scholarly journals Type 5 phosphodiesterase expression is a critical determinant of the endothelial cell angiogenic phenotype

2009 ◽  
Vol 296 (2) ◽  
pp. L220-L228 ◽  
Author(s):  
Bing Zhu ◽  
Li Zhang ◽  
Mikhail Alexeyev ◽  
Diego F. Alvarez ◽  
Samuel J. Strada ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Network Formation ◽  
Microvascular Endothelial Cells ◽  
Critical Determinant ◽  
Angiogenic Potential ◽  
Matrigel Plug ◽  
Microvascular Endothelial

Type 5 phosphodiesterase (PDE5) inhibitors increase endothelial cell cGMP and promote angiogenesis. However, not all endothelial cell phenotypes express PDE5. Indeed, whereas conduit endothelial cells express PDE5, microvascular endothelial cells do not express this enzyme, and they are rapidly angiogenic. These findings bring into question whether PDE5 activity is a critical determinant of the endothelial cell angiogenic potential. To address this question, human full-length PDE5A1 was stably expressed in pulmonary microvascular endothelial cells. hPDE5A1 expression reduced the basal and atrial natriuretic peptide (ANP)-stimulated cGMP concentrations in these cells. hPDE5A1-expressing cells displayed attenuated network formation on Matrigel in vitro and also produced fewer blood vessels in Matrigel plug assays in vivo; the inhibitory actions of hPDE5A1 were reversed using sildenafil. To examine whether endogenous PDE5 activity suppresses endothelial cell angiogenic potential, small interfering RNA (siRNA) constructs were stably expressed in pulmonary artery endothelial cells. siRNA selectively decreased PDE5 expression and increased basal and ANP-stimulated cGMP concentrations in these conduit cells. PDE5 downregulation increased network formation on Matrigel in vitro and increased blood vessel formation in Matrigel plug assays in vivo. Collectively, our results indicate that PDE5 activity is an essential determinant of angiogenesis and suggest that PDE5 downregulation in microvascular endothelium imparts a stable, enhanced angiogenic potential to this cell type.

scholarly journals Role of JNK in network formation of human lung microvascular endothelial cells

2008 ◽  
Vol 294 (4) ◽  
pp. L676-L685 ◽  
Author(s):  
Meetha Medhora ◽  
Anuradha Dhanasekaran ◽  
Phillip F. Pratt ◽  
Craig R. Cook ◽  
Laurel K. Dunn ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Network Formation ◽  
Human Lung ◽  
Dominant Negative ◽  
Microvascular Endothelial Cells ◽  
Erk Pathway ◽  
Jnk Pathway ◽  
Microvascular Endothelial

The signaling mechanisms in vasculogenesis and/or angiogenesis remain poorly understood, limiting the ability to regulate growth of new blood vessels in vitro and in vivo. Cultured human lung microvascular endothelial cells align into tubular networks in the three-dimensional matrix, Matrigel. Overexpression of MAPK phosphatase-1 (MKP-1), an enzyme that inactivates the ERK, JNK, and p38 pathways, inhibited network formation of these cells. Adenoviral-mediated overexpression of recombinant MKP-3 (a dual specificity phosphatase that specifically inactivates the ERK pathway) and dominant negative or constitutively active MEK did not attenuate network formation in Matrigel compared with negative controls. This result suggested that the ERK pathway may not be essential for tube assembly, a conclusion which was supported by the action of specific MEK inhibitor PD 184352, which also did not alter network formation. Inhibition of the JNK pathway using SP-600125 or l-stereoisomer (l-JNKI-1) blocked network formation, whereas the p38 MAPK blocker SB-203580 slightly enhanced it. Inhibition of JNK also attenuated the number of small vessel branches in the developing chick chorioallantoic membrane. Our results demonstrate a specific role for the JNK pathway in network formation of human lung endothelial cells in vitro while confirming that it is essential for the formation of new vessels in vivo.

