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Lysyl Oxidase-Like 2 Protects against Progressive and Aging Related Knee Joint Osteoarthritis in Mice

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Background: The goal of this study was to determine if adenovirus-delivered LOXL2 protects against progressive knee osteoarthritis (OA), assess its specific mechanism of action; and determine if the overexpression of LOXL2 in transgenic mice can protect against the development of OA-related cartilage damage and joint disability. Methods: Four-month-old Cho/+ male and female mice were intraperitoneally injected with either Adv-RFP-LOXL2 or an empty vector twice a month for four months. The proteoglycan levels and the expression of anabolic and catabolic genes were examined by immunostaining and qRT-PCR. The effect of LOXL2 expression on signaling was tested via the pro-inflammatory cytokine IL1β in the cartilage cell line ATDC5. Finally; the OA by monosodium iodoacetate (MIA) injection was also induced in transgenic mice with systemic overexpression of LOXL2 and examined gene expression and joint function by treadmill tests and assessment of allodynia. Results: The adenovirus treatment upregulated LOXL2; Sox9; Acan and Runx2 expression in both males and females. The Adv-RFP-LOXL2 injection; but not the empty vector injection increased proteoglycan staining and aggrecan expression but reduced MMP13 expression. LOXL2 attenuated IL-1β-induced phospho-NF-κB/p65 and rescued chondrogenic lineage-related genes in ATDC5 cells; demonstrating one potential protective mechanism. LOXL2 attenuated phospho-NF-κB independent of its enzymatic activity. Finally; LOXL2-overexpressing transgenic mice were protected from MIA-induced OA-related functional changes; including the time and distance traveled on the treadmill and allodynia. Conclusion: Our study demonstrates that systemic LOXL2 adenovirus or LOXL2 genetic overexpression in mice can protect against OA. These findings demonstrate the potential for LOXL2 gene therapy for knee-OA clinical treatment in the future.
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International Journal of
Molecular Sciences
Article
Lysyl Oxidase-Like 2 Protects against Progressive and
Aging Related Knee Joint Osteoarthritis in Mice
Mustafa Tashkandi 1, 2, , Faiza Ali 1,, Saqer Alsaqer 1, Thabet Alhousami 1, Amparo Cano 3,4,
Alberto Martin 3, 4,, Fernando Salvador 3,4, Francisco Portillo 3,4, Louis C. Gerstenfeld 5,
Mary B. Goldring 6and Manish V. Bais 1, *
1Department of Molecular and Cell Biology, Boston University Henry M. Goldman School of Dental
Medicine, Boston, MA 02118, USA; mtash@bu.edu (M.T.); fali7@bu.edu (F.A.); Alsaqer@bu.edu (S.A.);
thabet@bu.edu (T.A.)
2Department of Periodontology, Boston University Henry M. Goldman School of Dental Medicine, Boston,
MA 02118, USA
3Departamento de Bioquímica, Universidad Autónoma de Madrid, Instituto de Investigaciones Biomédicas
“Alberto Sols” CSIC-UAM, IdiPAZ, 28029 Madrid, Spain; acano@iib.uam.es (A.C.);
almmartin@isciii.es (A.M.); fernando.salvador@irbbarcelona.org (F.S.); fportillo@iib.uam.es (F.P.)
4Centro de Investigación Biomédica en Red Cáncer. Av Monforte de Lemos, 3-5, Pabellón 11, planta 0,
28029 Madrid, Spain
5Department of Orthopedic Surgery, School of Medicine, Boston University, Boston, MA 02118, USA;
lgersten@bu.edu
6Hospital for Special Surgery Research Institute, and Department of Cell and Developmental Biology,
Weill Cornell Medical College, New York, NY 10021, USA; mbgoldring@hss.edu
*Correspondence: bmanish@bu.edu; Tel.: +1-617-358-4880
These authors contributed equally to this work.
Present address: Instituto de Investigación de Enfermedades Raras (IIER), Instituto de Salud Carlos III,
Carretera Majadahonda- Pozuelo km. 2. Majadahonda, 28220 Madrid, Spain.
Received: 12 August 2019; Accepted: 26 September 2019; Published: 27 September 2019


Abstract:
Background: The goal of this study was to determine if adenovirus-delivered LOXL2
protects against progressive knee osteoarthritis (OA), assess its specific mechanism of action; and
determine if the overexpression of LOXL2 in transgenic mice can protect against the development
of OA-related cartilage damage and joint disability. Methods: Four-month-old Cho/+ male and
female mice were intraperitoneally injected with either Adv-RFP-LOXL2 or an empty vector twice a
month for four months. The proteoglycan levels and the expression of anabolic and catabolic genes
were examined by immunostaining and qRT-PCR. The eect of LOXL2 expression on signaling was
tested via the pro-inflammatory cytokine IL1
β
in the cartilage cell line ATDC5. Finally; the OA
by monosodium iodoacetate (MIA) injection was also induced in transgenic mice with systemic
overexpression of LOXL2 and examined gene expression and joint function by treadmill tests and
assessment of allodynia. Results: The adenovirus treatment upregulated LOXL2; Sox9; Acan and
Runx2 expression in both males and females. The Adv-RFP-LOXL2 injection; but not the empty vector
injection increased proteoglycan staining and aggrecan expression but reduced MMP13 expression.
LOXL2 attenuated IL-1
β
-induced phospho-NF-
κ
B/p65 and rescued chondrogenic lineage-related
genes in ATDC5 cells; demonstrating one potential protective mechanism. LOXL2 attenuated
phospho-NF-
κ
B independent of its enzymatic activity. Finally; LOXL2-overexpressing transgenic
mice were protected from MIA-induced OA-related functional changes; including the time and
distance traveled on the treadmill and allodynia. Conclusion: Our study demonstrates that systemic
LOXL2 adenovirus or LOXL2 genetic overexpression in mice can protect against OA. These findings
demonstrate the potential for LOXL2 gene therapy for knee-OA clinical treatment in the future.
