Human Cytomegalovirus Interleukin-10 Downregulates Metalloproteinase Activity and Impairs Endothelial Cell Migration and Placental Cytotrophoblast Invasiveness In Vitro

ABSTRACT At the uterine-placental interface, fetal cytotrophoblasts invade the decidua, breach maternal blood vessels, and form heterotypic contacts with uterine microvascular endothelial cells. In early gestation, differentiating- invading cytotrophoblasts produce high levels of matrix metalloproteinase 9 (MMP-9), which degrades the extracellular matrix and increases the invasion depth. By midgestation, when invasion is complete, MMP levels are reduced. Cytotrophoblasts also produce human interleukin-10 (hIL-10), a pleiotropic cytokine that modulates immune responses, helping to protect the fetal hemiallograft from rejection. Human cytomegalovirus (CMV) is often detected at the uterine-placental interface. CMV infection impairs cytotrophoblast differentiation and invasion, altering the expression of the cell adhesion and immune molecules. Here we report that infection with a clinical CMV strain, VR1814, but not a laboratory strain, AD169, downregulates MMP activity in uterine microvascular endothelial cells and differentiating-invading cytotrophoblasts. Infected cytotrophoblasts expressed CMV IL-10 (cmvIL-10) mRNA and secreted the viral cytokine, which upregulated hIL-10. Functional analyses showed that cmvIL-10 treatment impaired migration in endothelial cell wounding assays and cytotrophoblast invasion of Matrigel in vitro. Comparable changes occurred in cells that were exposed to recombinant hIL-10 or cmvIL-10. Our results show that cmvIL-10 decreases MMP activity and dysregulates the cell-cell and/or cell-matrix interactions of infected cytotrophoblasts and endothelial cells. Reduced MMP activity early in placental development could impair cytotrophoblast remodeling of the uterine vasculature and eventually restrict fetal growth in affected pregnancies.

Download Full-text

Isolation and characterization of human and rat cardiac microvascular endothelial cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1993.264.2.h639 ◽

1993 ◽

Vol 264 (2) ◽

pp. H639-H652 ◽

Cited By ~ 16

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.

Download Full-text

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

Scientific Reports ◽

10.1038/s41598-021-98906-9 ◽

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.

Download Full-text

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

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.90474.2008 ◽

2009 ◽

Vol 296 (2) ◽

pp. L220-L228 ◽

Cited By ~ 19

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.

Download Full-text

Isolation, culture, and characterization of rat lung microvascular endothelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1994.267.4.l433 ◽

1994 ◽

Vol 267 (4) ◽

pp. L433-L441 ◽

Cited By ~ 14

Author(s):

J. C. Magee ◽

A. E. Stone ◽

K. T. Oldham ◽

K. S. Guice

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Cell Function ◽

Primary Cultures ◽

Microvascular Endothelial Cells ◽

Rat Lung ◽

Simple Method ◽

Endothelial Cell Function ◽

Microvascular Endothelial

Highly pure primary cultures of rat lung microvascular endothelial cells were obtained from peripheral lung tissue using a combination of selective culture strategies. The cells had a characteristic morphology consistent with an endothelial origin and were positive for a number of endothelial cell markers, including uptake of fluorescent acetylated lactate dehydrogenase, binding of the lectin Bandeiraea simplicifolia I, and positive immunofluorescence staining with two endothelial cell monoclonal antibodies. The cells behaved as microvascular endothelial cells using an in vitro angiogenesis assay. This isolation method provides a simple method for culturing the pulmonary microvasculature of the rat and these studies support the idea that endothelial cells from different vessels exhibit phenotypic heterogeneity. This method should prove useful for studying specialized endothelial cell function and differentiation in vitro.

Download Full-text

Retroviral transformation of cerebral microvascular endothelial cells: macrophage-like and microvascular endothelial cell properties

Blood ◽

10.1182/blood.v77.2.294.bloodjournal772294 ◽

1991 ◽

Vol 77 (2) ◽

pp. 294-305

Author(s):

DH Robinson ◽

MK Warren ◽

LT Liang ◽

JJ Oprandy ◽

TB Nielsen ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Self Organization ◽

