Senescent Human Umbilical Vein Endothelial Cells Research Articles

Human salivary histatin 1 regulating IP3R1/GRP75/VDAC1 mediated mitochondrial-associated endoplasmic reticulum membranes (MAMs) inhibits cell senescence for diabetic wound repair

RationaleDifficulty in skin wound healing is a concern for diabetic patients across the world. Impaired mitochondrial dysfunction and aging-related vascular dysfunction in human umbilical vein endothelial cells (HUVECs) caused by oxidative stress are major impediments to diabetic wound healing. However, research on skin repair at the mechanistic level by improving mitochondrial function and inhibiting oxidative stress-induced HUVEC senescence remains lacking. Methods and resultsHuman saliva effectively inhibits the natural aging of HUVECs through immunodepletion experiments. Histatin 1 (Hst1), a short peptide comprising 38 amino acids, is the primary component of human saliva that prevents HUVEC aging. Based on in vitro findings, Hst1 decreased staining for senescence-associated β-galactosidase activity and expression of mediators of senescence signaling, including p53, p21, and p16. Mechanistically, HUVEC senescence is associated with Hst1-modulated nuclear factor Nrf2 signaling as Hst1 induces ERK-mediated Nrf2 nuclear translocation through NADPH oxidase-dependent ROS regulation, reinforced Nrf2 antioxidant response, and suppressed oxidative stress. RNA sequencing identified that the mitochondrial-related gene set was enriched in the Hst1 group. Coimmunoprecipitation indicated that Hst1 delayed hydrogen peroxide-induced HUVEC senescence by inhibiting mitochondria-associated endoplasmic reticulum (ER) membrane formation mediated by inositol 1,4,5-trisphosphate receptor 1-glucose-regulated protein 75-voltage-dependent anion channel 1 (VDAC1) complex interactions. Furthermore, in aging HUVECs, Hst1 treatment or VDAC1 silencing with small interfering RNA hindered calcium (Ca2+) transfer from the ER to the mitochondria, thereby ameliorating mitochondrial Ca2+ overload and restoring mitochondrial function. In an in vivo mouse model of diabetes mellitus skin defects, Hst1 facilitated wound healing by stimulating the new blood vessel formation and impeding the expression of senescent biomarkers. ConclusionsThis study proposes a theoretical solution that Hst1 can restore mitochondrial function by inhibiting oxidative stress or cellular senescence, thereby promoting angiogenesis and diabetic wound repair.

Andrographis paniculata Ethanol Extract Alleviates High Glucose-induced Senescence of Human Umbilical Vein Endothelial Cells via the Regulation of mTOR and SIRT1 Pathways

BACKGROUND: Chronic exposure of high glucose (HG) in endothelial cell induces senescence which may contribute to the development and progression of age-related diseases including insulin resistance. Andrographis paniculata improves insulin resistance in recent in vitro and in vivo studies. Anti-inflammatory and antioxidant properties of A. paniculata may be the new therapeutic approach to inhibiting premature senescence. However, the senolytic effect of A. paniculata on endothelial cells has not been investigated comprehensively. This study was conducted to evaluate the effect of A. paniculata extract on HG-induced endothelial cell senescence and the underlying mechanisms.METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with 33 mM HG and 7.5 μg/mL A. paniculata extract for 48 hours. The expressions of p16, p21, interleukin (IL)-6, IL-8, insulin receptor substrate (IRS)-1, mammalian target of rapamycin, and sirtuin 1 (SIRT1) were measured by performing real-time quantitative polymerase chain reaction (RT-qPCR). The senescence-associated-β-galactosidase (SA-β-gal) staining was performed to observe the positive-stained senescent cells, while the cell surface expression of IL-1α was examined with flow cytometry method.RESULTS: A. paniculata extract reversed senescence in HUVECs under HG conditions by reducing mRNA expressions of p16 and p21, the number of SA-β-gal-positive-stained cells, and the expression of IL-1α on cell surface, which decreased the activation of IL-6 and IL-8. In addition, A. paniculata extract decreased the mRNA expression of mTOR and increased the mRNA expressions of IRS-1 as well as SIRT1.CONCLUSION: A. paniculata extract ameliorated senescence and improved insulin sensitivity by regulating the mTOR, SIRT1, and IRS-1 mRNA expressions on HG-treated HUVECs.KEYWORDS: Andrographis paniculata, endothelial cell, senescence, high glucose, nutrient-sensing pathways

MiR-21-Mediated Endothelial Senescence and Dysfunction Are Involved in Cigarette Smoke-Induced Pulmonary Hypertension through Activation of PI3K/AKT/mTOR Signaling.

