Abstract 17225: Extracellular Signal-Regulated Kinase 1/2 is Essential for Lipocalin-2 Signaling in Endothelial Cells

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Markus Theurl ◽  
Andrea Schroll ◽  
Igor Theurl ◽  
Daniela Lener ◽  
Wolfgang-Michael Franz ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Vascular Function ◽  
Umbilical Vein ◽  
Brdu Incorporation ◽  
Lipocalin 2 ◽  
Vascular Disorders ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Stimulation Of

Introduction: Lipocalin-2 (Lcn2) is an acute phase protein and a marker of kidney injury. Recently, elevated Lcn2-levels have been reported in heart failure and myocardial infarction. Moreover, stimulation of breast cancer angiogenesis was observed. Thus, we hypothesized that Lcn2 may be a regulator of vascular function and a target for the treatment of ischemic vascular disorders. Methods/Results: In-vitro Lcn2 mediated proliferation of human umbilical vein endothelial cells (HUVEC; rel. proliferation Lcn2 10 nM vs. ctr.: 1.4±0.09, n=3, P<0.001) and human coronary artery endothelial cells (HCAEC; rel. proliferation Lcn2 10 nM vs. ctr.: 1.3±0.07, n=3, P<0.01) as determined by BrdU-incorporation. In the in-vitro matrigel assay stimulation of HUVEC (1.4 fold vs. ctr., n=3, P<0.01) and HCAEC (1.6 fold vs. ctr., n=3, P<0.001) with Lcn2 resulted in a significant induction of capillary like tube formation. All effects were similar to vascular endothelial growth factor (VEGF). Mechanistically these results can be traced back to phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). Real-time PCR analyses revealed expression of Lcn2 and its receptor by endothelial cells (EC) as well as a hypoxia-dependent up-regulation (rel. Lcn2 mRNA hypoxia vs. normoxia 1.6±0.2, P<0.05; rel. Lcn2-receptor mRNA hypoxia vs. normoxia 2.6±0.2, P<0.001). In the mouse aortic ring assay Lcn2-treatment resulted in a significant outgrowth of EC similar to VEGF. In the hind limb ischemia (HLI) model Lcn2 -/- mice showed an impressive phenotype. After induction of HLI we detected significantly more tissue defects compared to wild type (WT) mice. The ischemia-related lesions were more severe as determined by necrosis score (necrosis score Lcn2 -/- 1.8±0.2 vs. WT 0.7±0.2, n=5, P<0.01) and amputation rate was significantly higher. In ischemic hind limbs of Lcn2 -/- mice ERK1/2-phosphorylation was almost abrogated which might be an underlying mechanism. Transplantation of WT-bone marrow to irradiated Lcn2 -/- mice didn’t influence the outcome suggesting that observed effects are rather endothelium-dependent than influenced by an inflammatory response. Conclusion: Lcn2 might be a promising therapeutic factor for the treatment of ischemic vascular disorders.

scholarly journals Extracellular Signal-Regulated Kinase 5 is Required for Low-Concentration H2O2-Induced Angiogenesis of Human Umbilical Vein Endothelial Cells

2017 ◽  
Vol 2017 ◽  
pp. 1-13 ◽  
Author(s):  
Shan Jiang ◽  
Dongxin Zhang ◽  
Hong Huang ◽  
Yonghong Lei ◽  
Yan Han ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Tubule Formation ◽  
Human Umbilical Vein ◽  
Extracellular Signal Regulated Kinase ◽  
Knock Down ◽  
Low Concentrations ◽  
Extracellular Signal ◽  
Umbilical Vein Endothelial Cells

Background. The aim of this study was to assess the effects of low concentrations of H2O2 on angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro and explore the underlying mechanisms. Methods. HUVECs were cultured and stimulated with different concentrations of H2O2. Flow cytometric analysis was used to select an optimal concentration of H2O2 for the following experiments. Cell proliferation, migration, and tubule formation were evaluated by Cell Counting Kit-8 (CCK-8) assays, scratch wound assays, and Matrigel tubule formation assays, respectively. For gain and loss of function studies, constitutively active MEK5 (CA-MEK5) and ERK5 shRNA lentiviruses were used to activate or knock down extracellular signal-regulated kinase 5 (ERK5). Results. We found that low concentrations of H2O2 promoted HUVECs proliferation, migration, and tubule formation. ERK5 in HUVECs was significantly activated by H2O2. Enhanced ERK5 activity significantly amplified the proangiogenic effects of H2O2; in contrast, ERK5 knock-down abrogated the effects of H2O2. Conclusions. Our results confirmed that low concentrations of H2O2 promoted HUVECs angiogenesis in vitro, and ERK5 is an essential mediator of this process. Therefore, ERK5 may be a potential therapeutic target for promoting angiogenesis and improving graft survival.

