Abstract 259: Sam68 Regulates Endothelin-Converting Enzyme-1b Expression and Blood Pressure Homeostasis

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Junlan Zhou ◽  
Gangjian Qin
Keyword(s):  
Blood Pressure ◽  
Cell Cycle ◽  
Endothelial Cells ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Umbilical Vein ◽  
Converting Enzyme ◽  
Proteomic Approach ◽  
Endothelin Converting Enzyme

We have previously reported that E2F2, a transcription factor known for its role in cell cycle, regulates the expression of endothelin-converting enzyme-1b (ECE-1b) in endothelial cells and contributes to the maintenance of vascular contractility and blood pressure (BP). However, the molecular mechanisms underlying this novel, cell-cycle independent function of E2F2 remain largely elusive. In this study, we profiled E2F2 interactome in the nucleus of human umbilical vein endothelial cells by utilizing a proteomic approach. We identified that Sam68, a classic RNA-binding protein and Src kinase substrate, acts as an E2F2-interacting protein; co-immunoprecipitation analyses confirmed that both endogenous and ectopically-expressed Sam68 interact with E2F2. Overexpression of Sam68 repressed whereas knockdown of Sam68 increased E2F2-induced ECE-1b promoter activity and mRNA expression. Chromatin immunoprecipitation assays further confirmed that E2F2 and Sam68 co-localize on ECE-1b promoter, indicating that ECE-1b is a direct transcriptional target of E2F2 and Sam68. In vivo, Sam68 knockout (KO) mice displayed a significantly lowered BP as compared to WT littermates (tail-cuff measurements of mean BP, KO vs. WT: 104 ± 6 vs. 123 ± 7 mmHg, n=25 males of age 4-6 months, P<0.05). Together, our studies have revealed a previously unknown function of Sam68 as a transcriptional co-repressor of E2F2 and a critical regulator of BP homeostasis.

Expression of human endothelin-converting enzyme isoforms: role of angiotensin IIThis article is one of a selection of papers published in the special issue (part 1 of 2) on Forefronts in Endothelin.

2008 ◽  
Vol 86 (6) ◽  
pp. 299-309 ◽  
Author(s):  
W. Goettsch ◽  
A. Schubert ◽  
H. Morawietz
Keyword(s):  
Endothelial Cells ◽  
Mrna Expression ◽  
Umbilical Vein ◽  
Artery Bypass ◽  
Control Group ◽  
Ang Ii ◽  
Converting Enzyme ◽  
Mammary Arteries ◽  
Endothelin Converting Enzyme ◽  
Internal Mammary

A key step in endothelin-1 (ET-1) synthesis is the proteolytic cleavage of big ET-1 by the endothelin-converting enzyme-1 (ECE-1). Four alternatively spliced isoforms, ECE-1a to ECE-1d, have been discovered; however, regulation of the expression of specific ECE-1 isoforms is not well understood. Therefore, we stimulated primary human umbilical vein endothelial cells (HUVECs) with angiotensin II (Ang II). Furthermore, expression of ECE-1 isoforms was determined in internal mammary arteries of patients undergoing coronary artery bypass grafting surgery. Patients had received one of 4 therapies: angiotensin-converting enzyme inhibitors (ACE-I), Ang II type 1 receptor blockers (ARB), HMG-CoA reductase inhibitors (statins), and a control group that had received neither ACE-I, ARB (that is, treatment not interfering in the renin–angiotensin system), nor statins. Under control conditions, ECE-1a is the dominant isoform in HUVECs (4.5 ± 2.8 amol/μg RNA), followed by ECE-1c (2.7 ± 1.0 amol/μg), ECE-1d (0.49 ± 0.17 amol/μg), and ECE-1b (0.17 ± 0.04 amol/μg). Stimulation with Ang II did not change the ECE-1 expression pattern or the ET-1 release. We found that ECE-1 mRNA expression was higher in patients treated with statins than in patients treated with ARB therapy (5.8 ± 0.76 RU versus 3.0 ± 0.4 RU), mainly attributed to ECE-1a. In addition, ECE-1a mRNA expression was higher in patients receiving ACE-I therapy than in patients receiving ARB therapy (1.68 ± 0.27 RU versus 0.83 ± 0.07 RU). We conclude that ECE-1a is the major ECE-1 isoform in primary human endothelial cells. Its expression in internal mammary arteries can be regulated by statin therapy and differs between patients with ACE-I and ARB therapy.