Microvascular Endothelial Cells ◽

Microvascular Endothelial Cell ◽

Focus Formation ◽

Transformed Cell ◽

L Cell ◽

Microvascular Endothelial

We report that L-cell-conditioned medium (LCM) transforms porcine cerebral microvascular (PCMV) endothelial cells into cells with macrophage-like properties. LCM is known to contain both cytokine(s) and the L-cell virus, a murine retrovirus found in the L929 cell and LCM. Our evidence suggests that both LCM cytokine(s) and the L-cell virus are involved in this PCMV endothelial cell transformation. Criteria for transformation include focus formation, decreased serum requirements for growth, changes in morphology including nonadherence, propagation in suspension culture, and a decreased growth response to stimulation with a known endothelial cell mitogen. Macrophage-like characteristics of this transformed cell, designated as RVTE, include pinocytosis of low-density lipoprotein, Fc receptor-mediated phagocytosis, phagocytosis of bacteria and zymosan, the expression of macrophage enzyme markers, and constitutive production of colony- stimulating factor 1. However, the transformed cell retains several properties of the nontransformed cell including the expression of FVIII:RAg and in vitro self-organization into capillary-like structures. Cloning of RVTE cells clearly shows that both macrophage- like and cerebral microvascular endothelial cell properties are present in the same cell. During self-organization, nontransformed cells express morphologic and functional characteristics classically associated with the macrophage. These findings suggest that some brain capillary pathophysiologies could involve macrophage-like cerebral microvascular endothelial cells. Furthermore, the “reticuloendothelial” phenotypic repertoire expressed by this transformed cerebral microvascular endothelial cell may show that the cerebral capillary endothelial cell in vivo is derived from a hematopoietic and/or phagocytic precursor.

Download Full-text

Retroviral transformation of cerebral microvascular endothelial cells: macrophage-like and microvascular endothelial cell properties

Blood ◽

10.1182/blood.v77.2.294.294 ◽

1991 ◽

Vol 77 (2) ◽

pp. 294-305 ◽

Cited By ~ 7

Author(s):

DH Robinson ◽

MK Warren ◽

LT Liang ◽

JJ Oprandy ◽

TB Nielsen ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Self Organization ◽

Microvascular Endothelial Cells ◽

Microvascular Endothelial Cell ◽

Focus Formation ◽

Transformed Cell ◽

L Cell ◽

Microvascular Endothelial

Abstract We report that L-cell-conditioned medium (LCM) transforms porcine cerebral microvascular (PCMV) endothelial cells into cells with macrophage-like properties. LCM is known to contain both cytokine(s) and the L-cell virus, a murine retrovirus found in the L929 cell and LCM. Our evidence suggests that both LCM cytokine(s) and the L-cell virus are involved in this PCMV endothelial cell transformation. Criteria for transformation include focus formation, decreased serum requirements for growth, changes in morphology including nonadherence, propagation in suspension culture, and a decreased growth response to stimulation with a known endothelial cell mitogen. Macrophage-like characteristics of this transformed cell, designated as RVTE, include pinocytosis of low-density lipoprotein, Fc receptor-mediated phagocytosis, phagocytosis of bacteria and zymosan, the expression of macrophage enzyme markers, and constitutive production of colony- stimulating factor 1. However, the transformed cell retains several properties of the nontransformed cell including the expression of FVIII:RAg and in vitro self-organization into capillary-like structures. Cloning of RVTE cells clearly shows that both macrophage- like and cerebral microvascular endothelial cell properties are present in the same cell. During self-organization, nontransformed cells express morphologic and functional characteristics classically associated with the macrophage. These findings suggest that some brain capillary pathophysiologies could involve macrophage-like cerebral microvascular endothelial cells. Furthermore, the “reticuloendothelial” phenotypic repertoire expressed by this transformed cerebral microvascular endothelial cell may show that the cerebral capillary endothelial cell in vivo is derived from a hematopoietic and/or phagocytic precursor.

Download Full-text

Tumor cell surface beta 1-4-linked galactose binds to lectin(s) on microvascular endothelial cells and contributes to organ colonization.

