Thrombopoietin Protects Neural Cells and Endothelial Cells from Apoptosis Via PI3K/AKT Pathway

Background: Thrombopoietin (TPO) is a hematopoietic growth factor that regulates the production of platelets and stimulates production and differentiation. The expression of TPO and TPO receptor (c-mpl) in the central nervous system (CNS) has been identified. However, the role of TPO in neural cells and endothelial cells were not clear. Methods: C17.2 and human umbilical vein endothelial (HUVEC) cells were treated with CoCl2, TPO, or TPO + CoCl2. TPO was added into the culture medium 48 h before CoCl2 treatment. The cell viability and apoptosis of each group were tested by Cell Counter Kit 8 (CCK-8) assay and flow cytometry. The expression of Caspase-3 and mitochondrial membrane potential (MMP) were then determined by flow cytometry with Caspase-3-PE and JC-1. The effect of TPO in the PI3K/AKT pathway was detected by using Western blot. Results: TPO has a dose-dependent effect on the growth of C17.2 cells. LY-294002 pretreatment suppressed the TPO-induced AKT activation and abolished the prosurvival effect of TPO. Via the Bcl-2/BAX signaling pathway, TPO exerted an anti-apoptotic effect by suppressing mitochondria membrane potentials. We also investigated the protective effect of TPO on human endothelial cells. The cell viability of HUVECs decreased gradually with the enhancement of CoCl2 at a gradient of chemical concentrations (r= -0.997). CoCl2 dramatically increased apoptosis of HUVECs, whereas pre-treatment with TPO rescued cell apoptosis induced by CoCl2 (P<0.01). Further investigation found that TPO decreased the expression of Caspase-3 and inhibited the reduction of MMP induced by CoCl2 (P<0.05). TPO increased the activation of PI3K/AKT pathway in HUVECs. Conclusion: TPO has a protective effect against apoptosis of neural cells and endothelial cells through activating the PI3K/AKT pathway, thus decreasing the expression of apoptosis protease Caspase-3 and inhibiting the reduction of MMP. Disclosures No relevant conflicts of interest to declare.

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Abstract 15183: Thrombopoietin Has a Protective Effect Against Apoptosis of Endothelial Cells Through Activating Pi3k/akt Pathway

Circulation ◽

10.1161/circ.142.suppl_3.15183 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Liang Li ◽

Junyan Wang ◽

kuan zeng ◽

hua zhang ◽

Yanqi Yang ◽

...

Keyword(s):

Flow Cytometry ◽

Endothelial Cells ◽

Protective Effect ◽

Cell Viability ◽

Signaling Pathway ◽

Caspase 3 ◽

Umbilical Vein ◽

Neonatal Rat ◽

Akt Pathway ◽

Neural Cells

Introduction: Thrombopoietin (TPO) is a hematopoietic growth factor for platelet lineage. TPO was found to be neuroprotective in hypoxic-ischemic neonatal rat brain models and treatment with TPO reduced brain damage and improved sensorimotor functions (Li L et al, Aging-US, 2020). However, the underlying mechanism of TPO in this model, and its role in neural cells and endothelial cells were still unclear. Methods: C17.2 cells were divided into control (0.5% FCS), Normal (10% FCS), TPO and TPO + LY294002 groups. Human umbilical vein endothelial (HUVEC) cells were divided in to normal, CoCl 2 , TPO and TPO + CoCl2 groups. The expressed of TPO and c-mpl was tested by RT-PCR. The cell viability and apoptosis of each group were tested by Cell Counter Kit 8 (CCK-8) assay and flow cytometry. The expression of Caspase-3 and mitochondrial membrane potential (MMP) were then determined by flow cytometry with Caspase-3-PE and JC-1. The effect of TPO in PI3K/AKT pathway was detected by using Western blot. Results: Both TPO and c-mpl are expressed in the neurons of the human CNS. TPO was also detected in human cerebrospinal fluid. TPO promoted C17.2 cell proliferation through activation of the PI3K/Akt signaling pathway. Via the Bcl-2/BAX signaling pathway, TPO exerted an anti-apoptotic effect by suppressing mitochondria membrane potentials. We also investigated the protective effect of TPO on human endothelial cells. CoCl 2 significantly inhibited the growth of HUVECs. The cell viability of HUVECs decreased gradually with the enhancement of CoCl2 at a gradient of chemical concentrations (r= -0.997). CoCl 2 dramatically increased apoptosis of HUVECs, whereas pre-treatment with TPO rescued cell apoptosis induced by CoCl 2 (P<0.001). Further investigation found that TPO decreased the expression of Caspase-3 and inhibited the reduction of MMP induced by CoCl 2 (P<0.05). TPO increased the activation of PI3K/AKT pathway in HUVECs. Conclusions: TPO has a protective effect against apoptosis of neural cells and endothelial cells through activating the PI3K/AKT pathway, thus decreasing the expression of apoptosis protease Caspase-3 and inhibiting the reduction of MMP.

