Quantitative proteomic analysis reveals induction of premature senescence in human umbilical vein endothelial cells exposed to chronic low‐dose rate gamma radiation

Chronic low‐dose ionizing radiation induces cardiovascular disease in human populations but the mechanism is largely unknown. We suggested that chronic radiation exposure may induce endothelial cell senescence that is associated with vascular damage in vivo. We investigated whether chronic radiation exposure is causing a change in the onset of senescence in endothelial cells in vitro. Indeed, when exposed to continuous low‐dose rate gamma radiation (4.1 mGy/h), primary human umbilical vein endothelial cells (HUVECs) initiated senescence much earlier than the nonirradiated control cells. We investigated the changes in the protein expression of HUVECs before and during the onset of radiation‐induced senescence. Cellular proteins were quantified using isotope‐coded protein label technology after 1, 3, and 6 weeks of radiation exposure. Several senescence‐related biological pathways were influenced by radiation, including cytoskeletal organization, cell–cell communication and adhesion, and inflammation. Immunoblot analysis showed an activation of the p53/p21 pathway corresponding to the progressing senescence. Our data suggest that chronic radiation‐induced DNA damage and oxidative stress result in induction of p53/p21 pathway that inhibits the replicative potential of HUVECs and leads to premature senescence. This study contributes to the understanding of the increased risk of cardiovascular diseases seen in populations exposed to chronic low‐dose irradiation.     

血管内皮细胞衰老与血管疾病

细胞衰老是指细胞在各种应激条件下出现周期阻滞,不可逆地丧失增殖能力,其形态、基因表达和功能都发生特定变化的过程。研究表明,血管内皮细胞衰老可以通过削弱血管功能,促进衰老相关血管疾病的发生发展。然而,有关内皮细胞衰老的发生机制以及内皮细胞衰老影响血管功能及衰老相关血管疾病的潜在机制尚待挖掘。本文从血管内皮细胞衰老相关的信号通路,以及血管内皮细胞衰老与血管功能和血管相关疾病（动脉粥样硬化、高血压和糖尿病血管并发症）的最新研究进展进行综述,为进一步认识血管疾病的发病机制,延缓血管衰老提供新的思路。     

Vascular endothelial cells senescence is associated with NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation via reactive oxygen species (ROS)/thioredoxin-interacting protein (TXNIP) pathway

Endothelial dysfunction caused by endothelial cells senescence and chronic inflammation is tightly linked to the development of cardiovascular diseases. NLRP3 (NOD-like receptor family pyrin domain-containing3) inflammasome plays a central role in inflammatory response that is associated with diverse inflammatory diseases. This study explores the effects and possible mechanisms of NLRP3 inflammasome in endothelial cells senescence. Results show an increment of pro-inflammatory cytokine interleukin (IL) −1β secretion and caspase-1 activation during the senescence of endothelial cells induced by bleomycin. Moreover, secreted IL-1β promoted endothelial cells senescence through up-regulation of p53/p21 protein expression. NLRP3 inflammasome was found to mediate IL-1β secretion through the production of ROS (reactive oxygen species) during the senescence of endothelial cells. Furthermore, the association of TXNIP (thioredoxin-interacting protein) with NLRP3 induced by ROS promoted NLRP3 inflammasome activation in senescent endothelial cells. In addition, the expressions of NLRP3 inflammasome related genes, ASC (apoptosis associated speck-like protein containing a CARD), TXNIP, cleaved caspase-1 and IL-1β, were also increased in vitro and in vivo studies. These findings indicate that endothelial senescence could be mediated through ROS and NLRP3 inflammasome signaling pathways, suggesting a potential target for the prevention of endothelial senescence-related cardiovascular diseases.     

