■尽管处于富氧环境中,内皮细胞(ECs)使用无氧糖酵解(Warburg效应)作为细胞能量需求的主要代谢途径。PFKFB(6-磷酸果糖-2-激酶/果糖-2,6-双磷酸酶)-3调节糖酵解中的关键酶检查点,并已显示出诱导血管生成。这项研究建立在我们努力确定缺血性血管生成的代谢调节和缺血肌肉中的灌注恢复的基础上。
■使用低氧血清饥饿(HSS)作为体外外周动脉疾病(PAD)模型,以股动脉结扎切除后肢缺血作为临床前PAD模型。
■尽管增加了PFKFB3依赖性糖酵解,HSS显著降低缺血性ECs的血管生成能力。有趣的是,抑制PFKFB3显著诱导HSS-ECs的血管生成能力。由于缺血诱发的PFKFB3水平在后肢缺血肌肉与非缺血,我们希望确定缺血肌肉中葡萄糖的生物利用度(而不是PFKFB3的表达)是否是血管生成受损的限制因素.然而,用肌内递送D-葡萄糖或L-葡萄糖(渗透压对照)治疗缺血肌肉在灌注恢复中没有显着差异,表明葡萄糖生物利用度不是实验性PAD中诱导缺血性血管生成的限制因素。出乎意料的是,我们发现,与对照shRNA相比,在缺血肌肉中shRNA介导的PFKFB3抑制导致灌注恢复的数值增加和血管密度显著增加(与PFKFB3沉默HSS-ECs的血管生成能力增加一致).基于这些数据,我们假设在缺血性ECs中抑制HSS诱导的PFKFB3激活了实验性PAD中缺血性肌肉血运重建的替代代谢途径.PFKFB3沉默的HSS-ECs中的全面葡萄糖代谢基因qPCR阵列,和PFKFB3抑制的缺血肌肉与各自对照鉴定的UGP2(尿苷二磷酸-葡萄糖焦磷酸化酶2),蛋白质糖基化和糖原合成的调节剂,在体外和体内通过PFKFB3抑制诱导。与IgG对照相比,抗体介导的缺血肌肉中UGP2的抑制显着损害了灌注恢复。机械上,补充尿苷二磷酸葡萄糖,UGP2活性的代谢产物,通过增加蛋白质糖基化(而不是糖原合成),在体外显着诱导HSS-EC血管生成能力,并在体内增强灌注恢复。
■我们的数据表明,在HSS-EC中抑制适应不良的PFKFB3驱动的糖酵解是必要的,以促进UGP2-尿苷二磷酸-葡萄糖轴,从而增强实验性PAD中的缺血性血管生成和灌注恢复。
UNASSIGNED: Despite being in an oxygen-rich environment, endothelial cells (ECs) use anaerobic glycolysis (Warburg effect) as the primary metabolic pathway for cellular energy needs. PFKFB (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase)-3 regulates a critical enzymatic checkpoint in glycolysis and has been shown to induce angiogenesis. This study builds on our efforts to determine the metabolic regulation of ischemic angiogenesis and perfusion recovery in the ischemic muscle.
UNASSIGNED: Hypoxia serum starvation (HSS) was used as an in vitro peripheral artery disease (PAD) model, and hind limb ischemia by femoral artery ligation and resection was used as a preclinical PAD model.
UNASSIGNED: Despite increasing PFKFB3-dependent glycolysis, HSS significantly decreased the angiogenic capacity of ischemic ECs. Interestingly, inhibiting PFKFB3 significantly induced the angiogenic capacity of HSS-ECs. Since ischemia induced a significant in PFKFB3 levels in hind limb ischemia muscle versus nonischemic, we wanted to determine whether glucose bioavailability (rather than PFKFB3 expression) in the ischemic muscle is a limiting factor behind impaired angiogenesis. However, treating the ischemic muscle with intramuscular delivery of D-glucose or L-glucose (osmolar control) showed no significant differences in the perfusion recovery, indicating that glucose bioavailability is not a limiting factor to induce ischemic angiogenesis in experimental PAD. Unexpectedly, we found that shRNA-mediated PFKFB3 inhibition in the ischemic muscle resulted in an increased perfusion recovery and higher vascular density compared with control shRNA (consistent with the increased angiogenic capacity of PFKFB3 silenced HSS-ECs). Based on these data, we hypothesized that inhibiting HSS-induced PFKFB3 expression/levels in ischemic ECs activates alternative metabolic pathways that revascularize the ischemic muscle in experimental PAD. A comprehensive glucose metabolic gene qPCR arrays in PFKFB3 silenced HSS-ECs, and PFKFB3-knock-down ischemic muscle versus respective controls identified UGP2 (uridine diphosphate-glucose pyrophosphorylase 2), a regulator of protein glycosylation and glycogen synthesis, is induced upon PFKFB3 inhibition in vitro and in vivo. Antibody-mediated inhibition of UGP2 in the ischemic muscle significantly impaired perfusion recovery versus IgG control. Mechanistically, supplementing uridine diphosphate-glucose, a metabolite of UGP2 activity, significantly induced HSS-EC angiogenic capacity in vitro and enhanced perfusion recovery in vivo by increasing protein glycosylation (but not glycogen synthesis).
UNASSIGNED: Our data present that inhibition of maladaptive PFKFB3-driven glycolysis in HSS-ECs is necessary to promote the UGP2-uridine diphosphate-glucose axis that enhances ischemic angiogenesis and perfusion recovery in experimental PAD.