Premature Endothelial Senescence Research Articles

Activated factor X stimulates atrial endothelial cells and tissues to promote remodeling responses through AT1R/NADPH oxidases/SGLT1/2.

Atrial fibrillation (AF), the most common cardiac arrhythmia favoring ischemic stroke and heart failure involves left atrial remodeling, fibrosis and a complex interplay between cardiovascular risk factors. This study examined whether activated factor X (FXa) induces pro-remodeling and pro-fibrotic responses in atrial endothelial cells (AECs) and human atrial tissues and determined the underlying mechanisms. AECs were from porcine hearts and human right atrial appendages (RAA) from patients undergoing heart surgery. Protein expression levels were assessed by Western blot and immunofluorescence staining, mRNA levels by RT-qPCR, formation of reactive oxygen species (ROS) and NO using fluorescent probes, thrombin and angiotensin II generation by specific assays, fibrosis by Sirius red staining and senescence by senescence-associated beta-galactosidase (SA-β-gal) activity.In AECs, FXa increased ROS formation, senescence (SA-β-gal activity, p53, p21), angiotensin II generation and the expression of pro-inflammatory (VCAM-1, MCP-1), pro-thrombotic (tissue factor), pro-fibrotic (TGF-β and collagen-1/3a) and pro-remodeling (MMP-2/9) markers whereas eNOS levels and NO formation were reduced. These effects were prevented by inhibitors of FXa but not thrombin, protease-activated receptors antagonists (PAR-1/2) and inhibitors of NADPH oxidases, ACE, AT1R, SGLT1/SGLT2. FXa also increased expression levels of ACE1, AT1R, SGLT1/2 proteins which was prevented by SGLT1/2 inhibitors. Human RAA showed tissue factor mRNA levels that correlated with markers of endothelial activation, pro-remodeling and pro-fibrotic responses and SGLT1/2 mRNA levels. They also showed protein expression levels of ACE1, AT1R, p22phox, SGLT1/2, and immunofluorescence signals of nitrotyrosine and SGLT1/2 colocalized with those of CD31. FXa increased oxidative stress levels which were prevented by inhibitors of the AT1R/NADPH oxidases/SGLT1/2 pathway. FXa promotes oxidative stress triggering premature endothelial senescence and dysfunction associated with pro-thrombotic, pro-remodeling and pro-fibrotic responses in AECs and in human RAA involving the AT1R/NADPH oxidases/SGLT1/2 pro-oxidant pathway. Targeting this pathway may be of interest to prevent atrial remodeling and the progression of atrial fibrillation substrate.

Mitochondria-targeted esculetin and metformin delay endothelial senescence by promoting fatty acid β-oxidation: Relevance in age-associated atherosclerosis

Impaired mitochondrial fatty acid β-oxidation (FAO) plays a role in the onset of several age-associated diseases, including atherosclerosis. In the current work, we investigated the efficacies of mitochondria-targeted esculetin (Mito-Esc) and metformin in enhancing FAO in human aortic endothelial cells (HAECs), and its relevance in the delay of cellular senescence and age-associated atherosclerotic plaque formation in Apoe-/- mice. Chronic culturing of HAECs with either Mito-Esc or metformin increased oxygen consumption rates (OCR), and caused delay in senescence features. Conversely, etomoxir (CPT1 inhibitor) reversed Mito-Esc- and metformin-induced OCR, and caused premature endothelial senescence. Interestingly, Mito-Esc, unlike metformin, in the presence of etomoxir failed to preserve OCR. Thereby, underscoring Mito-Esc’s exclusive reliance on FAO as an energy source. Mechanistically, chronic culturing of HAECs with either Mito-Esc or metformin led to AMPK activation, increased CPT1 activity, and acetyl-CoA levels along with a concomitant reduction in malonyl-CoA levels, and lipid accumulation. Similar results were observed in Apoe-/- mice aorta and liver tissue with a parallel reduction in age-associated atherosclerotic plaque formation and degeneration of liver with either Mito-Esc or metformin administration. Together, Mito-Esc and metformin by potentiating FAO, may have a role in the delay of cellular senescence by modulating mitochondrial function.

Of Soldiers and Their Ghosts: Are We Ready for a Review of PTSD Evidence?

