Hydroxysafflor Yellow A promotes angiogenesis of brain microvascular endothelial cells from ischemia/reperfusion injury via glycolysis pathway in vitro

Background: Angiogenesis of brain microvascular endothelial cells (BMECs) after cerebral ischemia was conducive to improving the blood supply of ischemia tissues, which was upregulated by glycolysis. Hydroxysafflor Yellow A (HSYA) mends damaged tissues through increasing angiogenesis.

Methods: HSYA treated proliferation, migration and angiogenesis of BMECs in vitro in vitro during OGD/R. HSYA regulated the key enzymes of glycolysis, such as hexokinase 2 (HK2) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), glucose uptake and products (pyruvate, ATP and lactate) were detected by western blot and kits, respectively. Scratch wound assay, transwell, tube formation and spheroid sprouting were used to explore the pathway that HSYA recovered migration and angiogenesis of BMECs. We evaluated the potential target of HSYA promoting glycolysis via molecular docking, drug affinity responsive target stability (DARTS) and cellular thermal shift assay (CETSA).

Results: HSYA promoted the proliferation, migration, tube formation and spheroid sprouting of BMECs during OGD/R, and stimulated the expression of tip phenotype marker protein (CD34), and the receptor (Notch-1) that regulated the differentiation of endothelial cells into tip/stalk phenotype. In glycolysis, PFKFB3 expression was upregulated by HSYA; HSYA also improved ATP and pyruvate levels, as well as lactate release after OGD/R. Finally, upregulating VEGFA and p-VEGFR2 of HSYA was weakened because of suppressing glycolysis; the HSYA's improvement of BMECs migration and angiogenesis was attenuated under the inhibition of glycolysis, which confirmed that HSYA were upregulating angiogenesis and expression of VEGFA/VEGFR2 by glycolysis pathway. The result about molecular docking, DARTS and CETSA suggested that PFKFB3 was the possible target of HSYA.

Conclusion: HSYA promotes angiogenesis of BMECs in vitro through the glycolysis mediated VEGFA/VEGFR2 pathway, and PFKFB3 is the potential target of HSYA to heighten glycolysis.