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Superficial zone protein (SZP)/lubricin/PRG4 functions as a boundary lubricant in articular cartilage to decrease friction and wear. As articular cartilage lubrication is critical for normal joint function, the accumulation of SZP at the surface of cartilage is important for joint homeostasis. Recently, a heterocyclic compound called kartogenin (KGN) was found to induce chondrogenic differentiation and enhance mRNA expression of lubricin. The objective of this study was to determine whether KGN can stimulate synthesis of SZP in superficial zone, articular chondrocytes.
We investigated the effects of KGN and transforming growth factor-β1 (TGF-β1) on articular cartilage and synovium of the bovine knee joint by evaluating SZP secretion by enzyme-linked immunosorbent assay analysis. Monolayer, micromass, and explant cultures of articular cartilage, and monolayer culture of synoviocytes, were treated with KGN. SZP accumulation in the medium was evaluated and mRNA expression was measured through quantitative polymerase chain reaction.
TGF-β1 stimulated SZP secretion by superficial zone chondrocytes in monolayer, explant, and micromass cultures as expected. In addition, SZP secretion was inhibited by IL-1β in explant cultures, and enhanced by TGF-β1 in synoviocyte monolayer cultures. Although KGN elicited a 1.2-fold increase in SZP mRNA expression in combination with TGF-β1, KGN neither stimulated any significant increases in SZP synthesis nor prevented catabolic decreases in SZP production from IL-1β.
These data suggest that the chondrogenic effects of KGN depend on cellular phenotype and differentiation status, as KGN did not alter SZP synthesis in differentiated, superficial zone articular chondrocytes.
Osteoarthritis (OA) is a disease involving degradation of articular cartilage with attendant pain and impaired mobility. It is reported that OA affects more than 27 million people in the United States (2005) and it is estimated that by the age of 65 years, 80% of the U.S. population will exhibit radiographic evidence of OA.1 Normal articular cartilage on the ends of long bones in diarthrodial joints maintains a well-lubricated surface with an extremely low coefficient of friction for optimal joint mobility.2 Weight bearing of loads up to 18 MPa is accomplished through an extracellular matrix architecture that combines the compressive properties of hydrated glycosaminoglycans (GAG) with the tensile strength of crosslinked type II collagen fibrils.3 Since articular cartilage is an avascular tissue with limited, innate potential for repair and regeneration,4 several therapeutic strategies such as autologous chondrocyte implantation, mosaicplasty, and microfracture are used to repair articular cartilage. However, these treatments fail to regenerate tissue that resembles native articular cartilage, with zonal organization and functional lubrication.5 Articular cartilage is an anisotropic structure with a zonal design and consists of surface or superficial, middle, and deep zones. Superficial zone protein (SZP), also known as lubricin or proteoglycan 4 (PRG4), is an ~345 kDa glycoprotein that is secreted into synovial fluid by surface zone chondrocytes and synoviocytes.6,7 SZP localized in the superficial zone of articular cartilage reduces the coefficient of friction and wear in articular cartilage through a sacrificial, boundary lubrication mechanism.8-10 Moreover, mutation of the SZP gene, prg4, has been linked to camptodactyly-arthropathy-coxa vara-pericarditis (CACP) syndrome, an autosomal recessive disease characterized by synovial hyperplasia, fouling of the articular surface, and precocious joint failure.11 Mice lacking functional copies of the prg4 gene also demonstrated precocious failure of joint function,12 leading to OA.13 Therefore, SZP plays an important role in cartilage homeostasis. Given that superficial zone chondrocytes and synovium are the predominant sources of SZP in the synovial joint, we previously evaluated SZP production by protein analysis in various culture systems of cartilage and synovium, such as monolayer,7,14,15 micromass,16 explant,17-19 and pellet cultures.20
Recently, Johnson et al.21 reported the discovery of a heterocyclic compound, kartogenin (KGN), from 22,000 structurally diverse, drug-like molecules using a high-throughput screening system. This small molecule binds to the carboxyl end of filamin A and disrupts its interaction with the transcription factor, core-binding factor β subunit (CBFβ), and induces chondrogenic differentiation by regulating the CBFβ-RUNX1 transcriptional program.21 KGN has been observed to enhance the expression of lubricin, aggrecan, and type II collagen in human mesenchymal stem cells (MSCs), and to have a chondroprotective, anti-catabolic effect in both in vivo and in vitro studies.22-24
It is well established that morphogens, such as bone morphogenetic protein-7 (BMP-7) and transforming growth factor–β1 (TGF-β1), and cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor–α (TNF-α), play important roles in regulating SZP production.15,17,25 For example, TGF-β1 stimulates SZP production while IL-1β inhibits it. As SZP is secreted extracellularly into the synovial fluid in vivo, SZP synthesis is commonly measured by accumulation in the culture media in vitro.6 Motivated by previously published reports of KGN, the first aim of this investigation was to investigate the effect of KGN on SZP secretion in different tissue culture platforms such as monolayer, micromass, and cartilage explant cultures. The second objective was to investigate any potential interactions between KGN and the aforementioned regulatory cytokines, TGF-β1 and IL-1β, in these culture systems.