Psychosocial trauma has accompanied mankind since time immemorial and has been sufficiently portrayed in art and literature to suggest that posttraumatic stress disorder may be as old as combat itself. Since war is more frequent in human history than peace, public health measures are confined to mitigating the detrimental impact of battlefield experiences on combat participants. At present, PTSD outcome studies show mixed results, marked by high nonresponse rates, therapy dropout, and completed suicide, suggesting that novel strategies are urgently needed. Those of us who work routinely with combat veterans have noted an increasing trend of patients preferring mindfulness-based therapies as opposed to trauma-centered treatments, such as prolonged exposure or trauma-focused cognitive behavioral therapy. Preference for mindfulness over trauma-based therapies appears to coincide with the shift in research focus from the amygdala and fear to the insular cortex and interoceptive awareness. Therefore, rethinking PTSD as insular pathology is driven by the recent findings that neurons in this cortical area not only regulate cardiac rhythm but also record past intestinal inflammations. These discoveries likely explain the high comorbidity of stress-related disorders with premature endothelial senescence and a dysfunctional intestinal barrier. Moreover, the identification of the cholinergic anti-inflammatory pathway and the revelation that endothelial cells express alpha-7 nicotinic receptors has brought PTSD prevention and early detection within reach. In this narrative review, we discuss the relationship between early vascular aging, gut barrier disruption, and PTSD. We also examine the link between this pathology and faulty interoceptive awareness, surmising that hypertension and decreased heart rate variability are PTSD risk factors, while lipopolysaccharide, lipopolysaccharide binding protein, soluble CD14, microbial cell-free DNA, acyloxyacyl hydrolase, and IL22 comprise early detection markers of this disorder.

Disrupted Binding of Cystathionine γ-Lyase to p53 Promotes Endothelial Senescence.

Advanced age is unequivocally linked to the development of cardiovascular disease; however, the mechanisms resulting in reduced endothelial cell regeneration remain poorly understood. Here, we investigated novel mechanisms involved in endothelial cell senescence that impact endothelial cell transcription and vascular repair after injury. Native endothelial cells were isolated from young (20±3.4 years) and aged (80±2.3 years) individuals and subjected to molecular analyses to assess global transcriptional and metabolic changes. In vitro studies were conducted using primary human and murine endothelial cells. A murine aortic re-endothelialization model was used to examine endothelial cell regenerative capacity in vivo. RNA sequencing of native endothelial cells revealed that aging resulted in p53-mediated reprogramming to express senescence-associated genes and suppress glycolysis. Reduced glucose uptake and ATP contributed to attenuated assembly of the telomerase complex, which was required for endothelial cell proliferation. Enhanced p53 activity in aging was linked to its acetylation on K120 due to enhanced activity of the acetyltransferase MOZ (monocytic leukemic zinc finger). Mechanistically, p53 acetylation and translocation were, at least partially, attributed to the loss of the vasoprotective enzyme, CSE (cystathionine γ-lyase). CSE physically anchored p53 in the cytosol to prevent its nuclear translocation and CSE absence inhibited AKT (Protein kinase B)-mediated MOZ phosphorylation, which in turn increased MOZ activity and subsequently p53 acetylation. In mice, the endothelial cell-specific deletion of CSE activated p53, induced premature endothelial senescence, and arrested vascular repair after injury. In contrast, the adeno-associated virus 9-mediated re-expression of an active CSE mutant retained p53 in the cytosol, maintained endothelial glucose metabolism and proliferation, and prevented endothelial cell senescence. Adenoviral overexpression of CSE in native endothelial cells from aged individuals maintained low p53 activity and reactivated telomerase to revert endothelial cell senescence. Aging-associated impairment of vascular repair is partly determined by the vasoprotective enzyme CSE.

γ-Oryzanol ameliorates fine dust-induced premature endothelial senescence and dysfunction via attenuating oxidative stress.

Various cardiovascular diseases are associated with endothelial senescence, and a recent study showed that fine dust (FD)-induced premature endothelial senescence and dysfunction is associated with increased oxidative stress. The aim of the present study was to investigate protective effect of rice bran extract (RBE) and its major component of γ-Oryzanol (γ-Ory) against FD-induced premature endothelial senescence. Porcine coronary artery endothelial cells (PCAECs) were treated with FD alone or with RBE or γ-Ory. Senescence-associated β-galactosidase (SA-β-gal) activity, expression of cell cycle regulatory proteins, and oxidative stress levels were evaluated. The results indicated that SA-β-gal activity in the FD-treated PCAECs was attenuated by RBE and γ-Ory. Additionally, γ-Ory inhibited FD-induced cell cycle arrest, restored cell proliferation, and reduced the expression of cell cycle regulatory proteins. γ-Ory also inhibited oxidative stress and prevented senescence-associated NADPH oxidase and LAS activity in FD-exposed ECs suggesting that γ-Ory could protect against FD-induced ECs senescence and dysfunction.

