Abstract 1433: The Cardioregulatory Peptide Apelin is Preferentially Expressed by Endothelial Cells and Upregulated in Myocardial Disease States

Introduction: The APJ receptor and its ligand, apelin, comprise a homeostatic, cardio-regulatory pathway. Although cardiac apelin expression levels are altered in humans with cardiac failure, the cell type responsible for apelin expression and modulation in disease states remains unknown. Hypothesis: Apelin production is restricted to the endothelial compartment and is upregulated in states of cardiovascular stress. Methods: Transgenic apelin-LacZ reporter mice (SVJ background) were created by insertion of the nuclear localizing bacterial LacZ gene immediately downstream of the apelin promoter. Mice (n=12) were randomized to left anterior descending coronary artery (LAD) ligation, thoracic aortic constriction (TAC) or sham groups. Hearts were harvested 3 and 8 weeks post-TAC or LAD ligation, respectively. Localization of apelin expression was determined by Xgal staining. Endothelial phenotype of lacZ positive cells was confirmed by CD31 co-staining. Apelin expressing cells were quantified by histology. Apelin reporter results were confirmed by quantitating apelin expression in WT animals following LAD ligation (n=11) or sham (n=11) procedure by RT-PCR. Results: Extensive immunohistochemistry studies of heart tissue revealed lacZ reporter gene expression to be restricted to the coronary venous and capillary endothelium, with no expression by cardiomyocytes. Following both LAD ligation and TAC, the number of LacZ-apelin (+) endothelial cells significantly increased (p<0.002) in all chambers of the heart (Table ), with no evidence of apelin expression by other cell types. Evaluation of WT hearts by RT-PCR for the apelin gene confirmed the reporter gene findings with 1.3±0.3 fold increase (p<0.05) of apelin expression induced by LAD ligation compared to sham. Conclusions: Apelin is primary expressed by endothelial cells within the heart and is upregulated in response to myocardial stress. Apelin-LacZ Expressing Endothelial Cells are Increased Following Myocardial Injury

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Human endothelial cells produce orosomucoid, an important component of the capillary barrier

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1999.276.2.h530 ◽

1999 ◽

Vol 276 (2) ◽

pp. H530-H534 ◽

Cited By ~ 16

Author(s):

Jenny Sörensson ◽

Göran L. Matejka ◽

Maria Ohlson ◽

Börje Haraldsson

Keyword(s):

Endothelial Cells ◽

Primary Cultures ◽

Rnase Protection Assay ◽

Cell Types ◽

Endothelial Glycocalyx ◽

Rt Pcr ◽

Rnase Protection ◽

Total Rna ◽

Main Site ◽

Charge Selectivity

The serum protein orosomucoid (α1-acid glycoprotein) is needed to maintain the high capillary permselectivity required for normal homeostasis. It is not known how the protein executes its action, but it seems to contribute to the charge barrier. Moreover, recent studies suggest that the endothelial glycocalyx is essential for the charge barrier. The main site of orosomucoid synthesis is the liver, but we wanted to explore the possibility that orosomucoid was synthesized in endothelial cells. Primary cultures of human microvascular endothelial cells (HMVEC) from dermal tissue were established. Human liver cells were used as positive controls, and total RNA was prepared from both cell types. Reverse transcription-polymerase chain reaction (RT-PCR) was performed and demonstrated orosomucoid expression. After RT-PCR, the identities of the PCR products were confirmed by sequencing. RNase protection assay performed on total RNA from the HMVEC confirmed the results from the RT-PCR, i.e., orosomucoid mRNA is expressed by endothelial cells. Synthesis of orosomucoid in both liver and endothelial cells was demonstrated by immunoprecipitation. In conclusion, endothelial cells normally produce orosomucoid, which is essential for capillary charge selectivity. We suggest that orosomucoid exerts its effect by interacting with other components of the endothelial glycocalyx.

