AN ABSTRACT OF THE THESIS OF
Tara L. Riddick for the degree of Master of Science in Veterinary Science presented
on June 20, 2011.
Title: Molecular Characterization of Early Osteochondrosis in Prepubescent Foals
Abstract approved:
_________________________________________________________________
Stacy A. Semevolos
Osteochondrosis (OC) is a major source of lameness in horses, involving abnormal
maturation of cartilage during the first year of life. Previous studies have shown that
cartilage canals may be associated with early OC lesions with accumulation of large
numbers of small rounded chondrocytes surrounding the canals. Altered expression of
paracrine factors, parathyroid hormone-related peptide (PTH-rP) and Indian hedgehog
(Ihh), as well as the additional factors, vascular endothelial growth factor (VEGF),
platelet derived growth factor (PDGF), and matrix metalloproteinase-13 (MMP-13),
may play a role in the development of OC. The objective of this study was to elucidate
the expression of Ihh, PTH-rP, VEGF, PDGF, and MMP-13 in cartilage of
prepubescent foals to determine how this process becomes disrupted in early
osteochondrosis prior to the onset of clinical signs. The hypothesis was that
osteochondrosis develops as a result of increased expression of these regulatory
molecules via cartilage canals during development, leading to failure of cells
surrounding cartilage canals to mature and calcify.
Cartilage was harvested from femoropatellar joints of foals ≤6 months old. Lasercapture microdissection was used to capture cells surrounding the cartilage canals and
osteochondral junction. Equine-specific Ihh, PTH-rP, VEGF, PDGF-A, MMP-3,
MMP-13 and 18S mRNA expression levels were evaluated by real-time qPCR. Whole
cartilage RNA expression levels were also evaluated. Spatial tissue protein expression
was determined by immunohistochemistry.
There was significantly increased Ihh, MMP-3, and MMP-13 whole cartilage gene
expression in early OC cartilage compared to normal controls. Protein expression of
VEGF, PDGF-A, MMP-13, Ihh, and PTH-rP was mainly observed in the superficial
and deep cartilage layers, as well as along the osteochondral junction and cartilage
canals. In laser-captured samples, there was significantly increased MMP-13 and
PDGF-A gene expression in chondrocytes adjacent to cartilage canals and increased
PDGF-A gene expression in osteochondral junction chondrocytes of OCD-affected
foals compared to controls. Increased Ihh, MMP-3, and MMP-13 gene expression in
early OC whole cartilage provided stronger evidence for their association with OC,
while significantly increased PDGF-A and MMP-13 gene expression surrounding
OCD cartilage canals and increased PDGF-A gene expression at the osteochondral
junction in this study suggests that pathways involving endochondral maturation and
invasion of the ossification front are altered in early osteochondrosis.
Molecular Characterization of Early Osteochondrosis in Prepubescent Foals
by
Tara L. Riddick
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Presented June 20, 2011
Commencement June 2012
Master of Science thesis of Tara L. Riddick presented on June 20, 2011.
APPROVED:
____________________________________________
Major Professor, representing Veterinary Science
____________________________________________
Head of the College of Veterinary Medicine
____________________________________________
Dean of the Graduate School
I understand that my thesis will become part of the permanent collection of Oregon
State University libraries. My signature below authorizes release of my thesis to any
reader upon request.
____________________________________________
Tara L. Riddick. Author
ACKNOWLEDGEMENTS
Thanks go to my co-authors, Dr. Stacy Semevolos and Dr. Katja Zellmer, for their
assistance in sample and data collection, as well as manuscript preparation.
The authors would like to thank Dr. Chris Corless for use of the laser capture
microdissection unit at Oregon Health Sciences University.
The technical assistance of Mary Lou Norman with histologic processing at Cornell
University is gratefully appreciated.
Financial support was provided by the American Quarter Horse Foundation.
CONTRIBUTION OF AUTHORS
Dr. Stacy Semevolos assisted with initial sample and data collection and was involved
in manuscript preparation.
Dr. Katja Zellmer assisted with initial sample collection, performed laser capture
microdissection of cartilage samples, and assisted with manuscript preparation.
TABLE OF CONTENTS
Chapter 1: Introduction.…………………………………………
Page
2
Articular Cartilage Structure……………………………..
2
Endochondral Ossification……………………………….
4
Molecular Characterization of Endochondral
Ossification………………………………
6
Osteochondrosis………………………………………….
10
Proposed Etiologies………………………………
10
Chapter 2: Increased Gene Expression of Indian Hedgehog,
Matrix Metalloproteinase-3, and Matrix
Metalloproteinase-13 in Early Equine Osteochondrosis….
15
Introduction……………………………………………….
16
Materials and Methods……………………………………
18
Animals……………………………………………
18
Sample Collection…………………………………
18
RNA Isolation…………………………………….
19
Real-time quantitative RT-PCR…………………...
19
Immunohistochemistry……………………………
20
Sample Evaluation and Classification…………….. 20
Scoring and Statistical Analysis…………………...
21
Results……………………………………………………..
22
Discussion…………………………………………………
24
TABLE OF CONTENTS (Continued)
Figures …………..………………………………………...
Page
30
Tables ……………………………………………………..
36
Chapter 3: Molecular Expression of Cartilage Canal and
Osteochondral Junction Chondrocytes in Subclinical
Juvenile Osteochondritis Dissecans (OCD)………………
38
Introduction……………………………………………….
39
Methods……………...……………………………………
41
Sample Collection…………………………………
41
Laser-capture microdissection…………………….
41
RNA Isolation……………………………………..
42
Real-time quantitative RT-PCR…………………...
42
Immunohistochemistry……………………………
43
Sample Evaluation and Classification…………….. 44
Scoring and Statistical Analysis…………………...
45
Results……………………………………………………..
45
Discussion…………………………………………………
47
Figures ………….………………………………………...
52
Tables …………………………………………………….
62
Chapter 4: Conclusion …………………………….……………..
64
Footnotes………………………………………………………….
71
References………………………………………………………...
72
LIST OF FIGURES
Figure
Page
Figure 2.1: Photomicrographs following H&E staining of
osteochondral sections, showing middle to deep articular
cartilage layers and the osteochondral junction. ……...
30
Figure 2.2: Photomicrographs following immunohistochemical
localization using antibodies directed against Ihh. ……...
31
Figure 2.3: Photomicrographs following immunohistochemical
localization using antibodies directed against VEGF. ......
32
Figure 2.4: Photomicrographs following immunohistochemical
localization using antibodies directed against PDGF.…...
33
Figure 2.5: Photomicrographs following immunohistochemical
localization using antibodies directed against MMP-3......
34
Figure 2.6: Photomicrographs following immunohistochemical
localization using antibodies directed against MMP-13....
35
Figure 3.1: Photomicrograph of hematoxylin-stained
osteochondral section showing approximate regions
extracted by LCM. …….………………………………...
52
Figure 3.2: Photomicrograph of a frozen cartilage canal section
from a 4-month-old foal. ………………………………...
53
Figure 3.3: Gene expression of laser-captured cartilage canal
chondrocytes using real-time PCR.…………….....……...
54
Figure 3.4: Gene expression of laser-captured osteochondral
junction chondrocytes using real-time PCR.………….....
55
Figure 3.5: Gene expression comparisons of cartilage canal
chondrocytes vs. osteochondral junction chondrocytes
using real-time PCR.………………..……………………
56
LIST OF FIGURES (Continued)
Figure
Page
Figure 3.6: Photomicrographs of osteochondral sections
following immunohistochemical localization using
antibodies directed against VEGF.…………….………...
57
Figure 3.7: Photomicrographs of osteochondral sections
following immunohistochemical localization using
antibodies directed against PDGF.……………….……...
58
Figure 3.8: Photomicrographs of osteochondral sections
following immunohistochemical localization using
antibodies directed against MMP-13.…………….……...
59
Figure 3.9: Photomicrographs of osteochondral sections
following immunohistochemical localization using
antibodies directed against Ihh. ………………...
60
Figure 3.10: Photomicrographs of osteochondral sections
following immunohistochemical localization using
antibodies directed against PTH-rP. ..………...………...
61
LIST OF TABLES
Table
Page
Table 2.1: Mean gene expression +/- SEM quantified by
real-time RT-PCR using whole cartilage from early OC
and normal control horses. ...……………………..……..
36
Table 2.2: Mean total cartilage scores +/- SEM following
immunohistochemistry of osteochondral sections from
early OC and normal control horses. ..……...…………..
37
Table 3.1: Summary of Animals and Samples. ……………….
62
Table 3.2: Mean protein expression scores +/- SEM following
immunohistochemistry of osteochondral sections from
juvenile OCD and normal controls. ……………..
63
Molecular Characterization of Early Osteochondrosis in Prepubescent Foals
2
Chapter 1: Introduction
Osteochondrosis (OC) is a developmental orthopedic disease affecting both
veterinary and human patients. It is a major source of lameness in young horses,
resulting from a disruption of normal endochondral ossification and articular cartilage
development in the first year of life. This lameness leads to loss of use of domestic
species, incurring significant economic losses. Although the direct etiology of OC
remains unknown, many factors have been associated with the development of the
disease, such as rapid growth rate, a high plane of nutrition, heredity, and cartilage
trauma.1,2
Articular Cartilage Structure
Composed of less than 10% chondrocytes, the remainder of articular cartilage
consists of extracellular matrix (ECM). The ECM can be further broken down into
water, collagen, and proteoglycan, as well as minor components of lipids,
phospholipids, proteins, and glycoproteins.3 Within the ECM, water contributes to a
significant percentage of the weight of the ECM, providing approximately 65-80% of
the total weight.3 Of the remaining constituents, collagens and proteoglycans serve as
the load-bearing components. The vast majority of the collagen within articular
cartilage is type II collagen; however, articular cartilage also contains small amounts
of types V, VI, IX, X, and XI collagen.3,4 Covalent crosslinks exist between the
collagen fibrils to add stability to the cartilage, with type II collagen adding significant
3
tensile strength to the cartilage.3,4 The short chain collagens provide additional tensile
strength and create a meshwork that traps proteoglycans within the ECM.4
Proteoglycans are produced by chondrocytes and separate components of
glycosaminoglycan (GAG) side chains bound to a protein core can be identified.3 The
proteoglycan aggrecan then binds to hyaluronan through a link protein to form an
aggregate.3 Aggrecans compose approximately 90% of cartilage matrix, and they
provide the cartilage with compressive strength.3,4
While the composition of articular cartilage changes through the stages of
development, the final product is hyaline cartilage which provides shock absorption
and a friction-free surface for joint mobility. Within a section of articular cartilage are
four recognizable zones with distinct characteristics. The superficial zone faces the
articular surface. It is composed of elongated chondrocytes found with the longest
dimension oriented parallel to the articular surface. The collagen fibrils in this zone
are also arranged parallel to the surface.3 This zone has the lowest proteoglycan
content and the highest water content.3 The transitional zone is found deep to the
superficial zone. The transitional, or middle, zone is composed of more rounded
chondrocytes that are more haphazardly spaced, as well as obliquely oriented collagen
fibrils of larger diameter.3 Deep to the transitional zone is the deep zone. In this zone,
the collagen fibrils are larger yet in diameter and perpendicular to the articular
surface.3 The spherical chondrocytes are arranged in columns perpendicular to the
articular surface.3 The proteoglycan content is highest in the deep zone, and the water
4
content is lowest.3 The deepest zone of articular cartilage is the calcified zone. The
demarcation between the deep cartilage and the calcified cartilage is known as the
tidemark. Specifically, the tidemark is a histologic finding that becomes apparent in
adult cartilage. At this time, significant stain uptake can be seen as a line between the
deep and calcified zones.
