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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 20, 2004 as doi:10.1096/fj.04-2225fje. |
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* Rational Drug Design Program, Biomedicum and Transplantation Laboratory, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland;
Departments of Pediatric Transplantation and Biochemistry, Stanford University, Stanford, California, USA; and
Department of Surgery, Stellenbosch University, Cape Town, South Africa
1Correspondence: Transplantation Laboratory, PO Box 21, Haartmaninkatu 3, University of Helsinki, Helsinki 00014, Finland. E-mail: pekka.hayry{at}helsinki.fi
SPECIFIC AIMS
The purpose of the study was to establish a timeline for gene expression after vascular injury and to align human vascular specimens along this timeline. A model was established using baboon (Papio ursinus) vessels, which closely resemble human vessels in structure and response to injury.
PRINCIPAL FINDINGS
We performed left carotid artery overstretch injury of baboon carotid artery, investigated the time course of vascular expression of 41,000 human cDNA clones, and correlated these changes with carotid histology and function.
Key events after vascular injury are apoptosis and cell necrosis, adherence of platelets and inflammatory cells on the luminar surface, vasoconstriction, and vasodilatation, followed by a short wave of cell proliferation, sustained migration, and gradual deposition of extracellular matrix.
1. Microarray hybridization
Analysis revealed 20,788 differentially regulated cDNA clones (Web supplement 1). High stringency data selection yielded a file of 1629 clones representing 1510 genes of known function. Hierarchical clustering suggested 5 postinjury expression patterns over time (Fig. 1
).
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2. Functional grouping of genes
Genes corresponding to functional and anatomical alterations in the injured carotid wall were further aligned to functional groups according to Gene Ontology classification (supplementary data available in Web supplement 2).
Most proapoptotic genes (n=25) were up-regulated promptly on day 2. Some (red, n=10) remained up-regulated until days 30–90. The up-regulation of proapoptotic genes was followed with rapid up-regulation of antiapoptotic genes (red and blue, n=16).
Equally promptly up-regulated were several genes related to vasoconstriction (blue, n=6), peaking on days 2–3 after injury, and returning to predenudation level on days 14–30. This was followed by up-regulation of vasodilatatory genes (blue, n=5) from day 3 onward. Expression of endothelial nitric oxide synthetase gene was down-regulated on day 2 (likely as a consequence of endothelial removal), but the depression was sustained.
Some genes classically related to inflammatory events (red and blue, n=43) were up-regulated promptly and peaked on day 3, but the event was mostly short lasting.
Concomitant with re-endothelialization of the luminal surface, several genes characteristic to endothelial cells (green and purple, n=11), were repressed on day 2; their expression returned to close to predenudation level on days 3–4.
Genes characteristic to smooth muscle cells (green and purple, n=34) were repressed on day 2 and the repression was sustained. Retinoic acid-related genes (green, n=11) showed early repression on day 2, but had a more rapid recovery.
Early and sustained up-regulation from days 2–30 was observed with a number of proproliferative genes (red, blue, yellow, n=43). Concurrently and shortly thereafter, other antiproliferative genes with possible antiproliferative or tumor-suppressive functions were regulated. One group of these genes (red, n=9) showed sustained up-regulation; other groups (blue and yellow, n=7) normalized after day 14 or earlier.
Genes (n=21) that could be related to cell migration followed two expression patterns. One set (red, n=9), was up-regulated on day 2 and sustained; another group (blue, n=11) was activated on day 2 but turned down on day 14.
Regulation of genes related to increased metabolic activity such as those related to functions of Golgi complex, endoplasmic reticulum (n=13), and energy production (n=22) is characteristic of vascular genomics throughout the postoperative course from days 3–14.
Expression of extracellular matrix-related genes (n=59) followed several patterns. One group of genes (red, n=11) displayed an early and sustained overexpression from days 2–90. Another group (blue, n=17) either normalized after initial activation or was down-regulated by day 90. A fairly large group of extracellular matrix-related genes (green, n=21) was down-regulated on day 2 and sustained. A smaller group down-regulated connective tissue genes (purple, n=10) showed temporary activation after initial down-regulation on day 2.
Genomic analysis suggests a sustained abnormality in vascular wall functions even 90 days postinjury: 
4/5 of all genes remained abnormal in their expression level and only 1/5 normalized back to predenudation level.
3. Transcriptome analysis
The hierarchical clustering analysis left behind those clones that were not regulated (>2.5x) on >80% of measurements (5 of 6 time points). Transcriptome analysis was performed. All cDNAs expressing >2x the background at any time were included. The number of cDNAs expressed 
5 times was 3364, of which 805 were clones representing known genes and 2559 were ESTs (Table 1
, upper section).
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Genes expressed once (n=143) were expressed mostly on days 2–14, those expressed twice at consecutive times on days 4 and 14, and those expressed three times on days 3, 4, and 14 (Table 1)
. Genes related to proliferation, migration, and differentiation excluded from the original hierarchical clustering analysis were included (see Web supplement 4).
CONCLUSIONS AND SIGNIFICANCE
We chose a 2.5-fold cutoff for the expression level difference between any two time points. Web supplement #1 may be used for further analysis of the data with different cutoff limits.
The time zero transformation applied to the dataset allows a study of genes showing significant deviation in expression level from baseline preinjury level. A short list of the 1629 differentially regulated clones corresponding to 1510 genes of known function was generated. Genes corresponding to functional and anatomical alterations in the injured carotid wall were further aligned into functional groups according to gene ontology classification. This is the same approach used to correlate gene expression profiles to clinical status of the transplant recipient and in time course studies.
Gene expression patterns closely followed functional and anatomical alterations in the injured vessel wall. Short-lasting apoptosis and media cell necrosis after injury, coinciding with the up-regulation of proapoptotic genes, were blunted by rapid up-regulation of antiapoptotic genes. Vasoconstriction after endothelial removal, coinciding with up-regulation of vasoconstrictive genes, was followed by vasodilatation together with re-endothelialization and up-regulation of vasodilatatory genes, particularly the heme-oxygenases. Endothelial events were closely reflected via initial repression and quick normalization of genes related to endothelial function (e.g., the von Willebrand factor gene). Sustained repression of smooth muscle cell-related genes may be related to the influx of cells with a more immature phenotype.
The proliferative burst in baboon carotid after overstretch injury, as in humans, is short lasting and coincided in our study with the overexpression of proproliferative genes. Several antiproliferative genes or genes related to tumor suppressor function were shortly repressed and quickly normalized or overexpressed, blunting the proliferative burst.
Cell migration into injured baboon carotid occurs mostly between days 2 and 14, after which the intima cell number stabilizes. Some promigratory genes were repressed and sustained and another set (elastase, thrombospondin, and CD36) was up-regulated. Migratory and proliferative events were reflected in the regulation of genes related particularly to the Golgi complex, endoplasmic reticulum, and energy production.
Deposition of platelets and inflammatory cells on the denuded intima (mostly lymphocytes and monocytes) was observed on days 2–14. Coinciding with this, a variety of proinflammatory genes was expressed. The cell-rich character of baboon carotid neointima turns more fibrillar from day 14 postinjury onward. This coincided with the progressive and often sustained expression of several connective tissue genes.
This extensive analysis of postinjury vascular genomics in baboon has established a timeline that aligns sporadic human genomic findings. Even with this extended 90 day observation, the molecular structure of the vessel does not regain the properties of a healthy vessel, as earlier shown in morphological studies. Expression of several smooth muscle cell-related mRNAs remains low. This emphasizes the need for early pharmacological intervention with vasculoprotective compounds.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2225fje;
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