Bone morphogenetic protein 7 increased vascular endothelial growth factor (VEGF)-a expression in human granulosa cells and VEGF receptor expression in endothelial cells

Abstract

The formation of an individual capillary network in the theca cell layer is required for ovarian folliculogenesis. Although vascular endothelial growth factor (VEGF) is critical for this process, the regulation of VEGF has been unclear. In the present study, the relationship between VEGF and intraovarian cytokine, bone morphogenetic protein 7 (BMP-7) was investigated. Granulosa cells (GC), obtained from in vitro fertilization patients, were cultured with BMP-7 followed by RNA extraction. Human umbilical vein endothelial cells (HUVECs) were also cultured with BMP-7 followed by RNA extraction, tube formation assay, or cell count analysis. The BMP-7 stimulated VEGF messenger RNA (mRNA) and protein expression in GC significantly. In HUVEC, BMP-7 increased an approximately 1.8-fold in the cell number and induced the tube formation significantly compared to control. The BMP-7 also induced a 2-fold increase in VEGF receptor mRNA transcript relative abundance in HUVEC. The BMP-7, a theca cell-derived factor, may stimulate endothelial cell to form vasculature in the follicle via 2 distinct mechanisms, induction of VEGF expression in GC and increased sensitivity of endothelial cells to VEGF.

Keywords: BMP-7; VEGF; angiogenesis; folliculogenesis.

Figure 1.

Bone morphogenetic protein (BMP)-7 induced vascular endothelial growth factor (VEGF) A expression in human granulosa cells (GCs). Cultured human GCs were stimulated with BMP-7 (1, 10, and 100 ng/mL) for 24 hours (A) or with BMP-7 (100 ng/mL) for 6 to 24 hours (B). Total RNA was extracted from the cells and subjected to real-time polymerase chain reaction (PCR) to determine the VEGF-A messenger RNA (mRNA) levels. Data were normalized by GAPDH mRNA levels to show the relative abundance. C, VEGF-A concentration in cultured supernatant stimulated with BMP-7 (100 ng/mL, 24 hours) was measured using enzyme-linked immunosorbent assay. Data from at least 4 different experiments were combined and shown as the mean ± standard error of the mean relative to an adjusted value of 1.0 for the mean value of the each control. *P < .01 (vs control).

Figure 2.

The role of bone morphogenetic protein (BMP)-7 in human umbilical vein endothelial cells (HUVECs). A, Preconfluent-cultured HUVEC in 96-well plates were treated with vehicle (0.1% BSA + 4 mmol/L HCl) or BMP-7 (100 ng/mL) in DMEM/F12 without serum. After 48 hours of treatment, HUVEC number was determined using a cell counting kit. B, Tube formation assay; Matrigel (growth factor reduced) was spread onto 48-well chamber slides. HUVEC (3 × 10⁴/well) were plated and incubated with or without BMP-7 (100 ng/mL). After 18 hours, the number of tubes formed was counted in 3 fields. Mean data from 10 different experiments were shown as the mean ± standard error of the mean relative to an adjusted value of 1.0 for the mean value of the control. *P < .01 (vs control). Representative pictures of control and BMP-7 (100 ng/mL) were shown.

Figure 3.

Bone morphogenetic protein (BMP) 7 induced vascular endothelial growth factor (VEGF) receptor transcript relative abundance in human umbilical vein endothelial cells (HUVECs). Cultured HUVECs were stimulated with BMP-7 (100 ng/mL) for 24 hours. Total RNA was extracted from the cells and subjected to real-time polymerase chain reaction (PCR) to determine the VEGF receptor messenger RNA (mRNA) transcript relative abundance. Data were normalized by GAPDH mRNA levels. Data from 3 different experiments were combined and shown as the mean ± standard error of the mean relative to an adjusted value of 1.0 for the mean value of the control. *P < .01 (vs control).