Thrombin stimulates albumin transcytosis in lung microvascular endothelial cells via activation of acid sphingomyelinase

Transcellular albumin transport occurs via caveolae that are abundant in lung microvascular endothelial cells. Stimulation of albumin transcytosis by proinflammatory mediators may contribute to alveolar protein leak in lung injury, yet the regulation of albumin transport and its underlying molecular mechanisms are so far incompletely understood. Here we tested the hypothesis that thrombin may stimulate transcellular albumin transport across lung microvascular endothelial cells in an acid-sphingomyelinase dependent manner. Thrombin increased the transport of fluorescently labeled albumin across confluent human lung microvascular endothelial cell (HMVEC-L) monolayers to an extent that markedly exceeds the rate of passive diffusion. Thrombin activated acid sphingomyelinase (ASM) and increased ceramide production in HMVEC-L, but not in bovine pulmonary artery cells, which showed little albumin transport in response to thrombin. Thrombin increased total caveolin-1 (cav-1) content in both whole cell lysates and lipid rafts from HMVEC-L, and this effect was blocked by inhibition of ASM or de novo protein biosynthesis. Thrombin-induced uptake of albumin into lung microvascular endothelial cells was confirmed in isolated-perfused lungs by real-time fluorescence imaging and electron microscopy of gold-labeled albumin. Inhibition of ASM attenuated thrombin-induced albumin transport both in confluent HMVEC-L and in intact lungs, whereas HMVEC-L treatment with exogenous ASM increased albumin transport and enriched lipid rafts in cav-1. Our findings indicate that thrombin stimulates transcellular albumin transport in an acid sphingomyelinase-dependent manner by inducing de novo synthesis of cav-1 and its recruitment to membrane lipid rafts.

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Insulin Stimulates Tetrahydrobiopterin Synthesis in Mouse Brain Microvascular Endothelial Cells

Pteridines ◽

10.1515/pteridines.1999.10.4.213 ◽

1999 ◽

Vol 10 (4) ◽

pp. 213-216

Author(s):

Masakazu Ishi ◽

Shunichi Shimizu ◽

Tsutomu Nagai ◽

Yuji Kiuchi ◽

Toshinori Yamamoto

Keyword(s):

Endothelial Cells ◽

Mouse Brain ◽

De Novo ◽

Selective Inhibitor ◽

Microvascular Endothelial Cells ◽

Dependent Manner ◽

Brain Microvascular Endothelial Cells ◽

Gtp Cyclohydrolase I ◽

Concentration Dependent Manner ◽

Microvascular Endothelial

Summary We examined the effects of insulin on tetrahydrobiopterin (BH4) synthesis in mouse brain microvascular endothelial cells (MBMECs). Treatment of MBMECs with insulin increased the intracellular BH4 content in a time- and concentration-dependent manner. The insulin-induced increase in BH4 content was inhibited by treatment with 2,4-diamino-6-hydroxypyrimidine, a selective inhibitor of GTP cyclohydrolase I, and Nacetylserotonin, a selective inhibitor of sepiapterin reductase. These findings indicate that insulin stimulates BH4 synthesis in MBMECs through a de novo synthetic pathway of BH4.

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Stimulation of Tetrahydrobiopterin Synthesis by 17β-Estradiol in Brain Microvascular Endothelial Cells

Pteridines ◽

10.1515/pteridines.2000.11.4.129 ◽

2000 ◽

Vol 11 (4) ◽

pp. 129-132

Author(s):

Kazuhiro Shiota ◽

Masakazu Ishii ◽

Toshinori Yamamoto ◽

Shunichi Shimizu ◽

Yuji Kiuchi

Keyword(s):