The ECM can also be divided into regions surrounding the chondrocytes. The
pericellular matrix forms a thin layer around the cell membrane of the chondrocyte.
While this layer of ECM contains proteoglycan and matrix components, it contains
almost no collagen component.3 The territorial matrix surrounds the pericellular
matrix and contains thin collagen fibrils that form a mesh-like network at the outer
edge of this layer of matrix.3 A unit containing the chondrocyte, pericellular matrix,
and territorial matrix, is known as the chondron.3 The interterritorial region includes
the entire matrix between the individual territorial matrices and is the largest of the
three matrix regions.3 Large collagen fibrils and most of the proteoglycans are
contained within the interterritorial region.3
Endochondral Ossification
During development of the growing skeleton, two regions of growth cartilage
exist near the ends of long bones. These regions are the physeal cartilage and the
epiphyseal cartilage. The physeal cartilage, also known as the growth plate, is present
on either side of the primary center of ossification and is responsible for longitudinal
5
growth.2 The epiphyseal cartilage exists between the secondary centers of ossification
and the articular surface and provides the shape of the end of the bone and articular
cartilage.2 In both of these regions, a sequence of events, referred to as endochondral
ossification, takes place in which the cartilage is replaced by bone.5
Prior to endochondral ossification, pre-cartilage condensation must take place.6
This occurs when mesenchymal cells from the lateral plate mesoderm migrate to the
limb and form aggregates of pre-cartilage cells by condensing without proliferating.
The mesenchymal cells then begin to create an ECM, composed of collagen type I,
hyaluronan, tenascin, and fibronectin. As the mesenchymal cells differentiate into
chondrocytes, the composition of the ECM shifts. The chondrocytes begin to produce
collagen type II, collagen type IX, collagen type XI, Gla protein, proteoglycan,
aggrecan, and link protein. It is at this time that expression of collagen type I is halted
and endochondral ossification begins.6
As the chondrocytes become surrounded by the ECM, they take on a rounded
morphology.2,7 These rounded chondrocytes become the resting zone, or pool, of
chondrocytes. 2,7 They divide infrequently but serve as precursors to the proliferating
zone, which consists of flat, proliferating chondrocytes.2,7 In the physeal growth plate,
the proliferating chondrocytes form columns; however, in the epiphseal growth
cartilage, the proliferating chondrocytes form clusters.2 These chondrocytes further
differentiate and hypertrophy, forming the hypertrophic zone of cartilage.2,8
Hypertrophic chondrocytes produce and maintain matrix until they undergo
6
apoptosis.2,6,8 Chondroclasts remove septa between the lacunae created by terminal
chondrocytes.2 The resulting cartilage is then vascularized by vascular loops from the
perichondrium.2 Osteoblasts are transported into the cartilage by the blood vessels, or
cartilage canals, and the cartilage begins to be replaced with mineralized bone.2,6,8
Although multiple molecular mediators of endochondral ossification have been
identified, research continues to further elucidate the roles of these factors, as well as
identify additional factors.
Molecular Characterization of Endochondral Ossification
Among the many factors involved in endochondral ossification are Indian
hedgehog (Ihh), parathyroid hormone related protein (PTH-rP), platelet derived
growth factor (PDGF), vascular endothelial growth factor (VEGF), and the matrix
metalloproteinase (MMP) family. These are a few of the factors believed to play a
critical role in development of postnatal cartilage during endochondral ossification.
Ihh plays multiple roles in the process of endochondral ossification, including
promotion of chondrocyte proliferation and maturation, osteoblast development, and
vascular invasion of cartilage.9 Through the use of chimeric mice, it was shown that
Ihh, synthesized by prehypertrophic and hypertrophic chondrocytes, served to couple
the process of chondrogenesis to osteogenesis by signaling the periarticular growth
plate.8 This regulated hypertrophic differentiation and determined the site of bone
collar formation in the nearby perichondrium.8 In vivo, Ihh null chondrocytes
7
exhibited delayed hypertrophy, slowing cartilage maturation.10 Additionally, Gli3, a
Gli transcription factor and Ihh inhibitor, has also been evaluated. In a study
evaluating Ihh null mice, removal of Gli3 restored normal proliferation and maturation
of chondrocytes, but only partial repair of the defects of cartilage vascularization was
seen.9
Produced by proliferative chondrocytes in the periarticular region and the
perichondrium near the growth plate, PTH-rP influences chondrocytic differentiation.8
PTH-rP forms a negative feedback loop with Ihh to regulate the progression of
chondrocytic terminal differentiation in growth plate cartilage.7,11 As Ihh promotes
chondrocyte proliferation and release of PTH-rP, PTH-rP downregulates Ihh and
inhibits chondrocytic terminal differentiation. In a study involving PTH-rP null mice,
lack of PTH-rP resulted in a much thinner epiphyseal plate with fewer chondrocytes.
These chondrocytes failed to hypertrophy.12 It is through the balance between Ihh and
PTH-rP that normal growth plate cartilage is formed. Although it is unknown whether
this feedback mechanism exists for articular cartilage, the existence of this feedback
loop in the growth plate provides compelling evidence for the importance of the roles
of Ihh and PTH-rP in proper progression of endochondral ossification.
Produced by hypertrophic chondrocytes, as well as osteoblasts-like cells,2,13
VEGF promotes vascularization of cartilage during endochondral ossification. In fact,
blockage of VEGF in mice fed a soluble chimeric VEGF-A receptor resulted in
inhibition of blood vessels to invade the hypertrophic zone of growth plates, impaired
8
trabecular bone formation, and expansion of the hypertrophic zone.14 Another study
evaluating protein expression via immunohistochemistry of mammalian and avian
embryo long bone showed low levels of expression of VEGF in prehypertrophic cells
and strong expression in hypertrophic chondrocytes.15 As the presence of
prehypertrophic and hypertrophic chondrocytes are associated with cartilage
developmental stages that precede vascular invasion of cartilage, these findings
suggest an important role of VEGF in promoting vessel ingrowth. Additionally,
hypertrophic chondrocytes grown in culture expressed VEGF, and VEGF was
expressed in developing muscle tissue just prior to tissue vascularization.15
Expressed by osteoblasts and stored in bone, PDGF increases the pool of
osteochondroprogenitor cells.16 A study evaluating the response of rat costochondral
resting zone chondrocytes to a variety of doses of PDGF found that the presence of
this substance increased chondrocyte proliferation and proteoglycan production, while
halting progression of those chondrocytes through further stages of endochondral
ossification.16,17 In addition, when PDGF was applied to resting zone chondrocytes
placed on a scaffold and implanted into rat muscle tissue, it promoted the growth of
hyaline type cartilage more than chondrocyte scaffolds not treated with PDGF.18
PDGF also acts as a strong mitogenic factor for mesenchymal cells, including
chondrocytes.19
The MMPs compose an extensive group of calcium and zinc dependent
endopeptidases that influence a variety of processes, including angiogenesis, wound
9
healing, and extracellular matrix degradation.20-22 MMP-1, 8, and 13 are capable of
initiating intrahelical cleavage of triple helical collagen in vivo, while MMP-3 cleaves
type II collagen in the nonhelical domain.23 Additionally, a variety of MMPs have
been identified in association with synovial fluid or tissues of osteoarthritic joints,
including MMP-1, 2, 3, 7, 13, 14, and 17.22-28 Although their role in developing
cartilage is not as well understood, there is some evidence that MMPs play an
important role in endochondral ossification, as well. While not as severe as the
vascular inhibition seen in regions devoid of VEGF, lack of MMP-9 has been shown
to create excess hypertrophic cartilage, inhibit vascular invasion of the growth plate,
and stop hypertrophic chondrocytes from progressing toward terminal differentiation
and apoptosis, suggesting that the presence of MMP-9 is needed to couple
vascularization of the growth plate with apoptosis of hypertrophic chondrocytes.21 A
study using MMP-13 null mice showed that absence of MMP-13 resulted in
accumulation of interstitial collagen, increased chondrocyte hypertrophy, delay of
vascularization, and inhibition of endochondral ossification.20 This may occur due to
a lack of MMP-13 present to promote normal collagen cleavage prior to invasion of
vasculature during endochondral ossification.20 Using in situ hybridization and RTPCR, MMP-8 has been shown to be more broadly expressed than MMP-13 in
osteoblastic progenitors, differentiated osteoblasts, osteocytes, and chondrocytes.29
Despite these findings, the complete role of MMPs within endochondral ossification
has not been fully elucidated.
10
Osteochondrosis
Developmental orthopedic diseases encompass such juvenile diseases as
physitis, cuboidal bone hypoplasia, cervical vertebral malformation, and
osteochondritis dissecans.1 Osteochondrosis (OC) is a disease involving disruption of
the endochondral ossification process, leading to retained cartilage cores, an irregular
ossification front, osteochondral separation, and in some cases cartilage necrosis.
Osteochondritis dissecans (OCD) is a clinical manifestation of OC characterized by
cartilage flap or osteochondral fragment formation within the joint.
Proposed Etiologies
Although a definitive cause of OC and OCD has not been determined, it is
believed to be multifactorial with a variety of potential causes being implicated.
Among the proposed predisposing factors of OC are high nutritional plane, rapid
growth rate, genetic predisposition, traumatic insult to the cartilage, ischemia, and
aberrant signaling during endochondral ossification.1,2 It is likely that multiple
predisposing factors allow for development of OC and hinder healing of the cartilage,
eventually leading to OCD.
