Macrophage based drug delivery: Key challenges and strategies.

As a natural immune cell and antigen presenting cell, macrophages have been studied and engineered to treat human diseases. Macrophages are well-suited for use as drug carriers because of their biological characteristics, such as excellent biocompatibility, long circulation, intrinsic inflammatory homing and phagocytosis. Meanwhile, macrophages' uniquely high plasticity and easy re-education polarization facilitates their use as part of efficacious therapeutics for the treatment of inflammatory diseases or tumors. Although recent studies have demonstrated promising advances in macrophage-based drug delivery, several challenges currently hinder further improvement of therapeutic effect and clinical application. This article focuses on the main challenges of utilizing macrophage-based drug delivery, from the selection of macrophage sources, drug loading, and maintenance of macrophage phenotypes, to drug migration and release at target sites. In addition, corresponding strategies and insights related to these challenges are described. Finally, we also provide perspective on shortcomings on the road to clinical translation and production.

Iron biology.

Iron is a fundamental element for biological life, starting from bacteria till humans. Iron is essential for cell function and survival, energy production and metabolism, whereas increased levels cause oxidative stress. It is also a constituent of haemoglobin and thus it is necessary for oxygen transportation through the body. Given these multiple functions, the regulation of iron metabolism is complex and tight coupled with oxygen homeostasis at tissue and cellular levels, thanks to the interaction with the hypoxia inducible factor (HIF) system. In patients with chronic kidney disease (CKD), iron deficiency significantly contributes to anaemia development. This frequently overlaps with chronic inflammation, causing iron- restricted erythropoiesis. To add further complexity, metabolic hyperferritinemia may, on one side, increase the risk for CKD and, on the other, overlaps with functional iron deficiency. Excessive intracellular iron in certain cell types during CKD can also mediate cellular death (called ferroptosis), and contribute to the pathogenesis of kidney damage, atherosclerosis and vascular calcifications. This review is aimed at broadening the perspective of iron metabolism in the setting of CKD not just as a contributor to anaemia in CKD patients, but also as an important player with an impact on cell metabolism, renal fibrosis, and the cardiovascular system.

Downregulation of hepcidin by norcantharidin in macrophage.

Norcantharidin (NCTD) is a demethylated analogue of cantharidin. It was recently demonstrated that NCTD reduces iron contents in the liver and spleen of mice in vivo, indicating that NCTD can affect iron metabolism via hepcidin. Here, we investigated the effects of NCTD on expression of iron storage protein ferritin-light chain (Ft-L), transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1), ferroportin 1 (Fpn1), hepcidin, iron regulatory protein 1 (IRP1), IL-6, p-JAK2 and p-STAT3 in lipopolysaccharides (LPS)-treated RAW264.7 cells in vitro via Real-time PCR and Western blotting analysis. We demonstrate that NCTD down-regulates Ft-L, hepcidin, IL-6, pJAK2, pSTAT3 and up-regulates TfR1, DMT1, Fpn1 and IRP1 expression in LPS treated cells, showing that NCTD can inhibit hepcidin via the IL-6/JAK2/STAT3 signalling pathway and also increase TfR1, DMT1 and Fpn1 expression via down-regulating hepcidin and up-regulating IRP1. Our findings provide further evidence in vitro for the role of NCTD in iron metabolism.

HMGB1 Mediates Inflammation-Induced DMT1 Increase and Dopaminergic Neurodegeneration in the Early Stage of Parkinsonism.

