scholarly journals Enhancement of endothelial cell adhesion through treatment with CEACAM6 or TNF-? [Medical College of Wisconsin]

2019 ◽  
Author(s):  
Harsimran Kalsi ◽  
Brandon Tefft
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Cell Adhesion ◽  
Endothelial Cell ◽  
Statistical Significance ◽  
Lower Percentage ◽  
Cell Retention ◽  
Average Cell ◽  
Coronary Interventions ◽  
Endothelial Cell Adhesion

610,000 Americans die of heart disease every year and over 1 million Americans undergo coronary interventions each year. Risks of these coronary interventions include thrombosis, embolization, infection, and re-narrowing of the artery. However, it is currently believed that improving the adhesion capabilities of endothelial cells may significantly reduce the associated risks of coronary interventions, especially with regards to cardiovascular stent and graft placement procedures. In the present study, we investigated a method to biochemically facilitate endothelial cell adhesion. Using the Reactome database, we identified biochemical methods/targets which could be modified to promote endothelial cell adhesion. Here we describe a potential method of enhancing endothelial cell adhesion through controlled application of two treatment conditions: Carcinoembryonic Antigen-related Cell Adhesion Molecule 6 (CEACAM6) with Fibronectin-1 (CEACAM6+, FN1+) and Tumor Necrosis Factor-? (TNF-?) with Fibronectin-1 (TNF-?+, FN1+). DAPI stained porcine blood outgrowth endothelial cells (BOECs) were exposed to these treatments or a control treatment (FN1+) for one hour. Each treatment/control condition was then placed into a validated parallel plate flow chamber (PPFC) apparatus (flow rate accuracy and laminar flow were both validated) and exposed to a shear stress of 15 dyn/cm2 (to replicate in vivo shear stress conditions) for 30 minutes using saline solution. Fluorescent images were taken of all replicates before and after shear stress testing in order to determine the effects of treatments on cell adhesion. Statistical analysis of preliminary trials indicated that the treatment groups demonstrated a trend towards higher percentage cell retention (or lower percentage cell loss) with 82.3%, 70.2%, and 50.2% average cell retention for TNF-?, CEACAM6, and the control, respectively; however, statistical significance could not be reached using n=2 and p<0.05. Preliminary results of the TNF-? treatment are promising. In the future, an experiment with a larger sample size and a longer shear stress exposure time (e.g. 1 hour) is needed.

scholarly journals Thrombospondin-1, a natural inhibitor of angiogenesis, regulates platelet-endothelial cell adhesion molecule-1 expression and endothelial cell morphogenesis.

1997 ◽  
Vol 8 (7) ◽  
pp. 1329-1341 ◽  
Author(s):  
N Sheibani ◽  
P J Newman ◽  
W A Frazier
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Endothelial Cell ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Cell Contact ◽  
Platelet Endothelial Cell Adhesion ◽  
Cell Adhesion Molecule 1 ◽  
Endothelial Cell Adhesion ◽  
Cell Cell

Expression of thrombospondin-1 (TS1) in polyoma middle-sized T (tumor)-transformed mouse brain endothelial cells (bEND.3) restores a normal phenotype and suppresses their ability to form hemangiomas in mice. We show that TS1 expression results in complete suppression of platelet-endothelial cell adhesion molecule-1 (PECAM-1) expression and altered cell-cell interactions in bEND.3 cells. To further investigate the role of PECAM-1 in regulation of endothelial cell-cell interactions and morphogenesis, we expressed human (full length) or murine (delta 15) PECAM-1 isoforms in TS1-transfected bEND.3 (bEND/TS) cells. Expression of either human or murine PECAM-1 resulted in an enhanced ability to organize and form networks of cords on Matrigel, an effect that was specifically blocked by antibodies to PECAM-1. Anti-PECAM-1 antibodies also inhibited tube formation in Matrigel by normal human umbilical vein endothelial cells. However, PECAM-1-transfected bEND/TS cells did not regain the ability to form hemangiomas in mice and the expressed PECAM-1, unlike the endogenous PECAM-1 expressed in bEND.3 cells, failed to localize to sites of cell-cell contact. This may be, in part, attributed to the different isoforms of PECAM-1 expressed in bEND.3 cells. Using reverse transcription-polymerase chain reaction, we determined that bEND.3 cells express mRNA encoding six different PECAM-1 isoforms, the isoform lacking both exons 14 and 15 (delta 14&15) being most abundant. Expression of the murine delta 14&15 PECAM-1 isoform in bEND/TS cells resulted in a similar phenotype to that described for the full-length human or murine delta 15 PECAM-1 isoform. The delta 14&15 isoform, despite the lack of exon 14, failed to localize to sites of cell-cell contact even in clones that expressed it at very high levels. Thus, contrary to recent reports, lack of exon 14 is not sufficient to result in junctional localization of PECAM-1 isoforms in bEND/TS cells.