Increased planes of nutrition, high growth rate, and nutritional imbalances have
been blamed for development of OC in many species.1,2 Copper deficiency has been
commonly implicated in horses, and some benefit may be gained by supplementing
pregnant mares with copper.1 However, a significant difference between
11
supplemented and un-supplemented mares and foals was unable to be found in one
study that evaluated both gross and histologic lesions.30 Other possible nutritional
factors in the development of equine OC are low calcium or selenium levels, as well
as increased phosphorus, zinc, or molybdenum.1 While changes in mare and foal
nutrition are believed to have altered the incidence of OC through decreased growth
rate of foals and more balanced mineral supplementation, the incidence of OC still
remains approximately 10%.1,31
Heritability of OC has long been suspected, and genetic testing has become a
recent target of study. When evaluating the incidence of radiographically evident
OCD lesions in 350 Marremano horse offspring of 75 sires, it was estimated through
simulation of 5 generations that active selection could decrease the incidence from
16% to 2 %.32 In addition, a study of the heritability of fetlock and hock OCD in 167
South German Coldblood horses suggested a genetic component in the development of
OCD in the population of horses evaluated.33 Following these studies, genetic testing
has identified individual genes thought to be associated with occurrence of OCD in
certain genetic lines of horses. Initial studies identified quantitative trait loci on
equine chromosomes 2, 3, 4, 5, 8, 10, 12,15,16,18,19, and 21 that could be associated
with the presence of OC.34-40 Further genetic mapping led to the identification of the
candidate gene XIRP2 on equine chromosome 18, which has been associated with
higher risk of fetlock and hock OC.41
12
Another proposed mechanism for development of OC involves traumatic insult
to the developing cartilage. In normal developing cartilage, cartilage canals provide
vasculature and bone precursors to the ossification center that develops between the
epiphyseal growth cartilage and the growth plate.42-44 In piglets, it has been shown
that OC lesions occur in regions where cartilage canals cross the osteochondral
junction, suggesting that an inherent weakness exists in the area of the cartilage
canals.45 Histologic evaluation of these samples revealed cartilage canals containing
lightly staining vessel walls without cell nuclei and lumens found to be empty or
containing lysed red blood cells and fibrinoid material.45 In association with these
regions, necrotic cartilage was found, characterized by pale-staining cartilage matrix
and containing shrunken chondrocytes with condensed nuclei, and lesions thought to
be chronic exhibited retained cartilage cores extending into the bone plate.45
Mechanical trauma to the cartilage may cause damage to the vasculature, leading to
ischemia and development of chondronecrosis in these regions. A similar process has
been suggested in foals,46 and an association with hock, fetlock, and stifle OC has
been identified in regions surrounding the cartilage canals in foals.47-50 In these foals,
histologic findings similar to those found in piglets were seen. However, it is still
unproven whether trauma alone can be implicated as a cause of OC, and resulting
OCD, in foals.
Altered concentrations of the autocrine and paracrine factors involved in
endochondral ossification are also thought to play an important role in the
13
development of OC. In fact, it has been proposed that these alterations may be more
severe surrounding the cartilage canals, causing a similar distribution of lesions as that
seen with vessel trauma.51,52 Multiple methods of analysis have been utilized,
identifying a variety of changes, ranging from alterations in serum biomarkers to
increased articular cartilage gene and protein expression of factors involved in the
endochondral ossification pathway. Using whole cartilage RNA isolation and PCR
evaluation, increased gene expression of IGF-I was found to be significantly increased
in OC cartilage, and a trend toward increased TGF-β1 and collagen type I in OC
cartilage was noted.53 Serum biomarkers have been evaluated to determine predictive
value for OC, and osteocalcin was found to be significantly increased in animals with
OC.54 Although these findings are not directly related to a cause of OC, they have
fueled further research into the possible changes occurring in endochondral
ossification signaling factors. A study using PCR evaluation, found that MMP-13,
type I collagen, type X collagen, and Runx2 were all significantly increased in OC
cartilage.52 Ihh and PTH-rP have also been shown to be elevated in advanced OC
lesions.55,56 Additionally, VEGF has been shown to be increased in OC cartilage.57
Also of interest, PDGF has been shown to decrease the osteoinduction and
chondrogenesis that takes place in demineralized bone matrix implanted into mouse
muscle.58 While the alteration of these factors as it relates to endochondral ossification
and development of early OC remains to be determined, the findings of these previous
studies indicate a critical signaling role of many of these factors in the endochondral
14
ossification pathway, providing the impetuous to further evaluate these factors and
their association with early OC.
To determine how cartilage differentiation becomes disrupted in early
osteochondrosis prior to the onset of clinical signs, the gene and protein expression of
Ihh, PTH-rP, VEGF, PDGF-A, and MMP-1, 3, and 13 in articular cartilage of
prepubescent foals with early osteochondrosis was evaluated and compared to normal
controls. It was hypothesized that osteochondrosis develops as a result of increased
expression of regulatory molecules via cartilage canals during early development, and
that this leads to failure of cartilage surrounding the cartilage canals to mature and
calcify.
15
Chapter 2: Increased Gene Expression of Indian Hedgehog, Matrix
Metalloproteinase-3, and Matrix Metalloproteinase-13 in Early Equine
Osteochondrosis
Tara L. Riddick, DVM; Stacy A. Semevolos, DVM, MS, Dipl. ACVS; Katja
Duesterdieck-Zellmer, Dr. med. vet., MS, PhD, Dipl ACVS
From the Department of Clinical Sciences, College of Veterinary Medicine, Oregon
State University, Corvallis, OR 97331.
Presented in part as a podium presentation at the 37th Annual Conference of the
Veterinary Research Society, Breckenridge, CO, 2010.
Presented in part as a podium presentation at the 2010 ACVS Veterinary Symposium,
Seattle, Washington.
16
Introduction
Osteochondrosis (OC) is a major source of lameness in young horses,
involving abnormal maturation of cartilage during the first year of life. Despite
extensive research into the possible causes of OC, little work has been done to
characterize early OC in young horses before the disease becomes clinically apparent.
Although it is thought that normal endochondral ossification is altered in the
development of OC, the exact disruption in this process remains unknown.
Regulation of autocrine and paracrine factors within the deep cartilage layers likely
play a role in this process.
Indian hedgehog (Ihh) and parathyroid hormone-related peptide (PTH-rP) are
two paracrine factors that regulate the progression of chondrocytic terminal
differentiation in growth plate cartilage, forming a negative feedback loop.7,11 Ihh
promotes chondrocyte proliferation and release of PTH-rP, while PTH-rP
downregulates Ihh and inhibits chondrocytic terminal differentiation. Ihh and PTH-rP
are also expressed in articular cartilage and their expression has been shown to
gradually decrease during postnatal maturation of normal equine articular cartilage.59
Increased expression of Ihh and PTH-rP has been found in advanced OC lesions,55,56
but it is not known if their expression is altered in early OC. A recent study that
profiled gene expression of circulating leukocytes has revealed further evidence for
dysregulation of the Ihh pathway in advanced OC.60
17
Other factors have also been implicated in OC, including vascular endothelial
growth factor (VEGF) and platelet derived growth factor (PDGF).57 VEGF is an
angiogenic factor necessary for vascular invasion of cartilage and has been found to be
increased with OC.57 When VEGF was blocked with a soluble VEGF receptor protein
in mice, hypertrophic, avascular regions of cartilage were induced that healed upon
return of VEGF to the area.14 PDGF increases chondrocyte proliferation and
proteoglycan production, and has been shown to inhibit endochondral ossification in
vitro.16,17 Chondrocyte-derived PDGF may also play a paracrine role within different
zones of cartilage.61
Matrix metalloproteinases (MMPs) are catabolic enzymes that have been
shown to be increased in cartilage, synovial fluid, and synovium of human patients
with osteoarthritis.23 MMP-1, -8, and -13 are each capable of initiating intrahelical
cleavage of triple helical collagen, while MMP-3 cleaves type II collagen in the
nonhelical domain.23 In a study evaluating the effect of MMP-1 and -3 on collagen
degradation in rabbit cartilage explants, polyclonal anti-MMP-1 antibodies stopped
degradation of collagen, while polyclonal anti-MMP-3 antibodies prevented
approximately 40% of collagen degradation.62 In another ex vivo explant study,
increased collagen type II cleavage by collagenase (MMP) was found in advanced OC
cartilage lesions of young horses.63 MMP-13 gene expression has been shown to be
increased in advanced OC of older horses,64 as well as in earlier OC lesions.57 MMP-
18
13 is a hypertrophic cartilage marker that is required for resorption of hypertrophic
cartilage during endochondral ossification.65
The objective of this study was to elucidate gene and protein expression of Ihh,
PTH-rP, VEGF, PDGF, and MMP-1, 3, and 13 in articular cartilage of prepubescent
foals, to determine if these molecules have a strong correlative relationship with early
OC. The hypothesis was that there would be increased expression of these regulatory
molecules in the deep layer of early OC articular cartilage compared to normal control
cartilage.
Materials and Methods
Animals—Fifteen foals ≤6 months of age (7 foals with early OC, 8 normal controls)
were evaluated. There were 6 intact males and 9 females included in the study. The
study was approved by the institutional animal care and use committee.
Sample Collection— Foals were euthanized for reasons unrelated to lameness or joint
sepsis (sodium pentobarbital, 105 to 187 mg/kg IV). Osteochondral samples were
sharply dissected from the lateral and medial trochlear ridges of both distal femurs and
examined for any abnormalities. Whole cartilage samples were then sharply dissected
from the femoral trochlea, snap frozen in liquid nitrogen, and stored at -80C prior to
RNA isolation. Osteochondral sections were fixed in 4% paraformaldehyde for 48
hours and transferred to 10% EDTA solution for decalcification. Decalcified samples
19
were then embedded in paraffin and sectioned for immunohistochemistry and H&E
staining.a
RNA Isolation—Whole cartilage specimens for total RNA isolation were pulverized in
a freezer millb prior to guanidine isothiocyanatec denaturation, chloroform extraction,
isopropanol precipitation, and RNA purification.d Purity and concentration were
assessed by agarose gel electrophoresis and UV spectrophotometry.e
Real-time quantitative RT-PCR— Two-step quantitative real time RT-PCR was used
to evaluate expression of equine-specific VEGF, PDGF-A, MMP-1, -3, and -13, Ihh,
PTH-rP, collagen type IIB, and aggrecan mRNA, using the ABI StepOnePlus realtime PCR system and software.f Briefly, first strand cDNA synthesis was
accomplished with reverse transcription, using random hexamers and equine total
RNA from the cartilage samples isolated previously. Probes were labeled with FAM
(6-carboxy-fluoroscein) (reporter dye) and TAMRA (6-carboxytetramethylrhodamine) (quencher dye). For each experimental sample, the amount of
target cDNA was determined by a relative standard curve, constructed with six 10-fold
serial dilutions of a calibrator sample of cartilage cDNA (starting concentration:
100ng). The same calibrator sample was used for all experiments. PCR was
performed in duplicate, and 18S RNA was used as the housekeeping gene for
normalizing sample gene expression.