Vascular smooth muscle cell senescence and age-related diseases: State of the art

Aging is a worldwide challenge, and it is accompanied by the accumulation of senescent cells. Cellular senescence is traditionally defined as permanent cell growth arrest and currently includes the senescence-associated secretory phenotype (SASP). There are two main types of cellular senescence, including telomere-dependent replicative senescence and stress-induced premature senescence. The process of cellular senescence is mainly controlled by two effector pathways, namely, the p53-p21 and p16-retinoblastoma protein (pRB) pathways. Vascular smooth muscle cells (VSMCs) are integral parts of arteries and play an important role in vascular structure and function. VSMC senescence may be triggered by many factors, such as angiotensin II, oxidative stress, inflammation, DNA damage, and small molecule compounds. These inducers are able to genetically and epigenetically regulate VSMC senescence. The senescence of VSMCs together with the SASP contributes to chronic vascular inflammation, the loss of arterial function, and the development of age-related diseases. Current evidence suggests that the senescence of VSMCs might be harmful to individual health, whereas its influence on the lifespan is not clear. The purpose of this paper was to review the current knowledge regarding VSMC senescence and its relevance to hypertension, atherosclerosis, and diabetes, as well as the potential mechanisms responsible for VSMC senescence in these age-related diseases.     

Cellular lifespan and senescence signaling in embryonic stem cells

Most mammalian cells when placed in culture will undergo a limited number of cell divisions before entering an unresponsive non-proliferating state termed senescence. However, several pathways that are activated singly or in concert can allow cells to bypass senescence at least for limited periods. These include the telomerase pathway required to maintain telomere ends, the p53 and Rb pathways required to direct senescence in response to DNA damage, telomere shortening and mitogenic signals, and the insulin-like growth factor--Akt pathway that may regulate lifespan and cell proliferation. In this review, we summarize recent findings related to these pathways in embryonic stem (ES) cells and suggest that ES cells are immortal because these pathways are tightly regulated.     

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.     

The PI3K/Akt/mTOR Pathway Is Implicated in the Premature Senescence of Primary Human Endothelial Cells Exposed to Chronic Radiation

The etiology of radiation-induced cardiovascular disease (CVD) after chronic exposure to low doses of ionizing radiation is only marginally understood. We have previously shown that a chronic low-dose rate exposure (4.1 mGy/h) causes human umbilical vein endothelial cells (HUVECs) to prematurely senesce. We now show that a dose rate of 2.4 mGy/h is also able to trigger premature senescence in HUVECs, primarily indicated by a loss of growth potential and the appearance of the senescence-associated markers ß-galactosidase (SA-ß-gal) and p21. In contrast, a lower dose rate of 1.4 mGy/h was not sufficient to inhibit cellular growth or increase SA-ß-gal-staining despite an increased expression of p21. We used reverse phase protein arrays and triplex Isotope Coded Protein Labeling with LC-ESI-MS/MS to study the proteomic changes associated with chronic radiation-induced senescence. Both technologies identified inactivation of the PI3K/Akt/mTOR pathway accompanying premature senescence. In addition, expression of proteins involved in cytoskeletal structure and EIF2 signaling was reduced. Age-related diseases such as CVD have been previously associated with increased endothelial cell senescence. We postulate that a similar endothelial aging may contribute to the increased rate of CVD seen in populations chronically exposed to low-dose-rate radiation.     

Glioma-associated endothelial cells show evidence of replicative senescence

The innately programmed process of replicative senescence has been studied extensively with respect to cancer, but primarily from the perspective of tumor cells overcoming this stringent innate barrier and acquiring the capacity for unlimited proliferation. In this study, we focus on the potential role of replicative senescence affecting the non-transformed endothelial cells of the blood vessels within the tumor microenvironment. Based on the well-documented aberrant structural and functional features of blood vessels within solid tumors, we hypothesized that tumor-derived factors may lead to premature replicative senescence in tumor-associated brain endothelial cells (TuBEC). We show here that glioma tissue, but not normal brain tissue, contains cells that express the signature of replicative senescence, senescence-associated beta-galactosidase (SA-beta-gal), on CD31-positive endothelial cells. Primary cultures of human TuBEC stain for SA-beta-gal and exhibit characteristics of replicative senescence, including increased levels of the cell cycle inhibitors p21 and p27, increased resistance to cytotoxic drugs, increased growth factor production, and inability to proliferate. These data provide the first demonstration that tumor-derived brain endothelial cells may have reached an end-stage of differentiation known as replicative senescence and underscore the need for anti-angiogenic therapies to target this unique tumor-associated endothelial cell population.     

