Cystatin C Expression is Promoted by VEGFA Blocking, With Inhibitory Effects on Endothelial Cell Angiogenic Functions Including Proliferation, Migration, and Chorioallantoic Membrane Angiogenesis

Abstract

Background Vascular development, including vasculogenesis and angiogenesis, is involved in many diseases. Cystatin C ( CST 3) is a commonly used marker of renal dysfunction, and we have previously reported that its expression level is associated with variations in the gerbil circle of Willis. Thus, we hypothesized that CST 3 may affect endothelial function and angiogenic capacity. In the current study, we sought to determine the influence of CST 3 on endothelial function and explore its potential regulatory pathway. Methods and Results We analyzed CST 3 and vascular endothelial growth factor A ( VEGFA) levels in different developmental stages of gerbils using ELISA s and immunofluorescence (to examine the relationship between CST 3 and VEGFA . We used a real-time cell analyzer, cytotoxicity assays, and the chorioallantoic membrane assay to investigate the function of CST 3 in endothelial cells and the chorioallantoic membrane. Additionally, we used Western blotting to explore the downstream targets of CST 3. The expression levels of both CST 3 and VEGFA were at their highest on day 10 of the embryonic stage. CST 3 inhibited endothelial cell proliferation, migration, tube formation, and permeability, as well as vascular development in the chorioallantoic membrane. Blocking of VEGFA dose-dependently increased CST 3 expression in arterial and venous endothelial cells. Furthermore, overexpression and knockdown of CST 3 significantly affected the protein levels of p53 and CAPN10 (calpain 10), suggesting that CST 3 might play a role in vascular development through these proteins. Conclusions CST 3 may be associated with vascular development and angiogenesis, and this effect could be promoted by blocking VEGFA .

Keywords: cystatin C; stroke; vascular endothelial function; vascular endothelial growth factor.

Figure 1

The mRNA levels of CST3 and VEGFA in different developmental stages of gerbils (embryo [prenatal], brain and heart [postnatal]). A and B, mRNA levels of CST3 and VEGFA in different developmental stages of gerbil’ brains. C and D, mRNA levels of CST3 and VEGFA in different developmental stages of gerbil hearts. P‐embryo (prenatal), G#N‐brain (postnatal), G#X‐heart (postnatal). Sample sizes: n=3. CST3 indicates cystatin 3; VEGFA, vascular endothelial growth factor A.

Figure 2

CAM assays. A, The effect of CST3 on vascular development in the CAM assay. B and C, The statistical results of the CAM assay. Sample sizes: n=3. Comparisons between different groups were conducted using one‐way ANOVA. Bar charts show the mean±SEM. CAM indicates chorioallantoic membrane; CTL, control; CST3, cystatin 3; VEGFA, vascular endothelial growth factor A. *P≤ 0.05 and ^† P≤0.01 showed significant difference.

Figure 3

Cell proliferation ability as determined by RTCA analysis. A, HUVEC and RBMEC proliferation following treatment with CST3 and/or VEGF proteins. B, HUVEC and RBMEC proliferation following treatment with CST3 protein and/or VEGF blocking peptide. C, HUVEC proliferation following treatment with CST3 blocking peptide and/or VEGF protein. D, HUVEC and RBMEC proliferation following treatment with CST3 and/or VEGF blocking peptides. Sample sizes: n=3. Comparisons between different groups were conducted using repeated measures ANOVA. CTL indicates control; CST3, cystatin 3; VEGFA, vascular endothelial growth factor A. HUVECs indicate human umbilical vein endothelial cells; RBMECs, rat brain microvessel endothelial cells, RTCA, real‐time cell analyzer; VEGFA, vascular endothelial growth factor A.

Figure 4

Cytotoxicity as determined by the MTT test. A, Percentage of viable HUVECs and RBMECs treated with CST3 and/or VEGF proteins. B, Percentage of viable HUVECs and RBMECs treated with CST3 protein and/or VEGF blocking peptide. C, Percentage of viable HUVECs and RBMECs treated with CST3 blocking peptide and/or VEGF protein. D, Percentage of viable HUVECs and RBMECs treated with CST3 and/or VEGF blocking peptide. Sample sizes: n=8. Comparisons between different groups were conducted using one‐way ANOVA. Bar charts show the mean±SEM. CTL indicates control; CST3, cystatin 3; HUVEC, human umbilical vein endothelial cells; MTT, 3‐(4,5‐dimethyl‐thiazoyl)‐2,5‐diphenyl‐SH‐tetrazolium bromide; RBMECs, rat brain microvessel endothelial cells; VEGFA, vascular endothelial growth factor A. *P≤0.05 and ^† P≤0.01 showed significant difference.