Long COVID as a Tauopathy: Of "Brain Fog" and "Fusogen Storms".

Long COVID, also called post-acute sequelae of SARS-CoV-2, is characterized by a multitude of lingering symptoms, including impaired cognition, that can last for many months. This symptom, often called "brain fog", affects the life quality of numerous individuals, increasing medical complications as well as healthcare expenditures. The etiopathogenesis of SARS-CoV-2-induced cognitive deficit is unclear, but the most likely cause is chronic inflammation maintained by a viral remnant thriving in select body reservoirs. These viral sanctuaries are likely comprised of fused, senescent cells, including microglia and astrocytes, that the pathogen can convert into neurotoxic phenotypes. Moreover, as the enteric nervous system contains neurons and glia, the virus likely lingers in the gastrointestinal tract as well, accounting for the intestinal symptoms of long COVID. Fusogens are proteins that can overcome the repulsive forces between cell membranes, allowing the virus to coalesce with host cells and enter the cytoplasm. In the intracellular compartment, the pathogen hijacks the actin cytoskeleton, fusing host cells with each other and engendering pathological syncytia. Cell-cell fusion enables the virus to infect the healthy neighboring cells. We surmise that syncytia formation drives cognitive impairment by facilitating the "seeding" of hyperphosphorylated Tau, documented in COVID-19. In our previous work, we hypothesized that the SARS-CoV-2 virus induces premature endothelial senescence, increasing the permeability of the intestinal and blood-brain barrier. This enables the migration of gastrointestinal tract microbes and/or their components into the host circulation, eventually reaching the brain where they may induce cognitive dysfunction. For example, translocated lipopolysaccharides or microbial DNA can induce Tau hyperphosphorylation, likely accounting for memory problems. In this perspective article, we examine the pathogenetic mechanisms and potential biomarkers of long COVID, including microbial cell-free DNA, interleukin 22, and phosphorylated Tau, as well as the beneficial effect of transcutaneous vagal nerve stimulation.

Aerobic Exercise-Mimetic Effects of Resveratrol on the Prevention of Vascular Endothelial Senescence

PURPOSE: Regular exercise has been shown to have anti-aging effects on the vascular system by improving endothelial function and diminishing oxidative stress. However, adherence to an exercise regimen can be difficult for some individuals, particularly those with limited mobility. This study investigated the effects of resveratrol as an exercise mimetic for preventing and improving oxidative stressinduced senescence in vascular endothelial cells.METHODS: Cultured human umbilical vein endothelial cells were used to investigate the reversal effects of laminar shear stress and the protective effects of resveratrol on oxidative stress-induced premature senescence. Senescence was estimated using SA-β-gal staining and western blot analysis, whereas oxidative stress levels were evaluated using MitoSOX staining. Matrigel tube formation and scratch assays were also performed to assess endothelial angiogenic and migration functions.RESULTS: The study showed that laminar shear stress could partly normalize premature endothelial senescence. Resveratrol pretreatment attenuated reactive oxygen species production and prevented senescence in H2O2-induced premature aging. Additionally, resveratrol contributed to maintaining endothelial cells’ angiogenic and migratory functions.CONCLUSIONS: This study demonstrated that laminar shear stress can mitigate the effects of oxidative stress-induced premature senescence. Resveratrol may be a potential substitute for exercise to prevent age-related damage to the endothelium by increasing resistance to oxidative stress and improving endothelial function.

SGLT2 inhibition ameliorates nano plastics-induced premature endothelial senescence and dysfunction

Nano plastics (NPs) have been a significant threat to human health and are known to cause premature endothelial senescence. Endothelial senescence is considered one of the primary risk factors contributing to numerous cardiovascular disorders. Recent studies have suggested that inhibition of sodium glucose co-transporter (SGLT2) ameliorates endothelial senescence and dysfunction. Therefore, our study intends to explore the role of SGLT2 in NPs-induced endothelial senescence and dysfunction. Porcine coronary artery and its endothelial cells were treated with NPs in the presence or absence of Enavogliflozin (ENA), a SGLT2 inhibitor and then SGLTs expression, senescence markers and vascular function were evaluated. NPs significantly up-regulated SGLT2 and ENA significantly decreased NPs-induced senescence-associated-β‐gal activity, cell‐cycle arrest, and senescence markers p53 and p21 suggesting that inhibition of SGLT2 prevents NPs-induced endothelial senescence. In addition, ENA decreased the formation of reactive oxygen species with the downregulation of Nox2, and p22phox. Furthermore, SGLT2 inhibition also up regulated the endothelial nitric oxide synthase expression along with improving vascular function. In conclusion, premature endothelial senescence by NPs is, at least in part, associated with SGLT2 and it could be a potential therapeutic target for preventing and/or treating environmental pollutants-induced cardiovascular disorders mediated by endothelial senescence and dysfunction.