Both neuroinflammation and iron accumulation play roles in the pathogenesis of Parkinson's disease (PD). However, whether inflammation induces iron dyshomeostasis in dopaminergic neurons at an early stage of PD, at which no quantifiable dopaminergic neuron loss can be observed, is still unknown. As for the inflammation mediators, although several cytokines have been reported to increase in PD, the functions of these cytokines in the SN are double-edged and controversial. In this study, whether inflammation could induce iron dyshomeostasis in dopaminergic neurons through high mobility group protein B1 (HMGB1) in the early stage of PD is explored. Lipopolysaccharide (LPS), a toxin that primarily activates glia cells, and 6-hydroxydopamine (6-OHDA), the neurotoxin that firstly impacts dopaminergic neurons, were utilized to mimic PD in rats. We found a common and exceedingly early over-production of HMGB1, followed by an increase of divalent metal transporter 1 with iron responsive element (DMT1+) in the dopaminergic neurons before quantifiable neuronal loss. HMGB1 neutralizing antibody suppressed inflammation in the SN, DMT1+ elevation in dopaminergic neurons, and dopaminergic neuronal loss in both LPS and 6-OHDA administration- induced PD models. On the contrary, interleukin-1ß inhibitor diacerein failed to suppress these outcomes induced by 6-OHDA. Our findings not only demonstrate that inflammation could be one of the causes of DMT1+ increase in dopaminergic neurons, but also highlight HMGB1 as a pivotal early mediator of inflammation-induced iron increase and subsequent neurodegeneration, thereby HMGB1 could serve as a potential target for early-stage PD treatment.

Pharmacological mechanisms of norcantharidin against hepatocellular carcinoma.

Norcantharidin (NCTD) is a water-soluble synthetic small molecule drug that has been approved by the Chinese FDA for the treatment of cancer in China. Among these NCTD-treated cancers, hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide and one of the most extensively studied. Research over the past few decades has made great strides in understanding how NCTD induces mitotic arrest, anti-proliferation, anti-metastasis, apoptosis and cytotoxic autophagy or autophagic cell death in HCC. In this article, we review recent progress in the application of NCTD for the treatment of HCC, with emphasis on the pharmacological mechanism of NCTD against hepatocellular carcinoma. The accumulated results show that NCTD has the ability to induce mitotic arrest, anti-proliferation, anti-metastasis, apoptosis and cytotoxic autophagy or autophagic cell death in HCC by down-regulating the expression of ISG15, MMP-9, u-PA, Mcl-1 and the accumulation of regulatory T cells, up-regulating the expression of FAM46C, miR-214 and the expression and phosphorylation of p21Cip1/Waf1 and CDC25C, and by inhibiting the c-Met-mTOR and JAK/STAT3 signaling pathways, reversing the methylation of RASSF1A gene, and activating TRAIL-R2/DR5 signal transduction.

Apolipoprotein E is required for brain iron homeostasis in mice.

BACKGROUND: Apolipoprotein E deficiency (ApoE-/-) increases progressively iron in the liver, spleen and aortic tissues with age in mice. However, it is unknown whether ApoE affects brain iron. METHODS: We investigated iron contents, expression of transferrin receptor 1 (TfR1), ferroportin 1 (Fpn1), iron regulatory proteins (IRPs), aconitase, hepcidin, Aß42, MAP2, reactive oxygen species (ROS), cytokines and glutathione peroxidase 4 (Gpx4) in the brain of ApoE-/- mice. RESULTS: We demonstrated that ApoE-/- induced a significant increase in iron, TfR1 and IRPs and a reduction in Fpn1, aconitase and hepcidin in the hippocampus and basal ganglia. We also showed that replenishment of ApoE absent partly reversed the iron-related phenotype in ApoE-/- mice at 24-months old. In addition, ApoE-/- induced a significant increase in Aß42, MDA, 8-isoprostane, IL-1ß, IL-6, and TNFα and a reduction in MAP2 and Gpx4 in hippocampus, basal ganglia and/or cortex of mice at 24-months old. CONCLUSIONS: Our findings implied that ApoE is required for brain iron homeostasis and ApoE-/--induced increase in brain iron is due to the increased IRP/TfR1-mediated cell-iron uptake as well as the reduced IRP/Fpn1 associated cell-iron export and suggested that ApoE-/- induced neuronal injury resulted mainly from the increased iron and subsequently ROS, inflammation and ferroptosis.

Ferroportin1 in the brain.