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Tanshinone IIA Attenuates H2O2-Induced Injury in Human Umbilical Vein Endothelial Cells

The American Journal of Chinese Medicine ◽

10.1142/s0192415x12500966 ◽

2012 ◽

Vol 40 (06) ◽

pp. 1307-1319 ◽

Cited By ~ 15

Author(s):

Paul Chan ◽

Yen-Cheng Chen ◽

Li-Jen Lin ◽

Tzu-Hurng Cheng ◽

Kazunori Anzai ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Protective Effect ◽

Cell Apoptosis ◽

Caspase 3 ◽

Umbilical Vein ◽

Tanshinone Iia ◽

Cardiovascular Disorders ◽

P53 Expression ◽

Umbilical Vein Endothelial Cells

The injury of endothelial cell is the critical event of vascular disease. In endothelial cell, oxidative stress is regarded as critical to pathogenic factors in endothelial cell injury and apoptosis. Tanshinone IIA is the main effective component of Salvia miltiorrhiza known as "Danshen" in traditional Chinese medicine for treating cardiovascular disorders, but the mechanism by which it exerts the protective effect is not well established. The present study was designed to test the hypothesis that tanshinone IIA can inhibit hydrogen peroxide ( H2O2 )-induced injury and unravel its intracellular mechanism in human umbilical vein endothelial cells (HUVECs). In this study, HUVECs were treated with tanshinone IIA in the presence/absence of H2O2 . The protective effects of tanshinone IIA against H2O2 were evaluated. Our results show that HUVECs incubated with 200 μM H2O2 had significantly decreased the viability of endothelial cells, which was accompanied with apparent cell apoptosis, the activation of caspase-3 and the upregulation of p53 expression, which was known to play a key role in H2O2 -induced cell apoptosis. However, pretreatment with tanshinone IIA (3–10 μM) resulted in a significant resistance to H2O2 -induced apoptosis. In addition, pretreatment with tanshinone IIA decreased the activity of caspase-3 and p53 expression. Tanshinone IIA also induced activating transcription factor (ATF) 3 expression; while knockdown of ATF-3 with ATF-3 siRNAsignificantly reduced tanshinone IIA's protective effect. In conclusion, the present study shows that tanshinone IIA can protect endothelial cells against oxidative injury induced by H2O2 , suggesting that this compound may constitute a promising intervention against cardiovascular disorders and ATF-3 may play an important role in this process.

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N-acetylcysteine resists high-glucose-induced injury of human umbilical vein endothelial cells by inhibiting the leptin/leptin receptor

10.21203/rs.3.rs-18647/v1 ◽

2020 ◽

Author(s):

