biomolecules

Article
Inhibiting Extracellular Cathepsin D Reduces Hepatic
Steatosis in Sprague–Dawley Rats †
Princy Khurana 1,‡ , Tulasi Yadati 2,‡ , Sandeep Goyal 1 , Atul Dolas 1 , Tom Houben 2 ,
Yvonne Oligschlaeger 2 , Anil K. Agarwal 3 , Aditya Kulkarni 1,4 and Ronit Shiri-Sverdlov 2, *
1 Aten Porus Lifesciences Pvt Ltd., Bengaluru, Karnataka 560068, India; [email protected] (P.K.);
[email protected] (S.G.); [email protected] (A.D.); [email protected] (A.K.)
2 Department of Molecular Genetics, School of Nutrition and Translational Research in Metabolism (NUTRIM),
Maastricht University, 6229 ER Maastricht, The Netherlands; [email protected] (T.Y.);
[email protected] (T.H.); [email protected] (Y.O.)
3 Department of Chemistry, CHRIST (Deemed to be University), Bengaluru, Karnataka 560029, India;
[email protected]
4 Avaliv Therapeutics, Naples, FL 34120, USA
* Correspondence: [email protected]; Tel.: +31-43-388-1746; Fax: +31-43-388-4574
† Short title: Extracellular cathepsin D inhibitor for fatty liver disease.
‡ These authors contributed equally to this work.




Received: 26 March 2019; Accepted: 2 May 2019; Published: 4 May 2019


Abstract: Dietary and lifestyle changes are leading to an increased occurrence of non-alcoholic fatty
liver disease (NAFLD). Using a hyperlipidemic murine model for non-alcoholic steatohepatitis (NASH),
we have previously demonstrated that the lysosomal protease cathepsin D (CTSD) is involved with
lipid dysregulation and inflammation. However, despite identifying CTSD as a major player in
NAFLD pathogenesis, the specific role of extracellular CTSD in NAFLD has not yet been investigated.
Given that inhibition of intracellular CTSD is highly unfavorable due to its fundamental physiological
function, we here investigated the impact of a highly specific and potent small-molecule inhibitor
of extracellular CTSD (CTD-002) in the context of NAFLD. Treatment of bone marrow-derived
macrophages with CTD-002, and incubation of hepatic HepG2 cells with a conditioned medium
derived from CTD-002-treated macrophages, resulted in reduced levels of inflammation and improved
cholesterol metabolism. Treatment with CTD-002 improved hepatic steatosis in high fat diet-fed rats.
Additionally, plasma levels of insulin and hepatic transaminases were significantly reduced upon
CTD-002 administration. Collectively, our findings demonstrate for the first time that modulation of
extracellular CTSD can serve as a novel therapeutic modality for NAFLD.

Keywords: NAFLD; lysosomal enzyme; extracellular cathepsin D; small-molecule inhibitor

1. Introduction
Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases in the world
with an estimated prevalence of 25% in the adult population [1]. Histologically, NAFLD ranges from
steatosis, which is the benign accumulation of macro-vesicular and micro-vesicular lipid droplets in
hepatocytes, to the more severe form of NAFLD referred to as non-alcoholic steatohepatitis (NASH). NASH
is a combination of hepatic steatosis and inflammation with or without fibrosis [2], which can progress
further to severe conditions such as liver cirrhosis and liver failure [3,4]. Due to the lack of mechanistic
insights into the pathogenesis of NAFLD [5], clinically-approved treatment options do not yet exist.
NAFLD is characterized by an impaired hepatic lipid profile, which is in turn characterized
by dysregulation of cholesterol and triglyceride metabolism [6]. It has been well-established that

Biomolecules 2019, 9, 171; doi:10.3390/biom9050171 www.mdpi.com/journal/biomolecules

Biomolecules 2019, 9, 171 2 of 12

lysosomes, in particular their lysosomal proteases such as cathepsins, play a crucial role in maintaining
metabolic processes such as lipid homeostasis [7–10]. Specifically, it has been shown that changes
in the expression of the lysosomal enzyme cathepsin D (CTSD) are associated with differences in
cholesterol metabolism [11–13]. Relevantly, though CTSD is mainly functional within the intracellular,
acidic milieu of lysosomes, in response to certain physiological and pathological conditions, CTSD is
secreted extracellularly [11]. Strikingly, and similar to its intracellular fraction, it has been shown
that CTSD is also proteolytically active upon secretion into the extracellular space [14,15]. Indeed,
given that CTSD retains its catalytic activity at neutral pH [16], these data indicate a potential role for
extracellular CTSD in lipid-related disorders such as NAFLD [17,18].
Recently, we have demonstrated that targeting the proteolytic activity of CTSD using pepstatin-A
(Pep-A), a known inhibitor of CTSD, remarkably reduced steatohepatitis in a hyperlipidemic mouse
model, thereby pointing towards a role for CTSD activity in NAFLD [19]. However, the observed
effects in this study were the result of a targeted inhibition of intracellular and extracellular CTSD.
Moreover, Pep-A is a potent but relatively unspecific inhibitor of aspartic proteases [20]. Hence, in the
current study, we developed a highly specific and potent small-molecule inhibitor of extracellular
CTSD, referred to as CTD-002 (IC50 = 28 nM) in order to investigate its efficacy in treating NAFLD.
We hypothesized that specific inhibition of extracellular CTSD activity has a therapeutic benefit for
NAFLD. In order to test our hypothesis, Sprague–Dawley (SD) rats fed a high-fat diet (HFD), known as
the Lieber–Decarli diet, were used as an in vivo NAFLD model [21] and were injected with the CTD-002.
In addition, wild-type (Wt) bone marrow-derived macrophages (BMDMs) treated with CTD-002 were
used as an in vitro model to assess the ability of CTD-002 to reduce inflammation. Moreover,
to investigate whether the CTD-002 inhibitor influences the inflammatory crosstalk between hepatic
immune and parenchymal cells, a conditioned medium derived from BMDMs was transferred to HepG2
liver cells. Treating BMDMs with CTD-002 resulted in a decrease in inflammation and improvement in
lipid metabolism. In addition, incubating hepatic HepG2 cells with the conditioned medium derived
from CTD-002-treated BMDMs showed a decreasing trend in inflammation compared to control
cells. Sprague–Dawley rats fed an HFD showed decreased hepatic steatosis and inflammation upon
treatment with CTD-002. Altogether, these data suggest that targeting extracellular CTSD potentially
represents a novel and effective therapeutic strategy for NAFLD.