Int. J. Mol. Sci. 2019,20, 4798; doi:10.3390/ijms20194798 www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2019,20, 4798 2 of 13
Keywords:
Lysyl oxidase like-2; adenovirus delivery; knee joint; articular cartilage; regeneration;
osteoarthritis; anabolic response
1. Introduction
The progressive joint disease osteoarthritis (OA) aects a significant portion of the US population
but has a few therapeutic options. The estimated lifetime risk of knee OA is 14% [
1
], with annual
healthcare costs exceeding $185 billion in the United States [
2
], and the prevalence of OA is increasing [
3
].
However, no anabolic agent has been approved for clinical application.
Using transcriptomics in a mouse model, the authors previously demonstrated that copper-dependent
amine oxidase lysyl oxidase-like 2 (LOXL2) is specifically upregulated during fracture healing and mediates
endochondral ossification [
4
]. Our follow-up study also showed that LOXL2 is a critical regulator of the
chondrogenic lineage [
5
]. Further, LOXL2 also was found to have a novel anabolic function in OA
cartilage [
6
,
7
]. The microarray analysis demonstrates that the transduction of human OA articular
chondrocytes with Adv-LOXL2 increases the expression of anabolic genes [
6
]. Thus, LOXL2 is a
mediator of both the chondrogenic lineage and cartilagenous changes aected by OA and thus may
have therapeutic potential.
Our previous data inspired the following questions. (1) Can LOXL2 attenuate progressive
knee-OA-related degenerative changes? (2) If LOXL2 exerts a protective function, can it be exploited
in pre-clinical models to uncover new therapeutic strategies for OA?
The recent advances and success of gene therapy for delivering therapeutic molecules [
8
] has
stimulated interest in this field, and the application of adenovirus and lentivirus by direct intra-articular
joint and intraperitoneal injections in preclinical and clinical studies is now trending [
9
]. The studies
show that adenovirus-mediated administration of bFGF, IL-1Ra, or IGF-1 is eective in a rat OA
model [
10
]. Therefore, this study aimed to determine if adenoviral LOXL2 acts via an anabolic
mechanism to retard the progressive knee OA and if the overexpression of LOXL2 protects against
OA-related cartilage damage and joint dysfunction. Considering the benefits of the adenovirus delivery
system [
11
], this study used this system to deliver LOXL2 into an
in vitro
OA model or, using the
systemic injection, into knee joint OA mouse models.
The three models used to uncover the chondroprotective eect of LOXL2 are: Chondrodysplasia
(Cho/+) mice, a cartilage cell line exposed to the inflammatory cytokine IL-1
β
, and monosodium
iodoacetate (MIA)-induced LOXL2 transgenic mice. The Cho/+ mouse, which carries a single-nucleotide
deletion resulting in the premature termination of the
α
1 chain of type XI collagen [
12
], is a good
model to study the progressive OA changes that develop with aging. The type XI collagen expression
is reduced in the articular cartilage of older individuals [
13
], and the data from human and animal
experiments show that the decreased type XI collagen levels in cartilage might be one initiating factor
of tempoormandibular joint [
14
] and knee joint-OA pathogenesis [
15
]. Next,
in vitro
IL-1
β
-induced
catabolic changes and molecular signaling mechanisms were assessed in a cartilage cell line. Of note,
the IL-1
β
inhibitors have been implicated as a therapy for OA [
16
,
17
]. Finally, this study tested
whether the endogenous overexpression of LOXL2 can protect against MIA-induced knee OA and
alleviate OA-related structural and functional consequences, including the proteoglycan and aggrecan
expression, joint mobility, and allodynia. The MIA-induced model is extensively used for knee
OA-related studies in both mice and rats. This model allowed the assessment of the ecacy of the
genetic overexpression of LOXL2 in attenuating OA-related functions.
2. Results
2.1. Adenoviral LOXL2 Protects Against Progressive OA in Cho/+ Mice
To test whether LOXL2 induces anabolic responses in progressive mouse knee-OA, Cho/+ mice
injected with Adv-RFP-LOXL2 were compared to mice injected with Adv-RFP-Empty. First, this study
Int. J. Mol. Sci. 2019,20, 4798 3 of 13
evaluated if the adenovirus vector induced any adverse eects, such as the cytotoxicity or immune
reaction in the mice. The data showed that the adenovirus vector injection does not have any adverse
eects on the total LDH release of ATDC5 cells. Next, serum samples from mice injected with vehicle
or the adenovirus vector (Adv-RFP-Empty) was evaluated and found to have no significant dierences
in serum total LDH (Figure S1). The evaluation of tissues for the expression of inflammatory cytokines,
such as TNF
α
and IL6, does not show any eect due to the adenovirus vector injection compared to the
vehicle injected group. As shown in Figure 1A, the equal numbers of male and female Cho/+ mice were
injected intraperitoneally with either Adv-RFP-Empty or Adv-RFP-LOXL2 twice monthly for 12 weeks.
Safranin-O/Fast Green staining showed significantly increased proteoglycan deposition (Figure 1B).
Immunostaining revealed that Adv-RFP-LOXL2 increased the expression of LOXL2 aggrecan and
Col2 but reduced expression of Mmp13 and Adamts5 (Figure 1C–I). The knee joints from both male
and female mice injected with Adv-RFP-LOXL2 showed the higher expression of LOXL2,Acan,Sox9,
Col2a1, and Runx2 than knee joints from mice injected with Adv-RFP-Empty (Figure 2). The data also
shows that TNF
α
and IL6 expression was not aected significantly. The forced expression of LOXL2
significantly increased mRNA levels of anabolic genes, indicating that LOXL2 promotes anabolism
in general. For both male and female mice, the expression of Vegf-b and Col10 were not aected by
LOXL2-forced expression. However, LOXL2-forced expression caused a non-significant increase in
Mmp13 and Rankl in females only (Figure 2). Overall, these data suggest that the expression of LOXL2
protects against proteoglycan loss and OA progression and may stimulate a protective response in
knee joint articular cartilage via the factors secreted in the OA joint.
Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 3 of 14
any adverse effects on the total LDH release of ATDC5 cells. Next, serum samples from mice injected
with vehicle or the adenovirus vector (Adv-RFP-Empty) was evaluated and found to have no
significant differences in serum total LDH (Figure S1). The evaluation of tissues for the expression of
inflammatory cytokines, such as TNFα and IL6, does not show any effect due to the adenovirus vector
injection compared to the vehicle injected group. As shown in Figure 1A, the equal numbers of male
and female Cho/+ mice were injected intraperitoneally with either Adv-RFP-Empty or Adv-RFP-
LOXL2 twice monthly for 12 weeks. Safranin-O/Fast Green staining showed significantly increased
proteoglycan deposition (Figure 1B). Immunostaining revealed that Adv-RFP-LOXL2 increased the
expression of LOXL2 aggrecan and Col2 but reduced expression of Mmp13 and Adamts5 (Figure 1C–
I). The knee joints from both male and female mice injected with Adv-RFP-LOXL2 showed the higher
expression of LOXL2, Acan, Sox9, Col2a1, and Runx2 than knee joints from mice injected with Adv-
RFP-Empty (Figure 2). The data also shows that TNFα and IL6 expression was not affected
significantly. The forced expression of LOXL2 significantly increased mRNA levels of anabolic genes,
indicating that LOXL2 promotes anabolism in general. For both male and female mice, the expression
of Vegf-b and Col10 were not affected by LOXL2-forced expression. However, LOXL2-forced
expression caused a non-significant increase in Mmp13 and Rankl in females only (Figure 2). Overall,
these data suggest that the expression of LOXL2 protects against proteoglycan loss and OA
progression and may stimulate a protective response in knee joint articular cartilage via the factors
secreted in the OA joint.
Figure 1. LOXL2 induces a protective response in Cho/+ mice knee joint articular cartilage. (A) Scheme
of experimental groups and the treatment. (B) Safranin-O/Fast green staining of TMJ condylar
cartilage in Adv-RFP-Empty compared to Adv-RFP-LOXL2 adenovirus-injected mice, quantification.
Immunostaining and quantification of (C) LOXL2; (D) Acan; (E) MMP13; (F) Col2; (G) Col10; (H)
Adamts5 and (I) RFP in Adv-RFP-Empty and Adv-RFP-LOXL2 treated mice. The statistically
Figure 1.
LOXL2 induces a protective response in Cho/+ mice knee joint articular cartilage. (
A
) Scheme
of experimental groups and the treatment. (
B
) Safranin-O/Fast green staining of TMJ condylar
cartilage in Adv-RFP-Empty compared to Adv-RFP-LOXL2 adenovirus-injected mice, quantification.
Immunostaining and quantification of (
C
) LOXL2; (
D
) Acan; (
E
) MMP13; (
F
) Col2; (
G
) Col10;
(
H
) Adamts5 and (
I
) RFP in Adv-RFP-Empty and Adv-RFP-LOXL2 treated mice. The statistically
significant dierences in immunostaining were evaluated by one-way ANOVA with Bonferroni
correction (* represent significant dierences; ** p<0.01, *** p<0.001; ANOVA).
Int. J. Mol. Sci. 2019,20, 4798 4 of 13
Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 4 of 14
significant differences in immunostaining were evaluated by one-way ANOVA with Bonferroni
correction (* represent significant differences; **p < 0.01, ***p < 0.001; ANOVA).
Figure 2. LOXL2 induces mRNA expression of anabolic genes in the Cho/+ mouse model. Each panel
shows fold-change in mRNA levels of differentially regulated genes in male and female Cho/+ mice
injected with Adv-RFP-Empty (dot-male; triangle- female) and Adv-RFP-LOXL2 (square-male;
Figure 2.
LOXL2 induces mRNA expression of anabolic genes in the Cho/+ mouse model. Each
panel shows fold-change in mRNA levels of dierentially regulated genes in male and female Cho/+
mice injected with Adv-RFP-Empty (dot-male; triangle- female) and Adv-RFP-LOXL2 (square-male;
rhombus-female). The statistically significant dierences between groups for males (gold color line) and
females (green color line) were evaluated by two-way ANOVA with Bonferroni correction (* represent
significant dierences; * p<0.05, ** p<0.01, and *** p<0.001; ANOVA).
Int. J. Mol. Sci. 2019,20, 4798 5 of 13
2.2. LOXL2 Attenuates IL-1βInduced NF-κB in Cartilage
IL-1
β
is a pro-inflammatory factor secreted in the knee joint and promotes catabolism. To test if
LOXL2 acts partly by attenuating IL-1
β
-induced catabolic responses, ATDC5 cartilage cells were treated
with IL-1
β
alone or in combination with Adv-RFP-LOXL2, and molecular signaling and gene expression
were assessed (Figure 3). IL-1
β
reduced mRNA levels of Acan and Sox9, but LOXL2 blunted this eect.
Finally, LOXL2 attenuated the expression of inducers of IL-1
β
, including Admts4/5 and MMP13 after 1
and/or 3 days of treatment (Figure 3). Thus, LOXL2 may protect against pro-inflammatory signaling
mechanisms and restore the expression of cartilage-related genes.
Figure 3.
