Lan Wenya, Yang Fang, Li Zhuangli, Liu Ling, Sang Hongfei, Jiang Yongjun, Xiong Yunyun, Zhang Renliang
Department of Neurology, Jinling Hospital, Medical School of Nanjing University, Nanjing, China; Department of Geriatric Neurology, Nanjing Brain Hospital Affiliated to Nanjing Medical University, Nanjing, China.
Department of Neurology, Jinling Hospital, Medical School of Nanjing University, Nanjing, China.
J Vasc Surg. 2016 Oct;64(4):1074-83. doi: 10.1016/j.jvs.2015.04.433. Epub 2015 Jun 6.
Effective treatments against restenosis after percutaneous transluminal angioplasty and stenting are largely lacking. Human tissue kallikrein gene transfer has been shown to be able to attenuate neointima formation induced by balloon catheter. As a tissue kallikrein in vivo, human urinary kininogenase (HUK) is widely used to prevent ischemia-reperfusion injury. However, the effects of HUK on neointima formation have not been explored. We therefore investigated whether HUK could alleviate balloon catheter-induced intimal hyperplasia in rabbits fed with high-fat diets.
The effects of HUK on neointima and atherosclerosis formation were analyzed by hematoxylin-eosin staining and immunohistochemical staining in balloon-injured carotid arteries of rabbits. Local inflammatory response was evaluated by detecting the gene expression of tumor necrosis factor α and interleukin 1β with real-time quantitative polymerase chain reaction plus the invasion of macrophages with immunohistochemical staining. Western blotting was employed to investigate the effects of HUK on activities of endothelial nitric oxide synthase (eNOS), transforming growth factor β1 (TGF-β1), and Smad signaling pathway. The long-term effect of HUK on intimal hyperplasia of the injured carotid artery was assessed by angiography.
Quantitative image analysis showed that intravenous administration of HUK for 14 days significantly decreased the intimal areas and intima area/media area ratios (day 14, 54% decrease in intimal area and 58% decrease in intima area/media area ratios; day 28, 63% and 85%). Significant decreases were also noted in macrophage foam cell-positive area after 7-day or 14-day administration of HUK (day 7, 69% decrease in intimal area and 78% decrease in media area; day 14, 79% and 60%; day 28, 68% and 44%). Actin staining for smooth muscle cells in neointima at 2 months showed similar results (vascular smooth muscle cell-positive area of neointima, 28.21% ± 5.58% vs 43.78% ± 8.36%; P < .05). Real-time quantitative polymerase chain reaction or Western blot analysis showed that HUK reduced expression of tumor necrosis factor α, interleukin 1β, TGF-β1, and p-Smad2/3 but increased the expression of p-eNOS. Angiography analysis showed that 14-day administration of HUK significantly decreased the degree of stenosis (26.8% ± 7.1% vs 47.9% ± 5.7%; P < .01) at 2 months after balloon injury.
Our results indicate that HUK is able to attenuate atherosclerosis formation and to inhibit intimal hyperplasia by downregulating TGF-β1 expression and Smad2/3 phosphorylation, upregulating eNOS activity. HUK may be a potential therapeutic agent to prevent stenosis after vascular injury.
经皮腔内血管成形术和支架置入术后，针对再狭窄的有效治疗方法极为匮乏。已证实人组织激肽释放酶基因转移能够减轻球囊导管诱导的新生内膜形成。作为一种体内组织激肽释放酶，人尿激肽原酶（HUK）被广泛用于预防缺血再灌注损伤。然而，HUK对新生内膜形成的影响尚未得到探究。因此，我们研究了HUK是否能减轻高脂饮食喂养家兔球囊导管诱导的内膜增生。
通过苏木精-伊红染色和免疫组化染色，分析HUK对家兔球囊损伤颈动脉新生内膜和动脉粥样硬化形成的影响。通过实时定量聚合酶链反应检测肿瘤坏死因子α和白细胞介素1β的基因表达，并采用免疫组化染色评估巨噬细胞浸润情况，以此评价局部炎症反应。采用蛋白质印迹法研究HUK对内皮型一氧化氮合酶（eNOS）、转化生长因子β1（TGF-β1）和Smad信号通路活性的影响。通过血管造影评估HUK对损伤颈动脉内膜增生的长期影响。
定量图像分析显示，静脉注射HUK 14天可显著降低内膜面积和内膜面积/中膜面积比值（第14天，内膜面积减少54%，内膜面积/中膜面积比值减少58%；第28天，分别减少63%和85%）。在给予HUK 7天或14天后，巨噬细胞泡沫细胞阳性面积也显著减少（第7天，内膜面积减少69%，中膜面积减少78%；第14天，分别减少79%和60%；第28天，分别减少68%和44%）。2个月时对新生内膜平滑肌细胞进行肌动蛋白染色显示了类似结果（新生内膜血管平滑肌细胞阳性面积，28.21% ± 5.58% 对43.78% ± 8.36%；P <.05）。实时定量聚合酶链反应或蛋白质印迹分析表明，HUK降低了肿瘤坏死因子α、白细胞介素1β、TGF-β1和p-Smad2/3的表达，但增加了p-eNOS的表达。血管造影分析显示，注射HUK 14天可显著降低球囊损伤后2个月时的狭窄程度（26.8% ± 7.1% 对47.9% ± 5.7%；P <.01）。
我们的结果表明，HUK能够通过下调TGF-β1表达和Smad2/3磷酸化、上调eNOS活性来减轻动脉粥样硬化形成并抑制内膜增生。HUK可能是预防血管损伤后狭窄的一种潜在治疗药物。