Smoking is a pathogenic factor for pulmonary hypertension (PH). Our previous study showed that serum miR-21 levels are elevated in smokers. miR-21 is considered as engaged in the PH process; however, its mechanisms remain unclear. In this investigation, we found that in the lung tissue of smoking-induced PH patients, the levels of miR-21 and aging markers (p21 and p16) were upregulated, and the function of pulmonary vascular endothelial cells was also impaired. Exposure of mice to cigarette smoke (CS) for four months caused similar changes in lung tissues and increased pulmonary arterial pressure, which were attenuated by knockout of miR-21. Further, human umbilical vein endothelial cells (HUVECs) exposed to cigarette smoke extract (CSE) revealed upregulation of miR-21 levels, depression of PTEN, activation of PI3K/AKT/mTOR signaling, an increase in senescence indexes, and enhanced dysfunction. Inhibiting miR-21 overexpression reversed the PTEN-mTOR signaling pathway and prevented senescence and dysfunction of HUVECs. In sum, our data indicate that miR-21-mediated endothelial senescence and dysfunction are involved in CS-induced PH through the activation of PI3K/AKT/mTOR signaling, which suggests that selective miR-21 inhibition offers the potential to attenuate PH.

MiRNA-142-3p aggravates hydrogen peroxide-induced human umbilical vein endothelial cell premature senescence by targeting SIRT1.

Vascular endothelial cell premature senescence plays an important part in stroke. Many microRNAs (miRNAs) are known to be involved in the pathological process of vascular endothelial cell premature senescence. The present study aimed to investigate the mechanism of hydrogen peroxide (H2O2)-induced premature senescence in human umbilical vein endothelial cells (HUVECs) and effect of miR-142-3p on hydrogen peroxide (H2O2)-induced premature senescence. HUVECs were exposed to H2O2 to establish a model premature senescence in endothelial cells. CCK-8 assay was performed to detect cell viability. Senescence-associated β-galactosidase staining assay and senescence-related proteins p16 and p21 were used to detect changes in the degree of cell senescence. RT-qPCR and Western blot were conducted to measure mRNA and protein levels, respectively. The scratch wound-healing assay, transwell assay, and EdU assay were performed to evaluate the ability of migration and proliferation, respectively. miRNA-142-3p and silencing information regulator 2 related enzyme 1 (SIRT1) binding was verified using Targetscan software and a dual-luciferase assay. We found that miRNA-142-3p is abnormally up-regulated in HUVECs treated with H2O2. Functionally, miRNA-142-3p inhibition may mitigate the degree of HUVEC senescence and improve HUVEC migration and proliferation. Mechanistically, SIRT1 was validated to be targeted by miRNA-142-3p in HUVECs. Moreover, SIRT1 inhibition reversed the effects of miRNA-142-3p inhibition on senescent HUVECs exposed to H2O2. To our knowledge, this is the first study to show that miRNA-142-3p ameliorates H2O2-induced HUVECs premature senescence by targeting SIRT1 and may shed light on the role of the miR-142-3p/SIRT1 axis in stroke treatment.

Lycium barbarum polysaccharide's protective effects against PM2.5-induced cellular senescence in HUVECs

Fine particulate matter (PM2.5) exposure is strongly associated with vascular endothelial senescence, a process implicated in cardiovascular diseases. While there is existing knowledge on the impact of Lycium barbarum polysaccharide (LBP) on vascular endothelial damage, the protective mechanism of LBP against PM2.5-induced vascular endothelial senescence remains unclear. In this study, we investigated the impact of PM2.5 exposure on vascular endothelial senescence and explored the intervention effects of LBP in human umbilical vein endothelial cells (HUVECs). We found that PM2.5 exposure dose-dependently reduced cell viability and proliferation in HUVECs while increasing the production of reactive oxygen species (ROS), malondialdehyde (MDA), and hydrogen peroxide (H2O2). Additionally, PM2.5 exposure inhibited the activity of superoxide dismutase (SOD). Notably, PM2.5 exposure induced autophagy impairments and cellular senescence. However, LBP mitigated PM2.5-induced cell damage. Further studies demonstrated that correcting autophagy impairment in HUVECs reduced the expression of the senescence markers P16 and P21 induced by PM2.5. This suggests the regulatory role of autophagy in cellular senescence and the potential of LBP in improving HUVECs senescence. These findings provide novel insights into the mechanisms underlying PM2.5-induced cardiovascular toxicity and highlight the potential of LBP as a therapeutic agent for improving vascular endothelial health.