20
Immunohistochemistry—Immunohistochemistry was performed on sections using 1:20
dilution of rabbit -human polyclonal (Ihh, PTH-rP, VEGF, PDGF) or mouse human monoclonal (MMP-3, MMP-13) primary antibodiesg and the Supersensitive
Link-label Multilink Immunohistochemistry System.h Positive tissue controls were
used to confirm specificity in equine tissue and were based on the recommendations of
the primary antibody manufacturer,g including equine samplesi of skin (PTH-rP,
MMP-13), hemangioma (VEGF), liver (Ihh), cartilage (MMP-3), and squamous cell
carcinoma (PDGF). Negative procedural controls were confirmed by using nonimmune serum in place of primary antibody. Following deparaffinization, the sections
were either incubated at 37oC for 60 minutes under a solution of testicular
hyaluronidase (cartilage samples) or for 5 minutes under pepsin (connective tissue
controls) to expose the antigen. Endogenous peroxidases were quenched with
hydrogen peroxide and methanol. Non-immune goat serum was applied for 30
minutes (polyclonal primary antibodies only), and the primary antibody was applied
for 60-90 minutes at room temperature. Secondary biotinylated multilink antibodies
were applied, followed by labeling with streptavidin conjugated peroxidase, and then
applying diaminobenzidine tetrachloride (DAB) as a chromogen for production of
color product. The sections were counterstained with Harris hematoxylin and
mounted for microscopy.
Sample Evaluation and Classification—All osteochondral samples were evaluated
grossly at the time of harvest and histologically following H&E staining in order to
21
classify them as early OC, advanced OC, or normal (Figure 1). Normal cartilage was
defined as having no gross or histologic abnormalities. Early OC was defined as
samples having altered endochondral ossification along the osteochondral junction
(locally thickened cartilage, loss of normal columnar arrangement of chondrocytes,
chondrones) without concurrent superficial cartilage lesions or separation (fissures,
necrosis) along the osteochondral junction without concurrent superficial cartilage
lesions. Advanced OC was defined as having separation (fissures, necrosis) along the
osteochondral junction with concurrent superficial lesions (OCD flaps, osteochondral
fragments, fibrocartilage formation).
Scoring and Statistical Analysis—Blinded immunohistochemistry samples were
evaluated for protein expression by scoring each of three cartilage layers: superficial,
middle, and deep. Each layer was scored as follows: 0 (no staining/expression), 1
(mild staining/expression), 2 moderate staining/expression), or 3 (strong
staining/expression). The scores of each layer were added together for a total cartilage
score which was then averaged between the two observers (SAS and TLR). The
averaged total cartilage scores and quantitative comparisons from real-time PCR
assays were compared between OC and normal horses using a rank sum (MannWhitney) test (p<0.05).
22
Results
Following histological evaluation of samples, 7 foals were determined to have
early OC, no foals had advanced OC, and 8 foals were normal (Figure 1).
There was significantly increased Ihh gene expression in early OC cartilage
compared to normal controls (p=0.037) (Table 1). Ihh protein expression was mainly
limited to the superficial and deep layers, with cellular staining along the
osteochondral junction (Figure 2). Immunostaining was variably present in cells and
matrix surrounding the cartilage canals. There was no significant difference in total
cartilage scores of Ihh immunostaining between OC and normal controls (p=0.23)
(Table 2).
No difference was found in PTH-rP gene expression between OC and normal
cartilage (p=0.34) (Table 1). Protein expression of PTH-rP was mild in the superficial
layer, minimal in the middle layer and moderate in the deep layer. Moderate to strong
immunostaining was present in some chondrocytes along the osteochondral junction.
In many samples, there was mild to moderate PTH-rP expression in some cells lining
the cartilage canals and extending to the osteochondral junction. No difference in total
cartilage scores of PTH-rP was found between OC and normal controls (p=0.63)
(Table 2).
There was no difference in VEGF gene expression between OC and normal
cartilage (p=0.13) (Table 1). Moderate to strong VEGF protein expression was
23
apparent in cells in the superficial and deep cartilage layers, with minimal expression
in the middle layer (Figure 3). Strong cellular expression of VEGF was present along
the osteochondral junction, and mild to moderate expression was noted in the cells
lining the cartilage canals. No difference was found in total cartilage scores for VEGF
immunohistochemistry between OC and normal horses (p=0.41) (Table 2).
There was no difference in PDGF-A gene expression between OC and normal
cartilage (p=0.13) (Table 1). There was diffuse PDGF immunostaining in the cartilage
matrix of the superficial and middle layers, becoming territorial in the deep zone
(Figure 4). Strong PDGF expression was found in chondroclasts/osteoclasts,
particularly along the osteochondral junction, and moderate expression was apparent
in the cells lining the cartilage canals. No difference was found in total cartilage
scores for PDGF immunohistochemistry between OC and normal horses (p=0.53)
(Table 2).
There was significantly increased MMP-3 (p=0.049) and MMP-13 (p=0.007)
gene expression in early OC cartilage compared to normal controls, while no
difference was found in MMP-1 expression (p=0.24) (Table 1). MMP-3
immunostaining was present mainly in the superficial and deep layers of articular
cartilage, with minimal MMP-3 protein expression along the cartilage canals and
osteochondral junction (Figure 5). In contrast, MMP-13 protein expression was strong
along the osteochondral junction, moderate in the superficial and deep layers of
24
articular cartilage, and mild within the cartilage canals (Figure 6). No difference was
found in total cartilage scores for MMP-3 (p=0.41) or MMP-13 (p=0.41)
immunohistochemistry between OC and normal horses (Table 2).
No difference was found in collagen type IIB (p=0.55) and aggrecan (p=0.24)
gene expression between OC and normal cartilage samples (Table 1).
Discussion
Despite an abundance of research into the etiology of OC in horses, the disease
remains poorly understood. We hypothesized that OC would be associated with
increased expression of molecules regulating cartilage differentiation in the deep layer
of articular cartilage. In this study, we found that Ihh, MMP-3, and MMP-13 gene
expression in early OC cartilage samples were each significantly increased compared
to normal cartilage samples, indicating that these molecules may be associated with
early OC development. In contrast, PTH-rP, VEGF, PDGF, and MMP-1 were not
associated with early OC, although they may play a role later in the pathogenesis of
this disease.
Increased Ihh gene expression in early OC cartilage of this study was similar to
previous reports describing advanced OC lesions in older foals,55,56 providing stronger
evidence for its role in OC. In the growth plate, Ihh has been identified in pre-
25
hypertrophic chondrocytes and acts to modulate the progression of cartilage
differentiation.7,11 In mice, induced overexpression of Ihh has led to a retained
cartilage core with lack of hypertrophic cartilage, mainly due to secondary
upregulation of PTH-rP and subsequent inhibition of terminal differentiation.11
However, the role of Ihh in articular cartilage has not been clearly characterized, and
normal articular cartilage has low expression of Ihh during development.59 The role of
increased Ihh expression in early OC is not clear, but its production by the superficial
and deep cartilage layers may alter the normal progression of cartilage through the
terminal differentiation pathway. Alternatively, hedgehog (sonic) signaling has been
shown to be involved in wound repair,66 and Ihh may play a role in healing of early
OC lesions.
Because PTH-rP gene expression was not altered in early OC cartilage, it
appears less likely that PTH-rP is involved in the etiology of this disease. This is in
contrast to a previous study that showed increased PTH-rP immunostaining in
advanced OC lesions.55 The pattern of stronger PTH-rP expression in the deep layer
of articular cartilage found in this study was similar to that reported previously.55,59
Co-localization of Ihh and PTH-rP in the deep cartilage layer suggests potential
paracrine interactions. However, lack of concurrent upregulation of PTH-rP with
increased Ihh expression does not support the presence of a feedback loop in this
location. It is possible that altered PTH-rP or Ihh receptor levels exist in early OC
cartilage, but further evaluation would be warranted to determine their expression.
26
No difference was found in gene expression of VEGF in early OC and normal
cartilage samples. This is in contrast to a previous study that found increased
expression of VEGF in early OC lesions,57 but is in agreement with another study that
found no difference in VEGF expression between normal and early OC cartilage.52
The definition of early OC may have varied between these studies, with slightly more
advanced lesions having increased VEGF. In more advanced OC lesions, increased
VEGF may have reflected a healing response to damaged cartilage rather than a
primary alteration. In the deep layer of normal cartilage, VEGF is necessary for
normal progression of the endochondral ossification front.14 Our findings support the
importance of VEGF, particularly in the deep cartilage layers, despite a lack of altered
expression between OC and normal cartilage samples.
No difference in the expression of PDGF-A between OC and normal cartilage
samples was identified in this study. However, the strong protein expression of PDGF
in cells lining the cartilage canals and along the osteochondral junction in all samples
supports its importance in the process of endochondral ossification. Previous studies
have shown that PDGF can enhance cartilage matrix production while also preventing
the progression of cells in endochondral maturation.16,17 PDGF may have a paracrine
role in articular cartilage as indicated by a previous study showing strong expression
of PDGF in areas of low receptor expression.61 PDGF has also been associated with
indirect stimulation of vascular endothelial cell proliferation and angiogenesis,61
thereby playing an important part in vascular invasion of cartilage and endochondral
27
ossification. However, the results of this study do not show a clear connection
between PDGF and the development of OC lesions.
The potential importance of MMPs in the development of OC lesions is
supported by their role in normal cartilage development and their ability to degrade the
extracellular matrix.26,67 MMP regulation is critical for normal cartilage matrix
assembly and plays a significant role in endochondral ossification by facilitating
matrix degradation and vascular invasion.26,67-69 Additionally, MMPs have been found
to be upregulated secondary to increased biomechanical forces68 and are primary
factors in osteoarthritis development.27,70 Type II collagen, responsible for tensile
strength of cartilage, is susceptible to degradation by MMP-1, -8, and -13.23 While
aggrecan, which provides compressive strength to cartilage, is vulnerable to
proteolysis by MMP-3.26 MMP-1 and -3 have been implicated in the progression of
cartilage degradation by previous studies.23,62 Increased MMP-3 and -13 gene
expression levels found in early OC of our study are similar to those of a previous
study in which foals fed a high energy diet to promote OC lesions resulted in elevated
MMP-13 expression.52 MMP-13 is essential for resorption of cartilage matrix during
chondrocyte hypertrophy, vascular invasion, and endochondral ossification.69
Increased MMP-13 expression levels may indicate increased resorption of cartilage,
possibly due to abnormal biomechanical forces along the osteochondral junction.
