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Loss of cells in the human disc due to programmed cell death (apoptosis) is a major factor in the aging and degenerating human intervertebral disc. Our objective here was to determine if thymosin β4 (TB4), a small, multifunctional 5 kDa protein with diverse activities, might block apoptosis in human annulus cells cultured in monolayer or three-dimensional (3D) culture. Apoptosis was induced in vitro using hydrogen peroxide or serum starvation. Annulus cells were processed for identification of apoptotic cells using the TUNEL method. The percentage of apoptotic cells was determined by cell counts. Annulus cells also were treated with TB4 for determination of proliferation, and proteoglycan production was assessed using cell titer and 1,2 dimethylmethylamine (DMB) assays and histological staining. A significant reduction in disc cell apoptosis occurred after TB4 treatment. The percentage of cells undergoing apoptosis decreased significantly in TB4 treated cells in both apoptosis induction designs. TB4 exposure did not alter proteoglycan production as assessed by either DMB measurement or histological staining. Our results indicate the need for further studies of the anti-apoptotic effect of TB4 and suggest that TB4 may have therapeutic application in future biological therapies for disc degeneration.
The objective of our study was to investigate the anti-apoptotic effect of thymosin β4 (TB4) on cultured human intervertebral annulus cells in vitro. TB4 is a small, multifunctional 5 kDa protein with diverse activities (Goldstein et al. 2005). It is an abundant intracellular protein that has been studied extensively as a G-actin monomer sequestering protein (Safer et al. 1991,Yarmola et al. 2007) involved in actin skeletal remodeling that allows for cell migration (Bubb 2003). TB4 stimulates migration and survival of cardiomyocytes, endothelial cells and corneal cells, and is an essential paracrine factor for embryonic endothelial progenitor cell-mediated cardioprotection (Bock-Marquette et al. 2004, Bubb 2003, Hinkel et al. 2008). TB4 is implicated in many diverse extracellular functions including signaling during wound healing (Philp et al. 2003, 2006), migration of hair follicle stem cells (Philp et al. 2007), and inhibition of cell proliferation (Bonnet et al. 1996, Leeanansaksiri et al. 2004).
Loss of cells due to programmed cell death (apoptosis) is a major factor in the aging and degenerating human intervertebral disc (Freemont 2009, LeMaitre et al. 2007, Park et al. 2005). The human intervertebral disc contains a small cell population that becomes even further reduced with the processes of aging and degeneration. This puts the remaining cell population at high risk for further decrease in cell function or cell numbers (Gruber et al. 2008). Our work and that of others has shown that apoptosis reduces disc cell numbers during aging and degeneration in vivo (Gruber and Hanley 1998, Wang et al. 2008). Previous studies have shown that selected cytokines, such as insulin-like growth factor-1 or platelet-derived growth factor, can be effective for preventing or ameliorating of apoptosis in human annulus cells in vitro (Gruber et al. 2000a). Recent evidence also shows that TB4 has anti-apoptotic effects in various cells (Choi et al. 2006, 2007, Sosne et al. 2004a). This attribute attracted our attention and led to our study of programmed cell death using cultured human annulus cells.
Human disc cell studies were performed following approval by our human subjects Institutional Review Board. Data are presented from annulus cells from lumbar discs of nine surgical patients (5 female, 4 male; mean age 42 years, range 25–55 years), Thompson grades II – IV (Thompson et al. 1990).
Disc tissue was transported to the laboratory in Minimal Essential Media (MEM, Gibco, Grand Island, NY) within 30 min of removal and established in culture as described previously (Gruber and Hanley 2000b). Briefly, primary cultures were grown in standard T-25 flasks (Costar, Cambridge, MA) containing 5 ml MEM with 20% fetal bovine serum (MEM20; Life Technologies, Grand Island, NY) with antibiotics added. Cells used in experiments were passages 1–3.
Recombinant human TB4 was purified as described previously (Yarmola et al. 2000, 2001). The TB4 contained an added C-terminal cysteine. Previous results confirmed that the C-terminal cysteine-modified TB4 and TB4 labeled on the C-terminus with tetramethylrhodamine 5-maleimide have identical actin-binding and extracellular properties as wild-type TB4. The concentration of TB4 was confirmed by amino acid analysis. The stocks of TB4 were kept frozen at −70°C and thawed immediately before use.