LOXL2 protects against IL-1
β
-induced eect on cartilage-specific gene expression. RT-qPCR
analysis of IL-1
β
treated ATDC5 cells reduces cartilage-specific gene expression (Acan, Sox9), and LOXL2
protects against this eect. The minus (
) sign represents absence; plus (+) presence of LOXL2 or
IL-1
β
in the respective group. The statistically significant dierences in protein or mRNA levels were
evaluated by one-way ANOVA with Bonferroni correction (** p<0.01; *** p<0.001; ANOVA).
Int. J. Mol. Sci. 2019,20, 4798 6 of 13
2.3. Enzymatically Inactive LOXL2 also Attenuates IL-1β-Induced NF-κB Activity
IL-1
β
induced phosphorylation of NF-
κ
B/p65, but this was attenuated by the overexpression
of LOXL2 (Figure 4A,B). To test if the anti-catabolic eect of LOXL2 is mediated by an enzymatic or
non-enzymatic mechanism, ATDC5 cells were treated with IL-1
β
and Adv-LOXL2 in the presence or
absence of the LOX family inhibitor
β
-aminoproprinytryl (BAPN). The abilitry of LOXL2 expression to
block NK-
κ
B phosporylation was not prevented by BAPN (Figure 4A,B). These data indicate that the
ability of LOXL2 to inhibit OA-related structural and functional changes could be indepenent of its
enzymatic activity.
Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 7 of 14
to block NK-κB phosporylation was not prevented by BAPN (Figure 4A,B). These data indicate that
the ability of LOXL2 to inhibit OA-related structural and functional changes could be indepenent of
its enzymatic activity.
Figure 4. LOXL2 protects against IL-1β-induced NF-κB signaling even in the presence of a LOX family
inhibitor. (A) IL-1β promotes phospho-NF-κB in ATDC5 cells, and this effect is attenuated by LOXL2
overexpression in the absence or presence of BAPN. The minus () sign represents absence wheras
plus (+) sign represents presence of LOXL2 or IL-1β in the respective group. (B) Quantification of this
effect. The statistically significant differences were evaluated by one-way ANOVA with Bonferroni
correction for p-NF-κB (*p < 0.01, ***p < 0.001; ANOVA).
2.4. LOXL2 Overexpression Protects Against Catabolic Changes in Aging Knee OA.
To evaluate if endogenous LOXL2 can alleviate knee-OA-related catabolic changes and
functional consequences in aging mice, 14-month-old LOXL2 homozygous transgenic mice intra-
articularly injected with MIA were used to assess histological and functional changes (Figure 5A).
The mice overexpressing LOXL2 were engineered and validated by Cano laboratory [18]. Safranin-O
staining showed that proteoglycan expression was similar in untreated LOXL2 and wild-type (WT)
littermates, but proteoglycan was depleted in knee joints injected with MIA in WT mice. This
depletion was ameliorated in LOXL2 transgenic mice (Figure 5B). Similar to LOXL2 expression
(Figure 5C), the Acan expression appeared to be higher in untreated LOXL2 transgenic and WT
littermates, but decreased in knee joints of injected mice with MIA (Figure 5D). Again, LOXL2
overexpression in the transgenic mice prevented this depletion. Thus, LOXL2 protects against MIA-
induced proteoglycan and aggrecan degradation as well as decreased Mmp13 expression in knee OA.
Figure 4.
LOXL2 protects against IL-1
β
-induced NF-
κ
B signaling even in the presence of a LOX family
inhibitor. (
A
) IL-1
β
promotes phospho-NF-
κ
B in ATDC5 cells, and this eect is attenuated by LOXL2
overexpression in the absence or presence of BAPN. The minus (
) sign represents absence wheras
plus (+) sign represents presence of LOXL2 or IL-1
β
in the respective group. (
B
) Quantification of this
eect. The statistically significant dierences were evaluated by one-way ANOVA with Bonferroni
correction for p-NF-κB (* p<0.01, *** p<0.001; ANOVA).
2.4. LOXL2 Overexpression Protects Against Catabolic Changes in Aging Knee OA
To evaluate if endogenous LOXL2 can alleviate knee-OA-related catabolic changes and functional
consequences in aging mice, 14-month-old LOXL2 homozygous transgenic mice intra-articularly
injected with MIA were used to assess histological and functional changes (Figure 5A). The mice
overexpressing LOXL2 were engineered and validated by Cano laboratory [
18
]. Safranin-O staining
showed that proteoglycan expression was similar in untreated LOXL2 and wild-type (WT) littermates,
but proteoglycan was depleted in knee joints injected with MIA in WT mice. This depletion was
ameliorated in LOXL2 transgenic mice (Figure 5B). Similar to LOXL2 expression (Figure 5C), the Acan
expression appeared to be higher in untreated LOXL2 transgenic and WT littermates, but decreased in
knee joints of injected mice with MIA (Figure 5D). Again, LOXL2 overexpression in the transgenic
mice prevented this depletion. Thus, LOXL2 protects against MIA-induced proteoglycan and aggrecan
degradation as well as decreased Mmp13 expression in knee OA.
Int. J. Mol. Sci. 2019,20, 4798 7 of 13
Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 8 of 14
Figure 5. LOXL2 overexpression in transgenic mice protects against MIA-induced OA-related
catabolic changes in knee joint articular cartilage. (A) Scheme of LOXL2 mice MIA injection groups
and its functional analysis; (B) Safranin-O staining in the indicated WT and LOXL2 transgenic groups,
its quantification and OARSI scoring. Immunostaining and quantification of (C) LOXL2 and (D) Acan
in MIA injected LOXL2 overexpressing or WT mice. The fold change differences in immunostaining
for LOXL2 and Acan expression in WT (dot), LOXL2 (square), WT/MIA (triangle) and MIA/LOXL2
(inverted triangles) are shown in adjacent figures. The statistically significant differences in
immunostaining were evaluated by one-way ANOVA with Bonferroni correction (***p < 0.001;
ANOVA).
