C-peptide protects against hyperglycemic memory and vascular endothelial cell apoptosis

C-peptide exerts protective effects against diabetic complications; however, its role in inhibiting hyperglycemic memory (HGM) has not been elucidated. We investigated the beneficial effect of C-peptide on HGM-induced vascular damage in vitro and in vivo using human umbilical vein endothelial cells and diabetic mice. HGM induced apoptosis by persistent generation of intracellular ROS and sustained formation of ONOO− and nitrotyrosine. These HGM-induced intracellular events were normalized by treatment with C-peptide, but not insulin, in endothelial cells. C-peptide also inhibited persistent upregulation of p53 and activation of mitochondrial adaptor p66shc after glucose normalization. Further, C-peptide replacement therapy prevented persistent generation of ROS and ONOO− in the aorta of diabetic mice whose glucose levels were normalized by the administration of insulin. C-peptide, but not insulin, also prevented HGM-induced endothelial apoptosis in the murine diabetic aorta. This study highlights a promising role for C-peptide in preventing HGM-induced intracellular events and diabetic vascular damage.

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DEPTOR Deficiency-Mediated mTORc1 Hyperactivation in Vascular Endothelial Cells Promotes Angiogenesis

Cellular Physiology and Biochemistry ◽

10.1159/000488619 ◽

2018 ◽

Vol 46 (2) ◽

pp. 520-531 ◽

Cited By ~ 7

Author(s):

Yan Ding ◽

Lanlan Shan ◽

Wenqing Nai ◽

Xiaojun Lin ◽

Ling Zhou ◽

...

Keyword(s):

Endothelial Cells ◽

Signaling Pathway ◽

Molecular Mechanisms ◽

Gene Knockout ◽

Umbilical Vein ◽

Vascular Endothelial Cell ◽

Tube Formation ◽

Vascular Endothelial

Background/Aims: The mechanistic target of rapamycin (mTOR) signaling pathway is essential for angiogenesis and embryonic development. DEP domain-containing mTOR-interacting protein (DEPTOR) is an mTOR binding protein that functions to inhibit the mTOR pathway In vitro experiments suggest that DEPTOR is crucial for vascular endothelial cell (EC) activation and angiogenic responses. However, knowledge of the effects of DEPTOR on angiogenesis in vivo is limited. This study aimed to determine the role of DEPTOR in tissue angiogenesis and to elucidate the molecular mechanisms. Methods: Cre/loxP conditional gene knockout strategy was used to delete the Deptor gene in mouse vascular ECs. The expression or distribution of cluster of differentiation 31 (CD31), vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1 alpha (HIF-1α) were detected by immunohistochemical staining or western blot. Tube formation assay was used to measure angiogenesis in vitro. Results: Deptor knockdown led to increased expression of CD31, VEGF and HIF-1α in heart, liver, kidney and aorta. After treatment with rapamycin, their expression was significantly down regulated. In vitro, human umbilical vein endothelial cells (HUVECs) were transfected with DEPTOR-specific small interfering RNA (siRNA), which resulted in a significant increase in endothelial tube formation and migration rates. In contrast, DEPTOR overexpression markedly reduced the expression of CD31, VEGF and HIF-1α. Conclusions: Our findings demonstrated that deletion of the Deptor gene in vascular ECs resulted in upregulated expression of CD31 and HIF-1α, and further stimulated the expression of VEGF which promoted angiogenesis, indicating that disruption of normal angiogenic pathways may occur through hyperactivation of the mTORC1/HIF-1α/VEGF signaling pathway.

