Background
Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage destruction, synovitis, subchondral bone sclerosis, and osteophyte formation; OA affects almost 15% of the world’s population [
1]. Unfortunately, the mechanisms of OA development remain unclear and there are no available therapeutic agents that effectively prevent or arrest progression of the disease [
2,
3]. Although the etiology of OA is still unclear, it is believed that tumor necrosis factor alpha (TNFα) exhibits an important effect in the pathological processes of OA [
4]. The level of TNFα is increased in OA patients’ articular cartilage compared with that of healthy controls and TNFα is thought to cause inflammatory destruction [
5]. Additionally, anti-TNFα drugs demonstrate preventative and therapeutic effect in various OA models as well as in clinical trials [
6].
Progranulin (PGRN) is a growth factor with a unique “beads-on-a-string” structure [
7,
8]. PGRN participates in many pathophysiological processes, including anti-inflammation, tissue repair, and wound healing [
9‐
12]. Importantly, PGRN is also a growth factor involved in regulating cartilage development and degradation, and the PGRN level is significantly increased in OA patients’ cartilage relative to that of healthy controls [
13,
14]. Previously, we found that PGRN binds to tumor necrosis factor receptors (TNFRs) and is therapeutic in multiple mouse models of inflammatory arthritis [
6,
15]; our recent studies, implementing surgically induced OA models, reveal that PGRN also protects against OA through TNFR signaling [
16].
Through screening a series of PGRN deletion mutants retaining TNFR binding ability, we have generated an engineered protein which appears to be the minimal molecule of PGRN that still has TNFR binding affinity [
6]. This molecule consists of three fragments of PGRN and we named it Atsttrin (antagonist of TNF/TNFR signaling via targeting to TNF receptors) [
17]. Importantly, Atsttrin has a stronger therapeutic effect than recombinant PGRN in inflammatory arthritis animal models [
6]. Recently, another group reported that intraarticular delivery of mesenchymal stem cells (MSCs) which were pretransduced with Atsttrin could protect against OA development [
18]. In this study, we examine whether the engineered protein Atsttrin could protect against OA, as well as the molecular mechanisms involved, through the use of human primary chondrocytes in vitro alongside multiple models of OA implemented in genetically modified mice and Sprague–Dawley rats in vivo.
Methods
Animals, human cartilage, and recombinant Atsttrin
We performed all animal studies under institutional guidelines. All of the protocols were approval by the Institutional Animal Care and Use Committee, New York University, NY, USA. We generated, maintained, and genotyped the mice with the genetic background of C57BL/6 wildtype (WT), PGRN-deficient (PGRN
–/–), TNFR1-deficient (TNFR1
–/–), and TNFR2-deficient (TNFR2
–/–) mice as reported previously [
6]. Sprague–Dawley rats were obtained from Charles River (Wilmington, MA, USA). Eight-week-old male mice and 14-week-old male rats were used for this experiment [
19,
20]. For human primary chondrocyte culture, human cartilage samples were harvested from patients receiving total knee joint replacement surgery from New York University, Hospital for Joint Diseases (NY, USA). Acquisition and use of human tissue was conducted in accordance with an Institutional Review Board (IRB#12758) approved protocol. Recombinant Atsttrin was manufactured and provided by Atreaon, Inc.
Noninvasive anterior crucial ligament rupture rat model
The noninvasive OA rat model was established as described previously [
20]. Animals were anesthetized and maintained using isoflurane, and the noninvasive anterior crucial ligament rupture model was established using the indicated machine: Electroforce 3200 (Bose Corp., MN, USA), Solidworks (Dassault Systemes, MA, USA), or Mojo 3D printer (Stratasys, MN, USA). After the model was established, we intraarticularly injected PBS or recombinant Atsttrin once a week for 4 weeks in total. After 4 weeks of treatment, the rats were sacrificed for histological evaluation.