2.5. Overexpression of LOXL2 Protects Against Functional Changes in Aging Knee OA
MIA-induced OA produces pain-depressed wheel running [19]. Treadmill behavior is useful for
preclinical behavioral assessment of chronic pain and inflammation [20]. These LOXL2 transgenic
mice were used to evaluate if the constitutive overexpression of LOXL2 protects against OA pain and
inflammation. The treadmill analysis was performed with a standard protocol at 28 days post-MIA
Figure 5.
LOXL2 overexpression in transgenic mice protects against MIA-induced OA-related catabolic
changes in knee joint articular cartilage. (
A
) Scheme of LOXL2 mice MIA injection groups and its
functional analysis; (
B
) Safranin-O staining in the indicated WT and LOXL2 transgenic groups, its
quantification and OARSI scoring. Immunostaining and quantification of (
C
) LOXL2 and (
D
) Acan in
MIA injected LOXL2 overexpressing or WT mice. The fold change dierences in immunostaining for
LOXL2 and Acan expression in WT (dot), LOXL2 (square), WT/MIA (triangle) and MIA/LOXL2 (inverted
triangles) are shown in adjacent figures. The statistically significant dierences in immunostaining
were evaluated by one-way ANOVA with Bonferroni correction (*** p<0.001; ANOVA).
2.5. Overexpression of LOXL2 Protects Against Functional Changes in Aging Knee OA
MIA-induced OA produces pain-depressed wheel running [
19
]. Treadmill behavior is useful for
preclinical behavioral assessment of chronic pain and inflammation [
20
]. These LOXL2 transgenic
mice were used to evaluate if the constitutive overexpression of LOXL2 protects against OA pain
and inflammation. The treadmill analysis was performed with a standard protocol at 28 days
Int. J. Mol. Sci. 2019,20, 4798 8 of 13
post-MIA injection. The LOXL2-overexpressing mice ran longer and farther than wild-type littermates
(Figure 6A,B). The LOXL2-overexpressing mice also exhibited maximal exercise capacity and improved
allodynia compared to WT littermates (Figure 6C). Thus, LOXL2 appears to protect mice from
MIA-induced OA and restore joint function.
Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 9 of 14
injection. The LOXL2-overexpressing mice ran longer and farther than wild-type littermates (Figure
6 A,B). The LOXL2-overexpressing mice also exhibited maximal exercise capacity and improved
allodynia compared to WT littermates (Figure 6C). Thus, LOXL2 appears to protect mice from MIA-
induced OA and restore joint function.
Figure 6. LOXL2 overexpression in transgenic mice protects against MIA-induced OA-related decline
in knee joint function. (A) Maximal time spent on the treadmill by MIA-induced LOXL2 transgenic
and WT mice. (B) Total distance covered on the treadmill by MIA-induced LOXL2 transgenic and WT
mice. (C) Quantification of average Von Frey hairs weight (gms) (as indicated in the graph, y-axis),
showing pain-sensitive allodynia of MIA-induced LOXL2 transgenic (square) and WT mice (dot). The
statistically significant differences were evaluated by one-way ANOVA with Bonferroni correction
(***p < 0.001; ANOVA).
3. Discussion
While seeking to understand how LOXL2 attenuates catabolic factors during age-related OA
pathogenesis, the overarching goal was to evaluate if adenoviral LOXL2 could be used for
translational research and future clinical applications in OA treatment. In a prior study, the authors
showed that LOXL2 could have chondroprotective and anabolic effects in OA [6], but the in vivo role
of LOXL2 in cartilage development, maintenance, and protection from OA is not known. The data
from our current study indicate that LOXL2 could induce a chondroprotective response by inhibiting
catabolic factors or IL-1β-induced NF-κB signaling pathways.
First, this study utilized 4-month-old Cho/+ mice, which developed progressive knee-OA
[14,15,21,22], to evaluate the protective effect of LOXL2 and understand the regulation of key
regulatory targets involved in knee-OA. The knee joints of 3-month old Cho/+ mice were normal but
exhibited progressive OA-like changes with age-associated increases in severity. By 6 months of age,
Figure 6.
LOXL2 overexpression in transgenic mice protects against MIA-induced OA-related decline
in knee joint function. (
A
) Maximal time spent on the treadmill by MIA-induced LOXL2 transgenic
and WT mice. (
B
) Total distance covered on the treadmill by MIA-induced LOXL2 transgenic and WT
mice. (
C
) Quantification of average Von Frey hairs weight (gms) (as indicated in the graph, y-axis),
showing pain-sensitive allodynia of MIA-induced LOXL2 transgenic (square) and WT mice (dot).
The statistically significant dierences were evaluated by one-way ANOVA with Bonferroni correction
(*** p<0.001; ANOVA).
3. Discussion
While seeking to understand how LOXL2 attenuates catabolic factors during age-related OA
pathogenesis, the overarching goal was to evaluate if adenoviral LOXL2 could be used for translational
research and future clinical applications in OA treatment. In a prior study, the authors showed that
LOXL2 could have chondroprotective and anabolic eects in OA [
6
], but the
in vivo
role of LOXL2 in
cartilage development, maintenance, and protection from OA is not known. The data from our current
study indicate that LOXL2 could induce a chondroprotective response by inhibiting catabolic factors
or IL-1β-induced NF-κB signaling pathways.
First, this study utilized 4-month-old Cho/+ mice, which developed progressive knee-OA [
14
,
15
,
21
,
22
],
to evaluate the protective eect of LOXL2 and understand the regulation of key regulatory targets
involved in knee-OA. The knee joints of 3-month old Cho/+ mice were normal but exhibited progressive
Int. J. Mol. Sci. 2019,20, 4798 9 of 13
OA-like changes with age-associated increases in severity. By 6 months of age, the proteoglycan staining
was lost in superficial zones, and by 9 months, the region of deficient proteoglycan staining extended
from superficial to deep layers. By 15 months of age, the typical OA-like knee joints, including the loss
of articular cartilage, misshaped meniscus, and the inflammation in synovial tissues, have developed
in Cho/+ mice, but not WT littermates [
15
]. At 3 and 6 months, the expression of ECM-degrading
proteins MMP-3 and MMP-13 were elevated, respectively, relative to WT levels in knee joints of Cho/+
mice. MMP-13 is implicated in OA pathogenesis [
23
], and our current study shows that LOXL2 can
attenuate MMP-13 expression in vitro and in vivo.