Zhenhua Wang ◽

Jun Chen ◽

Shuping Lian ◽

Kaibin Liao ◽

Lizhi Huang ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Viability ◽

High Glucose ◽

Caspase 3 ◽

Leptin Receptor ◽

Umbilical Vein ◽

Enzyme Linked Immunosorbent Assay ◽

Apoptotic Cells ◽

Human Umbilical Vein ◽

Umbilical Vein Endothelial Cells

Abstract Baclground The present study aimed to investigate whether N- acetylcysteine (NAC) protects human umbilical vein endothelial cells (HUVECs) against high glucose (HG)-induced injury by inhibiting leptin/leptin receptor (LEPR).Methods HUVECs were treated with 40 mmol/L glucose for 24 h to establish a model of HG-induced endothelial cell injury; The cell viability was examined by cell counter kit-8(CCK-8) assay; The expression levels of leptin, LEPR, cleaved caspase-3 and endothelial nitric oxide synthase (eNOS) were detected by western blot. The intracellular levels of reactive oxygen species (ROS) were tested by DCFH-DA staining followed by photofluorography. Tumor necrosis factor-α (TNF-α)、nuclear factor-kappa B (NF-κB) and intercellular adhesion molecule-1 (ICAM-1) were detected by enzyme-linked immunosorbent assay (ELISA). The number of apoptotic cells was observed by photofluorograph with Hoechst 33258 nuclear staining. Mitochondrial membrane potential (MMP) was obtained using JC-1. Results The expression of leptin and LEPR began to significantly increase after exposure to 40 mmol/L HG for 24 h. Pretreatment of HUVECs with 7 mmol/L NAC or 50 ng/mL leptin antagonists (LA) for 30min inhibited the increased expression of leptin and LEPR induced by HG in HUVECs. Furthermore, pretreatment with 7 mmol/L NAC or 50 ng/mL LA for 30 min also inhibited HG-induced injury, by increasing the cell viability and eNOS expression, and decreasing the inflammatory response and cleaved caspase-3 expression, the apoptotic cells and generation of intracellular ROS and a loss of MMP. Conclusions NAC protects the HUVECs against HG-induced injury by inhibiting leptin/LEPR.

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Down-Regulation of MicroRNA-137 Improves High Glucose-Induced Oxidative Stress Injury in Human Umbilical Vein Endothelial Cells by Up-Regulation of AMPKα1

Cellular Physiology and Biochemistry ◽

10.1159/000447795 ◽

2016 ◽

Vol 39 (3) ◽

pp. 847-859 ◽

Cited By ~ 26

Author(s):

Jie Li ◽

Junfeng Li ◽

Tingting Wei ◽

Junhua Li

Keyword(s):

Oxidative Stress ◽

Endothelial Cells ◽

Protective Effect ◽

Cell Viability ◽

Vascular Injury ◽

High Glucose ◽

Umbilical Vein ◽

Down Regulation ◽

Human Umbilical Vein ◽

Umbilical Vein Endothelial Cells

Background/Aims: To investigate the effects of miR-137 on high glucose (HG)-induced vascular injury, and to establish the mechanism underlying these effects. Methods: Human umbilical vein endothelial cells (HUVECs) were transfected with miR-137 inhibitor or mimic, and then treated with normal or high glucose. Cell viability and apoptosis were detected by using the Cell Counting Kit-8 (CCK-8) assay and flow cytometry, respectively. Reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD) were detected by fluorescent probe (DCFH-DA), thiobarbituric acid reaction, and the nitroblue tetrazolium assay, respectively. The mRNA and protein expressions of AMPKα1 were determined by qRT-PCR and Western blotting. Results: Down-regulation of miR-137 dramatically reverted HG-induced decreases in cell viability and SOD levels and increases in apoptosis, ROS and MDA levels. Moreover, bioinformatics analysis predicted that the AMPKα1 was a potential target gene of miR-137. Luciferase reporter assay demonstrated that miR-137 could directly target AMPKα1. AMPKα1 overexpression had the similar effect as miR-137 inhibition. Down-regulation of AMPKα1 in HUVECs transfected with miR-137 inhibitor partially reversed the protective effect of miR-137 inhibition on HG-induced oxidative stress in HUVECs. Conclusion: Down-regulation of miR-137 ameliorates HG-induced injury in HUVECs by overexpression of AMPKα1, leading to increasing cellular reductive reactions and decreasing oxidative stress. These results provide further evidence for protective effect of miR-137 inhibition on HG-induced vascular injury.

