腹主動脈瘤(AAA)是一種嚴重的慢性血管疾病，其特徵是由於主動脈壁的局部受損造成腹主動脈膨脹。而在AAA中若發生主動脈鈣化，會導致主動脈變硬，同時鈣化也顯著增加AAA破裂的風險，造成高死亡率。在探討血管鈣化機制中，血管平滑肌細胞(VSMC)是關鍵角色，VSMC可以藉由從收縮態(contractile)轉變為成骨態(osteogenic)來調控血管鈣化影響血管重塑。CCN1 (Cellular communication network factor 1)是一種基質細胞蛋白，可以透過與不同的整合素結合來調節不同細胞活動，包括細胞增殖、分化、遷移、黏附、凋亡和老化。我們先前的研究表明，CCN1會透過結合整合素α6β1參與AAA形成，從而促進發炎和氧化壓力。我們推測CCN1也會參與在血管鈣化中，藉由與整合素α6β1結合去調控VSMC的成骨轉化，從而促進AAA的鈣化。為了探討這個假設，我們使用高濃度鈣離子(+ 1.8 mM 氯化鈣) 培養基建立體外VSMC鈣化模型，使用茜素紅(ARS)染色確認VSMC鈣化。透過西方墨點法檢測到CCN1會在給予氯化鈣後1小時內被誘導，在12小時後下降；而成骨基因 RUNX2 在1-12小時內升高，去分化基因 KLF4 在3小時後被誘導，這些結果顯示高濃度鈣處理促使VSMC轉變為成骨態。為了測試CCN1是否透過整合素α6β1影響VSMC鈣化，我們使用特定的拮抗劑T1 peptide來阻斷CCN1和整合素α6β1之間的結合。結果顯示使用30 μM T1 peptide處理可以有效抑制VSMC鈣化，顯著降低RUNX2和KLF4表達，證實CCN1會透過整合素α6β1調控成骨基因RUNX2和去分化基因 KLF4並促進VSMC鈣化。在動物實驗中我們使用氯化鈣誘導AAA模型在WT 和Ccn1dm/dm小鼠(CCN1上α6β1的結合位點突變導致CCN1無法與α6β1結合)探討CCN1的作用。我們發現Ccn1dm/dm小鼠的AAA形成和鈣化皆受到抑制，同時發現Ccn1dm/dm小鼠中RUNX2缺乏表現，此結果相呼應細胞實驗。我們的研究結果支持針對拮抗CCN1和整合素α6β1相互作用發展新的治療策略，來抑制VSMC成骨分化，從而減輕AAA中血管鈣化負擔。

Abdominal aortic aneurysm (AAA) is a serious chronic vascular disease characterized by the localized weakening and bulging of the abdominal aorta. Calcification of the vessel wall is associated with arterial stiffening, significantly increasing the risk of rupture. Vascular smooth muscle cells (VSMCs) play a pivotal role in aneurysm formation. The aberrant phenotypic switching of VSMCs from a contractile to an osteogenic-like state is a critical mechanism underlying arterial remodeling and vascular calcification. CCN1 (Cellular communication network factor 1) is a matricellular protein that regulates a variety of cellular activities, including cell proliferation, differentiation, migration, adhesion, apoptosis, and senescence through binding to different integrin receptors. Our previous in vivo data indicate that CCN1 participates in AAA formation via its receptor integrin α6β1 to promote inflammation and oxidative stress. We hypothesized that CCN1 promotes calcification in AAA by modulating osteogenic transformation in VSMCs through activating integrin α6β1 signaling. To test this hypothesis, we established an in vitro VSMC calcification model using high-calcium (+1.8 mM CaCl2) media. Cellular calcification was revealed by Alizarin red S staining. CCN1 protein expression in VSMCs was induced within 1 hour and started to decline 12 hours after CaCl2 treatment, as examined by western blotting. The osteogenic gene Runx2 was elevated between 1-12 hours, and the dedifferentiation marker KLF4 was induced after 3 hours, indicating a reprogramming of VSMCs towards an osteogenic phenotype by CaCl2. To test the requirement of integrin α6β1, a specific antagonistic T1 peptide was used to block the binding between CCN1 and integrin α6β1 during VSMC calcification. T1 peptide (30 μM) effectively inhibited calcification and significantly reduced Runx2 and KLF4 expression, indicating that CCN1 promotes VSMC calcification through integrin α6β1, Runx2, and KLF4 signaling. In an in vivo study, we used the CaCl2-induced AAA model to investigate the role of CCN1 and used Ccn1dm/dm mice, which carry a gene encoding an α6β1-binding deficient CCN1 mutant, to block CCN1 function. We found that AAA formation and calcification were inhibited in Ccn1dm/dm mice compared to WT controls. The absence of Runx2 expression in Ccn1dm/dm mice suggests that CCN1 regulates Runx2 signaling in AAA calcification through binding to integrin α6β1. Our findings indicate that therapeutic strategies targeting the interaction between CCN1 and integrin α6β1 may be effective in modulating VSMC phenotypic switching, thereby reducing the vascular calcification burden in AAA.

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