Endothelial Cells ◽

De Novo ◽

Vascular Endothelial Cells ◽

Mrna Level ◽

Microvascular Endothelial Cells ◽

No Production ◽

Brain Microvascular Endothelial Cells ◽

Protective Effects ◽

17Β Estradiol ◽

Microvascular Endothelial

Abstract The purpose of this study was to examine whether 17β-estradiol stimulates the synthesis of tetrahydrobiopterin : BH4), which is one of the cofactors of nitric oxide (NO) synthase, in mouse brain microvascular endothelial cells. Addition of 17()-estradiol to endothelial cells time- and concentration-dependently increased intracellular BH4 level. 17β-Estradiol also stimulated the mRNA level of GTP-cyclohydrolase I (GTPCH), which is a rate-limiting enzyme of the de novo BH4 synthetic pathway. In addition, the 17β-estradiol-induced expression of GTPCH mRNA was strongly attenuated by treatment with an inhibitor of 17β-estradiol receptor 4-hydroxy-tamoxlfen. These results suggest that 17β-estradiol stimulates BH4 synthesis through the induction of GTPCH by tamoxifensensitive receptor in vascular endothelial cells. The 17β-estradiol-induced increase in BH4 level might be implicated in not only NO production, but also protective effects of 17β-estradiol against ischemic brain damage and atherosclerosis, since BH4 is an intracellular antioxidant.

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MALAT1 modulates miR-146’s protection of microvascular endothelial cells against LPS-induced NF-κB activation and inflammatory injury

Innate Immunity ◽

10.1177/1753425919861427 ◽

2019 ◽

Vol 25 (7) ◽

pp. 433-443

Author(s):

Lin-Lin Feng ◽

Wei-Na Xin ◽

Xiu-Li Tian

Keyword(s):

Endothelial Cells ◽

Cell Viability ◽

Combined Treatment ◽

Luciferase Assay ◽

Luciferase Reporter ◽

Microvascular Endothelial Cells ◽

Dependent Manner ◽

Inflammatory Injury ◽

Tnf Α ◽

Microvascular Endothelial

To investigate the role of miR-146 and its possible relationship with MALAT1 in LPS-induced inflammation in human microvascular endothelial cells (HMECs), HMEC-1 cells were treated with LPS to construct an inflammatory injury cell model, and the cell viability, TNF-α and IL-6 secretion and the expression levels of VCAM-1, SELE and ICAM-1 were analysed as markers of inflammatory injury. The regulation mechanisms of miR-146 interacted with MALAT1 and the downstream NF-κB signalling were also verified by dual-luciferase assay and knockdown technology. LPS significantly decreased the cell viability, increased levels of VCAM-1, SELE and ICAM-1 and also up-regulated miR-146a/b, TNF-α and IL-6 in a dose-dependent manner. Over-expression of miR-146a resulted in down-regulation of TNF-α and IL-6, as well as VCAM-1, SELE and ICAM-1, while inhibition of miR-146a led to opposite results. The dual-luciferase reporter assay showed both miR-146a and miR-146b directly targeted and negatively regulated the expression of MALAT1. Silencing of MALAT1 suppressed LPS-induced NF-κB activation and TNF-α and IL-6 secretion, reducing the cell inflammatory injury, but these changes were reversed after combined treatment with miR-146a inhibitor. Taken together, we demonstrate that miR-146 protects HMECs against inflammatory injury by inhibiting NF-κB activation. This process is modulated by MALAT1.

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Human microvascular endothelial cells resist Shiga toxins by IFN-γ treatment in vitro

Microbiology ◽

10.1099/mic.0.26142-0 ◽

2003 ◽

Vol 149 (9) ◽

pp. 2609-2614 ◽

Cited By ~ 2

Author(s):

Tomoaki Yoshida ◽

Tsuyoshi Sugiyama ◽

Naoki Koide ◽

Isamu Mori ◽

Takashi Yokochi

Keyword(s):

Endothelial Cells ◽

Cell Model ◽

Cell Damage ◽

Shigella Dysenteriae ◽

Microvascular Endothelial Cells ◽

Microvascular Endothelial Cell ◽

Shiga Toxins ◽

Resistant Phenotype ◽

Microvascular Endothelial

Shiga toxins (Stxs) produced by enterohaemorrhagic Escherichia coli or Shigella dysenteriae damage human endothelial cells predominantly in cooperation with pro-inflammatory cytokines, such as TNF-α. However, in this study, in vitro IFN-γ pre-treatment resulted in human lung microvascular endothelial cells becoming over 10 000-fold less sensitive to Stxs. In contrast, in their basal condition, they were extremely sensitive to Stxs. Interestingly, TNF-α addition to IFN-γ reverted the Stx-resistant phenotype, which corresponded with its well-established enhancing effect on Stx toxicity. Toxin binding to the cell was barely affected by IFN-γ. Also, the toxin uptake in the Stx-resistant phenotype was more than 100-fold greater than that of normal cells, when compared at Stx concentrations resulting in equivalent degrees of cell damage. Protein synthesis was inhibited by nearly 90 % in the Stx-resistant phenotype after 24 h toxin exposure. This indicated that the intracellular toxin was active as an N-glycosidase, while cells were still over 60 % viable, suggesting a possible unknown cytotoxic function of Stx. In conclusion, this study shows a unique effect of IFN-γ in the suppression of the toxicity of Stxs in a human microvascular endothelial cell model and the involvement of a novel mechanism in this suppression.