Stifle (knee) joints from 3-month-old calves were obtained from Research 87 (Boston, MA). The joints were dissected under aseptic conditions, exposing the femoral condyles and the suprapatellar pouch.
Surface zone, bovine articular chondrocytes were obtained as described previously.15 Briefly, the surface zone of the articular cartilage was harvested from the anterior half of the lateral and medial femoral condyles (~100 µm thick) using a dermatome. The articular cartilage from 6 to 8 bovine stifle joints from different animals were pooled. Then, osteochondral plugs were removed from the same femoral condyles using a coring reamer (Acuderm). Middle zone cartilage (1.25 mm slices) was removed from each plug using a custom, cutting jig. The synovium was harvested from the suprapatellar pouch. The cartilage slices and synovium were divided into small pieces with a razor blade, and digested with 0.2% collagenase P (Roche) in Dulbecco’s modified Eagle medium (DMEM)/F12 (Gibco, Carlsbad, CA, USA) along with a nutrient mixture of 50 μg/mL ascorbate-2-phosphate (Sigma-Aldrich, St. Louis, MO, USA), 0.1% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA), and antibiotics (Medium-A) with 3% fetal bovine serum (FBS; Gibco) for 2 hours at 37°C. Cells released from the tissues were filtered through a cell strainer (70 μm; BD Biosciences, San Jose, CA, USA) and rinsed with DMEM/F12. The isolated chondrocytes or synovium-derived cells (synoviocytes) were plated in a monolayer at a density of 1 × 105 cells/well, in 12-well culture plates (Corning, NY, USA) in Medium-A with 10% FBS, and incubated at 37°C in a humid atmosphere of 5% CO2 and 95% air. Following 24-hour equilibration in the culture medium, the medium was changed to fresh Medium-A with 1% ITS+ Premix (insulin-transferrin-selenium + linoleic acid; BD Biosciences) containing various concentrations of KGN (Tocris, Minneapolis, MN, USA) in the presence and absence of 3 ng/mL TGF-β1 (R&D Systems, Minneapolis, MN, USA). All control cell cultures received an equal volume of the equivalent vehicle solution at the time of treatment. Cells were cultured for periods of 4 and 7 days. The culture media were collected and stored at −80°C for later analysis of SZP accumulation.
Isolated chondrocytes were suspended at a high density of 1 × 105 cells/20 μL in Medium-A with 10% FBS, and incubated at 37°C in a humid atmosphere of 5% CO2 and 95% air.16,26 A 20 μL drop of cell suspension was gently placed in the center of each well of a 12-well plate. After waiting for 90 minutes, 1 mL of Medium-A with 10% FBS was slowly added to each well.16,26 Following overnight equilibration, the medium was changed to fresh Medium-A with 1% ITS+ Premix in the presence and absence of 3 ng/mL TGF-β1 and 10 µM KGN. The culture media were collected and stored at −80°C for later analysis of SZP accumulation. Cultures were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde (Sigma Aldrich), in PBS for 3 hours. After rinsing in 3% glacial acetic acid (pH 1) 3 times, cultures were stained overnight in 0.5% Alcian blue (Sigma-Aldrich) in 0.1 N HCl (pH 1), and then destained in 3% glacial acetic acid.