2. Materials and Methods

2.1. Design and Development of CTD-002

CTD-002 was designed using the Schrodinger Small Molecule Drug Design Suite and medicinal
chemistry efforts. In this process, a library of ~500,000 commercially available compounds were
screened in silico using the pharmacophore modelling [22,23] in PHASE module and docking the top
10,000 molecules via the GLIDE XP [24,25] module of the Schrodinger suite (v7.2, Schrödinger, LLC).
This resulted in the identification of a set of 100 molecules with diverse chemical structures. These
molecules were then further screened against CTSD in a cell-free assay to identify those compounds
that had micromolar or higher potency. The compounds with the lowest permeability and efflux were
selected as exerting most of its effects extracellularly. Finally, these compounds were further refined
and modified to create a library of compounds with nanomolar efficacy against CTSD, with CTD-002
being the most potent small-molecule inhibitor, showing a dose-dependent inhibition of CTSD activity
(Figure S1). While the compound may have minor effects of intracellular CTSD, toxicity studies in
Sprague–Dawley rats and BALB/c mice, a widely used mouse strain, showed no toxic reactions at the
therapeutic dose of 50 mg/kg, which was injected twice a week for six weeks.

2.2. Isolation and Culturing of Bone Marrow-Derived Macrophages

Tibiae and femurs of wild type mice belonging to C75Bl/6 strain were broken and used for
harvesting bone marrow-derived macrophages (BMDMs). The culture medium in which the BMDMs
Biomolecules 2019, 9, 171 3 of 12

were cultured was an RPMI medium (GIBCO Invitrogen, Breda, the Netherlands) supplemented with
10% heat-inactivated fetal calf serum (Bodinco B.V. Alkmaar, the Netherlands), penicillin (100 U/mL),
streptomycin (100 µg/mL) and L-glutamine 2 mM (all derived from GIBCO Invitrogen, Breda,
The Netherlands). To differentiate between monocytes and macrophages, the culture medium was
also supplemented with 20% L929-conditioned medium (LCM) for 9 days. At day 10, BMDMs were
cultured in a 24-well plate at 350,000 cells per well. Next, the BMDMs’ culture medium was enriched
with oxidized low-density lipoprotein (oxLDL) for 24 h (25 µg/mL; Alfa Aesar: J65591, Wardhill, MA,
USA). Then, the macrophages were treated with CTD-002 (Aten Porus Lifesciences Pvt Ltd., India) or
with carrier-control dimethyl sulphoxide (DMSO) for 4 h. In order to increase the effects on inflammation,
cells were washed and further stimulated with lipopolysaccharide (LPS; 100 ng/mL) for 4 h.
To explore the possible effects of secreted CTSD on neighboring cells, hepatic HepG2 (ATCC
HB-8065) cells were incubated with the conditioned medium from CTD-002-treated BMDMs,
or control-treated BMDMs, for 4 h. Subsequently, cells were washed and stimulated with LPS
(100 ng/mL) for 4 h, after which the supernatant was collected for protein measurements and cells
were lysed for mRNA expression analyses.

2.3. Rats, Diet and Intervention

Animal experiments were conducted at TheraIndx Lifesciences Private Limited, following all
ethical practices as formulated in the guidelines for animal care and approved by the Institutional
Animals Ethics Committee (IAEC; protocol no. IAEC/05/2017/064), India. SD rats were given free
access to food and water and were housed under standard conditions. Following an acclimatization
period of seven days, rats were fed with either low-fat (LFD) or HFD for three weeks. The composition
of the diets is listed in Table S1. To test its therapeutic efficacy, CTD-002 was administered twice a
week intraperitoneally (dose 50 mg/kg) for three weeks. The experimental conditions are also shown
in Figure S2. After three weeks, the animals were sacrificed using CO2 anesthesia. Following blood
collection, the rats were dissected, liver tissues were excised, weighed and cut into small pieces
weighing ~100 to 200 mg. These liver tissues were frozen immediately in liquid nitrogen and stored
at −80 ◦ C for further analyses. In addition, liver samples were also fixed in 4% formaldehyde for
histologic examination. Blood was collected by cardiac puncture followed by termination, centrifuged
and plasma was harvested for biochemical analyses.