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Role of Thrombopoietin in Stimulating Megakaryopoiesis of ITP Patients Via Rescuing Vascular Niche

Blood ◽

10.1182/blood-2019-125679 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1063-1063

Author(s):

Yingying Jiao ◽

Xiaoyuan Zeng ◽

Zongpeng Li ◽

Beng H Chong ◽

Yujiao Zhang ◽

...

Keyword(s):

Flow Cytometry ◽

Endothelial Cells ◽

Protective Effect ◽

Conflicts Of Interest ◽

Treated Group ◽

Tube Formation ◽

Control Group ◽

Endothelial Progenitor ◽

Vascular Niche ◽

And Migration

Background: Previous studies have shown that the vascular niche was damaged in patients with immune thrombocytopenia (ITP). It is mainly manifested that the migration and tube-formation ability of bone marrow endothelial progenitor cells (BM-EPCs) were significantly reduced. Endothelial cells as an important part of the vascular niche play an important role in regulation of megakaryocytes and platelet hematopoiesis. Our previous studies suggested that thrombopoietin (TPO) has a protective effect on human endothelial cells. This study aim to investigate whether TPO has a protective effect in EPCs of ITP patients and to explore its mechanism. Methods: BM-EPCs were isolated from ITP patients, and divided into two groups: TPO-treated group and TPO-free (control group). CCK8 was used to explore whether TPO has proliferative effect on BM-EPCs. BM-EPCs was identified by flow cytometry with co-expression of mouse anti-humanCD133, mouse anti-human CD34 and mouse anti-human vascular endothelial growth factor receptor (CD309) monoclonal antibodies. Moreover, the number of EPCs was evaluated with DiL-Ac-LDL uptake and FITC-UEA -I binding assay. Furthermore, the function of BM-EPCs was studied by using tube-formation and migration experiments. Results: The ITP patient-derived EPCs in TPO-treated group exhibited longer fusiform and elliptical shape after 5 days and cobblestone-like morphology after 7-10 days. The result of CCK-8 showed TPO could promote the proliferation of EPCs and the optional drug concentration is 100 ng/ml. Much higher expression of CD34/CD133/CD309 in TPO-treated group was detected by flow cytometry. The number of cells with immunofluorescence double staining in TPO group was significantly higher than of control group (n=4, p<0.05). The tube formation (n=5, p<0.05) and migration capacity (n=5, p<0.05) were significantly enhanced in TPO-treated group. Conclusion: Our studies indicates that TPO may exert megakaryopoiesis effect in ITP patients via protection of BM-EPCs. This may provide a supplemental explanation of how recombinant TPO and TPO receptor antagonist acts on ITP treatment. Keywords: thrombopoietin; vascular niche; endothelial progenitor cell, megakaryopoiesis; endothelial cell Disclosures No relevant conflicts of interest to declare.

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Salidroside protects endothelial cells against LPS-induced inflammatory injury by inhibiting NLRP3 and enhancing autophagy

BMC Complementary Medicine and Therapies ◽

10.1186/s12906-021-03307-0 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Lijiao You ◽

Di Zhang ◽

Huan Geng ◽

Fangyuan Sun ◽

Ming Lei

Keyword(s):