scholarly journals Role of glycocalyx in leukocyte-endothelial cell adhesion

2002 ◽  
Vol 283 (4) ◽  
pp. H1282-H1291 ◽  
Author(s):  
A. W. Mulivor ◽  
H. H. Lipowsky
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Endothelial Cell ◽  
Inflammatory Response ◽  
Topical Application ◽  
Binding Sites ◽  
Subsequent Application ◽  
Endothelial Cell Adhesion

The binding of fluorescently labeled microspheres (FLMs, 0.1-μm diameter) coated with antibody (1a29) to ICAM-1 was studied in postcapillary venules during topical application of the chemoattractant N-formylmethionyl-leucyl-phenylalanine (fMLP). FLM adhesion to endothelial cells (ECs) increased dramatically from 50 to 150 spheres per 100-μm length of venule after superfusion of the mesentery with fMLP and equaled or exceeded levels of leukocyte (WBC) adhesion. Removal of the EC glycocalyx by micropipette infusion of the venule with heparinase increased FLM-EC adhesion to levels attained with fMLP. Subsequent application of fMLP did not increase FLM adhesion further, suggesting that the FLMs saturated all ICAM-1 binding sites. Perfusion with heparinase after suffusion with fMLP significantly increased FLM-EC adhesion above levels attained with fMLP. However, WBC adhesion fell because of possible removal of selectins necessary to maintain WBC rolling at the wall. It is concluded that the glycocalyx serves as a barrier to adhesion and that its shedding during natural activation of ECs may be an essential part of the inflammatory response.

scholarly journals Down-Regulation of Platelet Endothelial Cell Adhesion Molecule-1 Results in Thrombospondin-1 Expression and Concerted Regulation of Endothelial Cell Phenotype

1998 ◽  
Vol 9 (4) ◽  
pp. 701-713 ◽  
Author(s):  
Nader Sheibani ◽  
William A. Frazier
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Endothelial Cell ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Down Regulation ◽  
Platelet Endothelial Cell Adhesion ◽  
Thrombospondin 1 ◽  
Cell Adhesion Molecule 1 ◽  
Endothelial Cell Adhesion

bEND.3 cells are polyoma middle T-transformed mouse brain endothelial cells that express very little or no thrombospondin-1, a natural inhibitor of angiogenesis, but express high levels of platelet endothelial cell adhesion molecule-1 (PECAM-1) that localizes to sites of cell–cell contact. Here, we have examined the role of PECAM-1 in regulation of bEND.3 cell proliferation, migration, morphogenesis, and hemangioma formation. We show that down-regulating PECAM-1 expression by antisense transfection of bEND.3 cells has a dramatic effect on their morphology, proliferation, and morphogenesis on Matrigel. There is an optimal level for PECAM-1 expression such that high levels of PECAM-1 inhibit, whereas moderate levels of PECAM-1 stimulate, endothelial cell morphogenesis. The down-regulation of PECAM-1 in bEND.3 cells resulted in reexpression of endogenous thrombospondin-1 and its antiangiogenic receptor CD36. The expression of the vascular endothelial growth factor receptors flk-1 and flt-1, as well as integrins and metalloproteinases (which are involved in angiogenesis), were also affected. These observations are consistent with the changes observed in proliferation, migration, and adhesion characteristics of the antisense-transfected bEND.3 cells as well as with their lack of ability to form hemangiomas in mice. Thus, a reciprocal relationship exists between thrombospondin-1 and PECAM-1 expression, such that these two molecules appear to be constituents of a “switch” that regulates in concert many components of the angiogenic and differentiated phenotypes of endothelial cells.