Despite years of research, it remains unclear why certain brain regions of patients with neurodegenerative diseases (NDs) have abnormally high levels of iron, although it has long been suggested that disrupted expression of iron-metabolizing proteins due to genetic or non-genetic factors is responsible for the enhancement in brain iron contents. In addition to the increased expression of cell-iron importers lactoferrin (lactotransferrin) receptor (LfR) in Parkinson's disease (PD) and melanotransferrin (p97) in Alzheimer's disease (AD), some investigations have suggested that cell-iron exporter ferroportin 1 (Fpn1) may be also associated with the elevated iron observed in the brain. The decreased expression of Fpn1 and the resulting decrease in the amount of iron excreted from brain cells has been thought to be able to enhance iron levels in the brain in AD, PD and other NDs. Cumulative results also suggest that the reduction of Fpn1 can be induced by hepcidin-dependent and -independent pathways. In this article, we discuss the current understanding of Fpn1 expression in the brain and cell lines of rats, mice and humans, with emphasis on the potential involvement of reduced Fpn1 in brain iron enhancement in patients with AD, PD and other NDs.

Iron metabolism and atherosclerosis.

Despite several decades of study, whether iron is involved in the development of atherosclerosis remains a controversial and unresolved issue. Here, we focus on the up-to-date advances in studies on role of iron in atherosclerosis and discuss possible reasons why patients with hereditary hemochromatosis (HH) do not show any increased incidence of atherosclerosis. In addition, we analyze conflicting results concerning the role of iron in atherogenesis from several epidemiological and animal studies. We argue that atherosclerosis is not observed in HH because iron homeostasis in the arterial wall, the actual location of atherosclerosis, is not significantly affected, and support a causal link between iron in the arterial wall and atherosclerosis.

The Role of Iron in Atherosclerosis in Apolipoprotein E Deficient Mice.

The role of iron in atherosclerosis is still a controversial and unsolved issue. Here, we investigated serum iron, expression of iron regulatory, transport and storage proteins, pro-inflammatory chemokines and cytokines in ApoE-/- mice. We demonstrated that ApoE-/- induced atherosclerosis and an increase in iron contents, expression of transferrin receptor 1 (TfR1), iron regulatory proteins (IRPs), heme oxygenase 1 (HO1), cellular adhesion molecules and pro-inflammatory cytokines, production of reactive oxygen species (ROS), and a reduction in expression of superoxide dismutase and glutathione peroxidase enzyme in aortic tissues. All of these changes induced by ApoE deficiency could be significantly abolished by deferoxamine. The data showed that the increased iron in aortic tissues was mainly due to the increased iron uptake via IRP/TfR1 upregulation. These findings plus a brief analysis of the controversial results reported previously showed that ApoE deficiency-induced atherosclerosis is partly mediated by the increased iron in aortic tissues.

Norcantharidin down-regulates iron contents in the liver and spleen of lipopolysaccharide-treated mice.

OBJECTIVE: The inhibiting effect of Norcantharidin (NCTD) on IL-6 (interleukin-6) and STAT3 and the involvement of the IL-6/STAT3 pathway in hepcidin expression prompted us to speculate that NCTD could affect iron metabolism.We examined the effects of NCTD on serum iron (SI) and transferrin (Tf) saturation, iron and ferritin light chain (FTL), transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1), ferroportin 1 (Fpn1), iron regulatory protein 1 (IRP1) and hepcidin, as well as IL-6 and STAT3 in the liver, spleen and duodenum of mice treated with lipopolysaccharide (LPS) in vivo, using RT-PCR, Western blotting and immunofluorescence analysis.NCTD could increase SI and Tf saturation and reduce tissue iron and FTL content by affecting expression of cell-iron transport proteins TfR1, DMT1 and Fpn1. The impact of NCTD on TfR1, DMT1 and Fpn1 expression is mediated by up-regulating IRP1 and down-regulating hepcidin expression, while NCTD-induced down-regulation of hepcidin is mediated by the IL-6/STAT3 signalling pathway in LPS-treated mice.NCTD affects iron metabolism by modifying the expression of IL-6/JAK2/STAT3/hepcidin and IRP1 and suggest that the ability of NCTD to reduce tissue iron contents may be a novel mechanism associated with the anti-cancer effects of NCTD.

Verapamil downregulates iron uptake and upregulates divalent metal transporter 1 expression in H9C2 cardiomyocytes.

