Sasagawa Shota, Nishimura Yuhei, Sawada Hirofumi, Zhang Erquan, Okabe Shiko, Murakami Soichiro, Ashikawa Yoshifumi, Yuge Mizuki, Kawaguchi Koki, Kawase Reiko, Mitani Yoshihide, Maruyama Kazuo, Tanaka Toshio
Department of Molecular and Cellular Pharmacology, Pharmacogenomics and Pharmacoinformatics, Mie University Graduate School of Medicine, Tsu Japan.
Department of Molecular and Cellular Pharmacology, Pharmacogenomics and Pharmacoinformatics, Mie University Graduate School of Medicine, TsuJapan; Mie University Medical Zebrafish Research Center, TsuJapan; Department of Systems Pharmacology, Mie University Graduate School of Medicine, TsuJapan; Department of Omics Medicine, Mie University Industrial Technology Innovation Institute, TsuJapan; Department of Bioinformatics, Mie University Life Science Research Center, TsuJapan.
Front Pharmacol. 2016 Jun 7;7:142. doi: 10.3389/fphar.2016.00142. eCollection 2016.
Pulmonary arterial hypertension (PAH) is a heterogeneous disorder associated with a progressive increase in pulmonary artery resistance and pressure. Although various therapies have been developed, the 5-year survival rate of PAH patients remains low. There is thus an important need to identify novel genes that are commonly dysregulated in PAH of various etiologies and could be used as biomarkers and/or therapeutic targets. In this study, we performed comparative transcriptome analysis of five mammalian PAH datasets downloaded from a public database. We identified 228 differentially expressed genes (DEGs) from a rat PAH model caused by inhibition of vascular endothelial growth factor receptor under hypoxic conditions, 379 DEGs from a mouse PAH model associated with systemic sclerosis, 850 DEGs from a mouse PAH model associated with schistosomiasis, 1598 DEGs from one cohort of human PAH patients, and 4260 DEGs from a second cohort of human PAH patients. Gene-by-gene comparison identified four genes that were differentially upregulated or downregulated in parallel in all five sets of DEGs. Expression of coiled-coil domain containing 80 (CCDC80) and anterior gradient two genes was significantly increased in the five datasets, whereas expression of SMAD family member six and granzyme A was significantly decreased. Weighted gene co-expression network analysis revealed a connection between CCDC80 and collagen type I alpha 1 (COL1A1) expression. To validate the function of CCDC80 in vivo, we knocked out ccdc80 in zebrafish using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. In vivo imaging of zebrafish expressing a fluorescent protein in endothelial cells showed that ccdc80 deletion significantly increased the diameter of the ventral artery, a vessel supplying blood to the gills. We also demonstrated that expression of col1a1 and endothelin-1 mRNA was significantly decreased in the ccdc80-knockout zebrafish. Finally, we examined Ccdc80 immunoreactivity in a rat PAHmodel and found increased expression in the hypertrophied media and adventitia of the pre-acinar pulmonary arteries (PAs) and in the thickened intima, media, and adventitia of the obstructed intra-acinar PAs. These results suggest that increased expression of CCDC80 may be involved in the pathogenesis of PAH, potentially by modulating the expression of endothelin-1 and COL1A1.
肺动脉高压（PAH）是一种异质性疾病，与肺动脉阻力和压力的逐渐升高相关。尽管已经开发了多种治疗方法，但PAH患者的5年生存率仍然很低。因此，迫切需要鉴定在各种病因的PAH中普遍失调的新基因，这些基因可作为生物标志物和/或治疗靶点。在本研究中，我们对从公共数据库下载的五个哺乳动物PAH数据集进行了比较转录组分析。我们从低氧条件下抑制血管内皮生长因子受体引起的大鼠PAH模型中鉴定出228个差异表达基因（DEG），从与系统性硬化症相关的小鼠PAH模型中鉴定出379个DEG，从与血吸虫病相关的小鼠PAH模型中鉴定出850个DEG，从一组人类PAH患者中鉴定出1598个DEG，从另一组人类PAH患者中鉴定出4260个DEG。逐基因比较确定了在所有五组DEG中平行上调或下调的四个基因。在五个数据集中，含卷曲螺旋结构域80（CCDC80）和前梯度2基因的表达显著增加，而SMAD家族成员6和颗粒酶A的表达显著降低。加权基因共表达网络分析揭示了CCDC80与I型胶原α1（COL1A1）表达之间的联系。为了在体内验证CCDC80的功能，我们使用成簇规律间隔短回文重复序列（CRISPR）/Cas9系统在斑马鱼中敲除了ccdc80。对在内皮细胞中表达荧光蛋白的斑马鱼进行体内成像显示，ccdc80缺失显著增加了腹侧动脉的直径，腹侧动脉是为鳃供血的血管。我们还证明，在ccdc80敲除的斑马鱼中，col1a1和内皮素-1 mRNA的表达显著降低。最后，我们在大鼠PAH模型中检测了Ccdc80免疫反应性，发现在腺泡前肺动脉（PA）肥厚的中膜和外膜以及阻塞的腺泡内PA增厚的内膜、中膜和外膜中表达增加。这些结果表明，CCDC80表达增加可能参与PAH的发病机制，可能是通过调节内皮素-1和COL1A1的表达。