H2O2 down-regulates SIRT7’s protective role of endothelial premature dysfunction via microRNA-335-5p

Endothelial senescence is believed to constitute the initial pathogenesis of the atherosclerotic cardiovascular disease (ASCVD). MicroRNA-335-5p (miR-335-5p) expression is significantly up-regulated in oxidative stress-induced endothelial cells (ECs). Sirtuin7 (SIRT7) is considered to prevent EC senescence, yet data on its response to ASCVD risk factors are limited. The present study analyzed the elevated levels of miR-335-5p and the decreased levels of SIRT7 in human umbilical vein endothelial cells (HUVECs), and found that high glucose, tumor necrosis factor-α (TNF-α), and H₂O₂ are the three contributing factors that induced cellular senescence. The current study also assessed premature endothelial senescence and decreased proliferation, adhesion, migration, and nitric oxide (NO) secretion in HUVECs with these risk factors together with SIRT7–siRNA transfection. It found that the miR-335-5p inhibitor attenuated the down-regulation of SIRT7 expression induced by oxidative stress in HUVECs, and SIRT7 overexpression exerts a rescue effect against miR-335-5p-induced endothelial dysfunction. Furthermore, the direct binding of miR-335-5p to SIRT7 was observed in human embryonic kidney cells 293T (HEK 293T). Therefore, it can be inferred that miR-335-5p down-regulates the expression of SIRT7 in human cells. Current findings may provide deeper insights into the underlying mechanisms of endothelial senescence and potential therapeutic targets of ASCVD as well as other age-related diseases.     

DNA damage caused by ionizing radiation in embryonic diploid fibroblasts WI-38 induces both apoptosis and senescence

Cellular response to ionizing radiation-induced damage depends on the cell type and the ability to repair DNA damage. Some types of cells undergo apoptosis, whereas others induce a permanent cell cycle arrest and do not proliferate. Our study demonstrates two types of response of embryonic diploid fibroblasts WI-38 to ionizing radiation. In the WI-38 cells p53 is activated, protein p21 increases, but the cells are arrested in G2 phase of cell cycle. Some of the cells die by apoptosis, but in remaining viable cells p16 increases, senescence associated DNA-damage foci occur, and senescence-associated beta-galactosidase activity increases, which indicate stress-induced premature senescence.     

Autophagy: The missing link between non-enzymatically glycated proteins inducing apoptosis and premature senescence of endothelial cells?

In a series of studies into the fate of endothelial cells exposed to non-enzymatically glycated collagen I, a model of cytotoxic molecules relevant to diabetic vasculopathy, we demonstrate that cells either undergo apoptosis or become prematurely senescent despite relatively spared telomeres and telomerase activity. Our most recent work shows that long-lived advanced glycation end product (AGE)-modified proteins induce 1) lysosomal permeabilization leading to the inefficiency of autophagy due to the reduced digestion (early) and non-fusion (later) of lysosomes with phagosomes—a frustrated autophagy; and 2) accumulation of lipid mediators, such as ceramide and sphingosine-1-phosphate, known to be involved in autophagic cell death. Under the experimental conditions described here, the seesaw relations between premature senescence and apoptosis are integrated by autophagy, which plays a novel function of a cellular switch between states of premature senescence and apoptosis.

Addendum to: Patschan SA, Chen J, Polotskaia A, Mendelev N, Cheng J, Patschan D, Goligorsky MS. Lipid mediators of autophagy in stress-induced premature senescence of endothelial cells. Am J Physiol Heart Circ Physiol 2008; In press.

and

Patschan S, Chen J, Gealekman O, Krupincza K, Wang M, Shu L, Shayman JA, Goligorsky MS. Mapping mechanisms and charting the time course of premature cell senescence and apoptosis: lysosomal dysfunction and ganglioside accumulation in endothelial cells. Am J Physiol Renal Physiol 2008; 294:F100-9.     