The assay for apoptosis detection was performed using the R & D systems (Minneapolis, MN) TACS TdT in situ Apoptosis Detection Kit (TUNEL) with modifications described previously (Gruber et al. 1999). Positive and negative controls were included with each TUNEL assay (Gruber et al. 1999). The positive control was established by treating cells with TACS-Nuclease to generate DNA breaks in every cell. This reagent is included in the kit and is the positive control recommended by the package insert. The negative control was established by eliminating the TdT enzyme from the labeling reaction mix.
Cells were seeded into eight-well Nunc slides (LabTek, Naperville, IL) at a density of 5,000 cells/well. Cells were fed TB4 (0, 30, 100 or 800 nM in MEM20) for 24 h. Both the serum starvation and hydrogen peroxide methods described below for inducing apoptosis in disc cells were based on methods described for the TB4 induced apoptosis in corneal cells (Ho et al. 2007, Sosne et al. 2004a).
After 24 h in TB4, medium was removed and fresh medium (MEM20) containing 50 µM hydrogen peroxide, was added for 2 h. Medium was removed and replaced with MEM20 and the cells were incubated for 48 h before terminating the experiment. Control treatments of cells exposed to peroxide without TB4 were included in each experiment. Cells were fixed in 10% neutral buffered formalin (NBF) for 10 min, then transferred to 70% ethanol and processed for apoptosis identification.
After 24 h in TB4, medium was removed and replaced with MEM without serum. Cells were incubated for 48 h before terminating the experiment. Control treatments of cells treated with serum starvation without TB4 were included in each experiment. Cells were fixed as described above.
At least 200 cells/treatment were counted. The percentage of apoptotic cells was determined by dividing the number of apoptotic cells by the total number of apoptotic and normal cells counted in each well, and multiplying by 100.
Sterile Gelfoam® (Pharmacia & Upjohn Co., Kalamazoo, MI), an absorbable collagen sponge prepared from purified porcine skin, used previously in our laboratory to grow intervertebral disc cells in 3D culture (Gruber et al. 2006), was employed as a 3D cell scaffold. Annulus cells were suspended in MEM20 at 1 × 107 cells/ml concentration. Ten microliter droplets containing 1 × 105 cells were injected into collagen sponges trimmed into 0.5 cm3 cubes; 1 × 105 cells/0.5 cm3 of collagen sponges previously have been found to be an optimal number for maximum proteoglycan production in collagen sponges (Gruber et al. 2006). Replicate collagen sponges were placed on Costar Transwell Clear Inserts (Corning Incorporated-Life Sciences, Lowell, MA) in 24-well plates and fed three times per week with 2.0 ml of medium (MSCBM) with TB4 (30 nM) or without TB4 (untreated control). The concentration of 30 nM TB4 was chosen based on previous observations of cytokine-like activity of TB4 at low doses in mast cells (Leeanansaksiri et al. 2004). Cells were grown for 2 weeks and assayed for proteoglycan production in the presence or absence of TB4. Cultures were terminated, fixed in 10% NBF for 1 h and embedded in paraffin. Collagen sponges were sectioned for histological analysis and stained for extracellular matrix proteoglycan production using toluidine blue (0.1% in distilled water; Sigma). Proteoglycan production also was assessed quantitatively using the 1,9-dimethylmethylene blue (DMB) assay described below.
Cells were grown in 3D culture for 14 days in the presence or absence of TB4 (30 nM) and assayed for sulfated proteoglycan production using the DMB assay (Muller and Hanschke 1996). Briefly, collagen sponges containing cells were rinsed once in Hank’s Balanced Salt Solution (HBSS). Replicates were placed in 1.5 ml microcentrifuge tubes and collagenase type V (Sigma) in HBSS was added to produce a final concentration of 1.0 Units/ml. Proteoglycan concentrations were determined from the standard curve, replicates averaged, and results expressed as micrograms S-GAG/ ml.