2.4. Histological Analyses

Formalin-fixed liver samples were stained with Hematoxylin-Eosin (H&E) in order to score fat
vesicles as minimal, mild, moderate and severe steatosis. Lobular inflammatory activity was scored as
follows: (1) focal collections of mononuclear inflammatory cells; (2) diffuse infiltrates of mononuclear
inflammatory cells and (3) focal collections of polymorphonuclear cells in addition to mononuclear cell
infiltrates. In addition to H&E, formalin-fixed liver samples stained with Chromotrope Aniline Blue and
Sirius Red were quantified for total hepatic fat percentage by means of digital image analysis in ImageJ.

2.5. Plasma Measurements

Plasma concentrations of alanine aminotransferase (ALT) [S.G.P.T ERBA kit, 120903; Baddi, India]
and aspartate aminotransferase (AST) [S.G.O.T ERBA kit, B081717; Baddi, India] were measured with
an enzymatic color test according to the manufacturer’s protocols and were measured using an ERBA
semi-automated biochemistry analyzer. Plasma insulin was measured using a rat enzyme-linked
immunosorbent assay kit [ab100578, Abcam; Cambridge, MA, USA].

2.6. Liver Tumor Necrosis Factor-Alpha Levels

The rat tumor necrosis factor-alpha (TNF-α) ELISA assay was performed on liver homogenates
according to the manufacturer’s instructions [ab100785, Abcam; Cambridge, MA, USA] using a
microplate reader.
Biomolecules 2019, 9, x 4 of 12
Biomolecules 2019, 9, 171 4 of 12
2.7. Statistical Analyses
Data were
2.7. Statistical statistically analyzed by performing two-tailed non-paired t-tests using GraphPad
Analyses
Prism, version 6.0 for Windows. Data were expressed as mean ± SEM and considered significant at p
Data
< 0.05. *, ** were
and ***statistically
indicate p <analyzed
0.05, 0.01by performing
and two-tailed non-paired t-tests using GraphPad
0.001, respectively.
Prism, version 6.0 for Windows. Data were expressed as mean ± SEM and considered significant at
p3.<Results
0.05. *, ** and *** indicate p < 0.05, 0.01 and 0.001, respectively.

3. Results
3.1. Decreased inflammation after inhibition of extracellular CTSD in oxLDL-loaded primary mouse
macrophages.
3.1. Decreased Inflammation after Inhibition of Extracellular CTSD in oxLDL-Loaded Primary
MouseWithMacrophages
the aim of exploring the influence of extracellular CTSD on inflammation in macrophages
and With
to test
thetheaimefficacy of thethe
of exploring small-molecule inhibitor CTD-002,
influence of extracellular CTSD onWt BMDMs were
inflammation isolated and
in macrophages
incubated with oxLDL for 24 h to stimulate the secretion of CTSD.
and to test the efficacy of the small-molecule inhibitor CTD-002, Wt BMDMs were isolated Subsequently, cells were treated
and
with either carrier control (DMSO) or CTD-002 (100 µ M) for 4 h, followed
incubated with oxLDL for 24 h to stimulate the secretion of CTSD. Subsequently, cells were treated by 4 h stimulation with
LPS. either
with Tumorcarrier
necrosis factor(DMSO)
control α (TNFα)orprotein
CTD-002 levels,
(100a µM)
pro-inflammatory
for 4 h, followed cytokine,
by 4 hmeasured
stimulationfrom the
with
medium
LPS. Tumorof necrosis
CTD-002-treated BMDMs,
factor α (TNFα) were levels,
protein significantly reduced compared
a pro-inflammatory to control
cytokine, measuredcellsfrom
(Figure
the
1A). Consistent with these results, gene expression levels of the pro-inflammatory
medium of CTD-002-treated BMDMs, were significantly reduced compared to control cells (Figure 1A). cytokines Tnfα and
chemokine with
Consistent (C-X-C motif)
these ligand-2
results, gene (Ccl2) significantly
expression levels ofdecreased in CTD-002-treated
the pro-inflammatory macrophages
cytokines Tnfα and
compared to control (DMSO)-treated cells (Figure 1B), confirming the
chemokine (C-X-C motif) ligand-2 (Ccl2) significantly decreased in CTD-002-treated macrophages reduction of inflammation
upon treatment
compared with CTD-002.
to control Additionally,
(DMSO)-treated extracellular
cells (Figure CTSD inhibition
1B), confirming also improved
the reduction cholesterol
of inflammation
metabolism, as evidenced by the upregulation of cytochrome P450 27a1 (Cyp27a)1,
upon treatment with CTD-002. Additionally, extracellular CTSD inhibition also improved cholesterol the main enzyme
responsible for
metabolism, the conversion
as evidenced by the of cholesterolofinto
upregulation bile acidsP450
cytochrome (Figure
27a1 1C). Similar
(Cyp27a)1, thedata
mainwere also
enzyme
observed in control conditions without oxLDL and LPS stimulation, showing reduced
responsible for the conversion of cholesterol into bile acids (Figure 1C). Similar data were also observed Tnfα and Ccl2
levels
in andconditions
control improved lipid oxLDL
without and energy
and LPS metabolism
stimulation, asshowing
evidenced by Cyp27a1,
reduced Tnfα and acetyl-CoA
Ccl2 levels
acetyltransferase 2 (Acat2), carnitine palmitoyltransferase1 (Cpt1) and
and improved lipid and energy metabolism as evidenced by Cyp27a1, acetyl-CoA acetyltransferase scavenger receptor A (Sr-a)2
expression after CTD-002 treatment (Figure S3). Collectively, these data indicate
(Acat2), carnitine palmitoyltransferase1 (Cpt1) and scavenger receptor A (Sr-a) expression after CTD-002 that modulating the
activity of(Figure
treatment extracellular CTSD has beneficial
S3). Collectively, these data effects
indicateon inflammation
that modulating andthe cholesterol
activity ofmetabolism,
extracellular at
least in vitro.
CTSD has beneficial effects on inflammation and cholesterol metabolism, at least in vitro.