Increased MMP-3 expression may result from cartilage injury.68 Increased MMP-3
and -13 expression levels in early OC may be the result of downstream signaling and
28
have the potential to lead to matrix alterations associated with a weakened
osteochondral junction.
Despite finding significant changes in gene expression between early OC and
normal cartilage, protein expression levels did not appear to be altered in this study.
Previous studies have shown similar discrepancies in gene and protein expression
levels,55,71 and may reflect intracellular regulation of mRNA prior to translation into
protein. Alternatively, the sensitivity and specificity of immunohistochemistry may be
insufficient to accurately quantify protein levels within cells. Indeed, scoring of
immunohistological samples converts a qualitative evaluation into a quantitative
assessment, and scoring by cartilage layers and converting to a total cartilage score
may have missed subtle changes within specific cell populations. Additionally, nonspecies specific primary antibodies (human) were used for immunohistochemistry in
this study; although positive species specific (equine) controls were used to confirm
tissue and species specificity in our samples. Future research using Western blots,
ELISA, or mass spectrometry may better quantify protein expression levels.
Classification of OC has been variable between studies, depending on whether
arthroscopy, MRI, radiography, or histology was used to determine the classification
scale.51,52,72-75 In this study, early OC lesions corresponded best to class 1, 2, and 3
histological lesions described by van Weeren and Barneveld.75 Early OC lesions were
defined as samples having altered endochondral ossification along the osteochondral
29
junction (locally thickened cartilage, loss of normal columnar arrangement of
chondrocytes, chondrones) without concurrent superficial cartilage lesions (class 1 and
2) or separation (fissures, necrosis) along the osteochondral junction without
concurrent superficial cartilage lesions (class 3).75 Because concurrent superficial
cartilage lesions were not present in these samples, secondary changes within the joint
(synovitis, fibrocartilage, osteoarthritis) were not present to confound the
pathogenesis.
In conclusion, increased gene expression in articular cartilage of foals having
early OC provides evidence of altered cartilage maturation. The presence of Ihh,
VEGF, and PDGF in cells lining the cartilage canals and strong expression of these
proteins and MMP-13 along the osteochondral junction reiterate their importance in
the process of endochondral ossification. Increased Ihh expression in early OC
samples may indicate upstream signaling, while increased MMP-3 and -13 gene
expression may reflect downstream catabolic alterations resulting in improper matrix
assembly and resultant breakdown. The discrepancy in gene and protein expression
levels in this study may be due to low sensitivity of immunohistochemistry to quantify
protein levels or the lack of clinically relevant protein alterations in these whole
cartilage samples. Future studies are necessary to better quantify protein levels and to
determine expression changes in specific cell populations in early OC cartilage.
30
Figures
Figure 1: Photomicrographs following H&E staining of osteochondral sections, showing middle to deep articular cartilage
layers and the osteochondral junction. (A) Normal osteochondral junction and endochondral ossification in a 4-month-old
filly. (B) Osteochondral separation (arrow) in a 4-month-old colt with early OC. (C) Abnormal endochondral ossification
near the osteochondral junction in a 1-month-old filly with early OC. (bar=100μm)
31
Figure 2: Photomicrographs following immunohistochemical localization using antibodies directed against Ihh. (A) Middle to
deep articular cartilage layers and the osteochondral junction from a normal 4-month-old filly showing mild Ihh expression in
the deep layer. (B) Whole articular cartilage from a 5-month-old colt having OC, showing moderate to strong Ihh expression
in the superficial and deep layers. (C) Negative control for (B) following substitution of rabbit polyclonal Ihh antibody with
normal rabbit serum. (bar=100μm)
32
Figure 3: Photomicrographs following immunohistochemical localization using antibodies directed against VEGF. (A)
Articular cartilage from a normal 6-month-old filly showing strong VEGF expression (arrows) in the superficial layer and
along the osteochondral junction. (B) Negative control for (A) following substitution of rabbit polyclonal VEGF antibody with
normal rabbit serum. (C) Osteochondral junction from horse in (A) showing strong VEGF immunostaining (arrow) in the cells
lining the trabeculae. (D) Articular cartilage from a 4-month-old colt having OC, showing mild VEGF immunostaining in the
superficial layer and moderate immunostaining along the osteochondral junction. (bar=100μm)
33
Figure 4: Photomicrographs following immunohistochemical localization using antibodies directed against PDGF. (A)
Articular cartilage from a normal 6-month-old filly showing moderate to strong PDGF expression in the superficial layer and
cells along a cartilage canal and the osteochondral junction. (B) Whole articular cartilage from a 4-month-old colt having OC,
showing strong PDGF expression in the superficial and deep layers and the osteochondral junction. Territorial
immunostaining is present in the deep cartilage layer (arrow). (C) Negative control for (B) following substitution of rabbit
polyclonal PDGF antibody with normal rabbit serum. (bar=100μm)
34
Figure 5: Photomicrographs following immunohistochemical localization using antibodies directed against MMP-3. (A)
Middle to deep layer of articular cartilage and osteochondral junction in a normal 6-month-old filly, with mild MMP-3
expression (arrow) in some chondrocytes. (B) Moderate to strong MMP-3 cellular and matrix protein expression (arrow) in
the deep cartilage layer of a 1-month-old filly with early OC. No immunostaining was present in cells adjacent to the cartilage
canals (arrowheads). (C) Mild to moderate cellular MMP-3 protein expression (arrow) in the deep cartilage layer of a 5month-old filly with early OC. No immunostaining was present in cells adjacent to the cartilage canals (arrowhead). (D)
Negative control for (C) following substitution of mouse monoclonal MMP-3 antibody with normal mouse serum.
(bar=100μm)
35
Figure 6: Photomicrographs following immunohistochemical localization using antibodies directed against MMP-13. (A)
Articular cartilage from a normal 3-month-old filly showing strong MMP-13 expression (arrow) in the superficial layer and
moderate expression along the osteochondral junction. (B) Osteochondral junction from horse in (A), showing moderate
immunostaining in cells lining the osteochondral junction (arrow) and a cartilage canal. (C) Negative control for (B)
following substitution of mouse monoclonal MMP-13 antibody with normal mouse serum. (D) Articular cartilage from a 4month-old filly having OC, showing strong MMP-13 immunostaining (arrow) in the deep cartilage layer along the
osteochondral junction (separated). (bar=100μm)
36
Tables
Table 1. Mean gene expression +/- SEM quantified by real-time RT-PCR using whole
cartilage from early OC and normal control horses. Relative gene expression was
normalized by dividing the target gene by 18S expression.
Target gene
Early OC
Normal Control
P-value
Ihh
2.66 +/- 0.89
0.83 +/- 0.27
0.037
PTH-rP
0.13 +/- 0.03
0.14 +/- 0.05
0.34
VEGF
0.71 +/- 0.12
0.48 +/- 0.09
0.13
PDGF-A
5.13 +/- 2.52
1.81 +/- 0.38
0.13
MMP-1
0.10 +/- 0.04
0.09 +/- 0.06
0.24
MMP-3
32.6 +/- 16.1
2.64 +/- 0.86
0.049
MMP-13
0.24 +/- 0.09
0.08 +/- 0.05
0.007
Collagen type IIB
0.71 +/- 0.07
0.69 +/- 0.10
0.55
Aggrecan
1.69 +/- 0.20
1.51 +/- 0.19
0.24
37
Table 2. Mean total cartilage scores +/- SEM following immunohistochemistry of
osteochondral sections from early OC and normal control horses.
Target protein
Early OC
Normal Control
P-value
Ihh
3.37 +/- 0.32
3.01 +/- 0.31
0.23
PTH-rP
3.16 +/- 0.55
3.09 +/- 0.21
0.63
VEGF
3.75 +/- 0.58
4.61 +/- 0.50
0.41
PDGF-A
5.71 +/- 0.56
5.93 +/- 0.19
0.53
MMP-3
4.35 +/- 0.62
3.97 +/- 0.31
0.41
MMP-13
4.65 +/- 0.12
4.34 +/- 0.32
0.41
38
Chapter 3: Molecular Expression of Cartilage Canal and Osteochondral Junction
Chondrocytes in Subclinical Juvenile Osteochondritis Dissecans (OCD)
Tara L. Riddick, DVM, Katja Duesterdieck-Zellmer, Dr. med. vet., MS, PhD, Dipl
ACVS, Stacy A. Semevolos, DVM, MS, Dipl. ACVS
From the Department of Clinical Sciences, College of Veterinary Medicine, Oregon
State University, Corvallis, OR 97331.
Presented in part as a podium presentation at the 2010 ACVS Veterinary Symposium,
Seattle, Washington.
Presented in part as a poster presentation at the 2011 Annual Meeting of the
Orthopaedic Research Society, Long Beach, CA.
39
Introduction
Juvenile osteochondritis dissecans (OCD) involves abnormal differentiation and
ossification of articular-epiphyseal cartilage occurring in early adolescence, most often
characterized by separation of articular cartilage from underlying subchondral bone
along the osteochondral junction. If left untreated, OCD can cause chronic
degenerative changes and knee pain in children and young animals.76 Previous studies
in humans have mainly focused on MRI findings and surgical vs. nonsurgical
treatment outcomes.76,77 A few studies have examined histological findings in
children,73,78 but none have specifically determined molecular changes occurring in
this disease.
In contrast, studies in animals have focused on determination of gene and protein
expression changes of regulatory molecules, including hedgehog, growth factors, and
extracellular matrix molecules in advanced OCD of horses, a species in which clinical
disease closely parallels that in humans.53,56,63,74 Increased expression of Indian
hedgehog and parathyroid-related peptide has been found in advanced OCD
lesions.56,55,60 However, these signaling pathways have yet to be fully explored in
early OCD lesions.
Because advanced OCD lesions are likely to have secondary changes not involved
in the actual etiology of the disease, it is imperative to study early OCD lesions prior
to onset of clinical signs in order to have a better chance of determining the underlying
cause. Much less, however, is known about early OCD prior to the development of
40
overt clinical signs. Several studies have begun to examine early OCD lesions;52,72
however, these OCD lesions were taken from older animals, and full-thickness
cartilage samples were used in the analysis.
