Retinoic acid (vitamin A1 acid) is an ester found in organic solvents, liver tissue, and richly green plants.1,2 It is derived from all-trans-retinol and has a terminal carboxyl group. Retinoic acid binds to the RA receptor and the retinoid X receptor where it functions as a ligand-inducible transcription factor to regulate the growth and delineation of cells.3,4 In this way, RA modifies gene expression, which is regulated by bio-availability and the balance of synthesis and degradation. Consequences of RA excess or deficiency can be severe, particularly during growth and development, because RA controls proliferation, differentiation, and apotosis of progenitor cells.1,2 Importantly, RA synthesis is dependent on ingested retinyl esters and β-carotene (provitamin A), which are converted to all-trans-retinol in the intestine, stored in the liver, and tissue activated to RA for local effects.2 Thus, dietary concentrations of RA can regulate body RA content and can also be used to treat RA deficiency.2
Retinoic acid has a dual role in chondrogenesis. It has induced hypertrophy and maturation of clonal TC6 cartilage-derived cells and differentiation of ATCD5 chondrogenic progenitor cells in vitro5–7; however, ligand-activated RA receptors can also inhibit chondrogenesis.8 Retinoic acid receptor antagonists cause an increase in chondrogenesis of prechondrogenic cells as well as prevent RA from inducing terminal chondrocyte maturation.7,9,10
Although an influence of RA on chondrogenesis is supported by the described in vitro studies in cell lines, the mechanism underlying RA regulation of chondrogenesis is not understood. However, the potential impact of RA in vivo is evidenced by the fact that RA-deficient mutant mice have a limb deformity attributable to growth plate and articular cartilage growth abnormality.1 Interestingly, horses commonly have an articular cartilage growth abnormality, OCD, that is defined as a derangement in the maturation and mineralization of cartilage.11–24 As such, it may be clinically relevant to study the effects of various concentrations of RA on primary equine chondrocytes and equine BMDMSCs and to investigate the effects of RA receptor antagonism on these cells in vitro.
Our laboratory group recently investigated the gene expression profiles of WBCs of horses with OCD of the distal aspect of the tibia or femoral trochlea.a In that study, significant (P < 0.002) downregulation of 2 retinoid-associated genes, RA receptor and retinoid-inducible serine carboxypeptidase, was detected. The RA receptor protein facilitates RA-mediated functions3,4; to date, the cellular functions and properties of retinoid-inducible serine carboxypeptidase, a lysosomal matrix protein, remain uncharacterized.25 The purpose of the study reported here was to determine the effects of various concentrations of RA and a synthetic RA receptor antagonist (LE135) on cultured equine chondrocytes and BMDMSCs exposed to chondrogenic stimuli, as measured by cellular proliferation, morphology, and gene expression. Our hypotheses were that RA would support chondrocyte morphology in chondrocytes and BMDMSCs and that LE135 would increase chondrocyte differentiation of equine BMDMSCs.
Materials and Methods
Sample collection—Articular cartilage was aseptically collected immediately after death from the femoropatellar joints of 3 clinically normal adult horses euthanized for reasons unrelated to this study. Bone marrow—derived mesenchymal stem cells were obtained aseptically from 2 clinically normal adult horses during a previous study26 and stored in a standard cryopreservative solution (90% fetal bovine serumb with 10% dimethyl sulfoxide) at −80°C until use. All procedures were approved by The Ohio State University Institutional Animal Use and Care Committee.
Culture and treatment of cells—Chondrocytes were isolated from articular cartilage samples following an 8-hour digestion in DMEM with 0.02% collagenase I added under standard cell culture conditions (37°C, 5% CO2, and 100% humidity). Chondrocytes were subsequently resuspended in DMEM supplemented with 10% heat-inactivated fetal bovine serum, L-glutamine (29.2 mg/mL), and 1% penicillin-streptomycin (ie, supplemented DMEM) and incubated under standard cell culture conditions for 4 days. These cells were used in experiments as primary expanded chondrocytes. Previously frozen first-passage BMDMSCs were rapidly thawed at 25°C, centrifuged for 1 minute at 489 × g, washed twice with Dulbecco PBS solution, and cultured in supplemented DMEM under standard cell culture conditions for 4 days. At 90% to 100% confluence, chondrocytes or BMDMSCs were treated with trypsin, resuspended in supplemented DMEM, seeded into eight 6-well plates/horse at a concentration of 200,000 cells/well, and cultured in monolayer under the described conditions.