Figure 1. Effects
Figure 1. Effects ofof CTD-002
CTD-002 on on inflammation
inflammation and and cholesterol
cholesterol metabolism
metabolism in in bone
bone marrow-derived
marrow-derived
macrophages.
macrophages. (A) (A) TNFα
TNFα protein
protein levels
levels measured
measured fromfrom supernatant
supernatant of of oxidized
oxidized low-density
low-density
lipoprotein (oxLDL)-loaded bone marrow-derived macrophages (BMDMs).
lipoprotein (oxLDL)-loaded bone marrow-derived macrophages (BMDMs). (B) Gene expression (B) Gene expression
of
of Tnfα and Ccl2
inflammation-related genes Tnfα and Ccl2 and (C) the cholesterol breakdown enzyme Cyp27a1 in
inflammation-related genes and (C) the cholesterol breakdown enzyme Cyp27a1 in
oxLDL-loaded
oxLDL-loaded bonebone marrow-derived
marrow-derived macrophages.
macrophages. Each
Each bar
bar represents
represents aa technical triplicate ±± SEM;
technical triplicate SEM;
meanspp<< 0.05,
**means ** pp << 0.01
0.05, ** 0.01 and ***pp <<0.001
and *** 0.001compared
compared to to dimethyl
dimethyl sulfoxide
sulfoxide (DMSO)-treated
(DMSO)-treated BMDMs
BMDMs
by means of two-tailed unpaired
by means of two-tailed unpaired t-test. t-test.
Biomolecules 2019, 9, 171 5 of 12
Biomolecules 2019, 9, x 5 of 12

3.2.
3.2. Inhibition of Macrophage-Derived Extracellular
Extracellular CTSD
CTSD Reduces
Reduces Inflammation
Inflammation in
in HepG2
HepG2 Cells
Cells
In
In order
order toto confirm
confirm whether
whether macrophage-derived
macrophage-derived extracellular
extracellular CTSD
CTSD influences
influences neighboring
neighboring
parenchymal
parenchymal cells,
cells, hepatic
hepatic HepG2 cells were were incubated
incubated with
with aa conditioned
conditioned medium
medium derived
derived from
from
macrophages
macrophages that that were
were treated
treated either
either with
with carrier-control
carrier-controlor orwith
withCTD-002
CTD-002(100 µ M)for
(100 µM) for 44 h.
h. To
To boost
boost
the
the effects
effectsononinflammation,
inflammation, HepG2
HepG2 cells cells
received LPS forLPS
received 4 h. for
HepG24 h.cells that received
HepG2 thereceived
cells that conditioned the
medium from macrophages that were treated with CTD-002 showed a
conditioned medium from macrophages that were treated with CTD-002 showed a trend towardstrend towards reduced TNFα
secretion
reduced TNFα(Figuresecretion
2A). Additionally,
(Figure 2A). HepG2 gene expression
Additionally, HepG2 levels of Ccl2 showed
gene expression levelsa of
decreasing
Ccl2 showedtrenda
compared
decreasingtotrend
HepG2 cells thattowere
compared HepG2incubated in the
cells that control-treated
were incubated inconditioned while Tnfα
medium,conditioned
the control-treated
did not show
medium, whileany effect
Tnfα did (Figure
not show 2B).
anyAdditionally,
effect (Figurewe also
2B). observed awe
Additionally, reduced expression
also observed of the
a reduced
cluster of differentiation
expression of the cluster 36 (Cd36) in hepatocytes
of differentiation 36 (Cd36)receiving the conditioned
in hepatocytes receivingmedium that wasmedium
the conditioned derived
from BMDMs
that was treated
derived with
from CTD-002,
BMDMs suggesting
treated an effect on
with CTD-002, hepatocyteanlipid
suggesting metabolism
effect (Figurelipid
on hepatocyte S4).
These in vitro(Figure
metabolism findings S4).suggest
These inthat macrophage-derived
vitro findings suggest that extracellular CTSD affectsextracellular
macrophage-derived the inflammatoryCTSD
status
affectsofthe
neighboring
inflammatory parenchymal cells and thatparenchymal
status of neighboring inhibiting extracellular CTSD
cells and that activity extracellular
inhibiting reduces this
inflammatory
CTSD activity response.
reduces this inflammatory response.

