The Effect of Stress-Induced Senescence on Aging Human Cord Blood-Derived Endothelial Cells

Abstract

Purpose We sought to determine the effect of stress-induced senescence on the permeability to albumin of aging endothelial progenitor cells. Methods Human umbilical cord blood derived endothelial cells (hCB-ECs) and human aortic endothelial cells (HAECs) were treated with 200 μM H2O2 and permeability to FITC-bovine serum albumin was measured. Some samples were subsequently treated with 100μM 8-pCPT-2'-O-Me-cAMP, a cAMP analog that activates the Epac1-Rap1 pathway. Cell proliferation was measured with the EdU assay. Phase contrast, and immunofluorescence images were taken to observe morphological changes in cells after exposure to H2O2. Results hCB-ECs exposed to H2O2 exhibited a significant increase in permeability, but their response differed from the HAECs. Low passage hCB-ECs had a permeability increase of about 82% (p<0.01) compared to aged cells which had a permeability increase of about 37% (p<0.05). This increase in permeability was reduced by treating the cells with 100 μM 8-pCPT-2'-O-Me-cAMP. The younger cells exhibited a significant decrease in proliferation after being subjected to various concentrations of H2O2 whereas the aged cells exhibited a more gradual decrease in the percent of cells in S-phase. These changes also correlated with changes in cell morphology and junction staining. When placed back in the original media, the morphology and permeability of the hCB-ECs returned to the control condition, while the HAECs did not. Conclusions The permeability of low and high passage hCB-ECs and HAECs initially increases in response to oxidative stress. hCB-ECs, but not HAECs, were able to recover from the stress 24 hours later. Early passage hCB-ECs were more susceptible to exogenous H2O2 than late passage hCB-ECs. The increase in permeability of hCB-ECs to H2O2 also correlated with decreased cell proliferation and changes in cell junctions.

Endothelial Cell Senescence Increases Traction Forces due to Age-Associated Changes in the Glycocalyx and SIRT1

“…Because elevating SIRT1 plays an important role in reversal of senescence-associated phenotypes 10, 11, 14, 40, 66 and regulation of shear stress-induced eNOS, 9 we sought to determine whether modulation of SIRT1 altered cell traction forces. Isolated aged ECs (> 44 PDL) had significantly higher traction forces compared to the untreated young (< 31 PDL) hCB-EC control (Figure 5, p<0.05).…”

“…Further, aging endothelium exhibits increased permeability to macromolecules 11 and increased sensitivity to oxidative stress. 10 A localized increase in the permeability of the endothelial layer to proteins 24 is one of the earliest events in atherosclerosis development and influences the progression of atherosclerosis. 36, 52 …”

“…In vitro, exposure of ECs to oxidative stress from H 2 O 2 treatment elevated their permeability. 5, 10, 38 Reactive oxygen species regulate the dynamics of the actin cytoskeleton 35 and cell traction forces. 32 Oxidative stress can disrupt the glycocalyx – a thin layer of glycoproteins and proteoglycans on the surface of endothelial cells both in vitro and in vivo .…”

“…9 Activation of SIRT1 reverses several age-associated phenotypes. 10, 11, 40, 66 In ApoE-knockout mice, overexpression of SIRT1 in ECs decreased atherosclerosis. 40 Furthermore, SIRT1 may regulate permeability through regulation of cell junction proteins, such as occludin.…”

Application of Oxidative Stress to a Tissue-Engineered Vascular Aging Model Induces Endothelial Cell Senescence and Activation

“…H 2 O 2 has been used in many studies to examine the effects of oxidative stress or stress-induced senescence on endothelial cells in vitro. In some studies, H 2 O 2 is applied at a high dose, often in excess of 250 µM H 2 O 2 for less than 12 h [18]. While this significantly increased the senescence of the endothelial cells, this dose of H 2 O 2 is nearly eight times the highest estimated plasma concentrations of H 2 O 2 [19].…”