# Expression of Phosphocitrate-Targeted Genes in Osteoarthritis Menisci

Authors: Yubo Sun; David R. Mauerhan; Nury M. Steuerwald; Jane Ingram; Jeffrey S. Kneisl; Edward N. Hanley
Journal: BioMed Research International
 (2014)

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## Abstract

Phosphocitrate (PC) inhibited calcium crystal-associated osteoarthritis (OA) in Hartley guinea pigs. However, the molecular mechanisms remain elusive. This study sought to determine PC targeted genes and the expression of select PC targeted genes in OA menisci to test hypothesis that PC exerts its disease modifying activity in part by reversing abnormal expressions of genes involved in OA. We found that PC downregulated the expression of numerous genes classified in immune response, inflammatory response, and angiogenesis, including chemokine (C-C motif) ligand 5, Fc fragment of IgG, low affinity IIIb receptor (FCGR3B), and leukocyte immunoglobulin-like receptor, subfamily B member 3 (LILRB3).  In contrast, PC upregulated the expression of many genes classified in skeletal development, including collagen type II alpha1, fibroblast growth factor receptor 3 (FGFR3), and SRY- (sex determining region Y-) box 9 (SOX-9).  Immunohistochemical examinations revealed higher levels of FCGR3B and LILRB3 and lower level of SOX-9 in OA menisci. These findings indicate that OA is a disease associated with immune system activation and decreased expression of SOX-9 gene in OA menisci.  PC exerts its disease modifying activity on OA, at least in part, by targeting immune system activation and the production of extracellular matrix and selecting chondroprotective proteins.


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## Body

## 1. Introduction

Osteoarthritis (OA) is one of the most prevalent causes of disability in the aging population and has enormous economic and social consequences. However, existing nonsurgical treatment options only provide symptomatic relief but have no effect on the progression of the disease. The lack of progress in the development of structural disease-modifying drugs for OA therapy is largely due to our limited understanding of the pathogenesis of OA and insufficient knowledge regarding the molecular targets or key OA disease genes for therapeutic intervention.OA is not merely an articular cartilage disease, but a disease of the whole joint. An important local factor to the health of the knee joints is the structural integrity and biochemical properties of the knee meniscus. Knee meniscus is a specialized tissue that plays a vital role in load transmission, shock absorption, and joint stability. In recent years there has been a dramatic advance in our understanding of the integral role of the meniscus for the knee functions and the consequences of meniscal abnormality in cartilage degeneration. Studies found that meniscal degeneration is a general feature of OA [1, 2]; meniscal lesions at baseline were more common in the knees that developed OA than in the knees that did not develop OA [3] and that OA meniscal cells displayed a distinct gene expression profile different from normal meniscal cells [4]. These findings indicate that meniscal changes or abnormalities are involved in the OA disease process. The involvement of meniscal changes or abnormalities in the OA disease process has also been highlighted by recent findings that meniscal extrusion, vascular penetration (angiogenesis), and calcification are associated with cartilage degeneration and subchondral lesions in OA [5–7]. Meniscal abnormalities such as meniscal degeneration, inflammation, and angiogenesis may represent as new targets for the development of disease-modifying drugs for OA therapy, especially for a subgroup of OA patients who develop severe meniscal lesions before developing severe cartilage degeneration [8, 9].Phosphocitrate (PC), a potent calcification inhibitor, is a naturally occurring compound originally identified in rat liver mitochondrial extract [10, 11]. PC prevented soft tissue calcification and inhibited calcium crystal-induced mitogenesis, crystal-induced expression of matrix metalloproteinases (MMPs), and crystal-induced cell death [12–15]. In Hartley guinea pig model of crystal-associated OA, PC inhibited meniscal calcification and reduced the severity of cartilage degeneration [16]. These findings provide support for the notion that calcification inhibitors are potentially disease modifying drugs for crystal-associated OA therapy. It is believed that PC exerts its disease modifying activity by inhibiting the formation of articular calcium crystals and the detrimental interaction between the crystals and cells [17]. However, two studies found that bisphosphonates, which are also potent calcification inhibitors, failed to inhibit cartilage degeneration in animal models of OA, including the Hartley guinea pig model of crystal-associated OA [18, 19], raising doubts as to whether calcification inhibitors are potentially disease-modifying drugs for OA therapy. An alternative mechanism underlying the disease modifying activity of PC may be present.We previously reported that PC downregulated the expression of many genes classified in inflammatory response and angiogenesis in OA fibroblast-like synoviocytes (FLSs) and OA meniscal cells in the absence of calcium crystals [20, 21]. These findings suggest that the molecular mechanism underlying the disease-modifying activity of PC is more complicated than originally thought. In this study, we sought to further investigate the gene expression-modulating activity of PC and determine the expressions of select PC-targeted genes in menisci derived from OA patients. The hypothesis to be tested is that PC exerts its disease modifying activity, at least in part, by modulating the abnormal expressions of genes involved in the OA disease process. The information gained is not only important for a better understanding of the molecular mechanisms underlying the disease modifying activity of PC but may also valuable for the identification of disease candidate genes involved in the OA disease process.


## 2. Materials and Methods

### 2.1. Materials

Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), stock antibiotic/antimycotic mixture are products of Invitrogen (Carlsbad, CA, USA). Superfrost-Plus microscope slides and neutral buffered formalin (10%) were obtained from Allegiance Inc. (McGaw Park, IL, USA). PC was prepared as described [22]. Antibodies specific to Fc fragment of IgG, low affinity IIIb receptor (FCGR3B), SRY (sex determining region Y)-box 9 (Sox-9), and fibroblast growth factor receptor 3 (FGFR-3) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Antibody specific to leukocyte immunoglobulin-like receptor, subfamily B member 3 (LILRB3) was obtained from Lifespan Biosciences (Seattle, WA, USA).


### 2.2. Meniscal Explant Culture and RNA Extraction

OA meniscal tissue specimens were minced into small pieces and cultured in a six well-cluster plate (350 mg per well) at 37°C in DMEM containing 1% FBS and 0.5% antibiotic/antimycotic solution. Twenty-four hours later, the medium in the top three wells was replaced with DMEM containing 1% FBS and 1 mM of PC and the medium in the bottom three wells was replaced with DMEM containing 1% FBS without PC as control. Every three days, the medium was changed. On the eighth day, the medium was changed again. Twenty-four hours later, the meniscal explants were collected, snap-freezed, and stored in −70°C freezer until use. Total RNA was extracted from these snap-freezed meniscal explants using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and purified using Oligotex kit (Qiagen, Valencia, CA, USA). These RNA samples were used for microarray analysis.Meniscal tissue specimens were collected with the approval of the authors’ Institutional Review Board from end-stage OA patients undergoing knee joint replacement surgery and osteosarcoma patients undergoing lower limb amputation surgery at Carolinas Medical Center. The need for informed consent was waived because these meniscal specimens were surgical waste and no private patient information was collected. Meniscal specimens were collected in sterilized containers filled with tissue culture medium and transported to the laboratory from operating room using an ice box.


### 2.3. Microarray

RNA samples extracted from two independent experiments were used for microarray analysis. Briefly, double stranded DNA was synthesized using SuperScript double stranded cDNA synthesis kit using these RNA samples (Invitrogen, San Diego, CA, USA). The DNA product was purified using GeneChip sample cleanup module (Affymetrix, Santa Clara, CA, USA). cRNA was synthesized and biotin labeled using BioArray high yield RNA transcript labeling kit (Enzo Life Sciences, Farmingdale, NY, USA). The cRNA product was purified using GeneChip sample cleanup module and subsequently chemically fragmented. The fragmented and biotinylated cRNA was hybridized to HG-U133_Plus_2 gene chip using Affymetrix Fluidics Station 400 (Affymetrix, Santa Clara, CA, USA). The fluorescent signal was quantified during two scans by Agilent Gene Array Scanner G2500A (Agilent Technologies, Palo Alto, CA) and GeneChip operating Software (Affymetrix, Santa Clara, CA, USA). Genesifter software (VizX Labs, Seattle, WA, USA) was used for the analysis of differential gene expression and gene ontology.


### 2.4. Real-Time RT-PCR

Briefly, cDNA was synthesized using TaqMan Reverse Transcription Reagents (Applied Biosystems, University Park, IL, USA) using the RNA samples described. Quantification of relative transcript levels for selected genes and the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was performed using ABI7000 Real Time PCR system (Applied Biosystems, University Park, IL, USA). TaqMan Gene Expression assay (Applied Biosystems, University Park, IL, USA) was used. CDNA samples were amplified with an initial Taq DNA polymerase activation step at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds and annealing at 60°C for one minute. Fold change was calculated and the expression level of the genes to be examined was normalized to the expression level of GAPDH. RT-PCR experiment was performed in triplicates using the same RNA sample for the microarray analysis.


### 2.5. Immunohistochemistry

Medial meniscal specimens derived from 6 end-stage OA patients and 3 osteosarcoma patients were used for examination. These meniscal specimens were fixed in 10% neutral buffered formalin for twenty-four hours and transferred to 70% ethyl alcohol. A portion of 5 mm wide specimen was transversely excised from the middle part of meniscus, embedded in Paraplast Plus paraffin, and sectioned with a Leica RM2025 microtome (Nussloch, Germany) to obtain 4μm serial transverse sections [23]. Sections were examined with immunohistochemical staining using specific antibodies. Briefly, paraffin-embedded sections were deparaffinized with xylene and rehydrated with graded ethanol. Endogenous peroxidase activity was blocked by incubation of the sections with freshly prepared 3% H2O2 in deionized water for 5 minutes at room temperature. Nonspecific binding was blocked by incubation of the sections with 100 μL of 10% normal horse serum diluted in base solution (4% BSA and 5% nonfat dry milk in PBS) for 20 minutes. These sections were incubatedwith a specific primary antibody (2 μg/mL) for 1 hour, followed with the secondary reagent specific for each antibody for 30 minutes (Immpress reagent kit, Vector, Inc., Burlingame, CA). Negative control was performed using mouse IgG to replace the primary specific antibody. Slides were rinsed in phosphate buffered saline three times and visualized using 3,3′-diaminobenzidine for 5 minutes. Slides were then counterstained with light green, dehydrated, and mounted with resinous mounting media. These immunostainings were graded on a scale of 0–3, where 0 = very weak staining; 1 = weak staining; 2 = moderate staining; 3 = strong staining.


### 2.6. Statistical Analysis

The difference between the immunostaining grades of the OA meniscal group and control group was analyzed using the Wilcoxon rank-sum test.P values less than 0.05 were considered significant. Statistical analysis was performed using the statistical analysis tool in the Sigma Plot software, version 12 (Systat software Inc., San Jose, CA).


## 2.1. Materials

Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), stock antibiotic/antimycotic mixture are products of Invitrogen (Carlsbad, CA, USA). Superfrost-Plus microscope slides and neutral buffered formalin (10%) were obtained from Allegiance Inc. (McGaw Park, IL, USA). PC was prepared as described [22]. Antibodies specific to Fc fragment of IgG, low affinity IIIb receptor (FCGR3B), SRY (sex determining region Y)-box 9 (Sox-9), and fibroblast growth factor receptor 3 (FGFR-3) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Antibody specific to leukocyte immunoglobulin-like receptor, subfamily B member 3 (LILRB3) was obtained from Lifespan Biosciences (Seattle, WA, USA).


