/- mice, two-month-old mice were placed
on an atherogenic diet for one month and then treated with
LIGHT (2 µg/kg of BW/day, Peprotech) or with vehicle for 28
days. The cytokine was administered via subcutaneous osmotic
mini-pumps (Alzet 2001, five days or Alzet 2004 for 28 days) im-
planted under inhalational anaesthesia (5 % and 2 % of isofluor-
ane). For in vivo proliferation analysis apoE-/- mice received an in-
traperitoneal injection of bromodeoxyuridine, BrdU (2 mg/200 µl
of saline) on the fourth day and 18 hours (h) later BrdU incorpor-
ation was determined by flow cytometry of leukocytes stained
with an anti-BrdU Alexa Fluor-488 antibody (clone MoBU-1,
B35130, Invitrogen).
Quantification of atherosclerosis burden
Mice were sacrificed by cervical dislocation and whole aorta and
hearts were harvested for atherosclerosis analysis. An operator,
blinded to the genotype, quantified the atherosclerosis by en face
computer-assisted morphometric analysis (SigmaScan, Pro5) of
whole-mounted aorta fixed with 4 % paraformaldehyde/PBS and
stained with Oil Red-O (Sigma, 0.2 % in 80 % Methanol). Hearts
were paraffin-embedded and sectioned for haematoxylin/eosin
staining and atherosclerosis analysis (30, 31).
Human subjects
The study was carried out in accordance with the ethical principles
of the Helsinki Declaration, and was approved by the ethical com-
© Schattauer 2016
 Andrés-Blasco, Vinué et al. HL gene ablation decreases inflammation in insulin resistance 3

Figure 1: Metabolic characterisation of

apoE-/-Irs2+/-HL+/+ and apoE-/-Irs2+/-HL-/- mice
fed an atherogenic diet for two months. A)
ApoB–cholesterol, HDL-cholesterol and triglyce-
rides plasma levels from both groups of mice. B)
BW, fasting plasma glucose and insulin levels. C)
Plasmatic glucose levels during the GTT (top
graph) and the area under the curve (AUCglucose)
generated from the glucose measurements at dif-
ferent time points of the test (right panel) for
both groups of mice. Plasmatic glucose-stimu-
lated insulin levels during the GTT (lower graph)
and the AUCinsulin generated from the insulin
measurements at different time points of the test
(right panel). D) Glucose levels (percentage
relative to initial glucose levels) during ITT in 4
h-fasted mice. Statistical analysis was performed
using Student’s t-test analysis.

mittee of the institution. The investigation was a cross-sectional
study of 121 unrelated individuals attending the outpatient clinic
(Hospital Clínico Universitario de Valencia-INCLIVA, Spain) over
a period of 12 months and all the subjects gave written informed
consent.
Statistical analysis
Quantitative variables appear as mean ± SEM, qualitative variables
as percentages and correlation studies as individual data points.
Differences in quantitative parameters were assessed by Student´s
t-test, qualitative variants by Chi-square and correlation studies by
the Spearman correlation coefficient (SPSS version 15). Differ-
ences were considered statistically significant when p≤0.05. Out-
liers identified by the Grubb’s test were not considered for quantifi-
cation (GraphPadPrism Software).
Results
Metabolic characterisation of apoE-/-Irs2+/-HL+/+ and
apoE-/-Irs2+/-HL-/- mice fed an atherogenic diet
Consistent with a role for HL in lipid metabolism, analysis of lipid
levels showed increased apolipoprotein B(apoB)-cholesterol and
triglyceride levels and decreased high density lipoprotein (HDL)-
cholesterol in apoE-/-Irs2+/-HL-/- male mice compared with apoE-/-
Irs2+/-HL+/+ mice (▶ Figure 1 A). No changes were observed in BW,
fasting basal glucose or insulin levels between genotypes (▶ Figure

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4 Andrés-Blasco, Vinué et al. HL gene ablation decreases inflammation in insulin resistance
1 B). Carbohydrate metabolism analysis by glucose tolerance test
(GTT, ▶ Figure 1 C, top panel), measured as the area under the
curve (glucose curve vs time, AUCglucose right graph), and of the
glucose-stimulated insulin release during the test (▶ Figure 1 C,
lower panel), expressed as AUCinsulin (insulin curve vs time, right
graph), demonstrated no differences between mice. Insulin sensi-
tivity assessed by insulin tolerance test (ITT), demonstrated no ef-
fect of HL-inactivation in apoE-/-Irs2+/- mice as revealed by the
AUCglucose parameters (▶ Figure 1 D).
To verify that apoE-/-Irs2+/-HL-/- and apoE-/-Irs2+/-HL+/+ mice
displayed glucose metabolism impairment, comparisons with their
respective non-IR controls, apoE-/-Irs2+/+HL-/- and apoE-/-
Irs2+/+HL+/+ mice, fed an atherogenic diet for two months, were
Thrombosis and Haemostasis 116.2/2016
Figure 2: Atherosclerotic lesion analysis of
apoE-/-Irs2+/-HL+/+ and apoE-/-Irs2+/-HL-/- mice
fed an atherogenic diet for two months. A)
En face atherosclerosis analysis in Oil Red-O
stained whole-mounted aorta from both groups
of mice. B) Atheroma lesion size measured as
intima-media ratio in aortic root and ascending
aorta cross sections (separated by a distance of
160 µm) of mice. C) Analysis of in vivo foam cells
determined as the percentage of peritoneal mac-
rophages exhibiting cytoplasmic lipid droplets
after Oil Red-O staining. Representative photo-
graphs of aortas (A), images from hematoxylin/
eosin-stained aortic cross-sections (B) and im-
ages from stained peritoneal macrophages (C)
are shown. The discontinuous lines delineate the
limits of the media. Statistical analysis was per-
formed using Student’s t-test analysis.
performed (Suppl. Figure 1A, available online at www.thrombosis-
online.com). Irs2 haploinsufficiency produced glucose intolerance,
as shown by the AUCglucose parameters, compared with mice with
intact Irs2 (apoE-/-Irs2+/+HL+/+ and apoE-/-Irs2+/+HL-/- mice p<0.05
and p<0.02, respectively), regardless of the HL presence. Thus, HL
does not have an additional effect on glucose metabolism in
apoE-/-Irs2+/- mice.
Analysis of female apoE-/-Irs2+/-HL-/- mice revealed increased
apoB-cholesterol and decreased HDL-cholesterol without changes
in BW, glucose tolerance or in triglyceride and glucose levels com-
pared with controls (Suppl. Figure 2, available online at www.
thrombosis-online.com).
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 Andrés-Blasco, Vinué et al. HL gene ablation decreases inflammation in insulin resistance 5