Figure 5

Cell migration ability as determined by RTCA analysis. A, Cell migration of HUVECs and RBMECs treated with CST3 and/or VEGF protein. B, Cell migration of HUVECs and RBMECs treated with CST3 protein and/or VEGF blocking peptide. C, Cell migration of HUVECs treated with CST3 blocking peptide and/or VEGF protein. D, Cell migration of HUVECs and RBMECs treated with CST3 and/or VEGF blocking peptide. Sample sizes: n=3. Comparisons between different groups were conducted using repeated measures ANOVA. CTL indicates control; CST3, cystatin 3, HUVEC, human umbilical vein endothelial cells; RBMECs, rat brain microvessel endothelial cells; RTCA, real‐time cell analyzer.

Figure 6

Permeability of HUVEC. Permeability of HUVEC treated with CST3 or VEGFA proteins or blocking peptides. Sample sizes: n=8. Comparisons between different groups were conducted using one‐way ANOVA. Bar charts show the mean±SEM. CTL indicates control; CST3, cystatin 3; ECs, endothelial cells; HUVEC, human umbilical vein endothelial cell; VEGFA, vascular endothelial growth factor A. *P≤0.05 showed significant difference.

Figure 7

The regulatory relationship between CST3 and VEGFA explored by ELISA. A, VEGFA secretion in the cell culture media of HUVECs and RBMECs treated with CST3 protein and blocking peptide. B, CST3 secretion in cell culture media of HUVECs and RBMECs treated with VEGFA protein and CST3 blocking peptide. C, CST3 secretion in cell culture media of HUVECs and RBMECs treated with different VEGFA protein concentrations. D, CST3 secretion in cell culture media of HUVECs and RBMECs treated with different CST3 blocking peptide concentrations. CTL‐control. Sample sizes: n=8. Comparisons between different groups were conducted using one‐way ANOVA. Bar charts show the mean±SEM. CTL, indicates control; CST3, cystatin 3; HUVEC, human umbilical vein endothelial cells; RBMECs, rat brain microvessel endothelial cells; VEGFA, vascular endothelial growth factor A. *P≤0.05 showed significant difference.

Figure 8

VEGFA blocking peptide increased CST3 protein expression level. A, Immunofluorescence staining of CST3 in HUVECs treated with different concentrations of VEGF protein and blocking peptide. B, Immunofluorescence staining of CST3 in RBMECs treated with different concentrations of VEGF protein and blocking peptide. C, The statistical results of immunofluorescence staining in HUVECs and RBMECs. Sample sizes: n=8. Comparisons between different groups were conducted using one‐way ANOVA. Bar charts show the mean±SEM. CTL indicates control; CST3, cystatin 3; HUVEC, human umbilical vein endothelial cells; RBMECs, rat brain microvessel endothelial cells; VEGFA, vascular endothelial growth factor A. *P≤0.05 showed significant difference.

Figure 9

VEGFA blocking peptide increased CST3 mRNA of CST3 expression level. After treated with VEGF blocking peptide, the mRNA level of CST3 increased in HUVEC. CTL‐control. Sample sizes: n=8. Comparisons between different groups were conducted using Student t test. Bar charts show the mean±SEM. CAM indicates chorioallantoic membrane; CTL, control; CST3, cystatin 3; HUVEC, human umbilical vein endothelial cell; VEGFA, vascular endothelial growth factor A. *P≤0.05 showed significant difference.

Figure 10

Proliferation and migration of CST3 overexpression and shRNA interference ECs. A, The mRNA levels of CST3 after lentiviral vector construction and infection in the CST3 overexpression and shRNA interference ECs. B, Protein levels of CST3 after lentiviral vector construction and infection in the CST3 overexpression and shRNA interference ECs. C, Migration ability of ECs by wound‐scratch assay. D, Migration ability of ECs by transwell. E, Proliferation ability of ECs by MTT. F, VEGF content in culture medium of ECs. CTL‐control. Sample sizes: n=8. Comparisons between different groups were conducted using one‐way ANOVA. Bar charts show the mean±SEM. CTL indicates control; CST3, cystatin 3; ECs, endothelial cells; MTT, 3‐(4,5‐dimethyl‐thiazoyl)‐2,5‐diphenyl‐SH‐tetrazolium bromide; RT‐PCR, reverse transcription‐polymerase chain reaction. *P≤0.05 showed significant difference.

Figure 11

Effects of CST3 overexpression and knockdown on protein levels of P53 and CAPN10. A and B, Effects of CST3 overexpression and knockdown on protein levels of P53. C and D, Effects of CST3 overexpression and knockdown on protein levels of CAPN10. CTL‐control. Sample sizes: n=8. Comparisons between different groups were conducted using Student t test. Bar charts show the mean±SEM. CTL indicates control; CST3, cystatin 3. *P≤0.05 showed significant difference.