Figure 2.2.Effect
Figure Effectof of
medium
medium derived fromfrom
derived CTD-002-treated BMDMs
CTD-002-treated on HepG2
BMDMs cells. (A)cells.
on HepG2 TNFα(A)cytokine
TNFα
secretion of HepG2 cells cultured in a macrophage-conditioned medium that was
cytokine secretion of HepG2 cells cultured in a macrophage-conditioned medium that was treatedtreated with or
without CTD-002.
with or without (B) Gene
CTD-002. (B)expression levels of
Gene expression Tnfαofand
levels Ccl2
Tnfα andmeasured in HepG2
Ccl2 measured cells. cells.
in HepG2 ErrorError
bars
represent ± SEM.
meanmean
bars represent ± SEM.

3.3. Improved Metabolic Features after Inhibition of Extracellular CTSD in HFD-Fed Sprague–Dawley Rats
3.3. Improved Metabolic Features after Inhibition of Extracellular CTSD in HFD-Fed Sprague–Dawley Rats
To elucidate the metabolic effects of extracellular CTSD inhibition in vivo, metabolic parameters
To elucidate the metabolic effects of extracellular CTSD inhibition in vivo, metabolic parameters
were tested in HFD-fed SD rats that were injected with or without the CTD-002 inhibitor. Though
were tested in HFD-fed SD rats that were injected with or without the CTD-002 inhibitor. Though
food consumption was statistically similar among all experimental groups, a trend towards reduced
food consumption was statistically similar among all experimental groups, a trend towards reduced
food intake was apparent (Figure S5). By performing an H&E staining, hepatic steatosis was assessed
food intake was apparent (Figure S5). By performing an H&E staining, hepatic steatosis was assessed
based on the scoring of fat vesicles in the liver (Figure S6). The impact of HFD CTD-002-treated
based on the scoring of fat vesicles in the liver (Figure S6). The impact of HFD CTD-002-treated HFD-
HFD-fed SD rats showed a significant reduction in the amount of fat vesicles relative to control HFD-fed
fed SD rats showed a significant reduction in the amount of fat vesicles relative to control HFD-fed
rats, demonstrating the therapeutic benefit of extracellular CTSD inhibition in the context of hepatic
rats, demonstrating the therapeutic benefit of extracellular CTSD inhibition in the context of hepatic
steatosis, a main feature of NAFLD (Figure 3A). The impact of the HFD on hepatic fat deposition
steatosis, a main feature of NAFLD (Figure 3A). The impact of the HFD on hepatic fat deposition was
was further confirmed by quantification of H&E, Chromotrope Aniline Blue and Sirius Red staining,
further confirmed by quantification of H&E, Chromotrope Aniline Blue and Sirius Red staining,
showing increased fat deposition with the HFD (Figure S7).
showing increased fat deposition with the HFD (Figure S7).
To further assess whether inhibition of extracellular CTSD influences pathological parameters
To further assess whether inhibition of extracellular CTSD influences pathological parameters
related to NAFLD, plasma insulin levels were measured as an indicator of insulin sensitivity.
related to NAFLD, plasma insulin levels were measured as an indicator of insulin sensitivity. Plasma
Plasma insulin levels were significantly increased in HFD-fed SD rats compared to LFD-fed rats
insulin levels were significantly increased in HFD-fed SD rats compared to LFD-fed rats (Figure 3B).
(Figure 3B). Further, CTD-002-treated HFD-fed rats had significantly lower plasma insulin levels
Further, CTD-002-treated HFD-fed rats had significantly lower plasma insulin levels compared to
compared to HFD-fed rats (Figure 3B). This finding suggests an improvement in insulin sensitivity,
HFD-fed rats (Figure 3B). This finding suggests an improvement in insulin sensitivity, further
emphasizing the metabolic benefit induced by inhibition of extracellular CTSD. Altogether, these
Biomolecules 2019, 9, 171 6 of 12
Biomolecules 2019, 9, x 6 of 12
Biomolecules 2019, 9, x 6 of 12
further emphasizing
data indicate
indicate the metabolic
that modulating
modulating benefit induced
extracellular by inhibition
CTSD activity
activity of extracellular
improves CTSD. Altogether,
metabolic features
features associated
data
these data that
indicate that extracellular
modulating CTSD
extracellular CTSD improves
activity metabolic
improves metabolic features associated
associated
with
with NAFLD.
NAFLD.
with NAFLD.