Matched pairs were obtained from 8 different bovine stifle joints for cartilage explant cultures. Four osteochondral plugs were obtained in close proximity, using a 5 mm diameter coring reamer. Because of the limited size of juvenile bovine stifle joints, cartilage explants were obtained and utilized as a group from either the anterior region of the medial femoral condyle or trochlear groove, in order to conserve the number of joints employed. Comparable levels of SZP synthesis from these 2 locations have been reported.27 From these 5 mm diameter cartilage disks, 2 mm diameter disks from the surface were obtained using an adjustable, custom-made jig. Explants were randomly assigned to control or treatment groups, pairwise. Cartilage disks (2 mm diameter) were submersed and equilibrated at 1 disk per 2 mL medium per well in 12-well culture plates in Medium-A with 1% ITS+ Premix, and incubated at 37°C in a moist atmosphere of 5% carbon dioxide and 95% air. The medium was replaced after a 24 hour equilibration period. Subsequently, the explants were switched to fresh Medium-A with 1% ITS+ Premix. Explants were pretreated with or without 10 µM KGN for 2 hours before the addition of 10 ng/mL TGF-β1 or 10 ng/mL IL-1β (R&D Systems). Explants obtained from the medial femoral condyle were used for TGF-β1 treatment, while explants obtained from the trochlear groove were used for IL-1β treatment. All non-treated explant cultures received an equal volume of the equivalent vehicle solution at the time of treatment. Explants were cultured for 72 hours and the culture media were collected and stored at −80°C for later analysis of SZP accumulation.
All KGN used in this study was obtained from a single lot, and was dissolved in dimethyl sulfoxide at a concentration of 10 mM. Recombinant, human TGF-β1 was reconstituted in a filter sterilized solution of 0.1% BSA in 5 mM hydrochloric acid at 100 µg/mL. Recombinant human IL-1β was reconstituted in a filter sterilized solution of 0.1% BSA in PBS at 10 µg/mL. Stock concentrations of all reagents were prepared and stored at −20°C, until they were diluted in culture media to their final concentrations.
Since the majority of SZP is secreted into the culture medium,5 the media was collected after treatment and SZP secretion was measured by an enzyme-linked immunosorbent assay (ELISA) with purified, bovine SZP as a standard.7 Briefly, each well of 96-well MaxiSorp plates (Nalge Nunc; Penfield, NY, USA) was coated with 1 μg/mL peanut lectin (EY Laboratories; San Mateo, CA, USA) in 50 mM sodium carbonate buffer (pH 9.5). The wells were then blocked with 1% BSA in the same buffer for at least 2 hours. Aliquots of culture medium were incubated in the wells. Thereafter, the wells were incubated overnight with mAb S6.79 (1:5,000; a generous gift from Dr. T. Schmid, Rush Medical College, Chicago, IL, USA) as the primary antibody at 4°C,28 and then incubated at room temperature for 1 hour with goat anti-mouse IgG conjugated with horseradish peroxidase (1:3000; Bio-Rad; Hercules, CA, USA) as the secondary antibody. SuperSignal ELISA Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific; Waltham, MA, USA) was added, and the results were quantified in a luminometer. The wells were washed with PBS containing 0.05% Tween 20 after each step. SZP concentrations were calculated using a bovine SZP standard, which was purified by affinity chromatography on a peanut lectin column. Purity was verified by immunoblot analysis. The concentration of the SZP standard was quantified using a Micro BCA Protein Assay Kit (Thermo Fisher Scientific).7,14
For immunoblot analysis, equal quantities of harvested culture medium were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% nonfat dry milk in TBST (25 mM Tris HCl, 125 mM NaCl, and 0.1% Tween 20) for 1 hour and incubated overnight at 4°C with the primary antibody mAb S6.79 at a dilution of 1:5,000. The membranes were then incubated for 1 hour at 4°C with a horse radish peroxidase–conjugated secondary antibody at a dilution of 1:3,000 (Bio-Rad), followed by a 1 minute incubation with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) for visualization.