Abstract 10442: Protective Effect of Inhibition of Sglt2 on Nanoplastics-Induced Premature Endothelial Senescence and Dysfunction

Background: Nanoplastics (NPs) are now widely acknowledged as a vital part of environmental pollution. However, the effects of NPs on the cardiovascular system are not well understood. We previously reported that NPs induce premature endothelial senescence and dysfunction via a redox-sensitive signaling pathway. Increasing evidence has shown that SGLT2 inhibitors (gliflozins) exhibit protective effects against the development of major cardiovascular diseases. Objective: This study aimed to investigate the role of SGLT2 on NPs-induced premature endothelial senescence and dysfunction, and, if so, to assess the effect of SGLT2 inhibition. Methods: Porcine coronary artery (PCA) rings and cultured endothelial cells (ECs) were exposed to NPs in the presence or absence of the SGLT2 inhibitor, Enavogliflozin (ENA), and the level of SGLTs expression, senescence-associated-beta galactosidase (SA-β-gal) activity, senescence markers, oxidative stress level, and vascular function were evaluated. Results: NPs were internalized in ECs in a time- and concentration-dependent manner. Exposure of PCAECs to NPs significantly up-regulated SGLT2 expression and increased SA-β-gal activity, one of the prominent senescence markers. ENA significantly reduced the NPs-induced increase of SA-β-gal activity in PCA and PCAECs. In addition, ENA prevented the NPs-induced up-regulation of senescence markers, p53, and p21 that promoted the inhibition of ECs proliferation. ENA prevented also the NPs-induced oxidative stress and up-regulation of NOX2 and p22phox in ECs. Exposure of coronary artery rings to NPs blunted endothelium-dependent relaxation and decreased the expression level of endothelial nitric oxide synthase level, and both of these effects were prevented by ENA. Conclusion: The present findings indicate that premature endothelial senescence and dysfunction induced by nanoplastics involve, at least in part, an upregulation of SGLT2, which is associated with increased levels of oxidative stress and the down-regulation of eNOS. They further raise the possibility that SGLT2 may be a potential target for preventing and/or treating environment pollution-associated cardiovascular diseases.

Long COVID and the Neuroendocrinology of Microbial Translocation Outside the GI Tract: Some Treatment Strategies

Similar to previous pandemics, COVID-19 has been succeeded by well-documented post-infectious sequelae, including chronic fatigue, cough, shortness of breath, myalgia, and concentration difficulties, which may last 5 to 12 weeks or longer after the acute phase of illness. Both the psychological stress of SARS-CoV-2 infection and being diagnosed with COVID-19 can upregulate cortisol, a stress hormone that disrupts the efferocytosis effectors, macrophages, and natural killer cells, leading to the excessive accumulation of senescent cells and disruption of biological barriers. This has been well-established in cancer patients who often experience unrelenting fatigue as well as gut and blood–brain barrier dysfunction upon treatment with senescence-inducing radiation or chemotherapy. In our previous research from 2020 and 2021, we linked COVID-19 to myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) via angiotensin II upregulation, premature endothelial senescence, intestinal barrier dysfunction, and microbial translocation from the gastrointestinal tract into the systemic circulation. In 2021 and 2022, these hypotheses were validated and SARS-CoV-2-induced cellular senescence as well as microbial translocation were documented in both acute SARS-CoV-2 infection, long COVID, and ME/CFS, connecting intestinal barrier dysfunction to disabling fatigue and specific infectious events. The purpose of this narrative review is to summarize what is currently known about host immune responses to translocated gut microbes and how these responses relate to fatiguing illnesses, including long COVID. To accomplish this goal, we examine the role of intestinal and blood–brain barriers in long COVID and other illnesses typified by chronic fatigue, with a special emphasis on commensal microbes functioning as viral reservoirs. Furthermore, we discuss the role of SARS-CoV-2/Mycoplasma coinfection in dysfunctional efferocytosis, emphasizing some potential novel treatment strategies, including the use of senotherapeutic drugs, HMGB1 inhibitors, Toll-like receptor 4 (TLR4) blockers, and membrane lipid replacement.

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.

Apolipoprotein C3-Rich Low-Density Lipoprotein Induces Endothelial Cell Senescence via FBXO31 and Its Inhibition by Sesamol In Vitro and In Vivo.

