Human Pericardial Fluid Contains Exosomes Enriched with Cardiovascular-Expressed MicroRNAs and Promotes Therapeutic Angiogenesis.

The pericardial fluid (PF) is contained in the pericardial sac surrounding the heart. MicroRNA (miRNA) exchange via exosomes (endogenous nanoparticles) contributes to cell-to-cell communication. We investigated the hypotheses that the PF is enriched with miRNAs secreted by the heart and that it mediates vascular responses through exosome exchange of miRNAs. The study was developed using leftover material from aortic valve surgery. We found that in comparison with peripheral plasma, the PF contains exosomes enriched with miRNAs co-expressed in patients' myocardium and vasculature. At a functional level, PF exosomes improved survival, proliferation, and networking of cultured endothelial cells (ECs) and restored the angiogenic capacity of ECs depleted (via Dicer silencing) of their endogenous miRNA content. Moreover, PF exosomes improved post-ischemic blood flow recovery and angiogenesis in mice. Mechanistically, (1) let-7b-5p is proangiogenic and inhibits its target gene, TGFBR1, in ECs; (2) PF exosomes transfer a functional let-7b-5p to ECs, thus reducing their TGFBR1 expression; and (3) let-7b-5p depletion in PF exosomes impairs the angiogenic response to these nanoparticles. Collectively, our data support the concept that PF exosomes orchestrate vascular repair via miRNA transfer.

Coronary Artery-Bypass-Graft Surgery Increases the Plasma Concentration of Exosomes Carrying a Cargo of Cardiac MicroRNAs: An Example of Exosome Trafficking Out of the Human Heart with Potential for Cardiac Biomarker Discovery.

INTRODUCTION: Exosome nanoparticles carry a composite cargo, including microRNAs (miRs). Cultured cardiovascular cells release miR-containing exosomes. The exosomal trafficking of miRNAs from the heart is largely unexplored. Working on clinical samples from coronary-artery by-pass graft (CABG) surgery, we investigated if: 1) exosomes containing cardiac miRs and hence putatively released by cardiac cells increase in the circulation after surgery; 2) circulating exosomes and exosomal cardiac miRs correlate with cardiac troponin (cTn), the current "gold standard" surrogate biomarker of myocardial damage. METHODS AND RESULTS: The concentration of exosome-sized nanoparticles was determined in serial plasma samples. Cardiac-expressed (miR-1, miR-24, miR-133a/b, miR-208a/b, miR-210), non-cardiovascular (miR-122) and quality control miRs were measured in whole plasma and in plasma exosomes. Linear regression analyses were employed to establish the extent to which the circulating individual miRs, exosomes and exosomal cardiac miR correlated with cTn-I. Cardiac-expressed miRs and the nanoparticle number increased in the plasma on completion of surgery for up to 48 hours. The exosomal concentration of cardiac miRs also increased after CABG. Cardiac miRs in the whole plasma did not correlate significantly with cTn-I. By contrast cTn-I was positively correlated with the plasma exosome level and the exosomal cardiac miRs. CONCLUSIONS: The plasma concentrations of exosomes and their cargo of cardiac miRs increased in patients undergoing CABG and were positively correlated with hs-cTnI. These data provide evidence that CABG induces the trafficking of exosomes from the heart to the peripheral circulation. Future studies are necessary to investigate the potential of circulating exosomes as clinical biomarkers in cardiac patients.

Expression of ABCG2 (BCRP) in mouse models with enhanced erythropoiesis.

Haem is a structural component of numerous cellular proteins which contributes significantly to iron metabolic processes in mammals but its toxicity demands that cellular levels must be tightly regulated. Breast Cancer Resistance Protein (BCRP/ABCG2), an ATP Binding Cassette G-member protein has been shown to possess porphyrin/haem efflux function. The current study evaluated the expression and regulation of Abcg2 mRNA and protein levels in mouse tissues involved in erythropoiesis. Abcg2 mRNA expression was enhanced in bone marrow hemopoietic progenitor cells from mice that were treated with phenylhydrazine (PHZ). Abcg2 mRNA expression was increased particularly in the extramedullary haematopoietic tissues from all the mice models with enhanced erythropoiesis. Haem oxygenase (ho1) levels tended to increase in the liver of mice with enhanced erythropoiesis and gene expression patterns differed from those observed in the spleen. Efflux of haem biosynthetic metabolites might be dependent on the relative abundance of Abcg2 or ho1 during erythropoiesis. Abcg2 appears to act principally as a safety valve regulating porphyrin levels during the early stages of erythropoiesis and its role in systemic haem metabolism and erythrophagocytosis, in particular, awaits further clarification.