After 48 hours, the medium for chondrocytes was changed to supplemented DMEM that contained RAc at concentrations of 0, 0.1, 1, or 10μM or contained LE135d at concentrations of 0, 0.1, 1, or 10μM (this was considered day 0). At the same point, the medium for BMDMSCs was changed to a commercially available chondrogenic mediume that included dexamethazone; ascorbate; an insulin, transferrin, and selenium solution with a manufacturer-supplied supplement; pyruvate; proline; an aqueous gentamicin sulfate and amphotericin-B solution; and L-glutamine; manufacturer-supplied rhTGF-β3 (20 μg/mL [1 μL in 2 mL of media]) was added to this solution (on day 0). Retinoic acid or LE135 was added to the chondrogenic medium as described for chondrocyte medium. Dimethyl sulfoxide was included in both types of media solutions at a final stock concentration of 1μM to dissolve the RA and LE135; all control cell cultures were also exposed to this concentration of dimethyl sulfoxide. All experiments were performed in triplicate for each horse for each time point. Media for all cultures were changed on days 4, 7, and 11, and used media were stored at −80°C. Cells were harvested on days 7 and 14.
DNA concentrations of cell cultures—Total DNA concentration for each cultured cell population was analyzed via a Hoescht 33258 fluorometric assay. Cells were treated with trypsin, harvested from the 6-well plates, and centrifuged at 489 × g for 4 minutes in 2-mL Eppendorf tubes. The supernatant was discarded, and cells were resuspended in 750 μL of papainf (dissolved at 125 μg/mL in sterile PBS solution with 5mM cysteine and 5 mM Na2EDTA) and incubated for 24 hours at 65°C. Digested samples were then centrifuged at 489 × g for 1 minute, and the supernatant was isolated. The DNA standards were created with double-stranded calf thymus DNAg dissolved in 1× buffer solution (TNE; 100 mM Tris, 2M NaCl, 10 mM EDTA; pH, 7.4). Serial dilutions were created to achieve final concentrations of 0.001 to 10 μg/mL. Fifty microliters of each papain-digested sample or standard was added to 150 μL of 0.2 μg/mL Hoescht 33258 dyeh and placed in triplicate in a 96-well assay plate.i The plates were read on a UV spectrometerj with an excitation of 360 nm, an emission of 460 nm, and a cutoff filter of 435 nm. The DNA content of the samples was determined via comparison with the standards.
Morphological assessment—To determine cellular morphology, media were removed and adherent cells in the 6-well plates were fixed with 800 μL of neutral-buffered 10% formalin for < 10 minutes. The formalin was poured off, and 50 μL of toluidine blue containing 1% ethanol was added to the wells and incubated for 20 minutes. The cells were gently rinsed with PBS solution until all excess stain was removed. Cells were examined via light microscopy and photographed at a final magnification of 100×. Chondrocytic morphology and features consistent with chondrogenesis (ie, chondral morphology) were assessed in chondrocytes and BMDMSCs, respectively, and were scored according to cell characteristics described during the formation and differentiation of cartilage.27,28 Samples were independently scored by 2 investigators (SEH and ALB) who were blinded to the source of each sample and to the treatments applied. A scale of 0 to 4 with 0.5-point increments was used for this assessment (0 = no chondral morphology [elongated, tapered, or spindle- or stellate-shaped cells, with relatively low cytoplasm-to-nucleus ratio; confluence often leads to mounding of cells]; 0.5 = < 25% of cells with early chondral morphology [cellular enlargement and rounded shape with greater cytoplasm-to-nucleus ratio]; 1 = 25% to < 50% of cells with early chondral morphology; 1.5 = 50% to 75% of cells with early chondral morphology; 2 = > 75% of cells with early chondral morphology; 2.5 = < 25% of cells with chondral morphology typical of a hypertrophied cell or chondroblast [large polyhedral-shaped cells in single-layer confluence with intercellular compression and a high cytoplasm-to-nucleus ratio]; 3 = 25% to < 50% of cells with chondral morphology typical of a hypertrophied cell or chondroblast; 3.5 = 50% to 75% of cells with chondral morphology typical of a hypertrophied cell or chondroblast; and 4 = > 75% of cells with chondral morphology typical of a hypertrophied cell or chondroblast). Hypertrophy was defined as an increase of the cell culture mass specifically caused by an increase or growth in cell size, as opposed to hyperplasia, defined as an increase of the cell culture mass caused by an increase in cell number.
Degenerate changes within cells were recorded but were not included in the scores. Cells with degenerate changes were defined as those that were shrunken, intensely stained (dense), irregular in shape, or lifted off the plate as a small round structure. Chondral morphology scores for cultured chondrocytes and BMDMSCs were not directly compared or correlated. The scores reflected minimal interevaluator variability, and both sets of scores were included in the statistical analyses.
