NBHA reduces acrolein-induced changes in ARPE-19 cells: possible involvement of TGFβ

Abstract

Purpose: Acrolein, a toxic, reactive aldehyde formed metabolically and environmentally, has been implicated in the damage to and dysfunction of the retinal pigment epithelium (RPE) that accompanies age-related macular degeneration (AMD). Our purpose was to investigate the potential of acrolein to influence the release of transforming growth factor beta-2 (TGFβ2) and vascular endothelial growth factor (VEGF), to assess the ability of N-benzylhydroxylamine (NBHA) to prevent the effect of acrolein on cytokine release and reduction of viable cells, and to explore the pathway by which acrolein might be causing the increase of VEGF.

Materials and methods: Confluent ARPE-19 cells were treated with acrolein and/or NBHA. They were also pretreated with SIS3, a specific inhibitor of SMAD 3, and ZM39923, a JAK3 inhibitor, before being treated with acrolein. Viable cells were counted; ELISA was used to measure the TGFβ2 and/or VEGF in the conditioned media.

Results: Acrolein was shown to reduce the number of viable ARPE-19 cells and to upregulate the release of the proangiogenic cytokines TGFβ2 and VEGF. Co-treatment with 200 μM NBHA significantly reduced the effects of acrolein on viable cell number and TGFβ2 release. Pretreatment of the cells with SIS3 partially blocked the action of acrolein on decreased viable cell number and VEGF upregulation, suggesting that part of the effects of acrolein are mediated by the increased levels of TGFβ and its signaling.

Conclusions: Our results suggest that the action of acrolein on the reduction of viability and VEGF increase by ARPE-19 cells is partially mediated by TGFβ2. By reducing the effects of acrolein, NBHA and SIS3 could be potential pharmacological agents in the prevention and progression of acrolein-induced damage to the RPE that relates to AMD.

FIGURE 1

Acrolein affects ARPE-19 cell viability, VEGF, and TGFβ2 release. Confluent ARPE-19 cells were treated with 12.5, 25, or 50 μM acrolein for 72hr. (A) Viable cells were counted using a hemacytometer with Trypan Blue. Results are average viable cell number ± SE (N = 3). (B) ELISA for VEGF was performed on the conditioned media from each well, and the results were divided by the viable cell counts in Figure 1A. Results are the mean pg VEGF per 10,000 viable cells ± SE (N = 3). (C) Confluent ARPE-19 were treated with 50 μM acrolein; the conditioned media was collected after 24, 48, and 72hr. Results were obtained by comparing treatment groups to serum-free time-matched controls. Results are the mean pg VEGF per 10,000 viable cells ± SE (N = 3). (d) ELISA for TGFβ2 was performed on the conditioned media from each well, and the results were divided by the viable cell counts in Figure 1A. Results are the mean pg TGFβ2 per 10,000 viable cells ± SE (N = 3). Asterisks indicate statistical significance of p ≤ 0.05.

FIGURE 2

NBHA reduces acrolein effects. Confluent ARPE-19 cells were treated with 50 μM acrolein, 200 μM NBHA, or a combination of 50 μM acrolein with 50,100, or 200 μM NBHA for 72hr. (A) Viable cells were counted using a hemacytometer with Trypan Blue. Results are average viable cell number ± SE (N = 3). (B) ELISA for VEGF was performed on the conditioned media from each well, and the results were divided by the viable cell counts in Figure 2A. Results are the mean pg VEGF per 10,000 viable cells ± SE (N = 3). (C) ELISA for TGFβ2 was performed on the conditioned media from each well, and the results were divided by the viable cell counts in Figure 2A. Results are mean pg TGFβ2 per 10,000 viable cells ± SE (N = 3). Asterisks indicate statistical significance of p ≤ 0.05.

FIGURE 3

SIS3 reduces acrolein effects. Confluent ARPE-19 cells were treated 2 μM SIS3 or ZM39923 (ZM) for 24hr before being treated with 50 μM acrolein for 72hr. (A) Viable cells were counted using a hemacytometer with Trypan Blue. Results are average viable cell number ± SE (N = 3). (B) ELISA for VEGF was performed on the conditioned media from each well, and the results were divided by the viable cell counts in Figure 3A. Results are the mean pg VEGF per 10,000 viable cells ± SE (N = 3). Asterisks indicate statistical significance of p ≤ 0.05 between the acrolein-treated group and those treated with acrolein plus SIS3 or ZM.