Gene expression after crush injury of human saphenous vein: using microarrays to define the transcriptional profile.

BACKGROUND: Vein graft stenosis is believed to be the pathophysiologic response of vascular tissue to injury and is the major cause of vein graft failure. Therapeutic interventions might improve with knowledge of the physiologic pathways involved in the hyperplastic response to vascular injury. In this study, our purpose was to identify induced, early pathways that might be important in the human response to vascular injury. STUDY DESIGN: Human saphenous vein from 7 patients was organ cultured or crush injured and cultured for 48 or 72 hours after harvest. Gene expression was determined for syngeneic veins at harvest and at the experimental time points and compared to determine which genes were induced or repressed. Expressed genes (the transcriptional profile) were then assigned to functional physiologic classes. RESULTS: At 72 hours, in both organ-cultured and crush-injured vein, the gene for the Wnt ligand protein (WNT5A) was induced. At 48 hours in the organ-cultured vein only, the gene for the Frizzled protein (FZD2), a subunit of the Wnt receptor complex, was repressed. At 72 hours in injured vein only, the gene for the product of Wnt signaling (WISP1) was induced; the gene for the Wnt-binding, soluble Frizzled-related protein (FRZB) was repressed; and the gene for Dickkopf (DKK1) protein, which binds to the low density lipoprotein receptor-related protein subunit of the Wnt receptor complex, was induced. CONCLUSIONS: Early induction of WNT5A, coupled with the coordinated induction and repression of genes that modulate the Wnt signaling pathway, led to the early, selective induction of WISP1 and no other Wnt-inducible genes. This early, selective expression of a limited gene set might characterize the human vascular response to injury, and could enable development of therapies to treat the clinical sequelae of this response.

The sequence of gene expression in cultured human saphenous vein after injury.

OBJECTIVE: The objective of this study was a description of changes in gene expression that occur in response to mechanical injury of cultured human saphenous vein. METHODS: Restriction fragment differential display (Display Systems Biotech, Vista, Calif) was used for the comparison of the gene expression profile in seven sets of vein, with the first set representing gene expression at the time of harvest of the vein and the other six sets representing different lengths of time in culture with or without crush injury. All seven sets were from a single, freshly harvested vein. Each set contained eight separate vein segments. The first set (Set 1) was taken from the freshly harvested vein, and the RNA was immediately extracted. This set reflects the in vivo gene expression profile at the time of harvest. Three sets of vein segments (Sets 2, 4, and 6) were cultured for 24, 48, or 72 hours after harvest (culture only). Three sets of vein segments (Sets 3, 5, and 7) were harvested, crush-injured, and then cultured for 24, 48, or 72 hours (crush injury + culture). The gene expression profiles of these six cultured sets of vein segments were compared with the gene expression profile of the set of vein segments that were obtained at harvest. RESULTS: The crush injury of the vein segments resulted in the up-regulated expression of 21 identified (including inducible nitric oxide synthase) and nine unknown genes and in the down-regulated expression of eight identified and seven unknown genes within the first 72 hours after harvest. The vein segments that were cultured without crush injury had the up-regulated expression of nine identified and seven unknown genes and the down-regulated expression of five identified (including platelet-derived growth factor-B and transforming growth factor-beta2) and seven unknown genes within the first 72 hours after harvest. The pattern of gene regulation after transmural crush injury revealed eight genes whose products support cell migration and seven genes whose products oppose cell proliferation. CONCLUSION: The comparison of gene expression between those vein segments designated culture only and those vein segments designated crush injury and culture shows that some cells of the vein segments express phenotypic changes that are consistent with cell migration. Further studies of gene expression changes in vitro may elucidate the endogenous response of vascular tissue to injury.

Validation of an in vitro model of human saphenous vein hyperplasia.

OBJECTIVE: The purpose of this study was the validation of the physiologic appropriateness of in vitro organ culture of human saphenous vein as a model with the demonstration of the occurrence of the processes of cell proliferation, remodeling, and hyperplasia. METHODS: Saphenous vein from 28 patients was cross-sectioned into seven 2-mm segments and maintained in organ culture for 2 days or 2 weeks. Three organ culture media were used: a chemically well-defined medium (RPMI-1640) and the same medium supplemented with the undefined protein-containing supplements fetal bovine serum (FBS) or pooled adult human plasma (type AB). The outcome measures at 2 days and 2 weeks were compared with measurements of segments from the same vein at the time of harvest. Excess saphenous vein harvested for arterial bypass grafting was obtained after approval of the study protocol by the Institutional Review Board. Cell proliferation was measured with immunostaining for proliferating cell nuclear antigen. Remodeling and intimal hyperplasia were measured with micromorphometric comparisons of vein segment cross-sectional area before and after organ culture. RESULTS: There was no evidence of cell death or tissue degeneration on histologic examination of the cultured vein segments. Cell proliferation, expressed as proliferation index (PI; positive proliferating cell nuclear antigen nuclei/total nuclei), significantly increased as compared with freshly harvested vein after 2 days of culture in undefined, protein-supplemented media (mean PI, 42.4 +/- 7.4%; P <.001). A significant increase in cell proliferation did not occur in the defined, unsupplemented medium until 2 weeks (mean PI, 16.2 +/- 7.1%; P <.001). The cross-sectional area of the vein wall increased during culture in all media. A statistically significant increase in the cross-sectional area of the vein wall occurred during culture with plasma (P <.001) and FBS supplementation (P =.002). The increase in the cross-sectional area of the vein in defined media was almost statistically significant (P =.089). A significant increase was seen in the cross-sectional area of the media (P =.006) and adventitia (P =.030) of veins cultured with plasma supplementation and in the cross-sectional area of the adventitia (P =.034) of veins cultured with FBS supplementation. CONCLUSION: These results show that human saphenous vein in culture is viable, shows cell proliferation, and exhibits remodeling of the layers of the vein wall. This is the first report to document hyperplasia in human vascular tissue cultured in a defined medium.