## 2.2. Meniscal Explant Culture and RNA Extraction

OA meniscal tissue specimens were minced into small pieces and cultured in a six well-cluster plate (350 mg per well) at 37°C in DMEM containing 1% FBS and 0.5% antibiotic/antimycotic solution. Twenty-four hours later, the medium in the top three wells was replaced with DMEM containing 1% FBS and 1 mM of PC and the medium in the bottom three wells was replaced with DMEM containing 1% FBS without PC as control. Every three days, the medium was changed. On the eighth day, the medium was changed again. Twenty-four hours later, the meniscal explants were collected, snap-freezed, and stored in −70°C freezer until use. Total RNA was extracted from these snap-freezed meniscal explants using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and purified using Oligotex kit (Qiagen, Valencia, CA, USA). These RNA samples were used for microarray analysis.Meniscal tissue specimens were collected with the approval of the authors’ Institutional Review Board from end-stage OA patients undergoing knee joint replacement surgery and osteosarcoma patients undergoing lower limb amputation surgery at Carolinas Medical Center. The need for informed consent was waived because these meniscal specimens were surgical waste and no private patient information was collected. Meniscal specimens were collected in sterilized containers filled with tissue culture medium and transported to the laboratory from operating room using an ice box.


## 2.3. Microarray

RNA samples extracted from two independent experiments were used for microarray analysis. Briefly, double stranded DNA was synthesized using SuperScript double stranded cDNA synthesis kit using these RNA samples (Invitrogen, San Diego, CA, USA). The DNA product was purified using GeneChip sample cleanup module (Affymetrix, Santa Clara, CA, USA). cRNA was synthesized and biotin labeled using BioArray high yield RNA transcript labeling kit (Enzo Life Sciences, Farmingdale, NY, USA). The cRNA product was purified using GeneChip sample cleanup module and subsequently chemically fragmented. The fragmented and biotinylated cRNA was hybridized to HG-U133_Plus_2 gene chip using Affymetrix Fluidics Station 400 (Affymetrix, Santa Clara, CA, USA). The fluorescent signal was quantified during two scans by Agilent Gene Array Scanner G2500A (Agilent Technologies, Palo Alto, CA) and GeneChip operating Software (Affymetrix, Santa Clara, CA, USA). Genesifter software (VizX Labs, Seattle, WA, USA) was used for the analysis of differential gene expression and gene ontology.


## 2.4. Real-Time RT-PCR

Briefly, cDNA was synthesized using TaqMan Reverse Transcription Reagents (Applied Biosystems, University Park, IL, USA) using the RNA samples described. Quantification of relative transcript levels for selected genes and the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was performed using ABI7000 Real Time PCR system (Applied Biosystems, University Park, IL, USA). TaqMan Gene Expression assay (Applied Biosystems, University Park, IL, USA) was used. CDNA samples were amplified with an initial Taq DNA polymerase activation step at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds and annealing at 60°C for one minute. Fold change was calculated and the expression level of the genes to be examined was normalized to the expression level of GAPDH. RT-PCR experiment was performed in triplicates using the same RNA sample for the microarray analysis.


## 2.5. Immunohistochemistry

Medial meniscal specimens derived from 6 end-stage OA patients and 3 osteosarcoma patients were used for examination. These meniscal specimens were fixed in 10% neutral buffered formalin for twenty-four hours and transferred to 70% ethyl alcohol. A portion of 5 mm wide specimen was transversely excised from the middle part of meniscus, embedded in Paraplast Plus paraffin, and sectioned with a Leica RM2025 microtome (Nussloch, Germany) to obtain 4μm serial transverse sections [23]. Sections were examined with immunohistochemical staining using specific antibodies. Briefly, paraffin-embedded sections were deparaffinized with xylene and rehydrated with graded ethanol. Endogenous peroxidase activity was blocked by incubation of the sections with freshly prepared 3% H2O2 in deionized water for 5 minutes at room temperature. Nonspecific binding was blocked by incubation of the sections with 100 μL of 10% normal horse serum diluted in base solution (4% BSA and 5% nonfat dry milk in PBS) for 20 minutes. These sections were incubatedwith a specific primary antibody (2 μg/mL) for 1 hour, followed with the secondary reagent specific for each antibody for 30 minutes (Immpress reagent kit, Vector, Inc., Burlingame, CA). Negative control was performed using mouse IgG to replace the primary specific antibody. Slides were rinsed in phosphate buffered saline three times and visualized using 3,3′-diaminobenzidine for 5 minutes. Slides were then counterstained with light green, dehydrated, and mounted with resinous mounting media. These immunostainings were graded on a scale of 0–3, where 0 = very weak staining; 1 = weak staining; 2 = moderate staining; 3 = strong staining.


## 2.6. Statistical Analysis

The difference between the immunostaining grades of the OA meniscal group and control group was analyzed using the Wilcoxon rank-sum test.P values less than 0.05 were considered significant. Statistical analysis was performed using the statistical analysis tool in the Sigma Plot software, version 12 (Systat software Inc., San Jose, CA).


