Sox13 is a novel flow-sensitive transcription factor that prevents inflammation by repressing chemokine expression in endothelial cells

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.

Fluid shear stress-modulated chromatin accessibility reveals the mechano-dependency of endothelial SMAD1/5-mediated gene transcription

“… 58 , 59 While SOX TFs have been described to act during differentiation as well as to act vasoprotective in the mature endothelium, GATA TFs were shown to be upregulated by atheroprone FSS. For instance, Sox18 has been shown to regulate EC barrier integrity upon application of FSS in pulmonary arterial ECs, 60 FSS-dependent upregulation of SOX13 was connected to suppression of pro-inflammatory gene expression 31 and SOX17 acts as a critical regulator of vascular homoeostasis, commonly mutated in pulmonary arterial hypertension patients. 61 In contrast, elevated levels of GATA3 were connected to endothelial-mesenchymal transition-mediated pulmonary arterial hypertension (PAH), 62 GATA2 was shown to directly repress atheroprotective TF KLF2, 63 and GATA1/4 expression was elevated by pulsatile oscillatory atheroprone shear stress in HUVECs.…”

“… 29 Recently, it was shown that SOX13, a member of the SOX pioneer TF family, 30 is robustly upregulated by FSS and suppresses pro-inflammatory gene expression. 31 Similarly, pioneer TF KLF4 mediates vasculo-protective gene expression in response to FSS. 32 On the other hand, GATA TFs have been shown to be upregulated by atheroprone FSS.…”

Flow-induced reprogramming of endothelial cells in atherosclerosis

“…A study published in 2022 identified SOX13 as a novel flow-sensitive transcription factor. Disturbed flow represses the expression of SOX13 , which in turn leads to a strong induction of pro-inflammatory cytokine and chemokine production, including CCL5 and CXCL10, resulting in endothelial inflammation 134 .…”

Unveiling the Impact of Hemodynamics on Endothelial Inflammation‐Mediated Hepatocellular Carcinoma Metastasis Using a Biomimetic Vascular Flow Model

“…[14,19,20] For instance, the application of oscillatory shear stress to endothelial cells will lead to NLRP3 (NLR family, pyrin domain containing 3) mediated inflammation by activating sterol regulatory element binding protein 2, [21] and flow-sensitive transcription factor SOX13 can prevent the inflammation by suppressing chemokine expression. [22] Nonetheless, the complexities of the vascular geometry in vivo coupled with the presence of confounding factors such as red blood cells, dynamic blood pressure, and immune responses, render the vascular model difficult to control in vitro. [23,24] Even the assessment of the effects requires substantial simplification of the in vivo context.…”