Mitochondrial DNA as a non-invasive biomarker: accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias.

Circulating mitochondrial DNA (MtDNA) is a potential non-invasive biomarker of cellular mitochondrial dysfunction, the latter known to be central to a wide range of human diseases. Changes in MtDNA are usually determined by quantification of MtDNA relative to nuclear DNA (Mt/N) using real time quantitative PCR. We propose that the methodology for measuring Mt/N needs to be improved and we have identified that current methods have at least one of the following three problems: (1) As much of the mitochondrial genome is duplicated in the nuclear genome, many commonly used MtDNA primers co-amplify homologous pseudogenes found in the nuclear genome; (2) use of regions from genes such as ß-actin and 18S rRNA which are repetitive and/or highly variable for qPCR of the nuclear genome leads to errors; and (3) the size difference of mitochondrial and nuclear genomes cause a "dilution bias" when template DNA is diluted. We describe a PCR-based method using unique regions in the human mitochondrial genome not duplicated in the nuclear genome; unique single copy region in the nuclear genome and template treatment to remove dilution bias, to accurately quantify MtDNA from human samples.

Iron absorption in hepcidin1 knockout mice.

Hepcidin, the Fe-regulatory peptide, has been shown to inhibit Fe absorption and reticuloendothelial Fe recycling. The present study was conducted to explore the mechanism of in vivo Fe regulation through genetic disruption of hepcidin1 and acute effects of hepcidin treatment in hepcidin1 knockout (Hepc1-/-) and heterozygous mice. Hepcidin1 disruption resulted in significantly increased intestinal Fe uptake. Hepcidin injection inhibited Fe absorption in both genotypes, but the effects were more evident in the knockout mice. Hepcidin administration was also associated with decreased membrane localisation of ferroportin in the duodenum, liver and, most significantly, in the spleen of Hepc1-/- mice. Hypoferraemia was induced in heterozygous mice by hepcidin treatment, but not in Hepc1-/- mice, 4 h after injection. Interestingly, Fe absorption and serum Fe levels in Hepc1-/- and heterozygous mice fed a low-Fe diet were not affected by hepcidin injection. The present study demonstrates that hepcidin deficiency causes increased Fe absorption. The effects of hepcidin were abolished by dietary Fe deficiency, indicating that the response to hepcidin may be influenced by dietary Fe level or Fe status.

Haem and folate transport by proton-coupled folate transporter/haem carrier protein 1 (SLC46A1).

Haem carrier protein 1 (HCP1) was originally identified and characterised as a mammalian haem transporter. However, recent evidence has shown that it is also a proton-coupled folate transporter (PCFT) and mutations in the gene cause hereditary folate deficiency in humans. We therefore investigated haem and folate transport characteristics of PCFT/HCP1 both in vivo and in vitro in CD-1 mice and in the presence or absence of a blocking antibody for PCFT/HCP1, and also in cultured cells (which express PCFT/HCP1 endogenously) to elucidate the specificity and selectivity of PCFT/HCP1. The in vivo study showed that the addition of folic acid inhibited 59Fe-labelled haem transport in hypoxic mice but had no effect in normal mice. Using in vitro methods, the results showed increased [3H]folate uptake into everted duodenum from hypoxic mice but uptake was reduced by the addition of haem or PCFT/HCP1 antibodies to the medium. Caco-2 cells transiently transfected with small interfering RNA (siRNA) PCFT/HCP1 duplex oligos resulted in a 69 % reduction in PCFT/HCP1 mRNA when compared with the control siRNA. Both haem and folate uptake were significantly (P < 0.05) reduced in cells transfected with PCFT/HCP1 siRNA; however, the magnitude of reduction with folic acid uptake was greater (48 %) than that of haem (22.5 %). Overall the data support PCFT/HCP1 as a primary folate transporter with a lower affinity for haem. PCFT/HCP1 could therefore play a physiological role in Fe nutrition and the data highlight the potential for the interaction of folate and haem at the level of intestinal absorption.