Contribution of p16INK4a and p21CIP1 pathways to induction of premature senescence of human endothelial cells: permissive role of p53

We have previously found that nonenzymatically glycated collagen I (GC), mimicking diabetic microenvironment, can induce senescent phenotype in early passage human umbilical vein endothelial cells (HUVECs). In the present study, we explored the functional involvement of cell cycle checkpoint pathways in initiating GC-induced premature endothelial cell senescence. When compared with native collagen, early passage HUVECs showed increased p53, p21(CIP1) (p21), and p16(INK4a) (p16) mRNA expression after exposure to GC. Twenty-four hours after transfection of p16, p21, and p53-enhanced green fluorescent protein (EGFP) recombinant plasmids, HUVECs entered G(1)-phase cell cycle arrest. By days 3 and 5, HUVECs transfected with p16-EGFP showed an increased proportion of senescent cells, and this increase was more prominent in the GFP-positive cell population, which exhibited 68% of senescent cells. Transfection of p21 also induced senescence but only by day 5. Cotransfection of p16 and p21 showed no additive effect. Transfection of p21 or p53 induced apoptosis in HUVECs. Next, we suppressed endogenous p53, p21, p16, or retinoblastoma (Rb) gene expression through small interference RNA strategy and investigated their influence in p16- and p21-initiated endothelial cell senescence. Analysis indicated that suppression of p53 expression can abolish senescence induced by p16 overexpression. Paradoxically, this effect was not observed when p21 was suppressed. On the other hand, suppression of Rb eliminated senescence initiated by either p16 or p21 overexpression. In summary, the p53/p21 pathway is mainly responsible for GC-induced apoptosis, but the coordinated activation of the p53/p21 and p16 pathway is responsible for GC-induced endothelial cell senescence through a Rb-dependent mechanism.     

Pak4 induces premature senescence via a pathway requiring p16INK4/p19ARF and mitogen-activated protein kinase signaling

Exposure of primary cells to mitogenic stimuli or oncogenes often causes them to undergo premature senescence. This is most likely a protective function that prevents uncontrolled proliferation. Pak4 is a target for the Rho GTPase Cdc42. Pak4 is overexpressed in human tumor cell lines, and it is the only member of the Pak family that is highly transforming in immortalized fibroblasts. Here we show that in primary fibroblasts, activated Pak4 inhibits cell proliferation and promotes premature senescence. Furthermore, Pak4 expression levels are upregulated in response to stimuli that promote senescence. Pak4-induced arrest appears to be mediated by a pathway that requires the ERK mitogen-activated protein kinase, as well as the cell cycle inhibitors p16^INK4 and p19^ARF. These new results describing a role for Pak4 in senescence are important for understanding why this protein is associated with cancer and how it promotes transformation in immortalized cells.     

Foam cell‐derived 4‐hydroxynonenal induces endothelial cell senescence in a TXNIP‐dependent manner