Surgically induced OA mouse models
For the surgically induced destabilization of medial meniscus (DMM) mouse model, we took advantage of 8-week-old male PGRN–/– mice and their age-matched WT control littermates. The medial meniscotibial ligament of the right knee joint was cut to generate a destabilized medial meniscus. Six mice were used in each group. After surgery, WT mice received local delivery of 6 μl PBS via intraarticular injection, while PGRN–/– mice received local delivery of 6 μl PBS or recombinant Atsttrin (1 μg/μl). Four weeks after model induction, mice were sacrificed and knee joints were collected. The tissues were then processed for histological analysis.
To investigate the preventative as well as the therapeutic effect of Atsttrin, we also established the anterior cruciate ligament transection (ACLT) mouse model. To determine which TNFR was predominantly responsible for Atsttrin’s effect, we established the ACLT mouse model in age-matched WT, TNFR1
–/–, or TNFR2
–/– male mice (
n = 6, respectively). To address the preventative potential of Atsttrin in OA, we intraarticularly injected 6 μl Atsttrin or PBS once a week for 4 weeks beginning on the day of surgery, as based on our previous study [
16]. For examination of Atsttrin’s therapeutic effect, 6 μl Atsttrin or PBS were intraarticularly injected once a week for 4 weeks beginning 4 weeks postoperatively, as based on our previous study [
16]. Ambulatory behavior of mice was monitored and recorded throughout the study. After 4 weeks of treatment, mice were sacrificed for dorsal root ganglia (DRG) harvest and histological evaluation.
Sandwich ELISA for cartilage oligomeric matrix protein
The serum concentration of cartilage oligomeric matrix protein (COMP) was analyzed by our established sandwich ELISA [
21]. Protein A agarose (Invitrogen) purified rabbit anti-COMP pAb and anti-COMP type III mAb 2127F5B6 were used as capture and detection antibodies, respectively. Anti-COMP type III mAb 2127F5B6 was labeled with horseradish peroxidase (HRP) using the Lightning-Link™ Horseradish Peroxidase Labeling Kit (Innova) as per the manufacturer’s protocol. Results were interpreted based on the linear range of the standard curve. All of the samples were assayed in triplicate.
Primary cultures of chondrocytes
Human articular chondrocytes were harvested by enzymatic digestion in accordance with established methodology [
16]. Briefly, human cartilage slices were cut into small pieces and washed several times with PBS, pH 7.4. Minced tissues were incubated with agitation in digestion medium comprised of 0.25% collagenase II in DMEM medium with 5% FBS in a spinner flask for 16 hours at 37 °C with 5% CO
2. After digestion, the suspended cells were collected and seeded into six-well plates for subsequent study. For mouse primary chondrocyte culture, knee joints were collected from 6-day-old WT, TNFR1
–/–, or TNFR2
–/– mice following sacrifice. Under magnification, the cartilage samples were isolated with special attention to avoid damaging the subchondral bone and tissues were rinsed completely three times in PBS. Primary cartilage samples were placed in a 10-cm dish containing the aforementioned digestion buffer and incubated for 16 hours at 37 °C with 5% CO
2. After full digestion, suspended cells were collected and seeded in a six-well plate for use. All chondrocytes used for experiments are first-generation cells.
von Frey test
von Frey filaments (Stoelting, Wood Dale, IL, USA) were applied with ascending force intensities on the plantar surface of the hind paw to determine the tactile pain threshold as based on a previous publication [
22]. Rapid withdrawal of the hind paw was recorded as a positive response. Hind paws were subjected to 10 trials at a given intensity with a 30-second interval maintained between trials. The number of positive responses for each von Frey filament’s stimulus was recorded. Animals were considered to have reached tactile threshold when five in 10 trials generated a positive response. The examiner was blinded to the groups.
Dorsal root ganglia isolation
Eight weeks after ACLT surgery, mice were sacrificed and L3–L5 DRG were isolated based on a previous publication [
23]. Briefly, mice were anesthetized using isoflurane and fur was cleared from the dorsal surface. A longitudinal incision was made, the spinal column was exposed, and L3–L5 DRG were extracted and tissues flash-frozen using liquid nitrogen. These tissues were processed using the Qiagen RNeasy kit (Qiagen, Valencia, CA, USA) for RNA extraction.