Messenger RNA (mRNA) expression levels were measured by quantitative polymerase chain reaction (q-PCR) according to the manufacturers’ protocols. Briefly, total RNA was isolated from monolayer cell cultures using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Genomic DNA was removed through an on-column DNase I (Qiagen) digestion. Complementary DNA (cDNA) was reverse transcribed from mRNA using the SuperScript First Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). q-PCR was utilized for relative quantification of the cDNA using SYBR Green PCR Master Mix on the ABI 7900HT Fast Real-Time PCR System (Applied Biosystems; Foster City, CA, USA). SZP (prg4), collagen II (col2a1), aggrecan (acan), and Sox9 (sox9) expression levels (Ct) were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (ΔCt), computed relative to the untreated (i.e., control) group (ΔΔCt), and presented in terms of fold change (2−ΔΔCt).29 Primers for bovine SZP(Forward:5′-AGAAAACCCGATGGCTATGA-3′; Reverse: 5′-TCGCCCATCAGTCTAAGGAC-3′), collagen II(Forward:5′-GCATTGCCTACCTGGACGAA-3′; Reverse: 5′-CGTTGGAGCCCTGGATGA-3′), aggrecan (Forward:5′-GCGGGTGCGGGTCAA-3′;Reverse: 5′-TAGAATCCCGGAGTCATTGGA-3′), Sox9 (Forward: 5′-ACGCCGAGCTCAGCAAGA-3′;Reverse:5′-CACGAACGGCCGCTTCT-3′),andGAPDH(Forward: 5′-GGCGCCAAGAGGGTCAT-3′;Reverse:5′-GTGGTTCACGCCCATCACA-3′) were designed through Primer Express (Applied Biosystems) to span at least 1 intron and prevent nonspecific fluorescence arising from contaminating genomic DNA.14,30
For all monolayer and micromass cultures (n = 6), a 1-way analysis of variance (ANOVA) was used. Protein measurements were evaluated by the Tukey-Kramer honestly significant difference post hoc test. Groups not connected by the same letter were determined to be significantly different. Gene expression levels were assessed using the Dunnett’s method post hoc test; groups significantly different than the untreated control were designated with an asterisk (*). Articular cartilage explant cultures (n = 8) were evaluated using a 2-way ANOVA to assess each treatment group as a factor, and utilized the Student t test as a post hoc test (2 treatment levels per factor). P-values less than 0.05 were considered statistically significant. All statistical analyses were performed using JMP 12 (SAS Institute, Cary, NC, USA). Data are presented as the mean ± standard deviation (SD).
After 4 days of monolayer culture, untreated superficial zone chondrocytes were flattened and acquired a spread, fibroblastic morphology ( Fig. 1A ). On the other hand, the vast majority of middle zone chondrocytes maintained a round cell shape ( Fig. 1B ). Compared with the No Growth Factor group, TGF-β1 treatment promoted increased cell spreading in superficial zone chondrocytes; whereas no differences were observed in middle zone chondrocytes with TGF-β1 treatment. No morphological alterations from treatment with KGN (10 nM, 100 nM, 1 µM) were observed during culture in both superficial and middle zone chondrocytes.
TGF-β1 treatment enhanced SZP secretion significantly (P < 0.0002) compared with No Growth Factor groups in monolayer cultures of superficial zone chondrocytes ( Fig. 2A ). KGN stimulated SZP accumulation neither in the No Growth Factor groups (0 nM KGN, 0.8 ± 0.3; 10 nM KGN, 0.8 ± 0.2; 100 nM KGN, 0.9 ± 0.3; and 1 µM KGN, 0.9 ± 0.3 μg/mL; P > 0.99) nor in TGF-β1-treated groups (0 nM KGN, 6.1 ± 1.9; 10 nM KGN, 4.8 ± 1.6; 100 nM KGN, 4.9 ± 1.6; and 1 µM KGN, 4.4 ± 1.2 μg/mL; P > 0.24), at all examined concentrations. In monolayer cultures of middle zone articular chondrocytes, no SZP was detected in the media as measured by immunoblot analysis (data not shown).
