Vascular Endothelial Growth Factor Receptor-1 Modulates Hypoxia-Mediated Endothelial Senescence and Cellular Membrane Stiffness via YAP-1 Pathways

Abstract

Hypoxia-induced endothelial cell (EC) dysfunction has been implicated as potential initiators of different pathogenesis, including Alzheimer's disease and vascular dementia. However, in-depth structural, mechanical, and molecular mechanisms leading to EC dysfunction and pathology need to be revealed. Here, we show that ECs exposed to hypoxic conditions readily enter a senescence phenotype. As expected, hypoxia upregulated the expression of vascular endothelial growth factor (VEGFs) and its receptors (VEGFRs) in the ECs. Interestingly, Knockdown of VEGFR-1 expression prior to hypoxia exposure prevented EC senescence, suggesting an important role of VEGFR-1 expression in the induction of EC senescence. Using atomic force microscopy, we showed that senescent ECs had a flattened cell morphology, decreased membrane ruffling, and increased membrane stiffness, demonstrating unique morphological and nanomechanical signatures. Furthermore, we show that hypoxia inhibited the Hippo pathway Yes-associated protein (YAP-1) expression and knockdown of YAP-1 induced senescence in the ECs, supporting a key role of YAP-1 expression in the induction of EC senescence. And importantly, VEGFR-1 Knockdown in the ECs modulated YAP-1 expression, suggesting a novel VEGFR-1-YAP-1 axis in the induction of hypoxia-mediated EC senescence. In conclusion, VEGFR-1 is overexpressed in ECs undergoing hypoxia-mediated senescence, and the knockdown of VEGFR-1 restores cellular structural and nanomechanical integrity by recovering YAP-1 expression.

Keywords: atomic force microscopy; endothelial cells; hippo pathway; hypoxia; nano mechanics; senescence.

FIGURE 1

Hypoxia induce senescence phenotype in HUVECs. (A) β-galactosidase staining showing HUVECs (Passage 3) treated with normoxia (Nor) and (Hyp) hypoxia for 72 h. (B) Quantitative analysis of the β-galactosidase staining from (A) (X-axis represents the treatment groups and the Y-axis represents the percentage of cells which are positive for β-galactosidase staining. Relative mRNA expression of VEGF and receptors genes in HUVECs treated with (C) normoxia (Nor) and 24 h hypoxia (Hyp 24 h). (D) with normoxia and 72 h hypoxia (Hyp 72 h). (E) Western blot showing protein expression after exposure to normoxia and hypoxia for 24 h. (F) Quantitative analysis of the western blot. (Nor, Normoxia and Hyp, Hypoxia). The error bars represent mean ± SD. These data represent 3 independent experiments. (*, p < 0.05 and **, p < 0.01, Students t-test). (Scale bar = 100 µm).

FIGURE 2

VEGFR-1 knockdown prevents hypoxia induced senescence in HUVECs. (A–C) β-galactosidase staining showing HUVECs (Passage 3) treated with normoxia and hypoxia for 72 h with and without VEGFR-1 siRNA, respectively. (D) Quantitative analysis of the β-galactosidase staining from (A–C) (X-axis represents the treatment groups and the Y-axis represents the percentage of cells which are positive for β-galactosidase staining). (E) HUVECs treated with siRNA targeting VEGFR-1 for 24 h, followed by 72 h exposure to hypoxia prevents the upregulation of senescence associated markers. Data shown are from three independent experiments. The error bars represent mean ± SD. (*. p < 0.05, ***, p < 0.01 and ***, p < 0.001, Student’s t-test). (Scale bar = 100 µm).

FIGURE 3

Morphology characterization of HUVECs upon hypoxia treatment. Representative Height image of HUVEC following (A) Normoxia. (B) 24 h hypoxia. (C) 48 h hypoxia. (D) 72 h hypoxia. Representative Peak force error image of HUVEC following (E) Normoxia. (F) 24 h hypoxia (G) 48 h hypoxia. (H) 72 h hypoxia. (I) Average height profile of cells (n = 8). (J) Average roughness over the nuclear membrane region (n = 8). (ns, not significant; **, p < 0.01, Students t-test) (Scale bars = 10 µm).

FIGURE 4

Comparative study of nanomechanical properties of HUVECs derived from the force-separation curves post various treatments. Force-separation curves for (A) Normoxia. (B) 24 h hypoxia. (C) 48 h hypoxia. (D) 72 h hypoxia. Nanomechanical properties determined from (n = 16) and comprising of (E) Young’s modulus. (F) Deformation. (G) Adhesion. (ns, not significant; ****, p < 0.0001, Students t-test).

FIGURE 5

Morphology and nanomechanical properties of HUVECs following VEGFR-1 knockdown. In the presence of normoxia, representative height image of (A) Control siRNA. (B) VEGFR-1 siRNA. In the presence of 72 h hypoxia, representative height image of (C) Control siRNA. (D) VEGFR-1 siRNA. In the presence of normoxia, representative peak force error image of (E) Control siRNA. (F) VEGFR-1 siRNA. In the presence of 72 h hypoxia, representative peak force error image of (G) Control siRNA. (H) VEGFR-1 siRNA. (I) Average height profile of cells (n = 8). (J) Average roughness over the nuclear membrane region (n = 8). Nanomechanical properties of VEGFR-1 siRNA knockdown HUVECs followed by various time duration exposure of hypoxia treatment (n = 16 cells). (K) Young’s modulus. (L) Deformation. (M) Adhesion. (ns, not significant; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, Students t-test). (Scale bar = 20 μm)

FIGURE 6

YAP-1 downregulation induces senescence in HUVECs. (A) Representative western blot image showing expression of YAP and phospho-YAP when HUVECs were exposed to hypoxia for 24, 48, and 72 h. (B). Quantification showing the fold change of the protein expression. (C) mRNA expression of YAP-1 in HUVEC treated with hypoxia for 24, 48, and 72 h. (D) β-gal staining of the HUVEC cells after YAP-1 siRNA treatment (Scale bar 200 µm) and (E) quantification of the β-gal staining. (F) Western blot result showing effect of YAP-1 siRNA knockdown on senescence marker p16 and p21 expression (G) Quantification of (F) showing significant upregulation of p21 and p16. (G) mRNA expressions of YAP1 pathways genes. Error bars indicates the mean ± SD. *, p < 0.05, Student’s t-test.) (Scale bars = 50 µm). AU = Arbitrary Units *, p < 0.05; **, p < 0.01, Student t-test). (Scale bar = 200 µm).

FIGURE 7

VEGFR-1 Knockdown prevents hypoxia mediated YAP-1 downregulation in HUVECs. (A–C) Representative immunofluorescence image showing the expression of YAP-1 in (A) Normoxia (Nor), (B) hypoxia for 72 h (Hyp) and (C) VEGFR-1 knockdown followed by 72 h hypoxia. (Insert shows higher magnification of the cells and the arrowheads indicate the representative cells used.) Green = YAP-1, Blue = DAPI (D) Quantification of the fluorescence intensity of YAP-1 staining from (A–E) Representative western blot showing VEGFR-1 and YAP-1 expression in ECs following VEGFR-1 siRNA and hypoxia treatment. (F) Quantification of the VEGFR-1 expression from the western blot from (E). (G) Quantification of the YAP-1 expression from the western blot from (E). (H) mRNA expressions of YAP1 pathways genes. Error bars indicates the mean ± SD. *, p < 0.05, Student’s t-test.) (Scale bars = 50 µm). AU = Arbitrary Units.