Gene expression—Cells were directly lysed and removed from the 6-well plates with 1 mL of an RNA isolation reagentk/well and stored at −80°C for subsequent real-time RT-PCR analysis. Complementary DNA was made from total RNA by use of a commercial RT kit,l and real-time RT-PCR was performed by use of a PCR mixm according to the manufacturer's protocol with universal cycling parameters. Equine-specific primers and validated probes were used to amplify and detect CI, CII, and aggrecan cDNA in triplicate for each sample.26 A commercially available gene expression assay for eukaryotic 18s rRNA was run simultaneously and used as an endogenous control to which each gene of interest was normalized.n The fold change for gene expression, relative to that of untreated control samples, was calculated by use of the comparative CT (2−ΔΔCT) method; a CII:CI expression ratio was also determined for these normalized values.29
Statistical analysis—Objective data were expressed as mean ± SEM. Scored data were reported as median (range); when the median value was between 2 numbers, the value was reported to 1 decimal point. Total cellular DNA content was analyzed by use of a general linear model (repeated-measures ANOVA) for differences between cell type, dose, and day of culture followed by pairwise comparisons with Tukey 95% confidence intervals. Individual horses were considered a random effect nested within cell type. Comparisons between real-time RT-PCR data were analyzed via this same statistical process. Nonparametric scored data were analyzed by use of the Kruskal-Wallis 1-way ANOVA on ranks, followed by a Dunn post hoc test. All analyses were performed by use of a commercially available statistical software programo; values of P ≤ 0.05 were considered significant.
Results
DNA concentrations of cell cultures—Treatment with 0.1, 1, or 10μM RA resulted in a significant (P = 0.02) decrease in concentrations of DNA detected in chondrocyte cultures between days 7 and 14; on day 14, the concentration of DNA was also significantly (P < 0.05) decreased in chondrocyte cultures treated with 0.1μM RA, compared with that of untreated control chondrocytes (Figure 1). Treatment with LE135 had no effect on the DNA content of chondrocyte cultures. The values for cultures of untreated control chondrocytes remained similar between days 7 and 14.
Mean ± SEM DNA concentrations of equine chondrocytes (A) or BMDMSCs (B) in monolayer cultures treated with various concentrations of RA or the RA receptor antagonist LE135. The day of initial treatment was considered day 0. In panel A, treatment with 0.1, 1, or 10μM RA, but not with LE135, resulted in a significant (P = 0.02) decrease in DNA concentrations detected in chondrocyte cultures between days 7 and 14. In panel B, treatment of BMDMSCs with 1 or 10μM RA or with 10μM LE135 decreased DNA content relative to untreated controls. *Values are significantly (P < 0.05) different for treated cells, compared with those of untreated control cells on the same day.
Citation: American Journal of Veterinary Research 72, 7; 10.2460/ajvr.72.7.884
Mean ± SEM DNA concentrations of equine chondrocytes (A) or BMDMSCs (B) in monolayer cultures treated with various concentrations of RA or the RA receptor antagonist LE135. The day of initial treatment was considered day 0. In panel A, treatment with 0.1, 1, or 10μM RA, but not with LE135, resulted in a significant (P = 0.02) decrease in DNA concentrations detected in chondrocyte cultures between days 7 and 14. In panel B, treatment of BMDMSCs with 1 or 10μM RA or with 10μM LE135 decreased DNA content relative to untreated controls. *Values are significantly (P < 0.05) different for treated cells, compared with those of untreated control cells on the same day.
Citation: American Journal of Veterinary Research 72, 7; 10.2460/ajvr.72.7.884
Mean ± SEM DNA concentrations of equine chondrocytes (A) or BMDMSCs (B) in monolayer cultures treated with various concentrations of RA or the RA receptor antagonist LE135. The day of initial treatment was considered day 0. In panel A, treatment with 0.1, 1, or 10μM RA, but not with LE135, resulted in a significant (P = 0.02) decrease in DNA concentrations detected in chondrocyte cultures between days 7 and 14. In panel B, treatment of BMDMSCs with 1 or 10μM RA or with 10μM LE135 decreased DNA content relative to untreated controls. *Values are significantly (P < 0.05) different for treated cells, compared with those of untreated control cells on the same day.
Citation: American Journal of Veterinary Research 72, 7; 10.2460/ajvr.72.7.884
Treatment of BMDMSCs with 1 or 10μM RA or 10μM LE135 caused a significant (P < 0.05) decrease in concentrations of DNA in BMDMSC cultures on days 7 and 14, compared with that of untreated control BMDMSCs (Figure 1). The values for cultures of untreated control BMDMSCs remained similar between days 7 and 14 and were not different from those of BMDMSC cultures treated with 0.1μM RA and 0.1 or 1μM LE135.
Cell morphology—Treatment with RA at all tested concentrations supported a more mature and sustained chondrocytic morphology of chondrocytes in monolayer cultures for 7 and 14 days, compared with untreated control chondrocytes (P < 0.01; Figure 2). Maturation of nearly all cells to a cellular appearance similar to hypertrophied chondroblast morphology developed by day 14, as evidenced by many large, polyhedral cells, particularly at 1 and 10μM concentrations of RA. Untreated control cells dedifferentiated into a fibroblastic morphology within this same period.
