The Effect of Mutations in Low-Density Lipoprotein Receptor Related Protein 6 (LRP6) on Osteoarthritis Progression in a Murine Injury Model
+1Joiner, D M; 1Scholten II, D J; 1Less, K D; 1Morris, M; 1Mason, J
+1Van Andel Research Institute Orthopaedic Cell and Tissue Mechanics Laboratory, Grand Rapids, MI
Danese.joiner@vai.org
Introduction: Osteoarthritis (OA) is a disease secondary to the
degradation of articular cartilage over time, resulting in pain and
suffering to the patient, and if left untreated, eventual loss of joint
function. Among U.S. adults, nearly 27 million have clinical
osteoarthritis [1]. Currently OA is surgically treated with engineered
implants which last 10-15 years and require revision surgery. These
patients need alternatives to the current treatment which requires a better
understanding of the progression of this pervasive disease. Wnt/βcatenin signaling plays an important role in the maintenance of bone and
cartilage and the ability of bone to respond to mechanical stimulation.
Studies suggest that activation or inactivation of Wnt/β-catenin signaling
can lead to the development of OA in murine models. However, these
models lack the context of an altered mechanical environment, through
ligament and meniscus damage, which is an important clinical trigger of
OA [2,3,4,5]. To investigate the role of decreased Wnt/β-catenin
signaling in the progression of OA in the context of injury and
subsequently altered local mechanical environment, Lrp6 knockout and
wild type (WT) mice were examined utilizing a ligament/meniscus
transection model. Significance: This study highlights Wnt/β-catenin
signaling as a potential therapeutic for OA within a clinically relevant
animal injury model and also incorporates the role of
mechanotransduction in OA progression.
Materials and Methods: Animals: Male Lrp6+/+ and Lrp6+/- mice were
generated as described previously as Lrp6-/- animals are perinatal lethal
and previous data suggests Lrp6+/- mice have a bone phenotype [6].
Ligament/Meniscus Transection: Ligament/meniscus transections
were performed on the right knee (Surgery) of eight 8 week old WT and
eight 8 week old Lrp6+/- animals as described previously [7]. The left
knees (Control) of each of these animals were untouched and served as a
contralateral control. The animal was anesthetized with an
intraperitoneal injection of Avertin at a dose of 0.03 ml/g. The joint
capsule was opened medial to the patellar tendon. The fat pad over the
intercondylar region was dissected to expose the joint. The medial
meniscotibial ligament was sectioned to destabilize the medial meniscus.
The anterior cruciate, posterior cruciate and lateral meniscotibial
ligaments were transected and the medial and lateral menisci were
removed. The incision was then closed. Animals resumed normal
ambulation within hours after surgery. Animals were euthanized 60
days after surgery for analysis. Micro CT: Knees from WT (n=5) and
Lrp6+/- (n=5) mice were scanned at a 10µm voxel size with a Skyscan
1172 system (Skyscan, Belgium, Germany). Images were reconstructed
and bone and joint structure were observed. Histology: Knees from WT
(n=5) and Lrp6+/- (n=5) mice were fixed for 48h in 10% Neutral
Buffered Formalin (NBF) and decalcified for 48h in Decal. Samples
were transferred to a Sakura VIP tissue processor where they were
infiltrated with a series of alcohol grades, cleared with xylene, and
finally infiltrated with paraffin. Samples were embedded on edge into a
paraffin mold using the Leica Embedding center and sectioned along the
coronal plane at 5um using a microm microtome. The 5um sections were
collected onto glass slides, deparaffinized, and hydrated in distilled
water. Sections were stained with Alcian Blue (1g Alcian Blue
8GX:100mL 3% acetic acid in distilled water pH 2.5 (Sigma Aldrich, St.
Louis, MO)) for 30 min., counterstained with Hematoxylin (Sigma
Aldrich, St. Louis, MO) for 1 min., dehydrated and coverslipped. Other
sections were incubated in 0.3% hydrogen peroxide for 30min., blocked
in 5% goat serum in Tris Buffered Saline with 0.01% Tween (TBS-T)
for 1h, and incubated with primary rabbit polyclonal antibody for type II
collagen (abcam Cambridge, MA) overnight at 4°C. Slides were then
incubated at RT with anti rabbit secondary antibody for 1h, A and B
reagents for 30 min., and Nova Red substrate until slides developed with
a Vectastain ABC kit according to manufacturer’s instructions (Vector
Laboratories Burlingame, CA). All images were taken at 10x
magnification. Western Blot: Knees from WT (n=3) and Lrp6+/- (n=3)
mice were homogenized in protein lysis buffer. Protein concentration
was determined with a BCA assay (Thermo Scientific Rockford, IL).