Involvement of retinoic acid‑inducible gene‑I in radiation‑induced senescence of human umbilical vein endothelial cells.

In 2012, the threshold radiation dose (0.5 Gy) for cardiovascular and cerebrovascular diseases was revised, and this threshold dose may be exceeded during procedures involving radiation such as interventional radiology. Therefore, in addition to regulating radiation dose, it is necessary to develop strategies to prevent and mitigate the development of cardiovascular disease. Cellular senescence is irreversible arrest of cell proliferation. Although cellular senescence is one of the mechanisms for suppressing cancer, it also has adverse effects. For example, senescence of vascular endothelial cells is involved in development of vascular disorders. However, the mechanisms underlying induction of cellular senescence are not fully understood. Therefore, the present study explored the factors involved in the radiation-induced senescence in human umbilical vein endothelial cells (HUVECs). The present study reanalyzed the gene expression data of senescent normal human endothelial cells and fibroblast after irradiation (NCBI Gene Expression Omnibus accession no. GSE130727) and microarray data of HUVECs 24 h after irradiation (NCBI Gene Expression Omnibus accession no. GSE76484). Numerous genes related to viral infection and inflammation were upregulated in radiation-induced senescent cells. In addition, the gene group involved in the retinoic acid-inducible gene-I (RIG-I)-like receptor (RLR) signaling pathway, which plays an important role to induce anti-viral response, was altered in irradiated HUVECs. Therefore, to investigate the involvement of RIG-I and melanoma differentiation-associated gene 5 (MDA5), which are RLRs, in radiation-induced senescence of HUVECs, the protein expression of RIG-I and MDA5 and the activity of senescence-associated β-galactosidase (SA-β-gal), a representative senescence marker, were analyzed. Of note, knockdown of RIG-I in HUVECs significantly decreased radiation-increased proportion of cells with high SA-β-gal activity (i.e., senescent cells), whereas this phenomenon was not observed in MDA5-knockdown cells. Taken together, the present results suggested that RIG-I, but not MDA5, was associated with radiation-induced senescence in HUVECs.

DHCR24 Insufficiency Promotes Vascular Endothelial Cell Senescence and Endothelial Dysfunction via Inhibition of Caveolin-1/ERK Signaling.

Endothelial cells (ECs) senescence is critical for vascular dysfunction, which leads to age-related disease. DHCR24, a 3β-hydroxysterol δ 24 reductase with multiple functions other than enzymatic activity, has been involved in age-related disease. However, little is known about the relationship between DHCR24 and vascular ECs senescence. We revealed that DHCR24 expression is chronologically decreased in senescent human umbilical vein endothelial cells (HUVECs) and the aortas of aged mice. ECs senescence in endothelium-specific DHCR24 knockout mice was characterized by increased P16 and senescence-associated secretory phenotype, decreased SIRT1 and cell proliferation, impaired endothelium-dependent relaxation, and elevated blood pressure. In vitro, DHCR24 knockdown in young HUVECs resulted in a similar senescence phenotype. DHCR24 deficiency impaired endothelial migration and tube formation and reduced nitric oxide (NO) levels. DHCR24 suppression also inhibited the caveolin-1/ERK signaling, probably responsible for increased reactive oxygen species production and decreased eNOS/NO. Conversely, DHCR24 overexpression enhanced this signaling pathway, blunted the senescence phenotype, and improved cellular function in senescent cells, effectively blocked by the ERK inhibitor U0126. Moreover, desmosterol accumulation induced by DHCR24 deficiency promoted HUVECs senescence and inhibited caveolin-1/ERK signaling. Our findings demonstrate that DHCR24 is essential in ECs senescence.

PM2.5-induced premature senescence in HUVECs through the SIRT1/PGC-1α/SIRT3 pathway

