Endothelial Progenitor Cells Senescence Research Articles

DNA Tetrahedron Delivering miR-21-5p Promotes Senescent Bone Defects Repair through Synergistic Regulation of Osteogenesis and Angiogenesis.

Compromised osteogenesis and angiogenesis is the character of stem cell senescence, which brought difficulties for bone defects repairing in senescent microenvironment. As the most abundant bone-related miRNA, miRNA-21-5p plays a crucial role in inducing osteogenic and angiogenic differentiation. However, highly efficient miR-21-5p delivery still confronts challenges including poor cellular uptake and easy degradation. Herein, TDN-miR-21-5p nanocomplex is constructed based on DNA tetrahedral (TDN) and has great potential in promoting osteogenesis and alleviating senescence of senescent bone marrow stem cells (O-BMSCs), simultaneously enhancing angiogenic capacity of senescent endothelial progenitor cells (O-EPCs). Of note, the activation of AKT and Erk signaling pathway may direct regulatory mechanism of TDN-miR-21-5p mediated osteogenesis and senescence of O-BMSCs. Also, TDN-miR-21-5p can indirectly mediate osteogenesis and senescence of O-BMSCs through pro-angiogenic growth factors secreted from O-EPCs. In addition, gelatin methacryloyl (GelMA) hydrogels are mixed with TDN and TDN-miR-21-5p to fabricate delivery scaffolds. TDN-miR-21-5p@GelMA scaffold exhibits greater bone repair with increased expression of osteogenic- and angiogenic-related markers in senescent critical-size cranial defects in vivo. Collectively, TDN-miR-21-5p can alleviate senescence and induce osteogenesis and angiogenesis in senescent microenvironment, which provides a novel candidate strategy for senescent bone repair and widen clinical application of TDNs-based gene therapy.

Hsa-miR-134-5p predicts cardiovascular risk in circulating mononuclear cells and improves angiogenic action of senescent endothelial progenitor cells.

This research explores the role of microRNA in senescence of human endothelial progenitor cells (EPCs) induced by replication. Hsa-miR-134-5p was found up-regulated in senescent EPCs where overexpression improved angiogenic activity. Hsa-miR-134-5p, which targeted transforming growth factor β-activated kinase 1-binding protein 1 (TAB1) gene, down-regulated TAB1 protein, and inhibited phosphorylation of p38 mitogen-activated protein kinase (p38) in hsa-miR-134-5p-overexpressed senescent EPCs. Treatment with siRNA specific to TAB1 (TAB1si) down-regulated TAB1 protein and subsequently inhibited p38 activation in senescent EPCs. Treatment with TAB1si and p38 inhibitor, respectively, showed angiogenic improvement. In parallel, transforming growth factor Beta 1 (TGF-β1) was down-regulated in hsa-miR-134-5p-overexpressed senescent EPCs and addition of TGF-β1 suppressed the angiogenic improvement. Analysis of peripheral blood mononuclear cells (PBMCs) disclosed expression levels of hsa-miR-134-5p altered in adult life, reaching a peak before 65 years, and then falling in advanced age. Calculation of the Framingham risk score showed the score inversely correlates with the hsa-miR-134-5p expression level. In summary, hsa-miR-134-5p is involved in the regulation of senescence-related change of angiogenic activity via TAB1-p38 signalling and via TGF-β1 reduction. Hsa-miR-134-5p has a potential cellular rejuvenation effect in human senescent EPCs. Detection of human PBMC-derived hsa-miR-134-5p predicts cardiovascular risk.

Nanoparticles–SIRT1 Prevents Endothelial Progenitor Cells Senescence by Modulating Nitric Oxide Expression and Reducing Poly ADP-ribose Polymerase: In vitro Study

