Exogenous thymosin beta4 prevents apoptosis in human intervertebral annulus cells in vitro

Abstract

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 beta(4) (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.

Fig. 1

Cultured human annulus cells 3 days after serum starvation or hydrogen peroxide treatment prior to fixation for apoptosis assay. A) Annulus cells untreated or treated with 800 nM TB4 previously exposed to serum starvation. B) Annulus cells treated or untreated with 800 nM TB4 exposed to hydrogen peroxide. Most cells show typical spindle shaped morphology. Some rounded, shrunken, cells also were present in the hydrogen peroxide treated cells. × 115.

Fig. 2

Anti-apoptotic effect of 800 nM TB4 on annulus cells. Annulus cells with or without TB4 treatment after induction of apoptosis by hydrogen peroxide. Apoptotic cells are stained brown and non-apoptotic cells are counterstained green. Positive and negative experimental controls also are shown. × 95. Graph shows percent of apoptotic cells in the treated and untreated cultures. Data from five TUNEL analyses were analyzed using repeated measure analysis of variance; data were significant, *p < 0.05.

Fig. 3

Anti-apoptotic effect of 800 nM TB4 on annulus cells. Annulus cells with or without TB4 treatment after induction of apoptosis with serum starvation. Apoptotic cells are stained brown and non-apoptotic cells are counterstained green. Positive and negative experimental controls are also shown. × 95. Graph shows percent of apoptotic cells in the treated and untreated cultures. Data from five TUNEL analyses were analyzed using repeated measure analysis of variance; data were significant, *p < 0.05.

Fig. 4

Effect of 30 nM TB4 on annulus cells in 3D culture. After 2 weeks in 3D culture, both untreated and TB4 treated annulus cells appeared healthy with the typical rounded morphology of annulus cells in 3D culture. Slight pink staining shows the presence of proteoglycans in the extracellular matrix produced by the annulus cells. × 95. Graph shows DMB analysis from three separate cultures. Statistical analysis using repeated measure analysis of variance showed no change in proteoglycan production with or without TB4.