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Identification of human thrombin-activatable fibrinolysis inhibitor in vascular and inflammatory cells

Thrombosis and Haemostasis ◽

10.1160/th10-06-0413 ◽

2011 ◽

Vol 105 (06) ◽

pp. 999-1009 ◽

Cited By ~ 9

Author(s):

Joellen Lin ◽

Mathieu Garand ◽

Branislava Zagorac ◽

Steven Schadinger ◽

Corey Scipione ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Line ◽

Cell Lines ◽

Umbilical Vein ◽

Inflammatory Cells ◽

Cell Types ◽

Rt Pcr ◽

Thrombin Activatable Fibrinolysis Inhibitor ◽

Gene Encoding ◽

Fibrinolysis Inhibitor

SummaryTAFI (thrombin-activatable fibrinolysis inhibitor) is a carboxypeptidase zymogen originally identified in plasma. The TAFI pathway helps to regulate the balance between the coagulation and fibrinolytic cascades. Activated TAFI (TAFIa) can also inactivate certain pro-inflammatory mediators, suggesting that the TAFI pathway may also regulate communication between coagulation and inflammation. Expression in the liver is considered to be the source of plasma TAFI. TAFI has also been identified in platelets and CPB2 (the gene encoding TAFI) mRNA has been detected in megakaryocytic cell lines as well as in endothelial cells. We have undertaken a quantitative analysis of CPB2 mRNA and TAFI protein in extrahepatic cell types relevant to vascular disease. Using RT-PCR and quantitative RT-PCR, we detected CPB2 mRNA in the human megakaryoblastic cell lines MEG-01 and Dami, the human monocytoid cell line THP-1 as well as THP-1 cells differentiated into a macrophage-like phenotype, and in primary human umbilical vein and coronary artery endothelial cells. CPB2 mRNA abundance in MEG-01, Dami, and THP-1 cells was modulated by the state of differentiation of these cells. Using a recently developed TAFIa assay, we detected TAFI protein in the lysates of the human hepatocellular carcinoma cell line HepG2 as well as in MEG-01 and Dami cells and in the conditioned medium of HepG2 cells, differentiated Dami cells, and THP-1 macrophages. We have obtained clear evidence for extrahepatic expression of TAFI, which has clear implications for the physiological and pathophysiological functions of the TAFI pathway.

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Differential nitric oxide production by microvascular and macrovascular endothelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1997.273.1.l275 ◽

1997 ◽

Vol 273 (1) ◽

pp. L275-L281 ◽

Cited By ~ 17

Author(s):

M. Geiger ◽

A. Stone ◽

S. N. Mason ◽

K. T. Oldham ◽

K. S. Guice

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Endothelial Cell ◽

Fold Increase ◽

Cell Types ◽

Microvascular Endothelial Cells ◽

Tnf Alpha ◽

Cell Populations ◽

Ifn Gamma ◽

Nitrite Production

Phenotypic heterogeneity among endothelial cell populations may account for important organ-specific behaviors. Experimental evidence suggests that endothelium-derived nitric oxide mediates certain of these unique responses. The purpose of these investigations was to compare rat pulmonary microvascular endothelial cells with pulmonary artery and aortic macrovascular endothelial cells in their ability to generate nitric oxide (NO). Cultures of these microvascular and macrovascular endothelial cells were incubated with interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and Salmonella typhimurium lipopolysaccharide (LPS) alone or in combination, and nitrite production was measured. Single-agent exposure with IFN-gamma (up to 1,000 U/ml), TNF-alpha (up to 60,000 U/ml), or LPS (up to 500 ng/ml) had little effect on nitrite generation. Nitrite production by rat aortic macrovascular endothelial cells (RAEC) was significantly greater than that by the rat lung microvascular endothelial cells (RLMVEC) when stimulated with TNF-alpha + IFN-gamma, LPS + IFN-gamma, or TNF-alpha + LPS. The maximal response by all endothelial cell types (approximately 15-fold increase in RAEC and 8-fold increase in RLMVEC) was observed with LPS + IFN-gamma. The nitrite generation from rat pulmonary artery endothelial cells was intermediate between RAEC and RLMVEC responses when stimulated with IFN-gamma + LPS or TNF-alpha. Similar patterns of heterogeneous inducible nitric oxide synthase mRNA induction occurred when Northern analysis of specimens from the cultured endothelial cell types was done. These data suggest that phenotypic heterogeneity between these endothelial cell populations is substantial and, by inference, that site-specific NO. generation may occur.

