Temporal genomics of vein bypass grafting through oligonucleotide microarray analysis.
OBJECTIVE
Autologous vein is the conduit of choice for small artery reconstruction. Despite excellent patency, these conduits undergo remodeling over time. The purpose of this study was to identify temporal gene expression in vein grafts versus control veins through microarray analysis.


METHODS
Cephalic vein grafts (n = 12) were used to bypass femoral arteries in canines. Vein grafts were harvested after 1, 7, 14, and 30 days. Normal contralateral cephalic vein served as control. Total RNA was isolated; its quantity and quality were confirmed with spectrophotometry and gel electrophoresis. Affymetrix U133A GeneChips, comprising approximately 15,000 genes, were used to analyze differential gene expression at each time point. Statistical analysis was performed with Affymetrix and dChip software to identify consistently upregulated and downregulated genes. Real-time, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and immunohistochemistry were used to validate microarray data.


RESULTS
Statistical analysis revealed that 49 genes were consistently upregulated and 31 genes were consistently downregulated in all three animals at various time points. qRT-PCR to quantitatively assess messenger RNA expression was performed on specific genes to validate the microarray data. Immunohistochemistry to qualitatively assess protein expression was used for further validation. Hierarchical clustering with dChip identified additional genes with similar temporal or functional expression patterns.


CONCLUSIONS
This is the first study to use microarray analysis with confirmatory qRT-PCR to identify altered genes after vein bypass grafting. Oligonucleotide microarrays and hierarchical clustering are powerful tools to generate hypotheses as the basis for additional research on gene expression in vein graft remodeling. Ultimately, identification of a temporal sequence of differential gene expression may provide insights not preferred into the molecular mechanisms of vein graft remodeling, but also into the pathways leading to intimal hyperplasia.
Department of Surgery, Division of Vascular Surgery, Beth Israel Deacones Medical Center, Boston, MA 02115, USA. jkalish@bidmc.harvard.edu
Jeffrey A
JA
Kalish
David J
DJ
Willis
Cheng
C
Li
Jeffrey J
JJ
Link
Evan R
ER
Deutsch
Mauricio A
MA
Contreras
William C
WC
Quist
Frank W
FW
Logerfo
D000818:Animals; D019917:Blood Vessel Prosthesis Implantation; D004285:Dogs; D015870:Gene Expression / Q000502:physiology*; D005786:Gene Expression Regulation / Q000502:physiology*; D023281:Genomics; D023421:Models, Animal; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014680:Veins / Q000502:physiology* / Q000637:transplantation*