One of cardiovascular diseases that accounts for a high global death rate is atherosclerosis. Apart from the known risk factors, the cause of this disease is uncertain. This disease is triggered by the rupture of atheroma. This condition is associated with oxidative stress, which can trigger endothelial progenitor cells (EPC) dysfunction. One of the causes of oxidative stress is hydrogen peroxide, which triggers deoxyribose nucleotide acid (DNA) damage, which will later trigger ageing in EPC. Sirtuin-1 (SIRT1) is known to be involved in EPC repair, but currently, no studies have attempted to increase SIRT1 expression in vitro. This study aimed to determine the effect of nanoparticles-SIRT1 (NPS1) on the expression of nitric-oxide (NO) and poly (ADP-ribose)-polymerase (PARP) in EPC cell cultures induced by hydrogen peroxide (H2O2). Tests were carried out in vitro using EPC cell cultures to determine the potential of SIRT1 (NPS1) nanoparticles as an ageing inhibitor. The research method used was pure quantitative experimental with a post-test-only control group. The expression of NO and PARP was studied using the flow cytometry method. The data obtained were analysed using parametric one-way ANOVA and post-hoc Tukey-HSD tests with a 95 % confidence interval (p ≤ 0.05). The results showed that administration of SIRT1 nanoparticles was able to increase NO expression in EPC culture cells that induced H2O2 by 14.61 % and could also increase NO levels in normal EPC cells that were only given NPS1 by 38.51 % and was able to reduce PARP expression when compared with the normal group. The NPS1 given was 9.03 % for the NPS1 group and 19.78 % for the NPS1+ H2O2 group. This study concludes that NPS1 added to EPC cells further helps increase cell repair to prevent senescence, through increasing NO and also increasing the performance of PARP. HIGHLIGHTS This study aims to determine the effect of Sirtuin-1 (SIRT1) nanoparticles on nitric oxide (NO) and poly (ADP-ribose)-polymerase (PARP) expression in their role as senescence blockers in endothelial progenitor cells (EPC) by maintaining SIRT1 levels to keep EPC cells alive and functioning optimally This research succeeded in modulating NO levels with a significant increase in NO. This study found that Nanoparticles–SIRT1 (NPS1) added to EPC cells further helps increase cell repair to prevent senescence, through increasing NO and also increasing the performance of PARP as an enzyme that functions in the DNA repair response at levels that are not excessive and does not exceed SIRT1 levels in cells. GRAPHICAL ABSTRACT

Ginseng-Sanqi-Chuanxiong (GSC) extracts attenuate d-galactose-induced vascular aging in mice via inhibition of endothelial progenitor cells senescence

Vascular aging is an independent risk factor for age-related diseases and a specific type of organic aging. Endothelial progenitor cells (EPCs), a type of bone marrow stem cell, has been linked to vascular aging. The purpose of this study is to investigate if Ginseng-Sanqi-Chuanxiong (GSC) extract, a traditional Chinese medicine, can delay aortic aging in mice by enhancing the performance and aging of EPCs in vivo and to analyze the potential mechanisms through a d-Galactose (D-gal)-induced vascular aging model in mice. Our study revealed that GSC extracts not only enhanced the aortic structure, endothelial function, oxidative stress levels, and aging in mice, but also enhanced the proliferation, migration, adhesion, and secretion of EPCs in vivo, while reducing the expression of p53, p21, and p16. To conclude, GSC can delay vascular senescence by enhancing the function and aging of EPCs, which could be linked to a decrease in p16 and p53/p21 signaling. Consequently, utilizing GSC extracts to enhance the function and senescence of autologous EPCs may present a novel avenue for enhancing autologous stem cells in alleviating senescence.

Tumor necrosis factor superfamily 14 (TNFSF14/ LIGHT) attenuates neovascularization by reducing circulating endothelial progenitor cells and function

Abstract Background Tumor necrosis factor superfamily 14 (TNFSF14) is also known as LT-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells (LIGHT) can bind to herpesvirus invasion mediator and lymphotoxin-β receptor to perform its biological activity. LIGHT signaling strengthens the synthesis of nitric oxide, reactive oxygen species, and cytokines and affects organ function. LIGHT also stimulates angiogenesis in tumors and induces synthesis of high endothelial venules, degrades extracellular matrix in thoracic aortic dissection, induces the expression of interleukin-8, cyclooxygenase-2, and cell adhesion molecules in endothelial cells. While LIGHT induces tissue inflammation, its effects on angiogenesis after tissue ischemia are unclear. Thus, we analyzed this effect in the current study. Methods C57BL/6 mice underwent hind limb ischemia surgery for model construction. Doppler ultrasound, immunohistochemical staining, and western blotting were employed to analyze the effects of LIGHT on angiogenesis. In addition, human endothelial progenitor cells (EPCs) were used for in vitro studies to analyze the possible mechanisms. Results In the mice, LIGHT injection inhibited angiogenesis in ischemic limbs. In vitro, LIGHT inhibited the expression of integrins and E-selectin; decreased migration and tube formation capabilities, mitochondrial respiration, and succinate dehydrogenase activity; and promoted senescence in EPCs. Western blotting revealed that the impairment of EPC function by LIGHT may be due to its effects on the normal operation of intracellular Akt signaling pathway and endothelial nitrite oxide synthase (eNOS) function. Conclusions LIGHT inhibits angiogenesis after tissue ischemia. LIGHT inhibition of intracellular Akt-eNOS signaling axis and mitochondrial respiration in EPCs impair cell function. These events are the possible causes of impaired angiogenesis.LIGHT decreases the recovery of ischemiaSenescence and dysfunction of EPC

Cigarette smoke extract induces the senescence of endothelial progenitor cells by upregulating p300.