Full-thickness cartilage expression studies assess heterogenous cell populations
rather than specific cell populations most likely to be involved in the disease. Unlike
previous studies that have used full-thickness cartilage samples to quantify gene
expression, this study determined gene expression of specific cells of interest. In
particular, cells surrounding cartilage canals and the osteochondral junction were
selected. Cartilage canals provide blood supply to developing epiphyseal cartilage,
and damage to these canals may result in ischemic chondronecrosis and
osteochondrotic lesions.50 Previous studies have also shown that areas of delayed
endochondral ossification along the osteochondral junction are often associated with
retained cartilage canals in OCD lesions.79 Laser capture microdissection (LCM) is a
recent technology that allows extraction of specific cells from tissue samples. We
have used this technology to collect chondrocytes surrounding cartilage canals and the
osteochondral junction in juvenile OCD and normal horses.
In this study, we collected osteochondral samples from clinically normal knee
(stifle) joints of pre-pubescent horses, in order to obtain subclinical OCD (stable
OCD) and normal control samples. The objective was to determine gene and protein
expression of vascular endothelial growth factor (VEGF), platelet-derived growth
factor-A (PDGF-A), matrix metalloproteinase-13 (MMP-13), Indian hedgehog (Ihh),
41
and parathyroid hormone-related peptide (PTH-rP) in chondrocytes surrounding
cartilage canals and the osteochondral junction, respectively. The hypothesis was that
gene and protein expression levels of these molecules would be increased in
chondrocytes adjacent to cartilage canals and the osteochondral junction in juvenile
OCD lesions, when compared to normal controls.
Methods
Sample Collection—Fourteen foals, 1-6 months of age (median=4 months),
including 6 foals with juvenile OCD (3 males, 3 females) and 8 normal controls (1
male, 7 females), were evaluated (Table 1). The study was approved by the
institutional animal care and use committee. Foals were euthanized for reasons
unrelated to lameness or joint sepsis (sodium pentobarbital, 105 to 187 mg/kg IV).
Osteochondral samples were sharply dissected from the lateral and medial trochlear
ridges of both distal femurs and examined for any abnormalities. Osteochondral
samples were either frozen in OCT mediumj and stored at -80oC, or fixed in 4%
paraformaldehyde for 48 hours and transferred to 10% EDTA solution for
decalcification. Decalcified samples were then embedded in paraffin and sectioned
for immunohistochemistry.a
Laser-capture microdissection—Frozen osteochondral sections for LCM were
mounted on slides using a tape transfer systemk and stored at -80oC. Immediately
42
prior to LCM, each slide was dehydrated in graded alcohol and xylene. LCM was
performed on osteochondral sections (Figure 1 and 2) with PIXCELL II Laser Capture
Microdissection Systeml using CapSure Macro LCM caps.f Cells were captured from
the cartilage immediately surrounding the cartilage canals and at separate sites, the
osteochondral junction of each animal. Up to 8 caps were combined for each site
(approximately 400-800 cells per site).
RNA Isolation— The PicoPure™ RNA Isolation Kitl was used for RNA isolation
with slight modifications. Briefly, the extraction buffer was used to detach and lyse
cells attached to the polymer membrane of the CapSure Macro LCM cap and the
resulting lysate from up to 8 caps was loaded onto a pre-conditioned RNA-purification
column.m The column was then washed and treated with RNase-free DNase prior to
RNA elution from the column. RNA quality control was performed using an Agilent
2100 Bioanalyzern and the RNA 6000 Pico LabChip kit.n
Real-time quantitative RT-PCR—Two-step quantitative real time RT-PCR was
used to evaluate expression of equine-specific VEGF (NM_001081821), PDGF-A
(XM_001915189), MMP-13 (NM_001081804), Ihh (AY112896), PTH-RP
(NM_001163981), and 18S mRNA, using the ABI StepOnePlus real-time PCR system
and software.f Probes were labeled with FAM (6-carboxy-fluoroscein) (reporter dye)
and TAMRA (6-carboxy-tetramethylrhodamine) (quencher dye). Briefly, first strand
cDNA synthesis was accomplished with reverse transcription, using random hexamers
43
and equine total RNA from LCM-isolated chondrocyte samples. Logarithmic preamplification (10 cycles), using pooled equine specific primer pairs (VEGF, PDGF-A,
MMP-13, Ihh, and PTH-rP) and the Taqman PreAmp Master Mix kit,f was performed
on cDNA samples.80,81 Real-time quantitative PCR of diluted (1:5) pre-amplified
samples was then performed. For each experimental sample, the amount of target
cDNA was determined by a relative standard curve, constructed with six 10-fold serial
dilutions of a calibrator sample of cartilage cDNA (starting concentration: 100ng).
The same calibrator sample was used for all experiments and 18S RNA was used as
the housekeeping gene for normalizing sample gene expression. PCR was performed
in duplicate using a 20µl final reaction mixture of 2X TaqMan® Gene Expression
Master Mix (PE Biosystems, Branchburg, NJ), 250nM probe, 900nM forward and
reverse primers, and 7.5µl pre-amplified sample cDNA. After 2 minute incubation at
50 ºC to activate uracil-DNA glycosylase (UDG) and 10 minute incubation at 95ºC to
deactivate UDG and activate AmpliTaq® Gold DNA polymerase, 40 PCR cycles of 15
seconds of 95ºC followed by 1 minute of 60ºC were run.
Immunohistochemistry—Immunohistochemistry was performed on sections using
1:20 dilution of rabbit -human polyclonal (Ihh, PTH-rP, VEGF, PDGF) or mouse human monoclonal (MMP-13) primary antibodiesg and the Supersensitive Link-label
Multilink Immunohistochemistry System.h Positive tissue controls were used to
confirm specificity in equine tissue and were based on the recommendations of the
44
primary antibody manufacturer,n including equine samplesi of skin (PTH-rP, MMP13), hemangioma (VEGF), liver (Ihh), and squamous cell carcinoma (PDGF).
Negative procedural controls were confirmed by using non-immune serum in place of
primary antibody. Following deparaffinization, the sections were either incubated at
37oC for 60 minutes under a solution of testicular hyaluronidase (cartilage samples) or
for 5 minutes under pepsin (connective tissue controls) to expose the antigen.
Endogenous peroxidases were quenched with hydrogen peroxide and methanol. Nonimmune goat serum was applied for 30 minutes (polyclonal primary antibodies only),
and the primary antibody was applied for 60-90 minutes at room temperature.
Secondary biotinylated multilink antibodies were applied, followed by labeling with
streptavidin conjugated peroxidase, and then applying diaminobenzidine tetrachloride
(DAB) as a chromogen for production of color product. The sections were
counterstained with Harris hematoxylin and mounted for microscopy.
Sample Evaluation and Classification—All osteochondral samples were evaluated
grossly at the time of harvest and histologically following H&E staining in order to
classify them as OCD or normal. Normal cartilage was defined as having no gross or
histologic abnormalities. OCD was defined as samples having separation (fissures,
necrosis) or altered endochondral ossification along the osteochondral junction
(locally thickened cartilage, loss of normal columnar arrangement of chondrocytes,
chondrones) without concurrent superficial cartilage lesions.
45
Scoring and Statistical Analysis—Blinded immunohistochemistry samples were
evaluated for protein expression by scoring 1) chondrocytes surrounding the cartilage
canals and 2) chondrocytes adjacent to the osteochondral junction. Each component
was scored from 0 to 3: 0 (no staining/expression), 1 (mild staining/expression), 2
(moderate staining/expression), or 3 (strong staining/expression). The scores of the
two observers (SAS and TLR) were averaged for combined scores at each location.
The immunohistochemistry scores and quantitative comparisons from real-time PCR
assays were compared between OCD and normal horses using a rank sum (MannWhitney) test (p<0.05). Paired comparisons of gene expression within each horse
between cartilage canal and osteochondral junction chondrocytes were made using the
Wilcoxon signed rank test (p <0.05).
Results
Following histological evaluation of samples, 6 foals were determined to have
OCD and 8 foals were normal (Table 1).
In laser captured samples, there was significantly increased MMP-13 (p=0.02) and
PDGF-A (p=0.03) gene expression in cells adjacent to the cartilage canals of OCD
cartilage compared to normal controls (Figure 2). PDGF-A expression was also
significantly increased in osteochondral junction chondrocytes of OCD samples
(p=0.04) (Figure 3). No other gene expression differences were found between OCD
and normal laser-captured samples.
46
Paired comparisons of gene expression within each horse between cartilage canal
and osteochondral junction chondrocytes revealed several significant differences
(Figure 4). Gene expression of Ihh (p=0.0002) and VEGF (p=0.001) was significantly
higher in osteochondral junction chondrocytes than cartilage canal chondrocytes. In
contrast, PTH-rP gene expression was significantly higher in cartilage canal
chondrocytes (p=0.01). There was no significant difference in gene expression of
PDGF-A or MMP-13 between osteochondral junction and cartilage canal
chondrocytes.
Moderate VEGF immunostaining was apparent in cells along the osteochondral
junction, and mild protein expression was noted in the deep cartilage layer and cells
lining the cartilage canals (Figure 6). No significant difference in VEGF protein
expression was found between early OC and normal samples. Moderate to strong
PDGF protein expression was found in chondroclasts/osteoclasts, particularly along
the osteochondral junction, and moderate expression was apparent in the cells lining
the cartilage canals (Figure 7). There was also diffuse PDGF immunostaining in the
cartilage matrix of the superficial and middle layers, becoming territorial in the deep
zone. No significant difference in PDGF protein expression was found between early
OC and normal samples.
MMP-13 protein expression was moderate to strong along
the osteochondral junction and mild to moderate in the cartilage canals (Figure 8). No
significant difference in MMP-13 protein expression was found between early OC and
47
normal samples. Mild to moderate Ihh immunostaining was mainly limited to the
osteochondral junction and cells lining the cartilage canals, in addition to superficial
matrix (Figure 9). No significant difference in Ihh expression was found between
early OC and normal samples (Table 2). Mild PTH-rP protein expression was
localized to the superficial matrix, deep cartilage layer and osteochondral junction
chondrocytes (Figure 10). There was significantly greater PTH-rP protein expression
in osteochondral junction chondrocytes of OCD samples compared to normal controls
(p=0.01) (Table 2). No significant difference was found in PTH-rP protein expression
of cartilage canal chondrocytes between OCD cartilage vs. controls (p=0.06) (Table
2).