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

AJP Cell Physiology ◽

10.1152/ajpcell.00336.2018 ◽

2019 ◽

Vol 316 (3) ◽

pp. C353-C364 ◽

Cited By ~ 4

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.

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CULTURED HUMAN MICROVASCULAR ENDOTHELIAL CELLS SYNTHESIZE CYCLOOXYGENASE METABOLITES FROM ARACHIDONIC ACID IN RESPONSE TO LEUKOTRIENES C4 AND D4

10.1055/s-0038-1642838 ◽

1987 ◽

Author(s):

L O Carreras ◽

J Maclouf ◽

G Tobelem ◽

J P Caen

Keyword(s):

Endothelial Cells ◽

Arachidonic Acid ◽

Time Course ◽

Umbilical Vein ◽

Calcium Ionophore ◽

Microvascular Endothelial Cells ◽

Ionophore A23187 ◽

Total Production ◽

Dependent Manner ◽

Microvascular Endothelial

Several investigators have demonstrated that endothelial cells have heterogeneous intrinsic properties depending on their vascular origin. In this respect, very limited knowledge exists concerning the production of eicosanoids by human microvascular endothelial cells (HMEC). The aim of this study was to determine: 1) the pattern of the production of cyclooxygenase metabolites by cultured HMEC from omental adipose tissue as compared to the classical study of human umbilical vein endothelial cells (HUVEC); 2) the modification of this metabolism upon leukotrienes (LTs) stimulation. Cultured HMEC produced prostaglandin (PG) E2, PGF2 , 6-keto-PGF1 , and PGD2 (measured by enzymoimmunoassay). In basal conditions, PGD2 was the main product released in the supernatant. Upon stimulation with thrombin, arachidonic acid and calcium ionophore A23187, a marked increase in the production of PGE2, PGF2 , and 6-keto-PGFj , was observed; these results were quite different from HUVEC. In contrast, PGD2 remained unchanged under our experimental conditions and thromboxane B2 was always undetectable. In all cases, the release of PGE2 and PGF2 , was higher than that of 6-keto-PGFj . A considerable amount of the metabolites produced remained cell-associated. The total production (release + cell bound) of cyclooxygenase products was stimulated by LTC4 and LTD4 in a dose-dependent manner (10-9 to 10-6 M). The production of PGD2 was unchanged. LTC4 and LTD4 were almost equally potent, but LTB4 was unable to stimulate PG synthesis (n=4). The production of metabolites induced by 1 uM LTC4 or LTD4 was even higher than that obtained in the presence of high concentrations of thrombin (5 U/ml). This contrasted with the more pronounced stimulation of thrombin on HUVEC as compared to LTs. In the kinetic studies (n=2) we have observed a slow time-course of release of PGE2 and 6-keto-PGF1 into the supernatant of LTs-stimulated HMEC (half-maximal formation at 14-15 min). The stimulatory activity of LTC4 and LTD4 on the production of vasoactive cyclooxygenase metabolites by HMEC could be relevant in inflammatory processes.

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HIV-1 Tat Mediated Effects on Focal Adhesion Assembly and Permeability in Brain Microvascular Endothelial Cells.