Endothelial progenitor cells (EPCs) are the main source of endothelial cells. The senescence of EPCs is involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). Cigarette smoke extract (CSE) can directly induce the dysfunction and increased expression of senescence-related markers in EPCs cultured in vitro. Histone acetyltransferase p300 is a transcriptional activator, and its changes can lead to cell senescence. The present study investigated whether CSE can induce the senescence of EPCs by upregulating p300. EPCs were isolated from bone marrow of C57BL/6J mice by density gradient centrifugation. The p300 inhibitor C646 and agonist CTPB were used to interfere with EPCs, cell cycle and apoptosis were detected by flow cytometry, the proportion of senile cells was counted by β-galactosidase staining, the protein expression of p300, H4K12, Cyclin D1, TERT and Ki67 were detected by western blot. Compared with the control group, the cell cycle of CSE group and CTPB group were blocked, the apoptosis rate and early apoptosis rate were increased, the proportion of senile cells counted by β-galactosidase staining was increased, the expression of p300 and H4K12 protein were increased, the expression of Cyclin D1, TERT and Ki67 protein were decreased. C646 could partly alleviate the damages caused by CSE. CSE may promote the apoptosis and senescence of EPCs by upregulating the expression of p300 and H4K12 protein, thus preventing the transition of EPCs from G1 phase to S phase, affecting telomerase synthesis, and reducing EPCs proliferation.

Pannexin 1 Modulates Angiogenic Activities of Human Endothelial Colony-Forming Cells Through IGF-1 Mechanism and Is a Marker of Senescence.

We examined the role of Panxs (pannexins) in human endothelial progenitor cell (EPC) senescence. Young and replication-induced senescent endothelial colony-forming cells (ECFCs) derived from human circulating EPCs were used to examine cellular activities and senescence-associated indicators after transfection of short interference RNA specific to Panx1 or lentivirus-mediated Panx1 overexpression. Hind limb ischemia mice were used as in vivo angiogenesis model. Protein and phospho-kinase arrays were used to determine underlying mechanisms. Panx1 was the predominant Panx isoform in human ECFCs and upregulated in both replication-induced senescent ECFCs and circulating EPCs from aged mice and humans. Cellular activities of the young ECFCs were enhanced by Panx1 downregulation but attenuated by its upregulation. In addition, reduction of Panx1 in the senescent ECFCs could rejuvenate cellular activities with reduced senescence-associated indicators, including senescence-associated β-galactosidase activity, p16INK4a (cyclin-dependent kinase inhibitor 2A), p21 (cyclin-dependent kinase inhibitor 1), acetyl-p53 (tumor protein P53), and phospho-histone H2A.X (histone family member X). In mouse ischemic hind limbs injected senescent ECFCs, blood perfusion ratio, salvaged limb outcome, and capillary density were all improved by Panx1 knockdown. IGF-1 (insulin-like growth factor 1) was significantly increased in the supernatant from senescent ECFCs after Panx1 knockdown. The enhanced activities and paracrine effects of Panx1 knockdown senescent ECFCs were completely inhibited by anti-IGF-1 antibodies. FAK (focal adhesion kinase), ERK (extracellular signal-regulated kinase), and STAT3 (signal transducer and activator of transcription 3) were activated in senescent ECFCs with Panx1 knockdown, in which the intracellular calcium level was reduced, and the activation was inhibited by supplemented calcium. The increased IGF-1 in Panx1-knockdown ECFCs was abrogated, respectively, by inhibitors of FAK (PF562271), ERK (U0126), and STAT3 (NSC74859) and supplemented calcium. Panx1 expression is upregulated in human ECFCs/EPCs with replication-induced senescence and during aging. Angiogenic potential of senescent ECFCs is improved by Panx1 reduction through increased IGF-1 production via activation of the FAK-ERK axis following calcium influx reduction. Our findings provide new strategies to evaluate EPC activities and rejuvenate senescent EPCs for therapeutic angiogenesis.

StemRegenin-1 Attenuates Endothelial Progenitor Cell Senescence by Regulating the AhR Pathway-Mediated CYP1A1 and ROS Generation.