Figure 3: Plaque composition analysis in

aortic cross sections from two months athe-
rogenic diet-fed apoE-/-Irs2+/-HL+/+ and
apoE-/-Irs2+/-HL-/- mice. Neointimal content of
(A) macrophages (percentage) and (B) T-lympho-
cytes (number of cells/mm2) using antibodies
against specific markers (Mac-3 and CD3, re-
spectively). Quantification of (C) neointimal col-
lagen content (percentage), (D) necrotic core area
(µm2) and (E) fibrous cap thickness (µm) quanti-
fied in Masson trichrome stainings. Photomicro-
graphs show representative images of the stain-
ings and inmunofluorescences. The discontinuous
lines delineate the limits of the media, solid lines
delineate the limits of representative necrotic
cores. Statistical analysis was performed using
Student’s t-test analysis.

Genetic disruption of hepatic lipase reduces
the atherosclerosis burden and changes plaque
composition in apoE-/-Irs2+/- mice fed an atherogenic
diet for two months
Atherosclerosis analysis demonstrated that, compared with apoE-/-
Irs2+/-HL+/+ male controls, HL-deficient male mice exhibited
smaller atheromas in the aortic arch (▶ Figure 2 A, p<0.01) and in
the thoracic aorta (▶ Figure 2 A, p<0.03). Lesion size, measured as
the intima-media ratio in the cross-sections, was also decreased in
the aortic root (▶ Figure 2 B, top panel, p<0.02) and ascending
aorta (▶ Figure 2 B, lower panel, p<0.04) of apoE-/-Irs2+/-HL-/- mice.
Peritoneal macrophage foam cells were reduced in apoE-/-
Irs2+/-HL-/- mice compared with apoE-/-Irs2+/-HL+/+ mice (▶ Figure
2 C, p=0.05). Similarly, female mice exhibited a decreased lesion
size in the aortic arch and thoracic aorta of apoE-/-Irs2+/-HL-/- mice
compared with apoE-/-Irs2+/-HL+/+ mice (Suppl. Figure 3A, avail-
able online at www.thrombosis-online.com, p<0.03 and p<0.04,
respectively). However, atheroma size in the aortic cross-sections
was not significantly changed (Suppl. Figure 3B, available online at
www.thrombosis-online.com). Thus, atheroprotection by HL-defi-
ciency is vascular region-dependent and gender-specific.
To evaluate the global impact of HL-deficiency in combination
with Irs2 haploinsufficiency, atherosclerosis analysis of apoE-/-
Irs2+/-HL-/- mice and apoE-/-Irs2+/-HL+/+ mice and their respective
controls expressing intact Irs2 were performed (Suppl. Figure 1B,
available online at www.thrombosis-online.com). Atherosclerosis
analysis showed that, as described (8), HL inactivation reduced

