Receptor pathways of glycated basic fibroblast growth factor affecting the proliferation and vascularization of human dermal microvascular endothelial cells

Objective To investigate the receptor pathways of glycated basic fibroblast growth factor (bFGF) on proliferation and vascularization of human dermal microvascular endothelial cells (HDMECs). Methods The experimental research method was used. Glycated bFGF stimulating solution was prepared with glucose and bFGF. HDMECs of the third to sixth passages were used in the experiment. Cells were divided into small interfering RNA (siRNA)-positive control group, siRNA-negative control group, siRNA-receptor for advanced glycation end product (RAGE) group, and siRNA-receptor for fibroblast growth factor (FGFR) group and transfected with siRNA-positive control glyceraldehyde-3-phosphate dehydrogenase, siRNA-negative control, siRNA-RAGE, and siRNA-FGFR for 4 to 6 hours, and then were added into HDMEC culture medium for routine culture. The transfection effect of siRNA was identified by reverse transcription polymerase chain reaction. The cells were divided into normal control group, glycated bFGF alone group, siRNA-RAGE alone group, and siRNA-RAGE+ glycated bFGF group, and seeded into 96-well plate and 6-well plate. Cells in siRNA-RAGE alone group and siRNA-RAGE+ glycated bFGF group were transfected with siRNA-RAGE and then were added into HDMEC culture medium for routine culture. After two days, the original HDMEC culture medium was discarded, and cells in siRNA-RAGE alone group were routinely cultured in HDMEC culture medium, cells in siRNA-RAGE+ glycated bFGF group were routinely cultured in glycated bFGF stimulating solution. Cells in normal control group were routinely cultured in HDMEC culture medium, and cells in glycated bFGF alone group were routinely cultured in glycated bFGF stimulating solution. After transfection with siRNA-RAGE, cells were seeded into 48-well plate and divided into siRNA-RAGE alone group and siRNA-RAGE+ glycated bFGF group. Another cells were directly seeded into 48-well plate without transfection and divided into normal control group and glycated bFGF alone group. Cells in the 4 groups were conducted with the corresponding treatment as above. Cells were divided into normal control group, glycated bFGF alone group, siRNA-FGFR alone group, and siRNA-FGFR+ glycated bFGF group and seeded into 96-, 6-, and 48-well plates, respectively, with the corresponding treatment the same as above. Only siRNA-RAGE was replaced by siRNA-FGFR. Cell counting kit 8 method was used to determine the proliferation of cells after 2 days of culture (sample number was 6), flow cytometry was used to detect the apoptosis of cells after 2 days of culture (sample number was 3), tube forming test was used to detect the angiogenesis of cells after 6 hours of culture (sample number was 4). Data were statistically analyzed with one-way analysis of variance and least significant difference

test. Results At the 200 bp band, there were no target genes in siRNA-positive control group, siRNA-RAGE group, or siRNA-FGFR group, but target genes were detected in siRNA-negative control group, indicating the success of siRNA transfection. After 2 days of culture, the absorbance value of cells in glycated bFGF alone group was significantly lower than that of normal control group (

=2.359,

<0.05); absorbance value of cells in siRNA-RAGE+ glycated bFGF group was significantly higher than that of glycated bFGF alone group (

=3.858,

<0.01), which was similar to that of siRNA-RAGE alone group (

=2.148,

>0.05). The absorbance value of cells in siRNA-FGFR+ glycated bFGF group was similar to that of glycated bFGF alone group (

=0.805,

>0.05), but significantly lower than that of siRNA-FGFR alone group (

=4.201,

<0.01). After 2 days of culture, the apoptotic rate of cells in glycated bFGF alone group was significantly higher than that of normal control group (

=2.416,

<0.05). The apoptotic rate of cells in siRNA-RAGE+ glycated bFGF group was significantly lower than the rates in glycated bFGF alone group and siRNA-RAGE alone group (

=3.861, 2.724,

<0.05 or

<0.01). There were no statistically significant differences in apoptosis rate of cells among normal control group, glycated bFGF alone group, siRNA-FGFR alone group, and siRNA-FGFR+ glycated bFGF group (

=2.218,

>0.05). After 6 hours of culture, the number of tubules of cells in normal control group (636±5) was significantly more than that of glycated bFGF alone group (580±8,

=10.825,

<0.01), and the number of tubules of cells in siRNA-RAGE+ glycated bFGF group (647±10) was significantly more than those of glycated bFGF alone group and siRNA-RAGE alone group (628±4,

=13.040, 3.641,

<0.01). After 6 hours of culture, the number of tubules of cells in siRNA-FGFR+ glycated bFGF group (619±5) was more than that of glycated bFGF alone group (

=9.000,

<0.01), but less than that of siRNA-FGFR alone group (632±3,

=2.814,

<0.05). Conclusions Glycated bFGF affects the proliferation and angiogenesis of HDMEC through RAGE pathway, which may be one of the reasons for impaired wound healing of diabetic skin.