Discussion
Chondrocyte signaling surrounding the cartilage canals and osteochondral junction
is critical to cartilage terminal differentiation and endochondral ossification. Altered
molecular expression within these cells has the potential to be involved in the
development of OCD. We hypothesized that gene and protein expression levels of
VEGF, PDGF-A, MMP-13, Ihh, and PTH-rP would be increased in chondrocytes
surrounding cartilage canals and the osteochondral junction of OCD cartilage
compared to normal controls. Based on our data, we found significantly higher gene
(but not protein) expression levels of PDGF-A in OCD cartilage canal and
osteochondral junction chondrocytes and MMP-13 in OCD cartilage canal
48
chondrocytes. In addition, we found significantly increased protein (but not gene)
expression of PTH-rP in OCD cartilage canal chondrocytes.
VEGF is an angiogenic factor necessary for vascular invasion of cartilage and has
been found to be increased with OCD cartilage of horses.57 In the deep layer of
normal cartilage, VEGF is necessary for normal progression of the endochondral
ossification front.14 Strong VEGF protein expression along the osteochondral junction
in this study is in agreement with this function. However, contrary to our hypothesis,
we did not find an increase of VEGF gene or protein expression in cartilage canals or
the osteochondral junction of OCD cartilage compared to normal cartilage. This
finding is similar to a previous study,52 in which no difference of VEGF expression
was found in early OCD full-thickness articular cartilage samples.
PDGF has been shown to enhance cartilage proteoglycan production and prevent
the progression of cells in endochondral maturation.16,17 Additionally, PDGF has been
associated with indirect stimulation of vascular endothelial cell proliferation and
angiogenesis,61 thereby playing an important part in vascular invasion of cartilage and
endochondral ossification. In accordance with our hypothesis, PDGF-A gene
expression was increased in chondrocytes adjacent to cartilage canals and along the
osteochondral junction of OCD cartilage when compared to normal cartilage.
Increased PDGF-A gene expression may have the potential to delay endochondral
49
maturation in OCD samples, although corresponding protein expression levels of
PDGF were not significantly different in this study.
MMP-13 is required for resorption of cartilage matrix during chondrocyte
hypertrophy, vascular invasion, and endochondral ossification.69 Previous studies
using MMP-13 null mice have shown that regions of marked cartilage hypertrophy
develop, and delays in endochondral ossification occur in the absence of MMP-13
activity.20,69 In agreement with our hypothesis, MMP-13 gene expression was found
to be increased in chondrocytes adjacent to cartilage canals of OCD cartilage.
However, in contrast to our hypothesis, MMP-13 gene expression was not found to be
increased along the osteochondral junction of OCD samples. Increased gene
expression of MMP-13 has also been found in a previous study of full-thickness
articular cartilage samples in foals with OCD.52 In addition, we have found increased
gene expression of MMP-13 in full-thickness articular cartilage samples from the
same foals having OCD as in this study.82 Retention of cartilage canals has been
previously associated with OC lesions,51 and increased expression levels of MMP-13
near cartilage canals seen in this study may represent increased resorption along the
cartilage canals. This upregulation of MMP-13 may relate to the resolution of early
OC lesions that is sometimes observed as foals grow.83
In growth cartilage, Ihh is responsible for modulating the progression of cells
entering hypertrophy, and participates in a negative feedback loop with PTH-rP.59 In
50
articular cartilage, low levels of Ihh gene and protein expression have been
documented during normal postnatal development.59 Increased gene and protein
expression of Ihh has been found in full-thickness articular cartilage samples from
older foals having advanced OCD.55,56 In addition, increased Ihh gene expression was
found in full-thickness articular cartilage samples from the same foals having OCD as
in this study.82 However, contrary to our hypothesis, Ihh gene and protein expression
was not altered in chondrocytes surrounding the cartilage canals or osteochondral
junction in OCD samples. This difference may be explained by the more specific
location used for RNA isolation in this study, compared to the previous study which
evaluated full-thickness articular cartilage samples. Immunohistochemistry analysis
of full thickness cartilage has revealed that the majority of Ihh protein expression
localized to the superficial and deep articular layers.82
As stated previously, PTH-rP helps to regulate chondrocytic terminal
differentiation in growth cartilage through its role in a negative feedback loop with
Ihh.7,11 The role of PTH-rP in normal articular cartilage is less certain, but its
expression has been shown to decrease over the course of postnatal articular cartilage
development.59,84 Previous studies in advanced OCD of horses have shown increased
PTH-rP protein expression levels in OCD, but no difference in PTH-rP gene
expression levels.55 Similarly, in the current study of subclinical OCD, PTH-rP
protein expression levels along the osteochondral junction were significantly increased
51
in OCD samples, while corresponding gene expression levels were not altered. This
finding partially concurs with our hypothesis of increased PTH-rP expression in OCD.
In contrast, a trend (p=0.06) for decreased PTH-rP protein expression was found in
cartilage canals of OCD samples, contrary to our original hypothesis.
In conclusion, laser capture microdissection provided an opportunity for
quantitative assessment of gene expression levels in specific cell populations from
subclinical OCD and normal osteochondral sections. Our findings in this study of
significantly increased PDGF-A and MMP-13 gene expression in OCD cartilage canal
chondrocytes and increased PDGF-A gene expression in OCD osteochondral junction
chondrocytes suggest that pathways involving endochondral maturation and resorption
of cartilage are altered in subclinical OCD. Discrepancies in gene and protein
expression levels are likely due to the relatively low sensitivity of
immunohistochemical scoring for protein quantification. Future studies directed at
improved protein quantification techniques in specific cell populations will be
important to determine the relevance of the gene expression differences between OCD
and normal cartilage found in this study.
52
Figures
Figure 1: Photomicrograph of hematoxylin-stained osteochondral section showing
approximate regions extracted by LCM, including cells surrounding a cartilage canal
(outer solid line and inner dashed line) and osteochondral junction chondrocytes
(dashed-dotted lines).
53
Figure 2: Photomicrograph of a frozen cartilage canal section from a 4-month-old foal (A). (B) Chondrocytes surrounding the
cartilage canal are adhered to the LCM cap following laser ignition. (C) Extracted chondrocytes from (B) remain in the cap
following LCM.
54
Figure 3: Gene expression of laser-captured cartilage canal chondrocytes using real-time PCR. Relative gene expression (log
scale) was determined using a relative standard curve and dividing the target gene by 18S RNA. There was significantly
increased gene expression of MMP-13 (p=0.02) and PDGF-A (p=0.03) in early OCD samples compared to normal controls.
55
Figure 4: Gene expression of laser-captured osteochondral junction chondrocytes using real-time PCR. Relative gene
expression (log scale) was determined using a relative standard curve and dividing the target gene by 18S RNA. There was
significantly increased gene expression of PDGF-A (p=0.04) in early OCD compared to normal controls.
56
Figure 5: Gene expression comparisons of cartilage canal chondrocytes vs. osteochondral junction chondrocytes using realtime PCR. Ihh (p=0.0002) and VEGF (p=0.001) gene expression was significantly higher in osteochondral junction
chondrocytes than cartilage canal chondrocytes. PTH-rP gene expression was significantly higher (p=0.01) in cartilage canal
chondrocytes.
57
Figure 6: Photomicrographs of osteochondral sections following immunohistochemical localization using antibodies directed
against VEGF. Strong VEGF immunostaining was present along the osteochondral junction (arrows) and minimal to mild
expression was found in the cartilage canals of: (A) 4-month-old male colt having OCD and (B) 6-month-old normal filly. (C)
Negative control for horse in (B). Bar=100µm.
58
Figure 7: Photomicrographs of osteochondral sections following immunohistochemical localization using antibodies directed
against PDGF. (A) Moderate PDGF immunostaining throughout the deep cartilage layer and cartilage canals and mild to
moderate immunostaining along the osteochondral junction in a 4-month-old colt having OCD. (B) Negative control for (A).
(C) Strong PDGF immunostaining in chondrocytes surrounding the cartilage canals, in the deep cartilage layer and along the
osteochondral junction in a 6-month-old normal filly. Bar=100µm.
59
Figure 8: Photomicrographs of osteochondral sections following immunohistochemical localization using antibodies directed
against MMP-13. (A) Strong MMP-13 protein expression along the osteochondral junction and moderate to strong expression
surrounding a cartilage canal in a 4-month-old colt having OCD. (B) Mild to moderate immunostaining in chondrocytes along
a cartilage canal and moderate staining in some cells near the osteochondral junction in a 6-month-old normal filly. (C)
Negative control for (B). Bar=100µm.
60
Figure 9: Photomicrographs of osteochondral sections following immunohistochemical localization using antibodies directed
against Ihh. (A) Moderate to strong immunostaining (arrows) in chondrocytes surrounding a cartilage canal (long arrow) and
deep cartilage cells near the osteochondral junction (short arrow) in a 4-month-old colt having OCD. (B) Negative control for
(A). (C) Mild to moderate immunostaining in chondrocytes in the deep cartilage layer near the osteochondral junction in a 4month-old normal filly. (D) Mild to moderate immunostaining in cells surrounding a cartilage canal (arrow) in the same horse
as (C). Bar=100µm.
61
Figure 10: Photomicrographs of osteochondral sections following immunohistochemical localization using antibodies directed
against PTH-rP. (A) Mild protein expression of chondrocytes surrounding the cartilage canal (arrow) and osteochondral
junction in a 4-month-old colt having OCD. (B) Mild immunostaining around a cartilage canal (long arrow) and moderate
staining in a few chondrocytes near the osteochondral junction (short arrow) in a 6-month-old normal filly. (C) Negative
control for (B). Bar=100µm.
62
Tables
Table 1. Summary of Animals and Samples. OCD – osteochondritis dissecans; M –
male; F – female; Y – test performed; N – test not performed
Animal
Classification
1
OCD
Agemo.
5
Sex
2
OCD
4
M
3
OCD
5
M
4
OCD
4
F
5
OCD
1
F
6
OCD
5
F
7
Normal
4
M
Cartilage easily separated from
subchondral bone,
chondronecrosis along
osteochondral junction
Thickened cartilage easily
separated from subchondral
bone
Thickened cartilage easily
separated from subchondral
bone
Cartilage easily separated from
subchondral bone, subchondral
bone hemorrhage
Thick cartilage with
osteochondral junction
containing fingerlike
projections of cartilage
Thin cartilage distal medial
trochlear ridge, delayed
ossification with extremely
thickened cartilage proximally,
cartilage easily separated from
subchondral bone,
chondronecrosis along
osteochondral junction
No lesions
8
Normal
4
F
No lesions
N
Y
Y
9
Normal
4
F
No lesions
Y
Y
Y
10
Normal
5
F
No lesions
Y
Y
Y
11
Normal
4
F
No lesions
Y
Y
Y
12
Normal
4
F
No lesions
Y
Y
Y
13
Normal
6
F
No lesions
N
N
Y
14
Normal
3
F
No lesions
Y
Y
Y
M
Description
LCM
-CC
N
LCM
-OCJ
N
IHC
Y
Y
Y
Y
Y
Y
N
N
Y
Y
Y
Y
Y
Y
Y
N
N
Y
Y
63
Table 2. Mean protein expression scores +/- SEM following immunohistochemistry
of osteochondral sections from juvenile OCD and normal controls. Each component
was scored from 0 (no staining/expression) to 3 (strong staining/expression), and the
scores of the two observers (SAS and TLR) were averaged.