Representative photomicrographs showing cellular morphology of equine chondrocytes treated with various concentrations of RA (A) or LE135 (B) in monolayer cultures. Numbers below each image indicate median (range) chondral morphology scores, assessed on a scale of 0 to 4 in 0.5-point increments, where 0 = no chondral morphology and 4 = > 75% of cells with chondral morphology typical of a hypertrophied cell or chondroblast. In panel A, chondral morphology scores were significantly higher for all RA-treated cells than for untreated control cells on days 7 and 14. In panel B, chondrocytes treated with 0.1 or 10μM LE135 had higher morphology scores, compared with those of untreated control cells, on day 7; however, these reverted to a more fibroblastic appearance (similar to that of controls) by day 14. Bar = 100 μm. †Valuesare significantly (P < 0.01) different, compared with those of untreated control cells on the same day. ‡For a given treatment, values are significantly (P < 0.001) different on days 7 and 14. White outlines depict cytoplasmic borders of individual chondrocytes; notice the cellular morphology. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 72, 7; 10.2460/ajvr.72.7.884
Representative photomicrographs showing cellular morphology of equine chondrocytes treated with various concentrations of RA (A) or LE135 (B) in monolayer cultures. Numbers below each image indicate median (range) chondral morphology scores, assessed on a scale of 0 to 4 in 0.5-point increments, where 0 = no chondral morphology and 4 = > 75% of cells with chondral morphology typical of a hypertrophied cell or chondroblast. In panel A, chondral morphology scores were significantly higher for all RA-treated cells than for untreated control cells on days 7 and 14. In panel B, chondrocytes treated with 0.1 or 10μM LE135 had higher morphology scores, compared with those of untreated control cells, on day 7; however, these reverted to a more fibroblastic appearance (similar to that of controls) by day 14. Bar = 100 μm. †Valuesare significantly (P < 0.01) different, compared with those of untreated control cells on the same day. ‡For a given treatment, values are significantly (P < 0.001) different on days 7 and 14. White outlines depict cytoplasmic borders of individual chondrocytes; notice the cellular morphology. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 72, 7; 10.2460/ajvr.72.7.884
Representative photomicrographs showing cellular morphology of equine chondrocytes treated with various concentrations of RA (A) or LE135 (B) in monolayer cultures. Numbers below each image indicate median (range) chondral morphology scores, assessed on a scale of 0 to 4 in 0.5-point increments, where 0 = no chondral morphology and 4 = > 75% of cells with chondral morphology typical of a hypertrophied cell or chondroblast. In panel A, chondral morphology scores were significantly higher for all RA-treated cells than for untreated control cells on days 7 and 14. In panel B, chondrocytes treated with 0.1 or 10μM LE135 had higher morphology scores, compared with those of untreated control cells, on day 7; however, these reverted to a more fibroblastic appearance (similar to that of controls) by day 14. Bar = 100 μm. †Valuesare significantly (P < 0.01) different, compared with those of untreated control cells on the same day. ‡For a given treatment, values are significantly (P < 0.001) different on days 7 and 14. White outlines depict cytoplasmic borders of individual chondrocytes; notice the cellular morphology. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 72, 7; 10.2460/ajvr.72.7.884
Treatment with LE135 did not sustain chondrocytic morphology in chondrocytes. Cultures of cells that were treated with 0.1 or 10μM LE135 and had significantly (P < 0.05) higher median chondral morphology scores than did untreated control chondrocytes on day 7 reverted to a fibroblastic phenotype similar to that of untreated control cells by day 14 (P < 0.001; Figure 2). Chondrocytes treated with 10μM LE135 had degenerate changes by day 14.
Analysis of cell morphology revealed a small but significant increase in median chondral morphology score for BMDMSCs treated with 1μM RA, compared with untreated control BMDMSCs and cells treated with other concentrations of RA, on days 7 (P < 0.05) and 14 (P < 0.01; Figure 3). The BMDMSCs treated with 0.1 and 1μM concentrations of LE135 had significantly (P < 0.05 and P < 0.01, respectively) greater median chondral morphology scores (≥ 2.0), compared with untreated control BMDMSCs, as early as day 7, and this persisted to day 14. However, BMDMSCs exposed to 10μM LE135 had evidence of pyknotic nuclei and cellular degeneration as well as a significantly (P < 0.05) decreased morphology score, compared with BMDMSCs that received any other treatment, on both days.
Representative photomicrographs showing cellular morphology of equine BMDMSCs treated with various concentrations of RA (A) or LE135 (B). On days 7 and 14, BMDMSCs treated with 1μM RA had higher morphology scores than did untreated control cells or cells treated with other concentrations of RA. In panel B, BMDMSCs treated with 0.1 or 1μM concentrations of LE135 had significantly higher chondral morphology scores than did untreated control cells. These scores were significantly (P < 0.05) decreased at 10μM LE135, and cells undergoing degenerative processes were identified. See Figures 1 and 2 for remainder of key.