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Protective Effects of Rhodiola Crenulata Extract on Hypoxia-Induced Endothelial Damage via Regulation of AMPK and ERK Pathways

International Journal of Molecular Sciences ◽

10.3390/ijms19082286 ◽

2018 ◽

Vol 19 (8) ◽

pp. 2286 ◽

Cited By ~ 6

Author(s):

Pi-Kai Chang ◽

I-Chuan Yen ◽

Wei-Cheng Tsai ◽

Tsu-Chung Chang ◽

Shih-Yu Lee

Keyword(s):

Oxidative Stress ◽

Endothelial Cells ◽

Umbilical Vein ◽

Hypoxic Exposure ◽

No Production ◽

Root Extract ◽

Protective Effects ◽

Rhodiola Crenulata

Rhodiola crenulata root extract (RCE) has been shown to possess protective activities against hypoxia both in vitro and in vivo. However, the effects of RCE on response to hypoxia in the endothelium remain unclear. In this study, we aimed to examine the effects of RCE in endothelial cells challenged with hypoxic exposure and to elucidate the underlying mechanisms. Human umbilical vein endothelial cells were pretreated with or without RCE and then exposed to hypoxia (1% O2) for 24 h. Cell viability, nitric oxide (NO) production, oxidative stress markers, as well as mechanistic readouts were studied. We found that hypoxia-induced cell death, impaired NO production, and oxidative stress. These responses were significantly attenuated by RCE treatment and were associated with the activation of AMP-activated kinase and extracellular signal-regulated kinase 1/2 signaling pathways. In summary, we showed that RCE protected endothelial cells from hypoxic insult and suggested that R. crenulata might be useful for the prevention of hypoxia-associated vascular dysfunction.

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Sirt6 regulates autophagy in AGE-treated endothelial cells via KLF4

European Heart Journal ◽

10.1093/ehjci/ehaa946.3769 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

J Tong ◽

F Chen ◽

J.N Tang ◽

Z Ye ◽

X.B Liu

Keyword(s):

Endothelial Cells ◽

Laser Scanning ◽

Umbilical Vein ◽

Funding Source ◽

Diabetic Mice ◽

Mice Model ◽

National Budget ◽

Aortic Intima

Abstract Aims To explore Sirt6 regulating autophagy in endothelial cells and the specific mechanism of this function with involvement of KLF4. Materials and methods Human umbilical vein endothelial cells were cultured with advanced glycation end products (AGE) treatment. Adv-Sirt6, LV-Sirt6 and LV-KLF4 were used to knockup Sirt6 and knockdown Sirt6 and KLF4 respectively. qPCR and Western Blotting were used to detect the mRNA and protein expression of Sirt6 and KLF4. Laser scanning confocal microscope was used to observe the LC3-II marked autophagosomes. Wildlife BALB/c mice were treated with STZ to produce diabetic mice model. AAV-Sirt6 was injected by tail vein injection to achieve Sirt6 knockdown. HE staining and scanning electron microscope were used to observe the aortic intima condition and autophagosomes number respectively. Results In AGE treated HUVECs, knockdown of Sirt6 led to impaired autophagy level along with less expression of autophagic markers LC3-II, Beclin-1, Lamp2 and autophagic marker p62. Knockdown and knockup of Sirt6 directly affected KLF4 expression level but KLF4 didn't have any effect on Sirt6 expression. Knockout of KLF4 offset the augmented autophagy caused by overexpression of Sirt6. In high-fat fed diabetic mice, downregulation of Sirt6 led to better cardiac function along with less autophagosomes and impaired aortic intima integrity. Conclusions Sirt6 improves autophagy both in vivo and in vitro and Sirt6 regulates autophagy via KLF4 in HUVECs. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): National Natural Science Foundation of China

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Desmopressin (DDAVP) Enhances Platelet Adhesion to the Extracellular Matrix of Cultured Human Endothelial Cells through Increased Expression of Tissue Factor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656088 ◽

1997 ◽

Vol 77 (05) ◽

pp. 0975-0980 ◽

Cited By ~ 23

Author(s):

Angel Gálvez ◽

Goretti Gómez-Ortiz ◽

Maribel Díaz-Ricart ◽

Ginés Escolar ◽

Rogelio González-Sarmiento ◽

...

