Enhanced P2X7 Activity in Human Fibroblasts From Diabetic Patients: A Possible Pathogenetic Mechanism for Vascular Damage in Diabetes
Enhanced P2X7 Activity in Human Fibroblasts From Diabetic Patients: A Possible Pathogenetic Mechanism for Vascular Damage in Diabetes
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DOI：
10.1161/01.atv.0000133193.11078.c0
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发表时间：
2004-07
期刊：
影响因子：
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通讯作者：
A. Solini;P. Chiozzi;A. Morelli;E. Adinolfi;R. Rizzo;O. Baricordi;F. Di Virgilio
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文献类型：
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作者：
A. Solini;P. Chiozzi;A. Morelli;E. Adinolfi;R. Rizzo;O. Baricordi;F. Di Virgilio
我们研究了P2 X7受体在健康受试者和2型糖尿病患者成纤维细胞中的表达和功能。方法和结果成纤维细胞从皮肤活检中分离。通过功能测定、RT-PCR、荧光激活细胞分选仪和免疫印迹法测定两种细胞群中的P2 X7受体表达。我们发现，糖尿病受试者的成纤维细胞的特征在于增强P2 X7介导的反应，如增加的形状变化，微泡形成，增强的纤连蛋白和白细胞介素6分泌，和加速凋亡。通过与P2 X阻断剂KN-62、氧化ATP或磷酸吡哆醛-6-偶氮（苯-2，4-二磺酸）预孵育来阻断这些反应。此外，我们还发现，与健康受试者相比，糖尿病患者成纤维细胞中自发性纤维连接蛋白分泌和凋亡水平较高。更高的纤连蛋白分泌基础水平和自发凋亡率可能归因于细胞周围ATP浓度的增加，因为与健康受试者的成纤维细胞相比，糖尿病受试者的成纤维细胞向上清液中释放的ATP是健康受试者的3倍。结论：我们得出结论，2型糖尿病患者的成纤维细胞的特征是基于更高水平的ATP释放或P2 X7反应性增加的过度活跃的嘌呤能回路。
Objective—We have investigated expression and function of the P2X7 receptor in fibroblasts from healthy subjects and patients with type 2 diabetes. Methods and Results—Fibroblasts were isolated from skin biopsies. P2X7 receptor expression in both cell populations was measured by functional assays, RT-PCR, fluorescence-activated cell sorter, and immunoblotting. We found that fibroblasts from diabetic subjects are characterized by enhanced P2X7-mediated responses as indicated by increased shape changes, microvesiculation, enhanced fibronectin and interleukin 6 secretion, and accelerated apoptosis. These responses were blocked by preincubation with the P2X blockers KN-62, oxidized ATP, or pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid). Furthermore, we also found a higher level of spontaneous fibronectin secretion and of apoptosis in fibroblasts from diabetic compared with healthy subjects. Both higher basal level of fibronectin secretion and spontaneous rate of apoptosis were likely attributable to the increased pericellular concentration of ATP because fibroblasts from diabetic subjects released 3× as much ATP into the supernatants compared with fibroblasts from healthy subjects. Conclusions—We conclude that fibroblasts from type 2 diabetes patients are characterized by a hyperactive purinergic loop based either on a higher level of ATP release or on increased P2X7 reactivity.