Citation: American Journal of Veterinary Research 72, 7; 10.2460/ajvr.72.7.884
Representative photomicrographs showing cellular morphology of equine BMDMSCs treated with various concentrations of RA (A) or LE135 (B). On days 7 and 14, BMDMSCs treated with 1μM RA had higher morphology scores than did untreated control cells or cells treated with other concentrations of RA. In panel B, BMDMSCs treated with 0.1 or 1μM concentrations of LE135 had significantly higher chondral morphology scores than did untreated control cells. These scores were significantly (P < 0.05) decreased at 10μM LE135, and cells undergoing degenerative processes were identified. See Figures 1 and 2 for remainder of key.
Citation: American Journal of Veterinary Research 72, 7; 10.2460/ajvr.72.7.884
Representative photomicrographs showing cellular morphology of equine BMDMSCs treated with various concentrations of RA (A) or LE135 (B). On days 7 and 14, BMDMSCs treated with 1μM RA had higher morphology scores than did untreated control cells or cells treated with other concentrations of RA. In panel B, BMDMSCs treated with 0.1 or 1μM concentrations of LE135 had significantly higher chondral morphology scores than did untreated control cells. These scores were significantly (P < 0.05) decreased at 10μM LE135, and cells undergoing degenerative processes were identified. See Figures 1 and 2 for remainder of key.
Citation: American Journal of Veterinary Research 72, 7; 10.2460/ajvr.72.7.884
Gene expression analysis—Normalized gene expression of CI and CII was significantly (P < 0.05) decreased in chondrocytes treated with 1 or 10μM RA by day 7 and in all RA-treated chondrocytes by day 14 (Table 1). No significant changes in CII:CI expression ratios were detected following RA treatment (Figure 4). Treatment with LE135 did not affect expression of CI or CII or the CII:CI expression ratio in chondrocytes, except for a transient decrease detected in CII gene expression on day 7 in cells treated with 10μM LE135.
Mean ± SEM fold change in expression of CI and CII genes in equine chondrocytes treated with various concentrations of RA or the RA receptor antagonist LE135.
| CI | CII | |||||||
|---|---|---|---|---|---|---|---|---|
| Day 7 | Day 14 | Day 7 | Day 14 | |||||
| Variable | RA | LE135 | RA | LE135 | RA | LE135 | RA | LE135 |
| Concentration | ||||||||
| 0μM | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 |
| 0.1μM | 0.7 ± 0.1 | 0.8 ± 0.1 | 0.1 ± 0.1* | 1.0 ± 0.1 | 0.7 ± 0.1 | 0.8 ± 0.1 | 0.08 ± 0.1* | 1.0 ± 0.1 |
| 1μM | 0.1 ± 0.1* | 1.0 ± 0.1 | 0.1 ± 0.1* | 0.8 ± 0.1 | 0.1 ± 0.1* | 1.0 ± 0.1 | 0.08 ± 0.1* | 0.8 ± 0.1 |
| 10μM | 0.1 ± 0.1* | 0.8 ± 0.1 | 0.02 ± 0.1* | 0.8 ± 0.1 | 0.1 ± 0.1* | 0.4 ± 0.1* | 0.02 ± 0.1* | 0.8 ± 0.1 |
Cells were cultured as monolayers in supplemented DMEM; the day of initial treatment was considered day 0. The fold change for gene expression, relative to that in untreated control (0μM) cells, was calculated by use of the comparative CT(2−ΔΔCT) method, where ACT represents relative expression of the gene of interest normalized to the 18S ribosomal subunit.
Within columns, values for treated cells were significantly (P, 0.05) different from those of untreated control cells.
Mean ± SEM ratios of CII:CI gene expression in equine chondrocytes cultured in supplemented DMEM(A)and BMDMSCs cultured in chondrogenic media (B) treated with various concentrations of RA or LE135. For all treatments, BMDMSCs had a significantly (P < 0.001) greater CII:CI ratio than did chondrocytes, indicating an active state of chondrogenesis induced by use of chondrogenic media, even in untreated control BMDMSCs.
Citation: American Journal of Veterinary Research 72, 7; 10.2460/ajvr.72.7.884
Mean ± SEM ratios of CII:CI gene expression in equine chondrocytes cultured in supplemented DMEM(A)and BMDMSCs cultured in chondrogenic media (B) treated with various concentrations of RA or LE135. For all treatments, BMDMSCs had a significantly (P < 0.001) greater CII:CI ratio than did chondrocytes, indicating an active state of chondrogenesis induced by use of chondrogenic media, even in untreated control BMDMSCs.