Effect of hypoxia on adherence of granulocytes to endothelial cells in vitro

1994 ◽  
Vol 267 (3) ◽  
pp. H874-H879 ◽  
Author(s):  
A. Pietersma ◽  
N. De Jong ◽  
J. F. Koster ◽  
W. Sluiter
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Calcium Ionophore ◽  
Intercellular Adhesion Molecule ◽  
Protective Mechanism ◽  
Hypoxic Conditions ◽  
Umbilical Vein Endothelial Cells ◽  
Stimulation Of

The objective of this study was to investigate the effect of hypoxia on the adhesiveness of endothelial cells for granulocytes. Human umbilical vein endothelial cells (HUVEC) were exposed to a PO2 of 7.5 mmHg (1.0 kPa), and the adherence of granulocytes was assessed under continuous hypoxia by means of a hypoxic incubator room. After 2 h of hypoxia the adherence of granulocytes decreased to 50% of the normoxic control, which was not due to a decreased viability of the endothelial cells nor to an increased generation of the antiadhesive factors nitric oxide, prostacyclin, and adenosine. Hypoxia also had no effect on the expression of intercellular adhesion molecule (ICAM)-1 or ICAM-2 on the endothelium. Although the mechanism of the action of hypoxia on the adhesiveness of endothelial cells remains unclear as yet, our data suggest that HUVEC possess a protective mechanism that prevents granulocyte adherence to endothelial cells under extreme hypoxic conditions. The decreased adherence seems paradoxical to the in vivo situation for which the increased margination of granulocytes within the vascular compartment of the ischemic tissue has been observed. However, hypoxia did not impair the potential adhesiveness of HUVEC, since stimulation of endothelial cells under hypoxic conditions with calcium ionophore or lipopolysaccharide increased the adherence of granulocytes in a similar fashion as under normoxic conditions. We therefore conclude that the increased margination of granulocytes during ischemia may be accomplished by the additional stimulation of hypoxic endothelial cells.

scholarly journals NPNT is Expressed by Osteoblasts and Mediates Angiogenesis via the Activation of Extracellular Signal-regulated Kinase

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Vincent Kuek ◽  
Zhifan Yang ◽  
Shek Man Chim ◽  
Sipin Zhu ◽  
Huazi Xu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Structure Formation ◽  
Ex Vivo ◽  
Bone Diseases ◽  
Angiogenic Factors ◽  
Endothelial Cell Migration ◽  
Angiogenic Factor ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal

Abstract Angiogenesis plays an important role in bone development and remodeling and is mediated by a plethora of potential angiogenic factors. However, data regarding specific angiogenic factors that are secreted within the bone microenvironment to regulate osteoporosis is lacking. Here, we report that Nephronectin (NPNT), a member of the epidermal growth factor (EGF) repeat superfamily proteins and a homologue of EGFL6, is expressed in osteoblasts. Intriguingly, the gene expression of NPNT is reduced in the bone of C57BL/6J ovariectomised mice and in osteoporosis patients. In addition, the protein levels of NPNT and CD31 are also found to be reduced in the tibias of OVX mice. Exogenous addition of mouse recombinant NPNT on endothelial cells stimulates migration and tube-like structure formation in vitro. Furthermore, NPNT promotes angiogenesis in an ex vivo fetal mouse metatarsal angiogenesis assay. We show that NPNT stimulates the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated kinase (MAPK) in endothelial cells. Inhibition of ERK1/2 impaired NPNT-induced endothelial cell migration, tube-like structure formation and angiogenesis. Taken together, these results demonstrate that NPNT is a paracrine angiogenic factor and may play a role in pathological osteoporosis. This may lead to new targets for treatment of bone diseases and injuries.