## 3. Results

### 3.1. Effect of PC on Gene Expressions

Microarrayanalysis revealed that of the more than 50,000 transcripts examined, 2561 transcripts displayed significant differential expressions (more than 2.0 fold) in PC-treated OA meniscal explants via untreated OA meniscal explants. A total of 1430 transcripts displayed decreased expressions and 1131 transcripts displayed increased expressions. The genes that fell into specific biological processes previously implicated in OA or suspected to have a role in OA are listed in Tables1, 2, and 3.Table 1
Genes classified in the immune response.
Biological process
Gene name
Gene ID
Differ expre*
Description
Immune response
CCL20
NM_004591
−103.53
Chemokine (C-C motif) ligand 20
CCL5
NM_002985
−3.54
Chemokine (C-C motif) ligand 5
CCR5
NM_000579
−2.03
Chemokine (C-C motif) receptor 5
CXCL3
NM_002090
−10.03
Chemokine (C-X-C motif) ligand 3
CXCL5
AK026546
−4.49
Chemokine (C-X-C motif) ligand 5
CXCL2
M57731
−2.94
Chemokine (C-X-C motif) ligand 2
CXCL1
NM_001511
−3.01
Chemokine (C-X-C motif) ligand 1
CXCL9
NM_002416
−2.65
Chemokine (C-X-C motif) ligand 9
CXCL13
NM_006419
−2.62
Chemokine (C-X-C motif) ligand 13 (B-cell chemoattractant)
CXCR4
AF348491
−2.63
Chemokine (C-X-C motif) receptor 4
IGKC
BC005332
−37.44
Netrin 2-like (chicken)
IL6
NM_000600
−32.07
Interleukin 6 (interferon, beta 2)
IL8
NM_000584
−8.17
Interleukin 8
IL23A
M15564
−7.89
Enhancer of polycomb homolog 1 (Drosophila)
IL24
NM_006850
−3.73
Interleukin 24
IL15
NM_000585
−3.70
Interleukin 15
IL7R
BE217880
−3.31
Interleukin 7 receptor
IL1B
NM_000576
−2.28
Interleukin 1, beta
IL1RN
BE563442
−2.80
Interleukin 1 receptor antagonist
MS4A2
NM_000139
−28.91
Membrane-spanning 4-domains, subfamily A, member 2
FCGR3B
NM_000570
−22.56
Fc fragment of IgG, low affinity IIIb, receptor (CD16b)
FCGR2B
U90940
−7.13
Fc fragment of IgG, low affinity IIc, receptor for (CD32)
FCGR1B
L03419
−3.30
Fc fragment of IgG, high affinity Ib, receptor (CD64)
FCER1A
BC005912
−5.19
Fc fragment of IgE, high affinity I, receptor for; alpha polypeptide
HLA-C
AW575927
−22.41
Major histocompatibility complex, class I, C
HLA-F
BE138825
−2.22
Major histocompatibility complex, class I, F
AQP9
NM_020980
−17.49
Aquaporin 9
GZMA
NM_006144
−14.63
Granzyme A
IGHG1
M87789
−14.43
Immunoglobulin heavy constant gamma 1
LBP
M35533
−11.36
Lipopolysaccharide binding protein
EREG
NM_001432
−8.60
Epiregulin
CD86
NM_006889
−7.11
CD86 molecule
CD74
M28590
−2.76
CD74 molecule
CD40
NM_001250
−3.68
CD40 molecule, TNF receptor superfamily member 5
CD1D
NM_001766
−5.17
CD1d molecule
CD8A
AW006735
−4.99
CD8a molecule
CD14
NM_000591
−3.25
CD14 molecule
CD209
AF290886
−2.50
CD209 molecule
KYNU
BC000879
−6.69
Kynureninase (L-kynurenine hydrolase)
PTPRC
NM_002838
−5.88
Protein tyrosine phosphatase, receptor type, C
TLR8
AW872374
−5.55
Toll-like receptor 8
TLR7
NM_016562
−3.34
Toll-like receptor 7
TLR5
AF051151
−3.00
Toll-like receptor 5
TLR4
NM_003266
−2.40
Toll-like receptor 4
TLR2
NM_003264
−2.20
Toll-like receptor 2
TLR1
AL050262
−2.66
Toll-like receptor 1
SLC11A1
L32185
−5.41
Solute carrier family 11, member 1
CFD
NM_001928
−4.99
Complement factor D (adipsin)
CFI
BC020718
−3.37
Complement factor I
CFB
NM_001710
−2.39
Complement factor B
C3
NM_000064
−2.32
Complement component 3
CR1
AI052659
−2.24
Complement component (3b/4b) receptor 1 (Knops blood group)
C1QA
NM_015991
−2.20
Complement component 1, q subcomponent, A chain
C1QB
NM_000491
−2.10
Complement component 1, q subcomponent, B chain
C1RL
NM_016546
2.01
Complement component 1, r subcomponent-like
FYB
BF679849
−4.64
FYN binding protein (FYB-120/130)
TREM1
NM_018643
−4.52
Triggering receptor expressed on myeloid cells 1
BMP6
NM_001718
−4.48
Bone morphogenetic protein 6
INPP5D
U53470
−4.47
Inositol polyphosphate-5-phosphatase, 145 kDa
LILRB1
NM_006669
−4.33
Leukocyte immunoglobulin-like receptor, subfamily B, member 1
LILRB2
AF004231
−4.32
Leukocyte immunoglobulin-like receptor, subfamily B, member 2
LILRB3
AF009634
−4.23
leukocyte immunoglobulin-like receptor, subfamily B, member 3
LILRB4
U82979
−2.06
Leukocyte immunoglobulin-like receptor, subfamily B, member 4
LILRB5
NM_006840
−2.31
Leukocyte immunoglobulin-like receptor, subfamily B, member 5
LILRA2
NM_006866
−2.15
Leukocyte immunoglobulin-like receptor, subfamily A, member 2
ZEB1
NM_030751
−4.12
Zinc finger E-box binding homeobox 1
PLA2G7
M80436
−3.95
Platelet-activating factor receptor
MASP1
AI274095
−3.67
Mannan-binding lectin serine peptidase 1
EBI2
NM_004951
−3.59
Epstein-Barr virus induced gene 2
NOD2
NM_022162
−3.57
Nucleotide-binding oligomerization domain containing 2
LAIR1
NM_021708
−3.54
Leukocyte-associated immunoglobulin-like receptor 1
HLA-DQA1
BG397856
−3.38
Major histocompatibility complex, class II, DQ alpha 1
HLA-DQB1
M17955
−2.99
Major histocompatibility complex, class II, DQ beta 1
HLA-DRA
M60333
−3.22
Major histocompatibility complex, class II, DR alpha
HLA-DPA1
M27487
−2.83
Major histocompatibility complex, class II, DP alpha 1
HLA-DPB1
NM_002121
−2.15
Major histocompatibility complex, class II, DP beta 1
HLA-DRB4
BC005312
−2.81
Major histocompatibility complex, class II, DR beta 4
HLA-DRB1
AJ297586
−2.72
Major histocompatibility complex, class II, DR beta 3
HLA-DMB
NM_002118
−2.68
Major histocompatibility complex, class II, DM beta
HLA-DMA
X76775
−2.19
major histocompatibility complex, class II, DM alpha
LIF
NM_002309
−3.16
Leukemia inhibitory factor (cholinergic differentiation factor)
NCF4
NM_000631
−2.88
Neutrophil cytosolic factor 4, 40 kDa
PAG1
BF589359
−2.86
Phosphoprotein associated with glycosphingolipid microdomains 1
FYN
AK090692
−2.85
FYN oncogene related to SRC, FGR, YES
TREM2
NM_018965
−2.84
Triggering receptor expressed on myeloid cells 2
BST2
NM_004335
−2.84
Bone marrow stromal cell antigen 2
CTSG
NM_001911
−2.78
Cathepsin G
CTSS
AK024855
−2.60
Cathepsin S
MS4A1
AW474852
−2.74
Membrane-spanning 4-domains, subfamily A, member 1
NCF2
BC001606
−2.70
Neutrophil cytosolic factor 2
GPR65
NM_003608
−2.68
G protein-coupled receptor 65
GBP4
BG260886
−2.59
Guanylate binding protein 4
VAV1
NM_005428
−2.53
Vav 1 guanine nucleotide exchange factor
LCK
NM_005356
−2.49
Lymphocyte-specific protein tyrosine kinase
SYK
NM_003177
−2.42
Spleen tyrosine kinase
LY86
NM_004271
−2.36
Lymphocyte antigen 86
TNFSF10
U57059
−2.36
Tumor necrosis factor (ligand) superfamily, member 10
TNFSF13B
AF134715
−2.29
Tumor necrosis factor (ligand) superfamily, member 13b
IRF8
AI073984
−2.36
Interferon regulatory factor 8
RELB
NM_006509
−2.33
V-rel reticuloendotheliosis viral oncogene homolog B
SMAD6
AI628464
−2.25
SMAD family member 6
MBP
N37023
−2.24
Myelin basic protein
BCL6
S67779
−2.17
B-cell CLL/lymphoma 6 (zinc finger protein 51)
IGKC
BG485135
−2.12
Netrin 2-like (chicken)
CLEC7A
AF313468
−2.12
C-type lectin domain family 7, member A
LCP2
AI123251
−2.05
Lymphocyte cytosolic protein 2
IL31RA
AI123586
6.61
Interleukin 31 receptor A
C4BPA
NM_000715
3.34
Complement component 4 binding protein, alpha
ULBP2
AA831769
3.07
UL16 binding protein 2
CLEC4E
NM_014358
2.61
C-type lectin domain family 4, member E
TNFSF9
NM_003811
2.43
Tumor necrosis factor (ligand) superfamily, member 9
C7
NM_000587
2.36
Complement component 7
TGFB2
NM_003238
2.36
Transforming growth factor, beta 2
FAS
X83493
2.36
Fas (TNF receptor superfamily, member 6)
LAG3
NM_002286
2.26
Lymphocyte-activation gene 3
IL27RA
NM_004843
2.20
Interleukin 27 receptor, alpha
CD276
NM_025240
2.19
CD276 molecule
IL26
NM_018402
2.03
Interleukin 26
OAS1
NM_016816
2.02
2,5-oligoadenylate synthetase 1, 40/46 kDa
*Negative number indicates decreased expression and positive number indicates increased expression (fold change) in PC-treated OA meniscal explants compared with untreated OA meniscal explants.Table 2
Genes classified in inflammatory response and angiogenesis.
Biological process
Gene name
Gene ID
Differ expre (fold)*
Description
Inflammatory  response
CCL20
NM_004591
−103.53
Chemokine (C-C motif) ligand 20
CCL5
NM_002985
−3.54
Chemokine (C-C motif) ligand 5
CCR5
NM_000579
−2.03
Chemokine (C-C motif) receptor 5
CXCL3
NM_002090
−10.03
Chemokine (C-X-C motif) ligand 3
CXCL2
M57731
−2.94
Chemokine (C-X-C motif) ligand 2
CXCL1
NM_001511
−3.01
Chemokine (C-X-C motif) ligand 1
CXCL9
NM_002416
−2.65
Chemokine (C-X-C motif) ligand 9
CXCL13
NM_006419
−2.62
Chemokine (C-X-C motif) ligand 13 (B-cell chemoattractant)
CXCR4
AF348491
−2.63
Chemokine (C-X-C motif) receptor 4
PTGS2
AY151286
−34.01
Prostaglandin-endoperoxide synthase 2
IL6
NM_000600
−32.07
Interleukin 6 (interferon, beta 2)
IL23A
AF043179
−16.44
Enhancer of polycomb homolog 1 (Drosophila)
IL8
NM_000584
−8.17
Interleukin 8
IL1B
NM_000576
−2.28
Interleukin 1, beta
IL1RN
BE563442
−2.80
Interleukin 1 receptor antagonist
S100A8
NM_002964
−16.53
S100 calcium binding protein A8
S100A9
NM_002965
−2.79
S100 calcium binding protein A9
LBP
M35533
−11.36
Lipopolysaccharide binding protein
APOE
NM_000041
−9.23
Apolipoprotein E
FABP4
AI766029
−6.98
Fatty acid binding protein 4, adipocyte
LYZ
U25677
−6.37
Lysozyme (renal amyloidosis)
ITIH4
AI004137
−6.33
Inter-alpha (globulin) inhibitor H4
BDKRB2
NM_000623
−6.01
Bradykinin receptor B2
TLR8
AW872374
−5.55
Toll-like receptor 8
TLR7
NM_016562
−3.34
Toll-like receptor 7
TLR5
AF051151
−3.00
Toll-like receptor 5
TLR4
NM_003266
−2.40
Toll-like receptor 4
TLR2
NM_003264
−2.20
Toll-like receptor 2
TLR1
AL050262
−2.66
Toll-like receptor 1
AOX1
AB046692
−5.34
Aldehyde oxidase 1
FCER1A
BC005912
−5.19
Fc fragment of IgE, high affinity I, receptor for; alpha polypeptide
CFD
NM_001928
−4.99
Complement factor D (adipsin)
CFI
BC020718
−3.37
Complement factor I
CFB
NM_001710
−2.39
Complement factor B
C3
NM_000064
−2.32
Complement component 3
CR1
AI052659
−2.24
Complement component (3b/4b) receptor 1 (Knops blood group)
C1RL
NM_016546
−2.01
Complement component 1, r subcomponent-like
C1QA
NM_015991
−2.20
Complement component 1, q subcomponent, A chain
C1QB
NM_000491
−2.10
Complement component 1, q subcomponent, B chain
AOAH
NM_001637
−4.89
Acyloxyacyl hydrolase (neutrophil)
AGT
NM_000029
−4.66
Angiotensinogen (serpin peptidase inhibitor, clade A, member 8)
BMP6
NM_001718
−4.48
Bone morphogenetic protein 6
PLA2G7
M80436
−3.95
Platelet-activating factor receptor
FOS
BC004490
−3.95
V-fos FBJ murine osteosarcoma viral oncogene homolog
CD40
NM_001250
−3.68
CD40 molecule, TNF receptor superfamily member 5
CD163
NM_004244
−3.68
CD163 molecule
CD14
NM_000591
−3.25
CD14 molecule
MASP1
AI274095
−3.67
Mannan-binding lectin serine peptidase 1
F11R
AF191495
−3.16
F11 receptor
ITGB2
L78790
−2.98
Integrin, beta 2 (complement component 3 receptor 3 and 4 subunit)
NLRC4
NM_021209
−2.92
NLR family, CARD domain containing 4
AIF1
U19713
−2.76
Allograft inflammatory factor 1
ALOX5
NM_000698
−2.71
Arachidonate 5-lipoxygenase
NOD1
AF126484
−2.66
Nucleotide-binding oligomerization domain containing 1
JMJD3
AI830331
−2.46
Jumonji domain containing 3, histone lysine demethylase
SIGLEC1
NM_023068
−2.46
Sialic acid binding Ig-like lectin 1, sialoadhesin
TNFAIP6
AW188198
−2.37
Tumor necrosis factor, alpha-induced protein 6
LY86
NM_004271
−2.36
Lymphocyte antigen 86
AOC3
NM_003734
−2.31
Amine oxidase, copper containing 3 (vascular adhesion protein 1)
TNFRSF1B
NM_001066
−2.21
Tumor necrosis factor receptor superfamily, member 1B
BCL6
S67779
−2.17
B-cell CLL/lymphoma 6 (zinc finger protein 51)
CLEC7A
AF313468
−2.12
C-type lectin domain family 7, member A
CDO1
NM_001801
−2.11
Cysteine dioxygenase, type I
NFATC4
AI806528
−2.04
NF of activated T-cells, cytoplasmic, calcineurin-dependent 4
SERPINA3
NM_001085
5.20
Serpin peptidase inhibitor, clade A, member 3
