Kenagy Richard D, Civelek Mete, Kikuchi Shinsuke, Chen Lihua, Grieff Anthony, Sobel Michael, Lusis Aldons J, Clowes Alexander W
Department of Surgery, University of Washington, Seattle, Wash.
Department of Medicine, University of California, Los Angeles, Los Angeles, Calif.
J Vasc Surg. 2016 Jul;64(1):202-209.e6. doi: 10.1016/j.jvs.2014.12.052. Epub 2015 Apr 30.
Approximately 30% of autogenous vein grafts develop luminal narrowing and fail because of intimal hyperplasia or negative remodeling. We previously found that vein graft cells from patients who later develop stenosis proliferate more in vitro in response to growth factors than cells from patients who maintain patent grafts. To discover novel determinants of vein graft outcome, we have analyzed gene expression profiles of these cells using a systems biology approach to cluster the genes into modules by their coexpression patterns and to correlate the results with growth data from our prior study and with new studies of migration and matrix remodeling.
RNA from 4-hour serum- or platelet-derived growth factor (PDGF)-BB-stimulated human saphenous vein cells obtained from the outer vein wall (20 cell lines) was used for microarray analysis of gene expression, followed by weighted gene coexpression network analysis. Cell migration in microchemotaxis chambers in response to PDGF-BB and cell-mediated collagen gel contraction in response to serum were also determined. Gene function was determined using short-interfering RNA to inhibit gene expression before subjecting cells to growth or collagen gel contraction assays. These cells were derived from samples of the vein grafts obtained at surgery, and the long-term fate of these bypass grafts was known.
Neither migration nor cell-mediated collagen gel contraction showed a correlation with graft outcome. Although 1188 and 1340 genes were differentially expressed in response to treatment with serum and PDGF, respectively, no single gene was differentially expressed in cells isolated from patients whose grafts stenosed compared with those that remained patent. Network analysis revealed four unique groups of genes, which we term modules, associated with PDGF responses, and 20 unique modules associated with serum responses. The "yellow" and "skyblue" modules, from PDGF and serum analyses, respectively, correlated with later graft stenosis (P = .005 and P = .02, respectively). In response to PDGF, yellow was also associated with increased cell growth. For serum, skyblue was also associated with inhibition of collagen gel contraction. The hub genes for yellow and skyblue (ie, the gene most connected to other genes in the module), scavenger receptor class A member 5 (SCARA5) and suprabasin (SBSN), respectively, were tested for effects on proliferation and collagen contraction. Knockdown of SCARA5 increased proliferation by 29.9% ± 7.8% (P < .01), whereas knockdown of SBSN had no effect. Knockdown of SBSN increased collagen gel contraction by 24.2% ± 8.6% (P < .05), whereas knockdown of SCARA5 had no effect.
Using weighted gene coexpression network analysis of cultured vein graft cell gene expression, we have discovered two small gene modules, which comprise 42 genes, that are associated with vein graft failure. Further experiments are needed to delineate the venous cells that express these genes in vivo and the roles these genes play in vein graft healing, starting with the module hub genes SCARA5 and SBSN, which have been shown to have modest effects on cell proliferation or collagen gel contraction.
约30%的自体静脉移植物会出现管腔狭窄并因内膜增生或负性重塑而失败。我们之前发现，与移植血管保持通畅的患者的静脉移植物细胞相比，后来发生狭窄的患者的静脉移植物细胞在体外对生长因子的反应中增殖更多。为了发现静脉移植物转归的新决定因素，我们采用系统生物学方法分析了这些细胞的基因表达谱，通过共表达模式将基因聚类成模块，并将结果与我们之前研究中的生长数据以及迁移和基质重塑的新研究相关联。
从外静脉壁获取的人隐静脉细胞（20个细胞系），在4小时血清或血小板衍生生长因子（PDGF）-BB刺激后提取RNA，用于基因表达的微阵列分析，随后进行加权基因共表达网络分析。还测定了细胞在微趋化室中对PDGF-BB的迁移以及对血清的细胞介导的胶原凝胶收缩。在对细胞进行生长或胶原凝胶收缩测定之前，使用小干扰RNA抑制基因表达来确定基因功能。这些细胞来自手术时获取的静脉移植物样本，并且这些旁路移植物的长期转归是已知的。
迁移和细胞介导的胶原凝胶收缩均与移植物转归无关。尽管分别有1188个和1340个基因在血清和PDGF处理后差异表达，但与移植物狭窄的患者分离出的细胞相比，在移植物保持通畅的患者分离出的细胞中没有单个基因差异表达。网络分析揭示了与PDGF反应相关的4个独特基因组（我们称之为模块）以及与血清反应相关的20个独特模块。分别来自PDGF和血清分析的“黄色”和“天蓝色”模块与后来的移植物狭窄相关（分别为P = 0.005和P = 0.02）。对于PDGF，黄色模块还与细胞生长增加相关。对于血清，天蓝色模块还与胶原凝胶收缩的抑制相关。分别测试了黄色和天蓝色模块的中心基因（即模块中与其他基因连接最多的基因）清道夫受体A类成员5（SCARA5）和基底层蛋白（SBSN）对增殖和胶原收缩的影响。敲低SCARA5使增殖增加29.9%±7.8%（P < 0.01），而敲低SBSN没有影响。敲低SBSN使胶原凝胶收缩增加24.2%±8.6%（P < 0.05），而敲低SCARA5没有影响。
通过对培养的静脉移植物细胞基因表达进行加权基因共表达网络分析，我们发现了两个由42个基因组成的小基因模块，它们与静脉移植物失败相关。需要进一步实验来确定体内表达这些基因的静脉细胞以及这些基因在静脉移植物愈合中所起的作用，首先从已显示对细胞增殖或胶原凝胶收缩有适度影响的模块中心基因SCARA5和SBSN开始。