Vascular endothelial cell senescence plays a pivotal role in the development of atherosclerosis. Recent studies have demonstrated that ambient fine particulate matter (PM2.5) induces stress-induced premature senescence (SIPS) in vascular endothelial cells. However, the precise mechanisms underlying this process remain to be fully elucidated. Cellular senescence is closely associated with reactive oxygen species (ROS), and emerging research has established a strong connection between the SIRT1/PGC-1α/SIRT3 signaling pathway and the antioxidant system in vascular endothelial cells. In this study, we aimed to investigate the impact of PM2.5 on vascular endothelial cell senescence and to elucidate the underlying mechanisms. Our findings revealed that PM2.5 exposure led to an increase in senescence-associated β-galactosidase (SA-β-gal) activity and the expression of the cell cycle-blocking proteins P53/P21 and P16 in human umbilical vein endothelial cells (HUVECs). Flow cytometry analysis demonstrated an elevated proportion of cells arrested in the G0/G1 phase after PM2.5 exposure. In addition, PM2.5-induced cellular senescence was attributed to the disruption of the cellular antioxidative defense system through the SIRT1/PGC-1α/SIRT3 signaling pathway. The expression of cellular senescence markers was reduced after targeted scavenging of mitochondrial ROS using MitoQ. Moreover, treatment with SRT1720, a SIRT1-specific activator, upregulated the SIRT1/PGC-1α/SIRT3 signaling pathway, restored the antioxidant system, and attenuated the expression of cellular senescence markers. Taken together, our results suggest that PM2.5 downregulates the SIRT1/PGC-1α/SIRT3 signaling pathway, resulting in impaired antioxidant defenses in HUVECs. This, in turn, allows for the accumulation of ROS, leading to inhibition of endothelial cell cycle progression and the onset of stress-induced senescence in HUVECs.

Loss of Endothelial Annexin A1 Aggravates Inflammation-Induched Vascular Aging.

Chronic inflammation is increasingly considered as the most important component of vascular aging, contributing to the progression of age-related cardiovascular diseases. To delay the process of vascular aging, anti-inflammation may be an effective measure. The anti-inflammatory factor annexin A1 (ANXA1) is shown to participate in several age-related diseases; however, its function during vascular aging remains unclear. Here, an ANXA1 knockout (ANXA1-/-) and an endothelial cell-specific ANXA1 deletionmouse (ANXA1△EC) model are used to investigate the role of ANXA1 in vascular aging. ANXA1 depletion exacerbates vascular remodeling and dysfunction while upregulatesage- and inflammation-related protein expression. Conversely,Ac2-26 (a mimetic peptide of ANXA1) supplementation reverses this phenomenon. Furthermore, long-term tumor necrosis factor-alpha (TNF-α) induction of human umbilical vein endothelial cells (HUVECs) increases cell senescence. Finally, the senescence-associated secretory phenotype and senescence-related protein expression, rates of senescence-β-galactosidasepositivity, cell cycle arrest, cell migration, and tube formation ability are observed in both ANXA1-knockdown HUVECs and overexpressed ANXA1-TNF-α induced senescent HUVECs. They also explore the impact of formylpeptide receptor 2 (a receptor of ANXA1) in an ANXA1 overexpression inflammatory model. These data provide compelling evidence that age-related inflammation in arteries contributes to senescent endothelial cells that promote vascular aging.

Protective effects of villi mesenchymal stem cells on human umbilical vein endothelial cells by inducing SPOCD1 expression in cases of gestational diabetes mellitus

BackgroundGestational diabetes mellitus (GDM) is characterized by a lack of response to insulin in pregnancies, and often accompanied by severe complications. GDM is associated with structural and functional alterations, particularly endothelial dysfunction, in various tissues. This study is aimed to investigate the effect of placental mesenchymal stem cells (MSCs) on the endothelial biological function of human umbilical vein endothelial cells (HUVECs) and their molecular mechanisms. MethodsVilli mesenchymal stem cells (VMSCs) were co-cultured with HUVECs, and transcriptomic analysis of differential genes was performed in HUVECs under high-glucose induction. Lentiviral transfection was performed to construct HUVECs with stable knockdown or overexpression of SPOCD1. The immunohistochemical assays were used to detect the expression of SPOCD1 in GDM patients. TUNEL fluorescence staining was applied for detection of the HUVEC apoptosis. β galactosidase staining assay was performed to detect the cell senescence. Electron microscopy was used to detect the cell pyroptosis. qRT-PCR and western blot assays were conducted for identifying the mRNA & protein expressions of genes. ResultsVMSCs, when co-cultured with HUVECs, could inhibit the apoptosis, pyroptosis and senescence induced by high-glucose condition in HUVECs. Transcriptomic results showed an upregulation of SPOCD1 expression induced by VMSCs in HUVECs. Overexpression of SPOCD1 inhibited high-level glucose-induced apoptosis, pyroptosis and senescence in HUVECs via the β-catenin pathway. ConclusionVMSCs induce β-catenin activation by upregulating the expression of SPOCD1 in HUVECs, which ultimately inhibits high-level glucose-induced apoptosis, pyroptosis and senescence in HUVECs. This observation provides potential therapeutic insight for future GDM treatment.

Senescent Endothelial Cells Sustain Their Senescence-Associated Secretory Phenotype (SASP) through Enhanced Fatty Acid Oxidation.