Keyword(s):

Extracellular Matrix ◽

Endothelial Cells ◽

Tissue Factor ◽

Platelet Adhesion ◽

Umbilical Vein ◽

Procoagulant Activity ◽

Experimental Conditions ◽

Chromogenic Assay

SummaryThe effect of desmopressin (DDAVP) on thrombogenicity, expression of tissue factor and procoagulant activity (PCA) of extracellular matrix (ECM) generated by human umbilical vein endothelial cells cultures (HUVEC), was studied under different experimental conditions. HUVEC were incubated with DDAVP (1, 5 and 30 ng/ml) and then detached from their ECM. The reactivity towards platelets of this ECM was tested in a perfusion system. Coverslips covered with DD A VP-treated ECMs were inserted in a parallel-plate chamber and exposed to normal blood anticoagulated with low molecular weight heparin (Fragmin®, 20 U/ml). Perfusions were run for 5 min at a shear rate of 800 s1. Deposition of platelets on ECMs was significantly increased with respect to control ECMs when DDAVP was used at 5 and 30 ng/ml (p <0.05 and p <0.01 respectively). The increase in platelet deposition was prevented by incubation of ECMs with an antibody against human tissue factor prior to perfusion. Immunofluorescence studies positively detected tissue factor antigen on DDAVP derived ECMs. A chromogenic assay performed under standardized conditions revealed a statistically significant increase in the procoagulant activity of the ECMs produced by ECs incubated with 30 ng/ml DDAVP (p <0.01 vs. control samples). Northern blot analysis revealed increased levels of tissue factor mRNA in extracts from ECs exposed to DDAVP. Our data indicate that DDAVP in vitro enhances platelet adhesion to the ECMs through increased expression of tissue factor. A similar increase in the expression of tissue factor might contribute to the in vivo hemostatic effect of DDAVP.

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Epinephrine Induces von Willebrand Factor Release from Cultured Endothelial Cells: Involvement of Cyclic AMP-dependent Signalling in Exocytosis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656135 ◽

1997 ◽

Vol 77 (06) ◽

pp. 1182-1188 ◽

Cited By ~ 74

Author(s):

Ulrich M Vischer ◽

Claes B Wollheinn

Keyword(s):

Endothelial Cells ◽

Adenylate Cyclase ◽

Von Willebrand Factor ◽

Umbilical Vein ◽

Free Calcium ◽

Camp Content ◽

Von Willebrand ◽

Willebrand Factor

Summaryvon Willebrand factor (vWf) is released from endothelial cell storage granules after stimulation with thrombin, histamine and several other agents that induce an increase in cytosolic free calcium ([Ca2+]i). In vivo, epinephrine and the vasopressin analog DDAVP increase vWf plasma levels, although they are thought not to induce vWf release from endothelial cells in vitro. Since these agents act via a cAMP-dependent pathway in responsive cells, we examined the role of cAMP in vWf secretion from cultured human umbilical vein endothelial cells. vWf release increased by 50% in response to forskolin, which activates adenylate cyclase. The response to forskolin was much stronger when cAMP degradation was blocked with IBMX, an inhibitor of phosphodiesterases (+200%), whereas IBMX alone had no effect. vWf release could also be induced by the cAMP analogs dibutyryl-cAMP (+40%) and 8-bromo-cAMP (+25%); although their effect was weak, they clearly potentiated the response to thrombin. Epinephrine (together with IBMX) caused a small, dose-dependent increase in vWf release, maximal at 10-6 M (+50%), and also potentiated the response to thrombin. This effect is mediated by adenylate cyclase-coupled β-adrenergic receptors, since it is inhibited by propranolol and mimicked by isoproterenol. In contrast to thrombin, neither forskolin nor epinephrine caused an increase in [Ca2+]j as measured by fura-2 fluorescence. In addition, the effects of forskolin and thrombin were additive, suggesting that they act through distinct signaling pathways. We found a close correlation between cellular cAMP content and vWf release after stimulation with epinephrine and forskolin. These results demonstrate that cAMP-dependent signaling events are involved in the control of exocytosis from endothelial cells (an effect not mediated by an increase in [Ca2+]i) and provide an explanation for epinephrine-induced vWf release.

