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Abstract：
Background: Shear stress-induced Dickkopf-1 (DKK1) secretion by endothelial cells (ECs) promotes EC dysfunction and accelerates atherosclerosis (AS). However, the paracrine role of endothelial DKK1 in modulating adjacent smooth muscle cells (SMCs) in atherosclerosis remains unclear. This study investigated the role of EC-secreted DKK1 in SMC-derived foam cell formation under shear stress, in vitro and in vivo. Methods: Parallel-plate co-culture flow system was used to explore the cellular communication between ECs and SMCs under shear stress in vitro. Endothelium-specific knockout of DKK1 (DKK1ECKO/APOE-/-) and endothelium-specific overexpression of DKK1 (DKK1ECTg) mice were constructed to investigate the role of endothelial DKK1 in atherosclerosis and SMC-derived foam cell formation in vivo. RNA sequencing (RNA-seq) was used to identify the downstream targets of DKK1. Reverse transcription quantitative polymerase chain reaction (RT-qPCR), western blot, coimmunoprecipitation (Co-IP) assays and chromatin immunoprecipitation (ChIP) experiments were conducted to explore the underlying regulatory mechanisms. Results: DKK1 is transcriptionally upregulated in ECs under conditions of low shear stress, but not in co-cultured SMCs. However, DKK1 protein in co-cultured SMCs is increased via uptake of low shear stress-induced endothelial DKK1, thereby promoting lipid uptake and foam cell formation in co-cultured SMCs via the post-translational upregulation of scavenger receptor-A (SR-A) verified in parallel-plate co-culture flow system, DKK1ECKO and DKK1ECTg mice. RNA sequencing revealed that DKK1-induced SR-A upregulation in SMCs is dependent on Ubiquitin-specific Protease 53 (USP53), which bound to SR-A via its USP domain and cysteine at position 41, exerting deubiquitination to maintain the stability of the SR-A protein by removing the K48 ubiquitin chain and preventing proteasomal pathway degradation, thereby mediating the effect of DKK1 on lipid uptake in SMCs. Moreover, DKK1 regulates the transcription of USP53 by facilitating the binding of transcription factor CREB to the USP53 promoter. SMC-specific overexpression of USP53 via adeno-associated virus serotype 2 vectors in DKK1ECKO/APOE-/- mice reversed the alleviation of atherosclerotic plaque burden, SR-A expression and lipid accumulation in SMCs within plaques resulting from DKK1 deficiency. Conclusions: Our findings demonstrate that, endothelial DKK1, induced by pathological low shear stress, acts as an intercellular mediator, promoted the foam cell formation of SMCs. These results suggest that targeted intervention with endothelial DKK1 may confer beneficial effects on atherosclerosis.
摘要：
背景：剪切应激诱导的内皮细胞（ECs）分泌Dickkopf-1（DKK1）促进EC功能障碍并加速动脉粥样硬化（AS）。然而,内皮DKK1在动脉粥样硬化中调节邻近平滑肌细胞(SMC)的旁分泌作用尚不清楚.这项研究调查了在剪切应力下EC分泌的DKK1在SMC衍生的泡沫细胞形成中的作用，在体外和体内。方法:采用平行板共培养流系统探讨体外切应力下ECs与SMCs之间的细胞通讯。构建DKK1的内皮特异性敲除(DKK1ECKO/APOE-/-)和DKK1的内皮特异性过表达(DKK1ECTg)小鼠,研究内皮DKK1在体内动脉粥样硬化和SMC源性泡沫细胞形成中的作用。RNA测序(RNA-seq)用于鉴定DKK1的下游靶标。逆转录定量聚合酶链反应（RT-qPCR），westernblot,进行了共免疫沉淀（Co-IP）测定和染色质免疫沉淀（ChIP）实验以探索潜在的调节机制。结果：在低剪切应力条件下，DKK1在EC中转录上调，但在共培养的SMC中没有。然而,共培养的SMC中的DKK1蛋白通过摄取低剪切应力诱导的内皮DKK1而增加，从而通过在平行板共培养流系统中验证的清道夫受体A（SR-A）的翻译后上调促进共培养的SMC中的脂质摄取和泡沫细胞形成，DKK1ECKO和DKK1ECTg小鼠。RNA测序显示，在SMC中DKK1诱导的SR-A上调依赖于泛素特异性蛋白酶53（USP53），其通过其USP结构域和位置41的半胱氨酸与SR-A结合，通过去除K48泛素链和防止蛋白酶体途径降解来发挥去泛素化以维持SR-A蛋白的稳定性，从而介导DKK1对SMC中脂质摄取的影响。此外，DKK1通过促进转录因子CREB与USP53启动子的结合来调节USP53的转录。在DKK1ECKO/APOE-/-小鼠中通过腺相关病毒血清型2载体SMC特异性过表达USP53逆转了动脉粥样硬化斑块负荷的减轻，由DKK1缺乏导致的斑块内SMC中的SR-A表达和脂质积累。结论：我们的研究结果表明，病理性低剪切应力诱导的内皮DKK1，充当细胞间中介，促进了SMC泡沫细胞的形成。这些结果表明，内皮DKK1的靶向干预可能对动脉粥样硬化产生有益作用。