Endothelial progenitor cell (EPC)-based stem cell therapy is a promising therapeutic strategy for vascular diseases. However, continuous in vitro expansion for clinical studies induces the loss of EPC functionality due to aging. In this study, we investigated the effects of StemRegenin-1 (SR-1), an antagonist of aryl hydrocarbon receptor (AhR), on replicative senescence in EPCs. We found that SR-1 maintained the expression of EPC surface markers, including stem cell markers, such as CD34, c-Kit, and CXCR4. Moreover, SR-1 long-term-treated EPCs preserved their characteristics. Subsequently, we demonstrated that SR-1 showed that aging phenotypes were reduced through senescence-associated phenotypes, such as β-galactosidase activity, SMP30, p21, p53, and senescence-associated secretory phenotype (SASP). SR-1 treatment also increased the proliferation, migration, and tube-forming capacity of senescent EPCs. SR-1 inhibited the AhR-mediated cytochrome P450 (CYP)1A1 expression, reactive-oxygen species (ROS) production, and DNA damage under oxidative stress conditions in EPCs. Furthermore, as a result of CYP1A1-induced ROS inhibition, it was found that accumulated intracellular ROS were decreased in senescent EPCs. Finally, an in vivo Matrigel plug assay demonstrated drastically enhanced blood vessel formation via SR-1-treated EPCs. In summary, our results suggest that SR-1 contributes to the protection of EPCs against cellular senescence.

Promotion of NAD+ recycling by the hypoxia-induced shift in the lactate dehydrogenase isozyme profile reduces the senescence of human bone marrow-derived endothelial progenitor cells

Expansion of bone marrow-derived endothelial progenitor cells (EPCs) in vitro to obtain required cell numbers for therapeutic applications faces the challenge of growing cell senescence under the traditional normoxic culture condition. We previously found that 1% O2 hypoxic culture condition is favorable for reducing senescence of EPCs, but the mechanisms underlying the favorability are still unclear. Here, we found that, compared with normoxia, hypoxia induced a shift in lactate dehydrogenase (LDH) isozyme profile, which manifested as decreased LDH2 and LDH1 and increased LDH5, LDH4 and total LDHs. Moreover, under hypoxia, EPCs presented higher LDH activity, which could promote the conversion of pyruvate to lactate, as well as a higher level of NAD+, Bcl2 interacting protein 3 (BNIP3) expression and mitophagy. Additionally, under hypoxia, knock-down of the LDHA subunit increased the LDH2 and LDH1 levels and knock-down of the LDHB subunit increased the LDH5 level, while the simultaneous knock-down of LDHA and LDHB reduced total LDHs and NAD+ level. Inhibition of NAD+ recycling reduced BNIP3 expression and mitophagy and promoted cell senescence. Taken together, these data demonstrated that 1% O2 hypoxia induces a shift in the LDH isozyme profile, promotes NAD+ recycling, increases BNIP3 expression and mitophagy, and reduces EPC senescence. Our findings contribute to a better understanding of the connection between hypoxic culture conditions and the senescence of bone marrow-derived EPCs and provide a novel strategy to improve in vitro expansion of EPCs.

Tumor Necrosis Factor Superfamily 14 (LIGHT) Restricts Neovascularization by Decreasing Circulating Endothelial Progenitor Cells and Function.

Tumor necrosis factor superfamily 14 (TNFSF14) is also known as the LT-related inducible ligand (LIGHT). It can bind to the herpesvirus invasion mediator and lymphotoxin-β receptor to perform its biological activity. LIGHT has multiple physiological functions, including strengthening the synthesis of nitric oxide, reactive oxygen species, and cytokines. LIGHT also stimulates angiogenesis in tumors and induces the synthesis of high endothelial venules; degrades the extracellular matrix in thoracic aortic dissection, and induces the expression of interleukin-8, cyclooxygenase-2, and cell adhesion molecules in endothelial cells. While LIGHT induces tissue inflammation, its effects on angiogenesis after tissue ischemia are unclear. Thus, we analyzed these effects in the current study. In this study, the animal model of hind limb ischemia surgery in C57BL/6 mice was performed. Doppler ultrasound, immunohistochemical staining, and Western blotting were employed to analyze the situation of angiogenesis. In addition, human endothelial progenitor cells (EPCs) were used for in vitro studies to analyze the possible mechanisms. The results in the animal study showed that LIGHT injection inhibited angiogenesis in ischemic limbs. For the in vitro studies, LIGHT inhibited the expression of integrins and E-selectin; decreased migration and tube formation capabilities, mitochondrial respiration, and succinate dehydrogenase activity; and promoted senescence in EPCs. Western blotting revealed that the impairment of EPC function by LIGHT may be due to its effects on the proper functioning of the intracellular Akt signaling pathway, endothelial nitrite oxide synthase (eNOS), and mitochondrial respiration. In conclusion, LIGHT inhibits angiogenesis after tissue ischemia. This may be related to the clamped EPC function.