Isolation and characterization of human and rat cardiac microvascular endothelial cells

1993 ◽  
Vol 264 (2) ◽  
pp. H639-H652 ◽  
Author(s):  
M. Nishida ◽  
W. W. Carley ◽  
M. E. Gerritsen ◽  
O. Ellingsen ◽  
R. A. Kelly ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Microvascular Endothelial Cells ◽  
Microvascular Endothelium ◽  
Adult Rat ◽  
Isolation And Characterization ◽  
Ventricular Tissue ◽  
Microvascular Endothelial ◽  
Cardiac Microvascular Endothelial Cells

Although reciprocal intercellular signaling may occur between endocardial or microvascular endothelium and cardiac myocytes, suitable in vitro models have not been well characterized. In this report, we describe the isolation and primary culture of cardiac microvascular endothelial cells (CMEC) from both adult rat and human ventricular tissue. Differential uptake of fluorescently labeled acetylated low-density lipoprotein (Ac-LDL) indicated that primary isolates of rat CMEC were quite homogeneous, unlike primary isolates of human ventricular tissue, which required cell sorting based on Ac-LDL uptake to create endothelial cell-enriched primary cultures. The endothelial phenotype of both primary isolates and postsort subcultured CMEC and their microvascular origin were determined by characteristic histochemical staining for a number of endothelial cell-specific markers, by the absence of cells with fibroblast or pericyte-specific cell surface antigens, and by rapid tube formation on purified basement membrane preparations. Importantly, [3H]-thymidine uptake was increased 2.3-fold in subconfluent rat microvascular endothelial cells 3 days after coculture with adult rat ventricular myocytes because of release of an endothelial cell mitogen(s) into the extracellular matrix, resulting in a 68% increase in cell number compared with CMEC in monoculture. Thus biologically relevant cell-to-cell interactions can be modeled with this in vitro system.

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 α1G T-type calcium channel determines the angiogenic potential of pulmonary microvascular endothelial cells

2019 ◽  
Vol 316 (3) ◽  
pp. C353-C364 ◽  
Author(s):  
Zhen Zheng ◽  
Hairu Chen ◽  
Peilin Xie ◽  
Carol A. Dickerson ◽  
Judy A. C. King ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Functional Role ◽  
Transient Receptor Potential ◽  
Network Formation ◽  
Microvascular Endothelial Cells ◽  
Dependent Manner ◽  
Channel Blockade ◽  
Angiogenic Potential ◽  
Pulmonary Microvascular Endothelial Cells ◽  
Microvascular Endothelial

Pulmonary microvascular endothelial cells (PMVECs) display a rapid angioproliferative phenotype, essential for maintaining homeostasis in steady-state and promoting vascular repair after injury. Although it has long been established that endothelial cytosolic Ca2+ ([Ca2+]i) transients are required for proliferation and angiogenesis, mechanisms underlying such regulation and the transmembrane channels mediating the relevant [Ca2+]i transients remain incompletely understood. In the present study, the functional role of the microvascular endothelial site-specific α1G T-type Ca2+ channel in angiogenesis was examined. PMVECs intrinsically possess an in vitro angiogenic “network formation” capacity. Depleting extracellular Ca2+ abolishes network formation, whereas blockade of vascular endothelial growth factor receptor or nitric oxide synthase has little or no effect, suggesting that the network formation is a [Ca2+]i-dependent process. Blockade of the T-type Ca2+ channel or silencing of α1G, the only voltage-gated Ca2+ channel subtype expressed in PMVECs, disrupts network formation. In contrast, blockade of canonical transient receptor potential (TRP) isoform 4 or TRP vanilloid 4, two other Ca2+ permeable channels expressed in PMVECs, has no effect on network formation. T-type Ca2+ channel blockade also reduces proliferation, cell-matrix adhesion, and migration, three major components of angiogenesis in PMVECs. An in vivo study demonstrated that the mice lacking α1G exhibited a profoundly impaired postinjury cell proliferation in the lungs following lipopolysaccharide challenge. Mechanistically, T-type Ca2+ channel blockade reduces Akt phosphorylation in a dose-dependent manner. Blockade of Akt or its upstream activator, phosphatidylinositol-3-kinase (PI3K), also impairs network formation. Altogether, these findings suggest a novel functional role for the α1G T-type Ca2+ channel to promote the cell’s angiogenic potential via a PI3K-Akt signaling pathway.