SZP mRNA expression was significantly upregulated (P < 0.05) 1.2-fold compared with non-treated control (0 μM KGN, No Growth Factor) by a combination of TGF-β1 and KGN, at KGN concentrations of 100 nM and 1 μM ( Fig. 2B ). Whereas TGF-β1-treated chondrocyte cultures experienced an upregulation of collagen II mRNA of over 2.7-fold relative to nontreated control ( Fig. 2C ), KGN appeared to elicit no response in collagen II expression. While mRNA expression of aggrecan ( Fig. 2D ) increased in response to 10 nM KGN in the presence of TGF-β1 (P < 0.0004), all other treatment combinations did not result in substantial changes (P > 0.19). Sox9 gene expression did not change (P > 0.08) in response to any of the applied treatments ( Fig. 2E ).
Alcian blue staining demonstrated that GAG production was enhanced in superficial ( Fig. 3A ) and middle ( Fig. 3B ) zone micromass cultures treated with the positive control treatment TGF-β1 (TGF-β1 and KGN + TGF-β1), irrespective of the presence of KGN. In addition, all middle zone micromass cultures displayed stronger GAG staining than their respective treatment groups in the superficial zone micromass cultures. KGN had no apparent effect on GAG accumulation in the presence or absence of TGF-β1. As in monolayer cultures, SZP secretion from superficial zone micromass cultures was increased by TGF-β1, regardless of KGN treatment. While a faint immunoblot band was observed in the No Growth Factor (No GF; i.e., control) treatment group of superficial zone micromass cultures ( Fig. 3A ), no SZP was detected in immunoblots of middle zone micromass cultures ( Fig. 3B ). As was the case with GAG accumulation, KGN elicited no changes in SZP synthesis in superficial zone micromass cultures.
SZP is known to be modulated by morphogens and cytokines.7,15,31,32 This abundant evidence strongly suggests that the stimulatory effects of TGF-β1 may be used as a positive control in experimental studies, as well as IL-1β as a negative control because of its inhibitory effects. Thus, cartilage explants were also treated with TGF-β1 ( Fig. 4A ) and IL-1β ( Fig. 4B ) in order to determine whether there were any interactions between KGN and these cytokines. A 2-way ANOVA revealed no significant interactions between KGN and neither TGF-β1 (P > 0.85) nor IL-1β (P > 0.83). The TGF-β1 treatment level was significantly greater (P < 0.017) than the No Cytokine treatment level ( Fig. 4A ). Similarly, the IL-1β treatment level was significantly lower (P < 0.05) than its respective No Cytokine level ( Fig. 4B ). In both cases, KGN had no effect (P > 0.12) as the 10 μM KGN level was not statistically different than the 0 μM KGN level.
In monolayer culture, synovial cells showed fibroblastic morphology regardless of treatment (data not shown). Although TGF-β1 treatment (0 μM KGN, 0.37 ± 0.16; 10 μM KGN, 0.32 ± 0.17 µg/mL) enhanced SZP accumulation significantly (P < 0.003) compared with No Growth Factor groups (0 μM KGN, 0.04 ± 0.02; 10 µM KGN, 0.03 ± 0.01 µg/mL) at Day 4 of culture, KGN had no effect (P > 0.89) on SZP synthesis from synoviocytes ( Fig. 5B ). The ELISA results were corroborated by immunoblot analysis ( Fig. 5A ).
The objective of this study was to determine the effects of KGN on SZP secretion in primary, superficial zone articular chondrocytes under different culture methods, and to investigate potential interactions between KGN and regulatory cytokines such as TGF-β1 and IL-1β. Using immunoblot and ELISA, KGN was determined to have no significant effect on altering SZP synthesis in superficial zone chondrocytes cultured in monolayer, micromass, and explant cultures. Synoviocytes, the other major contributor of SZP to boundary lubricants in the synovial fluid, were similarly unaffected by KGN. While bioactivity was confirmed through changes in SZP mRNA expression, KGN did not modulate SZP protein synthesis in combination with TGF-β1 or IL-1β treatments either. As KGN had no effect on SZP synthesis in primary, superficial zone articular chondrocytes, this evidence suggests that the observed, chondrogenic effects of KGN are dependent on cellular differentiation status.