Premature endothelial senescence decreases the atheroprotective capacity of the arterial endothelium. Apolipoprotein C3 (ApoC3) delays the catabolism of triglyceride-rich particles and plays a critical role in atherosclerosis progression. FBXO31 is required for the intracellular response to DNA damage, which is a significant cause of cellular senescence. Sesamol is a natural antioxidant with cardiovascular-protective properties. In this study, we aimed to examine the effects of ApoC3-rich low-density lipoprotein (AC3RL) mediated via FBXO31 on endothelial cell (EC) senescence and its inhibition by sesamol. AC3RL and ApoC3-free low-density lipoproteins (LDL) (AC3(-)L) were isolated from the plasma LDL of patients with ischemic stroke. Human aortic endothelial cells (HAECs) treated with AC3RL induced EC senescence in a dose-dependent manner. AC3RL induced HAEC senescence via DNA damage. However, silencing FBXO31 attenuated AC3RL-induced DNA damage and reduced cellular senescence. Thus, FBXO31 may be a novel therapeutic target for endothelial senescence-related cardiovascular diseases. Moreover, the aortic arch of hamsters fed a high-fat diet with sesamol showed a substantial reduction in their atherosclerotic lesion size. In addition to confirming the role of AC3RL in aging and atherosclerosis, we also identified AC3RL as a potential therapeutic target that can be used to combat atherosclerosis and the onset of cardiovascular disease in humans.

Laminar Shear Stress Protects Against Premature Endothelial Senescence by SIRT1-Dependent Mechanisms

PURPOSE:Accumulation of senescent endothelial cells (ECs) and development of vascular aging have been implicated in the etiology of vascular dysfunction and disease. Aerobic exercise has been recognized as the single most effective non-pharmacological anti-aging intervention via increased laminar shear stress (LSS). This study aimed to determine the protective effects of LSS against premature senescence and the underlying mechanism.METHODS: Carotid artery partial ligation surgery was performed on the left carotid arteries (LCAs) of C57BL/6J male mice to determine the effect of disturbed flow on the development of endothelial senescence. Senescence-associated β-galactosidase (SA-β-gal) staining was performed to measure cellular senescence. Expression levels of protein markers for cell senescence including p21, p16, and p53 were measured by western blotting.RESULTS: <i>En face</i> SA-β-gal staining was identified only in the partially ligated LCAs of voluntary wheel-running mice, suggesting a direct relevance of LSS on the prevention of vascular senescence. In the <i>in vitro</i> H<sub>2</sub>O<sub>2</sub>-induced premature senescence model, preconditioning of high-flow LSS (20 dyne/cm2, 36 hours) induced significant reduction in the percentage of SA-β-gal positive ECs. Expression of the molecular markers of cellular senescence such as p21, p16, and p53 was significantly decreased by LSS pretreatment. However, the protective effects of LSS against premature senescence were completely abolished by SIRT1 inhibition.CONCLUSIONS: The results suggest that high-flow LSS has protective effects against oxidative stress-induced premature endothelial senescence through a SIRT1-dependent mechanism.

Abstract 356: Rice Bran Extracts and Its Active Compound, γ-oryzanol, Prevent Particulate Matters-induced Endothelium Senescence

Particulate matters (PMs) derived from are known to be responsible for a wide range of diseases. Recent studies have indicated that long-term exposure PMs is associated with several cardiovascular diseases, at least in part, by increase of inflammation, oxidative stress and nitric oxide (NO) balance disturbance. The present study was to investigate the protective effect of rice bran and its active compound, γ-oryzanol (γ-Orz), on PMs-induced premature endothelial senescence, and if then, to explore the underlying mechanism. Premature senescence was induced in porcine coronary arterial endothelial cells (PCAECs) by incubating 30 μg/mL of PM10. Senescence-associated β-galactosidase (SA-β-gal) activity, oxidative stress, expression level of cell cycle regulatory proteins, nicotinamide adenine dinucleotide phosphate(NADPH) oxidase, and local angiotensin system (LAS) component were evaluated. The result showed that senescence associated-β-gal activity in the PMs-treated PCAECs was attenuated by rice bran extracts (RBEs) and its major constituent, γ-Orz. Pretreatment with γ-Orz inhibited PMs-induced oxidative stress in PCAECs. Similarly, γ-Orz restored the cell proliferation of PCAECs by inhibiting PMs -induced cell cycle arrest. Western blot analyses revealed that cell cycle regulatory protein such as p53, p16, and p21 in senescent PCAECs treated with γ-Orz were reduced from PMs-exposed cells. In addition, γ- Orz prevented senescence associated NADPH oxidase, LAS activity of PM10-exposed cells. Pretreatment of γ-Orz restored PM10-induced endothelial dysfunction by upregulation of endothelial NO synthase expression. The present study demonstrated that γ-Orz protects against PMs induced PCAECs senescence, potentially through abrogating oxidative stress pathway. Taken together, this study can serve as a pharmacological use of rice bran extracts and γ-Orz for the prevention and/or treatment of air pollution-associated coronary artery diseases.