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Generation of a Novel In Vitro Model to Study Endothelial Dysfunction from Atherothrombotic Specimens

Cardiovascular Drugs and Therapy ◽

10.1007/s10557-021-07151-9 ◽

2021 ◽

Author(s):

Susan Gallogly ◽

Takeshi Fujisawa ◽

John D. Hung ◽

Mairi Brittan ◽

Elizabeth M. Skinner ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Dysfunction ◽

In Vitro Model ◽

Umbilical Vein ◽

Coronary Syndrome ◽

Coronary Endothelial Cells ◽

Vitro Model ◽

Umbilical Vein Endothelial Cells

Abstract Purpose Endothelial dysfunction is central to the pathogenesis of acute coronary syndrome. The study of diseased endothelium is very challenging due to inherent difficulties in isolating endothelial cells from the coronary vascular bed. We sought to isolate and characterise coronary endothelial cells from patients undergoing thrombectomy for myocardial infarction to develop a patient-specific in vitro model of endothelial dysfunction. Methods In a prospective cohort study, 49 patients underwent percutaneous coronary intervention with thrombus aspiration. Specimens were cultured, and coronary endothelial outgrowth (CEO) cells were isolated. CEO cells, endothelial cells isolated from peripheral blood, explanted coronary arteries, and umbilical veins were phenotyped and assessed functionally in vitro and in vivo. Results CEO cells were obtained from 27/37 (73%) atherothrombotic specimens and gave rise to cells with cobblestone morphology expressing CD146 (94 ± 6%), CD31 (87 ± 14%), and von Willebrand factor (100 ± 1%). Proliferation of CEO cells was impaired compared to both coronary artery and umbilical vein endothelial cells (population doubling time, 2.5 ± 1.0 versus 1.6 ± 0.3 and 1.2 ± 0.3 days, respectively). Cell migration was also reduced compared to umbilical vein endothelial cells (29 ± 20% versus 85±19%). Importantly, unlike control endothelial cells, dysfunctional CEO cells did not incorporate into new vessels or promote angiogenesis in vivo. Conclusions CEO cells can be reliably isolated and cultured from thrombectomy specimens in patients with acute coronary syndrome. Compared to controls, patient-derived coronary endothelial cells had impaired capacity to proliferate, migrate, and contribute to angiogenesis. CEO cells could be used to identify novel therapeutic targets to enhance endothelial function and prevent acute coronary syndromes.

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Ethanol inhibits monocyte chemotactic protein-1 expression in interleukin-1β-activated human endothelial cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00106.2005 ◽

2005 ◽

Vol 289 (4) ◽

pp. H1669-H1675 ◽

Cited By ~ 12

Author(s):

John P. Cullen ◽

Shariq Sayeed ◽

Ying Jin ◽

Nicholas G. Theodorakis ◽

James V. Sitzmann ◽

...

Keyword(s):