Endothelial Cells ◽

Cell Viability ◽

Inflammatory Cytokines ◽

Caspase 3 ◽

Cell Injury ◽

Culture Media ◽

Umbilical Vein ◽

Receptor Protein ◽

Sod Activity ◽

Related Proteins

Abstract Background Salidroside (SAL) is a bioactive compound extracted from Rhodiola rosea with various biological properties. This study was designed to explore the functions of SAL on the endothelial damage induced by lipopolysaccharide (LPS) and its related mechanisms. Methods Human umbilical vein endothelial cells (HUVECs) were pretreated with SAL (0, 10, 25, 50, 100 μM), and then incubated with LPS (10 μg/mL). Cell viability was evaluated by MTT assay, cell injury by lactate dehydrogenase (LDH) release, and inflammatory cytokines release by ELISA assay. Oxidative stress was evaluated by malondialdehyde (MDA) and superoxide dismutase (SOD) in cell lysate. Apoptosis was detected by flow cytometry and caspase-3 activity. Western blot were performed to determine expression levels of autophagy and NOD-like receptor protein 3 (NLRP3) related proteins. Results SAL at 50 μM concentration showed no toxicity on HUVECs, but attenuated LPS-induced injury, as evidenced by increased cell viability, reduction in LDH level and inflammatory cytokines in culture media. SAL also reduced MDA level and increased SOD activity in HUVECs, and inhibited apoptosis rate and caspase-3 activity. (P < 0.05). Moreover, LPS enhanced HUVECs autophagy, and SAL pretreatment further enhanced autophagy, with increased Beclin-1 protein and decreased P62 protein. SAL also attenuated LPS-induced activation of NLRP3 inflammasome, reduced the protein expression of NLRP3-related proteins, including ASC and caspase-1. Autophagy inhibition by 3-MA markedly reversed SAL-modulated changes in cell viability and NLRP3 expression in LPS-stimulated HUVECs. Conclusion SAL protects endothelial cells against LPS-induced injury through inhibition of NLRP3 pathways and enhancing autophagy.

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Sirolimus and propranolol inhibit endothelial proliferation while detergent sclerosants induce endothelial activation, microparticle release and apoptosis in vitro

Phlebology The Journal of Venous Disease ◽

10.1177/0268355520913384 ◽

2020 ◽

Vol 35 (8) ◽

pp. 566-575 ◽

Cited By ~ 2

Author(s):

David E. Connor ◽

Jessica Gerbelli ◽

An-Ning Chew ◽

Osvaldo Cooley-Andrade ◽

Dulani Goonawardhana ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Proliferation ◽

Endothelial Cells ◽

Caspase 3 ◽

Umbilical Vein ◽

Endothelial Activation ◽

Endothelial Microparticles ◽

Sodium Tetradecyl Sulphate ◽

Umbilical Vein Endothelial Cells

Objectives To investigate the effects of detergent sclerosants, sodium tetradecyl sulphate and polidocanol, on endothelial cell activation and microparticle release and the effects of detergent sclerosants, sirolimus and propranolol, on apoptosis in vitro. Methods Cultured human umbilical vein endothelial cells and murine haemangioendothelioma (EOMA) cell lines were incubated with different concentrations of sodium tetradecyl sulphate and polidocanol, as well as sirolimus and propranolol. Endothelial activation was assessed using flow cytometry for CD62e (E-Selectin), CD54 (ICAM-1), CD105 (endoglin), CD144 (VE-Cadherin), CD146 (MCAM) and the release of endothelial microparticles. Cell proliferation was assessed using [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] and carboxyfluorescein succinimidyl ester assays. Apoptosis was assessed using flow cytometry for lactadherin/propidium iodide staining and for Caspase-3 expression. Results Sublytic concentrations of sodium tetradecyl sulphate and polidocanol (0.075%–0.3%) increased the expression of the activation markers CD62e and CD54. The expression of CD105 decreased in sclerosant treated cultured human umbilical vein endothelial cells. Both sodium tetradecyl sulphate and polidocanol induced the release of endothelial microparticles. All agents inhibited cell proliferation. Sodium tetradecyl sulphate and polidocanol-induced apoptosis as evidenced by increased phosphatidylserine exposure and caspase-3 expression, whereas sirolimus and propranolol increased caspase-3 expression only. Conclusion Sublytic concentrations of detergent sclerosants induce endothelial activation and the release of endothelial microparticles. All agents were anti-proliferative in EOMA cell lines, with sodium tetradecyl sulphate and polidocanol inducing cellular apoptosis.

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Protective effects and plausible mechanisms of antler-velvet polypeptide against hydrogen peroxide induced injury in human umbilical vein endothelial cells

Canadian Journal of Physiology and Pharmacology ◽

10.1139/cjpp-2016-0196 ◽

2017 ◽

Vol 95 (5) ◽

pp. 610-619 ◽

Cited By ~ 2

Author(s):

Wenhe Zhu ◽

Huiyan Wang ◽

Wei Zhang ◽

Na Xu ◽

Junjie Xu ◽

...

