Samira Lakhal-Littleton

Exosome-mediated delivery of siRNA in vitro and in vivo

The use of small interfering RNAs (siRNAs) to induce gene silencing has opened a new avenue in drug discovery. However, their therapeutic potential is hampered by inadequate tissue-specific delivery. Exosomes are promising tools for drug delivery across different biological barriers. Here we show how exosomes derived from cultured cells can be harnessed for delivery of siRNA in vitro and in vivo. This protocol first describes the generation of targeted exosomes through transfection of an expression vector, comprising an exosomal protein fused with a peptide ligand. Next, we explain how to purify and characterize exosomes from transfected cell supernatant. Next, we detail crucial steps for loading siRNA into exosomes. Finally, we outline how to use exosomes to efficiently deliver siRNA in vitro and in vivo in mouse brain. Examples of anticipated results in which exosome-mediated siRNA delivery is evaluated by functional assays and imaging are also provided. The entire protocol takes ∼3 weeks.

Cardiac ferroportin regulates cellular iron homeostasis and is important for cardiac function

Significance The iron-exporting protein ferroportin is recognized as central to systemic iron regulation, but its role in tissues other than those involved in iron handling is unknown. This study shows that ferroportin expression in cardiomyocytes is essential to intracellular iron homeostasis and to normal cardiac function. It also demonstrates that the site of iron accumulation in the iron-overloaded heart depends on whether ferroportin is expressed in the cardiomyocytes. It further shows that the functional significance of cardiac iron overload is highly dependent upon the site of iron accumulation. These findings change our understanding of intracellular iron homeostasis and have significant implications for the clinical management of cardiac dysfunction associated with iron imbalance. Iron is essential to the cell. Both iron deficiency and overload impinge negatively on cardiac health. Thus, effective iron homeostasis is important for cardiac function. Ferroportin (FPN), the only known mammalian iron-exporting protein, plays an essential role in iron homeostasis at the systemic level. It increases systemic iron availability by releasing iron from the cells of the duodenum, spleen, and liver, the sites of iron absorption, recycling, and storage respectively. However, FPN is also found in tissues with no known role in systemic iron handling, such as the heart, where its function remains unknown. To explore this function, we generated mice with a cardiomyocyte-specific deletion of Fpn. We show that these animals have severely impaired cardiac function, with a median survival of 22 wk, despite otherwise unaltered systemic iron status. We then compared their phenotype with that of ubiquitous hepcidin knockouts, a recognized model of the iron-loading disease hemochromatosis. The phenotype of the hepcidin knockouts was far milder, with normal survival up to 12 mo, despite far greater iron loading in the hearts. Histological examination demonstrated that, although cardiac iron accumulates within the cardiomyocytes of Fpn knockouts, it accumulates predominantly in other cell types in the hepcidin knockouts. We conclude, first, that cardiomyocyte FPN is essential for intracellular iron homeostasis and, second, that the site of deposition of iron within the heart determines the severity with which it affects cardiac function. Both findings have significant implications for the assessment and treatment of cardiac complications of iron dysregulation.

Hepcidin demonstrates a biphasic association with anemia in acute Plasmodium falciparum malaria.

Hepcidin levels are high and iron absorption is limited in acute malaria. The mechanism(s) that regulate hepcidin secretion remain undefined. We have measured hepcidin concentration and cytokines in 100 Kenyan children with acute falciparum malaria and different degrees of anemia. Hepcidin was increased on admission and fell significantly one week and one month after treatment. The association of hepcidin with hemoglobin was not linear and hepcidin was very low in severe malarial anemia. Parasite density, IL-10 and IL-6 were significantly associated with hepcidin concentration. Hepcidin response to acute malaria supports the notion of iron sequestration during acute malaria infection and suggests that iron administration during acute malaria is futile. These data suggest iron supplementation policies should take into account the high hepcidin levels and probable poor utilization of iron for up to one week after treatment for the majority of patients with acute malaria.