Dimethyloxalylglycine Attenuates Steroid-Associated Endothelial Progenitor Cell Impairment and Osteonecrosis of the Femoral Head by Regulating the HIF-1α Signaling Pathway

Endothelial impairment and dysfunction are closely related to the pathogenesis of steroid-associated osteonecrosis of the femoral head (SONFH). Recent studies have showed that hypoxia inducible factor-1α (HIF-1α) plays a crucial role in endothelial homeostasis maintenance. Dimethyloxalylglycine (DMOG) could suppress HIF-1 degradation and result in nucleus stabilization by repressing prolyl hydroxylase domain (PHD) enzymatic activity. Our results showed that methylprednisolone (MPS) remarkably undermined biological function of endothelial progenitor cells (EPC) by inhibiting colony formation, migration, angiogenesis, and stimulating senescence of EPCs, while DMOG treatment alleviated these effects by promoting HIF-1α signaling pathway, as evidenced by senescence-associated β-galactosidase (SA-β-Gal) staining, colony-forming unit, matrigel tube formation, and transwell assays. The levels of proteins related to angiogenesis were determined by ELISA and Western blotting. In addition, active HIF-1α bolstered the targeting and homing of endogenous EPCs to the injured endothelium in the femoral head. Histopathologically, our in vivo study showed that DMOG not only alleviated glucocorticoid-induced osteonecrosis but also promoted angiogenesis and osteogenesis in the femoral head as detected by microcomputed tomography (Micro-CT) analysis and histological staining of OCN, TRAP, and Factor Ⅷ. However, all of these effects were impaired by an HIF-1α inhibitor. These findings demonstrate that targeting HIF-1α in EPCs may constitute a novel therapeutic approach for the treatment of SONFH.

Hsa-miR-409-3p regulates endothelial progenitor senescence via PP2A-P38 and is a potential ageing marker in humans.

We explored the roles of hsa-microRNA (miR)-409-3p in senescence and signalling mechanism of human endothelial progenitor cells (EPCs). Hsa-miR-409-3p was found upregulated in senescent EPCs. Overexpression of miRNA mimics in young EPCs inhibited angiogenesis. In senescent EPCs, compared to young EPCs, protein phosphatase 2A (PP2A) was downregulated, with activation of p38/JNK by phosphorylation. Young EPCs treated with siPP2A caused inhibited angiogenesis with activation of p38/JNK, similar to findings in senescent EPCs. Time series analysis showed, in young EPCs treated with hsa-miR-409-3p mimics, PP2A was steadily downregulated for 72 h, while p38/JNK was activated with a peak at 48 hours. The inhibited angiogenesis of young EPCs after miRNA-409-3p mimics treatment was reversed by the p38 inhibitor. The effect of hsa-miR-409-3p on PP2A signalling was attenuated by exogenous VEGF. Analysis of human peripheral blood mononuclear cells (PBMCs) obtained from healthy people revealed hsa-miR-409-3p expression was higher in those older than 65 years, compared to those younger than 30 years, regardless of gender. In summary, hsa-miR-409-3p was upregulated in senescent EPCs and acted as a negative modulator of angiogenesis via targeting protein phosphatase 2 catalytic subunit alpha (PPP2CA) gene and regulating PP2A/p38 signalling. Data from human PBMCs suggested hsa-miR-409-3p a potential biomarker for human ageing.

S-38-3: LONG NONCODING RNA P21 ALLEVIATES ENDOTHELIAL PROGENITOR CELLS DAMAGE AND PROMOTES ENDOTHELIAL REPAIR IN HYPERTENSION THROUGH ENHANCING AUTOPHAGY

Objective: Vascular damage of hypertension has been the focus of hypertension treatment, and endothelial progenitor cells (EPCs) play an important role in the repair of vascular endothelial damage. Functional damage and decreased number of EPCs are observed in the peripheral circulation of hypertensive patients, but its mechanism is not yet elucidated. Methods: Peripheral circulating endothelial progenitor cells of hypertensive patients were labeled and detected by flow cytometry. At the same time, the endothelial progenitor cells of hypertensive patients were cultured in vitro to detect their cell function and aging. The rat bone marrow-derived endothelial progenitor cells were isolated and cultured in vitro. After the intervention of AngII, the cell function, autophagy, and cell senescence were detected. PCR and WB were used to measure the expression and phosphorylation of SESN2, AMPK and TSC2. Results: Our results showed that the number of EPCs in hypertensive patients is significantly lower than that of normal population, and the cell function decreases with a higher proportion of EPCs at later stages. A decrease in autophagy is responsible for the senescence and damage of EPCs induced by AngII. Moreover, lncRNA-p21 plays a critical regulator role in the senescence and dysfunction of EPCs. Furthermore, lncRNA-p21 activates SESN2/AMPK/TSC2 pathway by promoting the transcriptional activity of p53 and lncRNA-p21 overexpression enhanced autophagy via AMPK/TSC2/mTOR pathway to protect against AngII-induced EPC damage. EPCs with lncRNA-p21 knockdown showed poor adhesion ability and vascular repair function. These results suggest that lncRNA-p21 enhances autophagy to alleviate endothelial progenitor cells damage and promote endothelial repair in hypertension through SESN2/AMPK/TSC2 pathway. Conclusion: The data provide evidence that a reversal of decreased autophagy serves as the protective mechanism of EPC injury in hypertensive patients, and lncRNA-p21 is a new therapeutic target for vascular endothelial repair in hypertension.