Endothelial Cells ◽

Umbilical Vein ◽

Binding Activity ◽

Protective Effects ◽

Monocyte Adhesion ◽

Monocyte Chemotactic Protein ◽

Chemotactic Protein ◽

Subendothelial Space ◽

Umbilical Vein Endothelial Cells

The aim of this study was to determine the effect of ethanol (EtOH) on endothelial monocyte chemotactic protein-1 (MCP-1) expression. IL-1β increased the production of MCP-1 by human umbilical vein endothelial cells from undetectable levels to ∼900 pg/ml at 24 h. EtOH dose-dependently inhibited IL-1β-stimulated MCP-1 secretion as determined by ELISA: 25 ± 1%, 35 ± 7%, and 65 ± 5% inhibition for 1, 10, and 100 mM EtOH, respectively, concomitant with inhibition of monocyte adhesion to activated endothelial cells. Similarly, EtOH dose-dependently inhibited IL-1β-stimulated MCP-1 mRNA expression. Experiments with actinomycin D demonstrated that EtOH decreased the stability of MCP-1 mRNA. In addition, EtOH significantly reduced NF-κB and AP-1 binding activity induced by IL-1β and inhibited MCP-1 gene transcription. Binding of 125I-labeled MCP-1 to its receptor (CCR2) on THP-1 human monocytic cells was not affected by EtOH treatment. Modulation of the expression of MCP-1 represents a mechanism whereby EtOH could inhibit atherogenesis by blocking the crucial early step of monocyte adhesion and subsequent recruitment to the subendothelial space. These actions of EtOH may underlie, in part, its cardiovascular protective effects in vivo.

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Abstract 116: Arterial Endothelial Cell Has Stronger Angiogenic Potential Compared To Venous Endothelial Cell Through Up-regulation Of Endogenous FGF2 And FGF5 Expression In 3D Microfluidic System

Circulation Research ◽

10.1161/res.115.suppl_1.116 ◽

2014 ◽

Vol 115 (suppl_1) ◽

Author(s):

Ha-Rim Seo ◽

Hyo Eun Jeong ◽

Hyung Joon Joo ◽

Seung-Cheol Choi ◽

Jong-Ho Kim ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Molecular Mechanisms ◽

Umbilical Vein ◽

Assay System ◽

Vascular Density ◽

Angiogenic Potential ◽

Angiogenesis Assay

Background: Human body contains many kinds of different type of endothelial cells (EC). However, cellular difference of their angiogenic potential has been hardly understood. We compared in vitro angiogenic potential between arterial EC and venous EC and investigated its underlying molecular mechanisms. Method: Used human aortic endothelial cells (HAEC) which was indicated from arterial EC and human umbilical vein endothelial cells (HUVEC) indicated from venous EC. To explore angiogenic potential in detail, we adopted a novel 3D microfluidic angiogenesis assay system, which closely mimic in vivo angiogenesis. Results: In 3D microfluidic angiogenesis assay system, HAEC demonstrated stronger angiogenic potential compared to HUVEC. HAEC maintained its profound angiogenic property under different biophysical conditions. In mRNA microarray sorted on up- regulated or down-regulated genes, HAEC demonstrated significantly higher expression of gastrulation brain homeobox 2 (GBX2), fibroblast grow factor 2 (FGF2), FGF5 and collagen 8a1. Angiogenesis-related protein assay revealed that HAEC has higher secretion of endogenous FGF2 than HUVEC. HAEC has only up-regulated FGF2 and FGF5 in this part of FGF family. Furthermore, FGF5 expression under vascular endothelial growth factor-A (VEGF-A) stimulation was higher in HAEC compared to HUVEC although VEGF-A augmented FGF5 expression in both HAEC and HUVEC. Those data suggested that FGF5 expression in both HAEC and HUVEC is partially dependent to VEGF-A stimulate. HUVEC and HAEC reduced vascular density after FGF2 and FGF5 siRNA treat. Conclusion: HAEC has stronger angiogenic potential than HUVEC through up-regulation of endogenous FGF2 and FGF5 expression

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Abstract 116: Slit-Robo Signaling Regulates Lipopolysaccharide-induced Endothelial Inflammation and Expression of Slit/Robo is Dysregulated During Endotoxemia

Circulation Research ◽

10.1161/res.113.suppl_1.a116 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Helong Zhao ◽

Appakkudal Anand ◽

Ramesh Ganju

Keyword(s):