Kartogenin was identified in 2012 as a small, heterocyclic molecule capable of stimulating chondrogenesis in primary, human MSCs.21 Chondrogenic activity was also observed in bovine articular cartilage explants, where KGN was found to reduce GAG release into media.21 Most interestingly, Johnson and colleagues21 noted that KGN enhanced the gene expression of lubricin/superficial zone protein. These observations motivated this current investigation to examine the response of SZP protein synthesis to KGN treatment in primary, bovine, superficial zone articular chondrocytes. In combination with TGF-β1, KGN enhanced gene expression of SZP in monolayer cultures at concentrations of 100 nM and 1 μM ( Fig. 2B ). However, this increased gene expression did not lead to any changes in protein synthesis, as measured by SZP secretion in monolayer ( Fig. 2A ), micromass ( Fig. 3A ), and cartilage explant ( Fig. 4 ) cultures. The corresponding data from these three different in vitro culture systems suggest that the culture system, or cell context, is an unlikely contributor to the observed results. Likewise, equivalent results were obtained from both predominant producers of SZP in the synovial joint, superficial zone articular chondrocytes ( Fig. 2A ) and synoviocytes ( Fig. 5 ). In light of the relative maturity of the cells examined, the chondrogenic effects of KGN may depend on cell naivety, with cell responsiveness to KGN decreasing with cellular differentiation and maturation. This inference regarding the effectiveness of KGN in relation to the potency and differentiation status of a cell will require additional study.
The dissimilar responses to KGN from progenitor and differentiated cells have been observed elsewhere in the literature. Decker and coworkers33 observed increased GAG accumulation in micromass cultures of embryonic, pre-skeletal MSCs in response to KGN. Additionally, KGN augmented GAG synthesis in leporine MSCs and patellar tendon stem/progenitor cells.34 However, Ono et al.22 found no changes in GAG from KGN treatment in tissue engineered, neocartilage disks produced from adult, bovine articular chondrocytes. Following this same pattern, KGN upregulated aggrecan, collagen II, and Sox9 expression in leporine MSCs.34 However, in alignment with our results ( Fig. 2C-E ), in human and bovine articular chondrocytes, KGN-induced changes in the expression of these genes were not observed by Ono et al.22 Investigating these cell differentiation state-dependent effects of KGN may yield important insights into KGN’s mechanism of action.
SZP/lubricin/PRG4 is gaining prominence as a marker of articular cartilage. In addition to collagen II, aggrecan, and Sox9, investigations in the literature frequently assay SZP gene expression as an indicator of the chondrogenic phenotype. Indeed, SZP is a hallmark of the superficial zone of articular cartilage as it functions as a boundary lubricant, reducing friction and wear at the articular surface.6 However, it is important to note that SZP is a marker at the glycoprotein level, and needs to be measured as such. While middle and deep zone articular chondrocytes express the prg4 gene,35,36 these zonal phenotypes do not synthesize and secrete measurable amounts of the glycoprotein.7,37 The expression of prg4 in middle and deep zone articular chondrocytes has an another important implication; mRNA expression levels of SZP do not correlate to protein synthesis. While this general concept is well established,38-40 the results of this investigation ( Fig. 2 ) and previous studies demonstrate that it applies to SZP as well.14 In summary, meaningful measurements of SZP/lubricin/PRG4 must be performed at the glycoprotein level as its mRNA expression values do not necessarily correspond to protein synthesis.
In conclusion, the story of KGN appears to be incomplete as much remains to be elucidated and described. KGN did not stimulate SZP synthesis in synoviocytes, and superficial zone articular chondrocytes cultured under multiple conditions. Based on this investigation, and others described in the literature, the chondrogenic effects of KGN appear to be attenuated in differentiated cell phenotypes, such as articular chondrocytes. However, these apparent differences between progenitor and differentiated cell types in response to KGN merit additional investigation since important new, mechanistic insights into the effects of KGN may be revealed.
Acknowledgments and Funding: The authors thank Dr. Thomas M. Schmid of Rush University for his gift of the antibody S6.79. This investigation was supported by the Lawrence J. Ellison Chair in Musculoskeletal Molecular Biology at the University of California, Davis, and the National Institute of Arthritis and Musculoskeletal and Skin Diseases at the National Institutes of Health under Award Number AR061496. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Ethical Approval: Ethical approval was not sought for the present study because neither living animals, nor humans, nor human tissues were used in the conduct of this research.
Animal Welfare: Guidelines for humane animal treatment did not apply to the present study because no living animals were involved. All tissues were obtained from an abattoir.