P2253Intake of the omega 3 PUFAs formulation EPA:DHA 6:1 by aged rats reduced shedding of microvesicles from spleen-derived cultured leukocytes and their ability to promote senescence in endothelial cells

Abstract Introduction Ageing is associated with the appearance of endothelial senescence promoting endothelial dysfunction and, ultimately, cardiovascular events. Circulating microvesicles (MVs) of patients with acute coronary syndrome promoted premature endothelial senescence by stimulating the local angiotensin system. Omega 3 PUFAs have been shown to reduce the risk of cardiovascular disease in patients at high risk. Purpose This study investigated whether a 7-day intake of the omega 3 formulation EPA:DHA 6:1 by rats affects the level of MVs released by spleen-derived cultured leukocytes as well as their ability to promote premature senescence in target endothelial cells (ECs), and, if so, to clarify the underlying mechanism. Methods Middle-aged male Wistar rats (M, 48-week old) received 500 mg/kg/d of either EPA:DHA 6:1, EPA:DHA 1:1, or vehicle (CTL) for 7 days. Thereafter, spleen-derived leukocytes, a rich source of MVs, were prepared and cultured for 24 h. Cultured ECs were prepared from porcine coronary arteries. Senescence-associated β-galactosidase activity (SA-β-gal) was assessed by C12FDG, protein expression level by Western blot analysis, oxidative stress by dihydroethidium using confocal microscopy, and procoagulant MVs by prothrombinase assay. Spleen-derived leukocytes from untreated young (Y, 12-week) and old (O, 72-week) rats were also studied. Results Shedding of MVs by spleen-derived leukocytes significantly increased with increasing age. Incubation of ECs with leukocyte-derived MVs (10 nM Phtd Ser eq.) from M and O but not those from Y induced premature senescence after 48 h. The stimulatory effect of M-MVs was prevented by losartan and associated with oxidative stress. M-MVs induced an upregulation of senescence markers (p16, p21, p53), pro-atherothrombotic markers (VCAM-1, ICAM-1, tissue factor), the pro-inflammatory marker cyclooxygenase-2 (COX-2) but not COX-1, and of the angiotensin system (angiotensin-converting enzyme and type 1 angiotensin receptor), whereas endothelial NO synthase was down-regulated. A one-week intake of EPA:DHA 1:1 and 6:1 by M rats decreased the leukocyte-derived MVs shedding by about 14% and 24%, and EPA:DHA 6:1 reduced their ability to induce ECs senescence by 38%. The stimulatory effect of M-MVs on the expression of target proteins was also observed with those from the EPA:DHA 1:1 but not with those from the 6:1 group. Conclusion These findings indicate that ingestion of EPA:DHA 6:1 by middle-aged rats reduces not only the shedding of MVs by spleen-derived leukocytes but also their ability to induce pro-senescent, pro-thrombotic and pro-inflammatory responses in endothelial cells most likely by decreasing the local angiotensin system. They further suggest that EPA:DHA 6:1 may help to delay ageing-related endothelial dysfunction. Acknowledgement/Funding Unrestricted research grant from PIVOTAL Therapeutics Inc.

Chronic Nucleoside Reverse Transcriptase Inhibitors Disrupt Mitochondrial Homeostasis and Promote Premature Endothelial Senescence.

Combination antiretroviral therapy (cART) has improved the life expectancy of HIV patients, thus increasing the number of people living with HIV (PLWH). However, cardiovascular diseases (CVD) are now one of the most prevalent causes of death among PLWH. Nucleoside reverse transcriptase inhibitors (NRTIs) are the backbone of cART, and the emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF) coformulation is commonly used. In prior studies, acute NRTI treatment-induced endothelial dysfunction, increased reactive oxygen species production, and mitophagic activity, suggesting that mitochondrial dysfunction may be critical to NRTI-induced endothelial dysfunction. Mitochondrial dysfunction plays a causal role in endothelial senescence, whereas premature endothelial senescence can promote the development of CVD. We hypothesize that for chronic NRTI treatment, a disruption in mitochondrial homeostasis leads to premature endothelial senescence and predisposes PLWH to CVD. We used human aortic endothelial cells (HAEC) and HIV-1 transgenic (Tg26) mice to test the interrelationship between mitochondrial and vascular dysfunction after chronic NRTI treatment in vitro and in vivo. Mitochondrial DNA copy number was decreased in late-passage HAEC treated with NRTIs, and senescence-associated β-galactosidase accumulation was elevated. In late-passage HAEC, NRTIs decreased the activity of Parkin-mediated mitophagy. In Tg26 mice treated with FTC, plasma nitrite levels were decreased. Endothelium-dependent vasodilation in NRTI-treated Tg26 mice was also reduced. Our work suggests that long-term use of NRTI may disrupt mitochondrial homeostasis, induce premature endothelial senescence, and impair vascular function.

