Department of Orthopaedic Surgery, Graduate School of Medicine, Yokohama City University, Yokohama, Japan.
Department of Orthopaedic Surgery, Yokohama City University Medical Center, Yokohama, Japan.
PLoS One. 2023 Oct 31;18(10):e0293463. doi: 10.1371/journal.pone.0293463. eCollection 2023.
Previous work suggested that tenogenic differentiation of tendon stem/progenitor cells (TSPCs) was suppressed by upregulated expression of the angiogenic marker vascular endothelial growth factor (VEGF). The purpose of this study was to test the hypothesis that anti-VEGF antibody, bevacizumab, promotes in vitro tenogenic differentiation and maturation of two distinct types of TSPCs, tendon proper-derived cells (TDCs), and paratenon-derived cells (PDCs) originating from rat Achilles tendon. TDCs and PDCs were isolated from the tendon proper and the paratenon of rat Achilles tendons. TDCs and PDCs were cultured for 3 days on plates with or without VEGF. TDCs and PDCs were also cultured in collagen gel matrix, and the blocking effect of VEGF was examined by the addition of 100 ng/mL of bevacizumab. Effects of bevacizumab on tenogenic differentiation were assessed using real-time PCR, immunofluorescent staining, and western blotting. VEGF significantly attenuated expression of the Tnmd gene in both PDCs and TDCs (P<0.05). Expressions of the Scx, Tnmd, and Col1a1 genes were significantly upregulated by the addition of bevacizumab (P<0.05). Immunofluorescent staining showed that the percentage of tenomodulin-positive PDCs and TDCs was significantly higher with bevacizumab treatment than in control cultures (P<0.05). Western blotting showed that bevacizumab suppressed pVEGFR-2 protein expression in both PDCs and TDCs. Bevacizumab promoted the in vitro tenogenic differentiation and maturation of two distinct TSPCs derived from rat Achilles tendon. Since the previous studies demonstrated that TSPCs have a potential to contribute to tendon repair, attenuating VEGF levels in TSPCs by administration of bevacizumab is a novel candidate therapeutic option for promoting tendon repair.
先前的工作表明，肌腱干细胞/祖细胞 (TSPCs) 的成肌腱分化受到血管内皮生长因子 (VEGF) 表达上调的抑制。本研究的目的是验证这样一个假设，即抗 VEGF 抗体贝伐单抗促进两种不同类型的 TSPCs，即肌腱固有细胞 (TDCs) 和来自大鼠跟腱的腱旁细胞 (PDCs) 的体外成肌腱分化和成熟。TDCs 和 PDCs 从大鼠跟腱的肌腱固有部分和腱旁组织中分离出来。将 TDCs 和 PDCs 分别在含有或不含有 VEGF 的培养板上培养 3 天。将 TDCs 和 PDCs 也在胶原凝胶基质中培养，并通过添加 100ng/mL 的贝伐单抗来检测 VEGF 的阻断作用。使用实时 PCR、免疫荧光染色和 Western blot 评估贝伐单抗对成肌腱分化的影响。VEGF 显著降低了 PDCs 和 TDCs 中 Tnmd 基因的表达 (P<0.05)。添加贝伐单抗后，Scx、Tnmd 和 Col1a1 基因的表达显著上调 (P<0.05)。免疫荧光染色显示，贝伐单抗处理后的腱调蛋白阳性 PDCs 和 TDCs 的百分比明显高于对照组 (P<0.05)。Western blot 显示，贝伐单抗抑制了 PDCs 和 TDCs 中 pVEGFR-2 蛋白的表达。贝伐单抗促进了两种不同的源自大鼠跟腱的 TSPCs 的体外成肌腱分化和成熟。由于先前的研究表明 TSPCs 具有促进肌腱修复的潜力，因此通过给予贝伐单抗来降低 TSPCs 中的 VEGF 水平是促进肌腱修复的一种新的候选治疗方法。