Citation: American Journal of Veterinary Research 72, 7; 10.2460/ajvr.72.7.884
Mean ± SEM ratios of CII:CI gene expression in equine chondrocytes cultured in supplemented DMEM(A)and BMDMSCs cultured in chondrogenic media (B) treated with various concentrations of RA or LE135. For all treatments, BMDMSCs had a significantly (P < 0.001) greater CII:CI ratio than did chondrocytes, indicating an active state of chondrogenesis induced by use of chondrogenic media, even in untreated control BMDMSCs.
Citation: American Journal of Veterinary Research 72, 7; 10.2460/ajvr.72.7.884
Aggrecan gene expression in chondrocytes significantly decreased with increasing concentrations of RA on day 7 and was equivalently decreased in all RA-treated cells by day 14 (Figure 5). Aggrecan gene expression of LE135-treated chondrocytes decreased with increasing concentrations of LE135 on day 14 and was decreased at the 10μM concentration, compared with that of untreated control chondrocytes, on day 7.
Mean ± SEM aggrecan gene expression in equine chondrocytes exposed to various concentrations of RA and LE135. Expression for genes of interest was normalized to 18s rRNA, and fold changes for treated cells were calculated relative to aggrecan gene expression in untreated control cells. Significant decreases in aggrecan expression were detected in cells treated with any concentration of RA on days 7 and 14, with 10μM LE135 on day 7, and with any concentration of LE135 on day 14. These expression changes in RA-treated cells corresponded to maturation and hypertrophy of cells shown in Figure 2. Significant (*P < 0.05; †P < 0.01; ‡P < 0.001) differences between treated cells and untreated control cells are indicated by symbols. Within treatment concentrations, values were also significantly (P = 0.01) different for RA-treated cells between days 7 and 14.
Citation: American Journal of Veterinary Research 72, 7; 10.2460/ajvr.72.7.884
Mean ± SEM aggrecan gene expression in equine chondrocytes exposed to various concentrations of RA and LE135. Expression for genes of interest was normalized to 18s rRNA, and fold changes for treated cells were calculated relative to aggrecan gene expression in untreated control cells. Significant decreases in aggrecan expression were detected in cells treated with any concentration of RA on days 7 and 14, with 10μM LE135 on day 7, and with any concentration of LE135 on day 14. These expression changes in RA-treated cells corresponded to maturation and hypertrophy of cells shown in Figure 2. Significant (*P < 0.05; †P < 0.01; ‡P < 0.001) differences between treated cells and untreated control cells are indicated by symbols. Within treatment concentrations, values were also significantly (P = 0.01) different for RA-treated cells between days 7 and 14.
Citation: American Journal of Veterinary Research 72, 7; 10.2460/ajvr.72.7.884
Mean ± SEM aggrecan gene expression in equine chondrocytes exposed to various concentrations of RA and LE135. Expression for genes of interest was normalized to 18s rRNA, and fold changes for treated cells were calculated relative to aggrecan gene expression in untreated control cells. Significant decreases in aggrecan expression were detected in cells treated with any concentration of RA on days 7 and 14, with 10μM LE135 on day 7, and with any concentration of LE135 on day 14. These expression changes in RA-treated cells corresponded to maturation and hypertrophy of cells shown in Figure 2. Significant (*P < 0.05; †P < 0.01; ‡P < 0.001) differences between treated cells and untreated control cells are indicated by symbols. Within treatment concentrations, values were also significantly (P = 0.01) different for RA-treated cells between days 7 and 14.
Citation: American Journal of Veterinary Research 72, 7; 10.2460/ajvr.72.7.884
Normalized gene expression of CI and CII was significantly (P < 0.05) decreased in all RA-treated BMDMSCs, compared with that in untreated control BMDMSCs, on days 7 and 14 (Table 2). All BMDMSCs had a high CII:CI expression ratio. There was no significant effect of RA treatment on the CII:CI expression ratio of BMDMSCs (Figure 4). Treatment with LE135 at concentrations > 1μM or > 0. 1μM resulted in decreased gene expression of CI on days 7 and 14, respectively, compared with that in untreated control BMDMSCs; however, this was not sufficient to affect the CII:CI expression ratio in BMDMSCs. The BMDMSCs cultured in chondrogenic media had greater CII:CI expression ratios than did chondrocytes cultured in supplemented DMEM media (P < 0.001), indicating some initiation of chondrogenic phenotype in these stem cells when subjected to appropriate chondrogenic stimuli. Corresponding aggrecan gene expression was not detected in BMDMSCs despite repeated analyses.
