Bioflavonoid luteolin prevents sFlt-1 release via HIF-1α inhibition in cultured human placenta

Abstract

Preeclampsia (PE) is a serious hypertensive complication of pregnancy and is a leading cause of maternal death and major contributor to maternal and perinatal morbidity, including establishment of long-term complications. The continued prevalence of PE stresses the need for identification of novel treatments which can target prohypertensive factors implicated in the disease pathophysiology, such as soluble fms-like tyrosine kinase 1 (sFlt-1). We set out to identify novel compounds to reduce placental sFlt-1 and determine whether this occurs via hypoxia-inducible factor (HIF)-1α inhibition. We utilized a commercially available library of natural compounds to assess their ability to reduce sFlt-1 release from primary human placental cytotrophoblast cells (CTBs). Human placental explants from normotensive (NT) and preeclamptic (PE) pregnancies were treated with varying concentrations of luteolin. Protein and mRNA expression of sFlt-1 and upstream mediators were evaluated using ELISA, western blot, and real-time PCR. Of the natural compounds examined, luteolin showed the most potent inhibition of sFlt-1 release, with >95% reduction compared to vehicle-treated. Luteolin significantly inhibited sFlt-1 in cultured placental explants compared to vehicle-treated in a dose- and time-dependent manner. Additionally, significant decreases in HIF-1α expression were observed in luteolin-treated explants, suggesting a mechanism for sFlt-1 downregulation. The ability of luteolin to inhibit HIF-1α may be mediated through the Akt pathway, as inhibitors to Akt and its upstream regulator phosphatidylinositol-3 kinase (PI3K) resulted in significant HIF-1α reduction. Luteolin reduces anti-angiogenic sFlt-1 through inhibition of HIF-1α, making it a novel candidate for the treatment of PE.

Keywords: flavones; hypoxia; placenta; preeclampsia; sFlt-1.

Figure 1:. Natural product library to reduce sFlt-1 release from primary placental CTBs.

CTBs were isolated from normal placentas and plated in 96-well plates. CTBs were treated with each bioflavonoid at 20 μg/mL (~10–20 μM) or DMSO vehicle for 72 h in normoxia. sFlt-1 protein was quantified in the conditioned medium using ELISA. Data are the percentage of the sFlt-1 reduction compared to the control (vehicle-treated) conditioned medium.

Figure 2:. Luteolin inhibits sFlt-1 production in placental explants.

Representative Western blots of placental explants from normotensive (A) and preeclamptic (B) patients treated with luteolin for 72 hours show a dose-dependent decrease in sFlt-1, demonstrating significance with 5–10μM treatment compared to DMSO control. Over time, explants from normotensive patients have significantly increased sFlt-1 production when treated with DMSO control, but levels of sFlt-1 remain unchanged over time when treated with 5μM luteolin (C). Similarly, sFlt-1 released into the media from normotensive (D) and preeclamptic (E) explants are significantly decreased with 5μM luteolin treatment compared to DMSO control. N=6; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 3:. Luteolin decreases placental HIF-1α and sFlt-1 expression.

Representative Western blots of placental explants from normotensive (NT) or preeclamptic (PE) patients have significantly reduced levels of HIF-1α expression when treated with 5μM luteolin compared to DMSO control. This decrease in HIF-1α is associated with a near-significant decrease in sFlt-1 in normotensive explants and a significant sFlt-1 decrease in preeclamptic explants (A). RT-PCR of mRNA from NT explants treated with luteolin demonstrates a significant reduction in sFlt-1 expression and a non-significant decrease in HIF-1α (B). N=5; *p<0.05, **p<0.01.

Figure 4:. The effect of luteolin on phospho-Akt and total Akt expression.

Representative Western blots of placental explants from normotensive and preeclamptic patients treated with luteolin show a near-significant decrease in the ratio of Phospho-Akt to total Akt, suggesting that this pathway may be relevant in the inhibition of HIF-1α and sFlt-1. N=5.

Figure 5:. Luteolin decreases HIF-1α through Akt Pathway.

Representative Western blots of explants from normotensive patients were treated in normoxic and hypoxic conditions with luteolin, Akt Inhibitor IV, or a combination of the two (A). We also compared luteolin treatment with PI3K inhibitor LY294002 or a combination of the two (B). We confirmed that phospho-Akt was decreased as a result of the treatment, and HIF-1α expression was measured and normalized to β-Actin (samples in blots are in the same order as listed in the graph). As expected, there was a significant increase in HIF-1α in hypoxia. However, treatment with luteolin, Akt inhibitor, and both concentrations of LY294002 resulted in a significant decrease of HIF-1α expression in hypoxia. Of note, samples in blots are in the same order as listed in the figures, and separate blots were used for the measurement of phospho-Akt and total Akt. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; N=5.