Cellular senescence is closely linked to endothelial dysfunction, a key factor in age-related vascular diseases. Senescent endothelial cells exhibit a proinflammatory phenotype known as SASP, leading to chronic inflammation (inflammaging) and vascular impairments. Albeit in a state of permanent growth arrest, senescent cells paradoxically display a high metabolic activity. The relationship between metabolism and inflammation is complex and varies across cell types and senescence inductions. While some cell types shift towards glycolysis during senescence, others favor oxidative phosphorylation (OXPHOS). Despite the high availability of oxygen, quiescent endothelial cells (ECs) tend to rely on glycolysis for their bioenergetic needs. However, there are limited data on the metabolic behavior of senescent ECs. Here, we characterized the metabolic profiles of young and senescent human umbilical vein endothelial cells (HUVECs) to establish a possible link between the metabolic status and the proinflammatory phenotype of senescent ECs. Senescent ECs internalize a smaller amount of glucose, have a lower glycolytic rate, and produce/release less lactate than younger cells. On the other hand, an increased fatty acid oxidation activity was observed in senescent HUVECs, together with a greater intracellular content of ATP. Interestingly, blockade of glycolysis with 2-deoxy-D-glucose in young cells resulted in enhanced production of proinflammatory cytokines, while the inhibition of carnitine palmitoyltransferase 1 (CPT1), a key rate-limiting enzyme of fatty acid oxidation, ameliorated the SASP in senescent ECs. In summary, metabolic changes in senescent ECs are complex, and this research seeks to uncover potential strategies for modulating these metabolic pathways to influence the SASP.

Liuwei Dihuang Prevents Human Umbilical Vein Endothelial Cells Senescence via the JPX-STING-IRF3 Pathway.

Cellular senescence plays a crucial role in age-related diseases. Endothelial senescence is closely associated with age-related vascular disorders. This study aimed to reveal the role of traditional Chinese medicine Liuwei Dihuang (LWDH) in anti-endothelial cell senescence. Human umbilical vein endothelial cells (HUVECs) were exposed to LPS treatment to induce senescence. Senescence-associated β-galactosidase (SA-β-gal) positive staining, p53 and p16 expression, BrdU staining, and relative telomere length (RTL) experiments were conducted to estimate LPS-induced cellular senescence of HUVECs. Real-time qPCR analysis was performed to identify differentially expressed lncRNAs in LPS-induced senescent HUVECs before and after treatment with LWDH. Bioinformatics analysis and ChIP assay were conducted to explore the mechanism of JPX in the anti-endothelial cell aging effect of LWDH. We first discovered that lncRNA JPX and STING-IRF3 pathways are involved in the process of anti-endothelial senescence of LWDH. Mechanistically, LWDH could reverse abnormally elevated JPX induced by LPS and inhibit the activation of STING, as well as the interaction between JPX and STING. Overall, our study explores the potential therapeutic value of LWDH and provides key insights into the potential avenues for preventing and treating HUVECs senescence.

External Application of Human Umbilical Cord-Derived Mesenchymal Stem Cells in Hyaluronic Acid Gel Repairs Foot Wounds of Types I and II Diabetic Rats Through Paracrine Action Mode.

Diabetic foot ulcer (DFU) is a main diabetic complication with unmet treatment needs. This study applied human umbilical cord-derived mesenchymal stem cells-hyaluronic acid (hucMSCs-HA) gel to treat DFU in a noninvasive external way and investigated its paracrine action and mechanism. In this study, after analyzing the physical and biological properties of HA gel, hucMSCs-HA gel was applied in 2 in vivo models (types I and II DFU), and a molecular mechanism was investigated. To evaluate the paracrine action of hucMSCs, hucMSCs-conditional medium (MSC-CM) was collected to treat 1 in vivo model (type I DFU) and 2 in vitro models (high glucose (HG)-injured human umbilical vein endothelial cells (HUVECs) and human skin fibroblasts (HSFs)). The results indicated that HA gel with a porous microstructure underwent over 90% degradation and swelled to the maximum value within 48 h. In vivo, hucMSCs-HA gel accelerated wound healing of DFU rats by improving re-epithelialization, collagen deposition, and angiogenesis, in which a paracrine action of hucMSCs was confirmed and the phosphorylation of p38, ERK1/2, JNK, and Akt was increased. In vitro, MSC-CM improved cell viability, wound healing, migration, tube formation, cell senescence, and abnormal expressions (TNF-α, IL-1β, IL-6, ET-1, p16 genes, and PCNA protein) of HUVECs, also improved cell viability, wound healing, antioxidant stress, and abnormal expressions (COL1, COL3, COL4, SOD1, SOD2 genes, and PCNA protein) of HSFs. Summarily, noninvasive external application of hucMSCs-HA gel shows great perspective against DFU and exerts wound healing effects through the MAPK and Akt pathways-mediated paracrine mechanism.

