Vascular Endothelial Cell Senescence Research Articles

ENOS-Dependent Antisenscence Effect of a Calcium Channel Blocker in Human Endothelial Cells

Senescence of vascular endothelial cells is an important contributor to the pathogenesis of age-associated vascular disorders such as atherosclerosis. We investigated the effects of antihypertensive agents on high glucose-induced cellular senescence in human umbilical venous endothelial cells (HUVECs). Exposure of HUVECs to high glucose (22 mM) for 3 days increased senescence-associated- β-galactosidase (SA-β-gal) activity, a senescence marker, and decreased telomerase activity, a replicative senescence marker. The calcium channel blocker nifedipine, but not the β1-adrenergic blocking agent atenolol or the angiotensin-converting enzyme inhibitor perindopril, reduced SA-β-gal positive cells and prevented a decrease in telomerase activity in a high-glucose environment. This beneficial effect of nifedipine was associated with reduced reactive oxygen species (ROS) and increased endothelial nitric oxide synthase (eNOS) activity. Thus, nifedipine prevented high glucose-induced ROS generation and increased basal eNOS phosphorylation level at Ser-1177. Treatment with N G-nitro-L-arginine (L-NAME) and transfection of small interfering RNA (siRNA) targeting eNOS eliminated the anti-senscence effect of nifedipine. These results demonstrate that nifedipine can prevent endothelial cell senescence in an eNOS-dependent manner. The anti-senescence action of nifedipine may represent a novel mechanism by which it protects against atherosclerosis.

Interleukin-8 prevents oxidative stress-induced human endothelial cell senescence via telomerase activation

Senescence is an irreversible growth arrest which can be triggered by stresses such as oxidative reaction, telomere shortening, DNA damage, or oncogene signaling. Oxidative stress accelerates vascular endothelial cell senescence, and may promote atherosclerosis in humans. Interleukin-8 (IL-8) has been shown to play an important role in tumor growth, angiogenesis, and metastasis, and has close relationship with oxidative stress. The objective of this study was to determine if IL-8 might be able to prevent oxidative stress-induced senescence of endothelial cells and the mechanisms. Human umbilical vein endothelial cells (HUVECs) were cultured and stimulated with hydrogen peroxide in the absence or presence of IL-8. After ex vivo cultivation, HUVECs became senescent as determined by acidic beta-galactosidase staining. IL-8 dose-dependently inhibited the onset of HUVEC senescence. Western blots indicated that IL-8 attenuated the oxidative stress induced high-expression of cell cycle regulation protein and inhibited the activation of p38 and NF-κB pathway. IL-8 also increased telomerase activity which was accompanied with upregulation of the catalytic subunit, telomerase reverse transcriptase (TERT), whereas these effects were significantly attenuated by SB 225002 (selective non-peptide CXCR2 antagonist). In conclusion, IL-8 exerted protective effects against endothelial senescence, which may be related to the activation of telomerase.

Oncogene‐ and Oxidative Stress‐Induced Cellular Senescence Shows Distinct Expression Patterns of Proinflammatory Cytokines in Vascular Endothelial Cells

Senescent cells are metabolically active and produce a variety of proinflammatory cytokines. It was previously reported that atherosclerotic plaques contain senescent cells, suggesting that senescence may contribute to the progression of atherosclerosis. In this study, we induced cellular senescence in vascular endothelial cells (VECs) using hydrogen peroxide (H2O2) or an adenovirus that expresses a constitutively active mutant of Ras (AdRas12V) and studied the expression of cytokines. Both H2O2 treatment and AdRas12V infection induced senescence in VECs, as assessed by senescence-associated β-Gal activity and the expression of proteins such as p53 and p21CIP1. In addition, both treatments induced the expression of a variety of cytokines, including interleukin-1β (IL-1β) and nerve growth factor (NGF). AdRas12V infection induced IL-1β expression more significantly than H2O2 treatment, whereas both treatments induced comparable mRNA and protein expression levels of NGF. These results suggest that senescent cells express different patterns of proinflammatory cytokines, depending on the trigger that induced senescence. It is therefore possible that senescent cells can differentially induce inflammation in the surrounding tissues, depending on the cause of senescence.

