Untitled

“…The human microvascular endothelial cell line, HMEC-1 (29), was generously provided by Dr. Edwin Ades (Centers for Disease Control and Prevention), Francisco J. Candal (Centers for Disease Control and Prevention), and Dr. Thomas Lawley (Emory University) and was cultured in MCDB-131 complete media (VEC Technologies, Rensselear, NY). The HMEC-1 cell line has been extensively studied and has been shown to have morphological, phenotypical, and functional characteristics of human microvascular endothelial cells (29,30), making it an excellent in vitro model for our studies. HMEC-1 cells were transfected by electroporation using an AMAXA Nucleofector II (Lonza, Walkersville, MD …”

Section: Methodsmentioning

confidence: 99%

Recycling of the Ca2+-activated K+ Channel, KCa2.3, Is Dependent upon RME-1, Rab35/EPI64C, and an N-terminal Domain

Gao

Balut

Bailey

et al. 2010

Journal of Biological Chemistry

123

View full text Add to dashboard Cite

Regulation of the number ofFurthermore, the effect of EPI64C was dependent upon its GTPase-activating proteins activity. Co-immunoprecipitation studies confirmed an association between KCa2.3 and both Rab35 and RME-1. In contrast to KCa2.3, KCa3.1 was rapidly endocytosed and degraded in an RME-1 and Rab35-independent manner. A series of N-terminal deletions identified a 12-amino acid region, Gly 206 -Pro 217 , as being required for the rapid recycling of KCa2.3. Deletion of Gly 206 -Pro 217 had no effect on the association of KCa2.3 with Rab35 but significantly decreased the association with RME-1. These represent the first studies elucidating the mechanisms by which KCa2.3 is maintained at the plasma membrane.

show abstract

“…Human endothelial cell line EA.hy926, derived by fusion of human umbilical vein endothelial cells with continuous human lung carcinoma cell line A549 (12) was obtained as a gift from Professor Cora-Jean S. Edgell (Pathology Department, University of North Carolina at Chapel Hill). This endothelial hybrid cell line is presently the best characterized macrovascular EC line (for review, see Bouis et al (13)). …”

Section: Methodsmentioning

confidence: 99%

Binding of PAI-1 to Endothelial Cells Stimulated by Thymosin β4 and Modulation of Their Fibrinolytic Potential

Boncela

Smolarczyk

Wyroba

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

Our previous studies showed that thymosin ␤4 (T␤4) induced the synthesis of plasminogen activator inhibitor-1 (PAI-1) in cultured human umbilical vein endothelial cells (HUVECs) via the AP-1 dependent mechanism and its enhanced secretion. In this work we provide evidence that the released PAI-1 is accumulated on the surface of HUVECs, exclusively in its active form, in a complex with ␣1-acid glycoprotein (AGP) that is also up-regulated and released from the cells. This mechanism is supported by several lines of experiments, in which expression of both proteins was analyzed by flow cytometry and their colocalization supported by confocal microscopy. PAI-1 did not bind to quiescent cells but only to the T␤4-activated endothelial cells. In contrast, significant amounts of AGP were found to be associated with the cells overexpressing enhanced green fluorescent protein (EGFP)-␣1-acid glycoprotein (AGP) without T␤4 treatment. The AGP⅐PAI-1 complex was accumulated essentially at the basal surface of endothelial cells, and such cells showed (a) morphology characteristic for strongly adhered and spread cells and (b) significantly reduced plasmin formation. Taken together, these results provide the evidence supporting a novel mechanism by which active PAI-1 can be bound to the T␤4-activated endothelial cells, thus influencing their adhesive properties as well as their ability to generate plasmin.Thymosin ␤4 (T␤4) 2 promotes skin and corneal wound healing through its effects on cell migration, angiogenesis and possibly cell survival (1-3). However, the precise molecular mechanism through which it functions and its potential role in solid organ wound healing remains unknown. In our studies of the biological activity of T␤4 we found that this 43-amino acid peptide strongly activates endothelial cells to express and release plasminogen activator inhibitor type 1 (PAI-1). It occurs via the mechanism involving the signaling pathway leading to activation of the MAPK cascade and enhanced c-Fos/c-Jun DNA binding activity (4). Such a role of AP-1 in activation of transcriptional events by T␤4 was confirmed by independent studies (5). Recently, T␤4 was reported to stimulate migration of cardiomyocytes and endothelial cells and promote survival of cardiomyocytes by formation of a functional complex with PINCH and integrin-linked kinase, resulting in activation of the survival kinase Akt (6). These observations revealed that T␤4 affects cellular functions by activation of several signaling pathways and induces changes in the cell properties necessary for both migration and survival. Here, we show that T␤4 up-regulates expression and induces the surface accumulation of ␣1-acid glycoprotein (AGP) known to bind PAI-1 and prolong its inhibitory activity (7). AGP is expressed by activated endothelial cells and exerts its effect by interacting with components of the endothelial glycocalyx (8). In this study, we identify a previously unrecognized function of AGP, a direct and primary role in plasminogen activation. We provide evidence that t...

show abstract

Untitled

Cited by 324 publications

References 84 publications

Activation of Vascular Endothelial Growth Factor Receptor-3 and Its Downstream Signaling Promote Cell Survival under Oxidative Stress

Activation of Vascular Endothelial Growth Factor Receptor-3 and Its Downstream Signaling Promote Cell Survival under Oxidative Stress

Recycling of the Ca2+-activated K+ Channel, KCa2.3, Is Dependent upon RME-1, Rab35/EPI64C, and an N-terminal Domain

Binding of PAI-1 to Endothelial Cells Stimulated by Thymosin β4 and Modulation of Their Fibrinolytic Potential

Contact Info

Product

Resources

About