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6 Andrés-Blasco, Vinué et al. HL gene ablation decreases inflammation in insulin resistance
lesion size in apoE-/- mice (Suppl. Figure 1B, available online at
www.thrombosis-online.com, apoE-/-Irs2+/+HL-/- vs apoE-/-
Irs2+/+HL+/+ mice p=0.05 and p<0.007). Consistent with previous
results (30), lesion was significantly increased in the aortic arch of
IR apoE-/-Irs2+/-HL+/+ mice compared with that in apoE-/-
Irs2+/+HL+/+ mice (Suppl. Figure 1B, available online at www.
thrombosis-online.com, p<0.01). Consistent with the results
shown in ▶ Figure 2, HL- inactivation in IR mice diminished
atherosclerosis (Suppl. Figure 1B, available online at www.throm
bosis-online.com, apoE-/-Irs2+/-HL-/- vs apoE-/-Irs2+/+HL+/+,
p<0.0001 and p<0.003) indicating a proatherogenic role of HL in
MetS/IR states. Therefore, Irs2 haploinsufficiency increases
atherosclerosis in apoE-/- mice which is ameliorated by genetic ab-
lation of HL.
Thrombosis and Haemostasis 116.2/2016
Figure 4: Inflammation analysis in two
months atherogenic diet-fed apoE-/-
Irs2+/-HL+/+ and apoE-/-Irs2+/-HL-/- mice. A)
Circulating levels of MCP1, TNFα and IL6 in
mouse plasma. B) Circulating leukocyte, lympho-
cyte, CD115+ monocyte and neutrophil numbers
in both genotypes. C) Circulating CD115+Ly6Clow-
and CD115+Ly6Chi-monocyte subsets in mouse
blood. D) Circulating total and activated T-lymp-
hocytes identified as CD3+ cells and CD3+CD69+
cells, respectively. E) Quantification of CD4+ and
CD8+ T-cells and activated CD4+CD69+ and
CD8+CD69+ T-cells in mice. Statistical analysis
was performed using the Student’s t-test.
Atheroma plaque composition analysis revealed a reduced
macrophage (▶ Figure 3 A, p<0.03) and T-lymphocyte (▶ Figure
3 B, p<0.05) content in apoE-/-Irs2+/-HL-/- lesions compared with
apoE-/-Irs2+/-HL+/+ lesions. ApoE-/-Irs2+/-HL-/- mice exhibited de-
creased collagen content, smaller necrotic core areas (▶ Figure 3 C,
p<0.05 and ▶ Figure 3 D, p<0.05) and enhanced fibrous cap thick-
ness (▶ Figure 3 E, p<0.05).
Decreased atheroma lesion size in apoE-/-Irs2+/-HL-/-
mice is associated with reduced LIGHT/Lymphotoxin-β
receptor (LTβ-R) pathway and proliferation
Circulating MCP1, TNF-α and IL6 plasma levels were decreased in
apoE-/-Irs2+/-HL-/- mice compared with those in apoE-/-Irs2+/-HL+/+
mice (▶ Figure 4 A, p<0.01, p<0.03 and p<0.03, respectively). Leu-
© Schattauer 2016
 Andrés-Blasco, Vinué et al. HL gene ablation decreases inflammation in insulin resistance 7

Figure 5: Expression of the LIGHT/LTβ-R in-

flammatory axis and proliferation rate in
aortic cross sections of atherogenic diet-fed
apoE-/-Irs2+/-HL+/+ and apoE-/-Irs2+/-HL-/- mice.
Circulating plasma levels of LIGHT (A) and
quantitative analysis (B) of LTβ-R-positive cells in
lesions. Double immunofluorescence with
LTβ-R/F4/80 identifies macrophages expressing
LTβ-R. C) Representative images from the double
immunofluorescences quantified in B are shown.
Enlarged views of the boxed area in the images
are shown. D) Proliferating macrophages in
lesions expressed as the number of
Ki67/F4/80-positive cells for both groups of mice.
Dotted lines delimit the medial area of the vessel
wall. Statistical analysis was performed using the
Student´s t-test.

kocyte analysis demonstrated a diminished number of total leuko-
cytes, monocytes and lymphocytes in apoE-/-Irs2+/-HL-/- mice
(▶ Figure 4 B, p<0.03, p<0.003 and p<0.02 and Suppl. Figure 4,
available online at www.thrombosis-online.com). Compared with
apoE-/-Irs2+/-HL+/+ controls, apoE-/-Irs2+/-HL-/- mice had a reduced
number of Ly6Chi-monocyte subset (▶ Figure 4 C, p<0.0003) with-
out changes in the Ly6Clow-monocytes (▶ Figure 4 C). ApoE-/-
Irs2+/-HL-/- mice also exhibited decreased numbers of CD3+ (▶ Fig-
ure 4 D, p<0.006), CD3+CD69+ (▶ Figure 4 D, p<0.0003), CD4+
(▶ Figure 4 E, p<0.03), CD4+CD69+ (▶ Figure 4 E, p<0.001), CD8+
(▶ Figure 4 E, p<0.002) and CD8+CD69+ (▶ Figure 4 E, p<0.0001)
cells.
HL expression has been associated with LIGHT/Lymphotox-
in-β receptor (LTβ-R) (28, 32) pathway, therefore this axis was in-
vestigated. Circulating plasmatic levels of LIGHT were decreased
in apoE-/-Irs2+/-HL-/- mice compared with apoE-/-Irs2+/-HL+/+ mouse
levels (▶ Figure 5 A, p<0.02). Consistently, LTβ-R-positive cell
content in atheromas was reduced in apoE-/-Irs2+/-HL-/- compared
with that in apoE-/-Irs2+/-HL+/+ controls (▶ Figure 5 B, p<0.03). Im-
munofluorescence for LTβ-R/F4/80 demonstrated expression of
the receptor in macrophages in lesions from both genotypes (im-
ages in 5C). Moreover, Light and Ltβ-r mRNA levels were de-
creased in aorta from apoE-/-Irs2+/-HL-/- mice compared with
apoE-/-Irs2+/-HL+/+ controls (Suppl. Figure 5A, available online at
www.thrombosis-online.com, p<0.007 and p<0.02, respectively).
Mcp1 and Tnfα mRNA levels were also diminished in aorta from
apoE-/-Irs2+/-HL-/- mice (Suppl. Figure 5B, available online at www.
thrombosis-online.com, p<0.02 and p<0.03, respectively). There-
fore, reduced lesion size in apoE-/-Irs2+/-HL-/- mice is associated
with decreased inflammation and LIGHT/LTβ-R axis.