Belgrade rats have a defect in divalent metal transport 1 (DMT1) with a reduced heart iron, indicating that DMT1 plays a physiological role in non-transferrin-bound iron (NTBI) uptake by cardiomyocytes. However, L-type voltage-dependent Ca2+ channel (LVDCC) blockers were recently demonstrated to significantly reduce NTBI uptake by cardiomyocytes, implying that LVDCC plays a dominant role in NTBI uptake by cardiomyocytes under iron-overloaded conditions. These findings led us to hypothesize that the LVDCC blocker-induced reduction in NTBI uptake might result not only from the inhibition of LVDCC-mediated NTBI uptake but also from the suppression of DMT1-mediated NTBI uptake. We therefore investigated the effects of the LVDCC blocker verapamil on NTBI uptake as well as DMT1 expression in H9C2 cells by the measurement of radio-labeled 55 Fe(II), reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis. We demonstrated that verapamil induced a significant reduction in NTBI uptake by H9C2 cells but also unexpectedly a remarkable increase rather than decrease in the expression of DMT1 mRNA and protein in H9C2 cells. Our findings imply that the verapamil-induced reduction in NTBI uptake by H9C2 cells is not associated with DMT1 and also indicate that verapamil stimulates rather than inhibits DMT1 expression and DMT1-mediated iron uptake by heart cells.

Signaling Integration of Hydrogen Sulfide and Iron on Cellular Functions.

Significance: Hydrogen sulfide (H2S) is an endogenous signaling molecule, regulating numerous physiological functions from vasorelaxation to neuromodulation. Iron is a well-known bioactive metal ion, being the central component of hemoglobin for oxygen transportation and participating in biomolecule degradation, redox balance, and enzymatic actions. The interplay between H2S and iron metabolisms and functions impacts significantly on the fate and wellness of different types of cells. Recent Advances: Iron level in vivo affects the production of H2S via nonenzymatic reactions. On the contrary, H2S quenches excessive iron inside the cells and regulates the redox status of iron. Critical Issues: Abnormal metabolisms of both iron and H2S are associated with various conditions and diseases such as iron overload, anemia, oxidative stress, and cardiovascular and neurodegenerative diseases. The molecular mechanisms for the interactions between H2S and iron are unsettled yet. Here we review signaling links of the production, metabolism, and their respective and integrative functions of H2S and iron in normalcy and diseases. Future Directions: Physiological and pathophysiological importance of H2S and iron as well as their therapeutic applications should be evaluated jointly, not separately. Future investigation should expand from iron-rich cells and tissues to the others, in which H2S and iron interaction has not received due attention. Antioxid. Redox Signal. 36, 275-293.

Central Nervous System Inflammation Induced by Lipopolysaccharide Up-Regulates Hepatic Hepcidin Expression by Activating the IL-6/JAK2/STAT3 Pathway in Mice.

It is known that lipopolysaccharide (LPS) triggers inflammatory response after intracerebroventricular (ICV) injection and elevates the expression of hepcidin through the interleukin 6/janus kinase 2/transducer and activator of the transcription 3 (IL-6/JAK2/STAT3) signaling pathway in the brain. This study was conducted to determine whether LPS ICV injection can regulate peripheral hepatic hepcidin expression and iron metabolism. Here, we studied the hepcidin expression in the liver, as well as serum iron and transferrin saturation, after LPS ICV injection. We also demonstrated the role of the IL-6/JAK2/STAT3 pathway in hepcidin expression in the livers of IL-6 knockout (IL-6-/- mice) and IL-6+/+ mice. AG490 was used to verify the effect of the IL-6/JAK2/STAT3 pathway on hepatic hepcidin expression. Our present study demonstrated that LPS ICV injection up-regulated hepatic hepcidin expression. This finding provides further evidence for highlighting the importance of the central inflammation on hepatic hepcidin expression and peripheral iron metabolism.

PRKAA/AMPKα phosphorylation switches the role of RASAL2 from a suppressor to an activator of autophagy.

