■动脉粥样硬化是哈钦森-吉尔福德早衰综合征的主要医学问题,由突变的lamin-A蛋白孕激素引起的一种罕见的早衰疾病。最近,我们发现,在Apoe缺陷小鼠中,将早衰蛋白的表达限制在血管平滑肌细胞(VSMC)足以加速动脉粥样硬化和死亡.然而,孕激素驱动的VSMC缺陷对内皮细胞(ECs)的影响尚不清楚.
■Apo-或Ldlr缺陷型C57BL/6J小鼠,VSMC-,在Hutchinson-Gilfordprogeria综合征相关的动脉粥样硬化期间,使用正常或高脂肪饮食的EC或髓样特异性progerin表达来研究内皮表型。通过静脉注射荧光标记的人低密度脂蛋白和主动脉的共聚焦显微镜分析来评估内皮对低密度脂蛋白的通透性。通过面对面免疫荧光评估白细胞向主动脉壁的募集。通过定量聚合酶链反应和RNA测序在主动脉内膜中以及在主动脉根部切片中通过免疫荧光评估内皮-间质转化(EndMT)。通过多重免疫测定分析血清中的TGFβ(转化生长因子β)信号,通过主动脉中的蛋白质印迹,并通过在主动脉根部切片中的免疫荧光。通过腹膜内注射SIS3(SMAD3的特异性抑制剂)在小鼠中评估TGFβ1/SMAD3途径抑制的治疗益处,通过油红O染色评估血管表型,组织学,以及主动脉和主动脉根部的免疫荧光。
■Apoe-null小鼠中普遍存在的和VSMC特异性的progerin表达均引起主动脉EC的改变,包括低密度脂蛋白通透性增加和白细胞募集。这些孕激素小鼠模型中的动脉粥样硬化病变,但在EC和骨髓特异性早衰模型中没有,含有丰富的结合内皮和间充质特征的细胞,表明功能失调的VSMC触发了广泛的EndMT。因此,动脉粥样硬化发作时普遍存在的和VSMC特异性早孕模型的内膜呈现EndMT相关基因的表达增加,特别是那些特异于成纤维细胞和细胞外基质的。两个模型中的主动脉均显示TGFβ1/SMAD3通路的激活,EndMT的主要触发因素,用SIS3治疗VSMC特异性孕激素小鼠可减轻主动脉表型。
■Progerin诱导的VSMC改变通过TGFβ1/SMAD3促进EC功能障碍和EndMT,将该过程确定为Hutchinson-Gilford早衰综合征治疗的候选靶标。这些发现还为EndMT在动脉粥样硬化形成过程中的复杂作用提供了见解。
UNASSIGNED: Atherosclerosis is the main medical problem in Hutchinson-Gilford
progeria syndrome, a rare premature aging disorder caused by the mutant lamin-A protein progerin. Recently, we found that limiting progerin expression to vascular smooth muscle cells (VSMCs) is sufficient to hasten atherosclerosis and death in Apoe-deficient mice. However, the impact of progerin-driven VSMC defects on endothelial cells (ECs) remained unclear.
UNASSIGNED: Apoe- or Ldlr-deficient C57BL/6J mice with ubiquitous, VSMC-, EC- or myeloid-specific progerin expression fed a normal or high-fat diet were used to study endothelial phenotype during Hutchinson-Gilford
progeria syndrome-associated atherosclerosis. Endothelial permeability to low-density lipoproteins was assessed by intravenous injection of fluorescently labeled human low-density lipoprotein and confocal microscopy analysis of the aorta. Leukocyte recruitment to the aortic wall was evaluated by en face immunofluorescence. Endothelial-to-mesenchymal transition (EndMT) was assessed by quantitative polymerase chain reaction and RNA sequencing in the aortic intima and by immunofluorescence in aortic root sections. TGFβ (transforming growth factor β) signaling was analyzed by multiplex immunoassay in serum, by Western blot in the aorta, and by immunofluorescence in aortic root sections. The therapeutic benefit of TGFβ1/SMAD3 pathway inhibition was evaluated in mice by intraperitoneal injection of SIS3 (specific inhibitor of SMAD3), and vascular phenotype was assessed by Oil Red O staining, histology, and immunofluorescence in the aorta and the aortic root.
UNASSIGNED: Both ubiquitous and VSMC-specific progerin expression in Apoe-null mice provoked alterations in aortic ECs, including increased permeability to low-density lipoprotein and leukocyte recruitment. Atherosclerotic lesions in these progeroid mouse models, but not in EC- and myeloid-specific
progeria models, contained abundant cells combining endothelial and mesenchymal features, indicating extensive EndMT triggered by dysfunctional VSMCs. Accordingly, the intima of ubiquitous and VSMC-specific progeroid models at the onset of atherosclerosis presented increased expression of EndMT-linked genes, especially those specific to fibroblasts and extracellular matrix. Aorta in both models showed activation of the TGFβ1/SMAD3 pathway, a major trigger of EndMT, and treatment of VSMC-specific progeroid mice with SIS3 alleviated the aortic phenotype.
UNASSIGNED: Progerin-induced VSMC alterations promote EC dysfunction and EndMT through TGFβ1/SMAD3, identifying this process as a candidate target for Hutchinson-Gilford
progeria syndrome treatment. These findings also provide insight into the complex role of EndMT during atherogenesis.