A Novel Role of Hyaluronic Acid and Proteoglycan Link Protein 1 (HAPLN1) in Delaying Vascular Endothelial Cell Senescence.

Cardiovascular diseases (CVDs) are the most common cardiovascular system disorders. Cellular senescence is a key mechanism associated with dysfunction of aged vascular endothelium. Hyaluronic acid and proteoglycan link protein 1 (HAPLN1) has been known to non-covalently link hyaluronic acid (HA) and proteoglycans (PGs), and forms and stabilizes HAPLN1-containing aggregates as a major component of extracellular matrix. Our previous study showed that serum levels of HAPLN1 decrease with aging. Here, we found that the HAPLN1 gene expression was reduced in senescent human umbilical vein endothelial cells (HUVECs). Moreover, a recombinant human HAPLN1 (rhHAPLN1) decreased the activity of senescence-associated β-gal and inhibited the production of senescence-associated secretory phenotypes, including IL-1β, CCL2, and IL-6. rhHAPLN1 also down-regulated IL-17A levels, which is known to play a key role in vascular endothelial senescence. In addition, rhHAPLN1 protected senescent HUVECs from oxidative stress by reducing cellular reactive oxygen species levels, thus promoting the function and survival of HUVECs and leading to cellular proliferation, migration, and angiogenesis. We also found that rhHAPLN1 not only increases the sirtuin 1 (SIRT1) levels, but also reduces the cellular senescence markers levels, such as p53, p21, and p16. Taken together, our data indicate that rhHAPLN1 delays or inhibits the endothelial senescence induced by various aging factors, such as replicative, IL-17A, and oxidative stress-induced senescence, thus suggesting that rhHAPLN1 may be a promising therapeutic for CVD and atherosclerosis.

Autophagy in homocysteine‑induced HUVEC senescence.

The senescence of vascular endothelial cells (VECs) drives the occurrence and development of cardiovascular disease (CVD). Homocysteine (HCY) is a general risk factor for age-associated CVDs. Autophagy, an evolutionarily conserved lysosomal protein degradation pathway, serves a part in VEC senescence. The purpose of this study was to investigate the role of autophagy in HCY-induced endothelial cell senescence and explore novel mechanisms and therapeutic approaches for related CVDs. Human umbilical vein endothelial cells (HUVECs) were isolated from fresh umbilical cords of healthy pregnancies. Cell Counting Kit-8, flow cytometry and senescence-associated (SA) β-galactosidase (Gal) staining demonstrated that HCY induced HUVEC senescence by decreasing cell proliferation, arresting cell cycle and increasing the number of SA-β-Gal-positive cells. Stub-RFP-Sens-GFP-LC3 autophagy-related double fluorescence lentivirus revealed that HCY increased autophagic flux. Further, inhibition of autophagy using 3-methyladenine increased HCY-induced HUVEC senescence. By contrast, the induction of autophagy via rapamycin alleviated HCY-induced HUVEC senescence. Finally, the detection of reactive oxygen species (ROS) with ROS kit showed that HCY increased intracellular ROS, whereas induction of autophagy reduced intracellular ROS. In conclusion, HCY increased HUVEC senescence and upregulated autophagy; moderate autophagy could reverse HCY-induced cell senescence. Autophagy may alleviate HCY-induced cell senescence by decreasing intracellular ROS. This provides insight into the underlying mechanism of HCY-induced VEC senescence and potential treatments for age-associated CVDs.

Echinacoside ameliorates 5-fluorouracil-induced endothelial injury and senescence through SIRT1 activation

Echinacoside (ECH) is a natural bioactive component with antioxidant, anti-inflammatory, anti-apoptosis, and anti-tumor properties. In the current study, we explore the ECH-mediated protective effect and underlying mechanism of 5-fluorouracil (5-FU)-induced endothelial injury and senescence in the Human umbilical vein endothelial cells (HUVECs). In HUVECs, Cell viability, Apoptosis and Senescence assays evaluated 5-fluorouracil-induced endothelial injury and senescence. Protein expressions were assessed using RT-qPCR and Western blotting. Our results showed that 5-FU-induced endothelial injury and endothelial cell senescence could be improved when treated with ECH in HUVECs. ECH treatment potentially attenuated oxidative stress and ROS production in HUVECs. In addition, the effect of ECH on autophagy markedly reduced the percentage of HUVECs with LC3-II dots and suppressed the Beclin-1 and ATG7 mRNA expression but enhanced the p62 mRNA expression. Besides, ECH treatment significantly increased migrated cells and suppressed the adhesion of THP-1 monocytes in HUVECs. Furthermore, ECH treatment activated the SIRT1 pathway, and its related proteins (SIRT1, p-AMPK and eNOS) expression increased. Nicotinamide (NAM), an inhibitor of SIRT1, significantly attenuated the ECH-induced decrease in the apoptotic rate, increased SA-β-gal-positive cells and significantly reversed the ECH-induced reduction of endothelial senescence. Our results demonstrated that ECH employed endothelial injury and senescence in HUVECs via activation of the SIRT1 pathway.

