Effects of age-dependent changes in cell size on endothelial cell proliferation and senescence through YAP1

Abstract

Angiogenesis - the growth of new blood capillaries- is impaired in aging animals. Biophysical factors such as changes in cell size control endothelial cell (EC) proliferation and differentiation. However, the effects of aging on EC size and the mechanism by which changes in cell size control age-dependent decline in EC proliferation are largely unknown. Here, we have demonstrated that aged ECs are larger than young ECs and that age-dependent increases in EC size control EC proliferation and senescence through CDC42-Yes-associated protein (YAP1) signaling. Reduction of aged EC size by culturing on single-cell sized fibronectin-coated smaller islands decreases CDC42 activity, stimulates YAP1 nuclear translocation and attenuates EC senescence. Stimulation of YAP1 or inhibition of CDC42 activity in aged ECs also restores blood vessel formation. Age-dependent changes in EC size and/or CDC42 and YAP1 activity may be the key control point of age-related decline in angiogenesis.

Keywords: aging; angiogenesis; cell proliferation; cell size; senescence.

Figure 1

Age-dependent changes in human adipose EC size, proliferation and senescence. (A) Silver nitrate-stained <50 years old (<50 y.o.) and >50 years old (>50 y.o.) human adipose tissue blood vessels. Scale bar, 20 μm. Graphs showing quantification of cell area (left) and cell density (right) in blood vessels dissecting from <50 y.o. and >50 y.o. human adipose tissues (n=27, 28, mean ± s.e.m., *, p<0.05). (B) Immunofluorescence (IF) micrographs showing VE-cadherin-positive cell-cell junctions and DAPI (top) and paxillin-positive focal adhesions and actin stress fiber formation (bottom). Scale bar, 20 μm. Graphs showing quantification of cell area (left) and nuclear size (right) of ECs isolated from <50 y.o. and >50 y.o. human adipose tissues (n=5, mean ± s.e.m., *, p<0.05). (C) IF micrographs showing BrdU⁺ ECs isolated from <50 y.o. and >50 y.o. human adipose tissues (top). IF micrographs showing P16^INK4A-positive ECs isolated from <50 y.o. and >50 y.o. human adipose tissues (middle). Micrographs showing SAβGal-stained ECs isolated from <50 y.o. and >50 y.o. human adipose tissues (bottom). Scale bar, 20 μm. Graphs showing quantification of BrdU⁺, P16^INK4A+, and SAβGal-stained ECs isolated from <50 y.o. and >50 y.o. human adipose tissues (n=5, mean ± s.e.m., *, p<0.05). (D) Graph showing P16^INK4A mRNA levels in ECs isolated from <50 y.o. and >50 y.o. human adipose tissues (n=7, mean ± s.e.m., *, p<0.05).

Figure 2

Age-dependent changes in YAP1 and CDC42 activity in human adipose ECs. (A) Representative immunoblots showing YAP1, YAP1S127 phosphorylation and β-actin protein levels in ECs isolated from <50 y.o. and >50 y.o. human adipose tissues (top). Graphs showing the quantification of immunoblots (bottom, n=6, mean ± s.e.m., *, p<0.05). (B) Representative immunoblots showing GTP-CDC42 and total CDC42 protein levels in ECs isolated from <50 y.o. and >50 y.o. human adipose tissues (top). Graph showing the quantification of immunoblots (bottom, n=6, mean ± s.e.m., *, p<0.05). (C) IF micrographs showing the levels of GTP-CDC42 in ECs isolated from <50 y.o. or >50 y.o. human adipose tissues (top). Graph showing quantification of the GTP-CDC42 levels in ECs isolated from <50 y.o. and >50 y.o. human adipose tissues (n=5, mean ± s.e.m., *, p<0.05). (D) IF micrographs showing YAP1 nuclear localization (green), actin (magenta), and DAPI (blue, top), GTP-CDC42 levels (green), actin (magenta) and DAPI (blue, middle), and P16^INK4A (green) and DAPI (blue, bottom) in ECs isolated from <50 y.o. or >50 y.o. human adipose tissues cultured on FN-coated island of different sizes. Scale bar, 10 μm. Graphs showing quantification of nuclear YAP1 (left top), GTP-CDC42 integrated density (left bottom) , and P16^INK4A integrated density (right top) (n=7, mean ± s.e.m., *, p<0.05). (E) Graph showing quantification of EdU-positive cells (n=7, mean ± s.e.m., *, p<0.05).