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

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

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

2018 ◽  
Vol 46 (2) ◽  
pp. 520-531 ◽  
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.

scholarly journals Transformer 2β Regulates the Alternative Splicing of Cell Cycle Regulator Genes to Promote Ovarian Cancer Cell Progression

2021 ◽  
Author(s):  
Peiying Fu ◽  
Ting Zhou ◽  
Dong Chen ◽  
ShiXuan Wang ◽  
Ronghua Liu
Keyword(s):  
Cell Cycle ◽  
Ovarian Cancer ◽  
Alternative Splicing ◽  
Cancer Progression ◽  
Poor Prognosis ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Expression Level

Abstract Background: Late-stage ovarian cancer (OV) has a poor prognosis and a high metastasis rate, but the underlying molecular mechanism is ambiguous. RNA binding proteins (RBPs) play important roles in posttranscriptional regulation in the contexts of neoplasia and tumor metastasis. Results: In this study, we explored the molecular functions of a canonical RBP, TRA2B, in cancer cells. TRA2B knockdown in HeLa cells and whole-transcriptome sequencing (RNA-seq) experiments revealed that the TRA2B-regulated alternative splicing (AS) profile was tightly associated with the mitotic cell cycle, apoptosis, and several cancer pathways. Moreover, hundreds of genes were regulated by TRA2B at the expression level, and their functions were enriched in cell proliferation, cell adhesion and angiogenesis, which are related to cancer progression. We also observed that AS regulation and expression regulation occurred independently by integrating the alternatively spliced and differentially expressed genes. We then explored and validated the carcinogenic functions of TRA2B by knocking down its expression in OV cells. In vivo, a high expression level of TRA2B was associated with a poor prognosis in OV patients. Conclusions: We demonstrated the important roles of TRA2B in ovarian neoplasia and OV progression and identified the underlying molecular mechanisms, facilitating the targeted treatment of OV in the future.

Endothelial cells internalize monoclonal antibody to angiotensin-converting enzyme

1996 ◽  
Vol 270 (5) ◽  
pp. L704-L713 ◽  
Author(s):  
V. R. Muzykantov ◽  
E. N. Atochina ◽  
A. Kuo ◽  
E. S. Barnathan ◽  
K. Notarfrancesco ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Endothelial Cells ◽  
Angiotensin Converting Enzyme ◽  
Umbilical Vein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Affinity Constant ◽  
Converting Enzyme

We investigated the fate of MAb 9B9, a monoclonal antibody to angiotensin-converting enzyme (ACE), which binds to endothelium both in vitro and in vivo. Using cultured human umbilical vein endothelial cells (HUVEC) and isolated perfused rat lungs (IPL), we demonstrated specific and saturable binding of 125I-labeled MAb 9B9 at 4 degrees C [affinity constant (Kd) = 20-50 nM, maximal number of binding sites (Bmax) = 1.5-3.0 x 10(5) sites/cell]. When 125I-MAb 9B9 was bound to HUVEC at 37 degrees C, only 40% of cell-associated radioactivity was acid elutable, suggesting antibody internalization. This was confirmed by finding that 1) the amount of MAb 9B9 uptake at 37 degrees C was higher than at 4 degrees C both in HUVEC and IPL; 2) binding of 125I-labeled streptavidin with HUVEC and IPL pretreated with biotinylated MAb 9B9 (b-MAb 9B9) was diminished in a temperature- and time-dependent fashion at 37 degrees C; and 3) b-MAb 9B9 bound to HUVEC at 37 degrees C was found intracellularly by ultrastructural analysis using streptavidin gold. Intracellular 125I-MAb 9B9 was found in microsomal fractions of lung homogenate from IPL and after intravenous (iv) injections in rats. Degradation of internalized MAb 9B9 was minimal, since > 90% of cell-associated 125I label remained precipitable by trichloracetic acid in HUVEC, IPL, and in vivo. Autoradiography of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of lung homogenates made as late as several days after iv injections of 125I-MAb 9B9 in rats demonstrated a predominant band above 140 kDa. These data indicate that endothelial cells either in vitro or in vivo internalize the ACE ligand MAb 9B9 without significant intracellular degradation. Therefore MAb 9B9 may be useful for selective intracellular delivery of drugs to the pulmonary vascular endothelium after systemic administration.