SERPINA1
AF119873
3.58
Serpin peptidase inhibitor, clade A, member 1
C4BPA
NM_000715
3.34
Complement component 4 binding protein, alpha
FN1
AJ276395
3.03
Fibronectin 1
B4GALT1
D29805
2.99
UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 1
GPR68
AI805006
2.40
G protein-coupled receptor 68
C7
NM_000587
2.36
Complement component 7
ANXA1
AU155094
2.23
Annexin A1
KLKB1
NM_000892
2.21
Cytochrome P450, family 4, subfamily V, polypeptide 2
Angiogenesis
PTGS2
AY151286
−34.00
Prostaglandin-endoperoxide synthase 2
BAI3
NM_001704
−33.48
Brain-specific angiogenesis inhibitor 3
IL6
NM_000600
−32.08
Interleukin 6 (interferon, beta 2)
IL8
NM_000584
−8.18
Interleukin 8
IL1B
NM_000576
−2.28
Interleukin 1, beta
SFRP1
AF017987
−17.55
Secreted frizzled-related protein 1
ANGPTL4
AF169312
−11.43
Angiopoietin-like 4
ANGPT2
BE501356
−2.56
Angiopoietin 2
EREG
NM_001432
−8.60
Epiregulin
VEGFA
M27281
−7.91
Vascular endothelial growth factor A
VASH1
AU152507
−4.24
Vasohibin 1
FLT1
U01134
−3.62
Fms-related tyrosine kinase 1
PTPRB
AL080103
−3.48
Protein tyrosine phosphatase, receptor type, B
FGF10
NM_004465
−3.34
Fibroblast growth factor 10
LIF
NM_002309
−3.16
Leukemia inhibitory factor (cholinergic differentiation factor)
BTG1
BC009050
−3.11
B-cell translocation gene 1, anti-proliferative
DLL4
AB036931
−2.85
Delta-like 4 (Drosophila)
SOX17
NM_022454
−2.45
SRY (sex determining region Y)-box 17
EPAS1
NM_001430
−2.43
Endothelial PAS domain protein 1
CTNNB1
AB062292
−2.35
Catenin (cadherin-associated protein), beta 1, 88 kDa
TGFBR2
NM_003242
−2.35
Transforming growth factor, beta receptor II (70/80 kDa)
TYMP
NM_001953
−2.34
Thymidine phosphorylase
C3
NM_000064
−2.32
Complement component 3
NRP1
AF280547
−2.19
Neuropilin 1
NOTCH4
AI743713
−2.17
Notch homolog 4 (Drosophila)
TSPAN12
AI056699
−2.16
Tetraspanin 12
ADAM8
NM_001109
−2.15
ADAM metallopeptidase domain 8
RHOB
AI263909
−2.12
Ras homolog gene family, member B
HHEX
NM_001529
−2.12
Hematopoietically expressed homeobox
THBS1
BF055462
−2.05
Thrombospondin 1
KDR
NM_002253
−2.03
Kinase insert domain receptor (a type III receptor tyrosine kinase)
GREM1
NM_013372
15.86
Gremlin 1, cysteine knot superfamily, homolog (Xenopus laevis)
FGFR2
AB030078
4.49
Fibroblast growth factor receptor 2
FGF1
X59065
2.39
fibroblast growth factor 1 (acidic)
FGF18
AI798863
2.33
Fibroblast growth factor 18
COL8A2
AI806793
4.46
Collagen, type VIII, alpha 2
COL8A1
BE877796
3.30
Collagen, type VIII, alpha 1
ARHGAP22
NM_021226
4.45
Rho GTPase activating protein 22
TNFRSF12A
NM_016639
3.23
Tumor necrosis factor receptor superfamily, member 12A
CSPG4
BE857703
3.14
Chondroitin sulfate proteoglycan 4
WNT5B
NM_030775
3.10
Wingless-type MMTV integration site family, member 5B
FN1
AJ276395
3.03
Fibronectin 1
SFRP2
AF311912
3.00
Secreted frizzled-related protein 2
B4GALT1
D29805
2.99
UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 1
ANGPT1
NM_001146
2.62
Angiopoietin 1
MFGE8
BC003610
2.39
Milk fat globule-EGF factor 8 protein
TGFB2
NM_003238
2.36
Transforming growth factor, beta 2
CYR61
NM_001554
2.30
Cysteine-rich, angiogenic inducer, 61
MEOX2
NM_005924
2.15
Mesenchyme homeobox 2
PLXDC1
NM_020405
2.09
Plexin domain containing 1
TBXA2R
NM_001060
2.09
Thromboxane A2 receptor
*Negative number indicates decreased expression and positive number indicates increased expression (fold change).Table 3
Genes classified in skeletal development, steroid biosynthetic process, and DNA repair.
Biological process
Gene name
Gene ID
Differ expre (fold)*
Description
Skeletal  development
COL2A1
X06268
21.29
Collagen, type II, alpha 1
COL11A1
J04177
9.86
Collagen, type XI, alpha 1
COL1A1
AI743621
2.59
Collagen, type I, alpha 1
ACAN
NM_001135
9.17
Aggrecan
POSTN
AW137148
5.57
Periostin, osteoblast specific factor
PAX1
AA725078
4.73
Paired box 1
FRZB
U91903
3.22
Frizzled-related protein
MMP9
NM_004994
2.58
Matrix metallopeptidase 9
GLI2
NM_030379
2.54
GLI-Kruppel family member GLI2
FGFR3
NM_000142
2.40
Fibroblast growth factor receptor 3
FGF18
AI798863
2.33
Fibroblast growth factor 18
TGFB2
NM_003238
2.36
Transforming growth factor, beta 2
TRPS1
AK000948
2.32
Trichorhinophalangeal syndrome I
RUNX2
AW469546
2.24
Runt-related transcription factor 2
PRELP
U41344
2.15
Proline/arginine-rich end leucine-rich repeat protein
PAPSS2
AW299958
2.03
3-phosphoadenosine 5-phosphosulfate synthase 2
SOX9
NM_000346
2.00
SRY (sex determining region Y)-box 9
MEPE
NM_020203
−13.5
Matrix, extracellular phosphoglycoprotein with ASARM motif
PTHLH
BC005961
−6.79
Parathyroid hormone-like hormone
CHRDL2
AF332891
−6.20
Chordin-like 2
COL10A
X98568
−5.40
collagen, type X, alpha 1 (Schmid metaphyseal chondrodysplasia)
BMP6
NM_001718
−4.48
Bone morphogenetic protein 6
GDF10
NM_004962
−2.99
Growth differentiation factor 10
BMP8B
AA610122
−2.70
Bone morphogenetic protein 8b
TGFBR2
NM_003242
−2.35
Transforming growth factor, beta receptor II (70/80 kDa)
IGFBP4
NM_001552
−2.30
Insulin-like growth factor binding protein 4
Steroid biosynthetic process
SQLE
AA639705
3.81
Squalene epoxidase
SRD5A1
NM_001047
3.75
Steroid-5-alpha-reductase, alpha polypeptide 1
FDXR
NM_004110
3.26
Ferredoxin reductase
HMGCS1
NM_002130
3.12
3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (soluble)
HMGCR
AL518627
2.15
3-hydroxy-3-methylglutaryl-Coenzyme A reductase
LSS
D63807
3.04
Lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase)
DHCR24
NM_014762
2.66
24-dehydrocholesterol reductase
DHCR7
NM_001360
2.57
7-dehydrocholesterol reductase
OPRS1
NM_005866
2.62
Opioid receptor, sigma 1
CYB5R2
NM_016229
2.37
Cytochrome b5 reductase 2
SC5D
D85181
2.06
Sterol-C5-desaturase
BMP6
NM_001718
−4.48
Bone morphogenetic protein 6
CYP39A1
NM_016593
−2.21
Cytochrome P450, family 39, subfamily A, polypeptide 1
HSD3B7
BC004929
−2.10
3 beta-hydroxysteroid dehydrogenase type 7
ADM
NM_001124
−2.06
Adrenomedullin
ABCG1
NM_004915
−2.04
ATP-binding cassette, sub-family G (WHITE), member 1
DNA repair
TOP2A
AU159942
3.75
Topoisomerase (DNA) II alpha 170 kDa
RAD52B
AF125949
2.83
RAD52 homolog (S. cerevisiae)
RAD54B
NM_012415
2.05
RAD54 homolog B (S. cerevisiae)
TYMS
NM_001071
2.40
Thymidylate synthetase
RFC5
BG260658
2.39
Replication factor C (activator 1) 5, 36.5 kDa
SOD2
AL050388
−9.58
Superoxide dismutase 2, mitochondrial
REV3L
NM_002912
−3.08
REV3-like, catalytic subunit of DNA polymerase zeta (yeast)
*Negative number indicates decreased expression and positive number indicates increased expression (fold change).As shown in Table1, the expressions of numerous genes classified in the immune response were downregulated by PC. Of the 120 differentially-expressed genes classified in the immune response, the expressions of 106 genes, including many genes encoding chemokines and cytokines, such as chemokine (C-C motif) ligand 20 (CCL20, −103.53 fold change), chemokine (C-C motif) ligand 5 (CCL5, −3.54 fold change), chemokine (C-C motif) receptor 5 (CCR5, −2.03 fold change), chemokine (C-X-C motif) ligand 3 (CXCL3, −10.03 fold), interleukin 6 (IL-6, −32.07 fold change), interleukin 7 receptor (IL-7R. −3.31 fold change), IL-8 (−8.17 fold change), IL-23, alpha subunit (IL23A, −5.46 fold change), and IL-1 beta (IL-1β, −2.28 fold change) genes, were downregulated by PC.The genes downregulated by PC also included many Fc fragments of IgG receptors (FCGRs), leukocyte immunoglobulin-like receptors (LILRs), toll-like receptors (TLRs), and major histocompatibility complex (MHC) class II molecules genes, such as FCGR3B (−22.56 fold change), FCGR2B (−7.13 fold change), LILRB3 (−4.23 fold change), LILRB2 (−4.32 fold change), LILRB1 (−4.33 fold change), TLR8 (−5.55 fold change), TLR7 (−3.34 fold change), MHC class II, DP alpha 1 (HLA-DPA1, −2.83 fold change), MHC class II, DQ alpha 1 (HLA-DQA1, −3.39 fold), MHC class II, DR beta 1 (HLA-DRB1, −2.72 fold change), and MHC class II, DR beta 4 genes (HLA-DRB4, −2.81 fold change).The expressions of many genes classified in inflammatory response and angiogenesis were also downregulated by PC. As shown in Table2, of the 73 differentially-expressed genes classified in inflammatory response, the expressions of 64 genes, including prostaglandin-endoperoxide synthase 2 (PTGS2/Cox-2, −34.01 fold change), S100 calcium binding protein A8 (S100A8, −16.53 fold change), complement factor D (CFD, −4.99 fold change), and allograft inflammatory factor 1 (AIF1, −2.76 fold change) genes, were downregulated by PC. Of the 51 differentially-expressed genes classified in angiogenesis, the expressions of 31 genes, including brain-specific angiogenesis inhibitor 3 (BAI3, −33.48 fold change), angiopoietin-like 4 (ANGPTL4, −11.43 fold change), and vascular endothelial growth factor A genes (VEGFA, −7.91 fold change), were downregulated by PC.In contrast, the expressions of many genes classified in skeletal development, steroid biosynthetic process, and DNA repair were upregulated by PC. As shown in Table3, of the 26 differentially-expressed genes classified in skeletal development, the expressions of 17 genes, including collagen type II, alpha 1 (COL2A1, 21.29 fold change), collagen type XI, alpha 1 (COL11A1, 9.86 fold change), aggrecan (ACAN, 9.17 fold change), FGFR3 (2.40 fold change), FGF18 (2.33 fold change), and SOX-9 (2.00 fold change) genes, were upregulated by PC. Of the 16 differentially expressed genes classified in steroid biosynthetic process, the expressions of 11 genes, including squalene epoxidase (SQLE, 3.81 fold change) and steroid-5-alpha-reductase and alpha polypeptide 1 (SRD5A1, 3.75 fold change) genes, were upregulated by PC. Of the 7 differentially expressed genes classified in DNA repair, the expressions of 5 genes, including topoisomerase (DNA) II alpha 170 kDa (TOP2A, 3.75 fold change) and RAD52 homolog (RAD52B, 2.83 fold change) genes, were upregulated by PC.We performed another independent microarray analysis using RNA samples extracted from meniscal explants derived from a different OA patient. Results from the two microarray analyses for select genes were listed in Table4. As shown, the results from the two microarray analyses were consistent. We also examined the expressions of selected genes using real-time quantitative RT-PCR. The results from the real-time RT-PCR for the genes examined were consistent with the results from the microarray analyses (Table 4).Table 4
Differential expression of selected genes.
Gene name
Gene ID
Differential expression* microarray
Differential expression** microarray
Differential expression*** real-time RT-PCR
CCL5
NM_002985
−3.54
−6.28
−3.69
CCR5
NM_000579
−2.03
−2.95
FCGR3B
NM_000570
−6.49
−2.09
FCGR2B
M31933
−3.45
−3.72
IL6
NM_000600
−32.07
−1.85
IL7R
BE217880
−3.31
−11.0
−2.85
IL8
NM_000584
−8.17
−2.25
IL23A
M15564
−7.89
−8.98
LILRB1
NM_006669
−4.33
−3.06
LILRB3
AF009634
−4.23
−3.38
TLR8
AW872374
−5.55
−3.55
TLR7
NM_016562
−3.34
−2.54
HLA-DRB1
AJ297586
−2.72
−3.41
−1.97
S100A8
NM_002964
−16.53
−9.95
S100A9
NM_002965
−2.79
−4.70
PTPRC
NM_002838
−5.88
−2.85
SYK
NM_003177
−2.42
−2.99
BMP6
NM_001718
−4.48
−3.00
CPA3
NM_001870
−15.23
−9.37
CPM
BE878495
−3.17
−2.74
ADAMTS5
BI254089
−2.84
−1.98
−2.65
ADAM28
NM_021778
−14.27
−3.59
MMP10
NM_002425
−2.41
−1.56
MMP1
NM_002421
−2.15
−1.83
−2.01
FGFR3
NM_000142
2.40
1.52
SOX-9
NM_000346
2.00
1.70
POSTN
AW137148
5.57
2.76
COL2A1
X06268
21.29
1.76
3.11
COL11A1
J04177
9.86
2.29
COL1A1
AI743621
2.59
1.79
ACAN
NM_001135
9.17
2.02
2.65
*Negative number indicates decreased expression and positive number indicates increased expression (fold change).
**Second microarray using different RNA samples extracted from meniscal explants derived from a different OA patient.
***Results of real-time RC-PCR.


