中文摘要

背景与目的

脑血管畸形是引发脑出血与神经功能障碍的关键因素之一，目前临床上缺乏有效的药物治疗手段。甲状腺激素（Thyroid hormones, THs），主要包含甲状腺素（T4）和三碘甲状腺原氨酸（T3），在正常血管发育过程中发挥着不可或缺的作用。然而，它们是否参与脑血管畸形的病理过程目前尚不清楚。课题组前期研究通过对人类脑动静脉畸形（Arteriovenous malformation, AVM）和脑海绵状血管畸形（Cerebral cavernous malformation, CCM）病变组织进行单细胞测序分析，发现成纤维细胞中碘甲状腺原氨酸脱碘酶2（Iodothyronine deiodinase 2， DIO2）的表达水平显著升高。Metascape基因富集分析显示，在CCM和AVM的成纤维细胞亚群中，细胞外基质重塑、免疫炎症反应等生物学功能呈高度富集状态。基因集富集分析（GSEA）也表明，在血管畸形组中，THs相关信号通路也被显著激活。虽然THs与多种心血管疾病存在关联，但其在脑血管系统中的具体作用机制尚未得到深入研究。本研究旨在探讨DIO2（一种将T4转化为活性T3的酶）与THs信号在脑血管畸形中的作用。

研究方法和结果

本研究首先通过免疫荧光技术证实了单细胞测序分析结果。在CCM和脑AVM患者组织切片中的成纤维细胞中，DIO2的表达升高。本研究构建内皮特异性Pdcd10敲除和内皮特异性Kras^G12D突变的两种脑血管畸形小鼠模型，在分离出的血管成纤维细胞中，Dio2的蛋白水平升高。

本研究随后通过眶后静脉窦注射AAV9-pLum-Dio2腺相关病毒建立起在成纤维细胞中特异性过表达Dio2的小鼠模型，并对其表型进行检测。研究结果显示过表达Dio2降低了Pdcd10 KO和Kras^G12D小鼠脑出血、细胞外基质重塑、血管通透性，显著改善了小鼠脑血管畸形相关表型。

本研究采用腹腔注射T3的方式，深入探究THs信号通路在脑血管畸形中所发挥的作用。研究结果表明，给予T3处理后，Pdcd10 KO和Kras^G12D小鼠的脑出血状况、血管细胞外基质重塑过程以及血管通透性等脑血管畸形相关表型均得到改善。

在分子机制的研究过程中，JASPAR预测提示Foxk1与Dio2的启动子结合，本研究应用了染色质免疫共沉淀实验、免疫荧光实验、蛋白印记实验、实时荧光定量PCR实验等技术手段，发现在分离出的小鼠脑血管成纤维细胞中Dio2受PI3K-Akt-mTOR-Foxk1信号通路调控。

本研究最后探究T3给药改善小鼠脑血管畸形的分子机制。实时荧光定量PCR实验显示T3显著抑制小鼠脑血管中炎症因子mRNA水平的表达。同时T3也显著降低脑血管细胞中ROS含量、改善线粒体膜电位损伤以及超微结构、降低血管细胞耗氧率。实验结果说明T3通过Pgc1a-Sod2/Prdx3/Gpx1信号通路改善小鼠脑血管畸形,T3对Pdcd10 KO/Kras^G12D小鼠的脑血管的这种修复作用依赖于Pgc1a。

结论

在人CCM和脑AVM单细胞样本的成纤维细胞中，TH信号明显激活，伴随DIO2表达升高。在Pdcd10 KO/Kras^G12D小鼠和患者的脑血管成纤维细胞中也观察到DIO2显著上调。外源性Dio2过表达或T3治疗有效减少了Pdcd10 KO/Kras^G12D小鼠的脑出血、细胞外基质重塑和血管通透性。机制上，转录因子Foxk1被确定与Dio2启动子区域相互作用。Pdcd10 KO/Kras^G12D小鼠中成纤维细胞PI3K-Akt-mTOR信号的激活触发了Foxk1核转位以促进Dio2转录。T3通过激活Pgc1a-Sod2/Prdx3/Gpx1信号通路减少ROS积聚，减轻畸形脑血管的炎症浸润，使线粒体形态正常化。

Exploring the Role and Underlying Mechanisms of DIO2 in the Development of Cerebrovascular malformation