The influence of flow on vascular endothelial cell senescence

The influence of flow on vascular endothelial cell senescence

Testosterone Deficiency Accelerates Neuronal and Vascular Aging of SAMP8 Mice: Protective Role of eNOS and SIRT1

Oxidative stress and atherosclerosis-related vascular disorders are risk factors for cognitive decline with aging. In a small clinical study in men, testosterone improved cognitive function; however, it is unknown how testosterone ameliorates the pathogenesis of cognitive decline with aging. Here, we investigated whether the cognitive decline in senescence-accelerated mouse prone 8 (SAMP8), which exhibits cognitive impairment and hypogonadism, could be reversed by testosterone, and the mechanism by which testosterone inhibits cognitive decline. We found that treatment with testosterone ameliorated cognitive function and inhibited senescence of hippocampal vascular endothelial cells of SAMP8. Notably, SAMP8 showed enhancement of oxidative stress in the hippocampus. We observed that an NAD+-dependent deacetylase, SIRT1, played an important role in the protective effect of testosterone against oxidative stress-induced endothelial senescence. Testosterone increased eNOS activity and subsequently induced SIRT1 expression. SIRT1 inhibited endothelial senescence via up-regulation of eNOS. Finally, we showed, using co-culture system, that senescent endothelial cells promoted neuronal senescence through humoral factors. Our results suggest a critical role of testosterone and SIRT1 in the prevention of vascular and neuronal aging.

Identification and characterisation of ataxia telangiectasia mutated (ATM) in regulation of oxidative stress-induced vascular endothelial cells senescence

BackgroundAging is known to be a major cardiovascular risk factor. Recent studies provide evidence for a critical role of cellular senescence in the process of vascular aging including hypertension and...

Effects of Rhein Lysinate on H2O2-induced cellular senescence of human umbilical vascular endothelial cells

To observe the effect of Rhein lysinate (RHL) on cellular senescence of human umbilical vascular endothelial cells (HUVECs) and elucidate its action mechanism. Cell viability was determined using MTT assay. The expression levels of Sirt1 mRNA and protein were measured by RT-PCR and Western blot, respectively. Senescence associated (SA)-β-galactosidase activity was detected to evaluate cell senescence. Apoptosis and cell cycle progression were determined using flow cytometry. Treatment with RHL (10 μmol/L) for 48 h significantly increased the proliferation of HUVECs. In contrast, treatment with H2O2 (25, 50 and 100 μmol/L) for 6 d dose-dependently increased β-galactosidase positive cells. Spontaneous cell senescence appeared as the cell passage increased. Pre-treatment with RHL (10 μmol/L) reversed H2O2 or increased cell passage-induced cell senescence. H2O2(100 μmol/L) significantly arrested HUVECs at G(1) phase (73.8% vs 64.6% in the vehicle group), which was blocked by RHL (10 μmol/L). RHL (5 and 10 μmol/L) enhanced both mRNA transcription and protein expression of Sirt1. H2O2 (100 μmol/L) significantly decreased Sirt1 expression, and induced up-regulation of p53 acetylation and p16(INK4a), which were blocked by pre-treatment with RHL (10 μmol/L). Interference with siRNA for Sirt1 abolished the effect of RHL. H2O2 (100 μmol/L) did not induce HUVEC apoptosis. The expression of apoptosis-associated proteins, such as p53, p21, Bcl-2, and Bax, did not significantly change in the presence of H(2)O(2) (100 μmol/L) or RHL (10 μmol/L). RHL protected HUVECs against cellular senescence induced by H2O2, via up-regulation of Sirt1 expression and down-regulation of the expression of acetyl-p53 and p16(INK4a).