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8 Andrés-Blasco, Vinué et al. HL gene ablation decreases inflammation in insulin resistance
Macrophage proliferation is a key event in atheroma growth,
thus, this event was evaluated. In vivo analysis demonstrated a re-
duced number of proliferating Ki67+/F4/80+-macrophages in
apoE-/-Irs2+/-HL-/- mouse lesions compared with those of apoE-/-
Irs2+/-HL+/+ controls (▶ Figure 5 D, p<0.04). Isolated macrophages
from apoE-/-Irs2+/-HL-/- mice also exhibited a decreased prolifer-
ative rate compared with that in apoE-/-Irs2+/-HL+/+ controls
(Suppl. Figure 6A, available online at www.thrombosis-online.
com, p<0.003). Consistent with reduced proliferation, mRNA lev-
els of the CDK4/6 kinase cell-cycle inhibitor p15Ink4b in apoE-/-
Irs2+/-HL-/- macrophages were increased (Suppl. Figure 6B, avail-
able online at www.thrombosis-online.com, p<0.04) without
changes in the other cell-cycle inhibitor p16Ink4a. Moreover, apoE-/-
Irs2+/-HL-/- macrophages had diminished migratory capacity, as
Thrombosis and Haemostasis 116.2/2016
Figure 6: Exposure of atherogenic diet-fed
apoE-/-Irs2+/-HL-/- mice to murine recombi-
nant LIGHT aggravates atherosclerosis. A)
En face atherosclerosis analysis in Oil Red-O
stained whole-mounted aorta from apoE-/-
Irs2+/-HL-/- mice treated with vehicle or with
LIGHT. B) Circulating leukocyte, lymphocyte,
CD115+ monocyte and neutrophil numbers in
both groups of mice. C) Circulating
CD115+Ly6Clow- and CD115+Ly6Chi-monocyte
subset analysis by flow cytometry in vehicle- and
LIGHT-treated mice. D) Total and activated circu-
lating T-lymphocytes identified as CD3+ and
CD3+CD69+. E) T-cell CD4+- and CD8+-subsets
and their respective activated subpopulations,
CD4+CD69+ and CD8+69+. Representative photo-
graphs of whole mounted aortas are shown.
Statistical analysis was performed using the Stu-
dent´s t-test.
demonstrated by the presence of higher wound-healing area at 24
h of migration compared with apoE-/-Irs2+/-HL+/+ controls (Suppl.
Figure 6C, available online at www.thrombosis-online.com,
p < 0.04). Therefore, decreased proliferation and migration pro-
voked by HL-deficiency might account for the decreased macro-
phage lesion content.
Treatment with LIGHT increases lesion development
and proinflammatory circulating Ly6Chi pool in
apoE-/-Irs2+/-HL-/- mice
The effect of LIGHT-treatment on apoE-/-Irs2+/-HL-/- mice was next
evaluated (▶ Figure 6). Treatment of apoE-/-Irs2+/-HL-/- mice with
murine LIGHT enhanced atheroma lesion size compared with ve-
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 Andrés-Blasco, Vinué et al. HL gene ablation decreases inflammation in insulin resistance 9

Figure 7: In vivo effect of acute exposure of

control diet-fed apoE-/- mice to murine rec-
ombinant LIGHT on leukocyte activation
and proliferation. A) Circulating leukocyte,
lymphocyte, CD115+ monocyte and neutrophil
numbers in both groups of mice. B) Circulating
CD115+Ly6Clow- and CD115+Ly6Chi-monocyte
subsets by flow cytometry in vehicle- and LIGHT-
treated mice. C) Total and activated circulating
T-lymphocytes identified as CD3+ and
CD3+CD69+. D) T-cell CD4+- and CD8+-subsets
and their respective activated subpopulations,
CD4+CD69+ and CD8+69+. E) Proliferating circu-
lating monocytes in vehicle- and LIGHT-treated
apoE-/- mice detected as doubly CD115+BrdU+
cells (left panel) and CD115+Ly6Chi+BrdU+ cells
(right panel). Representative flow cytometry plots
are shown. Statistical analysis was performed
using the Student´s t-test.

hicle-treated apoE-/-Irs2+/-HL-/- mice (▶ Figure 6 A, p<0.02 and
Suppl. Figure 7A, available online at www.thrombosis-online.com,
p<0.008). Lesion characterisation showed no differences in macro-
phage content but revealed increased T-cell number in LIGHT-
treated mice (Suppl. Figure 7B-C, available online at www.throm
bosis-online.com, p<0.05). LIGHT-treated apoE-/-Irs2+/-HL-/- mice
displayed increased leukocytes (▶ Figure 6 B, p<0.001) and neut-
rophils (▶ Figure 6 B, p<0.001) with no effect on total lymphocytes
in and monocytes. However, Ly6Chi-monocytes (▶ Figure 6 C,
p< 0.05), CD3+T-cells (▶ Figure 6 D, p< 0.001), CD4+T-cells
(▶ Figure 6 E, p< 0.0008) and CD8+T-cells (▶ Figure 6 E, p< 0.003)
were augmented in LIGHT-treated apoE-/-Irs2+/-HL-/- mice com-
pared with vehicle-treated mice. Thus, LIGHT increases the circu-
lating pool of Ly6Chi-monocytes, T-cells and lesion development
in vivo.
Decreased LIGHT was associated with lower proliferation in
atheromas, therefore, the effect of LIGHT in this process was
evaluated in regular diet-fed apoE-/- mice. Acute LIGHT-treatment
in apoE-/- mice did not change leukocyte, lymphocyte, monocyte
or neutrophil numbers (▶ Figure 7 A) but Ly6Chi-monocytes
(▶ Figure 7 B, p<0.04) and CD8+CD69+ T-lymphocytes (▶ Figure
7 D, p<0.04) were significantly increased. Monocyte proliferation
analysis revealed enhanced BrdU+CD115+Ly6Chi monocytes in
LIGHT-treated mice compared with controls (▶ Figure 7 E,
p<0.04). Therefore, LIGHT might enhance Ly6Chi-monocyte pool
by increasing monocyte proliferation.