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Antibodies to mouse lung capillary endothelium.

Journal of Histochemistry & Cytochemistry ◽

10.1177/36.7.3290332 ◽

1988 ◽

Vol 36 (7) ◽

pp. 741-749 ◽

Cited By ~ 20

Author(s):

M C Rorvik ◽

D P Allison ◽

J A Hotchkiss ◽

H P Witschi ◽

S J Kennel

Keyword(s):

Endothelial Cells ◽

Cell Types ◽

Interstitial Cells ◽

Mouse Lung ◽

Capillary Endothelium ◽

Specific Cell ◽

Alveolar Wall ◽

Gold Particles ◽

Capillary Endothelial Cells ◽

Quantitative Analyses

We are interested in developing monoclonal antibodies (MoAbs) that recognize specific cell types in the lung of BALB/c mice. Normal mouse lung homogenate was used to immunize F344 rats and hybridomas were produced by fusion of rat spleen cells with mouse myeloma SP 2/0. Two hybridomas were selected which produced MoAbs active in immunohistochemistry of lung cells. MoAb 273-34A and 411-201B both show extensive peroxidase staining of capillary endothelial cells within alveolar walls of lungs at the light microscopic level. To demonstrate cell specificity, immunoelectron microscopy with gold-labeled antibody was performed. Lightly fixed lungs were frozen and thin-sectioned before staining with MoAb and 5-nm gold particles coupled to secondary antibody. Quantitative analyses of these cryosections show that both antibodies, used at optimal concentrations, are specific for binding to capillary endothelial cells. More than 95% of the gold particles are associated with capillary endothelial cells on the thin side of the alveolar wall. When capillaries adjoined thick septa containing interstitial cells, about two thirds of the gold particles were associated with endothelial cells and about one quarter with interstitial cells. These MoAbs should be useful in studying the role of endothelial cells in toxic lung injury.

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Coordinated Adenine Nucleotide Phosphohydrolysis and Nucleoside Signaling in Posthypoxic Endothelium

Journal of Experimental Medicine ◽

10.1084/jem.20030891 ◽

2003 ◽

Vol 198 (5) ◽

pp. 783-796 ◽

Cited By ~ 343

Author(s):

Holger K. Eltzschig ◽

Juan C. Ibla ◽

Glenn T. Furuta ◽

Martin O. Leonard ◽

Kenneth A. Jacobson ◽

...

Keyword(s):

Endothelial Cells ◽

Vascular Permeability ◽

Adenine Nucleotide ◽

Endothelial Permeability ◽

Fold Increase ◽

Endothelial Cell Surface ◽

Disease States ◽

Critical Control Points ◽

Soluble Mediator

Limited oxygen delivery to tissues (hypoxia) is common in a variety of disease states. A number of parallels exist between hypoxia and acute inflammation, including the observation that both influence vascular permeability. As such, we compared the functional influence of activated polymorphonuclear leukocytes (PMN) on normoxic and posthypoxic endothelial cells. Initial studies indicated that activated PMN preferentially promote endothelial barrier function in posthypoxic endothelial cells (>60% increase over normoxia). Extension of these findings identified at least one soluble mediator as extracellular adenosine triphosphate (ATP). Subsequent studies revealed that ATP is coordinately hydrolyzed to adenosine at the endothelial cell surface by hypoxia-induced CD39 and CD73 (>20-and >12-fold increase in mRNA, respectively). Studies in vitro and in cd39-null mice identified these surface ecto-enzymes as critical control points for posthypoxia-associated protection of vascular permeability. Furthermore, insight gained through microarray analysis revealed that the adenosine A2B receptor (AdoRA2B) is selectively up-regulated by hypoxia (>5-fold increase in mRNA), and that AdoRA2B antagonists effectively neutralize ATP-mediated changes in posthypoxic endothelial permeability. Taken together, these results demonstrate transcription coordination of adenine nucleotide and nucleoside signaling at the vascular interface during hypoxia.