Figure 3. Metabolic parameters

Figure parameters of of high-fat
high-fat diet
diet (HFD)-fed Sprague–Dawley
Sprague–Dawley (SD) (SD) rats
rats treated
treated with or or
Figure 3.3.Metabolic
Metabolic parameters of high-fat (HFD)-fed
diet (HFD)-fed Sprague–Dawley (SD) rats withtreated
without the
without the extracellular
extracellular inhibitor
inhibitor CTD-002.
CTD-002. (A) (A) Scoring of of hepatic
hepatic steatosis
steatosis by by means
means of of
with or without the extracellular inhibitor CTD-002.Scoring (A) Scoring of hepatic steatosis by means
Hematoxylin-Eosin
Hematoxylin-Eosin
of Hematoxylin-Eosin (H&E)
(H&E)
(H&E) staining.
staining.
staining. (B) Plasma
(B)(B)
Plasma
Plasma levels
levels of insulin.
of insulin.
levels Error
Error
of insulin. bars represent
barsbars
Error represent mean
mean
represent ± SEM;
± SEM;
± SEM;
mean n=
n=
6n6 for
=for6 each
each group;
for each ** represents
group;
group; represents pp << p0.05
* represents 0.05 andand
< 0.05
and ***p***
***p << 0.001
0.001 compared
p < 0.001 compared
compared to rats
to rats on HFD;
to rats
on HFD;
on HFD;### ###
### represents pp <<
represents
represents
p0.001
< 0.001
0.001 compared
compared
compared to the
to the ratsrats
to rats
the on low-fat
on low-fat
on low-fat dietdiet
diet (LFD)
(LFD)(LFD) by means
by means
by meansof two-tailed
of two-tailed
of two-tailedunpaired
unpaired
unpaired t-test.
t-test.
t-test.

3.4. Reduced Liver Damage after Inhibition of Extracellular CTSD in HFD-Fed Sprague–Dawley Sprague–Dawley Rats Rats
Rats
3.4. Reduced Liver Damage after Inhibition of Extracellular CTSD in HFD-Fed Sprague–Dawley
To determine
To determine
determine the effects
the effects
effects of extracellular
of extracellular
extracellular CTSDCTSD inhibition
inhibition on hepatic
on hepatic
hepatic inflammation,
inflammation, the protein
the protein
protein levels
To the of CTSD inhibition on inflammation, the levels
levels of hepatic
of hepatic
hepatic TNFα wereTNFαmeasured
were were measured in HFD-fed
in HFD-fed
HFD-fed SD rats SD rats
rats that
that werethat werewith
treated treated with orthe
or without
without without
CTD-002the
of TNFα measured in SD were treated with or the CTD-002
CTD-002
inhibitor inhibitor
of of extracellular
extracellular CTSD. CTSD. CTD-002-treated
CTD-002-treated HFD-fed HFD-fed
SD rats SD ratsashowed
showed trend a trend reduced
towards towards
inhibitor of extracellular CTSD. CTD-002-treated HFD-fed SD rats showed a trend towards reduced
reduced hepaticlevels
hepatic TNFα
TNFα TNFα(plevels = 0.06) compared
(pcompared to the HFD-fed group
(Figure(Figure 4A), thereby suggesting
hepatic levels (p == 0.06)
0.06) compared to to the
the HFD-fed
HFD-fed group (Figure
group 4A), thereby
4A), thereby suggesting
suggesting an
an
an improvement
improvement in in hepatic
hepatic inflammation.
inflammation. ToTo further
further assessthe
assess theeffects
effectsofofextracellular
extracellularCTSDCTSDinhibition
inhibition
improvement in hepatic inflammation. To further assess the effects of extracellular CTSD inhibition
on liver damage,
liver damage, plasma
plasma aspartate
damage, plasma aspartateaminotransferase
aminotransferase(AST) (AST)and
andalanine
alanineaminotransferase
aminotransferase(ALT) (ALT)levels
on liver aspartate aminotransferase (AST) and alanine aminotransferase (ALT)
were
levelsmeasured. Upon Upon
were measured.
measured. treatment with CTD-002,
treatment HFD-fed
with CTD-002,
CTD-002, SD rats
HFD-fed SDshowed
rats showeda significant
showed reduction in
levels were Upon treatment with HFD-fed SD rats aa significant
significant
the aspartate
reduction in transaminase/alanine
the aspartate transaminase
transaminase/alanine (AST/ALT) ratio
transaminase (Figureratio
(AST/ALT) 4B), (Figure
pointing towards
4B), pointingan
reduction in the aspartate transaminase/alanine transaminase (AST/ALT) ratio (Figure 4B), pointing
improvement
towards an in liver damage.
improvement in liver damage.
towards an improvement in liver damage.

Figure 4.
Figure
Figure 4. Hepatic
4. Hepatic TNFα
Hepatic TNFα levels
TNFα levels and
levels and aspartate
and aspartate transaminase/alanine
aspartate transaminase/alanine transaminase
transaminase/alanine transaminase (AST/ALT)
transaminase (AST/ALT) ratio
(AST/ALT) ratio in
ratio in
in
HFD-fed
HFD-fed SD
SD rats
rats treated
treated with
with or
or without
without the
the extracellular
extracellular inhibitor
inhibitor of
of cathepsin
cathepsin D
D (CTSD).
(CTSD).
HFD-fed SD rats treated with or without the extracellular inhibitor of cathepsin D (CTSD). (A) Hepatic (A)
(A) Hepatic
Hepatic
TNFα levels.
TNFα
TNFα levels. (B)
levels. (B) Plasma
Plasma transaminase
transaminase levels.
levels. Error
Error bars
bars represent
represent mean
represent mean ±±
mean SEM; nn ==
± SEM; = 666for
for each
foreach group.
eachgroup.
indicates ppp <<< 0.05
*** indicates
indicates 0.05 compared
compared to
to rats
rats on
on the
the HFD.
HFD.