Target
protein
Site
Juvenile OCD
Normal
Control
Pvalue
VEGF
Cartilage Canal
1.00 +/- 0.32
1.04 +/- 0.31
0.53
Osteochondral Junction
1.83 +/- 0.49
2.14 +/- 0.33
0.48
Cartilage Canal
1.84 +/- 0.19
1.78 +/- 0.34
0.47
Osteochondral Junction
2.21 +/- 0.27
2.36 +/- 0.20
0.45
Cartilage Canal
1.50 +/- 0.21
1.56 +/- 0.14
0.53
Osteochondral Junction
2.46 +/- 0.15
2.23 +/- 0.14
0.24
Cartilage Canal
0.84 +/- 0.14
1.37 +/- 0.24
0.08
Osteochondral Junction
1.77 +/- 0.21
1.75 +/- 0.11
0.48
Cartilage Canal
0.95 +/- 0.14
1.42 +/- 0.20
0.06
Osteochondral Junction
2.23 +/- 0.15
1.83 +/- 0.06
0.01
PDGF
MMP-13
Ihh
PTH-rP
64
Chapter 4: Conclusion
In these studies, the protein and gene expression of factors believed to play a
role in the process of endochondral ossification were evaluated in articular cartilage.
While other predisposing factors have been implicated, such as a high plane of
nutrition, rapid growth rate, genetic predisposition, traumatic insult to the cartilage,
and ischemia,1,2 previous studies have shown alterations in molecular factors in
association with OC lesions.52-57 Additionally, foals with and without OC of a wide
range of ages have been evaluated for a multitude of predisposing factors.24, 30,32-41, 4757,59,60,63,74,75,79,83
While ischemic injury to vasculature within cartilage canals has been
implicated in the etiology of OC,46-50 it has also been noted that altered cartilage
development occurs surrounding cartilage canals that show no evidence of trauma or
ischemic change.51,52 In light of this, the age of the foals evaluated in this study was
limited to 6-months of age or less, as cartilage canals are present until this age.
Additionally, early changes in factors could be detected prior to the onset of clinical
OC, indicating changes that may be more causative in nature.
As hypothesized, gene expression of Ihh was increased in whole OC cartilage
when compared to normal whole cartilage samples. This is similar to previous reports
describing advanced OC lesions in older foals,55,56 providing stronger evidence for its
role in OC. In growth plate cartilage, Ihh is responsible for modulating the
progression of cells entering hypertrophy, and participates in a negative feedback loop
65
with PTH-rP.59 In articular cartilage, low levels of Ihh gene and protein expression
have been documented during normal postnatal development.59 However, contrary to
our hypothesis, Ihh gene and protein expression was not altered in chondrocytes
surrounding the cartilage canals or osteochondral junction in OCD samples. This
difference may be explained by the more specific location used for RNA isolation in
the laser-captured samples compared to the whole cartilage samples.
Immunohistochemistry analysis of full thickness cartilage has revealed that the
majority of Ihh protein expression localized to the superficial and deep articular
layers.82 The role of increased Ihh expression in early OC is not clear, but its
production by the superficial and deep cartilage layers may alter the normal
progression of cartilage through the terminal differentiation pathway. Alternatively,
hedgehog (sonic) signaling has been shown to be involved in wound repair,66 and Ihh
may play a role in healing of early OC lesions.
As stated previously, PTH-rP helps to regulate chondrocytic terminal
differentiation in growth cartilage through its role in a negative feedback loop with
Ihh.7,11 The role of PTH-rP in normal articular cartilage is less certain, but its
expression has been shown to decrease over the course of postnatal articular cartilage
development.59,84 Although PTH-rP gene expression was not altered in early OC
cartilage, PTH-rP protein expression was significantly increased along the
osteochondral junction of subclinical OCD cartilage. This agrees with a previous
66
study that showed increased PTH-rP immunostaining in advanced OC lesions.55 The
pattern of stronger PTH-rP expression in the deep layer of articular cartilage found in
this study was similar to that reported previously.55,59 Co-localization of Ihh and PTHrP in the deep cartilage layer suggests potential paracrine interactions. However, lack
of concurrent upregulation of PTH-rP with increased Ihh gene expression does not
support the presence of a feedback loop in articular cartilage. It is possible that altered
PTH-rP or Ihh receptor levels exist in early OC cartilage, but further evaluation would
be warranted to determine their expression.
VEGF is an angiogenic factor necessary for vascular invasion of cartilage and
has been found to be increased with OCD cartilage of horses.57 In the deep layer of
normal cartilage, VEGF is necessary for normal progression of the endochondral
ossification front.14 Strong VEGF protein expression along the osteochondral junction
in this study is in agreement with this function. However, contrary to our hypothesis,
we did not find an increase of VEGF gene or protein expression in whole cartilage
samples, cartilage canals, or the osteochondral junction of OCD cartilage compared to
normal cartilage. This finding is similar to another study that found no difference in
VEGF expression between normal and early OC cartilage.52 The definition of early
OC may have varied between these studies, with slightly more advanced lesions
having increased VEGF. In more advanced OC lesions, increased VEGF may have
reflected a healing response to damaged cartilage rather than a primary alteration. Our
67
findings support the importance of VEGF, particularly in the deep cartilage layers,
despite a lack of altered expression between OC and normal cartilage samples.
PDGF has been shown to enhance cartilage proteoglycan production and prevent
the progression of cells in endochondral maturation.16,17 Additionally, PDGF has been
associated with indirect stimulation of vascular endothelial cell proliferation and
angiogenesis,61 thereby playing an important part in vascular invasion of cartilage and
endochondral ossification. In accordance with our hypothesis, PDGF-A gene
expression was increased in chondrocytes adjacent to cartilage canals and along the
osteochondral junction of OCD cartilage when compared to normal cartilage.
Increased PDGF-A gene expression may have the potential to delay endochondral
maturation in OCD samples. Although corresponding protein expression levels of
PDGF were not significantly different in this study, the importance of PDGF in the
endochondral ossification pathway was highlighted by strong protein expression of
PDGF in cells lining the cartilage canals and along the osteochondral junction in all.
The potential importance of MMPs in the development of OC lesions is
supported by their role in normal cartilage development and their ability to degrade the
extracellular matrix.26,67 MMP regulation is critical for normal cartilage matrix
assembly and plays a significant role in endochondral ossification by facilitating
matrix degradation and vascular invasion.26,67-69 Additionally, MMPs have been found
to be upregulated secondary to increased biomechanical forces68 and are primary
68
factors in osteoarthritis development.27,70 Type II collagen, responsible for tensile
strength of cartilage, is susceptible to degradation by MMP-1, -8, and -13.23 While
aggrecan, which provides compressive strength to cartilage, is vulnerable to
proteolysis by MMP-3.26 MMP-1 and -3 have been implicated in the progression of
cartilage degradation by previous studies.23,62 Increased MMP-3 and -13 gene
expression levels found in early OC of our study are similar to those of a previous
study in which foals fed a high energy diet to promote OC lesions resulted in elevated
MMP-13 expression.52 Additionally, MMP-13 was found to be increased in
chondrocytes along the cartilage canals in OCD cartilage. MMP-13 is essential for
resorption of cartilage matrix during chondrocyte hypertrophy, vascular invasion, and
endochondral ossification.69 Increased MMP-13 expression levels may indicate
increased resorption of cartilage, possibly due to abnormal biomechanical forces along
the osteochondral junction. Increased MMP-3 expression may result from cartilage
injury.68 Increased MMP-3 and -13 expression levels in early OC may be the result of
downstream signaling and have the potential to lead to matrix alterations associated
with a weakened osteochondral junction.
Despite finding significant changes in gene expression between early OC and
normal cartilage, protein expression levels did not appear to be altered in this study.
Previous studies have shown similar discrepancies in gene and protein expression
levels,55,71 and may reflect intracellular regulation of mRNA prior to translation into
69
protein. Alternatively, the sensitivity and specificity of immunohistochemistry may be
insufficient to accurately quantify protein levels within cells. Indeed, scoring of
immunohistological samples converts a qualitative evaluation into a quantitative
assessment, and scoring by cartilage layers and converting to a total cartilage score
may have missed subtle changes within specific cell populations. Additionally, nonspecies specific primary antibodies (human) were used for immunohistochemistry in
this study; although positive species specific (equine) controls were used to confirm
tissue and species specificity in our samples. Future research using Western blots,
ELISA, or mass spectrometry may better quantify protein expression levels.
In conclusion, laser capture microdissection provided an opportunity for
quantitative assessment of gene expression levels in specific cell populations from
subclinical OCD and normal osteochondral sections. Our findings in this study of
significantly increased Ihh, MMP-3, and MMP-13 gene expression in whole OCD
cartilage, as well as increased PDGF-A and MMP-13 gene expression in OCD
cartilage canal chondrocytes and increased PDGF-A gene expression in OCD
osteochondral junction chondrocytes suggest that pathways involving endochondral
maturation and resorption of cartilage are altered in subclinical OCD. Discrepancies
in gene and protein expression levels are likely due to the relatively low sensitivity of
immunohistochemical scoring for protein quantification. Future studies directed at
improved protein quantification techniques in specific cell populations will be
70
important to determine the relevance of the gene expression differences between OCD
and normal cartilage found in this study.
71
Footnotes
a.
Clinical Sciences Histology Laboratory, Cornell University, Ithaca, NY
b.
Spex Certi Prep, Metuchen, NJ
c.
Trizol,® Invitrogen, Carlsbad, CA
d.
Rneasy,® QIAGEN, Valencia, CA
e.
NanoDrop ND 1000, Thermo Fischer Scientific, Wilmington, DE
f.
Applied Biosystems, Foster City, CA
g.
Research Diagnostics, Inc., Flanders, NJ
h.
Biogenex, San Ramon, CA
i.
Veterinary Diagnostic Laboratory, Oregon State University, Corvallis, OR
j.
Tissue Tek OCT compound, VWR International, West Chester, PA
k.
CryoJane, Instrumedics, Hackensack, NJ
l.
Arcturus Bioscience, Mountain View, CA
m.
Qiagen, Valencia, CA
n.
Agilent Technologies, Palo Alto, CA
72
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