Tumour necrosis factor alpha causes hypoferraemia and reduced intestinal iron absorption in mice.

Cytokines are implicated in the anaemia of chronic disease by reducing erythropoiesis and increasing iron sequestration in the reticuloendotheial system. However, the effect of cytokines, in particular TNFalpha (tumour necrosis factor alpha), on small bowel iron uptake and iron-transporter expression remains unclear. In the present study, we subjected CD1 male mice to intraperitoneal injection with TNFalpha (10 ng/mouse) and then examined the expression and localization of DMT1 (divalent metal transporter 1), IREG1 (iron-regulated protein 1) and ferritin in duodenum. Liver and spleen samples were used to determine hepcidin mRNA expression. Changes in serum iron and iron loading of duodenum, spleen and liver were also determined. We found a significant (P<0.05) fall in serum iron 3 h post-TNFalpha exposure. This was coincident with increased iron deposition in the spleen. After 24 h of exposure, there was a significant decrease in duodenal iron transfer (P<0.05) coincident with increased enterocyte ferritin expression (P<0.05) and re-localization of IREG1 from the basolateral enterocyte membrane. Hepatic hepcidin mRNA levels remained unchanged, whereas splenic hepcidin mRNA expression was reduced at 24 h. In conclusion, we provide evidence that TNFalpha may contribute to anaemia of chronic disease by iron sequestration in the spleen and by reduced duodenal iron transfer, which seems to be due to increased enterocyte iron binding by ferritin and a loss of IREG1 function. These observations were independent of hepcidin mRNA levels.

Identification of an intestinal heme transporter.

Dietary heme iron is an important nutritional source of iron in carnivores and omnivores that is more readily absorbed than non-heme iron derived from vegetables and grain. Most heme is absorbed in the proximal intestine, with absorptive capacity decreasing distally. We utilized a subtractive hybridization approach to isolate a heme transporter from duodenum by taking advantage of the intestinal gradient for heme absorption. Here we show a membrane protein named HCP 1 (heme carrier protein 1), with homology to bacterial metal-tetracycline transporters, mediates heme uptake by cells in a temperature-dependent and saturable manner. HCP 1 mRNA was highly expressed in duodenum and regulated by hypoxia. HCP 1 protein was iron regulated and localized to the brush-border membrane of duodenal enterocytes in iron deficiency. Our data indicate that HCP 1 is the long-sought intestinal heme transporter.

A role for tumour necrosis factor alpha in human small bowel iron transport.

Cytokines are integral to the development of anaemia of chronic inflammation. Cytokines modulate hepcidin expression and iron sequestration by the reticuloendothelial system but their direct effects on small bowel iron transport are not well characterized. The aim of the present study was to examine the local effects of TNFalpha (tumour necrosis factor alpha) on small bowel iron transport and on iron transporter expression in the absence of hepcidin. The effects of TNFalpha on iron transport were determined using radiolabelled iron in an established Caco-2 cell model. The effect of TNFalpha on the expression and localization of the enterocyte iron transporters DMT-1 (divalent metal transporter 1), IREG-1 (iron-regulated transporter 1) and ferritin was determined utilizing Caco-2 cells and in a human ex vivo small bowel culture system. TNFalpha mediated an early induction in both iron import and iron export, which were associated with increased DMT-1 and IREG-1 mRNA and protein expression (P<0.05). However, by 24 h, both iron import and iron export were significantly inhibited, coinciding with an induction of ferritin heavy chain (P<0.05) and a decrease in DMT-1 and IREG-1 to baseline levels. In addition, there was a relocalization of IREG-1 away from the basolateral cell border and increased iron deposition in villous enterocytes. In conclusion, TNFalpha has a direct effect on small bowel iron transporter expression and function, leading to an inhibition of iron transport.

Expression of iron absorption genes in mouse large intestine.

