Short Hairpin RNA Silencing of PHD-2 Improves Neovascularization and Functional Outcomes in Diabetic Wounds and Ischemic Limbs.
Short Hairpin RNA Silencing of PHD-2 Improves Neovascularization and Functional Outcomes in Diabetic Wounds and Ischemic Limbs.
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DOI：
10.1371/journal.pone.0150927
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Wan DC
中科院分区：
文献类型：
--
作者：
Paik KJ;Maan ZN;Zielins ER;Duscher D;Whittam AJ;Morrison SD;Brett EA;Ransom RC;Hu MS;Wu JC;Gurtner GC;Longaker MT;Wan DC
转录因子低氧诱导因子1-α(HIF-1α)负责调控60多个基因的下游表达，这些基因调节细胞在低氧条件下的生存和代谢，以及那些促进血管生成以缓解缺氧的基因。然而，在常氧条件下，缺氧诱导因子-1的α被脯氨酸羟基酶2羟化，随后被降解，生物半衰期不到5分钟。在这里，我们研究了在糖尿病创面和肢体缺血的背景下，通过沉默PHD-2的短发夹状α来抑制HIF-1RNA降解的治疗潜力。体外培养的糖尿病小鼠成纤维细胞经shPHD-2处理后，定量逆转录聚合酶链式反应检测到PHD-2基因转录水平下降，Western印迹检测到缺氧诱导因子-1α表达水平升高，下游血管生成基因sdf-1和血管内皮生长因子α表达水平升高。在体内，与shScr对照组相比，shPHD-2促进了糖尿病小鼠全层切除创面的愈合(14.33±0.45天比19±0.33天)，并与血管密度增加有关。与shScr相比，注射shPHD-2还可以改善缺血后肢的血流灌注，防止远端指尖坏死，并增加肌肉组织的存活率。通过shRNA治疗下调PHD-2基因有可能通过HIF-1α的过表达和HIF-1α下游促血管生成基因的上调来刺激血管生成，这可能是一种可行的、非病毒的用于缺血相关应用的基因治疗方法。
The transcription factor hypoxia-inducible factor 1-alpha (HIF-1α) is responsible for the downstream expression of over 60 genes that regulate cell survival and metabolism in hypoxic conditions as well as those that enhance angiogenesis to alleviate hypoxia. However, under normoxic conditions, HIF-1α is hydroxylated by prolyl hydroxylase 2, and subsequently degraded, with a biological half-life of less than five minutes. Here we investigated the therapeutic potential of inhibiting HIF-1α degradation through short hairpin RNA silencing of PHD-2 in the setting of diabetic wounds and limb ischemia. Treatment of diabetic mouse fibroblasts with shPHD-2 in vitro resulted in decreased levels of PHD-2 transcript demonstrated by qRT-PCR, higher levels of HIF-1α as measured by western blot, and higher expression of the downstream angiogenic genes SDF-1 and VEGFα, as measured by qRT-PCR. In vivo, shPHD-2 accelerated healing of full thickness excisional wounds in diabetic mice compared to shScr control, (14.33 ± 0.45 days vs. 19 ± 0.33 days) and was associated with an increased vascular density. Delivery of shPHD-2 also resulted in improved perfusion of ischemic hind limbs compared to shScr, prevention of distal digit tip necrosis, and increased survival of muscle tissue. Knockdown of PHD-2 through shRNA treatment has the potential to stimulate angiogenesis through overexpression of HIF-1α and upregulation of pro-angiogenic genes downstream of HIF-1α, and may represent a viable, non-viral approach to gene therapy for ischemia related applications.