Laboratory of Physiopharmacology, University of Antwerp, 2610 Antwerp, Belgium.
Aberdeen Cardiovascular and Diabetes Centre, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK.
Int J Mol Sci. 2021 May 31;22(11):5970. doi: 10.3390/ijms22115970.
Angiogenesis is the process of new blood vessel formation. In this complex orchestrated growth, many factors are included. Lately, focus has shifted to endothelial cell metabolism, particularly to the PFKFB3 protein, a key regulatory enzyme of the glycolytic pathway. A variety of inhibitors of this important target have been studied, and a plethora of biological effects related to the process of angiogenesis have been reported. However, recent studies have disputed their mechanism of action, questioning whether all the effects are indeed due to PFKFB3 inhibition. Remarkably, the most well-studied inhibitor, 3PO, does not bind to PFKFB3, raising questions about this target. In our study, we aimed to elucidate the effects of PFKFB3 inhibition in angiogenesis by using the small molecule AZ67. We used isothermal titration calorimetry and confirmed binding to PFKFB3. In vitro, AZ67 did not decrease lactate production in endothelial cells (ECs), nor ATP levels, but exhibited good inhibitory efficacy in the tube-formation assay. Surprisingly, this was independent of EC migratory and proliferative abilities, as this was not diminished upon treatment. Strikingly however, even the lowest dose of AZ67 demonstrated significant inhibition of angiogenesis in vivo. To our knowledge, this is the first study to demonstrate that the process of angiogenesis can be disrupted by targeting PFKFB3 independently of glycolysis inhibition.
血管生成是新血管形成的过程。在这个复杂的协调生长过程中，包含了许多因素。最近，研究的重点已经转移到内皮细胞代谢上，特别是 PFKFB3 蛋白，它是糖酵解途径的关键调节酶。已经研究了这种重要靶标的各种抑制剂，并且已经报道了与血管生成过程相关的大量生物学效应。然而，最近的研究对其作用机制提出了质疑，质疑所有这些效应是否确实归因于 PFKFB3 的抑制。值得注意的是，研究最多的抑制剂 3PO 并不与 PFKFB3 结合，这引发了对该靶标的质疑。在我们的研究中，我们旨在通过使用小分子 AZ67 来阐明 PFKFB3 抑制在血管生成中的作用。我们使用等温滴定量热法并证实了与 PFKFB3 的结合。在体外，AZ67 不会减少内皮细胞（EC）中的乳酸产生，也不会降低 ATP 水平，但在管形成测定中表现出良好的抑制效果。令人惊讶的是，这与 EC 的迁移和增殖能力无关，因为在治疗后并没有减少。然而，令人惊讶的是，即使是最低剂量的 AZ67 也能显著抑制体内的血管生成。据我们所知，这是第一项证明可以通过靶向 PFKFB3 而不抑制糖酵解来破坏血管生成过程的研究。