Next, the above histological and molecular findings were validated using an additional mouse
model and extended our study to examine joint function. The LOXL2-overexpressing transgenic mice
were more resistant to MIA-induced OA, indicating that LOXL2 has a chondroprotective function
and can inhibit disability as evaluated by the treadmill test and allodynia. A previous study showed
that LOXL2-induced collagen cross-linking enhances the tensile strength of articular cartilage and the
resistance to collagen proteolysis [
24
]. However, that study did not evaluate the specific mechanism
underlying that behavior.
This study sought to evaluate a possible mechanism by examining the eect of the LOXL2
expression on the catabolic IL-1
β
-induced signaling pathway. Of note, IL-1
β
is a target of various
therapeutic interventions. In chondrocytes, IL-1
β
activates the canonical NF-
κ
B pathway, which is
dependent upon IKK
β
, leading to increased gene expression of MMPs, ADAMTSs, inflammatory
mediators (COX2, NO, PGE2), chemokines (IL-8), and cytokines (IL-1
β
, TNF-
α
, IL-6) involved in
cartilage destruction. In contrast, the non-canonical NF-
κ
B activating kinase promotes hypertrophy [
25
].
Inhibiting the IL-1
β
-induced NF-
κ
B pathway can attenuate OA [
16
,
17
], and IL-1
β
promotes ADAMT5
and MMP13 in OA. It has been determined that LOXL2 inhibits IL-1
β
-induced NF-
κ
B and catabolic
mediator pathways, which are potential mechanisms for progressive OA and aging cartilage.
Although our data show that LOXL2 has a protective function in knee OA, many questions remain.
It is unknown whether defective LOXL2 function leads to OA. The mutation of collagen XI (Cho/+) [
15
]
or Col2a1 [
26
] in mice disrupts the cartilage matrix, increasing susceptibility to degradation [
27
].
However, it is not known if there is a direct correlation between functional dysregulation of LOXL2 and
human OA, which needs to be evaluated in the future. Of note, our study shows that the enzymatic
inhibition of LOXL2 does not block its ability to attenuate NF-
κ
B activity, demonstrating that its
anti-catabolic eect could be mediated by a non-enzymatic region of LOXL2. This needs to be further
evaluated, but is in line with other LOXL2 actions previously reported as independent of its catalytic
activity [28,29].
With the initial success of gene therapy, the development of specific gene therapy approaches for
several diseases and conditions is trending. Our study showed that adenovirus LOXL2 delivery at a
specific concentration protected against knee-OA with no adverse eects. However, there is a need to
establish a dose-response and safe delivery methods for clinical application. An earlier study showed
that LOXL2 overexpressing mice does not develop cancer, however, the application of carcinogen
could promote cancer growth or metastasis in breast cancer models [
30
]. Our study evaluated the
systemic delivery of adenovirus LOXL2. However, local delivery could be tested in preclinical and
clinical studies in the future. The understanding of detailed molecular and cellular mechanisms could
be very promising for future applications. Thus, this study provides a foundation for several future
mechanistic and translational studies.
In conclusion, our studies found that: 1) Adenoviral LOXL2 attenuates the eect of OA-related
catabolic factors released during progressive OA in the Cho/+ mouse model; 2) the constitutive
expression of LOXL2 has no adverse eects and attenuates knee-OA-related changes and preserves
normal joint function in aged mice; 3) LOXL2 attenuates IL-1
β
-induced signaling, which could be a
potential mechanism for its protective eect(s). This study demonstrates the importance of developing
a more detailed understanding of a LOXL2 function and that adenovirus LOXL2 is a candidate for
future translational applications.
Int. J. Mol. Sci. 2019,20, 4798 10 of 13
4. Materials and Methods
4.1. Animal Experiments
All mouse experiments were performed with the guidance, regulation, and approval of the Boston
University Institutional Animal Care and Use Committee (IACUC; approval number AN-15387, dated
24 August 2016). The animal study conformed to ARRIVE guidelines.
4.2. Preparation of Adenovirus
Adenoviruses for LOXL2 expression (Ad-CMV-RFP-CMV-hLOXL2-His, referred to as Ad-RFP-LOXL2)
and empty vector control (Ad-RFP-Empty) were custom synthesized (ADV-214438) by Vector Biolabs.
The adenovirus particles were amplified in 293T cells and quantified by using an adenovirus
quantification kit (Cell Biolabs, Inc., San Diego, CA, USA).
4.3. Cho/+ Mouse Experiments
The Cho/+ mice were obtained from Dr. Yefu Li at Harvard Medical School and bred with
C57/BL6 mice to maintain a heterozygous mouse colony. The mice were genotyped by PCR using
standard protocols. The sixteen-week-old Cho/+ mice were divided into 2 groups: Adv-RFP-Empty
was administered to 14 male and 14 female mice, and Adv-RFP-LOXL2 was administered to 14 male
and 14 female mice via twice monthly intraperitoneal injection (100
µ
L; 10
13
infectious particles/mL)
for 3 months. The knee joints were harvested and processed for RT-qPCR and histology.