© Schattauer 2016 Thrombosis and Haemostasis 116.2/2016

10 Andrés-Blasco, Vinué et al. HL gene ablation decreases inflammation in insulin resistance
Treatment with LIGHT increases activation of mitogen
activated protein kinase p38 in apoE-/- mice
LIGHT effects are partly mediated through the mitogen-activated
protein kinase (MAPK) p38 pathway, thus activation of this was
evaluated. Upon LIGHT-treatment, activation of p38 was aug-
mented in apoE-/- liver and macrophages (▶ Figure 8 A, p<0.04
and p<0.01). LIGHT-treatment also enhanced the mRNA and pro-
tein levels of HL in apoE-/- mouse liver (▶ Figure 8 B, p<0.01 and
p<0.04) and macrophages (▶ Figure 8 B, p<0.03 and p<0.03). Con-
sistent with a proinflammatory role for LIGHT and increased
Ly6Chi-monocytes, cytokine treatment augmented Tnfα mRNA
levels in apoE-/- mouse macrophages (▶ Figure 8 C, p<0.05). Inter-
estingly, HL-deficient mouse macrophages revealed defective
LIGHT-induced p38 activation compared with macrophages ex-
pressing intact HL (▶ Figure 8 D). These results indicate that
Thrombosis and Haemostasis 116.2/2016
Figure 8: Effect of LIGHT treatment on liver
and macrophage expression. A) Quantifi-
cation of p38 and activated phospho(p)p38 pro-
tein levels from vehicle- and LIGHT-treated
apoE-/- mouse liver and apoE-/- mouse macro-
phages. B) Quantification of mRNA and protein
HL levels in vehicle- and LIGHT-treated apoE-/-
mouse liver and apoE-/- mouse macrophages. C)
mRNA levels of Mcp1 and Tnfα in vehicle- and
LIGHT-treated apoE-/- mouse macrophages. D)
Quantification of p38 and activated phosp-
ho(p)p38 protein levels from vehicle- and LIGHT-
treated WT and HL-/- mouse macrophages. mRNA
levels were normalised to the endogenous cyclo-
philin expression and relativised to vehicle-
treated apoE-/- mouse macrophages. Phosphory-
lated p38 protein levels were normalised to total
p38 levels and HL protein levels were normalised
to β-actin protein levels. Statistical analysis was
performed using the Student´s t-test.
LIGHT induces a proinflammatory phenotype (enhanced Ly6Chigh
and TNFα) in apoE-/- mice via a p38-dependent mechanism and
that this is associated with enhanced HL macrophage expression.
However, HL-deficiency impairs activation of the p38-pathway.
Given the previously described effect of LIGHT on lipid levels,
these were analysed. Analysis showed no effect of LIGHT-treat-
ment in apoB-cholesterol, HDL-cholesterol or triglyceride levels in
apoE-/- or apoE-/-Irs2+/-HL-/- mice (Suppl. Figure V8A-B, available
online at www.thrombosis-online.com).
To understand the connection between LIGHT and the effect of
HL on monocyte/macrophages, experiments with isolated human
monocytes were performed. LIGHT-treatment increased the Mcp1
and Tnfα mRNA levels (Suppl. Figure 9A, available online at www.
thrombosis-online.com, p<0.0003 and p<0.005). However, silenc-
ing of HL gene (LIPC) with LIPC-siRNA in macrophages de-
creased LIGHT-induced expression of Mcp1 and Tnfα cytokine
© Schattauer 2016
 Andrés-Blasco, Vinué et al. HL gene ablation decreases inflammation in insulin resistance 11

Figure 9: Atherosclerosis burden analysis

and leukocyte expression in MetS and
MetS-IR subjects. A) The common carotid inti-
ma-media thickness (CC-IMT) (left panel) and
percentage of atherosclerotic plaques (right
panel) analysed by ultrasound in MetS and MetS-
IR subjects. B) Circulating plasma levels of sol-
uble LIGHT and Ltβ-r mRNA expression levels in
WBMCs from both groups of patients. C) Number
of classical CD14++CD16-, intermediate
CD14++CD16+ and alternative CD14+CD16++
monocytes in both groups of patients. D) Hl
mRNA expression levels in WBMCs in MetS and
MetS-IR subjects. E) Correlation studies between
Hl mRNA levels and HOMA index and insulin.
mRNA levels were normalised with endogenous
gapdh mRNA levels and were relativised to MetS
mRNA levels. Representative plots of the flow cy-
tometry are shown. Data are shown as mean ±
sem (A left panel, B, C, D), as a percentage (A
right panel) and as individual data points for cor-
relation studies (E). Statistical analysis was done
using the Student’s t-test (A left panel, B, C, D),
Chi-square test (A right panel), and the Spear-
man correlation coefficient (E).