The Journal of Cell Biology ◽

10.1083/jcb.111.2.773 ◽

1990 ◽

Vol 111 (2) ◽

pp. 773-781 ◽

Cited By ~ 31

Author(s):

I Cornil ◽

R S Kerbel ◽

J W Dennis

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Cell Surface ◽

Tumor Cell ◽

Tumor Cells ◽

Tumor Cell Line ◽

Microvascular Endothelial Cells ◽

Class 1 ◽

Microvascular Endothelial

Cell surface carbohydrate structures acting as ligands for tissue specific mammalian lectins have been implicated in cell-cell interactions during embryogenesis, lymphocyte homing, and tumor cell metastasis. In this report, we provide evidence that beta 1-4 linked galactose (Gal) residues in N-linked oligosaccharides on the surface of blood born tumor cells serve as a ligand for binding to microvascular endothelial cells. D36W25, a class 1 glycosylation mutant of the MDAY-D2 lymphoreticular tumor cell line, lacks sialic acid and Gal in cellular glycans due to a defect in the Golgi UDP-Gal transporter. Using UDP-Gal and bovine galactosyltransferase in vitro, beta 1-4 Gal was restored to the surface of the cells and 70% of the galactosylated glycans persisted for 8 h in vitro at 37 degrees C. Compared to mock-treated D36W25 cells, galactosylated D36W25 cells showed an 80% increase in binding to microvascular endothelial cell monolayers in vitro. The enhanced binding of galactosylated D36W25 cells to endothelial cell was inhibited by the addition of lactosamine-conjugated albumin to the assay. Consistent with these observations, swainsonine and castinospermine, two inhibitors of N-linked processing that result in loss of lactosamine antennae inhibited the binding of wild-type MDAY-D2 cells to endothelial cells in vitro. Injection of radiolabeled tumor cells into the circulation of syngeneic mice, showed that galactosylation of D36W25 cells resulted in 2-3 more tumor cells retained in the lungs and livers. In addition, galactosylation of D36W25 cells increased by 30-fold the number of visible liver metastases on inspection 4 wk after tumor cell injection. These results suggest that beta 1-4Gal-binding lectins on microvascular endothelial cells can contribute to retention and secondary tumor formation of blood born tumor cells. With the increasing availability of purified glycosyltransferases, reconstruction of a variety of carbohydrate sequences on the surface of class 1 mutants provides a controlled means of studying carbohydrate-lectin interactions on viable cells.

Download Full-text

Neutrophil-Derived Microvesicle Induced Dysfunction of Brain Microvascular Endothelial Cells In Vitro

International Journal of Molecular Sciences ◽

10.3390/ijms20205227 ◽

2019 ◽

Vol 20 (20) ◽

pp. 5227 ◽

Cited By ~ 7

Author(s):

Anjana Ajikumar ◽

Merete B. Long ◽

Paul R. Heath ◽

Stephen B. Wharton ◽

Paul G. Ince ◽

...

Keyword(s):

Gene Expression ◽

Endothelial Cells ◽

Endothelial Cell ◽

The Body ◽

Confocal Imaging ◽

Microvascular Endothelial Cells ◽

Microvascular Endothelial Cell ◽

Brain Microvascular Endothelial Cells ◽

Microvascular Endothelial

The blood-brain barrier (BBB), composed of brain microvascular endothelial cells (BMEC) that are tightly linked by tight junction (TJ) proteins, restricts the movement of molecules between the periphery and the central nervous system. Elevated systemic levels of neutrophils have been detected in patients with altered BBB function, but the role of neutrophils in BMEC dysfunction is unknown. Neutrophils are key players of the immune response and, when activated, produce neutrophil-derived microvesicles (NMV). NMV have been shown to impact the integrity of endothelial cells throughout the body and we hypothesize that NMV released from circulating neutrophils interact with BMEC and induce endothelial cell dysfunction. Therefore, the current study investigated the interaction of NMV with human BMEC and determined whether they altered gene expression and function in vitro. Using flow cytometry and confocal imaging, NMV were shown to be internalized by the human cerebral microvascular endothelial cell line hCMEC/D3 via a variety of energy-dependent mechanisms, including endocytosis and macropinocytosis. The internalization of NMV significantly altered the transcriptomic profile of hCMEC/D3, specifically inducing the dysregulation of genes associated with TJ, ubiquitin-mediated proteolysis and vesicular transport. Functional studies confirmed NMV significantly increased permeability and decreased the transendothelial electrical resistance (TEER) of a confluent monolayer of hCMEC/D3. These findings indicate that NMV interact with and affect gene expression of BMEC as well as impacting their integrity. We conclude that NMV may play an important role in modulating the permeability of BBB during an infection.

