Laminar shear stress differentially modulates gene expression of p120 catenin, Kaiso transcription factor, and vascular endothelial cadherin in human coronary artery endothelial cells.
We demonstrated previously that laminar shear stress (LSS) enhances human coronary artery endothelial cell (HCAEC) wound closure via a vascular endothelial cadherin (VE-cadherin)-dependent mechanism. VE-cadherin can interact with p120 catenin (p120(ctn)) to mediate cell locomotion and proliferation. In this study, we hypothesized that p120(ctn) and an interacting protein, Kaiso, a transcriptional factor with which p120(ctn) may interact, would be expressed differentially at the wound border and away from the wound border in HCAEC exposed to LSS. One of the major goals in this study was to assess the differential gene expression of p120(ctn), Kaiso, and VE-cadherin in HCAEC at specific locations along the wound border to further our understanding of the molecular mechanisms involved in wound closure. We combined the technique of laser capture microdissection with quantitative real time PCR to compare p120(ctn), Kaiso, and VE-cadherin mRNA expression in HCAEC at and away from the wound border under LSS. Total RNA was isolated from 200-1,000 laser-captured HCAEC and reverse transcribed into cDNA. Detection of p120(ctn), Kaiso, and VE-cadherin mRNA was carried out using quantitative real time PCR. Normalization of cDNA templates was achieved by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) quantification. Quantitative real time PCR analysis revealed p120(ctn):GAPDH ratios, Kaiso: GAPDH ratios, and VE-cadherin:GAPDH ratios, relative to static control for each set, of 0.99-4.18 (mean +/- S.E., 1.94 +/- 0.404), 1.0-5.24 (2.11 +/- 0.51), and 0.99-1.42 (1.09 +/- 0.09) after 3 h of LSS, respectively. With these techniques, we found that p120(ctn) and Kaiso transcripts were increased in laser-captured HCAEC at the wound border compared with HCAEC away from the wound border. In addition, differential expression of p120(ctn) and Kaiso mRNA was observed in HCAEC depending on how LSS was applied in relation to the wounding process. These techniques may have wide applicability for studying wound healing because gene expression of key adhesion molecules in HCAEC may now be determined from select regions of the endothelial wound border.
Kondapalli
Jyothisri
J
Critical Care Laboratory of Vascular Research, Division of Critical Care Medicine, Children's Memorial Hospital, Chicago, Illinois, USA.
Flozak
Annette S
AS
Albuquerque
Maria Luiza C
ML
D015703:Antigens, CD; D001483:Base Sequence; D015820:Cadherins / Q000235:genetics*; D051177:Catenins; D015815:Cell Adhesion Molecules / Q000235:genetics*; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D017931:DNA Primers; D018076:DNA, Complementary; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000502:physiology*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D006801:Humans; D010750:Phosphoproteins / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013314:Stress, Mechanical; D014157:Transcription Factors / Q000235:genetics*; D000095002:Delta Catenin; D000099152:Cadherin 5
D015703:Antigens, CD; D015820:Cadherins; D051177:Catenins; D015815:Cell Adhesion Molecules; D017931:DNA Primers; D018076:DNA, Complementary; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010750:Phosphoproteins; D012333:RNA, Messenger; D014157:Transcription Factors; D000095002:Delta Catenin; D000099152:Cadherin 5; C492254:ZBTB33 protein, human; C000722347:CTNND1 protein, human