An essential cell-autonomous role for hepcidin in cardiac iron homeostasis

Hepcidin is the master regulator of systemic iron homeostasis. Derived primarily from the liver, it inhibits the iron exporter ferroportin in the gut and spleen, the sites of iron absorption and recycling respectively. Recently, we demonstrated that ferroportin is also found in cardiomyocytes, and that its cardiac-specific deletion leads to fatal cardiac iron overload. Hepcidin is also expressed in cardiomyocytes, where its function remains unknown. To define the function of cardiomyocyte hepcidin, we generated mice with cardiomyocyte-specific deletion of hepcidin, or knock-in of hepcidin-resistant ferroportin. We find that while both models maintain normal systemic iron homeostasis, they nonetheless develop fatal contractile and metabolic dysfunction as a consequence of cardiomyocyte iron deficiency. These findings are the first demonstration of a cell-autonomous role for hepcidin in iron homeostasis. They raise the possibility that such function may also be important in other tissues that express both hepcidin and ferroportin, such as the kidney and the brain. DOI: http://dx.doi.org/10.7554/eLife.19804.001

Induced Disruption of the Iron-Regulatory Hormone Hepcidin Inhibits Acute Inflammatory Hypoferraemia

Withdrawal of iron from serum (hypoferraemia) is a conserved innate immune antimicrobial strategy that can withhold this critical nutrient from invading pathogens, impairing their growth. Hepcidin (Hamp1) is the master regulator of iron and its expression is induced by inflammation. Mice lacking Hamp1 from birth rapidly accumulate iron and are susceptible to infection by blood-dwelling siderophilic bacteria such as Vibrio vulnificus. In order to study the innate immune role of hepcidin against a background of normal iron status, we developed a transgenic mouse model of tamoxifen-sensitive conditional Hamp1 deletion (termed iHamp1-KO mice). These mice attain adulthood with an iron status indistinguishable from littermate controls. Hamp1 disruption and the consequent decline of serum hepcidin concentrations occurred within hours of a single tamoxifen dose. We found that the TLR ligands LPS and Pam3CSK4 and heat-killed Brucella abortus caused an equivalent induction of inflammation in control and iHamp1-KO mice. Pam3CSK4 and B. abortus only caused a drop in serum iron in control mice, while hypoferraemia due to LPS was evident but substantially blunted in iHamp1-KO mice. Our results characterise a powerful new model of rapidly inducible hepcidin disruption, and demonstrate the critical contribution of hepcidin to the hypoferraemia of inflammation.

Suppression of plasma hepcidin by venesection during steady-state hypoxia

To the editor: Hepcidin inhibits iron uptake from the gut and release of iron from macrophages in the reticuloendothelial system. Suppression of plasma hepcidin occurs in lowlanders after ascent to high altitude,[1][1],[2][2] reflecting the increased iron demand for erythropoiesis, but the

The interplay between iron and oxygen homeostasis with a particular focus on the heart

Iron is subject to tight homeostatic control in mammals. At the systemic level, iron homeostasis is controlled by the liver-derived hormone hepcidin acting on its target ferroportin in the gut, spleen, and liver, which form the sites of iron uptake, recycling, and storage, respectively. At the cellular level, iron homeostasis is dependent on the iron regulatory proteins IRP1/IRP2. Unique chemical properties of iron underpin its importance in biochemical reactions involving oxygen. As such, it is not surprising that there are reciprocal regulatory links between iron and oxygen homeostasis, operating both at the systemic and cellular levels. Hypoxia activates the IRP pathway, and in addition suppresses liver hepcidin through endocrine factors that have yet to be fully elucidated. This review summarizes current knowledge on the interplay between oxygen and iron homeostasis and describes recent insights gained into this interaction in the context of the heart. These include the recognition that the hepcidin/ferroportin axis plays a vital role in the regulation of intracellular iron homeostasis as well as regulating systemic iron availability. As is the case for other aspects of iron homeostasis, hypoxia significantly modulates the function of the hepcidin/ferroportin pathway in the heart. Key areas still to understand are the interactions between cardiac iron and diseases of the heart where hypoxia is a recognized component.