4.4. Cytotoxicity and Immune Eect of Adenovirus Vector
The
in vitro
cytotoxicity was evaluated in ATDC5 cells by treating the cells with vehicle and
adenovirus vector (Adv-RFP-Empty) by using Lactate dehydrogenase (LDH) assay as per the
manufacturers’ instruction (Abcam Inc, Cambridge, MA, USA, ab102526). The serum samples
of the mice injected with vehicle or adenovirus vector (Adv-RFP-Empty) were dilated 1:10 and 50 uL
of serum samples may be evaluated for LDH assay (Abcam, Inc.). In order to evaluate the eect of
the adenovirus vector on cytokine expression, RNA from the knee joint from the mice injected with
vehicle or adenovirus vector (Adv-RFP-Empty) were subjected to RT-qPCR analysis (Taqman gene
expression assay, Applied Biosystems, Waltham, MA, USA) for the expression of TNFαand IL6.
4.5. RNA Isolation and Analysis
The total RNA was extracted by Trizol and RNAeasy protocol according to the manufacturer’s
instructions (Qiagen, Germantown MD, USA). The quantitative real-time PCR (RT-qPCR) analysis
was performed using TaqMan gene expression assays from Life Technologies, according to a standard
protocol [31].
4.6. Histology and Immunostaining
The knee joints from Cho/+ mice or LOXL2 transgenic mice were paran embedded, decalcified,
and subjected to histological analysis and immunostaining. Safranin-O/Fast green (American Mastertek
Inc., Lodi, CA, USA) staining was performed as described [
6
]. The OARSI scoring was performed
as per the recommendations. Three sections from 4 mice/group/condition were de-paranized and
immunostained with specific antibodies to detect RFP, LOXL2, aggrecan, and MMP13 (Abcam) and
visualized with HRP-linked anti-rabbit antibodies. The stained tissues were scanned with a digital
slide scanner (panoramic MIDI, 3D Histech, Budapest, Hungary).
4.7. MIA Mouse Model and Treadmill Running in Aging Mice
The mice with a conditionally targeted Loxl2 transgene (loxP-PGK-neo-stop-loxP-LOXL2-
IRES-eGFP) were crossed to ROSA26-Cre to express LOXL2 constitutively, as shown in our collaborator’s
Int. J. Mol. Sci. 2019,20, 4798 11 of 13
study [
18
]. To evaluate if the constitutive overexpression of LOXL2 in mice protects from OA pain and
inflammation, MIA (0.5 mg) was injected into the right knees (n=12 per condition) of 13-month-old
(aged) homozygous LOXL2-overexpressing mice or wild-type littermates (WT).
Treadmill behavior is useful for preclinical behavioral assessment of chronic pain and
inflammation [
20
]. The treadmill analysis was performed with a standard protocol [
32
] at 28 days
post-MIA injection. The mice were acclimatized to treadmill running (TSE Systems) on 3 consecutive
days followed by resting for 1 day before evaluating performance [
32
]. The acclimatization consisted of
a 5-min rest on the treadmill conveyor belt followed by 5 min of running at 7.2 m/s and 5 min at 9.6 m/s.
On day 0, the mice were subjected to a graded maximal running test consisting of an initial 5-min rest,
after which the running protocol commenced at 4.8 m/min, gradually increasing by 2.4 m/min every
2 min. At all times, the belt was kept at a 5-degree incline. The maximal running speed was defined as
the fastest speed at which the mice were able to run for 5 consecutive seconds without touching the
electric shock grid at the back of the treadmill. The allodynia was evaluated by Von Frey Hairs test.
The researcher conducting the test was blinded to experimental groups.
Supplementary Materials:
Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/19/
4798/s1.
Author Contributions:
M.V.B.: conception and design, data collection, performing experiments, analysis and
interpretation of data, and manuscript writing. M.T., F.A., S.A.,T.A: performing experiments and conception. A.C.,
A.M., F.P., F.S. and L.C.G.: providing resources, conception, and manuscript editing. M.B.G.: conception and
design, interpretation of data, providing resources, and manuscript editing.
Funding:
The authors acknowledge NIH/NIDCR grants R03DE025274 and R21DE026892 as well as CTSI pilot
funding to Manish V. Bais.
Acknowledgments:
The authors also acknowledge Philip Trackman for suggestions, reading the manuscript,
and resources. The authors thank Marie Demay and the CSR Histology Core (supported by NIAMS P30 grant
AR075042) at Mass General Hospital for histology services. M.T. acknowleges the sponosorship from Umm
Al-Qura University College of Dentistry, Makkah, Saudi Arabia Ministry of Education.
Conflicts of Interest:
All authors declare that they have no conflicts of interest regarding the contents of
this manuscript.
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©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
... Subsequently, we determined that LOXL2 expression is critical for chondrogenic differentiation and proteoglycan deposition in a chondrocyte culture model 18 . More recently, we identified LOXL2 as an anabolic effector that attenuates pro-inflammatory signaling in OA cartilage of the TMJ and knee joint in vitro and in vivo 19,20 . We developed a model in which human TMJ cartilage was implanted in nude mice in combination with a specialized matrix of the basement membrane. ...
... RNA isolation and analysis. Total RNA was extracted by the Trizol protocol according to the manufacturer's instructions (Qiagen) as described earlier 19,20 . RT-qPCR analysis was performed using TaqMan gene expression assays from Life Technologies, according to a standard protocol 33 . ...
... The LOXL2-induced proteoglycan network in human cartilage implants is critical for maintaining normal joint homeostasis. For example, in TMJ-OA, collagen crosslinking is defective, and collagen is more susceptible to degradation 54 , and a single point mutation in Col11a1, as seen in Cho/+ mice 29 , or Col2a1 deficiency in mice 55 leads to OA. Intracellular roles for LOXL2 in molecular signaling and epigenetic modification may also affect the development of OA 19,20 . Whether defective LOXL2 function leads to TMJ-OA has not been evaluated. ...