mRNAs compared with Control-siRNA macrophages (Suppl. Fig-
ure 9A, available online at www.thrombosis-online.com, p<0.0003
and p<0.008, respectively). Consistent with decreased lesion size
and inflammation in HL-deficient mice, HL inactivation decreases
inflammatory macrophage phenotype.
Atherosclerosis progression is associated with
increased LIGHT/LTβ-R pathway activity and hepatic
lipase expression in metabolic syndrome-insulin
resistance patients
To analyse the clinical impact of the LIGHT/LTβ-R pathway and
HL, human MetS and MetS-IR subjects were studied. The demo-
graphic and clinical characteristics of these patients are summa-
rised in Suppl. Table 1 (available online at www.thrombosis-on
line.com). No differences in gender or age distribution were found
but MetS-IR subjects had higher BMI-index, waist circumference,
homeostatic model assessment (HOMA)-index score, and en-
hanced plasma levels of glucose, insulin, and triglycerides than
MetS individuals. Conversely, HDL-c levels were lower in MetS-
IR. Diabetes was more prevalent in MetS-IR patients and these had
a higher frequency of oral hypoglycaemic medication and a lower
frequency of lipid-lowering therapy than MetS controls.
Consistent with previous studies (20, 33), examination of the
human carotid artery revealed an increase in the common carotid
artery intima-media thickness (CC-IMT) in MetS-IR compared