Figure 3

CDC42-YAP1 mediates cell size-dependent changes in EC senescence in aged ECs. (A) Representative immunoblots showing YAP1, YAP1S127 phosphorylation, and β-actin protein levels in ECs isolated from >50 y.o. human adipose tissues treated with retrovirus overexpressing full-length YAP1 or YAP1S127A (left). Graph showing the quantification of immunoblots (right, n=3, *, p<0.05). (B) IF micrographs showing P16^INK4A expression and DAPI in ECs isolated from >50 y.o. human adipose tissues treated with retrovirus overexpressing full-length YAP1 or YAP1S127A, cultured on FN-coated island of different sizes. Scale bar, 10 μm. (C) Graph showing quantification of P16^INK4A integrated density (n=7, mean ± s.e.m., *, p<0.05). (D) IF micrographs showing the GTP-CDC42 levels and DAPI in ECs isolated from >50 y.o. human adipose tissues treated with ML141 (500 nM). Scale bar, 10 μm. Graph showing quantification of GTP-CDC42 integrated density (n=7, mean ± s.e.m., *, p<0.05). (E) Representative immunoblots showing YAP1, YAP1S127 phosphorylation, and β-actin protein levels in ECs isolated from >50 y.o. human adipose tissues treated with ML-141 (top). Graph showing the quantification of immunoblots (bottom, n=3, *, p<0.05). (F) IF micrographs showing P16^INK4A expression (green) and DAPI (blue, top) and YAP1 localization (green), actin structure (magenta), and DAPI (blue, bottom) in ECs isolated from >50 y.o. human adipose tissues treated with ML141 and cultured on FN-coated island of different sizes. Scale bar, 10 μm. Graphs showing quantification of P16^INK4A integrated density (bottom left) and nuclear YAP1 (bottom right) (n=7, mean ± s.e.m., *, p<0.05).

Figure 4

CDC42-YAP1 signaling mediates age-dependent decline in blood vessel formation in subcutaneously implanted gel. (A) IF micrographs showing vascular structures formed in the subcutaneously implanted fibrin gel supplemented with GFP-labeled ECs isolated from <50 y.o. or >50 y.o. human adipose tissues or in combination with treatment with ML141 (500 nM). Scale bar, 10 μm. Graphs showing quantification of vessel length (top) and vessel area (bottom) in the gel (n=7, mean ± s.e.m., *, p<0.05). (B) IF micrographs showing low MW fluorescently labeled dextran leakage (magenta) and GFP-labeled blood vessel formation (green) in the subcutaneously implanted fibrin gel supplemented with GFP-labeled ECs isolated from <50 y.o. or >50 y.o. human adipose tissues or in combination with treatment with ML141 (500 nM). Scale bar, 10 μm. Graph showing quantification of fluorescently labeled dextran leakage in the gel (n=7, mean ± s.e.m., *, p<0.05). (C) IF micrographs showing vascular structures formed in the subcutaneously implanted fibrin gel supplemented with GFP-labeled ECs isolated from >50 y.o. human adipose tissues in combination with treatment with retrovirus overexpressing full-length YAP1 or YAP1S127A mutant construct. Scale bar, 10 μm. Graphs showing quantification of vessel length (top) and vessel area (bottom) in the gel (n=7, mean ± s.e.m., *, p<0.05).