Blood ◽

10.1182/blood.v104.11.3105.3105 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3105-3105

Author(s):

Shalom Avraham ◽

Shuxian Jiang ◽

Tae-Hee Lee ◽

O. Prakash ◽

Hava Karsenty Avraham

Keyword(s):

Endothelial Cells ◽

Focal Adhesion ◽

Microvascular Endothelial Cells ◽

Mutant Form ◽

Dependent Manner ◽

Brain Microvascular Endothelial Cells ◽

Adhesion Sites ◽

Permeability Changes ◽

Microvascular Endothelial ◽

Hiv 1

Abstract The blood-brain barrier (BBB) is a network formed mainly by brain microvascular endothelial cells. The integrity of the BBB is critical for brain function. Breakdown of the BBB is commonly seen in AIDS patients with HIV-1-associated dementia (HAD), despite the lack of productive HIV-infection of the brain endothelium. The processes by which HIV causes these pathological conditions are not well understood. Here, we characterized the molecular mechanisms by which Tat mediates its pathogenic effects in-vitro on primary human brain microvascular endothelial cells (HBMECs). Tat treatment of HBMECs stimulated cytoskeletal organization and increased focal adhesion sites as compared to control cells or cells treated with heat-inactivated Tat. Pretreatment with Tat antibodies or with the specific inhibitor SU-1498, which interferes with VEGFR-2 (Flk-1/KDR) receptor phosphorylation, blocked the ability of Tat to stimulate focal adhesion assembly and the migration of HBMECs. Focal adhesion kinase (FAK) was tyrosine-phosphorylated by Tat and found to be an important component of focal adhesion sites. Inhibition of FAK by the dominant-interfering mutant form FRNK (FAK-related non-kinase) significantly blocked HBMEC migration and disrupted focal adhesions upon Tat activation. Furthermore, HIV-Tat induced permeability changes in HBMECs in a time dependent manner. Tat also impaired BBB permeability as observed in HIV-1 Tat transgenic mice. These studies define a mechanism for HIV-1 Tat in focal adhesion complex assembly in HBMECs, via activation of FAK, leading to cytoskeletal reorganization and permeability changes.

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Interleukin 15 Induces Endothelial Hyaluronan Expression in Vitro and Promotes Activated T Cell Extravasation through a Cd44-Dependent Pathway in Vivo

Journal of Experimental Medicine ◽

10.1084/jem.190.1.9 ◽

1999 ◽

Vol 190 (1) ◽

pp. 9-20 ◽

Cited By ~ 52

Author(s):

Pila Estess ◽

Animesh Nandi ◽

Mansour Mohamadzadeh ◽

Mark H. Siegelman

Keyword(s):

T Cells ◽

Endothelial Cells ◽

Endothelial Cell ◽

T Cell ◽

Microvascular Endothelial Cell ◽

Dependent Manner ◽

Cell Recruitment ◽

Microvascular Endothelial ◽

Dependent Pathway

T cell recruitment to extralymphoid tissues is fundamental to the initiation and perpetuation of the inflammatory state during immune and autoimmune responses. Interleukin (IL)-15 is a proinflammatory cytokine whose described functions largely overlap with those of IL-2. The latter is attributable in large part to its binding of the heterotrimeric receptor that contains the β and γ chains of the IL-2R in combination with an unique IL-15Rα chain. However, unlike IL-2, IL-15 and its receptor have a wide tissue and cell type distribution, including endothelial cells. Here, we examine the effect of IL-15 on hyaluronan expression by endothelial cells, and investigate its role in vivo in promoting the extravasation of antigen-activated T cells through a CD44-dependent pathway. The expression of hyaluronan on primary endothelial cells and microvascular endothelial cell lines is induced by IL-15, whereas IL-2 has no such activity. Moreover, intraperitoneal administration of IL-15 or TNF-α in the absence of other exogenous proinflammatory stimuli allows the extravasation of superantigen-stimulated T cells into this site in vivo in a CD44-dependent manner. T cell recruitment induced by IL-15 requires expression of an intact IL-2Rβ chain, indicating that IL-15 operates in this context through the traditional IL-15R. The results suggest that IL-15 can regulate endothelial cell function and thereby enables a CD44-initiated adhesion pathway that facilitates entry of activated T lymphocytes into inflammatory sites. They further demonstrate a novel role for IL-15 (distinct from any of IL-2) in regulating microvascular endothelial cell adhesive function, help to understand the role of IL-15R expression on endothelium, and further support a central position for this cytokine in orchestrating multiple sequential aspects of T cell effector function and therefore chronic inflammatory processes.

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Role of CPI-17 in the regulation of endothelial cytoskeleton

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00398.2003 ◽

2004 ◽

Vol 287 (5) ◽

pp. L970-L980 ◽

Cited By ~ 26

Author(s):

Irina A. Kolosova ◽

Shwu-Fan Ma ◽

Djanybek M. Adyshev ◽

Peyi Wang ◽

Motoi Ohba ◽

...