Mechanisms of cardiac iron homeostasis and their importance to heart function

Heart disease is a common manifestation in conditions of iron imbalance. Normal heart function requires coupling of iron supply for oxidative phosphorylation and redox signalling with tight control of intracellular iron to below levels at which excessive ROS are generated. Iron supply to the heart is dependent on systemic iron availability which is controlled by the systemic hepcidin/ferroportin axis. Intracellular iron in cardiomyocytes is controlled in part by the iron regulatory proteins IRP1/2. This mini-review summarises current understanding of how cardiac cells regulate intracellular iron levels, and of the mechanisms linking cardiac dysfunction with iron imbalance. It also highlights a newly-recognised mechanism of intracellular iron homeostasis in cardiomyocytes, based on a cell-autonomous cardiac hepcidin/ferroportin axis. This new understanding raises pertinent questions on the interplay between systemic and local iron control in the context of heart disease, and the effects on heart function of therapies targeting the systemic hepcidin/ferroportin axis.

Environmental influences on mouse embryonic heart development

myogenesis. Although specific CT transcription factors have been characterized as important regulators of muscle development, secreted factors have not been yet identified in the dialogue between CT and muscle cells during embryogenesis. We have observed in the chick embryonic limb that the CXCL12 chemokine was expressed in CT, while myogenic cells expressed its receptor CXCR7, leading us to suggest that the CXCL12/CXCR7 axis might be implicated in the cross-talk between CT and muscle during limb development. Using gainand loss-of-function experiments, we first assessed the involvement of the CXCL12/CXCR7 signaling on in vitro myogenesis using primary cultures of chick fetal muscle cells. Expression of a dominant-negative form of CXCR7 in myoblasts induced a decrease in the expression of myogenic markers, while overexpression of the receptor conversely led to an increase in the expression of myogenic genes. Interestingly, we observed that disruption of CXCR7 expression in vitro is associated withmuscle fusion defects, highlighting a putative role for CXCL12/CXCR7 signalling in the process of myoblasts fusion during myogenesis. In addition, we demonstrated that CXCL12 positively regulates the expression of collagens and CT markers in primary cultures of embryonic chick fibroblasts. In order to determine the role of CXCL12/CXCR7 axis during limb development in vivo, we performed gainand loss-of-functions approaches in chick embryonic limbs. Both overexpression and inactivation of components of the CXCL12/CXCR7 axis resulted in altered morphogenesis and mispatterning of limb muscles. Taken together, these results indicate that CXCL12 controls the differentiation of CT and signals to myogenic cells expressing CXCR7 to regulate muscle morphogenesis during limb development.

204 The cardiac hepcidin/ferroportin axis is essntial for cardiac iron homeostasis and function

Background Iron deficiency and chronic heart failure are two of the most common disorders worldwide. Recent evidence has demonstrated that they are linked. Moreover, clinical trials have demonstrated the benefits of intravenous iron supplementation in chronic heart failure. However, cardiac iron homeostasis remains unexplored. Recently, our laboratory demonstrated that cardiac-specific deletion of the ?iron-?exporting protein ferroportin causes fatal cardiac iron overload1. Ferroportin is known to be downregulated by the liver-derived hormone hepcidin. But hepcidin is also found in cardiomyocytes where its function remains unknown. Methods and results To explore the function of cardiomyocyte hepcidin, we generated mice with a cardiomyocyte-specific deletion of hepcidin or with a cardiomyocyte-specific knock-in of a hepcidin-resistant ferroportin mutant. While both models maintain normal systemic iron homeostasis, they nevertheless develop cardiomyocyte metabolic dysfunction followed by fatal contractile impairment as a consequence of cardiomyocyte iron deficiency. Intravenous iron supplementation prevents both the development of metabolic dysfunction and contractile impairment.2 Conclusions We conclude that regulation of iron export from cardiomyocytes by the cardiac hepcidin/ferroportin axis is essential to cardiomyocyte iron homeostasis and that its disruption leads to fatal cardiac dysfunction, even against a background of intact systemic iron homeostasis. These findings raise the possibility that hepcidin agonists/antagonists developed for disorders of systemic iron homeostasis could also modulate cardiac function. References 1. Lakhal-Littletonet al. An essential cell-autonomous role for hepcidin in cardiac iron homeostasis. Elife. 2016Nov 29;5. pii: e19804. doi: 10.7554/eLife.19804. 2. Lakhal-Littletonet al. Cardiac ferroportin regulates cellular iron homeostasis and is important for cardiac function. PNAS, 2015;112, 3164–3169.