Department of Internal Medicine, Division of Cardiovascular, Kyung Hee University Hospital, Kyung Hee University, Seoul, Korea.
Department of Biotechnology, Cha University, Pocheon, Korea.
PLoS One. 2024 May 20;19(5):e0303758. doi: 10.1371/journal.pone.0303758. eCollection 2024.
Nitric oxide (NO) promotes angiogenesis via various mechanisms; however, the effective transmission of NO in ischemic diseases is unclear. Herein, we tested whether NO-releasing nanofibers modulate therapeutic angiogenesis in an animal hindlimb ischemia model. Male wild-type C57BL/6 mice with surgically-induced hindlimb ischemia were treated with NO-releasing 3-methylaminopropyltrimethoxysilane (MAP3)-derived or control (i.e., non-NO-releasing) nanofibers, by applying them to the wound for 20 min, three times every two days. The amount of NO from the nanofiber into tissues was assessed by NO fluorometric assay. The activity of cGMP-dependent protein kinase (PKG) was determined by western blot analysis. Perfusion ratios were measured 2, 4, and 14 days after inducing ischemia using laser doppler imaging. On day 4, Immunohistochemistry (IHC) with F4/80 and gelatin zymography were performed. IHC with CD31 was performed on day 14. To determine the angiogenic potential of NO-releasing nanofibers, aorta-ring explants were treated with MAP3 or control fiber for 20 min, and the sprout lengths were examined after 6 days. As per either LDPI (Laser doppler perfusion image) ratio or CD31 capillary density measurement, angiogenesis in the ischemic hindlimb was improved in the MAP3 nanofiber group; further, the total nitrate/nitrite concentration in the adduct muscle increased. The number of macrophage infiltrations and matrix metalloproteinase-9 (MMP-9) activity decreased. Vasodilator-stimulated phosphoprotein (VASP), one of the major substrates for PKG, increased phosphorylation in the MAP3 group. MAP3 nanofiber or NO donor SNAP (s-nitroso-n-acetyl penicillamine)-treated aortic explants showed enhanced sprouting in an ex vivo aortic ring assay, which was partially abrogated by KT5823, a potent inhibitor of PKG. These findings suggest that the novel NO-releasing nanofiber, MAP3 activates PKG and promotes therapeutic angiogenesis in response to hindlimb ischemia.
一氧化氮（NO）通过多种机制促进血管生成；然而，NO 在缺血性疾病中的有效传递尚不清楚。在此，我们测试了释放一氧化氮的纳米纤维是否能调节动物后肢缺血模型中的治疗性血管生成。通过将 NO 释放的 3-甲基氨丙基三甲氧基硅烷（MAP3）衍生的纳米纤维或对照（即非 NO 释放）纳米纤维应用于伤口 20 分钟，每两天三次，对手术诱导后肢缺血的雄性野生型 C57BL/6 小鼠进行处理。通过 NO 荧光测定法评估纳米纤维进入组织的 NO 量。通过 Western blot 分析测定 cGMP 依赖性蛋白激酶（PKG）的活性。用激光多普勒成像术在缺血后 2、4 和 14 天测量灌注比。在第 4 天，进行 F4/80 和明胶酶谱免疫组织化学（IHC）。在第 14 天进行 CD31 的 IHC。为了确定释放一氧化氮的纳米纤维的血管生成潜力，用 MAP3 或对照纤维处理主动脉环外植体 20 分钟，然后在 6 天后检查芽的长度。根据激光多普勒灌注图像（LDPI）比率或 CD31 毛细血管密度测量，缺血后肢的血管生成在 MAP3 纳米纤维组中得到改善；此外，附加肌肉中的总硝酸盐/亚硝酸盐浓度增加。巨噬细胞浸润和基质金属蛋白酶-9（MMP-9）活性减少。血管扩张刺激磷蛋白（VASP），PKG 的主要底物之一，在 MAP3 组中磷酸化增加。MAP3 纳米纤维或 NO 供体 SNAP（s-亚硝基-n-乙酰青霉胺）处理的主动脉外植体在体外主动脉环测定中显示出增强的发芽，这在一定程度上被 PKG 的强效抑制剂 KT5823 阻断。这些发现表明，新型的 NO 释放纳米纤维 MAP3 通过激活 PKG 来促进对后肢缺血的治疗性血管生成。