Melatonin Improves Ischemia-Induced Circulation Recovery Impairment in Mice with Streptozotocin-Induced Diabetes by Improving the Endothelial Progenitor Cells Functioning.

Patients with diabetes mellitus tend to develop ischemia-related complications and have compromised endothelial progenitor cell (EPC) function. Melatonin protects against ischemic injury, possibly via EPC modulation. We investigated whether melatonin pretreatment could restore EPC function impairment and improve circulation recovery in a diabetic critical limb ischemia mouse model. Under 25 mM high-glucose medium in vitro, EPC proliferation, nitric oxide production, tube formation, and endothelial nitric oxide synthase (eNOS) phosphorylation were significantly suppressed. Hyperglycemia promoted EPC senescence and apoptosis as well as increased reactive oxygen species (ROS) production. Melatonin treatment reversed the harmful effects of hyperglycemia on EPC through adenosine monophosphate–activated protein kinase-related mechanisms to increase eNOS phosphorylation and heme oxygenase-1 expression. In an in-vivo study, after a 4-week surgical induction of hindlimb ischemia, mice with streptozotocin (STZ)-induced diabetes showed significant reductions in new vessel formation, tissue reperfusion, and EPC mobilization in ischemic hindlimbs compared to non-diabetic mice. Mice with STZ-induced diabetes that received melatonin treatment (10 mg/kg/day, intraperitoneal) had significantly improved blood perfusion ratios of ischemic to non-ischemic limb, EPC mobilization, and densities of capillaries. In addition, a murine bone marrow transplantation model to support these findings demonstrated that melatonin stimulated bone marrow-originated EPCs to differentiate into vascular endothelial cells in femoral ligation-induced ischemic muscles. In summary, this study suggests that melatonin treatment augments EPC function along with neovascularization in response to ischemia in diabetic mice. We illustrated the protective effects of melatonin on EPC H2O2 production, senescence, and migration through melatonin receptors and modulating eNOS, AMPK, and HO-1 activities at the cellular level. Thus, melatonin might be used to treat the impairment of EPC mobilization and circulation recuperation in response to ischemic injury caused by chronic hyperglycemia. Additional studies are needed to elucidate the applicability of the results in humans.

Inhibition of oxidative stress delays senescence and augments functional capacity of endothelial progenitor cells

Ageing is characterised by a progressive loss of vascular endothelial function and integrity. Endothelial progenitor cells (EPCs) play an integral role in endothelial regeneration but are prone to age-dependent changes which may accelerate their senescence and diminish their availability and functionality. Considering these, we firstly investigated the quantity of circulating EPCs in older (73.3 ± 7.2 years) and younger (40.2 ± 14.3 years) healthy volunteers and showed sharp declines in the number of EPCs expressing stemness markers (CD34 + and/or CD133 + ) in older people. These coincided with the decreases in total anti-oxidant capacity (TAC) and concomitant increases in plasma levels of pro-inflammatory cytokine, TNF-α and anti-angiogenic factor, endostatin and thrombospondin-1. The subsequent experimental studies to scrutinise the effect of ageing on molecular and functional properties of outgrowth endothelial cells (OECs), the functional subtype of EPCs, showed that chronological ageing, mimicked by replicative senescence, profoundly impaired proliferation, migration, tubulogenesis, and blood–brain barrier (BBB)-forming capacity of these cells. Similar to those seen in the clinical observational studies, senescent OECs also manifested decreased TAC and increased pro-oxidant NADPH oxidase activity and endostatin level. Suppressing oxidative stress level using structurally and functionally distinct anti-oxidants, namely vitamin C or VAS2870, an NADPH oxidase inhibitor, delayed OEC senescence and restored their tubulogenic and BBB-forming capacities. In conclusion, the enhanced oxidative stress level that develops during physiological ageing may promote EPC senescence and evoke endothelial dysfunction. Effective control of oxidative stress using either compound somewhat delays both phenomena and augments EPC functionality.