Purple sweet potato color attenuated NLRP3 inflammasome by inducing autophagy to delay endothelial senescence.

Autophagy is a vital negative factor regulating cellular senescence. Purple sweet potato color (PSPC), one type of flavonoid, has been demonstrated to suppress endothelial senescence and restore endothelial function in diabetic mice by inhibiting the nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing protein 3 (NLRP3) inflammasome. However, the roles of autophagy in the inflammatory response during endothelial senescence are unknown. Here, we found that PSPC augmented autophagy to restrict high-glucose-induced premature endothelial senescence. In addition, PSPC administration impaired endothelium aging in diabetic mice by increasing autophagy. Inhibition of autophagy accelerated endothelial senescence, while enhancement of autophagy delayed senescence. Moreover, deactivation of the NLRP3 inflammasome triggered by PSPC was autophagy-dependent. Autophagy receptor microtubule-associated protein 1 light chain 3 and p62 interacted with the inflammasome component NLRP3, suggesting that autophagosomes target the NLRP3 inflammasome and deliver it to the lysosome for degradation. Altogether, PSPC amplified cellular autophagy, subsequently attenuated NLRP3 inflammasome activity and finally delayed endothelial senescence to ameliorate cardiovascular complication. These results suggest a potential therapeutic target in senescence-related cardiovascular diseases.

Leukocyte-derived microparticles exaggerate endothelial senescence and vascular dysfunction induced by high glucose

Microparticles (MPs) are plasma membrane vesicles and vascular effectors. High levels of pro-inflammatory cytokines and procoagulant endothelial-derived MPs circulate in diabetic patients. We have shown that (i) leukocyte-derived MPs shed in response to stress are pro-inflammatory, procoagulant and prosenescent endothelial effectors; (ii) high glucose induces premature endothelial senescence. To determine the possibility that leukocyte-derived MPs affect endothelial senescence and vascular function in response to high glucose. Leukocyte MPs were isolated from rat splenocytes with either 5 mg/ml LPS (MPLPS), 25 ng/ml PMA/1 mM A23187 ionophore (MPPMAi), or vehicle (MPCTL). Porcine coronary artery endothelial cells (ECs) at passage 1 were incubated for 48 h with 1–30 nM MPs in high or low glucose concentration (HG 25 mM, NG 5.5 mM). Senescence-associated β-galactosidase (SA-ß-GAL) activity was assessed by C12FDG, protein expression by Western blot analysis. Pig coronary artery rings were pre-incubated with HG or NG for 12 h prior to addition of 1–30 nM MPs for 12 h. Bradykinin (BK)-induced endothelium-dependent relaxations were assessed in organ chambers, and staining of target proteins by confocal microscopy. At 10 nM, MPLPS and MPPMAi enhanced SA-b-GAL activity both by about 2-fold in NG, and respectively 3 and 3.7-fold in HG. The expression of senescence markers p21, p16 doubled and that of eNOS decreased 2-fold. MPPMAi and MPLPS induced a concentration-dependent inhibition of BK-induced relaxation, inhibition by respectively 10 nM and 30 nM,being 65% in NG, amounting to about 85% in HG, whereas 30 nM MPCTL had no effect. MPPMAi and MPLPS reduced eNOS expression by 60% in NG and 80% in HG. Conversely, VCAM-1, COX-2 were up-regulated. Leukocyte-derived MPs enhance HG-induced alteration of the endothelial function by inducing premature senescence and might contribute to vascular dysfunction in diabetes patients.

Functional Gene Analysis Reveals Cell Cycle Changes and Inflammation in Endothelial Cells Irradiated with a Single X-ray Dose.