### 3.2. Immunohistochemical Staining

To investigate whether PC-targeted genes were associated with OA, we decided to examine the expressions of selected PC-targeted genes in OA and normal menisci. Medial menisci derived from 6 end-stage OA patients and 3 osteosarcoma patients were used for the examinations. Representative images of the normal meniscus and OA menisci are shown in Figure1.Figure 1
Images of normal and OA menisci. Meniscus derived from a male osteosarcoma patient, a male OA patient and a female OA patient. Menisci derived from OA patients exhibited discoloration and rough surface, with clear signs of degeneration whereas the normal control menisci had a smooth and glistening surface, with no signs of degeneration.The expressions of genes we selected for immunohistochemical examinations included FCGR3B, LILRB3, FGFR3 and SOX-9. FCGR3B and LILRB3 are two genes classified in the immune and inflammatory responses. FGFR3 and SOX-9 were two genes classified in the skeletal development. We first tested the antibodies specific to these proteins using human tissues known for their expression before using these antibodies to examine the meniscal tissues.As shown in Figure2, positive immunostainings were observed when these primary antibodies specific to these proteins were used whereas no immunostainings were observed when these primary antibodies were replaced by mouse IgG, confirming that these antibodies could be used to examine the expressions of FCGR3B, LILRB3, FGFR3, and SOX-9 in human tissues. We then used these antibodies to examine medial meniscal specimens derived from 6 end-stage OA patients (diseased tissues) and 3 osteosarcoma patients (control tissues). Representative images of FCGR3B immunostaining are provided in Figure 2(a).(a) Positive FCGR3B immunostaining in human tonsil tissue, positive LILRB3 immunostaining in human tonsil tissue, positive FGFR3 immunostaining in human skin tissue, and positive SOX-9 immunostaining in human cartilage tissue. (b) Negative immunostaining of these proteins when primary antibodies were replaced by mouse IgG.
(a)
(b)As shown, FCGR3B protein was detected in the surface zone of normal menisci. In contrast, FCGR3B was detected in both the surface and middle zones of OA menisci. It was clear that OA menisci contained more FCGR3B immunostaining-positive cells than the normal menisci, suggesting infiltration of inflammatory cells within the OA menisci. Representative images of LILRB3 immunostaining are provided in Figure3(b). As shown, LILRB3 protein was detected in all 3 zones (surface, middle, and deep zones) of the normal and OA menisci. OA menisci not only contained more LILRB3 immunostaining-positive cells but also displayed more intensified LILRB3 immunostaining compared to the normal menisci.Representative images of FCGR3B and LILR3B immunostaining (magnification 10x). (a) FCGR3B immunostaining of normal menisci and OA menisci. (b) LILRB3 immunostaining of normal menisci and OA menisci.
(a)
(b)Representative images of FGFR3 and SOX-9 immunostaining are provided in Figure4. As shown in Figure 4(a), few FGFR3 immunostaining-positive cells were detected in the normal menisci and only a small number of FGFR3 immunostaining-positive cells were detected in the OA menisci. In contrast, strong SOX-9 immunostaining was detected in the normal menisci and the intensity of SOX-9 immunostaining and number of SOX-9 immunostaining-positive cells were decreased significantly in the OA menisci compared to the normal menisci (Figure 4(b)).Representative images of FGFR3 and SOX-9 immunostaining (magnification 10x). (a) FGFR3 immunostaining of normal menisci and OA menisci. (b) SOX-9 immunostaining of normal menisci and OA menisci.
(a)
(b)The immunostainings of FCGR3B, LILRB3, FGFR3, and SOX-9 were graded according to the scale described in Methods. The results along with the demographic patient information are listed in Table5. As shown, the mean grades of FCGR3B, LILRB3, FGFR3, and SOX-9 immunostainings for the normal menisci were 0.33, 1.33, 0.00, and 3.00, respectively, and the mean grades of FCGR3B, LILRB3, FGFR3, and SOX-9 immunostaining for the OA menisci were 2.00, 2.67, 1.17, and 1.50, respectively. The difference between the mean grades of FCGR3B, LILRB3, FGFR3, and SOX-9 immunostaining of the OA menisci and the normal menisci were statistically significant (P
=
0.023, 0.010, 0.002, and 0.024, resp.).Table 5
Grades of immunostaining of FCGR3B, LILRB3, FGFR3, and SOX-9.
Control12 F
Control43 M
Control 39 F
OA77 M
OA49 F
OA66 F
OA70 F
OA57 M
OA65 F
FCGR3B
0
1
0
3
3
2
2
1
1
Mean (FCGR3B)
0.33
2.00
LILRB3
1
2
1
2
3
3
2
3
3
Mean (LILRB3)
1.33
2.67
FGFR3
0
0
0
1
1
1
1
2
1
Mean (FGFR3)
0.00
1.17
SOX-9
3
3
3
2
1
1
1
2
2
Mean (SOX-9)
3.00
1.50
Ages of the patients are listed in years; M = male; F = female.