Keyword(s):

Oxidative Stress ◽

Flow Cytometry ◽

Endothelial Cells ◽

Membrane Potential ◽

Cell Viability ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Umbilical Vein ◽

Protective Effects ◽

Umbilical Vein Endothelial Cells

Antler velvet polypeptide (VAP) is a prominent bioactive component of antler velvet. Whereas uncharacterized crude extracts have typically been used in pharmacological studies, in this study, the velvet polypeptide was isolated and purified by acid water extraction, ethanol precipitation, ammonium sulfate fractionation and precipitation, and chromatography, progressively. Human umbilical vein endothelial cells (HUVECs) were induced with H2O2 followed purified polypeptide treatment. Cell viability was evaluated by MTT assay. The apoptosis of cells was detected by fluorescence microscopy and flow cytometry. A cell analyzer was used to measure the mitochondrial membrane potential. The intracellular reactive oxygen species (ROS) levels were determined by flow cytometry. Oxidative stress related biochemical parameters were detected, and the expression of apoptosis-related proteins was examined by Western blot analysis. The results indicated that a 7.0 kDa polypeptide (VAP II) was isolated from antler velvet. VAP II enhanced cell viability, decreased cell apoptosis, reversed depolarization of mitochondrial membrane potential, decreased ROS levels, inhibited oxidative stress, and regulated the downstream signaling apoptotic cascade expression caused by H2O2. The protective effects of VAP II on HUVECs suggests a potential strategy for the treatment of cardiovascular disease.

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N-acetylcysteine resists high-glucose-induced injury of human umbilical vein endothelial cells by inhibiting the leptin/leptin receptor

10.21203/rs.3.rs-18647/v2 ◽

2020 ◽

Author(s):

Zhenhua Wang ◽

Jun Chen ◽

Shuping Lian ◽

Kaibin Liao ◽

Lizhi Huang ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Viability ◽

High Glucose ◽

Caspase 3 ◽

Leptin Receptor ◽

Umbilical Vein ◽

Enzyme Linked Immunosorbent Assay ◽

Apoptotic Cells ◽

Human Umbilical Vein ◽

Umbilical Vein Endothelial Cells

Abstract Background The present study aimed to investigate whether N- acetylcysteine (NAC) protects human umbilical vein endothelial cells (HUVECs) against high glucose (HG)-induced injury by inhibiting leptin/leptin receptor (LEPR). Methods HUVECs were treated with 40 mmol/L glucose for 24 h to establish a model of HG-induced endothelial cell injury; The cell viability was examined by cell counter kit-8 (CCK-8) assay; The expression levels of leptin, LEPR, cleaved caspase-3 and endothelial nitric oxide synthase (eNOS) were detected by western blot. The intracellular levels of reactive oxygen species (ROS) were tested by DCFH-DA staining followed by photofluorography. Tumor necrosis factor-α (TNF-α)、nuclear factor-kappa B (NF-κB) and intercellular adhesion molecule-1 (ICAM-1) were detected by enzyme-linked immunosorbent assay (ELISA). The number of apoptotic cells was observed by photofluorograph with Hoechst 33258 nuclear staining. Mitochondrial membrane potential (MMP) was obtained using JC-1.Results The expression of leptin and LEPR began to significantly increase after exposure to 40 mmol/L HG for 24 h. Pretreatment of HUVECs with 7 mmol/L NAC or 50 ng/mL leptin antagonists (LA) for 30min inhibited the increased expression of leptin and LEPR induced by HG in HUVECs. Furthermore, pretreatment with 7 mmol/L NAC or 50 ng/mL LA for 30 min also inhibited HG-induced injury, by increasing the cell viability and eNOS expression, and decreasing the inflammatory response and cleaved caspase-3 expression, the apoptotic cells and generation of intracellular ROS and a loss of MMP. Conclusions NAC protects the HUVECs against HG-induced injury by inhibiting leptin/LEPR.

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