RASAL2 (RAS protein activator like 2), a RASGTPase activating protein, can catalyze the hydrolysis of RAS-GTP into RAS-GDP to inactivate the RAS pathway in various types of cancer cells. However, the cellular function of RASAL2 remains elusive. Here we showed that RASAL2 can attenuate PRKAA/AMPKα phosphorylation by recruiting phosphatase PPM1B/pp2cß, thus inhibiting the initiation of basal autophagy under normal conditions. In addition, we found that glucose starvation could induce dissociation of PPM1B from RASAL2 and then RASAL2 at S351 be phosphorylated by PRKAA, followed by the binding of phosphorylated-RASAL2 with to PIK3C3/VPS34-ATG14-BECN1/Beclin1 complex to increase PIK3C3 activity and autophagy. Furthermore, RASAL2 S351 phosphorylation facilitated breast tumor growth and correlated to poor clinical outcomes in breast cancer patients. Our study demonstrated that the phosphorylation status of RASAL2 S351 can function as a molecular switch to either suppress or promote AMPK-mediated autophagy. Inhibition of RASAL2 S351 phosphorylation might be a potential therapeutic strategy to overcome the resistance of AMPK-activation agents.Abbreviations: AICAR: aminoimidazole carboxamide ribonucleotide; AMPK: adenosine 5'-monophosphate (AMP)-activated protein kinase; ATG14: autophagy related 14; C.C: compound C; CQ: chloroquine; DKO: double-knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15: phosphoinositide-3-kinase regulatory subunit 4; PPM1B/pp2cß: protein phosphatase, Mg2+/Mn2+ dependent 1B; PRKAA/AMPKα: protein kinase AMP-activated catalytic subunit alpha; PtdIns: phosphatidylinositol; PtdIns3P: phosphatidylinositol-3-phosphate; RASAL2: RAS protein activator like 2; RasGAPs: RasGTPase activating proteins; SQSTM1/p62: sequestosome 1; TNBC: triple-negative breast cancer.

Hepcidin inhibits autophagy in intracerebral hemorrhage models in vitro and in vivo.

Iron has a key role in the activation of the autophagic pathway in rats with intracerebral hemorrhage (ICH), and hepcidin has the ability to reduce brain iron in ICH-rats. We therefore hypothesized that hepcidin might be able to inhibit autophagy by reducing iron in an ICH brain. Here, we investigated the effects of Ad-hepcidin and/or hepcidin peptide on autophagic activities in ICH models in vitro and in vivo. We demonstrated that ad-hepcidin and hepcidin peptide both inhibited hemin-induced increase in LC3-II/LC3-I conversion ratio and reversed the reduction in p62 content in cortical neurons in vitro. We also showed that ad-hepcidin inhibited ICH-induced increase in LC3-II/LC3-I conversion ratio and reversed ICH-induced reduction in p62 content in the brain cortex of rats in vivo. Based on these findings plus previous data on the effects of ad-hepcidin and/or hepcidin peptide on iron contents in ICH models, we suggested that hepcidin-induced inhibition of autophagy might be mediated via reducing iron in hemin-treated neurons in vitro and ICH-rat brain in vivo.

The involvement of nuclear factor-κB in astroprotection against ischemia-reperfusion injury by ischemia-preconditioned neurons.

Ischemic preconditioned (IP) neurons protect astrocytes against ischemia/reperfusion (I/R)-induced injury by inhibiting oxidative stress. However, the relevant mechanisms are unknown. Based on the role of nuclear factor-κB (NF-κB) in cell survival and adaption to oxidative stress, we hypothesized that NF-κB might be associated with astroprotection induced by IP neurons via upregulation of antioxidant enzymes. Here, we investigated the effects of IP neurons on NF-κB activation, cell viability, reactive oxygen species (ROS), expression of antioxidant enzymes, erythropoietin (EPO), and tumor necrosis factor α (TNF-α), in the presence or absence of BAY11-7082 (an NF-κB inhibitor), anti-EPO, and anti-TNF-α antibodies, in astrocytes treated with or without I/R. We found that IP neurons could keep NF-κB activation at a relatively higher but beneficial level, and in turn, upregulated the activity of antioxidant enzymes and hence enhanced cell viability and reduced ROS in I/R treated astrocytes. The results collectively indicated that IP neurons are able to significantly inhibit the I/R-induced NF-κB overactivation, probably via EPO and TNF-α, being essential for IP neuron-induced astroprotection under the conditions of I/R. We concluded that NF-κB-mediated antioxidative stress is one of the mechanisms by which IP neurons protect astrocytes against I/R injury.

