Bone morphogenetic protein-4 enhances vascular endothelial growth factor secretion by human retinal pigment epithelial cells - PubMed (original) (raw)

Bone morphogenetic protein-4 enhances vascular endothelial growth factor secretion by human retinal pigment epithelial cells

Rhonda R Vogt et al. J Cell Biochem. 2006.

Abstract

Retinal pigment epithelial (RPE) cells secrete vascular endothelial growth factor (VEGF), a cytokine known to promote angiogenesis. Results from RNase protection assays (RPAs) show that RPE from non-diabetic human donors and from adult retinal pigment epithelium-19 (ARPE-19) cells expressed significant bone morphogenetic protein-4 (BMP-4) message. In addition, ARPE-19 cells cultured in high glucose (25 mM), compared to those in physiological glucose (5.5 mM) released significantly more BMP-4 into the conditioned media (CM). However, the effect of BMP-4 on the release of VEGF by ARPE-19 cells has not been studied. Accordingly, ARPE-19 cells were treated with BMP-4 to determine VEGF secretion. BMP-4 and VEGF levels in the CM and cell lysates were measured by enzyme-linked immunosorbent assay (ELISA). Cells treated with exogenous BMP-4 had higher VEGF in the CM and this treatment effect was dose- and time-dependent, while cell lysates had low levels of VEGF. Addition of cycloheximide (CHX) or actinomycin-D (ACT) significantly reduced VEGF secretion from cells treated with BMP-4, suggesting that the BMP-4-induced secretion of VEGF requires new RNA and protein synthesis. Our results suggest that BMP-4 may play a role in the regulation of ocular angiogenesis associated with diabetic retinopathy (DR) by stimulating VEGF release from RPE cells.

(c) 2006 Wiley-Liss, Inc.

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Figures

Fig. 1

Fig. 1

BMP mRNA expression in ARPE-19 cells and in RPE cells from a non-diabetic human donor. Total RNA was isolated using the TRI reagent. Twenty micrograms of total RNA was used for the measurement of BMP mRNA expression by the RPA. The protected RNA fragments were fractionated on 5% polyacrylamide gels containing 8 M urea and detected by PhosphorImaging. Positions of labeled probes for the different BMPs and a housekeeping gene control (ribosomal protein L32) are marked on the left of the image. The protected fragments are indicated on the right with arrows.

Fig. 2

Fig. 2

Effect of glucose treatment on BMP-4 secretion by ARPE-19 cells. Cells were grown to confluence in media containing physiological glucose (5.5 mM) before being harvested and re-seeded into different media for 24 h. After cells were treated for 5 days, CM was collected for ELISA measurement of BMP-4. No change in total cell protein (ranged from 0.48 to 0.66 mg/well) was observed in glucose treatment. Mean and standard error from results of three experiments are indicated in the diagram. In each experiment, five observations were made in each treatment group. *Indicates statistically significant difference between BMP-4 concentration in CM from cells grown in 25 mM glucose and those from cells in 5.5 mM glucose+ 19.5 mM mannitol as compared to those from cells grown in 5 mM glucose, P ≤ 0.05.

Fig. 3

Fig. 3

Effect of exogenous BMP-4 concentration on VEGF secretion by ARPE-19 cells. ARPE-19 cells were grown to confluence in SF media containing 5.5 mM glucose before the addition of exogenous BMP-4 at 0.1, 1.0, or 10 ng/ml. For detailed treatment paradigm, see Materials and Methods. CM and cells were collected after a 24-h period of BMP-4 treatments. VEGF levels in the CM and cell lysate were measured using ELISA. Results of VEGF in the CM are mean and standard error from two representative experiments (with triplicate observations per concentration per experiment). Results of VEGF in the cell lysates were derived from one experiment (with triplicate measurements for each concentration).

Fig. 4

Fig. 4

Effect of exogenous BMP-4 treatment duration on VEGF secretion by ARPE-19 cells. ARPE-19 cells were grown to confluence in SF media containing 5.5 mM glucose before the addition of exogenous BMP-4 (10 ng/ml). For detailed treatment paradigm, see Materials and Methods. CM was collected from cells after the specified period of BMP-4 treatments. VEGF levels in the CM and cell lysate were measured using ELISA. Results of VEGF in the CM are mean and standard error from two representative experiments (with triplicate observations per time point per experiment). Results of VEGF in the cell lysates were derived from one experiment (with triplicate measurements for each time point).

Fig. 5

Fig. 5

Effect of CHX and ACT on VEGF secretion by ARPE-19 cells. ARPE-19 cells were grown to confluence in SF media containing 5 mM glucose before the addition of exogenous BMP-4 (10 ng/ml) and CHX (3.6 μM) or ACT (0.5 μM). CM was collected from cells at the 5th day of treatment with BMP-4. VEGF in the CM was assayed using ELISA. One experiment was performed and four observations were made per treatment group. The mean ± SE are indicated. *Indicates significant difference between VEGF concentration in CM without inhibitors compared to those with CHX and with ACT.

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