Luciferase assay
Luciferase assay was performed as reported previously [
24]. Lipofectamine2000 DNA transfection reagent was used to cotransfect NF-κB and renilla plasmids in C28I2 cells according to the manufacturer’s protocol (Life Technologies). Eighteen hours after transfection, C28I2 cells were treated without or with 10 ng/ml TNFα in the absence or presence of 200 ng/ml recombinant Atsttrin. After 24-hour incubation, we measured luciferase activity using the Reporter Assay System of Dual-Luciferase® in accordance with the manufacturer's instructions (Promega).
Histological analysis and immunostaining
Histological analysis was conducted as described previously [
16]. Briefly, knee joints were fixed immediately after sacrifice in 4% PFA at room temperature for 48 hours. After washing three times in PBS, the tissues were decalcified at 4 °C in 10% w/v EDTA for 2 weeks. Tissues were measured using a vernier caliper before paraffin processing. Knee joints were dehydrated and embedded; the blocks were trimmed to the midpoint as calculated previously from caliper measurements and serial 5-μm sections were placed on slides for staining. H&E or Safranin O/fast green staining was performed following the established protocol. The extent of synovitis was determined using a graded scale based on H&E staining: grade 0, no signs of inflammation; grade 1, mild inflammation with hyperplasia of the synovial lining without cartilage destruction; and grades 2–4, increasing degrees of inflammatory cell infiltrate and cartilage/bone destruction. For immunohistochemistry staining, sections were pretreated with 0.1% trypsin for 30 minutes at 37 °C. Sections were washed with PBS three times, followed by treatment with 0.25 U/ml chondroitinase ABC (Sigma-Aldrich) for 1 hour and then 1 U/ml hyaluronidase (Sigma-Aldrich) for 1 hour at 37 °C. To reduce nonspecific staining, sections were blocked at room temperature with 20% normal horse serum diluted in 3% BSA for 1 hour. Without washing after blocking, Col X antibody (1:200 dilution; DSHB), MMP-13 antibody (ab3208, 1:200 dilution; Abcam), and affinity-purified monoclonal anti-COMP fragments (1:200 dilution) were diluted in 20% normal horse serum with 3% BSA at 4 °C overnight. Sections were prepared for detection using the Vectastain Elite ABC kit following the manufacturer’s guidelines at 25 °C for 1 hour. Immunorecativity was visualized using 0.5 mg/ml 3,3′-diaminobenzidine (DAB) in 50 mM Tris–HCl substrate, pH 7.8. Methyl green (1%) was used for counterstaining.
Histological analysis and score
The articular cartilage proteoglycan content was defined on the basis of Safranin O staining. In this study, we used the well-accepted Osteoarthritis Research Society International (OARSI) scoring system [
25]: 0 = normal cartilage without any damage; 0.5 = loss of Safranin O staining with no detectable structural change; 1 = small fibrillation; 2 = vertical damage of cartilage limited to superficial layer; 3 = vertical damage, no more than 25% of the cartilage surface; 4 = vertical damage, 25–50% of the cartilage surface; 5 = vertical damage, 50–75% of the cartilage surface; and 6 = vertical damage, more than 75% of the cartilage surface.
Real-time RT-PCR
Total RNA were extracted from cultured chondrocytes using the RNeasy kit (Qiagen) and reverse transcribed into cDNA using the ImProm-II reverse transcription system (Promega). Data were normalized to the internal control, GAPDH. The primers for specific amplification of murine genes are as follows: 5′-AATGCTGGTACTCCAAACCC-3′ and 5′-CTGGATCGTTATCCAGCAAACAGC-3′ for Aggrecan; 5′-ACTAGTCATCCAGCAAACAGCCAGG-3′ and 5′-TTGGCTTTGGGAAGAGAC-3′ for Col II; 5′-AATCTCACAGCAGCACATCA-3′ and 5′-AAGGTGCTCATGTCCTCATC-3′ for IL-1β; 5′-ACAGGAGGGGTTAAAGCTGC-3′ and 5′-TTGTCTCCAAGGGACCAGG-3′ for NOS-2; 5′-GCATTGACGCATCCAAACCC-3′ and 5′-CGTGGTAGGTCCAGCAAACAGTTAC-3′ for ADAMTS-4; 5′-ACTTTGTTGCCAATTCCAGG-3′ and 5′-TTTGAGAACACGGGGAAGAC-3′ for MMP-13; 5′-CATAGCAGCCACCTTCATTCC-3′ and 5′-TCTCCTTGGCCACAATGGTC-3′ for MCP-1; 5′-AGAGAGCTGCAGCAAAAAGG-3′ and 5′-GGAAAGAGGCAGTTGCAAAG-3′ for CCR-2; and 5′-AGAACATCATCCCTGCATCC-3′ and 5′-AGTTGCTGTTGAAGTCGC-3′ for GAPDH. Melting curve analysis was used to verify the PCR product. Each experiment was repeated three times.