Apolipoprotein E deficiency induces a progressive increase in tissue iron contents with age in mice.

Association of both iron/hepcidin and apolipoprotein E (ApoE) with development of Alzheimer disease (AD) and atherosclerosis led us to hypothesize that ApoE might be required for body iron homeostasis. Here, we demonstrated that ApoE knock-out (KO) induced a progressive accumulation of iron with age in the liver and spleen of mice. Subsequent investigations showed that the increased iron in the liver and spleen was due to phosphorylated extracellular regulated protein kinases (pERK) mediated up-regulation of transferrin receptor 1 (TfR1), and nuclear factor erythroid 2-related factor-2 (Nrf2)-dependent down-regulation of ferroportin 1. Furthermore, replenishment of ApoE could partially reverse the iron-related phenotype in ApoE KO mice. The findings imply that ApoE may be essential for body iron homeostasis and also suggest that clinical late-onset diseases with unexplained iron abnormality may partly be related to deficiency or reduced expression of ApoE.

Hepcidin attenuates the iron-mediated secondary neuronal injury after intracerebral hemorrhage in rats.

Iron plays a key role in secondary neuronal injury after intracerebral hemorrhage (ICH), and hepcidin is able to reduce brain iron in iron-overloaded rats by down-regulating iron transport proteins including ferroportin 1 and transferrin receptor 1. These led us to hypothesize that hepcidin might reduce iron-mediated neurotoxicity by inhibiting iron accumulation in ICH brain. Here, we examined effects of Ad-hepcidin (hepcidin expression adenovirus) on the nonheme iron contents, expression of hepcidin, ferritin and iron transport proteins, neuronal cell survival, water contents in the brain and/or cerebrospinal fluid (CSF), and ICH-induced apoptosis, neurological deficit by RT-PCR, Western blot analysis, NeuN Immunofluorescence, TUNEL, Fluoro-Jade B staining, behavioral performance and Morris water-maze tests in 510 rats. We demonstrated that hepcidin could significantly suppress the ICH-induced increase in iron and ferritin in brain tissues and CSF by inhibiting expression of iron transport proteins, increase neuronal survival by attenuating ICH-induced apoptosis, reactive oxygen species, neurodegeneration and brain edema, as well as effectively improve ICH-induced behavioral and cognitive deficit in rats. The findings collectively showed that hepcidin could effectively attenuate iron-mediated secondary neuronal injury after ICH in rats. This naturally existing protein can potentially be developed into a therapeutic drug for the treatment of ICH patients.

Interaction among estrogen, IGF-1, and H2S on smooth muscle cell proliferation.

Both estrogen and hydrogen sulfide (H2S) inhibit the proliferation of vascular smooth muscle cells (SMCs) and development of atherosclerosis. In the absence of endogenous H2S as occurred in CSE-knockout (KO) mouse, however, estrogen stimulates the proliferation of vascular SMCs. The underlying mechanisms for this seemingly controversial vascular effect of estrogen are unclear. In the present study, we demonstrated that the stimulatory effect of estrogen on the proliferation of CSE-KO SMCs was suppressed by the inhibitor of insulin-like growth factor-1 receptor (IGF-1R) or knockdown of IGF-1R protein expression. Estrogen downregulated the expression of insulin-like growth factor-1 (IGF-1) and IGF-1R in aortic tissues or aortic SMCs isolated from WT and CSE-KO mice. Furthermore, endogenous H2S downregulated IGF-1R, but upregulated estrogen receptor (ER)-α, in aortic tissues or SMCs. ER-α and IGF-1R were co-located in SMCs and co-immunoprecipitated, which was decreased by H2S. Finally, both endogenous and exogenous H2S induced the S-sulfhydration of IGF-1R, but not ER-α, in WT-SMCs and CSE-KO SMCs, which underlies the decreased formation of IGF-1R/ER-α hybrid in the presence of H2S. Thus, the absence of H2S favors the interaction of estrogen with IGF-1R/ER-α hybrid to stimulate SMCs proliferation. The appreciation of a critical role of H2S in preventing estrogen-induced SMCs proliferation will help better understand the regulation of complex vascular effects of estrogen and sex-related cardiovascular diseases.