Institute of Biopharmaceutical Science, National Yang Ming Chiao Tung University, Taipei, Taiwan (T.-Y.T., Y.S.).
Departments of Medical Research (T.-Y.T., C.-L.H., B.-J.W., Y.-N.L.), MacKay Memorial Hospital, Taipei, Taiwan.
Arterioscler Thromb Vasc Biol. 2023 Oct;43(10):1935-1951. doi: 10.1161/ATVBAHA.123.319529. Epub 2023 Aug 17.
We examined the role of Panxs (pannexins) in human endothelial progenitor cell (EPC) senescence.
Young and replication-induced senescent endothelial colony-forming cells (ECFCs) derived from human circulating EPCs were used to examine cellular activities and senescence-associated indicators after transfection of short interference RNA specific to Panx1 or lentivirus-mediated Panx1 overexpression. Hind limb ischemia mice were used as in vivo angiogenesis model. Protein and phospho-kinase arrays were used to determine underlying mechanisms.
Panx1 was the predominant Panx isoform in human ECFCs and upregulated in both replication-induced senescent ECFCs and circulating EPCs from aged mice and humans. Cellular activities of the young ECFCs were enhanced by Panx1 downregulation but attenuated by its upregulation. In addition, reduction of Panx1 in the senescent ECFCs could rejuvenate cellular activities with reduced senescence-associated indicators, including senescence-associated β-galactosidase activity, p16 (cyclin-dependent kinase inhibitor 2A), p21 (cyclin-dependent kinase inhibitor 1), acetyl-p53 (tumor protein P53), and phospho-histone H2A.X (histone family member X). In mouse ischemic hind limbs injected senescent ECFCs, blood perfusion ratio, salvaged limb outcome, and capillary density were all improved by Panx1 knockdown. IGF-1 (insulin-like growth factor 1) was significantly increased in the supernatant from senescent ECFCs after Panx1 knockdown. The enhanced activities and paracrine effects of Panx1 knockdown senescent ECFCs were completely inhibited by anti-IGF-1 antibodies. FAK (focal adhesion kinase), ERK (extracellular signal-regulated kinase), and STAT3 (signal transducer and activator of transcription 3) were activated in senescent ECFCs with Panx1 knockdown, in which the intracellular calcium level was reduced, and the activation was inhibited by supplemented calcium. The increased IGF-1 in Panx1-knockdown ECFCs was abrogated, respectively, by inhibitors of FAK (PF562271), ERK (U0126), and STAT3 (NSC74859) and supplemented calcium.
Panx1 expression is upregulated in human ECFCs/EPCs with replication-induced senescence and during aging. Angiogenic potential of senescent ECFCs is improved by Panx1 reduction through increased IGF-1 production via activation of the FAK-ERK axis following calcium influx reduction. Our findings provide new strategies to evaluate EPC activities and rejuvenate senescent EPCs for therapeutic angiogenesis.
我们研究了 Panx（pannexin）在人内皮祖细胞（EPC）衰老中的作用。
从人循环 EPC 中分离得到的年轻和复制诱导衰老的内皮集落形成细胞（ECFC），在转染 Panx1 特异性短发夹 RNA 或慢病毒介导的 Panx1 过表达后，用于检测细胞活性和衰老相关指标。利用后肢缺血小鼠作为体内血管生成模型。使用蛋白质和磷酸激酶谱来确定潜在的机制。
Panx1 是人 ECFC 中主要的 Panx 同工型，在复制诱导的衰老 ECFC 以及来自老年小鼠和人类的循环 EPC 中均上调。Panx1 下调可增强年轻 ECFC 的细胞活性，而上调则减弱其活性。此外，衰老 ECFC 中 Panx1 的减少可恢复细胞活性，同时减少衰老相关指标，包括衰老相关β-半乳糖苷酶活性、p16（细胞周期蛋白依赖性激酶抑制剂 2A）、p21（细胞周期蛋白依赖性激酶抑制剂 1）、乙酰化-p53（肿瘤蛋白 P53）和磷酸化组蛋白 H2A.X（组蛋白家族成员 X）。在注射衰老 ECFC 的缺血后肢小鼠中，Panx1 敲低可改善血液灌注比、挽救肢体结局和毛细血管密度。Panx1 敲低后，衰老 ECFC 上清液中 IGF-1（胰岛素样生长因子 1）显著增加。抗 IGF-1 抗体完全抑制了 Panx1 敲低的衰老 ECFC 的增强活性和旁分泌作用。FAK（粘着斑激酶）、ERK（细胞外信号调节激酶）和 STAT3（信号转导和转录激活因子 3）在 Panx1 敲低的衰老 ECFC 中被激活，其中细胞内钙离子水平降低，并用补充钙抑制激活。在 Panx1 敲低的 ECFC 中，IGF-1 的增加分别被 FAK（PF562271）、ERK（U0126）和 STAT3（NSC74859）抑制剂和补充钙所阻断。
在复制诱导衰老和衰老过程中，人 ECFC/EPC 中 Panx1 的表达上调。通过减少 Panx1，通过减少细胞内钙离子流入，激活 FAK-ERK 轴来增加 IGF-1 的产生，从而改善衰老 ECFC 的血管生成潜力。我们的研究结果为评估 EPC 活性和恢复衰老 EPC 的治疗性血管生成提供了新的策略。