Cigarette smoke extract mediates cell premature senescence in chronic obstructive pulmonary disease patients by up-regulating USP7 to activate p300-p53/p21 pathway

ObjectivesTobacco hazard is one of the most severe public health issues in the world. It is believed that smoking is the most important factor leading to chronic obstructive pulmonary disease (COPD). Endothelial progenitor cells (EPCs) originate from the bone marrow and can effectively repair vascular endothelial damage and improve vascular endothelial function. Current studies suggest that EPCs senescence and EPCs depletion exist in smoking-related COPD, but the molecular mechanism remains unclear. MethodsCo-immunoprecipitation was used to detect the interaction between USP7 and p300. EPCs from smoking COPD patients were isolated, and the expressions of USP7 and p300 were detected by RT-PCR and Western Blot. Different concentrations of cigarette smoke extract (CSE) and USP7 or p300 inhibitors were used to treat EPCs, then the expression of p53, p53 target genes and aging-related genes were detected. Cell Counting Kit - 8 (CCK8) was used to detect cell proliferation, flow cytometry was used to detect cell cycle distribution, β-galactosidase (β-gal) staining and Lamp1 immunofluorescence was used to detect the proportion of aging cells. COPD mouse models were used to confirm the molecular mechanism. ResultsUSP7 and p300 interacted with each other, and USP7 affected the protein stability of p300 by regulating the ubiquitination of p300. There existed high expressions of USP7 and p300 proteins in EPCs of smoking COPD patients and COPD mouse model. CSE promoted the high expressions of USP7 and p300 in EPCs. Further studies showed that CSE mediated the USP7/p300-dependent high expression of p53 and activated the expression of p53 target genes especially p21. Activation of p53 - p21 pathway finally inhibited cell activity, led to cell cycle arrest and premature senescence of EPCs. ConclusionCSE mediated up-regulation of USP7 and p300 activated p53 - p21 pathway was a molecular mechanism that might lead to COPD.

Abstract 14015: Extracellular Vesicle-Associated microRNAs From the Plasma of HIV-Positive Individuals Enhance Atherosclerosis Burden

Introduction: People living with HIV (PLHIV) have a higher risk of cardiovascular diseases (CVD) compared to HIV negative (HIV - ) individuals. The molecular mechanisms underlying the increased CVD risk remain elusive. Extracellular vesicles (EVs) mediate cell-to-cell communication through cargo transfer, including microRNAs (EV-miRs). Hypothesis: HIV infection results in modification of plasma EVs cargo and these HIV positive EVs (HIV posEVs ), specifically EV-miRs, recapitulate the effects of HIV infection on CVD development. Methods: Atherosclerosis burden was assessed by carotid ultrasound in 74 PLHIV treated with antiretroviral therapy (ART) with viral loads <50 c/ml and 23 HIV - subjects. Circulating endothelial progenitor cell (EPC) levels were measured by colony forming unit (CFU) assay. PLHIV plasma was fractionated into plasma EVs (HIV posEVs ) and plasma-depleted of EVs (HIV plasmadepEVs ). These fractions, EVs from HIV - subjects (HIV negEVs ) and PBS were injected weekly into apoE -/- mice via tail vein for three months. Atherosclerosis burden (H&E and Oil Red O staining), bone marrow (BM) EPC senescence (β-galactosidase staining), proliferation (MTT assay) and migration (“scratch” assay) were examined. RNASeq was used to identify EV-miRs that mediated the effects of HIV posEVs . Tailored EVs (TEVs) loaded with antagomirs for top EV-miRs were generated to determine their capacity to reverse the adverse effects of HIV posEVs in mouse EPCs in vitro . Results: PLHIV had higher atherosclerosis burden and lower circulating EPC numbers. Mice treated with HIV posEVs showed increased atherosclerotic burden, higher BM EPC senescence and lower functionality. HIV posEVs also negatively affected mouse BM EPC senescence in vitro . RNAseq identified four top candidate EV-miRs upregulated in HIV posEVs : let-7b-5p, miR-16-5p, miR-423-5p, and miR-103a-2. TEVs loaded with the antagomirs for let-7b and miR103a-2 counteracted the effects of HIV posEVs on the accelerated senescence of mouse BM EPCs. Conclusion: 1) Plasma EVs from PLHIV reconstitute the effects of HIV infection on accelerated atherosclerosis via negatively affects EPC functionality; and 2) TEVs, targeting candidate EV-miRs counteracts the effects of HIV infection on EPCs.

