Competence Effect of Basic Fibroblast Growth Factor on Cell Cycle in Subcultured Endothelial Cells of Rat Aorta by Flow Cytometry

Abstract

ABSTRACT-With flow cytometry, we investigated the effect of basic fibroblast growth factor (bFGF)-induced competence in subcultured endothelial cells (EC) (4-9-passage) of rat thoracic aorta. The cell population in each phase of the cell cycle was determined by a double staining technique with fluorescein isothiocyanate-conjugated mouse monoclonal antibody against the proliferation-associated nucleus antigen Ki-67 and propidium iodide for total DNA content. EC were cultured in medium containing 5% fetal bo vine serum (FBS) for 6 days. After serum-starvation for 2 days, the treatment with bFGF (3-10 ng/ml) for 12 hr promoted the entry of cells into the G1 phase from the G0 phase concentration-dependently.bFGF (10 ng/ml) increased the cell population in the G1 phase by 50% of the total EC, compared with the control culture without bFGF. A further 12-15-hr culture with 1% FBS after bFGF treatment promoted the entry of the cell into the S phase. Thus flow cytometric analysis demonstrates that bFGF stimulates the entry of EC into the G1 phase from the Go phase. Keywords:Basic fibroblast growth factor, Flow cytometry, Rat aortic endothelial cell, Ki-67 antigen, Propidium iodideAbnormal proliferation of vascular endothelial cells (EC) causes many diseases such as diabetic retinopathy (1) and atherosclerosis (2). Development of these angio genic diseases are reported to be induced several growth factors such as basic fibroblast growth factor (bFGF), platelet-derived growth factor and interleukin-8 (3 5). Growth factors are classified into two types, a compe tence factor and a progression factor, based on their modes of enhancement of cell proliferation (6). The com petence factors advance the cell cycle from the Go to G1 phase, whereas the progression factors promote entrance to the S phase from the G1 phase (7). bFGF, a compe tence factor not a progression factor, can induce DNA synthesis only in combination with a progression factor containing platelet-poor plasma in Balb/3T3 fibroblast (7). bFGF is also a potent mitogen for EC proliferation since the bFGF effect is enhanced in an autocline manner in vitro (8). In the previous study, we demonstrated that bFGF hastens the starting time of [3H]-thymidine incor poration into subcultured EC of rat aorta, suggesting the competence effect of bFGF (9). However, it remains to be determined if bFGF is a competence factor in EC as well as in fibroblasts.A proliferation-associated nucleus antigen, Ki-67, is a useful tool for detecting the transition from the Go to G1 phase, because this antigen is expressed in actively grow ing cells but not in Go-arrested (quiescent) cells (10, 11). In the present study using flow cytometry with double staining of Ki-67 antigen and total DNA, we provided evidence that bFGF stimulates the competence in quies cent EC of rat thoracic aorta. MATERIALS AND METHODS Cell cultureRat aortic EC in primary culture were prepared by the method reported by Schwartz (12) and Nagaura et al. (9) as follows: The thoracic aorta of male Wistar rats (9 week-old, 24...

Platelet-Derived Growth Factor Blocks the Cell-Cycle Transition from the G0 to G1 Phase in Subcultured Angiogenic Endothelial Cells in Rat Thoracic Aorta.

“…After a 5-day culture with 10% FBS-DMEM and the subsequent 2-day culture under serum-starvation, most of EC population were distri buted at the Go phase of the cell cycle (Fig. 4a) by gating analysis of green fluorescence of FITC bound to Ki-67 and red fluorescence of PI bound to DNA as shown previously (8). The percentages of cell population at each phase relative to the total number of EC were estimated to be 84.9± 1.1 % at the Go phase, 4.6±0.4% at the G1 phase, 7.4± 1.0% at the S phase, 5.2±0.4% at the G2/M phases (n = 3).…”

“…PDGF induces the com petence in quiescent Balb/c 3T3 fibroblasts as well as bFGF (11). We have previously reported that bFGF stimulates the Go-arrested population of proliferative EC of rat aorta in the 5-day culture to promote enterance into the G1 phase, as a competence factor (8). In contrast, the present study shows that PDGF-BB inhibited the compe tence of the angiogenic EC growth by blocking the tran sition from the Go to G1 phase after a long-time culture (35 days).…”

“…To achieve cellular quiescence, these EC were arrested by serum starvation for 2 days (Fig. IA-a) (8). The EC were at the mixed state of cell cycles.…”