Exogenous Hydrogen Sulfide Activates PI3K/Akt/eNOS Pathway to Improve Replicative Senescence in Human Umbilical Vein Endothelial Cells.

Endothelial cell senescence is one of the key mechanistic factors in the pathogenesis of atherosclerosis. In terms of molecules, the phosphatidylinositol 3-kinase/protein kinase B/endothelial nitric oxide synthase (PI3K/Akt/eNOS) signaling plays an important role in the prevention and control of endothelial cell senescence, while hydrogen sulfide (H2S) improves the induced precocious senescence of endothelial cells through the PI3K/Akt/eNOS pathway. Comparatively, replicative senescence in endothelial cells is more in line with the actual physiological changes of human aging. This study aims to investigate the mechanism by which H2S improves endothelial cell replicative senescence and the involvement of the PI3K/Akt/eNOS pathway. we established a model of replicative senescence in human umbilical vein endothelial cells (HUVECs) and explored the effect of 200 μmol/L sodium hydrosulfide (NaHS; a donor of H2S) on senescence, which was determined by cell morphology, the expression level of plasminogen activator inhibitor 1 (PAI-1), and the positive rate of senescence-associated β-galactosidase (SA-β-Gal) staining. Cell viability was detected by MTT assay to evaluate the effect of NaHS and the PI3K inhibitor, LY294002. Meanwhile, the protein expression of PI3K, Akt, p-Akt, and eNOS in endothelial cells of each group was detected by Western blot. the replicative senescence model was established in HUVECs at the passage of 16 cumulative cell population doubling values (CPDL). Treatment with NaHS not only significantly reduced the expression of PAI-1 and the positive rate of SA-β-Gal in HUVEC's replicative senescence model but also notably increased the expression of PI3K, p-Akt, p-eNOS, and the content of nitric oxide(NO). However, the effects of NaHS on the expression of the pathway and the content of NO in HUVECs were abolished when LY294002 specifically inhibited PI3K. NaHS improves the replicative senescence of HUVECs with the contribution of the PI3K/Akt/eNOS pathway.

Abstract 4604: Effect of CDK 4/6 inhibition on anti-angiogenesis effect in gastric cancer

Abstract Angiogenesis, a hallmark of cancer, is a phenomenon in which new blood vessels are created from existing blood vessels. As the tumor grows, they secrete ligands such as vascular endothelial growth factor (VEGF). Therefore, anti-tumor effect could be achieved by preventing tumor-associated angiogenesis. In previous reports, CDK4/6 has been acknowledged to regulate VEGF transcription in addition to cell cycle regulation. There are few studies explaining the angiogenesis-related effects of CDK4/6 inhibitors in gastric cancer (GC). In this study, we aim to verify the anti-angiogenesis effect through CDK4/6 inhibition. First, we confirmed proliferation and angiogenesis in endothelial cells. Next, the change in VEGF expression induced by CDK4/6 inhibitor in GC cell lines was investigated. In-vitro angiogenesis research was conducted using human umbilical vein endothelial cells (HUVEC). Abemaciclib was used as a CDK4/6 inhibitor. To determine the efficacy of abemaciclib, HUVEC was treated with abemaciclib for 3 days and cell viability was assessed by CCK-8 assay. IC50 value was determined by the CalcuSyn software. Furthermore, tube formation assay, wound healing assay, cell cycle analysis and SA-β-gal assay were performed to explore effect of abemaciclib on HUVEC. VEGF-A expression was evaluated by ELISA. First, we identified direct anti-angiogenesis effect of abemaciclib on HUVEC. The IC50 value was 1.28 μM. At clinically achievable concentrations of abemaciclib (Cmax, 0.5 μM), HUVEC only showed a 20% growth inhibition. Abemaciclib did not suppress tube formation and migratory potential of HUVECs. However, abemaciclib induced a G1 arrest in HUVEC after 0.5 μM abemaciclib treatment for 24hr. To ensure whether the increase in the number of G1 phase cells is due to senescence, SA-β-gal assay was performed. The levels of SA-β-gal-positive cell staining in abemaciclib-treated group were higher than control, suggesting that abemaciclib induced cell senescence on HUVEC. While our results suggested that CDK4/6 inhibitor did not show direct anti-angiogenic effect on endothelial cells, we studied indirect anti-angiogenesis effect of abemaciclib in GC cell lines through regulation of VEGF transcription. Conditioned media of 49 GC cell lines had a wide range of VEGF secretion ranged from 0.146 to 22.676 ng/mL. (ave. 4.07 ng/mL). We have set the cut off of secreted VEGF as 10 ng/mL, at which angiogenesis can be induced. There were four cell lines (4/49, 8.2%) whose VEGF concentrations were more than the angiogenesis-inducing VEGF concentration (10 ng/mL). Interestingly, 3 of these 4 GC cell lines demonstrated a decrease in VEGF secretion, following 0.5 μM abemaciclib treatment. In conclusion, we did not observe inhibition of angiogenesis in HUVEC. However, CDK4/6 inhibition can cause senescence in HUVEC, inhibit tumor growth, and indirectly induce anti-angiogenic effects in gastric cancer. Citation Format: Sang Woo Cho, Woo Sun Kwon, Tae Soo Kim, Juin Park, Eun Seo Kim, Hyun Cheol Chung, Sun Young Rha. Effect of CDK 4/6 inhibition on anti-angiogenesis effect in gastric cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4604.