Long noncoding RNA p21 enhances autophagy to alleviate endothelial progenitor cells damage and promote endothelial repair in hypertension through SESN2/AMPK/TSC2 pathway

Vascular damage of hypertension has been the focus of hypertension treatment, and endothelial progenitor cells (EPCs) play an important role in the repair of vascular endothelial damage. Functional damage and decreased number of EPCs are observed in the peripheral circulation of hypertensive patients, but its mechanism is not yet elucidated. Here, we show that the number of EPCs in hypertensive patients is significantly lower than that of normal population, and the cell function decreases with a higher proportion of EPCs at later stages. A decrease in autophagy is responsible for the senescence and damage of EPCs induced by AngII. Moreover, lncRNA-p21 plays a critical regulator role in EPCs’ senescence and dysfunction. Furthermore, lncRNA-p21 activates SESN2/AMPK/TSC2 pathway by promoting the transcriptional activity of p53 and enhances autophagy to protect against AngII-induced EPC damage. The data provide evidence that a reversal of decreased autophagy serves as the protective mechanism of EPC injury in hypertensive patients, and lncRNA-p21 is a new therapeutic target for vascular endothelial repair in hypertension.

Bradykinin Protects Human Endothelial Progenitor Cells from High-Glucose-Induced Senescence through B2 Receptor-Mediated Activation of the Akt/eNOS Signalling Pathway.

Background Circulating endothelial progenitor cells (EPCs) play important roles in vascular repair. However, the mechanisms of high-glucose- (HG-) induced cord blood EPC senescence and the role of B2 receptor (B2R) remain unknown. Methods Cord blood samples from 26 patients with gestational diabetes mellitus (GDM) and samples from 26 healthy controls were collected. B2R expression on circulating CD34+ cells of cord blood mononuclear cells (CBMCs) was detected using flow cytometry. The plasma concentrations of 8-isoprostaglandin F2α (8-iso-PGF2α) and nitric oxide (NO) were measured. EPCs were treated with HG (40 mM) alone or with bradykinin (BK) (1 nM). The B2R and eNOS small interfering RNAs (siRNAs) and the PI3K antagonist LY294002 were added to block B2R, eNOS, and PI3K separately. To determine the number of senescent cells, senescence-associated β-galactosidase (SA-β-gal) staining was performed. The level of mitochondrial reactive oxygen species (ROS) in EPCs was assessed by Mito-Sox staining. Cell viability was evaluated by Cell Counting Kit-8 (CCK-8) assays. Mitochondrial DNA (mtDNA) copy number and the relative length of telomeres were detected by real time-PCR. The distribution of human telomerase reverse transcriptase (hTERT) in the nucleus, cytosol, and mitochondria of EPCs was detected by immunofluorescence. The expression of B2R, p16, p21, p53, P-Ser473AKT, T-AKT, eNOS, and hTERT was demonstrated by Western blot. Results B2R expression on circulating CD34+ cells of CBMCs was significantly reduced in patients with GDM compared to healthy controls. Furthermore, B2R expression on circulating CD34+ cells of CBMCs was inversely correlated with plasma 8-iso-PGF2α concentrations and positively correlated with plasma NO levels. BK treatment decreased EPC senescence and ROS generation. Furthermore, BK treatment of HG-exposed cells led to elevated P-Ser473AKT and eNOS protein expression compared with HG treatment alone. BK reduced hTERT translocation in HG-induced senescent EPCs. B2R siRNA, eNOS siRNA, and antagonist of the PI3K signalling pathway blocked the protective effects of BK. Conclusion BK, acting through PI3K-AKT-eNOS signalling pathways, reduced hTERT translocation, increased the relative length of telomeres while reducing mtDNA copy number, and finally protected against EPC senescence induced by HG.

Deferoxamine accelerates endothelial progenitor cell senescence and compromises angiogenesis.

Senescence reduces the circulating number and angiogenic activity of endothelial progenitor cells (EPCs), and is associated with aging-related vascular diseases. However, it is very time-consuming to obtain aged cells (~1 month of repeated replication) or animals (~2 years) for senescence studies. Here, we established an accelerated senescence model by treating EPCs with deferoxamine (DFO), an FDA-approved iron chelator. Four days of low-dose (3 μM) DFO induced senescent phenotypes in EPCs, including a senescent pattern of protein expression, impaired mitochondrial bioenergetics, altered mitochondrial protein levels and compromised angiogenic activity. DFO-treated early EPCs from young and old donors (< 35 vs. > 70 years old) displayed similar senescent phenotypes, including elevated senescence-associated β-galactosidase activity and reduced relative telomere lengths, colony-forming units and adenosine triphosphate levels. To validate this accelerated senescence model in vivo, we intraperitoneally injected Sprague-Dawley rats with DFO for 4 weeks. Early EPCs from DFO-treated rats displayed profoundly senescent phenotypes compared to those from control rats. Additionally, in hind-limb ischemic mice, DFO pretreatment compromised EPC angiogenesis by reducing both blood perfusion and capillary density. DFO thus accelerates EPC senescence and appears to hasten model development for cellular senescence studies.