© Schattauer 2016 Thrombosis and Haemostasis 116.2/2016

12 Andrés-Blasco, Vinué et al. HL gene ablation decreases inflammation in insulin resistance
with MetS subjects (▶ Figure 9 A, 0.616 vs 0.704, p<0.002). The
atheroma plaque incidence was enhanced, but not significantly, in
MetS-IR compared with MetS patients (▶ Figure 9 A, 35.3 % vs
20.8 %, p=0.07).
Analysis of the LIGHT/LTβ-R pathway demonstrated increased
circulating LIGHT plasma levels and enhanced Ltβ-r mRNA levels
in white blood mononuclear cells (WBMC) in MetS-IR subjects
compared with MetS controls (▶ Figure 9 B, p<0.002 and p<0.05).
Monocyte analysis using CD14 and CD16 markers (34) demon-
strated higher number of the inflammatory intermediate
CD14++CD16+-monocytes in MetS-IR subjects compared with
those in MetS individuals (▶ Figure 9 C, p<0.01). Thus, MetS-IR
subjects display increased inflammation.
Compared with MetS, MetS-IR subjects had higher HL mRNA
levels in WBMC (▶ Figure 9 D, p<0.0003). Moreover, HL mRNA
levels from all MetS were positively correlated with the HOMA
index (▶ Figure 9 E, p<0.04) indicating that WBMC-derived HL is
modulated by the severity of IR. Consistent with the results in the
mouse model of MetS/IR, increased atherosclerosis in MetS-IR
subjects associates with up-regulation of the LIGHT/LTβ-R path-
way, increased inflammatory monocytes and augmented HL gene
expression.
Discussion
Coexistence of IR and MetS in humans increases the risk of CVD
and there is a medical need to reduce the residual CV risk of these
patients (4). HL is a classic metabolic enzyme that modulates lipo-
protein and lipid metabolism (7, 10, 11) but has an ill-defined role
in inflammatory cells. In this study, we demonstrate that HL ex-
pression is increased in circulating leukocytes from MetS-IR sub-
jects, who also display enhanced number of inflammatory mono-
cytes, which predict CV events (34), and enhanced atherosclerosis.
HL gene ablation in a mouse model of MetS/IR markedly reduced
inflammation, T-cell activation, proinflammatory monocytes and
atherosclerosis progression, despite increased lipid levels. Lesions
in HL-deficient MetS/IR mice, showed decreased macrophage and
T-lymphocyte infiltration, a reduced necrotic core area and in-
creased fibrous cap thickness. Notably, apoE-/-Irs2+/-HL-/- mice ex-
hibited reduced LIGHT/LTβ-R pathway expression, an axis pre-
viously involved in T-cell maturation, macrophage activation, in-
flammation and HL expression (28, 35). In addition, the levels of
proinflammatory Ly6Chi-monocytes and activated CD4+ and
CD8+ T-lymphocytes were markedly decreased in apoE-/-
Irs2+/-HL-/- mice which also displayed diminished circulating levels
of MCP1, TNFα and IL6. Interestingly, macrophage proliferation,
a key process in plaque expansion, was reduced in lesions and in
macrophages from apoE-/-Irs2+/-HL-/- mice. Consistently, treatment
of apoE-/-Irs2+/-HL-/- mice with murine LIGHT increased Ly6Chi-
monocytes and lesion size indicating a proatherogenic role of
LIGHT in inflammation and lesion progression. In addition, treat-
ment of apoE-/- mice with LIGHT augmented the number of prolif-
erating Ly6Chi-monocytes suggesting that LIGHT/LTβ-R axis
might promote lesion formation by enhancing the proliferation
Thrombosis and Haemostasis 116.2/2016
rate of these invasive monocytes. LIGHT mediated its effects in
vivo by a p38 MAPK-dependent mechanism as activation levels of
this kinase were increased upon LIGHT treatments. In agreement
with a proatherogenic role for LIGHT/LTβ-R pathway, increased
atherosclerosis in MetS-IR subjects was associated with its up-
regulation indicating that our findings are highly clinically rel-
evant. Altogether these results indicate that genetic inactivation of
HL in MetS/IR apoE-/-Irs2+/- mice reduces atheroma plaque pro-
gression by decreasing systemic inflammation and macrophage
proliferation which may be due to diminished LIGHT/LTβ-R axis
expression.
High HL activity might be detrimental in the majority of the
cases with hypertriglyceridaemia and dyslipidaemia associated
with IR/MetS (7, 10, 11). Thus, low HL activity is highly desirable
in MetS-IR subjects as it would presumably prevent atheroma pro-
gression. We found that MetS-IR patients exhibiting increased
subclinical atherosclerosis and inflammatory monocytes had en-
hanced HL gene expression. HL expression was positively corre-
lated with the HOMA-index indicating that HL gene is up-regu-
lated by IR in leukocytes. In addition, HL-inactivation in a pre-
clinical mouse model diminished in vivo foam cell formation, de-
creased atherosclerosis and ameliorated inflammation. Our results
indicating that HL has a detrimental effect on MetS-IR are consist-
ent with previous studies showing that increased HL expression
and activity in a fructose-fed rodent model is associated with IR
(36). Moreover, decreased atherosclerosis in MetS/IR HL-deficient
mice was observed despite increased lipid levels, which is consist-
ent with investigations showing that apoE-/-HL-/- mice display re-
duced atherosclerosis but enhanced apoB-containing lipoproteins
(8, 13). Consistent with our findings, reduced abdominal obesity
in humans is associated with reduced HL activity and prevention
of T2DM (4, 13) and abdominal fat reduction modifies the effect
that HL polymorphisms have on myocardial infarction risk in
women (37). Together, these studies suggest that HL-inactivation
could be beneficial in metabolic alterations with a particularly
proinflammatory lipoprotein phenotype such as the one induced
by IR.
All subjects used in the study were high-risk patients and there-
fore, the confounding effects of high-dose medications and the use
of combined therapies in MetS-IR subjects could not be avoided.
The most frequent medications in MetS-IR subjects were met-
formin as an oral hypoglycaemia-inducing drug and statins as a
lipid-lowering drug therapy, which have been described as able to
exert anti-inflammatory action (38, 39). Despite the fact that these
treatments can modify inflammatory parameters, we observed
augmented LIGHT/LTβ-R axis and increased numbers of mono-
cytes in MetS-IR patients, indicating that these medications did
not affect the results or the conclusions we drew from them.
Accumulation of inflammatory cells and mediators beneath the
fibrous cap makes plaques unstable and promotes clinical syn-
dromes (17). HL-deficiency in MetS/IR mice decreased inflamma-
tory infiltration, lowered cell proliferation and migration, reduced
cytokine production and induced features of stable plaques (dim-
inished necrotic core area and increased fibrous cap thickness) (20,
40). Plaques also displayed reduced collagen content, whose pres-
© Schattauer 2016
 Andrés-Blasco, Vinué et al. HL gene ablation decreases inflammation in insulin resistance
ence provides stability, but in plaques with low inflammatory infil-
tration and decreased necrotic area, as in this case, are indicating
the presence of less-advanced plaques. Although lesional macro-
phages might effectively eliminate cellular debris, the macrophage
accumulation observed in apoE-/-Irs2+/-HL+/+ mice seems to be
representative of a chronic unresolved inflammatory process (16,
41) because atheroma size was exacerbated. Therefore, decreased
atheroma inflammation and leukocyte infiltration could be one of
the main mechanisms by which HL absence ameliorates plaque
progression.
Reduced atherosclerosis and leukocyte infiltration in apoE-/-
Irs2+/-HL-/- mice were associated with decreased LIGHT plasma
levels and reduced LIGHT/LTβ-R inflammatory axis in lesions.