Mean ± SEM fold change in expression of CI and CII genes in equine BMDMSCs treated with various concentrations of RA or LE135.
| CI | CII | |||||||
|---|---|---|---|---|---|---|---|---|
| Day 7 | Day 14 | Day 7 | Day 14 | |||||
| Variable | RA | LE135 | RA | LE135 | RA | LE135 | RA | LE135 |
| Concentration | ||||||||
| OμM | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 |
| 0.1μM | 0.3 ± 0.1* | 0.90 ± 0.10 | 0.5 ± 0.1* | 0.5 ± 0.1* | 0.4 ± 0.1* | 1.0 ± 0.1 | 0.5 ± 0.1* | 0.5 ± 0.1* |
| 1μM | 0.5 ± 0.1* | 0.10 ± 0.10* | 0.3 ± 0.1* | 0.3 ± 0.1* | 0.3 ± 0.1* | 1.0 ± 0.1 | 0.5 ± 0.1* | 0.8 ± 0.1 |
| 10μM | 0.6 ± 0.1* | 0.05 ± 0.10* | 0.4 ± 0.1* | 0.1 ± 0.1* | 0.5 ± 0.1* | 0.5 ± 0.1* | 0.5 ± 0.1* | 0.8 ± 0.1 |
See Table 1 for key.
Discussion
Concentrations of RA and the RA receptor antagonist LE135 used in the study reported here were selected on the basis of information in related studies6,9 performed in human mesenchymal stem cells and TC6 cells. Results of the present study indicated that equine chondrocytes in monolayer cultures treated with 0.1, 1, or 10μM RA significantly sustained chondrocyte morphology as evidenced by results of morphological analysis as well as detectable aggrecan gene expression and some, although declining, CII gene expression.
Hypertrophy of cells in chondrocyte monolayer cultures in the present study appeared similar to that of nonspecific chondroblasts in the differentiation of many forms of cartilage, such as elastic cartilage, fibrocartilage, and hyaline cartilage. In this process, stellate-shaped mesenchymal or fibroblastic cells morphologically progress to form first rounder, then larger, polyhedral chondroblasts that merge to form closely packed aggregates and begin to synthesize extracellular matrix as they transform to chondrocytes.27,28 Chondral morphology scores in the cell culture system in the present study reflected histologic maturation typical of these chondroblasts in differentiation toward a chondrocyte pathway. The described cellular hypertrophy corresponded to our other findings of significantly decreased DNA concentrations in the RA-treated chondrocyte cultures and reduction in aggrecan and collagen gene expression as cells matured and became confluent. It is also known that RA functions as a transcription factor to regulate growth and delineation of cells and may cause a nonspecific reduction of gene expression.3,4 As chondroblast-like cells form a layer of aggregated cells, proliferation and metabolic processes slow down. The results of the present study suggested that these hypertrophied cells, which have a collapsed intercellular space, may have a decreased need for extracellular matrix production. The significant decrease in aggrecan gene expression with RA treatment supports this interpretation. Chondrocytes treated with LE135 had taken on a predominantly fibroblastic appearance by day 14 with correspondingly reduced aggrecan gene expression. In summary, RA treatment prevented the fibro blastic dedifferentiation of chondrocytes that occurs in vitro and promoted the maturation of chondrocytes in monolayer cultures to hypertrophied chondroblast-like cells. Treatment with LE135 had no effect on the fibroblastic dedifferentiation of monolayer chondrocytes that occurred in vitro. Treatment with RA had a similar effect to that reported for transforming growth factor β or cartilage oligomeric matrix protein in monolayer cultures of mesenchymal stem cells30 or dermal fibroblasts,31 respectively. Cells in those studies had morphological changes similar to those described in the present study and had gene expression changes indicative of chondrogenesis within 1 to 2 weeks after culture initiation.
The study reported here also demonstrated that BMDMSCs treated with RA or LE135, particularly at a 1μM concentration, had significant morphological changes that included cellular enlargement and rounding, but had no detectable aggrecan production and no differences in CII:CI expression ratios, compared with untreated control BMDMSCs. We therefore concluded there was no significant effect of these treatments on chondrogenesis. The decrease in CI expression in LE135-treated BMDMSCs may have been an early indicator of a shift to less fibroplasia, but this could not be confirmed as a shift to chondrogenesis. All BMDMSCs were cultured in media that is known to drive chondrogenesis in stem cells under certain conditions. The high CII:CI expression ratio in these stem cells was indicative of chondrogenesis because CII production is unique to cartilage cells. Culture of BMDMSCs in pellets or 3-D cultures for a longer period may have resulted in detectable chondral differentiation. Despite the lack of evidence for an effect on chondrogenesis in BMDMSCs in monolayer cultures, treatment with RA at concentrations ≥ 1μM or LE135 at the 10μM concentration did significantly decrease DNA content in BMDMSC cultures. As in chondrocytes, this likely reflected a decrease in cell number during cell enlargement (although less extensive in BMDMSCs) but also may have been contributed to by the cellular degeneration detected in cultures treated with 10μM LE135. Given the morphological appearance of these stem cells that were mostly spindle shaped and not confluent, this may have represented a decrease in proliferation, an increase in apotosis, or both. At high concentrations (10μM) of LE135, degenerate changes indicated cell apotosis or death.