Telomere Dysfunction Causes Sustained Inflammation in Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is associated with chronic inflammation of unknown pathogenesis. To investigate whether telomere dysfunction and senescence of pulmonary vascular endothelial cells (P-ECs) induce inflammation in COPD. Prospective comparison of patients with COPD and age- and sex-matched control smokers. Investigation of mice null for telomerase reverse transcriptase (Tert) or telomerase RNA component (Terc) genes. In situ lung specimen studies showed a higher percentage of senescent P-ECs stained for p16 and p21 in patients with COPD than in control subjects. Cultured P-ECs from patients with COPD exhibited early replicative senescence, with decreased cell-population doublings, a higher percentage of β-galactosidase-positive cells, reduced telomerase activity, shorter telomeres, and higher p16 and p21 mRNA levels at an early cell passage compared with control subjects. Senescent P-ECs released cytokines and mediators: the levels of IL-6, IL-8, monocyte chemotactic protein (MCP)-1, Hu-GRO, and soluble intercellular adhesion molecule (sICAM)-1 were elevated in the media of P-ECs from patients compared with control subjects at an early cell passage, in proportion to the senescent P-EC increase and telomere shortening. Up-regulation of MCP-1 and sICAM-1 led to increased monocyte adherence and migration. The elevated MCP-1, IL-8, Hu-GROα, and ICAM-1 levels measured in lungs from patients compared with control subjects correlated with P-EC senescence criteria and telomere length. In Tert(-/-) and/or Terc(-/-) mouse lungs, levels of the corresponding cytokines (MCP-1, IL-8, Hu-GROα, and ICAM-1) were also altered, despite the absence of external stimuli and in proportion to telomere dysfunction. Telomere dysfunction and premature P-EC senescence are major processes perpetuating lung inflammation in COPD.

MiR-146a is modulated in human endothelial cell with aging

Background Increasing evidence has demonstrated that the senescence of vascular endothelial cells has critical roles in the pathogenesis of vascular dysfunction such as atherosclerosis and thrombosis. MicroRNA (miR) are small non-coding RNAs that inhibit gene expression by binding to complementary sequences in the 3′UTR of their target mRNAs. MiRs modulate a variety of biological functions such as cell development, cell differentiation, and apoptosis. Moreover, several miRs involved in endothelial cell function have been identified. Methods and results Through a microarray approach, we have identified a miR-146a that is progressively modulated in endothelial cells with aging. In young human umbilical vein endothelial cells, this miR is involved in a premature senescence-like phenotype through direct targeting of the NOX4 protein, implicated in cell senescence and aging. Conclusions and general significance Finding important factors that regulate endothelial cell senescence, like miR-146a, will help provide novel therapeutic strategies for vascular disorders.

Modulation of vascular endothelial cell senescence by integrin β4

Increasing evidence has demonstrated that the senescence of vascular endothelial cells (VECs) has critical roles in the pathogenesis of vascular dysfunction. Finding important factors that regulate VEC senescence will help provide novel therapeutic strategies for vascular disorders. Previously, we found that integrin β4 was involved in VEC senescence. However, the mechanism underlying VEC senescence mediated by integrin β4 remains poorly understand. In this study, we used a mouse in vivo model and showed that the level of integrin β4 in the endothelium of mouse thoracic aorta was increased during natural aging and atherosclerosis. Furthermore, we found that H-ras, caveolin-1, and AP-1 were implicated in the senescent signal pathway mediated by integrin β4 in human umbilical vein ECs (HUVECs). Knockdown of integrin β4 could attenuate HUVEC senescent features, including increased interleukin-8 (IL-8) release and decreased endothelial nitric oxide synthase (eNOS) and NO levels and mitochondrial membrane potential in vitro. Our findings provide new clues illustrating the mechanism of VEC senescence. Integrin β4 might be a potential target for therapy in cardiovascular diseases.

Ataxia Telangiectasia Mutated (ATM)-mediated DNA Damage Response in Oxidative Stress-induced Vascular Endothelial Cell Senescence

Oxidative stress regulates dysfunction and senescence of vascular endothelial cells. The DNA damage response and its main signaling pathway involving ataxia telangiectasia mutated (ATM) have been implicated in playing a central role in mediating the actions of oxidative stress; however, the role of the ATM signaling pathway in vascular pathogenesis has largely remained unclear. Here, we identify ATM to regulate oxidative stress-induced endothelial cell dysfunction and premature senescence. Oxidative stress induced senescence in endothelial cells through activation/phosphorylation of ATM by way of an Akt/p53/p21-mediated pathway. These actions were abrogated in cells in which ATM was knocked down by RNA interference or inhibited by specific inhibitory compounds. Furthermore, the in vivo significance of this regulatory pathway was confirmed using ATM knock-out mice in which induction of senescent endothelial cells in the aorta in a diabetic mouse model of endothelial dysfunction and senescence was attenuated in contrast to pathological changes seen in wild-type mice. Collectively, our results show that ATM through an ATM/Akt/p53/p21-dependent signaling pathway mediates an instructive role in oxidative stress-induced endothelial dysfunction and premature senescence.