Interestingly, this pathway has previously been linked to changes
in HL expression (28, 32). In agreement with a role for LIGHT in
T-lymphocyte activation and maturation (35), reduced LIGHT
levels were correlated with decreased activated T-cells in apoE-/-
Irs2+/-HL-/- mice. MCP1, TNF-α and IL6 plasma levels were also
diminished being consistent with studies showing reduced pro-
duction of cytokines in LIGHT-deficient macrophages (42) and
with other investigations indicating that LIGHT overexpression
leads to severe inflammation (35). Decreased LIGHT levels co-
incided with reduced Ly6Chi-monocyte subset, which have an in-
vasive phenotype in lesions (43) and whose recruitment is highly
dependent on MCP1 cytokine in atheromas (44). These findings
are consistent with a lower migration, proliferation and accumu-
lation of macrophages in atheromas in apoE-/-Irs2+/-HL-/- mice
therefore pointing to that atherosclerosis in apoE-/-Irs2+/-HL+/+
mice could be mediated by activation of the LIGHT/LTβ-R in-
flammatory pathway.
In support of the above findings, LIGHT-treatment increased
the pool of the inflammatory Ly6Chi-monocytes and T-lympho-
cytes and accelerated atherosclerosis in LIGHT-treated apoE-/-
Irs2+/-HL-/- mice. Notably, MetS-IR subjects exhibited increased
numbers of CD14++CD16+ proinflammatory (likely invasive)
monocytes, enhanced atherosclerosis, and augmented LIGHT/
LTβ-R inflammatory axis expression. Thus, atherosclerosis pro-
gression in MetS/IR subjects and augmented plaque size in mice,
might be partly caused by increased activation of the LIGHT/
LTβ-R pathway. Moreover, our results also suggest that LIGHT
might accelerate atherosclerosis by increasing T cells, proinflam-
matory monocytes and lesion proliferation. Consistent with our
findings, previous investigations in humans have shown increased
plasmatic LIGHT levels in patients with unstable angina (45) and
enhanced circulating LTβ-R levels in atherosclerosis (46). How-
ever, in experimental atherosclerosis, discrepant results have been
obtained in mice lacking LTβ-R (47, 48). In one of the studies inac-
tivation of the Ltβr gene in apoE-/- mice decreased atherosclerosis
by augmenting the Ly6Clow-monocyte subset pool (47), while in
the study by Hu and colleagues, apoE-/-Ltβr-/- mice displayed accel-
erated atherosclerosis and demonstrated that VSMC-LTβ-R path-
way is atheroprotective by maintaining T-cell homeostasis and the
size of aortic tertiary lymphoid organs (48). Although, our results
agree with one of these studies (47), Ltβr-inactivation and acute
LIGHT-treatment are very different approaches because they are
© Schattauer 2016
13
not exclusive partners (LTβR has another ligand, LTβ, and LIGHT
has another receptor, the herpesvirus entry mediator, HVEM). Al-
together, these studies unveil a complex role of LIGHT/LTβ-R-de-
pendent signalling in atherosclerosis, which might exert divergent
effects in different cell-types (VSMCS vs monocytes/macrophages)
and in different experimental settings. In addition, ours is the first
study to demonstrate involvement of the LIGHT/LTβ-R axis in
MetS/IR cardiovascular complications in mice as well as in hu-
mans.
Of note, LIGHT treatment induced a 30 % increase in athero-
sclerosis in apoE-/-Irs2+/-HL-/- mice while HL-deficiency reduced
atherosclerosis by 48 % in apoE-/-Irs2+/- mice indicating that addi-
tional HL-mediated proatherogenic mechanisms, independent of
LIGHT effects, contribute to plaque growth. In this sense, previous
studies have shown that HL activity might promote atherosclerosis
in apoE-/- mice by generating small dense LDL which are retained
in the subendothelial space and facilitate atheroma development
(8). Moreover, a role for macrophage-derived HL has been sug-
gested in foam-cell formation as HL-deficient macrophages dis-
play decreased oxLDL uptake in vitro (25) owing to its role facili-
tating lipoprotein uptake. In fact, decreased LIGHT/LTβ-R path-
way expression in HL-deficient apoE-/-Irs2+/- mice was associated
with diminished peritoneal foam cells, a process in which LIGHT/
LTβ-R axis has previously been involved (45). Thus, decreased HL
and reduced LIGHT/LTβ-R axis may also independently con-
tribute to foam-cell formation and therefore have additional ef-
fects on lesion progression
What is known about this topic?
• Type 2 diabetes mellitus (T2DM) and metabolic syndrome (MetS)
patients have a high risk of atherosclerosis and cardiovascular
disease (CVD).
• Human genetic studies have linked hepatic lipase (HL) gene poly-
morphisms to an increased risk of CVD in T2DM subjects.
• HL promotes atherosclerosis by its presence in leukocytes.
• Atherosclerosis progression induced by inflammatory mediators
or cells is accompanied by changes in HL expression and activity.
What does this paper add?
• Genetic ablation of HL, in a mouse model of MetS and insulin
resistance (IR), reduces inflammation and atherosclerosis pro-
gression.
• Decreased atherosclerosis, induced by HL inactivation, is partly
mediated by reduced LIGHT/Lymphotoxin β-receptor (LTβ-R) acti-
vation.
• MetS subjects with IR and subclinical atherosclerosis display
up-regulation of the LIGHT(TNFSF14)/LTβ-R axis, increased pro-
inflammatory monocytes and HL mRNA expression in circulating
leukocytes.
• This study demonstrates a main role of LIGHT(TNFSF14)/LTβ-R
pathway in atherosclerosis and inflammation associated with
atherosclerosis burden in MetS/IR.
Thrombosis and Haemostasis 116.2/2016
14 Andrés-Blasco, Vinué et al. HL gene ablation decreases inflammation in insulin resistance
Increased inflammation and proliferating Ly6Chi-monocytes
induced by LIGHT seemed to be mediated by p38 as LIGHT-treat-
ments in apoE-/- mice and macrophages augmented the activation
of this kinase. These results are consistent with a function of
LIGHT in VSMC proliferation and macrophage migration via a
p38-dependent mechanism (49) and with p38 playing a role in
amplifying inflammatory signals and macrophage survival (50).
Interestingly, a function in atherosclerosis has been suggested for
p38 because inhibition of this kinase decreases macrophage pha-
gocytic activity and atherosclerosis in apoE-/- mice (51). Interest-
ingly, HL-deficiency in macrophages blunted LIGHT-induced p38
activation and HL silencing in human macrophages also blocked
LIGHT-induced Mcp1 and Tnfα expression. Therefore, HL-defi-
ciency might limit atheroma progression in MetS/IR by restraining
inflammation, including decreased LIGHT expression, and by re-
ducing survival properties of macrophages. Given that HL defi-
ciency also decreased p38-MAPK activation, a potential hypoth-
esis for diminished LIGHT levels could be an impaired p38-me-
diated response in LIGHT-producing cells, a hypothesis that needs
to be further investigated.
Taken together our observations demonstrate that HL modu-
lates inflammation and macrophage proliferation in MetS/IR by a
LIGHT/LTβ-R axis-dependent mechanism and that HL inacti-
vation might be beneficial in MetS/IR. In addition, ours is the first
study to demonstrate an involvement of LIGHT/LTβ-R axis in
MetS/IR cardiovascular complications in mice and in humans.
Therefore, these studies suggest that inactivation of this inflamma-
tory pathway might limit monocyte/macrophage proliferation,
lymphocyte activation and atheroma progression in MetS/IR. Fu-
ture studies are warranted to fully assess whether these approaches
might be useful for preventing atherosclerosis.
Acknowledgements
We thank Guadalupe Herrera for flow cytometry and Ana Díaz for
animal care.
Conflicts of interest
None declared.
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