PDF(Pubmed)
Abstract：
Atherosclerosis is a chronic inflammatory disease and occurs preferentially in arterial regions exposed to disturbed blood flow (d-flow) while the stable flow (s-flow) regions are spared. D-flow induces endothelial inflammation and atherosclerosis by regulating endothelial gene expression partly through the flow-sensitive transcription factors (FSTFs). Most FSTFs, including the well-known Kruppel-like factors KLF2 and KLF4, have been identified from in vitro studies using cultured endothelial cells (ECs). Since many flow-sensitive genes and pathways are lost or dysregulated in ECs during culture, we hypothesized that many important FSTFs in ECs in vivo have not been identified. We tested the hypothesis by analyzing our recent gene array and single-cell RNA sequencing (scRNAseq) and chromatin accessibility sequencing (scATACseq) datasets generated using the mouse partial carotid ligation model. From the analyses, we identified 30 FSTFs, including the expected KLF2/4 and novel FSTFs. They were further validated in mouse arteries in vivo and cultured human aortic ECs (HAECs). These results revealed 8 FSTFs, SOX4, SOX13, SIX2, ZBTB46, CEBPβ, NFIL3, KLF2, and KLF4, that are conserved in mice and humans in vivo and in vitro. We selected SOX13 for further studies because of its robust flow-sensitive regulation, preferential expression in ECs, and unknown flow-dependent function. We found that siRNA-mediated knockdown of SOX13 increased endothelial inflammatory responses even under the unidirectional laminar shear stress (ULS, mimicking s-flow) condition. To understand the underlying mechanisms, we conducted an RNAseq study in HAECs treated with SOX13 siRNA under shear conditions (ULS vs. oscillatory shear mimicking d-flow). We found 94 downregulated and 40 upregulated genes that changed in a shear- and SOX13-dependent manner. Several cytokines, including CXCL10 and CCL5, were the most strongly upregulated genes in HAECs treated with SOX13 siRNA. The robust induction of CXCL10 and CCL5 was further validated by qPCR and ELISA in HAECs. Moreover, the treatment of HAECs with Met-CCL5, a specific CCL5 receptor antagonist, prevented the endothelial inflammation responses induced by siSOX13. In addition, SOX13 overexpression prevented the endothelial inflammation responses. In summary, SOX13 is a novel conserved FSTF, which represses the expression of pro-inflammatory chemokines in ECs under s-flow. Reduction of endothelial SOX13 triggers chemokine expression and inflammatory responses, a major proatherogenic pathway.
摘要：
动脉粥样硬化是一种慢性炎性疾病,并且优先发生在暴露于受干扰的血流(d流)的动脉区域中,而稳定的血流(s流)区域不受影响。D流通过部分通过流敏感转录因子（FSTFs）调节内皮基因表达来诱导内皮炎症和动脉粥样硬化。大多数FSTF,包括众所周知的Kruppel样因子KLF2和KLF4,已经从使用培养的内皮细胞(ECs)的体外研究中鉴定。由于许多流动敏感的基因和途径在培养过程中在ECs中丢失或失调，我们假设尚未发现体内ECs中许多重要的FSTFs。我们通过分析我们最近的基因阵列和使用小鼠部分颈动脉结扎模型生成的单细胞RNA测序（scRNAseq）和染色质可及性测序（scATACseq）数据集来测试假设。从分析来看，我们确定了30个FSTF，包括预期的KLF2/4和新的FSTF。它们在体内小鼠动脉和培养的人主动脉ECs(HAECs)中进一步验证。这些结果揭示了8个FSTF，SOX4，SOX13，SIX2，ZBTB46，CEBPβ，NFIL3、KLF2和KLF4在小鼠和人体内和体外保守。我们选择SOX13进行进一步研究，因为它具有强大的流量敏感调节功能，在EC中优先表达，和未知的流量相关函数。我们发现，即使在单向层流剪切应力下，siRNA介导的SOX13敲低也会增加内皮炎症反应（ULS，模仿s流)条件。要了解潜在的机制，我们在剪切条件下用SOX13siRNA处理的HAECs中进行了RNAseq研究（ULS与振荡剪切模拟d流)。我们发现94个下调和40个上调的基因以剪切和SOX13依赖性方式变化。几种细胞因子，在用SOX13siRNA处理的HAECs中,包括CXCL10和CCL5是最强烈上调的基因。通过qPCR和ELISA在HAECs中进一步验证CXCL10和CCL5的稳健诱导。此外，用一种特异性CCL5受体拮抗剂Met-CCL5治疗HAECs，阻止siSOX13诱导的内皮炎症反应。此外,SOX13过表达阻止了内皮炎症反应。总之,SOX13是一种新颖的保守FSTF，在s流下抑制ECs中促炎趋化因子的表达。内皮SOX13的减少触发趋化因子表达和炎症反应，主要的致动脉粥样硬化途径.