Ceramide induces endothelial cell senescence

Ceramide has been proposed to be a mediator of replicative senescence. Our aim was to determine whether ceramide induces senescence in vascular endothelial cells. Human umbilical vein endothelial cells were cultured to different population doubling levels and ceramide levels were quantitated. The endogenous levels of ceramide increased 2.4-fold with senescence onset. Low passage cells were chronically treated with exogenous C(6)-ceramide. This treatment induced a senescent phenotype as measured by an inhibition of cell proliferation and DNA replication while increasing senescence-associated beta-galactosidase expression. This is the second cell type in which ceramide induces senescence, thus implicating ceramide as a general mediator of cellular senescence.

Aging and vascular endothelial cell senescence: potential role of SIRT1 in development of inflammation and dysfunction

Aging leads to vascular endothelial dysfunction, but the mechanisms involved are incompletely understood. We assessed the effects of aging on endothelial cell (EC) senescence in vivo and the possible role of the anti‐senescence and anti‐inflammatory deacetylase, SIRT1, in vitro. Compared with young adult (5 mo) mice, old (30 mo) mice had reduced endothelium‐dependent dilation and a higher % of aortic EC that stained for the senescence marker p16INK4a (31 vs. 6). "Aged" cultures of human aortic EC (HAEC) (35±2 population doublings, PDL) had a higher % of cells stained for p16INK4a (46 vs. 7) and (beta)‐galactosidase, a 2nd marker of senescence (57 vs. 6), than "young" cultures (20±1 PDL). SIRT1 expression was 55% lower in senescent HAEC (0.45±0.08 vs. 1.00 ± 0.10 AUs, P<0.002). Senescent HAEC demonstrated reduced expression of the anti‐inflammatory proteins interleukin‐10 (0.6±0.05 vs. 1.0±0.2 AUs, P<0.05) and interleukin‐1 receptor‐α (0.7±0.05 vs. 1.0±0.2 AUs, P<0.05), and increased expression of the inflammatory protein monocyte chemoattractant protein‐1 (1.6±0.1 vs. 1.0±0.1 AUs, P<0.005). In conclusion, the % of senescent arterial EC in vivo is greater in old age. A decrease in SIRT1 may play an important role in EC senescence and contribute to development of vascular endothelial inflammation and dysfunction.Swedish Research Council; NIH AG006537, AG013038, AG022241, RR00051

Protective Effects of a Synthesized Butyrolactone Derivative against Chloroquine-Induced Autophagic Vesicle Accumulation and the Disturbance of Mitochondrial Membrane Potential and Na+,K+-ATPase Activity in Vascular Endothelial Cells

We previously found a butyrolactone derivative, 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one (3BDO), could inhibit vascular endothelial cell (VEC) apoptosis and senescence induced by a deprivation of serum and FGF-2. In this study, we aimed to investigate its actions in VEC autophagy induced by chloroquine (CQ). The measurement on the volume of acidic compartments (VAC) and autophagy analysis by acridine orange (AO) staining and microtubule-associated protein 1 light chain 3 (MAP1LC3) process revealed that 3BDO was an effective inhibitor of autophagic vesicle accumulation (vacuolation) induced by CQ in VECs. 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) was used for mitochondrial membrane potential (MMP) measurement. The results showed that CQ elevated MMP significantly and that 3BDO could significantly inhibit CQ-induced MMP increase. Na+,K+-ATPase activity assay showed that CQ inhibited this enzyme activity significantly and that 3BDO attenuated the alteration of Na+,K+-ATPase activity caused by CQ. We concluded that 3BDO was a promising inhibitor of CQ-induced accumulation of autophagic vesicles in VECs and could weaken the alterations of MMP and Na+,K+-ATPase activity induced by CQ. The data indicate that 3BDO will be a potential tool for investigating the mechanism of autophagy.