Background and Purpose: Epidemiological data suggests an excess risk of cardiovascular disease (CVD) at low doses (0.05 and 0.1 Gy) of ionizing radiation (IR). Furthermore, the underlying biological and molecular mechanisms of radiation-induced CVD are still unclear. Because damage to the endothelium could be critical in IR-related CVD, this study aimed to identify the effects of radiation on immortalized endothelial cells in the context of atherosclerosis.Material and Methods: Microarrays and RT-qPCR were used to compare the response of endothelial cells irradiated with a single X-ray dose (0.05, 0.1, 0.5, 2 Gy) measured after various post-irradiation (repair) times (1 day, 7 days, 14 days). To consolidate and mechanistically support the endothelial cell response to X-ray exposure identified via microarray analysis, DNA repair signaling (γH2AX/TP53BP1-foci quantification), cell cycle progression (BrdU/7AAD flow cytometric analysis), cellular senescence (β-galactosidase assay with CPRG and IGFBP7 quantification) and pro-inflammatory status (IL6 and CCL2) was assessed.Results: Microarray results indicated persistent changes in cell cycle progression and inflammation. Cells underwent G1 arrest in a dose-dependent manner after high doses (0.5 and 2 Gy), which was compensated by increased proliferation after 1 week and almost normalized after 2 weeks. However, at this point irradiated cells showed an increased β-Gal activity and IGFBP7 secretion, indicative of premature senescence. The production of pro-inflammatory cytokines IL6 and CCL2 was increased at early time points.Conclusions: IR induces pro-atherosclerotic processes in endothelial cells in a dose-dependent manner. These findings give an incentive for further research on the shape of the dose-response curve, as we show that even low doses of IR can induce premature endothelial senescence at later time points. Furthermore, our findings on the time- and dose-dependent response regarding differentially expressed genes, cell cycle progression, inflammation and senescence bring novel insights into the underlying molecular mechanisms of the endothelial response to X-ray radiation. This may in turn lead to the development of risk-reducing strategies to prevent IR-induced CVD, such as the use of cell cycle modulators and anti-inflammatory drugs as radioprotectors and/or radiation mitigators.

Endothelial Microparticles From Acute Coronary Syndrome Patients Induce Premature Coronary Artery Endothelial Cell Aging and Thrombogenicity: Role of the Ang II/AT1 Receptor/NADPH Oxidase-Mediated Activation of MAPKs and PI3-Kinase Pathways.

Microparticles (MPs) have emerged as a surrogate marker of endothelial dysfunction and cardiovascular risk. This study examined the potential of MPs from senescent endothelial cells (ECs) or from patients with acute coronary syndrome (ACS) to promote premature EC aging and thrombogenicity. Primary porcine coronary ECs were isolated from the left circumflex coronary artery. MPs were prepared from ECs and venous blood from patients with ACS (n=30) and from healthy volunteers (n=4) by sequential centrifugation. The level of endothelial senescence was assessed as senescence-associated β-galactosidase activity using flow cytometry, oxidative stress using the redox-sensitive probe dihydroethidium, tissue factor activity using an enzymatic Tenase assay, the level of target protein expression by Western blot analysis, platelet aggregation using an aggregometer, and shear stress using a cone-and-plate viscometer. Senescence, as assessed by senescence-associated β-galactosidase activity, was induced by the passaging of porcine coronary artery ECs from passage P1 to P4, and was associated with a progressive shedding of procoagulant MPs. Exposure of P1 ECs to MPs shed from senescent P3 cells or circulating MPs from ACS patients induced increased senescence-associated β-galactosidase activity, oxidative stress, early phosphorylation of mitogen-activated protein kinases and Akt, and upregulation of p53, p21, and p16. Ex vivo, the prosenescent effect of circulating MPs from ACS patients was evidenced only under conditions of low shear stress. Depletion of endothelial-derived MPs from ACS patients reduced the induction of senescence. Prosenescent MPs promoted EC thrombogenicity through tissue factor upregulation, shedding of procoagulant MPs, endothelial nitric oxide synthase downregulation, and reduced nitric oxide-mediated inhibition of platelet aggregation. These MPs exhibited angiotensin-converting enzyme activity and upregulated AT1 receptors and angiotensin-converting enzyme in P1 ECs. Losartan, an AT1 receptor antagonist, and inhibitors of either mitogen-activated protein kinases or phosphoinositide 3-kinase prevented the MP-induced endothelial senescence. These findings indicate that endothelial-derived MPs from ACS patients induce premature endothelial senescence under atheroprone low shear stress and thrombogenicity through angiotensin II-induced redox-sensitive activation of mitogen-activated protein kinases and phosphoinositide 3-kinase/Akt. They further suggest that targeting endothelial-derived MP shedding and their bioactivity may be a promising therapeutic strategy to limit the development of an endothelial dysfunction post-ACS.