Bloodletting has no effect on the blood pressure abnormalities of hyperandrogenic women taking oral contraceptives in a randomized clinical trial

Normoferritinemic women with functional hyperandrogenism show a mild iron overload. Iron excess, hyperandrogenism, and cardioautonomic dysfunction contribute to blood pressure (BP) abnormalities in these patients. Furthermore, combined oral contraceptives (COC) prescribed for hyperandrogenic symptoms may worse BP recordings. Iron depletion by phlebotomy appears to lower BP in other acquired iron overload conditions. We aimed to determine the effect of iron depletion on the office BP, ambulatory BP monitoring, and frequency of hypertension in patients with functional hyperandrogenism submitted to standard therapy with COC. We conducted a phase 2 randomized, controlled, parallel, open-label clinical trial (NCT02460445) in adult women with functional hyperandrogenism including hyperandrogenic polycystic ovary syndrome and idiopathic hyperandrogenism. After a 3-month run-in period of treatment with 35 µg ethinylestradiol plus 2 mg cyproterone acetate, participants were randomized (1:1) to three scheduled bloodlettings or observation for another 9 months. Main outcome measures were the changes in office BP, 24-h-ambulatory BP, and frequency of hypertension in both study arms. From June 2015 to June 2019, 33 women were included in the intention-to-treat analyses. We observed an increase in mean office systolic BP [mean of the differences (MD): 2.5 (0.3–4.8) mmHg] and night-time ambulatory systolic BP [MD 4.1 (1.4–6.8) mmHg] after 3 months on COC. The percentage of nocturnal BP non-dippers also increased, from 28.1 to 92.3% (P < 0.001). Office and ambulatory BP did not change throughout the experimental period of the trial, both when considering all women as a whole or as a function of the study arm. The frequency of the non-dipping pattern in BP decreased during the experimental period [OR 0.694 (0.577–0.835), P < 0.001], regardless of the study arm. Decreasing iron stores by scheduled bloodletting does not override the BP abnormalities caused by COC in women with functional hyperandrogenism.

Coordinate regulation of liver ferroportin degradation and de novo synthesis determines serum iron levels in mice

The iron hormone hepcidin is transcriptionally activated by iron or inflammation via distinct, partially overlapping pathways. We addressed how iron affects inflammatory hepcidin levels and the ensuing hypoferremic response. Dietary iron overload did not mitigate hepcidin induction in LPS-treated wt mice but prevented effective inflammatory hypoferremia. Likewise, LPS modestly decreased serum iron in hepcidin-deficient Hjv-/- mice, model of hemochromatosis. Synthetic hepcidin triggered hypoferremia only in control but not iron-loaded wt animals. Furthermore, it dramatically decreased hepatic and splenic ferroportin in Hjv-/- mice on standard or iron-deficient diet, but only triggered hypoferremia in the latter. Mechanistically, iron induced liver ferroportin mRNA translation, thereby antagonizing hepcidin-mediated hypoferremia. Conversely, iron depletion suppressed de novo ferroportin synthesis in Hjv-/- livers, allowing exogenous hepcidin to cause hypoferremia. Consequently, prolonged LPS treatment eliminating ferroportin mRNA permitted hepcidin-mediated hypoferremia in iron-loaded mice. Thus, liver ferroportin mRNA translation is critical determinant of serum iron and finetunes hepcidin-dependent functional outcomes. Our data indicate a crosstalk between hepcidin/ferroportin and IRE/IRP systems. Moreover, they suggest that hepcidin supplementation therapy is more efficient combined with iron depletion.

Iron homeostasis in the absence of ferricrocin and its consequences in fungal development and insect virulence in Beauveria bassiana

The putative ferricrocin synthetase gene ferS in the fungal entomopathogen Beauveria bassiana BCC 2660 was identified and characterized. The 14,445-bp ferS encodes a multimodular nonribosomal siderophore synthetase tightly clustered with Fusarium graminearum ferricrocin synthetase. Functional analysis of this gene was performed by disruption with the bar cassette. ΔferS mutants were verified by Southern and PCR analyses. HPLC and TLC analyses of crude extracts indicated that biosynthesis of ferricrocin was abolished in ΔferS. Insect bioassays surprisingly indicated that ΔferS killed the Spodoptera exigua larvae faster (LT50 59 h) than wild type (66 h). Growth and developmental assays of the mutant and wild type demonstrated that ΔferS had a significant increase in germination under iron depletion and radial growth and a decrease in conidiation. Mitotracker staining showed that the mitochondrial activity was enriched in ΔferS under both iron excess and iron depletion. Comparative transcriptomes between wild type and ΔferS indicated that the mutant was increased in the expression of eight cytochrome P450 genes and those in iron homeostasis, ferroptosis, oxidative stress response, ergosterol biosynthesis, and TCA cycle, compared to wild type. Our data suggested that ΔferS sensed the iron excess and the oxidative stress and, in turn, was up-regulated in the antioxidant-related genes and those in ergosterol biosynthesis and TCA cycle. These increased biological pathways help ΔferS grow and germinate faster than the wild type and caused higher insect mortality than the wild type in the early phase of infection.

Callyspongiolide kills cells by inducing mitochondrial dysfunction via cellular iron depletion

The highly cytotoxic marine natural product callyspongiolide holds great promise as a warhead of antibody-drug conjugate in cancer therapeutics; however, the mechanism underlying its cytotoxicity remains unclear. To elucidate how callyspongiolide kills cells, we employed label-free target identification with thermal stability-shift-based fluorescence difference in two-dimensional (2-D) gel electrophoresis (TS-FITGE), which allowed observation of a unique phenomenon of protein-spot separation on 2-D gels upon treatment with callyspongiolide at increasing temperatures. During our exploration of what proteins were associated with this phenomenon as well as why it happens, we found that callyspongiolide induces mitochondrial/lysosomal dysfunction and autophagy inhibition. Moreover, molecular biology studies revealed that callyspongiolide causes lysosomal dysfunction, which induces cellular iron depletion and leads to mitochondrial dysfunction and subsequent cytotoxicity. Notably, these effects were rescued through iron supplementation. Although our approach was unable to reveal the direct protein targets of callyspongiolide, unique phenomena observed only by TS-FITGE provided critical insight into the mechanism of action of callyspongiolide and specifically its cytotoxic activity via induction of mitochondrial dysfunction through cellular iron depletion caused by lysosomal deacidification, which occurred independent of known programmed cell death pathways.