Western blot analysis
Proteins extracted from chondrocytes were processed on 8% SDS-polyacrylamide gel, followed by electrotransfer to nitrocellulose membrane. The membrane was blocked in 3% BSA in 10 mM Tris–HCl, pH 8.0, 150 mM NaCl, and 0.5% Tween 20. After washing three times, blots were incubated at 4 °C overnight with polyclonal anti-Erk1/2 (#4695, 1:1000 dilution; Cell Signaling Technology), anti-phosphorylated Erk1/2 (#4370S, 1:1000 dilution; Cell Signaling Technology), polyclonal anti-Akt (#9272, 1:1000 dilution; Cell Signaling Technology), anti-phosphorylated Akt (#4058S, 1:1000 dilution; Cell Signaling Technology), polyclonal anti-MMP-3 (ab52915, 1:1000 dilution; Abcam), polyclonal anti-MMP-13 (ab3208, 1:1000 dilution; Abcam), polyclonal anti-ADAMTS-4 (PA1-1750, 1:1000 dilution; Thermo Fisher Scientific), polyclonal anti-NOS-2 (SC651, 1:1000 dilution; Santa Cruz Biotechnology), polyclonal anti-GAPDH (SC25778, 1:1000 dilution; Santa Cruz Biotechnology), polyclonal anti-tubulin (#5346, 1:1000 dilution; Cell Signaling Technology), or diluted polyclonal anti-lamin B (SC-6217, 1:500 dilution; Santa Cruz Biotechnology). After washing three times, blots were incubated with an appropriate HRP-conjugated anti-rabbit/mouse immunoglobulin secondary antibody at 25 °C for 1 hour. The bound antibody was visualized using an enhanced chemiluminescence system (Amersham Life Science, Arlington Heights, IL, USA).
Cartilage explant cultures
Cartilage explants were cultured as described previously [
16]. Briefly, mouse femoral head cartilage was isolated and finely minced to 1 mm diameter and 1 mm thickness. The cartilage explants were then dispensed into serum-free DMEM containing 25 mM HEPES and 2 mM glutamine, in the absence or presence of recombinant Atsttrin (200 ng/ml).
Dimethylmethylene blue assay of GAG
The mouse cartilage culture medium was collected and GAG release was quantified by dimethylmethylene blue assay (DMMB) (Polysciences, Warrington, PA, USA). Hyaluronidase (0.5 unit/ml; Seikagaku, Tokyo, Japan) was incubated with collected medium for 3 hours at 37 °C to remove hyaluronan in order to reduce inhibition of the DMMB assay. The DMMB signal from digests was measured at 520 nm using a SpectraMax 384 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). The GAG content was calculated based on linear regression of readings from chondroitin-6-sulfate standards from Shark cartilage (Sigma Aldrich, St. Louis, MO, USA). Each sample was read in triplicate. The average values of the triplicates were normalized to the standard curve.
Statistical analysis
Results were expressed as mean ± SEM. Statistical analysis included Student’s t test performed by SPSS software (SPSS Inc., Chicago, IL, USA). p < 0.05 was considered statistically significant.
