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.

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

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.

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Iron deficiency augments hypoxic pulmonary arterial pressure in healthy individuals and exacerbates pulmonary arterial hypertension (PAH) in patients, even without anemia. Conversely, iron supplementation has been shown to be beneficial in both settings. The mechanisms underlying the effects of iron availability are not known, due to lack of understanding of how cells of the pulmonary vasculature respond to changes in iron levels. The iron export protein ferroportin (FPN) and its antagonist peptide hepcidin control systemic iron levels by regulating release from the gut and spleen, the sites of absorption and recycling, respectively. We found FPN to be present in pulmonary arterial smooth muscle cells (PASMCs) and regulated by hepcidin cell autonomously. To interrogate the importance of this regulation, we generated mice with smooth muscle-specific knock in of the hepcidin-resistant isoform fpn C326Y. While retaining normal systemic iron levels, this model developed PAH and right heart failure as a consequence of intracellular iron deficiency and increased expression of the vasoconstrictor endothelin-1 (ET-1) within PASMCs. PAH was prevented and reversed by i.v. iron and by the ET receptor antagonist BQ-123. The regulation of ET-1 by iron was also demonstrated in healthy humans exposed to hypoxia and in PASMCs from PAH patients with mutations in bone morphogenetic protein receptor type II. Such mutations were further associated with dysregulation of the HAMP/FPN axis in PASMCs. This study presents evidence that intracellular iron deficiency specifically within PASMCs alters pulmonary vascular function. It offers a mechanistic underpinning for the known effects of iron availability in humans.