OBJECTIVE: The large intestine has been reported to have a capacity for iron absorption and expresses genes for iron absorption normally found in the duodenum. The importance and function of these genes in the large intestine are not understood. We therefore investigated the cellular localization and regulation of expression of these genes in mouse caecum and colon. MATERIAL AND METHODS: Gene expression was measured by real-time PCR using RNA extracted from iron-deficient and hypoxic mouse large intestine, compared to controls. Protein localization and regulation were measured by immunohistochemistry using frozen sections of the large intestine from the same mice. RESULTS: Dcytb (duodenal ferric reductase) was expressed at very low levels in the large intestine, compared to the duodenum, while Ireg1 and DMT1 were expressed at significant levels in the large intestine and were increased in iron-deficient caecum, proximal and distal colon, with the most significant increases seen in the distal colon. Hypoxia increased Ireg1 expression in the proximal colon. Immunohistochemistry detected significant levels of only IREG1, which was localized to the basolateral membrane of colonic epithelial cells. CONCLUSIONS: Iron absorption genes were expressed at lower levels in mouse caecum and colon than in the duodenum. They are regulated by body iron requirements. Colonic epithelial cells express basolateral IREG1in the same fashion as in the duodenum and this protein could regulate colonic epithelial cell iron levels.

The effects of inhibition of haem biosynthesis by griseofulvin on intestinal iron absorption.

The relationship between haem biosynthesis and intestinal iron absorption in mice was investigated by ascertaining the effect of the haem synthesis inhibitor, griseofulvin, on duodenal iron absorption using both in vivo and in vitro measurements. Urinary 5-aminolaevulinic acid levels were increased within 24 hr of feeding mice with griseofulvin diet (2.5% w/w), with more marked increases seen after 3-7 days. Urinary porphobilinogen levels also showed a similar trend. In vivo intestinal iron absorption was significantly reduced (P<0.05) in experimental mice, mainly due to reduction in the transfer of 59Fe from the enterocytes to the portal circulation. In vitro studies using isolated duodenal fragments also exhibited marked decreases in both iron uptake and Fe (III) reduction. Changes in mucosal Divalent Metal Transporter 1 (DMT-1), Dcytb and Ireg1 (iron regulated protein 1) mRNA levels paralleled the changes in iron absorption. The reduction in iron absorption after griseofulvin treatment was normalised when mice were simultaneously injected with haem-arginate. These data support the hypothesis that intermediates in haem biosynthesis, particularly 5-aminolaevulinic acid, regulate intestinal iron absorption.

Duodenal ascorbate levels are changed in mice with altered iron metabolism.

Ascorbate has long been thought to play an important role in intestinal iron absorption. The recent identification of a possible ascorbate-dependent duodenal ferric reductase suggests a role for intracellular ascorbate in the control of iron absorption. We set out to determine whether duodenal ascorbate concentrations are altered by treatments known to alter the rate of iron absorption and whether ascorbate levels affect duodenal reductase activity. Duodenal ascorbate was extracted and assayed by HPLC and/or a chemical assay. Ferric reductase was assayed in vitro with ferric nitrilotriacetate or nitroblue tetrazolium as substrates. Duodenal ascorbate concentrations were increased by iron deficiency, genetic hypotransferrinemia, and hypoxia. Parenteral iron overload increased iron stores but did not affect duodenal ascorbate concentrations. Hemolytic anemia induced in mice by phenylhydrazine injection also did not affect duodenal ascorbate concentrations. In vitro studies with incubated duodenum showed that decreased tissue ascorbate was associated with decreased mucosal ferric reductase activity, whereas incubation with dehydroascorbate prevented both the decrease in ascorbate concentration and reductase activity. Mouse duodenum ascorbate concentrations changed in response to treatments that altered iron absorption rates; in particular, ascorbate levels generally increased when iron absorption was increased by iron deficiency, hypoxia, or genetic hypotransferrinemia. We conclude that changes in ascorbate levels are associated with changes in ferric reductase activity. These findings are consistent with the proposal that duodenal ascorbate plays a role in intestinal iron absorption.

Effect of hepcidin on intestinal iron absorption in mice.