Abstract

Background and Purposes：

Cerebrovascular malformation is one of the key factors leading to cerebral hemorrhage and neurological dysfunction. At present, there is a lack of effective drug treatment in clinical practice. Thyroid hormones (Thyroid hormones, THs), mainly including thyroxine (T4) and triiodothyronine (T3), play an indispensable role in normal vascular development. However, whether they are involved in the pathological process of cerebrovascular malformations is currently unknown. In our previous study, we performed single-cell sequencing analysis of human brain arteriovenous malformation (Arteriovenous malformation, AVM) and cerebral cavernous malformation (Cerebral cavernous malformation, CCM) lesion tissues, and found that the expression level of iodothyronine deiodinase 2 (DIO2) in fibroblasts was significantly increased. Metascape gene enrichment analysis showed that biological functions such as extracellular matrix remodeling and immune inflammatory response were highly enriched in the fibroblast subsets of CCMs and AVMs. Gene set enrichment analysis (GSEA) also showed that THS-related signaling pathways were also significantly activated in the vascular malformation group. Although THs are associated with a variety of cardiovascular diseases, the specific mechanism of its action in the cerebrovascular system has not been thoroughly studied. The purpose of this study was to investigate the role of DIO2, an enzyme that converts T4 to active T3, and THs signaling in cerebrovascular malformations.

Methods and Results:

The results of single-cell sequencing analysis were confirmed by immunofluorescence. DIO2 expression was elevated in fibroblasts from tissue sections of CCM and AVM patients. In this study, two mouse models of endothelium-specific Pdcd10 knockout and endothelium-specific Kras^G12D mutation were constructed. The protein level of Dio2 was increased in isolated vascular fibroblasts.

A fibroblast-specific Dio2 overexpression mouse model was subsequently established by retro-orbital venous sinus injection of AAV9-pLum-Dio2 adeno-associated virus, and the phenotype was examined. Our results showed that Dio2 overexpression reduced cerebral hemorrhage, extracellular matrix remodeling, vascular permeability, and significantly improved cerebrovascular malformation-related phenotypes in Pdcd10 KO and Kras^G12D mice.

In this study, intraperitoneal injection of T3 was used to further explore the role of THs signaling pathway in cerebrovascular malformations. The results showed that after T3 treatment, the cerebral hemorrhage condition, the remodeling process of vascular extracellular matrix, and vascular permeability were improved in Pdcd10 KO and Kras^G12D mice.

In the process of studying the molecular mechanism, JASPAR predicted that Foxk1 bound to the promoter of Dio2. It was found that Dio2 was regulated by PI3K-Akt-mTOR-Foxk1 signaling pathway in isolated mouse cerebral vascular fibroblasts.

Finally, the molecular mechanisms underlying the improvement of cerebral vascular malformations by T3 administration in mice were investigated. Real-time fluorescence quantitative PCR showed that T3 significantly inhibited the mRNA expression of inflammatory factors in the cerebral vessels of mice. Meanwhile, T3 significantly reduced ROS content, improved mitochondrial membrane potential damage and ultrastructure, and reduced oxygen consumption rate of cerebral vascular cells. Western blot showed that T3 ameliorated cerebral vascular malformations in mice through Pgc1A-Sod2/Prdx3/Gpx1 signaling pathway.

Conclusions:

In fibroblasts from human CCM and AVM single-cell samples, TH signaling was clearly activated, accompanied by elevated DIO2 expression. A significant upregulation of DIO2 was also observed in cerebral vascular fibroblasts from Pdcd10 KO/Kras^G12D mice and patients. Exogenous Dio2 overexpression or T3 treatment effectively reduced intracerebral hemorrhage, extracellular matrix remodeling, and vascular permeability in Pdcd10 KO/Kras^G12D mice. Mechanistically, the transcription factor Foxk1 was identified to interact with the Dio2 promoter region. Activation of PI3K-Akt-mTOR signaling in fibroblasts in Pdcd10 KO/Kras^G12D mice triggers Foxk1 nuclear translocation to promote Dio2 transcription. T3 reduced ROS accumulation by activating Pgc1a-Sod2/Prdx3/Gpx1 signaling pathway, alleviated inflammatory infiltration of malformed cerebral vessels, and normalized mitochondrial morphology.