Retinoic acid has been shown to upregulate so-called marker proteins of hypertrophic chondroblasts and to simultaneously suppress cell proliferation in chondroprogenitor (TC6 and ATDC5) cells.6,7 Retinoic acid also profoundly promoted chondrocyte proliferation of costal cartilage, even at low concentrations.32 This is consistent with our findings, in that RA promoted chondral maturation, hypertrophy, and growth to confluence. Results of the present study and the previously published studies support the concept that RA promotes maturation of chondral cells and suppresses progenitor proliferation, survival rates, or both. By antagonizing RA receptors, LE135 may prevent the induction of specific factors necessary to induce hypertrophy and final differentiation of chondrocytes because LE135-treated cells reverted to a fibroblastic phenotype.
The timing and adequacy of RA signaling during cartilage growth may affect the development of normal mature cartilage in vivo. Our previous findingsa indicated that nucleated cells in blood of yearling Thoroughbreds with confirmed diagnoses of OCD, compared with those of age-matched horses on the same farm without OCD, had statistically significant reductions in the transcript expression of 2 RA-related genes (RA receptor and retinoid-inducible serine carboxypeptidase). This reduced expression in RA-related genes was statistically significant, suggesting that the change could be associated with clinical OCD. Results of the present study are compatible with the concept that reduced RA function could result in failure to promote chondrocyte maturation and simultaneous failure to slow down progenitor differentiation. This could lead to a dysregulation of chondrogenesis with the potential for superimposition of differentiating but nonmaturing chondrocyte precursor cells. Histologically, osteochondrosis lesions contain excessively thickened areas of chondrocytes within a disorganized cartilage structure compatible with this potential dysregulation.14,20,21
Investigation of the role of RA pathway actions from genetic or nutritional aspects warrants further study. Variation in expression of genes related to RA function could make certain horses or breeds more susceptible to development of cartilage growth abnormalities. In addition, a possible synergy between known genetic predispositions15–17 and RA deficiencies has the potential to exacerbate cartilage growth abnormalities. Retinoic acid is a vitamin A derivative that promotes not only chondrogenesis but also bone morphogenetic protein—mediated skeletogenesis.33 Although not investigated in the present study, an equally important role of RA in bone formation may contribute to joint growth abnormalities and cartilage lesions.
Retinoic acid is supplied primarily as vitamin A in the diet. Hay that is bleached, stored for long periods, or of low quality may contribute to a deficiency of RA in growing horses. Investigators of nutrition studies11,12 that evaluated energy values and protein concentrations in diets fed to young horses were unable to substantiate a direct cause-and-effect relationship between diet and orthopedic disease except in cases of severe deprivation, although a nutritional link was suspected. Retinoic acid should be investigated further for a potential nutritional link to the molecular signaling and histologic changes that develop in equine cartilage growth abnormalities.
Retinoic acid may be necessary to drive chondrocytes into a metabolic or physiologic pathway that results in hypertrophy and differentiation, whereas RA antagonism may drive progenitor cells into a pathway that initiates proliferation. Further investigations of the role of RA in chondrogenesis may help characterize a possible factor in the development of cartilage growth abnormalities in horses.
ABBREVIATIONS
| BMDMSC | Bone marrow—derived mesenchymal stem cell |
| CI | Collagen type Ia |
| CII | Collagen type II |
| CT | Cycle threshold |
| DMEM | Dulbecco modified Eagle medium |
| OCD | Osteochondrosis dessicans |
| RA | Retinoic acid |
| RT | Reverse transcription |
Bertone AL, Reed SM. Blood gene expression equine microarray profiles associated with osteochondrosis in yearling Thoroughbreds (abstr), in Proceedings. 3rd Int Workshop Equine Osteochondrosis 2008;26.
Fetal bovine serum, Sigma Aldrich, St Louis, Mo.
RA, Sigma Aldrich, St Louis, Mo.
LE135, Tocris Bioscience, Ellisville, Mo.
hMSC Mesenchymal Stem Cell Chondrocyte Differentiation Medium, Lonza, Allendale, NJ.
Papain, Acros Organics, Morris Plains, NJ.
DNA (cellulose double stranded from calf thymus DNA), Sigma Aldrich, St Louis, Mo.
Hoescht 33258 dye (20mM solution in water), AnaSpec Inc, Fremont, Calif.
Microtest 96-well assay plate, BD Biosciences, San Jose, Calif.
Spectramax M2 microplate reader, Molecular Devices, Sunnyvale, Calif.
TRIzol, Invitrogen, Carlsbad, Calif.
Applied Biosystems, Foster City, Calif.
Taqman Universal PCR Master Mix, Applied Biosystems, Foster City, Calif.
Eukaryotic 18s rRNA endogenous control (FAM dye/MGB probe, nonprimer limited), Applied Biosystems, Foster City, Calif.
Minitab Statistical Software, State College, Pa.
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