## 3.1. Effect of PC on Gene Expressions

Microarrayanalysis revealed that of the more than 50,000 transcripts examined, 2561 transcripts displayed significant differential expressions (more than 2.0 fold) in PC-treated OA meniscal explants via untreated OA meniscal explants. A total of 1430 transcripts displayed decreased expressions and 1131 transcripts displayed increased expressions. The genes that fell into specific biological processes previously implicated in OA or suspected to have a role in OA are listed in Tables1, 2, and 3.Table 1
Genes classified in the immune response.
Biological process
Gene name
Gene ID
Differ expre*
Description
Immune response
CCL20
NM_004591
−103.53
Chemokine (C-C motif) ligand 20
CCL5
NM_002985
−3.54
Chemokine (C-C motif) ligand 5
CCR5
NM_000579
−2.03
Chemokine (C-C motif) receptor 5
CXCL3
NM_002090
−10.03
Chemokine (C-X-C motif) ligand 3
CXCL5
AK026546
−4.49
Chemokine (C-X-C motif) ligand 5
CXCL2
M57731
−2.94
Chemokine (C-X-C motif) ligand 2
CXCL1
NM_001511
−3.01
Chemokine (C-X-C motif) ligand 1
CXCL9
NM_002416
−2.65
Chemokine (C-X-C motif) ligand 9
CXCL13
NM_006419
−2.62
Chemokine (C-X-C motif) ligand 13 (B-cell chemoattractant)
CXCR4
AF348491
−2.63
Chemokine (C-X-C motif) receptor 4
IGKC
BC005332
−37.44
Netrin 2-like (chicken)
IL6
NM_000600
−32.07
Interleukin 6 (interferon, beta 2)
IL8
NM_000584
−8.17
Interleukin 8
IL23A
M15564
−7.89
Enhancer of polycomb homolog 1 (Drosophila)
IL24
NM_006850
−3.73
Interleukin 24
IL15
NM_000585
−3.70
Interleukin 15
IL7R
BE217880
−3.31
Interleukin 7 receptor
IL1B
NM_000576
−2.28
Interleukin 1, beta
IL1RN
BE563442
−2.80
Interleukin 1 receptor antagonist
MS4A2
NM_000139
−28.91
Membrane-spanning 4-domains, subfamily A, member 2
FCGR3B
NM_000570
−22.56
Fc fragment of IgG, low affinity IIIb, receptor (CD16b)
FCGR2B
U90940
−7.13
Fc fragment of IgG, low affinity IIc, receptor for (CD32)
FCGR1B
L03419
−3.30
Fc fragment of IgG, high affinity Ib, receptor (CD64)
FCER1A
BC005912
−5.19
Fc fragment of IgE, high affinity I, receptor for; alpha polypeptide
HLA-C
AW575927
−22.41
Major histocompatibility complex, class I, C
HLA-F
BE138825
−2.22
Major histocompatibility complex, class I, F
AQP9
NM_020980
−17.49
Aquaporin 9
GZMA
NM_006144
−14.63
Granzyme A
IGHG1
M87789
−14.43
Immunoglobulin heavy constant gamma 1
LBP
M35533
−11.36
Lipopolysaccharide binding protein
EREG
NM_001432
−8.60
Epiregulin
CD86
NM_006889
−7.11
CD86 molecule
CD74
M28590
−2.76
CD74 molecule
CD40
NM_001250
−3.68
CD40 molecule, TNF receptor superfamily member 5
CD1D
NM_001766
−5.17
CD1d molecule
CD8A
AW006735
−4.99
CD8a molecule
CD14
NM_000591
−3.25
CD14 molecule
CD209
AF290886
−2.50
CD209 molecule
KYNU
BC000879
−6.69
Kynureninase (L-kynurenine hydrolase)
PTPRC
NM_002838
−5.88
Protein tyrosine phosphatase, receptor type, C
TLR8
AW872374
−5.55
Toll-like receptor 8
TLR7
NM_016562
−3.34
Toll-like receptor 7
TLR5
AF051151
−3.00
Toll-like receptor 5
TLR4
NM_003266
−2.40
Toll-like receptor 4
TLR2
NM_003264
−2.20
Toll-like receptor 2
TLR1
AL050262
−2.66
Toll-like receptor 1
SLC11A1
L32185
−5.41
Solute carrier family 11, member 1
CFD
NM_001928
−4.99
Complement factor D (adipsin)
CFI
BC020718
−3.37
Complement factor I
CFB
NM_001710
−2.39
Complement factor B
C3
NM_000064
−2.32
Complement component 3
CR1
AI052659
−2.24
Complement component (3b/4b) receptor 1 (Knops blood group)
C1QA
NM_015991
−2.20
Complement component 1, q subcomponent, A chain
C1QB
NM_000491
−2.10
Complement component 1, q subcomponent, B chain
C1RL
NM_016546
2.01
Complement component 1, r subcomponent-like
FYB
BF679849
−4.64
FYN binding protein (FYB-120/130)
TREM1
NM_018643
−4.52
Triggering receptor expressed on myeloid cells 1
BMP6
NM_001718
−4.48
Bone morphogenetic protein 6
INPP5D
U53470
−4.47
Inositol polyphosphate-5-phosphatase, 145 kDa
LILRB1
NM_006669
−4.33
Leukocyte immunoglobulin-like receptor, subfamily B, member 1
LILRB2
AF004231
−4.32
Leukocyte immunoglobulin-like receptor, subfamily B, member 2
LILRB3
AF009634
−4.23
leukocyte immunoglobulin-like receptor, subfamily B, member 3
LILRB4
U82979
−2.06
Leukocyte immunoglobulin-like receptor, subfamily B, member 4
LILRB5
NM_006840
−2.31
Leukocyte immunoglobulin-like receptor, subfamily B, member 5
LILRA2
NM_006866
−2.15
Leukocyte immunoglobulin-like receptor, subfamily A, member 2
ZEB1
NM_030751
−4.12
Zinc finger E-box binding homeobox 1
PLA2G7
M80436
−3.95
Platelet-activating factor receptor
MASP1
AI274095
−3.67
Mannan-binding lectin serine peptidase 1
EBI2
NM_004951
−3.59
Epstein-Barr virus induced gene 2
NOD2
NM_022162
−3.57
Nucleotide-binding oligomerization domain containing 2
LAIR1
NM_021708
−3.54
Leukocyte-associated immunoglobulin-like receptor 1
HLA-DQA1
BG397856
−3.38
Major histocompatibility complex, class II, DQ alpha 1
HLA-DQB1
M17955
−2.99
Major histocompatibility complex, class II, DQ beta 1
HLA-DRA
M60333
−3.22
Major histocompatibility complex, class II, DR alpha
HLA-DPA1
M27487
−2.83
Major histocompatibility complex, class II, DP alpha 1
HLA-DPB1
NM_002121
−2.15
Major histocompatibility complex, class II, DP beta 1
HLA-DRB4
BC005312
−2.81
Major histocompatibility complex, class II, DR beta 4
HLA-DRB1
AJ297586
−2.72
Major histocompatibility complex, class II, DR beta 3
HLA-DMB
NM_002118
−2.68
Major histocompatibility complex, class II, DM beta
HLA-DMA
X76775
−2.19
major histocompatibility complex, class II, DM alpha
LIF
NM_002309
−3.16
Leukemia inhibitory factor (cholinergic differentiation factor)
NCF4
NM_000631
−2.88
Neutrophil cytosolic factor 4, 40 kDa
PAG1
BF589359
−2.86
Phosphoprotein associated with glycosphingolipid microdomains 1
FYN
AK090692
−2.85
FYN oncogene related to SRC, FGR, YES
TREM2
NM_018965
−2.84
Triggering receptor expressed on myeloid cells 2
BST2
NM_004335
−2.84
Bone marrow stromal cell antigen 2
CTSG
NM_001911
−2.78
Cathepsin G
CTSS
AK024855
−2.60
Cathepsin S
MS4A1
AW474852
−2.74
Membrane-spanning 4-domains, subfamily A, member 1
NCF2
BC001606
−2.70
Neutrophil cytosolic factor 2
GPR65
NM_003608
−2.68
G protein-coupled receptor 65
GBP4
BG260886
−2.59
Guanylate binding protein 4
VAV1
NM_005428
−2.53
Vav 1 guanine nucleotide exchange factor
LCK
NM_005356
−2.49
Lymphocyte-specific protein tyrosine kinase
SYK
NM_003177
−2.42
Spleen tyrosine kinase
LY86
NM_004271
−2.36
Lymphocyte antigen 86
TNFSF10
U57059
−2.36
Tumor necrosis factor (ligand) superfamily, member 10
TNFSF13B
AF134715
−2.29
Tumor necrosis factor (ligand) superfamily, member 13b
IRF8
AI073984
−2.36
Interferon regulatory factor 8
RELB
NM_006509
−2.33
V-rel reticuloendotheliosis viral oncogene homolog B
SMAD6
AI628464
−2.25
SMAD family member 6
MBP
N37023
−2.24
Myelin basic protein
BCL6
S67779
−2.17
B-cell CLL/lymphoma 6 (zinc finger protein 51)
IGKC
BG485135
−2.12
Netrin 2-like (chicken)
CLEC7A
AF313468
−2.12
C-type lectin domain family 7, member A
LCP2
AI123251
−2.05
Lymphocyte cytosolic protein 2
IL31RA
AI123586
6.61
Interleukin 31 receptor A
C4BPA
NM_000715
3.34
Complement component 4 binding protein, alpha
ULBP2
AA831769
3.07
UL16 binding protein 2
CLEC4E
NM_014358
2.61
C-type lectin domain family 4, member E
TNFSF9
NM_003811
2.43
Tumor necrosis factor (ligand) superfamily, member 9
C7
NM_000587
2.36
Complement component 7
TGFB2
NM_003238
2.36
Transforming growth factor, beta 2
FAS
X83493
2.36
Fas (TNF receptor superfamily, member 6)
LAG3
NM_002286
2.26
Lymphocyte-activation gene 3
IL27RA
NM_004843
2.20
Interleukin 27 receptor, alpha
CD276
NM_025240
2.19
CD276 molecule
IL26
NM_018402
2.03
Interleukin 26
OAS1
NM_016816
2.02
2,5-oligoadenylate synthetase 1, 40/46 kDa
*Negative number indicates decreased expression and positive number indicates increased expression (fold change) in PC-treated OA meniscal explants compared with untreated OA meniscal explants.Table 2
Genes classified in inflammatory response and angiogenesis.
Biological process
Gene name
Gene ID
Differ expre (fold)*
Description
Inflammatory  response
CCL20
NM_004591
−103.53
Chemokine (C-C motif) ligand 20
CCL5
NM_002985
−3.54
Chemokine (C-C motif) ligand 5
CCR5
NM_000579
−2.03
Chemokine (C-C motif) receptor 5
CXCL3
NM_002090
−10.03
Chemokine (C-X-C motif) ligand 3
CXCL2
M57731
−2.94
Chemokine (C-X-C motif) ligand 2
CXCL1
NM_001511
−3.01
Chemokine (C-X-C motif) ligand 1
CXCL9
NM_002416
−2.65
Chemokine (C-X-C motif) ligand 9
CXCL13
NM_006419
−2.62
Chemokine (C-X-C motif) ligand 13 (B-cell chemoattractant)
CXCR4
AF348491
−2.63
Chemokine (C-X-C motif) receptor 4
PTGS2
AY151286
−34.01
Prostaglandin-endoperoxide synthase 2
IL6
NM_000600
−32.07
Interleukin 6 (interferon, beta 2)
IL23A
AF043179
−16.44
Enhancer of polycomb homolog 1 (Drosophila)
IL8
NM_000584
−8.17
Interleukin 8
IL1B
NM_000576
−2.28
Interleukin 1, beta
IL1RN
BE563442
−2.80
Interleukin 1 receptor antagonist
S100A8
NM_002964
−16.53
S100 calcium binding protein A8
S100A9
NM_002965
−2.79
S100 calcium binding protein A9
LBP
M35533
−11.36
Lipopolysaccharide binding protein
APOE
NM_000041
−9.23
Apolipoprotein E
FABP4
AI766029
−6.98
Fatty acid binding protein 4, adipocyte
LYZ
U25677
−6.37
Lysozyme (renal amyloidosis)
ITIH4
AI004137
−6.33
Inter-alpha (globulin) inhibitor H4
BDKRB2
NM_000623
−6.01
Bradykinin receptor B2
TLR8
AW872374
−5.55
Toll-like receptor 8
TLR7
NM_016562
−3.34
Toll-like receptor 7
TLR5
AF051151
−3.00
Toll-like receptor 5
TLR4
NM_003266
−2.40
Toll-like receptor 4
TLR2
NM_003264
−2.20
Toll-like receptor 2
TLR1
AL050262
−2.66
Toll-like receptor 1
AOX1
AB046692
−5.34
Aldehyde oxidase 1
FCER1A
BC005912
−5.19
Fc fragment of IgE, high affinity I, receptor for; alpha polypeptide
CFD
NM_001928
−4.99
Complement factor D (adipsin)
CFI
BC020718
−3.37
Complement factor I
CFB
NM_001710
−2.39
Complement factor B
C3
NM_000064
−2.32
Complement component 3
CR1
AI052659
−2.24
Complement component (3b/4b) receptor 1 (Knops blood group)
C1RL
NM_016546
−2.01
Complement component 1, r subcomponent-like
C1QA
NM_015991
−2.20
Complement component 1, q subcomponent, A chain
C1QB
NM_000491
−2.10
Complement component 1, q subcomponent, B chain
AOAH
NM_001637
−4.89
Acyloxyacyl hydrolase (neutrophil)
AGT
NM_000029
−4.66
Angiotensinogen (serpin peptidase inhibitor, clade A, member 8)
BMP6
NM_001718
−4.48
Bone morphogenetic protein 6
PLA2G7
M80436
−3.95
Platelet-activating factor receptor
FOS
BC004490
−3.95
V-fos FBJ murine osteosarcoma viral oncogene homolog
CD40
NM_001250
−3.68
CD40 molecule, TNF receptor superfamily member 5
CD163
NM_004244
−3.68
CD163 molecule
CD14
NM_000591
−3.25
CD14 molecule
MASP1
AI274095
−3.67
Mannan-binding lectin serine peptidase 1
F11R
AF191495
−3.16
F11 receptor
ITGB2
L78790
−2.98
Integrin, beta 2 (complement component 3 receptor 3 and 4 subunit)
NLRC4
NM_021209
−2.92
NLR family, CARD domain containing 4
AIF1
U19713
−2.76
Allograft inflammatory factor 1
ALOX5
NM_000698
−2.71
Arachidonate 5-lipoxygenase
NOD1
AF126484
−2.66
Nucleotide-binding oligomerization domain containing 1
JMJD3
AI830331
−2.46
Jumonji domain containing 3, histone lysine demethylase
SIGLEC1
NM_023068
−2.46
Sialic acid binding Ig-like lectin 1, sialoadhesin
TNFAIP6
AW188198
−2.37
Tumor necrosis factor, alpha-induced protein 6
LY86
NM_004271
−2.36
Lymphocyte antigen 86
AOC3
NM_003734
−2.31
Amine oxidase, copper containing 3 (vascular adhesion protein 1)
TNFRSF1B
NM_001066
−2.21
Tumor necrosis factor receptor superfamily, member 1B
BCL6
S67779
−2.17
B-cell CLL/lymphoma 6 (zinc finger protein 51)
CLEC7A
AF313468
−2.12
C-type lectin domain family 7, member A
CDO1
NM_001801
−2.11
Cysteine dioxygenase, type I
NFATC4
AI806528
−2.04
NF of activated T-cells, cytoplasmic, calcineurin-dependent 4
SERPINA3
NM_001085
5.20
Serpin peptidase inhibitor, clade A, member 3
SERPINA1