4. Discussion
Treating NAFLD is of critical importance to prevent further progression to liver cirrhosis and
end-stage liver disease. The present study provides the first evidence that modulating extracellular
CTSD activity can significantly improve metabolic parameters associated with NAFLD. These findings
suggest the potential therapeutic benefits of targeting extracellular CTSD within the context of metabolic
diseases and opens new perspectives for therapeutic interventions in NAFLD.
To elucidate the effects of extracellular CTSD inhibition in NAFLD, we have used SD rats that were
fed a high-fat diet for three weeks. Sprague–Dawley rats are known to develop micro-vesicular steatosis
with [21] or without inflammation [26], depending on the composition and duration of the HFD.
Here, SD rats developed hepatic steatosis. Similar to the findings of Lieber and Ahmed et al. [21,26],
Biomolecules 2019, 9, 171 7 of 12

no changes in relative liver weights were observed between different groups of rats (Table S2).
Comparable to human NAFLD patients, we observed that rats on an HFD developed insulin resistance
(IR) [27], as shown by elevated plasma insulin levels with the HFD. Indeed, it has been shown that
insulin resistance is commonly associated with obesity-induced hepatic steatosis [28,29]. Furthermore,
hepatic lipid accumulation is known to activate intrahepatic inflammatory pathways, which stimulate
the pro-inflammatory cytokine production, in turn leading to both hepatic and peripheral insulin
resistance [30,31]. Upon administration of the extracellular CTSD inhibitor, we observed that HFD-fed
rats showed a significant reduction in plasma insulin levels and improvement of steatosis, two important
characteristics of diet-induced NAFLD. Hence, these data provide initial evidence that extracellular
CTSD activity is involved with the regulation of lipid metabolism and thereby controls insulin
sensitivity. However, the trend towards reduced food intake suggests additional potential effects on
energy expenditure, which should be further investigated in the future. Additionally, though hepatic
damage and inflammation were not increased in SD rats fed an HFD, treatment with the extracellular
CTSD inhibitor showed reduced hepatic TNFα levels and AST/ALT ratio, indicating an improvement
in hepatic damage and inflammation. Moreover, we validated this finding in primary macrophages
and in an in vitro setup in which hepatic cells were incubated with a macrophage-derived conditioned
medium, an approach that has been previously used to investigate crosstalk between hepatic immune
and parenchymal cells [32,33]. As such, our findings suggest that macrophage-derived extracellular
CTSD activity influences hepatic lipid metabolism and inflammation. However, despite the promising
first results, the potential minor effects of intracellular CTSD and of the trend towards reduced food
intake in the inhibitor-treated group suggest additional non-beneficial effects related to intracellular
CTSD inhibition. To determine whether these non-beneficial metabolic effects are related directly to
intracellular CTSD, a comparison with a compound with high permeability and low efflux (indicating
that the compound mainly exerts its effects intracellularly) is necessary. Likewise, lower concentrations
of the compound should be investigated in several NAFLD models before transition to the clinic
is made.
Substantial evidence points towards the role of cathepsins in the context of NAFLD. Inactivation of
cathepsin B in a dietary murine model of NAFLD prevented the development of hepatic steatosis [34].
Additionally, cathepsin B was found to be involved in hepatic injury and in progression of liver
fibrosis [35,36]. Similarly, we have shown that lysosomal cholesterol accumulation inside Kupffer cells
of Ldlr-/- mice leads to increased CTSD activity in the liver [37]. CTSD is a key lysosomal protease
that affects many fundamental functions in the cell. A reduction in cellular CTSD expression or
catalytic activity leads to devastating neurodegenerative disorders [38,39]. In contrast, an increase in
extracellular CTSD expression and activity is associated with many types of cancers [40–42]. Owing
to this differential expression and secretion of CTSD in many pathophysiological processes, there is
increased awareness for the extracellular fraction of CTSD to play a role in health and disease. However,
to date, research primarily focuses on targeting the whole fraction of CTSD [43], which is known
to disturb physiological processes, resulting in serious side effects [44]. Hence, in the present study,
we specifically investigated the benefits of targeting the extracellular fraction of CTSD in the context
of NAFLD.
Though the mechanisms underlying the secretion of CTSD are not yet known, changes in the
lysosomal pH or lysosomal accumulation of poorly degradable lipids are known to cause mistargeting
of CTSD into the extracellular milieu [45,46]. Additionally, cholesterol-filled lysosomes have been
shown to induce disturbances in the lysosomal enzyme trafficking pathway that can potentially lead to
increased levels of lysosomal enzymes in the plasma [47,48]. In vitro, particularly, the intracellular
accumulation of the oxidized fraction of cholesterol has been shown to enhance extracellular secretion
of CTSD [49,50]. Building further on this knowledge, we found in this study that targeting the activity
of the extracellular fraction of CTSD in oxLDL-loaded macrophages improves inflammation and lipid
metabolism, suggesting that macrophage-derived extracellular CTSD has a key role in lipid metabolism
and inflammation.
Biomolecules 2019, 9, 171 8 of 12

