Inability of vascular smooth muscle cells to proceed beyond S phase of cell cycle, and increased apoptosis in symptomatic carotid artery disease
Inability of vascular smooth muscle cells to proceed beyond S phase of cell cycle, and increased apoptosis in symptomatic carotid artery disease
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DOI：
10.1016/s0741-5214(02)75463-3
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发表时间：
2003-07-01
影响因子：
4.3
通讯作者：
Agrawal, DK
中科院分区：
文献类型：
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作者：
Dhume, AS;Agrawal, DK
目标。颈动脉高度狭窄下游的微栓子通过大脑动脉可产生短暂性缺血症状，并可能导致中风。颈动脉斑块破裂是颈内动脉高度狭窄微栓子的主要来源。然而，斑块破裂的机制尚不清楚。我们假设与无症状患者相比，颈动脉狭窄患者斑块中的血管平滑肌细胞（VSMC）凋亡增加，增殖减少。在有颈动脉狭窄症状的患者（如半球短暂性脑缺血发作、黑朦或中风）和无症状的高度狭窄患者中，通过酶解方法从颈动脉内膜切除术去除的斑块中分离出VSMC。培养VSMC并用平滑肌a-actin和caldesmon抗体进行免疫染色以确保其纯度。TUNEL法和膜联蛋白V标记法鉴定凋亡的VSMC。用胎牛血清（FBS）刺激VSMC进行了[H-3]胸苷结合增殖实验，并用Vindelov试剂进行DNA染色，分析细胞周期谱。我们从有明显溃疡的症状斑块和无症状斑块中分离出VSMC。通过TUNEL检测，有症状斑块的VSMC细胞凋亡率为5.45% +/- 0.8%，无症状斑块的VSMC细胞凋亡率为1.20% +/- 0.2%。膜联蛋白V标记显示，有症状斑块VSMC中有26.8% +/- 3.8%的细胞被标记为磷脂酰丝氨酸，而无症状斑块中有4.8% +/- 0.3%的细胞被标记为磷脂酰丝氨酸。与有症状斑块相比，无症状斑块的VSMC在所有浓度的FBS下都明显增加了[H-3]胸苷嘧啶的摄取。在10% FBS存在的情况下，无症状斑块的VSMC进展到细胞周期的S期，而明显增加的有症状斑块的VSMC数量在S期被阻止。结论：与无症状斑块的VSMC相比，有症状斑块的VSMC数量增加并发生凋亡。这可能是由于VSMC不能从症状斑块发展到细胞周期的S期。在有症状的斑块中，由于细胞凋亡导致的VSMC增殖减少和损失增加可能导致斑块破裂，从而导致症状的发展。
Objective. Microemboli passing through the cerebral artery downstream from high-grade carotid artery stenosis produce transient ischemic symptoms and may result in stroke. Rupture of carotid artery plaque is the main source of microemboli in high-grade internal carotid artery stenosis. However, the mechanisms underlying plaque rupture are unclear. We hypothesized that vascular smooth muscle cells (VSMC) from plaque in patients with symptoms of carotid artery stenosis undergo increased apoptosis and decreased proliferation, compared with VSMC in patients without symptoms.Methods. VSMC were isolated by means of enzymatic dissociation from plaque removed at carotid endarterectomy in patients with symptoms of carotid artery stenosis, eg, hemispheric transient ischemic attacks, amaurosis fugax, or stroke, and patients with high-grade stenosis without symptoms. VSMC were cultured and immunostained with smooth muscle a-actin and caldesmon antibodies to ensure purity. TUNEL assay and annexin V labeling were performed to identify VSMC undergoing apoptosis. Proliferation assay with [H-3] thymidine incorporation was performed in VSMC stimulated with fetal bovine serum (FBS), and cell cycle profile was analyzed with DNA staining with Vindelov reagent.Results. We isolated VSMC from symptomatic plaque that showed gross ulceration, and asymptomatic plaque. Apoptosis, as measured with the TUNEL assay, in VSMC from symptomatic plaque was 5.45% +/- 0.8%, and in asymptomatic plaque was 1.20% +/- 0.2%. Annexin V labeling revealed that 26.8% +/- 3.8% cells were labeled for phosphatidylserine in VSMC in symptomatic plaque, compared with 4.8% +/- 0.3% cells in asymptomatic plaque. VSMC in asymptomatic plaque showed significantly increased uptake of [H-3] thymidine at all concentrations of FBS, compared with symptomatic plaque. In the presence of 10% FBS, VSMC from asymptomatic plaque progressed through the S phase of the cell cycle, whereas significantly increased numbers of VSMC from symptomatic plaque were arrested in the S phase.Conclusion: Increased numbers of VSMC from symptomatic plaque undergo apoptosis, compared with VSMC from asymptomatic plaque. This could be due to inability of VSMC from symptomatic plaque to progress beyond the S phase of the cell cycle. Decreased proliferation and increased loss of VSMC as a result of apoptosis in symptomatic plaque may result in plaque rupture, leading to development of symptoms.