Interferon-γ induces cellular senescence through p53-dependent DNA damage signaling in human endothelial cells

Cellular senescence is a stress-response phenomenon in which cells lose the ability to proliferate; it is induced by telomere shortening, activation of oncogenes or tumor suppressor genes, or exposure to a sub-lethal dose of DNA damaging agents or oxidative stresses. cDNA microarray analysis reveals that the levels of interferons (IFNs) and IFN-inducible genes were altered during replicative senescence in human umbilical vascular endothelial cells (HUVECs). However, the role of IFNs in cellular senescence of HUVECs remains unidentified. This study demonstrated that prolonged treatment with IFN-γ induced cellular senescence in HUVECs, as confirmed by G0/G1 cell cycle arrest, up-regulation of p53 and p21 protein levels, increased SA-β-gal staining, and the accumulation of phospho-H 2AX foci. IFN-γ-induced cellular senescence was observed only in p16-knockdown cells or p16-null mouse embryonic fibroblasts (MEFs), but not in p53-knockdown cells or p53-null MEFs. IFN-γ treatment increased ROS production, and an antioxidant, N-acetylcysteine, inhibited IFN-γ-induced cellular senescence. Knockdown of ATM kinase or IFI16 rescued IFN-γ-induced cellular senescence. Therefore, these results suggest that IFN-γ might play an important role in cellular senescence through a p53-dependent DNA damage pathway and contribute to the pathogenesis of atherosclerosis via its pro-senescent activity.

HTERT overexpression delays replicative senescence but not damage‐induced senescence of vascular endothelial cells isolated from patients with severe CAD

Overexpression of the catalytic subunit of telomerase (hTERT) immortalizes healthy human endothelial cells (EC) but its effect on senescence in pathological conditions is unknown. Our objective was to investigate whether hTERT overexpression would prevent senescence in EC isolated from patients with coronary artery disease (CAD). EC isolated from discarded internal mammary arteries of 11 patients undergoing CABG were infected or not with a lentivirus expressing hTERT and cultured in vitro. We quantified senescence by cytochemical detection of senescence‐associated b‐galactosidase, hTERT activity by real‐time TRAPassay, telomere length by Southern blotting, mRNA levels (p21 and ATM, a DNA damage response protein) by qPCR and oxidative stress by immunostaining of lipid peroxidation (HNE). Compared to control EC, lentiviral infection increased hTERT activity (p<0.05), elongated telomeres (p<0.05), decreased ATM and p21 mRNA levels (p<0.05), increased EC replicative potential (p<0.05) but only delayed the onset of senescence (p<0.05). HNE levels increased in both groups, with time (p<0.05). In conclusion, since no immortalization was observed, we hypothesize that hTERT overexpression delays replicative senescence but cannot bypass stress‐induced senescence associated with CAD.This study was supported by the Canadian Institutes of Health Research and the Fonds de la Recherche en Santé du Québec.

Replicative senescence of vascular endothelial cells isolated from coronary patients is worsened by oxidative stress associated with risk factors for cardiovascular disease

The p53‐p21 pathway can trigger senescence either by telomere shortening (replicative senescence via ATM) or following exposure to oxidative stress and DNA damages (stress‐induced senescence, SIS, via caveolin‐1). Our objective was to determine the main pathway in endothelial cells (EC) from coronary patients exposed to risk factors for cardiovascular diseases (CVD). EC were isolated from human internal mammary artery discarded during CABG (n = 56) and cultured in vitro until senescence. We quantified senescence by cytochemical detection of senescence‐associated b‐galactosidase, telomere length (RFL) by Southern blotting, gene expression of p53, p21, ATM and caveolin‐1 (Cav‐1) by qPCR and levels of lipid peroxidation (HNE) by immunofluorescence. Senescent EC displayed both telomeric instability markers (RFL shortening and increase in ATM, p<0.05) and oxidative stress markers (HNE and Cav‐1, p<0.05). mRNA levels of p53 were directly associated with the DNA damage response protein ATM and p21 (p<0.05) but not with Cav‐1 or HNE (p>0.05), suggesting that senescence is not exclusively directed by SIS. In conclusion, our data suggest that in human EC exposed to risk factors for CVD, there is a significant interplay between replicative senescence and SIS due to a state of cumulative cell damages. This study was supported by the Canadian Institutes of Health Research and the Fonds de la Recherche en Santé du Québec.