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MiR-126-3p Is Dynamically Regulated in Endothelial-to-Mesenchymal Transition during Fibrosis

International Journal of Molecular Sciences ◽

10.3390/ijms22168629 ◽

2021 ◽

Vol 22 (16) ◽

pp. 8629

Author(s):

Nina P. Jordan ◽

Samuel J. Tingle ◽

Victoria G. Shuttleworth ◽

Katie Cooke ◽

Rachael E. Redgrave ◽

...

Keyword(s):

Endothelial Cells ◽

Umbilical Vein ◽

Cell Types ◽

Heart Tissue ◽

Lineage Tracing ◽

Ischaemic Injury ◽

Kidney Fibrosis ◽

Mesenchymal Transition ◽

Over Expression ◽

Endothelial To Mesenchymal Transition

In fibrotic diseases, myofibroblasts derive from a range of cell types including endothelial-to-mesenchymal transition (EndMT). Increasing evidence suggests that miRNAs are key regulators in biological processes but their profile is relatively understudied in EndMT. In human umbilical vein endothelial cells (HUVEC), EndMT was induced by treatment with TGFβ2 and IL1β. A significant decrease in endothelial markers such as VE-cadherin, CD31 and an increase in mesenchymal markers such as fibronectin were observed. In parallel, miRNA profiling showed that miR-126-3p was down-regulated in HUVECs undergoing EndMT and over-expression of miR-126-3p prevented EndMT, maintaining CD31 and repressing fibronectin expression. EndMT was investigated using lineage tracing with transgenic Cdh5-Cre-ERT2; Rosa26R-stop-YFP mice in two established models of fibrosis: cardiac ischaemic injury and kidney ureteric occlusion. In both cardiac and kidney fibrosis, lineage tracing showed a significant subpopulation of endothelial-derived cells expressed mesenchymal markers, indicating they had undergone EndMT. In addition, miR-126-3p was restricted to endothelial cells and down-regulated in murine fibrotic kidney and heart tissue. These findings were confirmed in patient kidney biopsies. MiR-126-3p expression is restricted to endothelial cells and is down-regulated during EndMT. Over-expression of miR-126-3p reduces EndMT, therefore, it could be considered for miRNA-based therapeutics in fibrotic organs.

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TNF-α alters the release and transfer of microparticle-encapsulated miRNAs from endothelial cells

Physiological Genomics ◽

10.1152/physiolgenomics.00079.2014 ◽

2014 ◽

Vol 46 (22) ◽

pp. 833-840 ◽

Cited By ~ 42

Author(s):

Tamas Alexy ◽

Kimberly Rooney ◽

Martina Weber ◽

Warren D. Gray ◽

Charles D. Searles

Keyword(s):