Discussion
OA is one of the most common joint diseases; however, the exact pathological mechanism of OA is still largely unclear [
34]. Unfortunately, no drugs are able to prevent or halt the progression of OA [
35]. Regardless of the complicated etiology of OA, it is well accepted that cytokines are closely involved in initiating and aggravating OA. Our genome-wide screen found that PGRN was an OA-related growth factor [
6]; levels of PGRN were also significantly elevated in the joints of arthritic patients [
14]. The finding that PGRN deficiency accelerated OA while recombinant PGRN ameliorated OA prompted us to determine whether the PGRN-derived engineered protein Atsttrin could rescue enhanced OA brought on by PGRN deficiency. In the present study, we took advantage of the DMM model in WT and PGRN
–/– mice and found that Atsttrin effectively prevented PGRN deficiency-mediated OA, evidenced by less cartilage destruction and reduced serum level of COMP fragments. In our previous study, we found that, compared with WT littermates, PGRN deficiency led to more severe synovium inflammation as well as osteophyte formation. In line with this finding, Atsttrin could effectively reduce synovium inflammation. However, we failed to observe osteophyte formation in our current model in either PBS-treated or Atsttrin-treated mice, which may be attributable to the duration of our model. Observation and analysis of the osteophyte formation phenotype may require an extended time course.
The surgically induced mouse model is a well-accepted method to investigate OA pathogenesis in vivo [
36], whereas the noninvasive rat model can mimic closed-joint injury-induced OA [
20,
37]. Our finding that intra-articular delivery of recombinant Atsttrin could dramatically prevent cartilage destruction in both mouse and rat OA models is in line with reports that local delivery of Atsttrin-transduced MSCs is protective against OA-related cartilage destruction in vivo [
18]. OA is also characterized by progressive loss of extracellular matrix, leading to the breakdown of articular cartilage [
38]. COMP is a noncollagenous molecule of the extracellular matrix in articular cartilage and plays an important role in maintaining chondrocyte function and cartilage integrity. COMP is fragmented upon degradation and the elevated serum level of COMP fragments observed in the progression of OA is considered a biomarker of disease activity [
39]. Previously, we showed that Atsttrin dramatically inhibited COMP degradation in animal models of inflammatory arthritis [
6]. In the current study, we found that Atsttrin also prevented COMP degradation in OA progression. Furthermore, Atsttrin dramatically reduced MMP-13 expression, which is significantly increased in OA and is thought to be the major enzyme responsible for digesting major cartilage component Col II.
Underlying articular cartilage, subchondral bone provides nourishment for cartilage and subchondral bone deterioration is often observed alongside cartilage defects [
40]. Previous reports demonstrate that the thickness of subchondral bone gradually decreases during the early phase of the ACLT mouse model; in the late phase, the thickness of subchondral bone gradually increases but does not return to a thickness representative of a normal joint [
27]. Additionally, studies have indicated that molecules targeting subchondral bone demonstrate a therapeutic effect in OA [
30]. In the present study, we found that Atsttrin effectively inhibited subchondral bone loss. Notably, osteoclastogenesis plays an important role in subchondral bone remodeling in OA [
30] and we have shown previously that Atsttrin significantly inhibited osteoclastogenesis in vitro [
6]. Here, we report that Atsttrin also inhibited osteoclast activity in OA progression, ultimately lending to preservation of subchondral bone.
Besides pathological changes, the OA-related pain and biomechanical dysfunctions are the major complaints of patients [
41] and OA pain is associated with pathological structural severity [
42]. It is believed that anti-inflammatory drugs could reduce pain in the pathogenesis of OA [
43,
44]. Herein, we found that Atsttrin effectively relieved OA-associated pain by improving travel distance and tactile sensitivity in mice with experimental OA. Additionally, alteration of pain markers and inflammatory molecules in the sensory neurons of the DRG has been reported as a result of interactions between neuropathic pathways and OA tissues [
22]. Here we found the levels of mRNA for proinflammatory cytokines in DRG were also significantly suppressed by Atsttrin in OA progression. Besides, a recent study indicated that PGRN could attenuate pain by binding to ATG12 and regulating autophagy [
45]. Whether Atsttrin also functions through this mechanism needs further investigation.