Our observation that extracellular CTSD activity is involved with the pathogenesis of NAFLD
implies that CTSD remains active in the circulation. Indeed, modulating the vacuolar type H+ ATPase
pump by macrophages creates a more acidic pericellular space, in which lysosomal enzymes such
as CTSD can remain active [51]. Moreover, several findings have implicated that upon secretion,
anchorage of CTSD to the cell surface ameliorates its ability to interact with specific extracellular
substrates [52]. The combination of an acidic pericellular environment and localization proximal to the
cell proposes a potential mechanism by which extracellular CTSD activity can influence physiological
processes. However, the question remains of which underlying mechanism is responsible for the
observed improvements in lipid metabolism and inflammation upon inhibition of extracellular CTSD
activity. In the current study, extracellular inhibition of CTSD upregulated the expression of Cyp27a1 in
macrophages. Cyp27a1 is a gene encoding for an essential enzyme responsible for regulating cholesterol,
fatty acid and bile acid metabolism through modulation of the mitochondrial P450 enzyme sterol
27-hydroxylase. In the liver, sterol 27-hydroxylase catalyzes the first step of the alternative bile acid
biosynthetic pathway from cholesterol [53]. Previously, we have demonstrated the potential of Cyp27a1
to modulate intracellular cholesterol distribution in Kupffer cells [37]. Of note, Cyp27a1 has been
shown to regulate ATP-binding cassette transporter A1 (ABCA1), an exporter of cellular lipids [54].
Based on this evidence, a possible explanation for our current findings is that the extracellular CTSD
binds to one of the cell surface lipid transporters such as ABCA1 or scavenger receptors, which in turn
blocks cholesterol transport, preventing regulation of cellular cholesterol and efflux pathways. It is
therefore feasible that CTSD acts as an extracellular messenger interacting with an as yet unidentified
cell surface receptor and regulates lipid homeostasis in addition to inflammation. In this regard,
extracellular CTSD has recently been shown to be involved with ABCA1 regulation via influencing
the low-density lipoprotein receptor-related protein 1 (LRP1) [55]. Additionally, we here observed
differential expression of Cd36 and Sr-a upon extracellular CTSD inhibition, suggesting an involvement
of these scavenger receptors. Moreover, modulating proteases is known to influence the transcriptional
regulation of cholesterol handling via Srebp [56] and Nfe2l1 [57]. However, further studies are warranted
to decipher the exact underlying mechanism of extracellular CTSD function.
In conclusion, we provide for the first time evidence that extracellular CTSD has a central role in
the progression of NAFLD. In contrast to conventional therapeutic targeting of cathepsins, our data
demonstrate that inhibiting specifically the extracellular fraction of CTSD can be a valuable therapeutic
strategy for NAFLD. Further studies that investigate the downstream targets regulated by extracellular
CTSD will provide deeper understanding of the mechanisms of NAFLD pathogenesis.

Supplementary Materials: The following are available online at http://www.mdpi.com/2218-273X/9/5/171/s1,

Figure S1: Inhibitory activity of CTD-002 on CTSD activity. Figure S2: Schematic representation of the in vivo
setup. Figure S3: Effect of CTD-002 in bone marrow-derived macrophages under control conditions. Figure S4:
Cd36 gene expression levels of BMDMs and HepG2 cells. Figure S5: Food consumption of Sprague–Dawley rats.
Figure S6: Representative images of fat droplets stained by haematoxylin and eosin. Figure S7: Impact of high-fat
diet on hepatic fat deposition in Sprague–Dawley rats. Upper panels, Chromotrope Aniline Blue staining; bottom
panels, Sirius Red staining. Table S1: Composition of low and high fat diets. Table S2: Relative liver weight (per
100 g of body weight) of the experimental groups of rats.
Author Contributions: Conceptualization, T.H., A.K. and R.S.-S.; Data curation, P.K., T.Y., S.G. and A.D.; Formal
analysis, P.K., T.Y., T.H. and Y.O.; Funding acquisition, A.K. and R.S.-S.; Methodology, P.K., T.Y., S.G., A.D.,
T.H., A.A. and R.S.-S.; Project administration, A.K.; Resources, A.K. and R.S.-S.; Supervision, A.K. and R.S.-S.;
Writing—original draft, P.K. and T.Y.; Writing—review & editing, S.G., A.D., T.H., Y.O., A.A., A.K. and R.S.-S.
Funding: This work was jointly supported by Avaliv Therapeutics and the Netherlands Organisation for Scientific
Research (NWO) VIDI (grant number: 016.126.327), ASPASIA (grant no. 015.008.043) and by Top consortia for
Knowledge and Innovation (TKI; grant number: 40-41200-98-9306).
Conflicts of Interest: RSS is on the advisory board of Avaliv Therapeutics, which has filed intellectual property
protecting the inhibitors. The terms of this arrangement have been reviewed and approved by Maastricht
University in accordance with its policy on objectivity in research. AK is a co-founder of Avaliv Therapeutics.
Biomolecules 2019, 9, 171 9 of 12

Abbreviations
NAFLD non-alcoholic fatty liver disease
NASH non-alcoholic steatohepatitis
CTSD cathepsin D
HFD high-fat diet
LFD low-fat diet
BMDMs Bone Marrow-derived macrophages
oxLDL oxidized low-density lipoprotein
Wt wildtype
LDL(R) low-density lipoprotein (receptor)
SD Sprague–Dawley
ELISA enzyme-linked immune sorbent assay
LCM L929-conditioned medium
TNF tumor necrosis factor
LPS lipopolysaccharide
AST aspartate transaminase
ALT alanine transaminase
MetS metabolic syndrome
Pep-A Pepstatin-A

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