scholarly journals Tranilast inhibits the proliferation, chemotaxis and tube formation of human microvascular endothelial cells in vitro and angiogenesis in vivo

1997 ◽  
Vol 122 (6) ◽  
pp. 1061-1066 ◽  
Author(s):  
Masayuki Isaji ◽  
Hiroshi Miyata ◽  
Yoshiyuki Ajisawa ◽  
Yasuo Takehana ◽  
Nagahisa Yoshimura
Keyword(s):  
Endothelial Cells ◽  
Tube Formation ◽  
Microvascular Endothelial Cells ◽  
Microvascular Endothelial

scholarly journals A Genomic Comparison of in vivo and in vitro Brain Microvascular Endothelial Cells

2007 ◽  
Vol 28 (1) ◽  
pp. 135-148 ◽  
Author(s):  
Anthony R Calabria ◽  
Eric V Shusta
Keyword(s):  
Endothelial Cells ◽  
Quantitative Polymerase Chain Reaction ◽  
Genomic Analysis ◽  
Subtractive Hybridization ◽  
Gene Panel ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
Microvascular Endothelial

The blood—brain barrier (BBB) is composed of uniquely differentiated brain microvascular endothelial cells (BMEC). Often, it is of interest to replicate these attributes in the form of an in vitro model, and such models are widely used in the research community. However, the BMEC used to create in vitro BBB models de-differentiate in culture and lose many specialized characteristics. These changes are poorly understood at a molecular level, and little is known regarding the consequences of removing BMEC from their local in vivo microenvironment. To address these issues, suppression subtractive hybridization (SSH) was used to identify 25 gene transcripts that were differentially expressed between in vivo and in vitro BMEC. Genes affected included those involved in angiogenesis, transport and neurogenesis, and real-time quantitative polymerase chain reaction (qPCR) verified transcripts were primarily and significantly downregulated. Since this quantitative gene panel represented those BMEC characteristics lost upon culture, we used it to assess how culture manipulation, specifically BMEC purification and barrier induction by hydrocortisone, influenced the quality of in vitro models. Puromycin purification of BMEC elicited minimal differences compared with untreated BMEC, as assessed by qPCR. In contrast, qPCR-based gene panel analysis after induction with hydrocortisone indicated a modest shift of 10 of the 23 genes toward a more ‘ in vivo-like’ gene expression profile, which correlated with improved barrier phenotype. Genomic analysis of BMEC de-differentiation in culture has thus yielded a functionally diverse set of genes useful for comparing the in vitro and in vivo BBB.

scholarly journals EPAC2 acts as a negative regulator in Matrigel-driven tubulogenesis of human microvascular endothelial cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Takayuki Ikeda ◽  
Yoshino Yoshitake ◽  
Yasuo Yoshitomi ◽  
Hidehito Saito-Takatsuji ◽  
Yasuhito Ishigaki ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Morphology ◽  
Molecular Mechanisms ◽  
Network Formation ◽  
Tissue Growth ◽  
Negative Regulator ◽  
Endothelial Cell Migration ◽  
Microvascular Endothelial Cells ◽  
Microvascular Endothelial

AbstractAngiogenesis is physiologically essential for embryogenesis and development and reinitiated in adult animals during tissue growth and repair. Forming new vessels from the walls of existing vessels occurs as a multistep process coordinated by sprouting, branching, and a new lumenized network formation. However, little is known regarding the molecular mechanisms that form new tubular structures, especially molecules regulating the proper network density of newly formed capillaries. This study conducted microarray analyses in human primary microvascular endothelial cells (HMVECs) plated on Matrigel. The RAPGEF4 gene that encodes exchange proteins directly activated by cAMP 2 (EPAC2) proteins was increased in Matrigel-driven tubulogenesis. Tube formation was suppressed by the overexpression of EPAC2 and enhanced by EPAC2 knockdown in endothelial cells. Endothelial cell morphology was changed to round cell morphology by EPAC2 overexpression, while EPAC2 knockdown showed an elongated cell shape with filopodia-like protrusions. Furthermore, increased EPAC2 inhibited endothelial cell migration, and ablation of EPAC2 inversely enhanced cell mobility. These results suggest that EPAC2 affects the morphology and migration of microvascular endothelial cells and is involved in the termination and proper network formation of vascular tubes.

