Supplementary Material for: Cyclic Mechanical Stretch Induced Smooth Muscle Cell Changes in Cerebral Aneurysm Progress by Reducing Collagen Type IV and Collagen Type VI Levels

<b><i>Background/Aims:</i></b> Cerebral aneurysm growth is characterized by continuous structural weakness of local smooth muscle cells, though the mechanism is unclear. In this study, we examine protein changes in cerebral aneurysm and human brain vascular smooth muscle cells after cyclic mechanical stretch. We further explore the relationship between the smooth muscle cell changes and reductions in the levels of collagen types IV and VI. <b><i>Methods:</i></b> Saccular cerebral aneurysms (n=10) were collected, and temporal artery samples were used as controls. Quantitative proteomics were analyzed and histopathological changes were examined. Smooth muscle cells were cultured in a flexible silicone chamber and subjected to 15% cyclic mechanical stretch. The effect of stretch on the cell viability, function, gene and protein expression were further studied for the understanding the molecular mechanism of aneurysm development. <b><i>Results:</i></b> Proteomics analysis revealed 92 proteins with increased expression and 88 proteins with decreased expression compared to the controls (<i>p</i>&lt;0.05). KEGG pathway analysis showed that the change in focal adhesion and extracellular matrix-receptor interaction, suggesting the involvement of collagen type IV and VI. The aneurysm tissue exhibited fewer smooth muscle cells and lower levels of collagen type IV and VI. Human brain vascular smooth muscle cell culture showed spindle-like cells and obvious smooth muscle cell layer. Cell proteomics analysis showed that decreased expression of 118 proteins and increased expression of 32 proteins in smooth muscle cells after cyclic mechanical stretch. KEGG pathway analysis indicated that focal adhesion and ECM-receptor interaction were involved. After cyclic mechanical stretch, collagen type IV and IV expression were decreased. Moreover, the stretch induced MMP-1 and MMP-3 expression elevation. <b><i>Conclusion:</i></b> We demonstrated that collagen type IV and VI were decreased in cerebral aneurysms and continuous cyclic mechanical stretch induced smooth muscle cell changes. Smooth muscle cell protection provides an additional therapeutic option to prevent the growth of cerebral aneurysms.

**背景与目的**：脑动脉瘤的生长以局部平滑肌细胞持续性结构薄弱为特征，但其具体机制尚未阐明。本研究旨在探讨周期性机械牵张后，脑动脉瘤组织及人脑血管平滑肌细胞内的蛋白表达变化，并进一步阐明平滑肌细胞改变与IV型、VI型胶原蛋白水平降低之间的关联。
**方法**：收集囊状脑动脉瘤标本10例，以颞动脉标本作为对照。开展定量蛋白质组学分析，并观察组织病理学变化。将平滑肌细胞接种于柔性硅胶培养腔中，施加15%幅度的周期性机械牵张。进一步研究牵张对细胞活力、功能、基因及蛋白表达的影响，以阐明动脉瘤发生发展的分子机制。
**结果**：蛋白质组学分析显示，与对照组相比，共有92种蛋白表达上调、88种蛋白表达下调（*P*<0.05）。京都基因与基因组百科全书（KEGG）通路富集分析显示，黏着斑与细胞外基质-受体互作通路发生显著改变，提示IV型与VI型胶原蛋白可能参与动脉瘤病理过程。动脉瘤组织中平滑肌细胞数量减少，IV型及VI型胶原蛋白水平降低。人脑血管平滑肌细胞培养可见梭形细胞及完整的平滑肌细胞层。细胞蛋白质组学分析显示，周期性机械牵张后平滑肌细胞中有118种蛋白表达下调、32种蛋白表达上调。KEGG通路分析显示，黏着斑与细胞外基质（extracellular matrix, ECM）-受体互作通路被激活。周期性机械牵张后，IV型及VI型胶原蛋白表达水平降低。此外，牵张可诱导基质金属蛋白酶-1（matrix metalloproteinase, MMP-1）与基质金属蛋白酶-3（MMP-3）的表达上调。
**结论**：本研究证实，脑动脉瘤组织中IV型与VI型胶原蛋白表达降低，持续周期性机械牵张可诱导平滑肌细胞发生功能改变。保护平滑肌细胞可为预防脑动脉瘤生长提供新的治疗策略。