scholarly journals Vascular Network Formation by Human Microvascular Endothelial Cells in Modular Fibrin Microtissues

2016 ◽  
Vol 2 (11) ◽  
pp. 1914-1925 ◽  
Author(s):  
Ramkumar Tiruvannamalai Annamalai ◽  
Ana Y. Rioja ◽  
Andrew J. Putnam ◽  
Jan P. Stegemann
Keyword(s):  
Endothelial Cells ◽  
Network Formation ◽  
Vascular Network ◽  
Microvascular Endothelial Cells ◽  
Microvascular Endothelial

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 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 Extrinsic acidosis suppresses glycolysis and migration while increasing network formation in pulmonary microvascular endothelial cells

2019 ◽  
Vol 317 (2) ◽  
pp. L188-L201 ◽  
Author(s):  
Ji Young Lee ◽  
Mher Onanyan ◽  
Ian Garrison ◽  
Roderica White ◽  
Maura Crook ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Network Formation ◽  
Segment Length ◽  
Microvascular Endothelial Cells ◽  
Carbonic Anhydrases ◽  
Pulmonary Microvascular Endothelial Cells ◽  
Ca Ix ◽  
Microvascular Endothelial ◽  
And Migration ◽  
The Impact

Acidosis is common among critically ill patients, but current approaches to correct pH do not improve disease outcomes. During systemic acidosis, cells are either passively exposed to extracellular acidosis that other cells have generated (extrinsic acidosis) or they are exposed to acid that they generate and export into the extracellular space (intrinsic acidosis). Although endothelial repair following intrinsic acidosis has been studied, the impact of extrinsic acidosis on migration and angiogenesis is unclear. We hypothesized that extrinsic acidosis inhibits metabolism and migration but promotes capillary-like network formation in pulmonary microvascular endothelial cells (PMVECs). Extrinsic acidosis was modeled by titrating media pH. Two types of intrinsic acidosis were compared, including increasing cellular metabolism by chemically inhibiting carbonic anhydrases (CAs) IX and XII (SLC-0111) and with hypoxia. PMVECs maintained baseline intracellular pH for 24 h with both extrinsic and intrinsic acidosis. Whole cell CA IX protein expression was decreased by extrinsic acidosis but not affected by hypoxia. When extracellular pH was equally acidic, extrinsic acidosis suppressed glycolysis, whereas intrinsic acidosis did not. Extrinsic acidosis suppressed migration, but increased Matrigel network master junction and total segment length. CRISPR-Cas9 CA IX knockout PMVECs revealed an independent role of CA IX in promoting glycolysis, as loss of CA IX alone was accompanied by decreased hexokinase I and pyruvate dehydrogenase E1α expression and decreasing migration. 2-deoxy-d-glucose had no effect on migration but profoundly inhibited network formation and increased N-cadherin expression. Thus, we report that while extrinsic acidosis suppresses endothelial glycolysis and migration, it promotes network formation.

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 Dynamic Observation of Oxygenation-Induced Contraction of and Transient Fiber-Network Formation–Disassembly in Cultured Human Brain Microvascular Endothelial Cells

2003 ◽  
Vol 23 (7) ◽  
pp. 821-828 ◽  
Author(s):  
Kouji Inoue ◽  
Minoru Tomita ◽  
Yasuo Fukuuchi ◽  
Norio Tanahashi ◽  
Masahiro Kobari ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Human Brain ◽  
Network Formation ◽  
Pure Oxygen ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
Fiber Network ◽  
Rapid Formation ◽  
Oxygen Gas ◽  
Microvascular Endothelial

Oxygenation-induced contraction of nonconfluent cultured human brain microvascular endothelial cells (HBECs, n = 30) was examined by video-enhanced contrast-differential interferential contrast microscopy. After administering a continuous gentle blow of pure oxygen gas to the surface of the medium just above the flattened HBEC, the plasma membrane exhibited tensioning and wrinkling, resulting in a strong contraction of the cell body by 14 ± 7% ( P < 0.001). When the cell stopped contracting, transient formation of a fiber network starting from certain spots (possibly adhesion plaques, though these were not visible in the majority of cases) and expanding to the whole cell was observed. The occurrence of fiber network formation was statistically significant (26 of 30 separate cells, P < 0.05). After cessation of oxygen delivery, the observed network of fibers broke up rapidly (in a period of 3.3 ± 1.2 seconds) into small particles of <0.5 μm in diameter, which subsequently fused into the cellular structure. The HBEC completely recovered the control appearance. The sequential process was completed within 30 seconds and was reproduced in individual cells each time that oxygen gas was supplied. The authors conclude that the HBEC strongly contracts in response to a transient oxygenation stimulus, followed by rapid formation/disassembly of a network structure.