Download Full-text

H2S Pretreatment Is Promigratory and Decreases Ischemia/Reperfusion Injury in Human Microvascular Endothelial Cells

Oxidative Medicine and Cellular Longevity ◽

10.1155/2021/8886666 ◽

2021 ◽

Vol 2021 ◽

pp. 1-13

Author(s):

Elisa Zicola ◽

Elisa Arrigo ◽

Daniele Mancardi

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Reperfusion Injury ◽

Vascular Function ◽

Cell Injury ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Endothelial Cell Migration ◽

Microvascular Endothelial Cells ◽

Microvascular Endothelial

Endothelial cell injury and vascular function strongly correlate with cardiac function following ischemia/reperfusion injury. Several studies indicate that endothelial cells are more sensitive to ischemia/reperfusion compared to cardiomyocytes and are critical mediators of cardiac ischemia/reperfusion injury. H2S is involved in the regulation of cardiovascular system homeostasis and can act as a cytoprotectant during ischemia/reperfusion. Activation of ERK1/2 in endothelial cells after H2S stimulation exerts an enhancement of angiogenesis while its inhibition significantly decreases H2S cardioprotective effects. In this work, we investigated how H2S pretreatment for 24 hours prevents the ischemia/reperfusion injury and promotes angiogenesis on microvascular endothelial cells following an ischemia/reperfusion protocol in vitro, using a hypoxic chamber and ischemic buffer to simulate the ischemic event. H2S preconditioning positively affected cell viability and significantly increased endothelial cell migration when treated with 1 μM H2S. Furthermore, mitochondrial function was preserved when cells were preconditioned. Since ERK1/2 phosphorylation was extremely enhanced in ischemia/reperfusion condition, we inhibited ERK both directly and indirectly to verify how H2S triggers this pathway in endothelial cells. Taken together, our data suggest that H2S treatment 24 hours before the ischemic insult protects endothelial cells from ischemia/reperfusion injury and eventually decreases myocardial injury.

Download Full-text

Abstract W P407: Shear Stress and Co-culture with Astrocytes Determine Brain Microvascular Endothelial Cell Phenotype

Stroke ◽

10.1161/str.46.suppl_1.wp407 ◽

2015 ◽

Vol 46 (suppl_1) ◽

Author(s):

Sina Salehi Omran ◽

Fernando Garcia Polite ◽

Elazer Edelman ◽

Mercedes Balcells-Camps

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Endothelial Cell ◽

Immunocytochemical Staining ◽

Microvascular Endothelial Cells ◽

Cell Phenotype ◽

Brain Microvascular Endothelial Cells ◽

Glut 1 ◽

Microvascular Endothelial

Introduction: Dementia has classically been identified to be of either vascular or neural origin. These domains overlap and are complementary, thus we consider dementia a disease of the single cerebrovascular unit. Our objective was to generate a modular platform for co-culture of brain microvascular endothelial cells and astrocytes that also incorporates mechanical stresses, and to then use this model of the cerebrovascular unit microenvironment to study the unit in vitro . Hypothesis: We assessed the hypothesis that endothelial health and blood-brain barrier integrity are modulated by shear stress and co-culture with astrocytes. Methods: We lithographed polydimethylsiloxane substrate on Teflon negative molds with subjacent rectangular channels of 0.45 and 2 mm depth for seeding of human brain microvascular cells and astrocytes, respectively, separated by a polytetrafluoroethylene (0.45 μm pore size) membrane under no flow or physiologic flow (6.2 dynes/cm 2 ) for one week. Immunocytochemical staining for glial fibrillary acidic protein and CD31 was simultaneously visualized by confocal microscopy. Cells from each channel were detached via trypsinization, and expression of transport proteins P-glycoprotein (P-gp) and glucose transporter-1 (GLUT-1), in addition to junction proteins zona occludens-1 (ZO-1) and CD31, was measured by Western Blot. Results: We stably co-cultured brain microvascular endothelial cells and astrocytes with no chamber leakage or mixing. CD31 staining revealed endothelial cell alignment to direction of flow. Expression of ZO-1 by endothelial cells increased in presence of flow and co-culture independently, by 1.6-fold in combined conditions relative to static monoculture (p<0.05). For P-gp, the increase in combined conditions was 5.5-fold (p<0.05). GLUT-1 and CD31 levels did not change significantly with co-culture or flow. Conclusion: Cell biology devoid of microenvironmental cues provides limited insight, especially when considering whole tissues, on the impact of disease. A co-culture system that introduces multiple cells, flow, controlled stress and independent visualization and sampling of each cell domain adds deeper understanding and greater value to in vitro biological models and tissue biology.

Download Full-text