scholarly journals Endothelial specific YY1 deletion restricts tumor angiogenesis and tumor growth

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Huan Liu ◽  
Yikai Qiu ◽  
Xiuying Pei ◽  
Ramamurthy Chitteti ◽  
Rebbeca Steiner ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Tumor Growth ◽  
Tumor Angiogenesis ◽  
Molecular Mechanisms ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Vital Role ◽  
Vascular Endothelial

AbstractAngiogenesis is a physiological process for the formation of new blood vessels from the pre-existing vessels and it has a vital role in the survival and growth of neoplasms. During tumor angiogenesis, the activation of the gene transcriptions in vascular endothelial cells (ECs) plays an essential role in the promotion of EC proliferation, migration, and vascular network development. However, the molecular mechanisms underlying transcriptional regulation of EC and tumor angiogenesis remains to be fully elucidated. Here we report that the transcription factor Yin Yang 1 (YY1) in ECs is critically involved in tumor angiogenesis. First, we utilized a tamoxifen-inducible EC-specific YY1 deficient mouse model and showed that YY1 deletion in ECs inhibited the tumor growth and tumor angiogenesis. Using the in vivo matrigel plug assay, we then found that EC-specific YY1 ablation inhibited growth factor-induced angiogenesis. Furthermore, vascular endothelial growth factor (VEGF)-induced EC migration was diminished in YY1-depleted human umbilical vein endothelial cells (HUVECs). Finally, a rescue experiment revealed that YY1-regulated BMP6 expression in ECs was involved in EC migration. Collectively, our results demonstrate that endothelial YY1 has a crucial role in tumor angiogenesis and suggest that targeting endothelial YY1 could be a potential therapeutic strategy for cancer treatment.

scholarly journals Lysosome purinergic receptor P2X4 regulates neoangiogenesis induced by microvesicles from sarcoma patients

2021 ◽  
Vol 12 (9) ◽  
Author(s):  
Wulf Palinski ◽  
Maria Monti ◽  
Rosa Camerlingo ◽  
Ilaria Iacobucci ◽  
Serena Bocella ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Membrane ◽  
Purinergic Receptor ◽  
Umbilical Vein ◽  
Therapeutic Interventions ◽  
Vascular Networks ◽  
Proteomic Approach ◽  
Umbilical Vein Endothelial Cells

AbstractThe tumor microenvironment modulates cancer growth. Extracellular vesicles (EVs) have been identified as key mediators of intercellular communication, but their role in tumor growth is largely unexplored. Here, we demonstrate that EVs from sarcoma patients promote neoangiogenesis via a purinergic X receptor 4 (P2XR4) -dependent mechanism in vitro and in vivo. Using a proteomic approach, we analyzed the protein content of plasma EVs and identified critical activated pathways in human umbilical vein endothelial cells (HUVECs) and human progenitor hematopoietic cells (CD34+). We then showed that vessel formation was due to rapid mitochondrial activation, intracellular Ca2+ mobilization, increased extracellular ATP, and trafficking of the lysosomal P2XR4 to the cell membrane, which is required for cell motility and formation of stable branching vascular networks. Cell membrane translocation of P2XR4 was induced by proteins and chemokines contained in EVs (e.g. Del-1 and SDF-1). Del-1 was found expressed in many EVs from sarcoma tumors and several tumor types. P2XR4 blockade reduced EVs-induced vessels in angioreactors, as well as intratumor vascularization in mouse xenografts. Together, these findings identify P2XR4 as a key mediator of EVs-induced tumor angiogenesis via a signaling mediated by mitochondria-lysosome-sensing response in endothelial cells, and indicate a novel target for therapeutic interventions.

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