scholarly journals The LINC complex is required for endothelial cell adhesion and adaptation to shear stress and cyclic stretch

2021 ◽  
pp. mbc.E20-11-0698
Author(s):  
Kevin B. Denis ◽  
Jolene I. Cabe ◽  
Brooke E. Danielsson ◽  
Katie V. Tieu ◽  
Carl R. Mayer ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Cell Adhesion ◽  
Endothelial Cell ◽  
Cell Function ◽  
Dominant Negative ◽  
Cyclic Stretch ◽  
Linc Complex ◽  
Endothelial Cell Function ◽  
Altered Cell

The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex is a structure consisting of nesprin, SUN, and lamin proteins. A principal function of the LINC complex is anchoring the nucleus to the actin, microtubule, and intermediate filament cytoskeletons. The LINC complex is present in nearly all cell types, including endothelial cells. Endothelial cells line the inner most surfaces of blood vessels, and are critical for blood vessel barrier function. In addition, endothelial cells have specialized functions, including adaptation to the mechanical forces of blood flow. Previous studies have shown that depletion of individual nesprin isoforms results in impaired endothelial cell function. To further investigate the role of the LINC complex in endothelial cells we utilized dominant negative KASH (DN-KASH), a dominant negative protein which displaces endogenous nesprins from the nuclear envelope and disrupts nuclear-cytoskeletal connections. Endothelial cells expressing DN-KASH had altered cell-cell adhesion and barrier function, as well as altered cell-matrix adhesion and focal adhesion dynamics. In addition, cells expressing DN-KASH failed to properly adapt to shear stress or cyclic stretch. DN-KASH expressing cells exhibited impaired collective cell migration in wound healing and angiogenesis assays. Our results demonstrate the importance of an intact LINC complex in endothelial cell function and homeostasis. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Enhancing Endothelial Cell Retention on ePTFE Constructs by siRNA-Mediated SHP-1 Gene Silencing

2011 ◽  
Vol 2 (1) ◽  
Author(s):  
Brandon J. Tefft ◽  
Adrian M. Kopacz ◽  
Wing Kam Liu ◽  
Shu Q. Liu
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Endothelial Cell ◽  
Gene Silencing ◽  
Vascular Grafts ◽  
Sh2 Domain ◽  
Great Promise ◽  
Shear Stresses ◽  
Cell Retention ◽  
Stress Dependent

Polymeric vascular grafts hold great promise for vascular reconstruction, but the lack of endothelial cells renders these grafts susceptible to intimal hyperplasia and restenosis, precluding widespread clinical applications. The purpose of this study is to establish a stable endothelium on expanded polytetrafluoroethylene (ePTFE) membrane by small interfering RNA (siRNA)-induced suppression of the cell adhesion inhibitor SH2 domain-containing protein tyrosine phosphatase-1 (SHP-1). Human umbilical vein endothelial cells (HUVECs) were treated with scrambled siRNA as a control or SHP-1 specific siRNA. Treated cells were seeded onto fibronectin-coated ePTFE scaffolds and exposed to a physiological range of pulsatile fluid shear stresses for 1 h in a variable-width parallel plate flow chamber. Retention of cells was measured and compared between various shear stress levels and between groups treated with scrambled siRNA and SHP-1 specific siRNA. HUVECs seeded on ePTFE membrane exhibited shear stress-dependent retention. Exposure to physiological shear stress (10 dyn/cm2) induced a reduction in the retention of scrambled siRNA treated cells from 100% to 85% at 1 h. Increased shear stress (20 dyn/cm2) further reduced retention of scrambled siRNA treated cells to 55% at 1 h. SHP-1 knockdown mediated by siRNA enhanced endothelial cell retention from approximately 60% to 85% after 1 h of exposure to average shear stresses in the range of 15–30 dyn/cm2. This study demonstrates that siRNA-mediated gene silencing may be an effective strategy for improving the retention of endothelial cells within vascular grafts.

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