Keyword(s):

Endothelial Cells ◽

Molecular Mechanisms ◽

Rho Kinase ◽

Focal Adhesions ◽

Endothelial Permeability ◽

Dominant Negative ◽

Microvascular Endothelial Cell ◽

Microvascular Endothelial ◽

Lung Microvascular Endothelial Cell

We have previously shown that myosin light chain (MLC) phosphatase (MLCP) is critically involved in the regulation of agonist-mediated endothelial permeability and cytoskeletal organization (Verin AD, Patterson CE, Day MA, and Garcia JG. Am J Physiol Lung Cell Mol Physiol 269: L99–L108, 1995). The molecular mechanisms of endothelial MLCP regulation, however, are not completely understood. In this study we found that, similar to smooth muscle, lung microvascular endothelial cells expressed specific endogenous inhibitor of MLCP, CPI-17. To elucidate the role of CPI-17 in the regulation of endothelial cytoskeleton, full-length CPI-17 plasmid was transiently transfected into pulmonary artery endothelial cells, where the background of endogenous protein is low. CPI-17 had no effect on cytoskeleton under nonstimulating conditions. However, stimulation of transfected cells with direct PKC activator PMA caused a dramatic increase in F-actin stress fibers, focal adhesions, and MLC phosphorylation compared with untransfected cells. Inflammatory agonist histamine and, to a much lesser extent, thrombin were capable of activating CPI-17. Histamine caused stronger CPI-17 phosphorylation than thrombin. Inhibitory analysis revealed that PKC more significantly contributes to agonist-induced CPI-17 phosphorylation than Rho-kinase. Dominant-negative PKC-α abolished the effect of CPI-17 on actin cytoskeleton, suggesting that the PKC-α isoform is most likely responsible for CPI-17 activation in the endothelium. Depletion of endogenous CPI-17 in lung microvascular endothelial cell significantly attenuated histamine-induced increase in endothelial permeability. Together these data suggest the potential importance of PKC/CPI-17-mediated pathway in histamine-triggered cytoskeletal rearrangements leading to lung microvascular barrier compromise.

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C-Peptide Reduces Mitochondrial Superoxide Generation by Restoring Complex I Activity in High Glucose-Exposed Renal Microvascular Endothelial Cells

ISRN Endocrinology ◽

10.5402/2012/162802 ◽

2012 ◽

Vol 2012 ◽

pp. 1-10 ◽

Cited By ~ 7

Author(s):

Himani Vejandla ◽

John M. Hollander ◽

Anand Kothur ◽

Robert W. Brock

Keyword(s):

Endothelial Cells ◽

High Glucose ◽

Complex I ◽

Microvascular Endothelial Cells ◽

Microvascular Endothelial Cell ◽

Superoxide Generation ◽

C Peptide ◽

Mitochondrial Superoxide ◽

Microvascular Endothelial ◽

Complex I Activity

Hyperglycemia-mediated microvascular damage has been proposed to originate from excessive generation of mitochondrial superoxide in endothelial cells and is the suggested mechanism by which the pathogenesis of diabetes-induced renal damage occurs. C-peptide has been shown to ameliorate diabetes-induced renal impairment. Yet, the mechanisms underlying this protective benefit remain unclear. The objective of this study was to determine whether C-peptide affords protection to renal microvascular endothelial cell mitochondria during hyperglycemia. Conditionally immortalized murine renal microvascular endothelial cells (MECs) were exposed to low (5.5 mM) or high glucose (25 mM) media with either C-peptide (6.6 nM) or its scrambled sequence control peptide for 24 or 48 hours. Respiratory control ratio, a measure of mitochondrial electrochemical coupling, was significantly higher in high glucose renal MECs treated with C-peptide than those of high glucose alone. C-peptide also restored high glucose-induced renal MEC mitochondrial membrane potential changes back to their basal low glucose state. Moreover, C-peptide prevented the excessive mitochondrial superoxide generation and concomitant reductions in mitochondrial complex I activity which are mediated by the exposure of the renal MECs to high glucose. Together, these data demonstrate that C-peptide protects against high glucose-induced generation of mitochondrial superoxide in renal MECs via restoration of basal mitochondrial function.

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