AF119873
3.58
Serpin peptidase inhibitor, clade A, member 1
C4BPA
NM_000715
3.34
Complement component 4 binding protein, alpha
FN1
AJ276395
3.03
Fibronectin 1
B4GALT1
D29805
2.99
UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 1
GPR68
AI805006
2.40
G protein-coupled receptor 68
C7
NM_000587
2.36
Complement component 7
ANXA1
AU155094
2.23
Annexin A1
KLKB1
NM_000892
2.21
Cytochrome P450, family 4, subfamily V, polypeptide 2
Angiogenesis
PTGS2
AY151286
−34.00
Prostaglandin-endoperoxide synthase 2
BAI3
NM_001704
−33.48
Brain-specific angiogenesis inhibitor 3
IL6
NM_000600
−32.08
Interleukin 6 (interferon, beta 2)
IL8
NM_000584
−8.18
Interleukin 8
IL1B
NM_000576
−2.28
Interleukin 1, beta
SFRP1
AF017987
−17.55
Secreted frizzled-related protein 1
ANGPTL4
AF169312
−11.43
Angiopoietin-like 4
ANGPT2
BE501356
−2.56
Angiopoietin 2
EREG
NM_001432
−8.60
Epiregulin
VEGFA
M27281
−7.91
Vascular endothelial growth factor A
VASH1
AU152507
−4.24
Vasohibin 1
FLT1
U01134
−3.62
Fms-related tyrosine kinase 1
PTPRB
AL080103
−3.48
Protein tyrosine phosphatase, receptor type, B
FGF10
NM_004465
−3.34
Fibroblast growth factor 10
LIF
NM_002309
−3.16
Leukemia inhibitory factor (cholinergic differentiation factor)
BTG1
BC009050
−3.11
B-cell translocation gene 1, anti-proliferative
DLL4
AB036931
−2.85
Delta-like 4 (Drosophila)
SOX17
NM_022454
−2.45
SRY (sex determining region Y)-box 17
EPAS1
NM_001430
−2.43
Endothelial PAS domain protein 1
CTNNB1
AB062292
−2.35
Catenin (cadherin-associated protein), beta 1, 88 kDa
TGFBR2
NM_003242
−2.35
Transforming growth factor, beta receptor II (70/80 kDa)
TYMP
NM_001953
−2.34
Thymidine phosphorylase
C3
NM_000064
−2.32
Complement component 3
NRP1
AF280547
−2.19
Neuropilin 1
NOTCH4
AI743713
−2.17
Notch homolog 4 (Drosophila)
TSPAN12
AI056699
−2.16
Tetraspanin 12
ADAM8
NM_001109
−2.15
ADAM metallopeptidase domain 8
RHOB
AI263909
−2.12
Ras homolog gene family, member B
HHEX
NM_001529
−2.12
Hematopoietically expressed homeobox
THBS1
BF055462
−2.05
Thrombospondin 1
KDR
NM_002253
−2.03
Kinase insert domain receptor (a type III receptor tyrosine kinase)
GREM1
NM_013372
15.86
Gremlin 1, cysteine knot superfamily, homolog (Xenopus laevis)
FGFR2
AB030078
4.49
Fibroblast growth factor receptor 2
FGF1
X59065
2.39
fibroblast growth factor 1 (acidic)
FGF18
AI798863
2.33
Fibroblast growth factor 18
COL8A2
AI806793
4.46
Collagen, type VIII, alpha 2
COL8A1
BE877796
3.30
Collagen, type VIII, alpha 1
ARHGAP22
NM_021226
4.45
Rho GTPase activating protein 22
TNFRSF12A
NM_016639
3.23
Tumor necrosis factor receptor superfamily, member 12A
CSPG4
BE857703
3.14
Chondroitin sulfate proteoglycan 4
WNT5B
NM_030775
3.10
Wingless-type MMTV integration site family, member 5B
FN1
AJ276395
3.03
Fibronectin 1
SFRP2
AF311912
3.00
Secreted frizzled-related protein 2
B4GALT1
D29805
2.99
UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 1
ANGPT1
NM_001146
2.62
Angiopoietin 1
MFGE8
BC003610
2.39
Milk fat globule-EGF factor 8 protein
TGFB2
NM_003238
2.36
Transforming growth factor, beta 2
CYR61
NM_001554
2.30
Cysteine-rich, angiogenic inducer, 61
MEOX2
NM_005924
2.15
Mesenchyme homeobox 2
PLXDC1
NM_020405
2.09
Plexin domain containing 1
TBXA2R
NM_001060
2.09
Thromboxane A2 receptor
*Negative number indicates decreased expression and positive number indicates increased expression (fold change).Table 3
Genes classified in skeletal development, steroid biosynthetic process, and DNA repair.
Biological process
Gene name
Gene ID
Differ expre (fold)*
Description
Skeletal  development
COL2A1
X06268
21.29
Collagen, type II, alpha 1
COL11A1
J04177
9.86
Collagen, type XI, alpha 1
COL1A1
AI743621
2.59
Collagen, type I, alpha 1
ACAN
NM_001135
9.17
Aggrecan
POSTN
AW137148
5.57
Periostin, osteoblast specific factor
PAX1
AA725078
4.73
Paired box 1
FRZB
U91903
3.22
Frizzled-related protein
MMP9
NM_004994
2.58
Matrix metallopeptidase 9
GLI2
NM_030379
2.54
GLI-Kruppel family member GLI2
FGFR3
NM_000142
2.40
Fibroblast growth factor receptor 3
FGF18
AI798863
2.33
Fibroblast growth factor 18
TGFB2
NM_003238
2.36
Transforming growth factor, beta 2
TRPS1
AK000948
2.32
Trichorhinophalangeal syndrome I
RUNX2
AW469546
2.24
Runt-related transcription factor 2
PRELP
U41344
2.15
Proline/arginine-rich end leucine-rich repeat protein
PAPSS2
AW299958
2.03
3-phosphoadenosine 5-phosphosulfate synthase 2
SOX9
NM_000346
2.00
SRY (sex determining region Y)-box 9
MEPE
NM_020203
−13.5
Matrix, extracellular phosphoglycoprotein with ASARM motif
PTHLH
BC005961
−6.79
Parathyroid hormone-like hormone
CHRDL2
AF332891
−6.20
Chordin-like 2
COL10A
X98568
−5.40
collagen, type X, alpha 1 (Schmid metaphyseal chondrodysplasia)
BMP6
NM_001718
−4.48
Bone morphogenetic protein 6
GDF10
NM_004962
−2.99
Growth differentiation factor 10
BMP8B
AA610122
−2.70
Bone morphogenetic protein 8b
TGFBR2
NM_003242
−2.35
Transforming growth factor, beta receptor II (70/80 kDa)
IGFBP4
NM_001552
−2.30
Insulin-like growth factor binding protein 4
Steroid biosynthetic process
SQLE
AA639705
3.81
Squalene epoxidase
SRD5A1
NM_001047
3.75
Steroid-5-alpha-reductase, alpha polypeptide 1
FDXR
NM_004110
3.26
Ferredoxin reductase
HMGCS1
NM_002130
3.12
3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (soluble)
HMGCR
AL518627
2.15
3-hydroxy-3-methylglutaryl-Coenzyme A reductase
LSS
D63807
3.04
Lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase)
DHCR24
NM_014762
2.66
24-dehydrocholesterol reductase
DHCR7
NM_001360
2.57
7-dehydrocholesterol reductase
OPRS1
NM_005866
2.62
Opioid receptor, sigma 1
CYB5R2
NM_016229
2.37
Cytochrome b5 reductase 2
SC5D
D85181
2.06
Sterol-C5-desaturase
BMP6
NM_001718
−4.48
Bone morphogenetic protein 6
CYP39A1
NM_016593
−2.21
Cytochrome P450, family 39, subfamily A, polypeptide 1
HSD3B7
BC004929
−2.10
3 beta-hydroxysteroid dehydrogenase type 7
ADM
NM_001124
−2.06
Adrenomedullin
ABCG1
NM_004915
−2.04
ATP-binding cassette, sub-family G (WHITE), member 1
DNA repair
TOP2A
AU159942
3.75
Topoisomerase (DNA) II alpha 170 kDa
RAD52B
AF125949
2.83
RAD52 homolog (S. cerevisiae)
RAD54B
NM_012415
2.05
RAD54 homolog B (S. cerevisiae)
TYMS
NM_001071
2.40
Thymidylate synthetase
RFC5
BG260658
2.39
Replication factor C (activator 1) 5, 36.5 kDa
SOD2
AL050388
−9.58
Superoxide dismutase 2, mitochondrial
REV3L
NM_002912
−3.08
REV3-like, catalytic subunit of DNA polymerase zeta (yeast)
*Negative number indicates decreased expression and positive number indicates increased expression (fold change).As shown in Table1, the expressions of numerous genes classified in the immune response were downregulated by PC. Of the 120 differentially-expressed genes classified in the immune response, the expressions of 106 genes, including many genes encoding chemokines and cytokines, such as chemokine (C-C motif) ligand 20 (CCL20, −103.53 fold change), chemokine (C-C motif) ligand 5 (CCL5, −3.54 fold change), chemokine (C-C motif) receptor 5 (CCR5, −2.03 fold change), chemokine (C-X-C motif) ligand 3 (CXCL3, −10.03 fold), interleukin 6 (IL-6, −32.07 fold change), interleukin 7 receptor (IL-7R. −3.31 fold change), IL-8 (−8.17 fold change), IL-23, alpha subunit (IL23A, −5.46 fold change), and IL-1 beta (IL-1β, −2.28 fold change) genes, were downregulated by PC.The genes downregulated by PC also included many Fc fragments of IgG receptors (FCGRs), leukocyte immunoglobulin-like receptors (LILRs), toll-like receptors (TLRs), and major histocompatibility complex (MHC) class II molecules genes, such as FCGR3B (−22.56 fold change), FCGR2B (−7.13 fold change), LILRB3 (−4.23 fold change), LILRB2 (−4.32 fold change), LILRB1 (−4.33 fold change), TLR8 (−5.55 fold change), TLR7 (−3.34 fold change), MHC class II, DP alpha 1 (HLA-DPA1, −2.83 fold change), MHC class II, DQ alpha 1 (HLA-DQA1, −3.39 fold), MHC class II, DR beta 1 (HLA-DRB1, −2.72 fold change), and MHC class II, DR beta 4 genes (HLA-DRB4, −2.81 fold change).The expressions of many genes classified in inflammatory response and angiogenesis were also downregulated by PC. As shown in Table2, of the 73 differentially-expressed genes classified in inflammatory response, the expressions of 64 genes, including prostaglandin-endoperoxide synthase 2 (PTGS2/Cox-2, −34.01 fold change), S100 calcium binding protein A8 (S100A8, −16.53 fold change), complement factor D (CFD, −4.99 fold change), and allograft inflammatory factor 1 (AIF1, −2.76 fold change) genes, were downregulated by PC. Of the 51 differentially-expressed genes classified in angiogenesis, the expressions of 31 genes, including brain-specific angiogenesis inhibitor 3 (BAI3, −33.48 fold change), angiopoietin-like 4 (ANGPTL4, −11.43 fold change), and vascular endothelial growth factor A genes (VEGFA, −7.91 fold change), were downregulated by PC.In contrast, the expressions of many genes classified in skeletal development, steroid biosynthetic process, and DNA repair were upregulated by PC. As shown in Table3, of the 26 differentially-expressed genes classified in skeletal development, the expressions of 17 genes, including collagen type II, alpha 1 (COL2A1, 21.29 fold change), collagen type XI, alpha 1 (COL11A1, 9.86 fold change), aggrecan (ACAN, 9.17 fold change), FGFR3 (2.40 fold change), FGF18 (2.33 fold change), and SOX-9 (2.00 fold change) genes, were upregulated by PC. Of the 16 differentially expressed genes classified in steroid biosynthetic process, the expressions of 11 genes, including squalene epoxidase (SQLE, 3.81 fold change) and steroid-5-alpha-reductase and alpha polypeptide 1 (SRD5A1, 3.75 fold change) genes, were upregulated by PC. Of the 7 differentially expressed genes classified in DNA repair, the expressions of 5 genes, including topoisomerase (DNA) II alpha 170 kDa (TOP2A, 3.75 fold change) and RAD52 homolog (RAD52B, 2.83 fold change) genes, were upregulated by PC.We performed another independent microarray analysis using RNA samples extracted from meniscal explants derived from a different OA patient. Results from the two microarray analyses for select genes were listed in Table4. As shown, the results from the two microarray analyses were consistent. We also examined the expressions of selected genes using real-time quantitative RT-PCR. The results from the real-time RT-PCR for the genes examined were consistent with the results from the microarray analyses (Table 4).Table 4
Differential expression of selected genes.
Gene name
Gene ID
Differential expression* microarray
Differential expression** microarray
Differential expression*** real-time RT-PCR
CCL5
NM_002985
−3.54
−6.28
−3.69
CCR5
NM_000579
−2.03
−2.95
FCGR3B
NM_000570
−6.49
−2.09
FCGR2B
M31933
−3.45
−3.72
IL6
NM_000600
−32.07
−1.85
IL7R
BE217880
−3.31
−11.0
−2.85
IL8
NM_000584
−8.17
−2.25
IL23A
M15564
−7.89
−8.98
LILRB1
NM_006669
−4.33
−3.06
LILRB3
AF009634
−4.23
−3.38
TLR8
AW872374
−5.55
−3.55
TLR7
NM_016562
−3.34
−2.54
HLA-DRB1
AJ297586
−2.72
−3.41
−1.97
S100A8
NM_002964
−16.53
−9.95
S100A9
NM_002965
−2.79
−4.70
PTPRC
NM_002838
−5.88
−2.85
SYK
NM_003177
−2.42
−2.99
BMP6
NM_001718
−4.48
−3.00
CPA3
NM_001870
−15.23
−9.37
CPM
BE878495
−3.17
−2.74
ADAMTS5
BI254089
−2.84
−1.98
−2.65
ADAM28
NM_021778
−14.27
−3.59
MMP10
NM_002425
−2.41
−1.56
MMP1
NM_002421
−2.15
−1.83
−2.01
FGFR3
NM_000142
2.40
1.52
SOX-9
NM_000346
2.00
1.70
POSTN
AW137148
5.57
2.76
COL2A1
X06268
21.29
1.76
3.11
COL11A1
J04177
9.86
2.29
COL1A1
AI743621
2.59
1.79
ACAN
NM_001135
9.17
2.02
2.65
*Negative number indicates decreased expression and positive number indicates increased expression (fold change).
**Second microarray using different RNA samples extracted from meniscal explants derived from a different OA patient.
***Results of real-time RC-PCR.