40µg protein were loaded into the lanes of a 10% Mini-PROTEAN TGX
Precast gel (BioRad Hercules, CA). The gel was run at 125V for 90min.
and transferred at 80V for 40min. Membranes were blocked for 1h at
RT in 5% milk in TBS-T. After blocking, membranes were rinsed
3x10min. in TBS-T and incubated overnight at 4°C with primary
antibodies against Type II collagen (abcam Cambridge, MA),
ADAMTS-5 (abcam Cambridge, MA), and Beta Tubulin (Van Andel
Grand Rapids, MI). After primary antibody incubation, membranes were
rinsed 3x10min. in TBS-T and incubated with secondary antibody (GE
Healthcare Piscataway, NJ) for 2h at RT. Membranes were rinsed
3x10min. in TBS-T, covered in Amersham ECL Prime Western Blotting
Detecting Reagent and developed on Amersham Hyperfilm ECL (GE
Healthcare Piscataway, NJ). Membranes were stripped for 30min. at RT
with a harsh stripping buffer (abcam Cambridge, MA) and rinsed in
TBS-T 3x10min. between probes for each protein.
Results: Radiographic evidence of OA (tibia plateau erosion, joint space
narrowing, and femoral bony outgrowths) was observed in the surgery
knee of WT and Lrp6+/- mice compared to control knees (Fig. 1a).
ADAMTS-5 protein levels were higher in the transected knee of Lrp6+/mice compared to controls, however no apparent difference in
ADAMTS-5 was observed between the knees of WT animals (Fig. 1b).
There was decreased Type II Collagen protein in the surgery knees of
WT and Lrp6+/- animals compared to control knees and this was more
evident in the Lrp6+/- animals (Fig. 1b). Histological sections of the
knee joint stained with Alcian Blue for cartilage imaging show
hypercellularity and fibrous tissue in the joint space of transected knees
for both WT and Lrp6+/- mice (Fig. 2). Articular cartilage is degraded
and disorganized in the surgical knees of WT and Lrp6+/- animals
compared to control knees and appears more severe in Lrp6+/- mice (Fig.
2). There is less Type II collagen in the articular cartilage of transected
knees from WT and Lrp6+/- animals compared to control knees and tibia
plateau degradation is severe in the Lrp6+/- surgical knee (Fig. 3).
Fig. 1a Micro-CT Coronal Plane Images Fig. 1b Western Blots
Lrp6+/Lrp6+/+
Type II Collagen
ADAMTS-5
Beta Tubulin
Control
Surgery
Control
Surgery
Control Surgery Control Surgery
Lrp6+/- Lrp6+/+
Fig. 2 Alcian Blue Hematoxylin Images of the Knee Joint
Surgery
Lrp6+/-Control
Surgery
Lrp6+/+Control
Fig. 3 Type II Collagen Hematoxylin IHC Images of the Knee Joint
Surgery
Lrp6+/-Control
Surgery
Lrp6+/+Control
Discussion: Data from this study suggests OA development in knees of
WT and Lrp6+/- mice after damage to ligaments and meniscus, however
increased severity in heterozygous animals. This data is supported by a
study in which suppressed β-catenin signaling in articular chondrocytes
caused OA-like cartilage degradation. OA progression could also be
accelerated in the transected knees of Lrp6+/- animals due to changes in
the local mechanical environment. Lrp5-/- mice have a decreased
osteogenic response to mechanical loading, however the effect of
heterozygous mutations in Lrp6 on bone mechanotransduction has not
yet been elucidated [8]. This study highlights Lrp6 as a potential target
for the development of pharmaceutical treatments to slow, stop, or
reverses the progression of this pervasive disease.
Acknowledgements: The authors would like to thank Paula Davidson,
Travis Burgers, Alex Zhong, Cassie Diegel, Bart Williams, Tessa
Grabinski, and Lisa Turner.
References: [1]Lawrence, R. et al. 2008 [2]Zhu, M. et al. 2008 [3]Ning,
B. et al. 2011 [4]Zhu, M. et al. 2009 [5]Weng, L. et al. 2010 [6]Holmen,
S. et al. 2004 [7]Kamekura, S. et al. 2005 [8]Sawakami, K. et al. 2006.
Poster No. 0801 • ORS 2012 Annual Meeting