scholarly journals KLF2 Is a Novel Transcriptional Regulator of Endothelial Proinflammatory Activation

2004 ◽  
Vol 199 (10) ◽  
pp. 1305-1315 ◽  
Author(s):  
Sucharita SenBanerjee ◽  
Zhiyong Lin ◽  
G. Brandon Atkins ◽  
Daniel M. Greif ◽  
Ravi M. Rao ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Adhesion Molecule ◽  
Proinflammatory Cytokines ◽  
Vascular Function ◽  
Cell Attachment ◽  
Umbilical Vein ◽  
Laminar Shear Stress ◽  
Endothelial Adhesion Molecule ◽  
Umbilical Vein Endothelial Cells

The vascular endothelium is a critical regulator of vascular function. Diverse stimuli such as proinflammatory cytokines and hemodynamic forces modulate endothelial phenotype and thereby impact on the development of vascular disease states. Therefore, identification of the regulatory factors that mediate the effects of these stimuli on endothelial function is of considerable interest. Transcriptional profiling studies identified the Kruppel-like factor (KLF)2 as being inhibited by the inflammatory cytokine interleukin-1β and induced by laminar shear stress in cultured human umbilical vein endothelial cells. Overexpression of KLF2 in umbilical vein endothelial cells robustly induced endothelial nitric oxide synthase expression and total enzymatic activity. In addition, KLF2 overexpression potently inhibited the induction of vascular cell adhesion molecule-1 and endothelial adhesion molecule E-selectin in response to various proinflammatory cytokines. Consistent with these observations, in vitro flow assays demonstrate that T cell attachment and rolling are markedly attenuated in endothelial monolayers transduced with KLF2. Finally, our studies implicate recruitment by KLF2 of the transcriptional coactivator cyclic AMP response element–binding protein (CBP/p300) as a unifying mechanism for these various effects. These data implicate KLF2 as a novel regulator of endothelial activation in response to proinflammatory stimuli.

scholarly journals Positive versus negative effects of VEGF165 on Ca2+ signaling and NO production in human endothelial cells

2017 ◽  
Vol 312 (1) ◽  
pp. H173-H181 ◽  
Author(s):  
Derek S. Boeldt ◽  
Jennifer Krupp ◽  
Fu-Xian Yi ◽  
Nauman Khurshid ◽  
Dinesh M. Shah ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Vascular Function ◽  
Negative Impact ◽  
Ex Vivo ◽  
Umbilical Vein ◽  
No Production ◽  
Human Endothelial Cells ◽  
Direct Stimulation ◽  
Human Umbilical Vein ◽  
Stimulation Of

The role increased vascular endothelial growth factor (VEGF) plays in vascular function during normal vs. preeclamptic pregnancy has been a source of some controversy of late. In this study, we seek to understand how VEGF165 influences vasodilator production via Ca2+ signaling mechanisms in human endothelial cells. We utilize human umbilical vein endothelial cells (HUVEC) as well as intact ex vivo human umbilical vein (HUV Endo) to address direct stimulation of Ca2+ and NO by VEGF165 alone, as well as the effect of VEGF165 on subsequent ATP-stimulated Ca2+ signaling and NO production. We show that VEGF165 stimulates Ca2+ responses in both HUVEC and HUV Endo, which results in a corresponding increase in NO production in HUV Endo. Longer-term VEGF165 pretreatment then inhibits sustained Ca2+ burst responses to ATP in HUVEC and HUV Endo. This is paralleled by a corresponding drop in ATP-stimulated NO production in HUV Endo, likely through inhibition of Cx43 gap-junction function. Thus, although VEGF165 makes a small initial positive impact on vasodilator production via direct stimulation of Ca2+ responses, this is outweighed by the greater subsequent negative impact on Ca2+ bursts and vasodilator production promoted by more potent agonists such as ATP. Overall, elevated levels of VEGF165 associated with preeclampsia could contribute to the endothelial dysfunction by preventing Ca2+ bursts to other agonists including but not limited to ATP. NEW & NOTEWORTHY In this manuscript, we show that VEGF levels associated with preeclampsia are a net negative contributor to potential vasodilator production in both a human ex vivo and in vitro endothelial cell model. Therefore, pharmacological targeting of VEGF-stimulated signaling pathways could be a novel treatment modality for preeclampsia-related hypertension.

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