Vascular endothelial cell (VEC) senescence is considered an early event in the development of atherosclerotic lesions. Stressful stimuli, in particular oxidative stress, have been linked to premature senescence in the vasculature. Foam cells are a major source of reactive oxygen species and may play a role in the induction of VEC senescence; hence, we investigated their involvement in the induction of VEC senescence in a co‐culture transwell system. Primary bovine aortic endothelial cells, exposed to the secretome of THP‐1 monocyte‐derived foam cells, were analysed for the induction of senescence. Senescence associated β‐galactosidase activity and the expression of p16 and p21 were increased, whereas phosphorylated retinoblastoma protein was reduced. This senescent phenotype was mediated by 4‐hydroxnonenal (4‐HNE), a lipid peroxidation product secreted from foam cells; scavenging of 4‐HNE in the co‐culture medium blunted this effect. Furthermore, both foam cells and 4‐HNE increased the expression of the pro‐oxidant thioredoxin‐interacting protein (TXNIP). Molecular manipulation of TXNIP expression confirmed its involvement in foam cell‐induced senescence. Previous studies showed that peroxisome proliferator‐activated receptor (PPAR)δ was activated by 4‐hydroalkenals, such as 4‐HNE. Pharmacological interventions supported the involvement of the 4‐HNE‐PPARδ axis in the induction of TXNIP and VEC senescence. The association of TXNIP with VEC senescence was further supported by immunofluorescent staining of human carotid plaques in which the expression of both TXNIP and p21 was augmented in endothelial cells. Collectively, these findings suggest that foam cell‐released 4‐HNE activates PPARδ in VEC, leading to increased TXNIP expression and consequently to senescence.     

Autophagy: The missing link between non-enzymatically glycated proteins inducing apoptosis and premature senescence of endothelial cells?

In a series of studies into the fate of endothelial cells exposed to non-enzymatically glycated collagen I, a model of cytotoxic molecules relevant to diabetic vasculopathy, we demonstrate that cells either undergo apoptosis or become prematurely senescent despite relatively spared telomeres and telomerase activity. Our most recent work shows that long-lived advanced glycation end product (AGE)-modified proteins induce (1) lysosomal permeabilization leading to the inefficiency of autophagy due to the reduced digestion (early) and non-fusion (later) of lysosomes with phagosomes--a frustrated autophagy; and (2) accumulation of lipid mediators, such as ceramide and sphingosine-1-phosphate, known to be involved in autophagic cell death. Under the experimental conditions described here, the seesaw relations between premature senescence and apoptosis are integrated by autophagy, which plays a novel function of a cellular switch between states of premature senescence and apoptosis.     

Disc cell senescence in intervertebral disc degeneration: Causes and molecular pathways

The accumulation of senescent disc cells in degenerative intervertebral disc (IVD) suggests the detrimental roles of cell senescence in the pathogenesis of intervertebral disc degeneration (IDD). Disc cell senescence decreased the number of functional cells in IVD. Moreover, the senescent disc cells were supposed to accelerate the process of IDD via their aberrant paracrine effects by which senescent cells cause the senescence of neighboring cells and enhance the matrix catabolism and inflammation in IVD. Thus, anti-senescence has been proposed as a novel therapeutic target for IDD. However, the development of anti-senescence therapy is based on our understanding of the molecular mechanism of disc cell senescence. In this review, we focused on the molecular mechanism of disc cell senescence, including the causes and various molecular pathways. We found that, during the process of IDD, age-related damages together with degenerative external stimuli activated both p53-p21-Rb and p16-Rb pathways to induce disc cell senescence. Meanwhile, disc cell senescence was regulated by multiple signaling pathways, suggesting the complex regulating network of disc cell senescence. To understand the mechanism of disc cell senescence better contributes to developing the anti-senescence-based therapies for IDD.     

Phenotypic Alterations in Senescent Large-Vessel and Microvascular Endothelial Cells

Endothelial cell senescence likely plays a key role in age-associated vascular diseases. A close relationship between in vitro and in vivo senescence of endothelial cells has been established. Therefore, elucidating the structural and functional changes occurring during long-term cultures of endothelial cells would contribute to clarifying the pathogenesis of vascular disorders in the elderly. We investigated the effects of replicative senescence on the architecture of bovine aortic vs microvascular endothelial cells. A marked increase in cell area was observed in both cell types, whereas dramatic morphological alterations were detected in microvascular endothelial cells only. The latter also showed age-associated reorganization of the actin cytoskeleton. Finally, both aortic and microvascular endothelial cells lost their migratory response to basic fibroblast growth factor with age. Our results highlight dramatic structural and functional alterations in senescent endothelial cells. Such rearrangements might account for in vivo endothelial cell alterations involved in age-associated vascular dysfunction.