背景:由于特殊的血液动力学特征,与先天性心脏病(PAH-CHD)相关的肺动脉高压的肺血管疾病分为两个阶段:可逆和不可逆。到目前为止,从可逆阶段过渡到不可逆阶段的机制难以捉摸。此外,没有公认和可靠的评估来区分这两个阶段。此外,我们发现与对照和可逆性PAH相比,通过生物信息学分析,血小板反应蛋白-4(THBS4)在不可逆组中显著上调.因此,我们进一步验证和研究了THBS4在PAH-CHD中的表达和作用。
方法:我们建立了野百合碱加主动脉静脉分流术(MCT-AV)大鼠模型。我们测量了THBS4在MCT-AV大鼠肺组织中的表达。肺组织的THBS4和α-SMA(平滑肌细胞的生物标志物)或vWF(内皮细胞的生物标志物)的双重免疫荧光染色以鉴定THBS4在肺动脉中的位置。原代肺动脉平滑肌细胞(PASMC)培养,已识别,并在这项研究中使用。THBS4被siRNA和质粒抑制和过表达,分别,探讨THBS4对表型转化的影响,扩散,凋亡,和PASMC的迁移。通过抑制THBS4表达的腺相关病毒在体内评估THBS4对肺血管重塑的影响。通过ELISA测量PAH-CHD患者的THBS4循环水平。
结果:THBS4在MCT-AV大鼠肺组织中上调,并在严重肺血管病变中进一步上调。THBS4主要在PASMC中表达。当THBS4被抑制时,收缩标志物α-SMA和MYH11上调,而增殖标志物PCNA降低,内皮-间质转化标志物N-cad下调,前凋亡标记BAX增加。此外,PASMCs的增殖和迁移受到抑制,凋亡增加。相反,THBS4过表达导致相反的效果。THBS4对PASMC的影响可能是通过调节PI3K/AKT通路实现的。THBS4抑制减弱了肺血管重塑。此外,与单纯先天性心脏病和轻度PAH-CHD患者相比,重度PAH-CHD患者的THBS4循环水平较高.
结论:THBS4是一种有前途的生物标志物,可在修复分流之前区分可逆和不可逆的PAH-CHD。THBS4是PAH-CHD的潜在治疗靶点,尤其是在不可逆转的阶段。
BACKGROUND: Due to a special hemodynamic feature, pulmonary vascular disease in pulmonary arterial hypertension associated with congenital heart disease (PAH-CHD) has two stages: reversible and irreversible. So far, the mechanism involved in the transition from reversible to irreversible stage is elusive. Moreover, no recognized and reliable assessments to distinguish these two stages are available. Furthermore, we found that compared with control and reversible PAH, thrombospondin-4 (THBS4) was significantly upregulated in irreversible group by bioinformatic analysis. Hence, we further verify and investigate the expression and role of THBS4 in PAH-CHD.
METHODS: We established the monocrotaline plus aorto-cava shunt-induced (MCT-AV) rat model. We measured the expression of THBS4 in lung tissues from MCT-AV rats. Double immunofluorescence staining of lung tissue for THBS4 and α-SMA (biomarker of smooth muscle cells) or vWF (biomarker of endothelial cells) to identify the location of THBS4 in the pulmonary artery. Primary pulmonary artery smooth muscle cells (PASMCs) were cultivated, identified, and used in this study. THBS4 was inhibited and overexpressed by siRNA and plasmid, respectively, to explore the effect of THBS4 on phenotype transformation, proliferation, apoptosis, and migration of PASMCs. The effect of THBS4 on pulmonary vascular remodeling was evaluated in vivo by adeno-associated virus which suppressed THBS4 expression. Circulating level of THBS4 in patients with PAH-CHD was measured by ELISA.
RESULTS: THBS4 was upregulated in the lung tissues of MCT-AV rats, and was further upregulated in severe pulmonary vascular lesions. And THBS4 was expressed mainly in PASMCs. When THBS4 was inhibited, contractile markers α-SMA and MYH11 were upregulated, while the proliferative marker PCNA was decreased, the endothelial-mensenchymal transition marker N-cad was downregulated, proapototic marker BAX was increased. Additionally, proliferation and migration of PASMCs was inhibited and apoptosis was increased. Conversely, THBS4 overexpression resulted in opposite effects. And the impact of THBS4 on PASMCs was probably achieved through the regulation of the PI3K/AKT pathway. THBS4 suppression attenuated pulmonary vascular remodeling. Furthermore, compared with patients with simple congenital heart disease and mild PAH-CHD, the circulating level of THBS4 was higher in patients with severe PAH-CHD.
CONCLUSIONS: THBS4 is a promising biomarker to distinguish reversible from irreversible PAH-CHD before repairing the shunt. THBS4 is a potential treatment target in PAH-CHD, especially in irreversible stage.