The effect of the putative iron regulatory peptide hepcidin on iron absorption was investigated in mice. Hepcidin peptide was synthesized and injected into mice for up to 3 days, and in vivo iron absorption was measured with tied-off segments of duodenum. Liver hepcidin expression was measured by reverse transcriptase-polymerase chain reaction. Hepcidin significantly reduced mucosal iron uptake and transfer to the carcass at doses of at least 10 microg/mouse per day, the reduction in transfer to the carcass being proportional to the reduction in iron uptake. Synthetic hepcidin injections down-regulated endogenous liver hepcidin expression excluding the possibility that synthetic hepcidin was functioning by a secondary induction of endogenous hepcidin. The effect of hepcidin was significant at least 24 hours after injection of hepcidin. Liver iron stores and hemoglobin levels were unaffected by hepcidin injection. Similar effects of hepcidin on iron absorption were seen in iron-deficient and Hfe knockout mice. Hepcidin inhibited the uptake step of duodenal iron absorption but did not affect the proportion of iron transferred to the circulation. The effect was independent of iron status of mice and did not require Hfe gene product. The data support a key role for hepcidin in the regulation of intestinal iron uptake.

Hypoxic response of iron absorption is not affected by the Hfe gene knock-out in mice.

The effect of Hfe (haemochromatosis) gene deletion on the hypoxic response of iron absorption was investigated. Hfe knock-out mice were exposed to 0.5 atmospheres hypoxia for 3 d before in vivo iron absorption was measured. Both wild-type and Hfe knock-out mice had similar (two- to threefold) increases in iron absorption in response to hypoxia. We conclude that the Hfe gene product is not required for mice to increase iron absorption rates in response to hypoxia. The data further support the hypothesis that at least two independent mechanisms for the regulation of iron absorption exist, only one of which requires Hfe.

Effect of Tin-mesoporphyrin, an inhibitor of haem catabolism, on intestinal iron absorption.

Haem biosynthesis is the most important destination for absorbed iron, hence it can be hypothesized that iron absorption regulation should be integrated with haem metabolism. As an initial step to test this hypothesis, the effect on iron absorption of Tin-mesoporphyrin (SnMP), inhibitor of haem oxygenase, altering haem and its biosynthetic intermediates, was studied. Mice injected with SnMP (5-25 micro mol/kg daily for up to 3 d) showed dose-dependent increases in intestinal iron absorption measured in vivo and in vitro. In order to investigate the effects of SnMP, enzymes and intermediates of haem metabolism were measured. Hepatic 5-amino-laevulinate (ALA) synthase activity (pmol/min/mg protein) was significantly reduced in SnMP-treated mice (10 and 25 micro mol/kg daily for 3 d) (mean +/- standard deviation, control 11.2 +/- 2.6; treated 6.3 +/- 1.7; P < 0.01). Hepatic ALA dehydratase activity (pmol porphobilinogen/mg protein/min) showed significant reductions following SnMP treatment (control 180 +/- 60, treated 130 +/- 50; P < 0.05). The effect of SnMP on iron absorption was reversible, with absorption returning to normal after 3 d. Furthermore, the effect of SnMP on duodenal iron absorption was abolished by the simultaneous injection of ALA (6 micro mol/l). ALA alone had no effect on iron absorption. In-vitro studies using duodenal fragments isolated from mice treated with SnMP (10 micro mol/kg daily for 3 d), showed significant increases (P < 0.05) in both mucosal iron uptake and Fe(III) reducing activity. We conclude that intermediates in haem metabolism, in particular levels of ALA, may play a role in duodenal iron absorption.

Duodenal nonheme iron content correlates with iron stores in mice, but the relationship is altered by Hfe gene knock-out.

Hereditary hemochromatosis is a common iron-loading disorder found in populations of European descent. It has been proposed that mutations causing loss of function of HFE gene result in reduced iron incorporation into immature duodenal crypt cells. These cells then overexpress genes for iron absorption, leading to inappropriate cellular iron balance, a persistent iron deficiency of the duodenal mucosa, and increased iron absorption. The objective was to measure duodenal iron content in Hfe knock-out mice to test whether the mutation causes a persistent decrease in enterocyte iron concentration. In both normal and Hfe knock-out mice, duodenal nonheme iron content was found to correlate with liver iron stores (P <.001, r = 0.643 and 0.551, respectively), and this effect did not depend on dietary iron levels. However, duodenal iron content was reduced in Hfe knock-out mice for any given content of liver iron stores (P <.001).