Cell proliferation was evaluated by seeding replicate samples of disc cells, 5,000 cells per well, in monolayer in 48-well tissue culture plates and either treating or not with TB4 (0–800 nM). After 3 days of culture, 20 µl of kit reagent was added (Cell Titer 96 Aqueous One Solution Kit; Promega, Madison, WI) (Cory et al. 1991). Plates were wrapped in foil, shaken for 5 min, and incubated for 2 h at 37° C. Absorbance at 490 nm was measured using a plate reader (EL 800 Universal Micro Plate Reader; Bio-Tek Instruments, Inc). Data are expressed as means ± SD of 16 replicate wells per treatment.
Data were analyzed using SAS version 8.2. A p-value < 0.05 was considered statistically significant. Standard statistical methods were used including repeated measure analysis of variance. Data are presented as means ± SD (n).
Three days after exposure to the apoptosis inducing agents, human annulus cells induced to undergo apoptosis by hydrogen peroxide exposure showed both normal appearing spindle shaped cells and cells that were rounded (Fig. 1). Most cells under serum starvation conditions continued to be spindle shaped for the duration of the experiment.
Using the TUNEL apoptosis assay, apoptotic cells exhibiting DNA fragmentation were stained brown (Figs. 2 and and3)3) and were readily distinguished from non-apoptotic or necrotic cells, counterstained green. Positive and negative experimental control cells also are shown in Figs. 2 and and3.3. TB4 (800 nM) was found to prevent apoptosis induced by both hydrogen peroxide and serum starvation in the disc cells (Figs. 2 and and33).
In our initial studies, we evaluated TB4 in a dose-response experimental design. Preliminary experiments using lower concentrations of TB4 with the hydrogen peroxide treatment (30 and 100 nM) showed no significant reduction in hydrogen peroxide induced disc cell apoptosis (21.5% ± 6.2 (3) [mean ± S.D. (n)] in controls vs. 27.4% ± 8.2 (3) in 30 nM TB4 treated cells and 14.2% ± 11.6 (5) in controls vs. 8.7% ± 8.3 (5) in 100 nM TB4-treated cells). A significant reduction in disc cell apoptosis was seen after 800 nM TB4 treatment following either hydrogen peroxide (Fig. 2) or serum starvation (Fig. 3) induced apoptosis.
The percentage of cells undergoing apoptosis decreased significantly in TB4 treated cells for both apoptosis induction designs: hydrogen peroxide induced apoptosis, 46.9% ± 10.9 (5) in controls vs. 21.4% ± 6.1 (5) in TB4 treated cells (P = 0.0437) (Fig. 2); serum starvation design, 38.8% ± 8.1 (5) for controls vs. 12.3% ± 8.4 (5) in TB4 treated cells (P = 0.0347) (Fig. 3).
TB4 previously has been shown to stimulate production of laminin-5 in the extracellular matrix of human corneal epithelial cells (Sosne et al. 2004b, 2007). TB4 also has been shown to exert a cytokine-like activity at very low doses in mast cells (Leeanansaksiri et al. 2004). Two main classes of extracellular macromolecules make up the extracellular matrix: proteoglycans, and fibrous proteins including collagen and laminin, which have both structural and adhesive functions. To test whether TB4 at very low cytokine-like doses has an effect similar to cancer cells on annulus extracellular matrix production of proteoglycans, we grew the cells in 3D collagen culture and exposed them to 30 nM TB4 for 14 days. Fig. 4A shows the growth of disc cells in 3D collagen culture with or without 30 nM TB4. In 3D cultures, the rounded cells are clearly visible growing in and around the 3D scaffold (Fig. 4A). No significant differences were observed in the appearance of the TB4 treated cells. The 3D cultures were stained with toluidine blue, which stains proteoglycans pink. Slight pink staining is visible around the cells showing the presence of proteoglycans in the extracellular matrix produced by the disc cells (Fig. 4A). Although in this particular image more cells happen to be present in the TB4 treated cultures, no increase in the amount of proteoglycans around each cell is evident.
The presence of proteoglycans in 3D cultures treated or untreated with TB4 also was confirmed quantitatively using the DMB assay. No significant difference in proteoglycan production was observed when the cells were treated with TB4 (Fig. 4B) (0.81 mg/ml ± 0.44 (3) for controls vs. 0.94 ± 0.42 (3) for TB4 treated cells.