Endothelial Cells ◽

Cell Adhesion ◽

Inflammatory Responses ◽

Umbilical Vein ◽

Model Studies ◽

Endothelial Inflammation ◽

Anti Inflammatory ◽

Whole Heart

Abstract Introduction: Lipopolysaccharide (LPS) is one of the critical factors which induce endothelial inflammation during the pathogenesis of atherosclerosis, endocarditis and sepsis shock induced heart injury. The secretory Slit2 protein and its endothelial receptors Robo1 and Robo4 have been shown to regulate mobility and permeability of endothelial cells, which could be functional in regulating LPS induced endothelial inflammation. Hypothesis: We hypothesized that in addition to regulating permeability and migration of endothelial cells, Slit2-Robo1/4 signaling might regulate other LPS-induced endothelial inflammatory responses. Methods and Results: Using Human Umbilical Vein Endothelial Cells (HUVEC) culture, we observed that Slit2 treatment suppressed LPS-induced secretion of pro-inflammatory cytokines (including GM-CSF), cell adhesion molecule upregulation and monocyte (THP-1 cell) adhesion. With siRNA knock down techniques, we further confirmed that this anti-inflammatory effect is mediated by the interaction of Slit2 with its dominant receptor in endothelial cells, Robo4, though the much lesser expressed minor receptor Robo1 is pro-inflammatory. Our signaling studies showed that downstream of Robo4, Slit2 suppressed inflammatory gene expression by inhibiting the Pyk2 - NF-kB pathway following LPS-TLR4 interaction. In addition, Slit2 can induce a positive feedback to its expression and downregulate the pro-inflammatory Robo1 receptor via mediation of miR-218. Moreover, both in in vitro studies using HUVEC and in vivo mouse model studies indicated that LPS also causes endothelial inflammation by downregulating the anti-inflammatory Slit2 and Robo4 and upregulating the pro-inflammatory Robo1 during endotoxemia, especially in mouse arterial endothelial cells and whole heart. Conclusions: Slit2-Robo1/4 signaling is important in regulation of LPS induced endothelial inflammation, and LPS in turn causes inflammation by interfering with the expression of Slit2, Robo1 and Robo4. This implies that Slit2-Robo1/4 is a key regulator of endothelial inflammation and its dysregulation during endotoxemia is a novel mechanism for LPS induced cardiovascular pathogenesis.

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Abstract 531: Tie-2/Cre--Mediated Conditional Deletion of Lipid Phosphate Phosphatase-3 Reveals Its Role in Endothelial Cell Apoptosis

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.32.suppl_1.a531 ◽

2012 ◽

Vol 32 (suppl_1) ◽

Author(s):

Ishita Chatterjee ◽

Kishore K Wary

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Genome Wide Association Study ◽

Vascular Endothelial Cell ◽

Protein Levels ◽

Conditional Deletion ◽

Transmission Electron ◽

Increased Risk

Rationale: A recent genome-wide association study (GWAS) has linked a frequently occurring variation in the LPP3 (also known as PPAP2b) loci to increased risk of coronary heart disease (CAD). However, the in vivo function of LPP3 in vascular endothelial cell is incompletely understood. Goal: To address the endothelial cell (EC) specific function of Lpp3 in mice. Results: Tie-2/Cre mediated Lpp3 deletion did not affect normal vasculogenesis in early embryonic development, in contrast, in late embryonic stages it led to impaired angiogenesis associated with hemorrhage, edema and late embryonic lethal phenotype. Immunohistochemical staining followed by microscopic analyses of mutant embryos revealed reduced fibronectin and VE-cadherin expression throughout different vascular bed, and increased apoptosis in CD31+ vascular structures. Transmission electron microscopy (TEM) showed the presence of apoptotic endothelial cells and disruption of adherens junctions in mutant embryos. LPP3-knockdown in vitro showed an increase in p53 and p21 protein levels, with concomitant decrease in cell proliferation. LPP3-knockdown also decreased transendothelial electrical resistance (TER), interestingly re-expression of ß-catenin cDNA into LPP3-depleted endothelial cells partially restored the effect of loss of LPP3. Conclusion: These results suggest the ability of LPP3 to regulate survival and apoptotic activities of endothelial cells during patho/physiological angiogenesis.

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