Endothelial Cells ◽

Cell Communication ◽

Fold Increase ◽

Cell Types ◽

Rock Inhibitor ◽

Aortic Endothelial Cells ◽

Extracellular Release ◽

Tnf Α ◽

Different Cell Types ◽

Human Aortic Endothelial Cells

MicroRNAs (miRNAs) encapsulated within microparticles (MPs) are likely to have a role in cell-to-cell signaling in a variety of diseases, including atherosclerosis. However, little is known about the mechanisms by which different cell types release and transfer miRNAs. Here, we examined TNF-α-induced release of MP-encapsulated miR-126, miR-21, and miR-155 from human aortic endothelial cells (ECs) and their transfer to recipient cells. ECs were treated with TNF-α (100 ng/ml) in the presence or absence of inhibitors that target different MP production pathways. MPs released in response to TNF-α were characterized by: 1) 70–80% decrease in miRNA/MP levels for miR-126 and -21 but a significant increase in pre-miR-155 and miR-155 ( P < 0.05), 2) 50% reduction in uptake by recipient cells ( P < 0.05), and 3) diminished ability to transfer miRNA to recipient cells. Cotreatment of donor ECs with TNF-α and caspase inhibitor (Q-VD-OPH, 10 μM) produced MPs that had: 1) 1.5- to 2-fold increase in miRNA/MP loading, 2) enhanced uptake by recipient cells (2-fold), and 3) increased ability to transfer miR-155. Cotreatment of ECs with TNF-α and Rho-associated kinase (ROCK) inhibitor (10 μM) produced MPs with features similar to those produced by TNF-α treatment alone. Our data indicate that TNF-α induced the production of distinct MP populations: ROCK-dependent, miRNA-rich MPs that effectively transferred their cargo and were antiapoptotic, and caspase-dependent, miRNA-poor MPs that were proapoptotic. These data provide insight into the relationship between MP production and extracellular release of miRNA, as well as the potential of encapsulated miRNA for cell-to-cell communication.

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Putative Cyclooxygenase-3 Expression in Rat Brain Cells

Journal of Cerebral Blood Flow & Metabolism ◽

10.1097/01.wcb.0000090681.07515.81 ◽

2003 ◽

Vol 23 (11) ◽

pp. 1287-1292 ◽

Cited By ~ 57

Author(s):

Bela Kis ◽

James A Snipes ◽

Toyohi Isse ◽

Krisztina Nagy ◽

David W Busija

Keyword(s):

Endothelial Cells ◽

Rat Brain ◽

Primary Cultures ◽

Pcr Primers ◽

Cell Types ◽

Rt Pcr ◽

Lps Challenge ◽

Intron 1 ◽

Pcr Products ◽

Cox 1

Cyclooxygenase-3 (COX-3), a new acetaminophen-sensitive isoform of the COX family, has recently been cloned from canine tissues. Canine COX-3 apparently is identical to the full-length form of COX-1, with the exception that the COX-3 mRNA retains intron 1. Additionally, COX-3 mRNA expression is high in the brain. We investigated the expression of the putative rat COX-3 mRNA in primary cultures of neurons, astrocytes, endothelial cells, pericytes, and choroidal epithelial cells from the rat brain. Specific RT-PCR primers were designed to detect putative rat COX-3 mRNA, and the RT-PCR products were sequenced and compared to the known sequence of the rat COX-1 gene. Our results demonstrate that the mRNA of the putative COX-3 is expressed in all of the cell types except neurons. Cerebral endothelial cells showed the highest COX-3 expression. Whereas COX-2 expression increased several-fold after lipopolysaccharide (LPS) challenge, COX-1 and COX-3 expression did not change significantly, suggesting that cells constitutively express COX-3. In summary, we report, for the first time to our knowledge, that the putative COX-3 mRNA is detectable in rats and is differentially expressed in various cell types from rat brain, as well as that its expression is not stimulated by LPS.

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Endothelial cell–oligodendrocyte interactions in small vessel disease and aging

Clinical Science ◽

10.1042/cs20160618 ◽

2017 ◽

Vol 131 (5) ◽

pp. 369-379 ◽

Cited By ~ 23

Author(s):

Rikesh M. Rajani ◽

Anna Williams

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Neurological Disease ◽

Small Vessel Disease ◽

Cell Types ◽

Cerebral Small Vessel Disease ◽

Small Vessel ◽

Pathological Changes ◽

Vessel Disease ◽

Disease States

Cerebral small vessel disease (SVD) is a prevalent, neurological disease that significantly increases the risk of stroke and dementia. The main pathological changes are vascular, in the form of lipohyalinosis and arteriosclerosis, and in the white matter (WM), in the form of WM lesions. Despite this, it is unclear to what extent the key cell types involved–the endothelial cells (ECs) of the vasculature and the oligodendrocytes of the WM–interact. Here, we describe the work that has so far been carried out suggesting an interaction between ECs and oligodendrocytes in SVD. As these interactions have been studied in more detail in other disease states and in development, we explore these systems and discuss the role these mechanisms may play in SVD.

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