## 3.2. Immunohistochemical Staining

To investigate whether PC-targeted genes were associated with OA, we decided to examine the expressions of selected PC-targeted genes in OA and normal menisci. Medial menisci derived from 6 end-stage OA patients and 3 osteosarcoma patients were used for the examinations. Representative images of the normal meniscus and OA menisci are shown in Figure1.Figure 1
Images of normal and OA menisci. Meniscus derived from a male osteosarcoma patient, a male OA patient and a female OA patient. Menisci derived from OA patients exhibited discoloration and rough surface, with clear signs of degeneration whereas the normal control menisci had a smooth and glistening surface, with no signs of degeneration.The expressions of genes we selected for immunohistochemical examinations included FCGR3B, LILRB3, FGFR3 and SOX-9. FCGR3B and LILRB3 are two genes classified in the immune and inflammatory responses. FGFR3 and SOX-9 were two genes classified in the skeletal development. We first tested the antibodies specific to these proteins using human tissues known for their expression before using these antibodies to examine the meniscal tissues.As shown in Figure2, positive immunostainings were observed when these primary antibodies specific to these proteins were used whereas no immunostainings were observed when these primary antibodies were replaced by mouse IgG, confirming that these antibodies could be used to examine the expressions of FCGR3B, LILRB3, FGFR3, and SOX-9 in human tissues. We then used these antibodies to examine medial meniscal specimens derived from 6 end-stage OA patients (diseased tissues) and 3 osteosarcoma patients (control tissues). Representative images of FCGR3B immunostaining are provided in Figure 2(a).(a) Positive FCGR3B immunostaining in human tonsil tissue, positive LILRB3 immunostaining in human tonsil tissue, positive FGFR3 immunostaining in human skin tissue, and positive SOX-9 immunostaining in human cartilage tissue. (b) Negative immunostaining of these proteins when primary antibodies were replaced by mouse IgG.
(a)
(b)As shown, FCGR3B protein was detected in the surface zone of normal menisci. In contrast, FCGR3B was detected in both the surface and middle zones of OA menisci. It was clear that OA menisci contained more FCGR3B immunostaining-positive cells than the normal menisci, suggesting infiltration of inflammatory cells within the OA menisci. Representative images of LILRB3 immunostaining are provided in Figure3(b). As shown, LILRB3 protein was detected in all 3 zones (surface, middle, and deep zones) of the normal and OA menisci. OA menisci not only contained more LILRB3 immunostaining-positive cells but also displayed more intensified LILRB3 immunostaining compared to the normal menisci.Representative images of FCGR3B and LILR3B immunostaining (magnification 10x). (a) FCGR3B immunostaining of normal menisci and OA menisci. (b) LILRB3 immunostaining of normal menisci and OA menisci.
(a)
(b)Representative images of FGFR3 and SOX-9 immunostaining are provided in Figure4. As shown in Figure 4(a), few FGFR3 immunostaining-positive cells were detected in the normal menisci and only a small number of FGFR3 immunostaining-positive cells were detected in the OA menisci. In contrast, strong SOX-9 immunostaining was detected in the normal menisci and the intensity of SOX-9 immunostaining and number of SOX-9 immunostaining-positive cells were decreased significantly in the OA menisci compared to the normal menisci (Figure 4(b)).Representative images of FGFR3 and SOX-9 immunostaining (magnification 10x). (a) FGFR3 immunostaining of normal menisci and OA menisci. (b) SOX-9 immunostaining of normal menisci and OA menisci.
(a)
(b)The immunostainings of FCGR3B, LILRB3, FGFR3, and SOX-9 were graded according to the scale described in Methods. The results along with the demographic patient information are listed in Table5. As shown, the mean grades of FCGR3B, LILRB3, FGFR3, and SOX-9 immunostainings for the normal menisci were 0.33, 1.33, 0.00, and 3.00, respectively, and the mean grades of FCGR3B, LILRB3, FGFR3, and SOX-9 immunostaining for the OA menisci were 2.00, 2.67, 1.17, and 1.50, respectively. The difference between the mean grades of FCGR3B, LILRB3, FGFR3, and SOX-9 immunostaining of the OA menisci and the normal menisci were statistically significant (P
=
0.023, 0.010, 0.002, and 0.024, resp.).Table 5
Grades of immunostaining of FCGR3B, LILRB3, FGFR3, and SOX-9.
Control12 F
Control43 M
Control 39 F
OA77 M
OA49 F
OA66 F
OA70 F
OA57 M
OA65 F
FCGR3B
0
1
0
3
3
2
2
1
1
Mean (FCGR3B)
0.33
2.00
LILRB3
1
2
1
2
3
3
2
3
3
Mean (LILRB3)
1.33
2.67
FGFR3
0
0
0
1
1
1
1
2
1
Mean (FGFR3)
0.00
1.17
SOX-9
3
3
3
2
1
1
1
2
2
Mean (SOX-9)
3.00
1.50
Ages of the patients are listed in years; M = male; F = female.


## 4. Discussion

There is increasing evidence indicating the involvement of immune system in OA. The expressions of several TLRs genes, which play a key role in innate immune system, were increased in OA cartilage and correlated with the severity of cartilage degeneration [24, 25]. The expressions of several MHC class II genes were increased in degenerative menisci of older patients and OA meniscal cells compared to younger patients and normal meniscal cells [4, 26]. In this study, we demonstrated that PC, which inhibited cartilage degeneration in Hartley guinea pigs [16], downregulated the expression of numerous genes classified in the immune response, including many TLRs genes (Table 1, such as TLR-4 and TLR-8), MHC class II genes (such as HLA-DPA1, HLA-DRA, and HLA-DRB1), FCGRs genes (such as FCGR2B and FCGR3B), and LILRs genes (such as LILRA2, LILRB1, and LILRB3). These findings suggest that PC may be capable of reversing the abnormal expressions of many genes involved in immune system activation in OA menisci and cartilage.Studies found that the expression of FCGRs, which help to bridge the adaptive and innate immune responses, and the expression of LILRs, which exert influence on signaling pathways of both innate and adaptive immune systems, were increased in inflammatory arthritis such as rheumatoid arthritis (RA) [27–30]. In addition, studies found that the numbers of FCGRs- and LILRs-positive immune cells were decreased in RA patients who responded to treatment with anti-rheumatic drugs [31, 32]. These previous findings indicate that abnormal expressions of FCGRs and LILRs are associated with inflammatory arthritis. In this study, we demonstrated that the expressions of FCGR3B and LILRB3 genes were increased in OA menisci and that their expressions in OA meniscal explant culture were inhibited by PC. These findings indicate that abnormal expressions of FCGRs and LILRs are also associated with OA. PC exerts its disease-modifying activity on OA, at least in part, by targeting abnormal immune system activation in OA.Inflammation and angiogenesis are closely integrated processes in OA [33, 34]. A study demonstrated that inhibition of inflammation and angiogenesis reduced pain and retard joint damage in a rat model of OA [35]. In our study, we demonstrated that PC downregulated the expression of numerous genes classified in the inflammatory response and angiogenesis, including CCL5, CCR5, IL-8, IL-7R, IL-6, IL-1β, PTGS2/Cox-2, S100A8, ANGPTL4, and VEGFA. It is worth noting that abnormal expressions of these genes are associated with either OA or RA. For example, the protein levels of CCL5, IL-6, IL-8, IL-1β, S100A8, ANGPTL4, and VEGFA were increased in chondrocytes, cartilage, synovium, or synovial fluid derived from OA patients, which in turn stimulated the expression of MMPs [36–44]; the expression of IL-7R was elevated in RA FLSs and blockade of IL-7R reduced joint inflammation and cartilage destruction [45, 46]; PTGS2/Cox-2 is a key molecular target for the management of arthritis pain [47]. These findings together suggest that PC exerts its disease-modifying activity on OA, at least in part, by targeting abnormal inflammatory response and angiogenesis in OA. These findings also suggest that abnormal inflammatory response and angiogenesis in the menisci may be new target for OA intervention.PC upregulated the expressions of many genes classified in skeletal development, including putative chondroprotective proteins FGFR3 and SOX-9 [48, 49]. To identify which proteins might be involved in the OA disease process, we examined the protein levels of FGFR3 and SOX-9 in normal and OA menisci. We found that FGFR3 protein was barely detected in the normal menisci and was slightly increased in OA menisci, suggesting that FGFR3 gene is unlikely a key OA disease candidate gene. In contrast, the protein level of SOX-9 was very high in the normal menisci but was significantly decreased in the OA menisci. This finding is consistent with the previous findings that the expression of SOX-9 gene was significantly decreased in OA articular cartilage and chondrocytes [50–52]. Taken together, it suggests that SOX-9 may be an OA disease candidate gene and that PC exerts its disease modifying activity on OA in part by reversing the abnormal expression of SOX-9 in OA menisci or cartilage. Study with SOX-9 knok-in or knock-out using an animal model of OA may provide more clues about the role of SOX-9 in OA.A recent study reported that hundreds of genes were differentially expressed in degenerative menisci derived from older patients and younger patients [26]. The genes displayed higher expressions in the degenerative menisci derived from older patients compared to younger patients included HLA-DRB1 (15.01 fold change), FCER1A (4.15 fold change), and IL-7R (2.83 fold change) [26]. These findings are consistent with our findings and indicate that immune system activation occurs in the degenerative menisci. These findings, together with our findings, also suggest that increased expressions of HLA-DRB and IL-7R is likely a phenomenon associated with both the normal meniscal aging process and OA disease process whereas the increased expression of FCER1A is a phenomenon only associated with the normal meniscal aging process.The genes displayed lower expression in the degenerative menisci derived from older patients included COL2A1 (−10.38 fold change) and FGFR3 (−4.65 fold change) [26]. These findings, together with our findings [23], indicate that the decreased expression of COL2A1 is likely a phenomenon associated with both the normal meniscal aging process and OA disease process whereas the decreased expression of FGFR3 is a phenomenon associated with the meniscal aging process. Our findings presented in this study demonstrate that PC affects the expressions of many genes involved in both OA disease process and meniscal aging process in the absence of calcium crystals. This suggests that PC is not only potentially a disease-modifying drug for calcification-induced OA therapy but also potentially a disease-modifying drug for noncalcification-induced arthritis therapy such as posttraumatic OA.


## 5. Conclusions

OA is a disease associated with immune system activation and decreased expression of chondroprotective protein SOX-9. PC exerts its disease-modifying activity on OA, at least in part, by suppressing immune system activation and stimulating the production of extracellular cellular matrix proteins and chondroprotective proteins. PC is potentially a disease-modifying drug for noncalcification-induced arthritis therapy.



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*Source: 210469-2014-11-23.xml*