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... Further, studies have indicated the upregulation of LOXL2 in OA cartilage in response to injury, which may be considered as a naturally protective response that promotes anabolism while inhibiting specific catabolic response during OA pathophysiology (Alshenibr et al., 2017;Bais and Goldring, 2017). Likewise, a recent study further confirmed that systemic adenovirus-delivered LOXL2 expression or LOXL2 genetic overexpression both exhibited chondroprotective effects through inhibition of catabolic factors and IL-1β-induced NF-κB signaling in mice (Tashkandi et al., 2019). Also, Matrigel constructs of human chondrocytes from the knee joint and TMJ implanted in nude mice showed enhanced anabolic responses after LOXL2 transduction, including increased expression of sex determining region Y-box containing gene 9 (SOX9), aggrecan (ACAN), and COL2A1, whilst reduced the levels of extracellular matrix (ECM)-degrading enzymes matrix metalloproteinases and inhibited chondrocyte apoptosis (Alshenibr et al., 2017). ...
... The decreased expression of LOXs may attribute to reduction of HIF-1 activity in aging organisms (Rivard et al., 2000;Ceradini et al., 2004). Meanwhile, LOXL2 has recently been demonstrated as a potential chondroprotective factor in aging related joint osteoarthritis, mainly through inducing anabolic gene expression and attenuating catabolic genes (Bais and Goldring, 2017;Tashkandi et al., 2019). Thus, restoration of collagen and elastic fiber synthesis in juvenile ECM components though regulation of signaling pathways governing LOX expression may become a promising therapeutic approach for amelioration of aging-associated cartilage degeneration and enhanced cartilage regeneration. ...
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Articular cartilage remains among the most difficult tissues to regenerate due to its poor self-repair capacity. The lysyl oxidase family (LOX; also termed as protein-lysine 6-oxidase), mainly consists of lysyl oxidase (LO) and lysyl oxidase-like 1-4 (LOXL1-LOXL4), has been traditionally defined as cuproenzymes that are essential for stabilization of extracellular matrix, particularly cross-linking of collagen and elastin. LOX is essential in the musculoskeletal system, particularly cartilage. LOXs-mediated collagen cross-links are essential for the functional integrity of articular cartilage. Appropriate modulation of the expression or activity of certain LOX members selectively may become potential promising strategy for cartilage repair. In the current review, we summarized the advances of LOX in cartilage homeostasis and functioning, as well as copper-mediated activation of LOX through hypoxia-responsive signaling axis during recent decades. Also, the molecular signaling network governing LOX expression has been summarized, indicating that appropriate modulation of hypoxia-responsive-signaling-directed LOX expression through manipulation of bioavailability of copper and oxygen is promising for further clinical implications of cartilage regeneration, which has emerged as a potential therapeutic approach for cartilage rejuvenation in tissue engineering and regenerative medicine. Therefore, targeted regulation of copper-mediated hypoxia-responsive signalling axis for selective modulation of LOX expression may become potential effective therapeutics for enhanced cartilage regeneration and rejuvenation in future clinical implications.
... The copper-dependent amine oxidase lysis oxidase-like 2 (LOXL2) catalyzes the first step in the formation of crosslinks in collagens and elastin and has been shown to mediate endochondral ossification. Tashkandi et al. wanted to assess the potential of Admediated overexpression of LOXL2 for OA treatment (Table 5) (77). In an IL-1β stimulated ATDC5 cartilage cell line, LOXL2 overexpression blunted the decrease of aggrecan and SOX9, and attenuated both the IL-1β mediated expression of ADAMTS4/5, and MMP-13, as well as the IL-1β induced activity of NF-κB. ...
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... LOX family members, in particular LOX and LOXL2, are also involved in tumor progression through extracellular and intracellular actions [24][25][26][27]. Different lysyl oxidase genetic mouse models have been instrumental in characterizing their participation in several nontumor pathologies [28][29][30][31][32][33][34]. Regarding cancer, LOX and LOXL2 contribute to breast cancer metastasis [35,36], and LOXL2 is involved in the initiation and progression of head and neck squamous cell carcinoma [34]. ...
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... However, comparable levels of phosphorylated JNK1/2 and ERK1/2 were detected in HASMCs with or without LOXL2 knockdown ( Figure 3E). In addition, LOXL2 inhibits NF-κBp65 phosphorylation in ATDC5 cells, and NF-κBp65 activation is also involved in AD (23). Unexpectedly, the phosphorylation of NF-κBp65 was not regulated by LOXL2 in HASMCs ( Figure 3E). ...
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... Expression of collagen crosslinking enzymes lysyl oxidases (Lox, Loxl2, and loxl3) also decreased with age ( Figure S3C). Lysyl oxidases play a key role in physiological and pathological remodeling of extracellular matrix and it has been shown that systemic LOXL2 adenovirus or LOXL2 genetic overexpression in mice can protect against OA [34]. Procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 (Plod2), another collagen processing enzyme, also had significantly lower expression in old mice compared with young. ...
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The lysyl oxidase-like protein LOXL2 has been suggested to contribute to tumor progression and metastasis, but in vivo evidence has been lacking. Here we provide functional evidence that LOXL2 is a key driver of breast cancer metastasis in two conditional transgenic mouse models of PyMT-induced breast cancer. LOXL2 ablation in mammary tumor cells dramatically decreased lung metastasis, whereas LOXL2 overexpression promoted metastatic tumor growth. LOXL2 depletion or overexpression in tumor cells does not affect extracellular matrix stiffness or organization in primary and metastatic tumors, implying a function for LOXL2 independent of its conventional role in extracellular matrix remodeling. In support of this likelihood, cellular and molecular analyses revealed an association of LOXL2 action with elevated levels of the EMT regulatory transcription factor Snail1 and expression of several cytokines that promote pre-metastatic niche formation. Taken together, our findings established a pathophysiological role and new function for LOXL2 in breast cancer metastasis.
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