A novel role for YPEL2 in mediating endothelial cellular senescence via the p53/p21 pathway

Yippee-like 2 (YPEL2) is expressed in tissues and organs enriched in vascular networks, such as heart, kidney, and lung. However, the roles of YPEL2 in endothelial cell senescence and the expression of YPEL2 in atherosclerotic plaques have not yet been investigated. Here, we report the essential role of YPEL2 in promoting senescence in human umbilical vein endothelial cells (HUVECs) and the upregulation of YPEL2 in human atherosclerotic plaques. YPEL2 was significantly upregulated in both H2O2-induced senescent HUVECs and the arteries of aged mice. Endothelial YPEL2 deficiency significantly decreased H2O2-increased senescence-associated beta-galactosidase (SA-β-gal) activity and reversed H2O2-inhibited cell viability. Additionally, endothelial YPEL2 knockdown reduced H2O2-promoted THP-1 cell adhesion to HUVECs and downregulated ICAM1 and VCAM1 expression. Mechanistic studies divulged that the p53/p21 pathway was involved in YPEL2-induced cellular senescence. We conclude that YPEL2 promotes cellular senescence via the p53/p21 pathway and that YPEL2 expression is elevated in atherosclerosis. These findings reveal YPEL2 as a potential therapeutic target in aging-associated diseases.

Estrogen receptor alpha mediates 17β-estradiol, up-regulates autophagy and alleviates hydrogen peroxide-induced vascular senescence.

Atherosclerosis threatens human health by developing cardiovascular diseases, the deadliest disease world widely. The major mechanism contributing to the formation of atherosclerosis is mainly due to vascular endothelial cell (VECs) senescence. We have shown that 17β-estradiol (17β-E2) may protect VECs from senescence by upregulating autophagy. However, little is known about how 17β-E2 activates the autophagy pathway to alleviate cellular senescence. Therefore, the aim of this study is to determine the role of estrogen receptor (ER) α and β in the effects of 17β-E2 on vascular autophagy and aging through in vitro and in vivo models. Hydrogen peroxide (H2O2) was used to establish Human Umbilical Vein Endothelial Cells (HUVECs) senescence. Autophagy activity was measured through immunofluorescence and immunohistochemistry staining of light chain 3 (LC3) expression. Inhibition of ER activity was established using shRNA gene silencing and ER antagonist. Compared with ER-β knockdown, we found that knockdown of ER-α resulted in a significant increase in the extent of HUVEC senescence and senescence-associated secretory phenotype (SASP) secretion. ER-α-specific shRNA was found to reduce 17β-E2-induced autophagy, promote HUVEC senescence, disrupt the morphology of HUVECs, and increase the expression of Rb dephosphorylation and SASP. These in vitro findings were found consistent with the in vivo results. In conclusion, our data suggest that 17β-E2 activates the activity of ER-α and then increases the formation of autophagosomes (LC3 high expression) and decreases the fusion of lysosomes with autophagic vesicles (P62 low expression), which in turn serves to decrease the secretion of SASP caused by H2O2 and consequently inhibit H2O2-induced senescence in HUVEC cells.