There are two distinct receptors for TNFα: TNFR1 and TNFR2 [
46]. TNFα/TNFR1 signaling is the classical pathway and is thought to mediate inflammatory signaling [
47]. On the contrary, TNFR2 signaling is still largely unknown [
8]. Recent studies indicate that TNFR2 activates protective and proliferative pathways [
48,
49]. Specifically, studies indicate that TNFR2 effectively protects against TNFα-mediated heart failure [
50]. Additionally, TNFR2 has been shown to promote cancer growth [
51]. Furthermore, TNFR2 was found to be required for PGRN-mediated immune regulation, cartilage homeostasis, physiological bone formation, and inhibition of LPS-induced lung inflammation [
52]. Atsttrin exhibited much higher affinity for TNFR2, when compared to TNFα [
53]. Here, we found that Atsttrin’s protective effect against OA primarily depends on TNFR2, although inhibition of TNFR1 inflammatory signaling also partially accounts for Atsttrin’s therapeutic action in OA.
It is known that Erk1/2 and Akt signaling are involved in chondrocyte protection, and that PGRN slightly activates Akt signaling and strongly actives Erk1/2 signaling [
13]. In addition, PGRN-mediated chondroprotective effect primarily depends on TNFR2-Erk1/2 signaling [
16]. Although Atsttrin is derived from PGRN, it exhibits distinct signal activation patterns. In the present study, we found that Atsttrin strongly activates Akt signaling whereas Erk1/2 signaling is only slightly activated. This signaling activation was lost in TNFR2-deficient chondrocytes but maintained in TNFR1-deficient chondrocytes, which implies that TNFR2 plays a major role in mediating Atsttrin’s proanabolic effects. Furthermore, after applying specific inhibitors of Akt and Erk1/2 signaling, Atsttrin completely lost its proanabolic effect. Thus, together with in-vivo data, we found that the Atsttrin-mediated anabolic effect in OA depends on TNFR2-Akt/Erk1/2 signaling.
As an important proinflammatory cytokine, upregulated TNFα directly promotes inflammatory reactions and triggers chondrocyte death in OA [
54]. Furthermore, TNFα-induced metalloproteinases, such as ADAMTS-4, ADAMTS-7, as well as ADAMTS-12, degrade cartilage matrix, leading to deteriorative articular cartilage [
55]. TNFα inhibitors demonstrate a therapeutic effect in murine models [
56] and OA patients in clinical trials [
57,
58]. Additionally, a report from another group showed that Atsttrin promoted bone healing through TNF/TNFR signaling [
59]. In the present study, the Atsttrin-mediated chondroprotective effect occurred partially through TNFR1 in vivo. Additionally, a mechanistic study demonstrated that Atsttrin effectively inhibited TNFα-induced inflammatory catabolism in OA progression. Specifically, Atsttrin effectively inhibited TNFα-induced NF-κB phosphorylation, translocation, and activity and the expression of inflammatory catabolic markers such as NOS-2, COX2, as well as ADAMTS-4. Intriguingly, a recent study revealed that DR3, the highest homolog of TNFR1 in the TNFR family, is also capable of binding Atsttrin [
60]. TNF-like ligand 1A (TL1A) is the sole identified ligand for DR3. Importantly, Atsttrin could inhibit the interaction between DR3 and TL1A [
60]. Thus, whether the Atsttrin-mediated chondroprotective effect partially depends on the DR3 pathway needs to be further investigated.
Although Atsttrin was shown to be more effective than PGRN in preventing inflammatory arthritis [
6,
10], we did not find a significant difference between PGRN and Atsttrin in terms of protecting against OA development. Based on our previous studies, we surmise that this disease specificity results from the fact that Atsttrin has a better anti-inflammatory/anti-catabolic TNFα/TNFR1 effect, whereas PGRN has a better effect in activating the anabolic TNFR2 pathway.
It is noted that we used a similar OA model with the 2-month-old mice as we did in our previous publication [
16]. Atsttrin’s long-term chondroprotective effect in various animal models, including DMM model mice 4–6 months old and "aged" PGRN-deficient mice that spontaneously develop an OA-like phenotype [
16], warrants further investigation. We have shown that Atsttrin rescued the accelerated surgically induced OA phenotype seen in PGRN-deficient mice (Fig.
1) and we anticipate that Atsttrin would also be effective in preventing spontaneous OA in "aged" PGRN-deficient mice.