Vascular endothelial cell senescence mediated by integrin β4 in vitro

To understand whether integrin β4 is involved in vascular endothelial cell (VEC) senescence, we examined integrin β4 level changes, as well as P53 and reactive oxygen species (ROS) levels and alterations of phosphatidylcholine-specific phospholipase C (PC-PLC) activity before and after knocking-down integrin β4 by small interfering RNA. We found integrin β4, P53 and ROS levels increased significantly, while Ca 2+-independent PC-PLC activity obviously decreased during VEC senescence. On the other hand, integrin β4 down-regulation attenuated the senescence phenotype and reversed Ca 2+-independent PC-PLC activity, and P53 and ROS levels. The data suggested that integrin β4 might mediate VEC senescence through depressing Ca 2+-independent PC-PLC and elevating the levels of P53 and ROS.

Is Pathologic Intimal Thickening the Key to Understanding Early Plaque Progression in Human Atherosclerotic Disease?

The term “ P athologic I ntimal T hickening” (PIT) was recently introduced to define an early stage of atherosclerosis described in human coronary lesions found at autopsies of sudden death victims.1 This descriptive identifier is based on the AHA type III (intermediate) lesion and, as originally presented by Stary and collegues, it’s believed to be the morphological and chemical bridge to more advanced plaques.2 The precise histological features and clinical relevance of PIT remains unsettled, and use of the term is still far from widespread. In short, PIT identifies a lesion with an extracellular lipid pool with intimal smooth muscle cell loss typically adjacent to the medial wall in addition to varying degrees of macrophage infiltration near the lumen. These morphological features indicate a progressive lesion in the earlier stages of atherosclerosis, although there is yet the presence of a necrotic core. As recently studied by Nakashima and colleagues in this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , it may provide a key in settling the chicken-versus-egg debate of atherosclerotic plaque progression: does lipid come first, or do macrophages?3 Is PIT the precursor lesion of fibroatheroma? The study in this issue uses 3-dimensional histology to attempt to address some of these issues, and, in doing so, may raise as many questions as it answers. See page 1159 Although there are many detailed autopsy studies describing various lesion morphologies, little is known how human atherosclerosis progresses from early to more advanced plaques, marked by the formation of a necrotic core. This important question remains, in part, from a lack of direct experimental testing in prospective models of human disease. Moreover, potentially relevant finding in animals are difficult to associate with humans because the pathologic change of atherosclerosis in man cannot be definitely equated with animals. Although …

Both Senescence and Apoptosis Induced by Deprivation of Growth Factors Were Inhibited by a Novel Butyrolactone Derivative through Depressing Integrin β4 in Vascular Endothelial Cells

Both senescence and apoptosis of vascular endothelial cells (VECs) are involved in the development of cardiovascular diseases, including atherosclerosis. To understand the association between senescence and apoptosis in vascular endothelial cells, the authors first explored whether senescence and apoptosis took place at the same time in human umbilical vein endothelial cells (HUVECs) deprived of the growth factors. Integrin beta4 is a key factor in HUVEC apoptosis, to know whether this integrin is implicated in VEC senescence, the authors checked the changes of integrin beta4 level during HUVEC aging. Then the authors investigated the effects of 3BDO (3-benzyl-5-((2-nitrophenoxy)methyl)-dihydrofuran-2(3H)-one) on the senescence induced by deprivation of serum and fibroblast growth factor (FGF)-2. The results showed that deprivation of growth factors not only induced apoptosis, but also triggered senescence in HUVECs. The authors found that the level of integrin beta 4 was increased markedly during HUVEC senescence. 3BDO (20 to 60 microg/mL) could inhibit both senescence and apoptosis and depress integrin beta 4 level. The data suggested that integrin beta4 might be a pivotal factor in the relationship between senescence and apoptosis.