TB4 has been shown previously to inhibit cell proliferation in mast cells and hematopoietic stem cells (Bonnet et al. 1996, Leeanansaksiri et al. 2004). To determine the effect of TB4 on disc cell proliferation after exposure to TB4, we performed the cell titer colorimetric assay where light absorption at 490 nm is proportional to the number of cells present (Cory et al. 1991). No significant change in proliferation was observed following exposure to concentrations from 1 to 800 nM (1.66 nm ± 0.15 (3) for controls vs. 1.72 nm ± 0.14 (3) for TB4 treated cells.
In the study reported here, we have shown that exposure to 800 nM of exogenous TB4 exerts an anti-apoptotic effect on cultured annulus cells following application of the apoptotic inducing external stresses, hydrogen peroxide exposure or serum starvation. Although TB4 is known to be internalized by cells, the cell surface binding site of TB4 currently is unknown (Bubb 2003). When fibroblastic cells were induced to undergo apoptosis by anti-tumor drugs, intracellular TB4 levels decreased (Iguchi et al. 1999). Increased resistance to apoptosis in cells over-expressing TB4 also has been reported (Niu and Nachmias 2000).
Cell apoptosis is known to involve mechanisms such as activation of caspases-3/7, which are mediated extrinsically by common apoptosis inducing factors such as FasL, tumor necrosis factor alpha (TNF-α), and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) (Zhang et al. 2008) or by intrinsic mitochondrial pathways such as mitochondrial involvement in Fas-mediated apoptosis in disc cells (Park et al. 2005). In corneal cells, TB4 has been shown to function as an anti-apoptotic agent by inhibiting the release of cytochrome c from mitochondria and by suppressing the activation of caspases (Sosne et al. 2004a). Although the receptors involved in binding TB4 to cells are unknown, internalization is essential for the anti-apoptotic effects of TB4 (Ho et al. 2007). To date, the anti-apoptotic effect of TB4 on human disc cells has been unexplored.
Modulation of apoptosis with cytokines and other reagents is a potential therapeutic strategy for treating disc degeneration (Zhao et al. 2006). Degenerated discs have decreased proteoglycan content associated with loss of load bearing function. During early stages of disc degeneration, stimulation of matrix synthesis by disc cells may be feasible using a number of potential biological therapies (Gruber and Hanley 2003). TB4 has been implicated in wound healing and increased production of laminin-5, an important protein for cell-extracellular matrix adhesion, in the extracellular matrix of human corneal epithelial cells (Sosne et al. 2004b, 2007). TB4 has been found to induce both TGFβ and laminin-5 gamma 2 chain expression in corneal epithelial cells (Sosne et al. 2004b).
In our study, no change in proteoglycans in the extracellular matrix produced by disc cells exposed to low doses of TB4 was detectable by toluidine blue staining or quantitative proteoglycan assay. It is possible that future studies of synergistic interactions of TB4 with cytokines such as TGFβ may show stimulation of matrix production.
TB4 has been found to have varying effects on proliferation of different types of cells. The presence of TB4 did not effect the proliferation of corneal cells (Sosne et al. 2006), but has been shown previously to inhibit proliferation of hematopoietic progenitor cells and mast cells (Bonnet et al. 1996, Leeanansaksiri et al. 2004). We were unable to identify a positive effect of TB4 on disc cell proliferation.
Further studies of the effects of TB4 on cell survival and proliferation and of diverse cell processes along different cellular pathways (Sosne et al. 2004a) are warranted, as is further investigation into the mechanisms by which TB4 inhibits apoptosis in annulus cells.
Our work has shown that TB4 suppressed or prevented apoptosis in human intervertebral annulus cells following apoptotic stresses in vitro; however, TB4 did not influence cell proliferation or alter proteoglycan production by annulus cells. Our results suggest that TB4, possibly administered by injection to specific disc sites, may have future therapeutic potential for biological therapies for disc degeneration.
We gratefully acknowledge the technical assistance of Dr. Jim Norton and Ms Natalia Zinchenko. This work was funded by a grant from the Charlotte-Mecklenburg Health Services Foundation, the Carolinas Back Pain Research Endowment, the Medical Research Service of the Department of Veterans Affairs, the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), grant number 5K25AR048918 to EGY and the National Science Foundation grant number NSF-0316015 to MRB.