Role of interleukin-6 in human umbilical vein endothelial cell to mesenchymal cell transformation

Objective To observe the effect of interleukin-6 (IL-6) on the phenotype and function of human umbilical vein endothelial cells (HUVECs) and explore the role of IL-6 in the process of endothelial-to-mesenchymal transition (EndMT). Methods The experimental research method was used. Fresh umbilical cord discarded after normal maternal delivery was collected. On the second day of the primary cell isolation and cultivation, the cell morphology was observed under inverted phase contrast microscope. HUVECs of the 4th passage were identified by immunofluorescence method, and 2 batches of HUVECs ofthe 3rd to 5th passages were used for the subsequent experiments. The first batch of cells were divided into 6 groups according to the random number table (the same below): blank control group, 5 ng/mL IL-6 group, 10 ng/mL IL-6 group, 25 ng/mL IL-6 group, 50 ng/mL IL-6 group, and 100 ng/mL IL-6 group. The second batch of cells were divided into 4 groups: blank control group, 10 ng/mL IL-6 group, 25 ng/mL IL-6 group,and 50 ng/mL IL-6 group; the cells in blank control group was cultured with complete culture medium only, while the cells in the other groups were added with IL-6 of the corresponding final mass concentrations.Cells from the 1st batch were cultured for 72 hours after grouping, the morphology of HUVECS in the 6 groups was observed under inverted phase contrast microscope. At 72 h after grouping culture, the positive expressions of coagulation factor Ⅷ and α vascular smooth muscle actin (α-SMA) in HUVECs in the 6 groups were detected by immunofluorescence method, and the ratio of the number of double positive cells to the number of coagulation factor Ⅷ positive cells (the ratio of double positive cells for short) was calculated, with 6 samples per group; mRNA expression levels of vascular endothelial cadherin and α-SMA of HUVECs in 6 groups were detected by reverse transcription-polymerase chain reaction, with 3 samples per group.Cells from the 2nd batch were cultured 72 hours after grouping, the protein expression levels of vascular endothelial cadherin, α-SMA, and type Ⅰ collagen in the 4 groups were detected by Western blotting, with 3 samples per group. Data were statistically analyzed with one-way analysis of variance and Bonferroni correction. Results On the 2nd day after isolation and cultivation, the primary cells were in short spindle shape or polygon, cells of the 4th passage were identified as HUVECs by immunofluorescence method. At 72 hours of culture after grouping, the cells from the 1st batch in the 6 groups changed to long spindle shape morphologically along with the increase of IL-6 concentration, the intercellular connections decreased or disappeared with the gap between cells becoming larger. At 72 h after grouping culture, compared with that inblank control group, the ratio of double positive cells in 25 ng/mL IL-6 group, 50 ng/mL IL-6 group, and 100 ng/mL IL-6 group were significantly increased (

<0.01); compared with that in 5 ng/mL IL-6 group, the ratio of double positive cells in 25 ng/mL IL-6 group, 50 ng/mL IL-6 group, and 100 ng/mL IL-6 group were significantly increased (

<0.01); compared with that in 10 ng/mL IL-6 group, the ratio of double positive cells in 50 ng/mL IL-6 group and 100 ng/mL IL-6 group were significantly increased (

<0.01); the ratio of double positive cells in 100 ng/mL IL-6 group was significantly increased compared with those in 25 ng/mL IL-6 group and 50 ng/mL IL-6 group (

<0.01). At 72 h after grouping culture, compared with that in blank control group, the mRNA expression levels of vascular endothelial cadherin of cells in 25 ng/mL IL-6 group, 50 ng/mL IL-6 group, and 100 ng/mL IL-6 group were significantly decreased (

<0.01 or

<0.05); compared with that in 5 ng/mL IL-6 group, the mRNA expression levels of vascular endothelial cadherin of cells in 50 ng/mL IL-6 group and 100 ng/mL IL-6 group were significantly decreased (

<0.01); compared with that in 10 ng/mL IL-6 group, the mRNA expression levels of vascular endothelial cadherin of cells in 50 ng/mL IL-6 group and 100 ng/mL IL-6 group were significantly decreased (

<0.01); compared with that in 25 ng/mL IL-6 group, the mRNA expression levels of vascular endothelial cadherin of cells in 50 ng/mL IL-6 group and 100 ng/mL IL-6 group were significantly decreased (

<0.01). At 72 h after grouping culture, compared with that in blank control group, the mRNA expression levels of α-SMA of cells in 5 ng/mL IL-6 group, 10 ng/mL IL-6 group, 25 ng/mL IL-6 group, 50 ng/mL IL-6, group, and 100 ng/mL IL-6 group were significantly increased (

<0.05 or

<0.01). Cells from the 2nd batch were cultured for 72 hours after grouping. Compared with 1.391±0.026 in blank control group, the protein expressions of vascular endothelial cadherin of cells in 10 ng/mL IL-6 group (1.185±0.063), in 25 ng/mL IL-6 group (0.717±0.078), and in 50 ng/mL IL-6 group (0.239±0.064) were significantly decreased (

<0.05); compared with that in 10 ng/mL IL-6 group, the protein expressions of vascular endothelial cadherin of cells in 25 ng/mL IL-6 group and 50 ng/mL IL-6 group were significantly decreased (

<0.01); compared with that in 25 ng/mL IL-6 group, the protein expression of vascular endothelial cadherin of cells in 50 ng/mL IL-6 group was significantly decreased (

<0.01). At 72 h after grouping culture, compared with that in blank control group, the protein expression levels of α-SMA of cells in 10 ng/mL IL-6 group, 25 ng/mL IL-6 group, and 50 ng/mL IL-6 group were significantly increased (

<0.01); compared with that in 10 ng/mL IL-6 group, the protein expression levels of α-SMA of cells in 25 ng/mL IL-6 group and 50 ng/mL IL-6 group were significantly increased (

<0.01). At 72 h after grouping culture, compared with that in blank control group, the protein expressions of type Ⅰ collagen of cells in 25 ng/mL IL-6 group and 50 ng/mL IL-6 group were significantly increased (

<0.05). Conclusions After IL-6 treatment, the phenotype and function of HUVECS showed the characteristics of mesenchymal cells in a concentration-dependent manner. The inflammatory factor can promote the process of EndMT, and become one of the important factors regulating the mechanism of tissue fibrosis.