scholarly journals Bovine Organospecific Microvascular Endothelial Cell Lines as New and Relevant In Vitro Models to Study Viral Infections

2020 ◽  
Vol 21 (15) ◽  
pp. 5249 ◽  
Author(s):  
Anne-Claire Lagrée ◽  
Fabienne Fasani ◽  
Clotilde Rouxel ◽  
Marine Pivet ◽  
Marie Pourcelot ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Lines ◽  
T Antigen ◽  
In Vitro Studies ◽  
Microvascular Endothelial Cell ◽  
Target Cells ◽  
Microvascular Endothelial

Microvascular endothelial cells constitute potential targets for exogenous microorganisms, in particular for vector-borne pathogens. Their phenotypic and functional variations according to the organs they are coming from provide an explanation of the organ selectivity expressed in vivo by pathogens. In order to make available relevant tools for in vitro studies of infection mechanisms, our aim was to immortalize bovine organospecific endothelial cells but also to assess their permissivity to viral infection. Using transfection with SV40 large T antigen, six bovine microvascular endothelial cell lines from various organs and one macrovascular cell line from an umbilical cord were established. They display their own panel of endothelial progenitor/mature markers, as assessed by flow cytometry and RT-qPCR, as well as the typical angiogenesis capacity. Using both Bluetongue and foot-and-mouth disease viruses, we demonstrate that some cell lines are preferentially infected. In addition, they can be transfected and are able to express viral proteins such as BTV8-NS3. Such microvascular endothelial cell lines bring innovative tools for in vitro studies of infection by viruses or bacteria, allowing for the study of host-pathogen interaction mechanisms with the actual in vivo target cells. They are also suitable for applications linked to microvascularization, such as anti-angiogenic and anti-tumor research, growing fields in veterinary medicine.

Human Erythropoietin Induces a Pro-Angiogenic Phenotype in Cultured Endothelial Cells and Stimulates Neovascularization In Vivo

Blood ◽  
1999 ◽  
Vol 93 (8) ◽  
pp. 2627-2636 ◽  
Author(s):  
Domenico Ribatti ◽  
Marco Presta ◽  
Angelo Vacca ◽  
Roberto Ria ◽  
Roberta Giuliani ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Chorioallantoic Membrane ◽  
Janus Kinase ◽  
Angiogenic Factor ◽  
Matrix Metalloproteinase 2 ◽  
Angiogenic Potential ◽  
Angiogenic Response

Abstract Hematopoietic and endothelial cell lineages share common progenitors. Accordingly, cytokines formerly thought to be specific for the hematopoietic system have been shown to affect several functions in endothelial cells, including angiogenesis. In this study, we investigated the angiogenic potential of erythropoietin (Epo), the main hormone regulating proliferation, differentiation, and survival of erythroid cells. Epo receptors (EpoRs) have been identified in the human EA.hy926 endothelial cell line by Western blot analysis. Also, recombinant human Epo (rHuEpo) stimulates Janus Kinase-2 (JAK-2) phosphorylation, cell proliferation, and matrix metalloproteinase-2 (MMP-2) production in EA.hy926 cells and significantly enhances their differentiation into vascular structures when seeded on Matrigel. In vivo, rHuEpo induces a potent angiogenic response in the chick embryo chorioallantoic membrane (CAM). Accordingly, endothelial cells of the CAM vasculature express EpoRs, as shown by immunostaining with an anti-EpoR antibody. The angiogenic response of CAM blood vessels to rHuEpo was comparable to that elicited by the prototypic angiogenic cytokine basic fibroblast growth factor (FGF2), it occurred in the absence of a significant mononuclear cell infiltrate, and it was not mimicked by endothelin-1 (ET-1) treatment. Taken together, these data demonstrate the ability of Epo to interact directly with endothelial cells and to elicit an angiogenic response in vitro and in vivo and thus act as a bona fide direct angiogenic factor.

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