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. 2018 Jan;67(1):309-317.e7.
doi: 10.1016/j.jvs.2016.12.113. Epub 2017 May 16.

A single nucleotide polymorphism of cyclin-dependent kinase inhibitor 1B (p27Kip1) associated with human vein graft failure affects growth of human venous adventitial cells but not smooth muscle cells

Richard D Kenagy  1 , Shinsuke Kikuchi  2 , Lihua Chen  3 , Errol S Wijelath  4 , Andrew B Stergachis  5 , John Stamatoyannopoulos  5 , Gale L Tang  6 , Alexander W Clowes  3 , Michael Sobel  6
Affiliations

A single nucleotide polymorphism of cyclin-dependent kinase inhibitor 1B (p27Kip1) associated with human vein graft failure affects growth of human venous adventitial cells but not smooth muscle cells

Richard D Kenagy et al. J Vasc Surg. 2018 Jan.

Abstract

Background: Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a cell-cycle inhibitor whose -838C>A single nucleotide polymorphism (rs36228499; hereafter called p27 SNP) has been associated with the clinical failure of peripheral vein grafts, but the functional effects of this SNP have not been demonstrated.

Methods: Human saphenous vein adventitial cells and intimal/medial smooth muscle cells (SMCs) were derived from explants obtained at the time of lower extremity bypass operations. We determined the following in adventitial cells and SMCs as a function of the p27 SNP genotype: (1) p27 promoter activity, (2) p27 messenger (m)RNA and protein levels, and (3) growth and collagen gel contraction. Deoxyribonuclease I footprinting was also performed in adventitial cells and SMCs.

Results: p27 promoter activity, deoxyribonuclease I footprinting, p27 mRNA levels, and p27 protein levels demonstrated that the p27 SNP is functional in adventitial cells and SMCs. Both cell types with the graft failure protective AA genotype had more p27 mRNA and protein. As predicted because of higher levels of p27 protein, adventitial cells with the AA genotype grew slower than those of the CC genotype. Unexpectedly, SMCs did not show this genotype-dependent growth response.

Conclusions: These results support the functionality of the p27 SNP in venous SMCs and adventitial cells, but an effect of the SNP on cell proliferation is limited to only adventitial cells. These data point to a potential role for adventitial cells in human vein graft failure and also suggest that SMCs express factors that interfere with the activity of p27.

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Figures

Figure 1
Figure 1
Effect of the p27 SNP on p27 promoter activity in human venous SMCs and adventitial cells. Venous SMCs matched to the adventitial cells used in Conte et al were transfected with the A or C form of the p27 promoter-luciferase constructs. Luciferase activity is expressed as the ratio of A to C construct activity and is the mean of 4 independent experiments (P<.01, A vs C). Data for the matched adventitial cells was previously published.
Figure 2
Figure 2
(A) DNaseI hypersensitivity assay of ENCODE cell lines in the region between p27 (CDKN1B) and GPR19. (B) Map from the 1000 Genomes Project of SNPs in linkage to the p27 SNP (rs36228499) on chromosome 12. The p27 SNP is the first SNP upstream of p27 (CDKN1B) with the 6 upstream SNPs that are linked (CV >0.8) to the p27 SNP also indicated in figure 2A. (C) DNaseI hypersensitivity assay of a matched pair of human saphenous vein adventitial cells and SMCs showing the p27 SNP (rs36228499) and rs3759216 (the two SNPs in DNaseI hypersensitive sites in the ENCODE cell lines). Only the p27 SNP is in a DNaseI hypersensitive site.
Figure 3
Figure 3
The effect of siRNA-mediated knockdown of p27 mRNA on proliferation of adventitial cells (A) and SMCs (B) with either the AA or CC p27 SNP genotype in response to 2% serum, 15% serum, 2% serum with PDGF-BB, or commercial SMC growth medium. Data are presented as the mean ± SEM of 8 cell lines per group with 3 replicates per cell line. *P<.05 p27 siRNA vs scrambled.
Figure 3
Figure 3
The effect of siRNA-mediated knockdown of p27 mRNA on proliferation of adventitial cells (A) and SMCs (B) with either the AA or CC p27 SNP genotype in response to 2% serum, 15% serum, 2% serum with PDGF-BB, or commercial SMC growth medium. Data are presented as the mean ± SEM of 8 cell lines per group with 3 replicates per cell line. *P<.05 p27 siRNA vs scrambled.
Figure 4
Figure 4
The effect of p27 SNP genotype on PDGF-BB/2% serum-mediated proliferation of adventitial cells (A) and SMCs (B). Data are presented as the mean ± SEM of 8–11 cell lines per group with 3 replicates per cell line. * P<.05 AA vs CC.
Figure 5
Figure 5
The effect of p27 SNP genotype on p27 mRNA expression by matched adventitial cells (A) and SMCs (B) after treatment with PDGF-BB/2% serum. Data are presented as the mean ± SEM of 3 cell lines per group with 2 replicates per cell line. * P<.05 AA vs CC
Figure 6
Figure 6
The effect of p27 SNP genotype on levels of p27 protein in adventitial cells and SMCs after 72 hours in 2% fetal bovine serum alone (A) or with 10 ng/ml PDGF-BB (B). AA cells are represented by closed bars and CC cells by open bars. Results are the mean ± SEM relative to an arbitrary control of 8–9 paired adventitial/SMC lines for each genotype. * P<.05 AA vs CC
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Comment in

  • Invited commentary.
    Rzucidlo EM. Rzucidlo EM. J Vasc Surg. 2018 Jan;67(1):318. doi: 10.1016/j.jvs.2017.02.026. J Vasc Surg. 2018. PMID: 29268919 No abstract available.

References

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