CaMK4 Is a Downstream Effector of the α1G T-type Calcium Channel to Determine the Angiogenic Potential of Pulmonary Microvascular Endothelial Cells

2021 ◽  
Author(s):  
Zhen Zheng ◽  
Xuelin Wang ◽  
Yuxia Wang ◽  
Judy A.C. King ◽  
Peilin Xie ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Network Formation ◽  
Microvascular Endothelial Cells ◽  
Angiogenic Potential ◽  
Akt Phosphorylation ◽  
Pulmonary Microvascular Endothelial Cells ◽  
Downstream Effector ◽  
Phosphorylation Level ◽  
Microvascular Endothelial

Pulmonary microvascular endothelial cells (PMVECs) uniquely express an α1G-subtype of voltage-gated T-type Ca2+ channel. We have previously revealed that the α1G channel functions as a background Ca2+ entry pathway that is critical for the cell proliferation, migration, and angiogenic potential of PMVECs, a novel function attributed to the coupling between α1G-mediate Ca2+ entry and constitutive Akt phosphorylation and activation. Despite this significance, mechanism(s) that link the α1G-mediated Ca2+ entry to Akt phosphorylation remain incompletely understood. In the present study, we demonstrate that Ca2+/calmodulin-dependent protein kinase (CaMK) 4 serves as a downstream effector of the α1G-mediated Ca2+ entry to promote the angiogenic potential of PMVECs. Notably, CaMK2 and CaMK4 are both expressed in PMVECs. Pharmacological blockade or genetic knockdown of the α1G channel led to a significant reduction in the phosphorylation level of CaMK4 but not the phosphorylation level of CaMK2. Pharmacological inhibition as well as genetic knockdown of CaMK4 significantly decreased cell proliferation, migration, and network formation capacity in PMVECs. However, CaMK4 inhibition or knockdown did not alter Akt phosphorylation status in PMVECs, indicating that α1G/Ca2+/CaMK4 is independent of the α1G/Ca2+/Akt pathway in sustaining the cells' angiogenic potential. Altogether, these findings suggest a novel α1G-CaMK4 signaling complex that regulates the Ca2+-dominated angiogenic potential in PMVECs.

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.

Resveratrol Reduces Matrix Metalloproteinase-2 Activity Induced by Oxygen-Glucose Deprivation and Reoxygenation in Human Cerebral Microvascular Endothelial Cells

2012 ◽  
Vol 82 (4) ◽  
pp. 267-274 ◽  
Author(s):  
Zahide Cavdar ◽  
Mehtap Y. Egrilmez ◽  
Zekiye S. Altun ◽  
Nur Arslan ◽  
Nilgun Yener ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Neuroprotective Effect ◽  
Ischemia Reperfusion ◽  
Neurovascular Unit ◽  
Glucose Deprivation ◽  
Matrix Metalloproteinase 2 ◽  
Microvascular Endothelial Cells ◽  
Oxygen Glucose Deprivation ◽  
Microvascular Endothelial

The main pathophysiology in cerebral ischemia is the structural alteration in the neurovascular unit, coinciding with neurovascular matrix degradation. Among the human matrix metalloproteinases (MMPs), MMP-2 and -9, known as gelatinases, are the key enzymes for degrading type IV collagen, which is the major component of the basal membrane that surrounds the cerebral blood vessel. In the present study, we investigated the effects of resveratrol on cytotoxicity, reactive oxygen species (ROS), and gelatinases (MMP-2 and -9) in human cerebral microvascular endothelial cells exposed to 6 hours of oxygen-glucose deprivation and a subsequent 24 hours of reoxygenation with glucose (OGD/R), to mimic ischemia/reperfusion in vivo. Lactate dehydrogenase increased significantly, in comparison to that in the normoxia group. ROS was markedly increased in the OGD/R group, compared to normoxia. Correspondingly, ROS was significantly reduced with 50 μM of resveratrol. The proMMP-2 activity in the OGD/R group showed a statistically significant increase from the control cells. Resveratrol preconditioning decreased significantly the proMMP-2 in the cells exposed to OGD/R in comparison to that in the OGD/R group. Our results indicate that resveratrol regulates MMP-2 activity induced by OGD/R via its antioxidant effect, implying a possible mechanism related to the neuroprotective effect of resveratrol.

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