Two articles in the current issue focus on the interaction between food and the brain. Chuang and colleagues explain the molecular reasons why we seek comfort foods such as cheeseburgers after stressful experiences (page 2684), while Masino and colleagues show that increased activation of adenosine A1 receptors underlies the beneficial, seizure-suppressing effects of a high-fat ketogenic diet (page 2679).
The term evidence-based medicine is overused, abused, and is beginning to ring hollow. It is not that evidence (or at least of what most people in biomedicine think evidence-based medicine should strive to be) is a bad thing. Rather, there is more rhetoric about evidence than there is actual evidence to support the degree of talk.
Laurence A. Turka, Arthur Caplan
Over the past decade and a half, the biomedical community has uncovered a previously unappreciated reciprocal relationship between cells of the immune and skeletal systems. Work in this field, which has been termed “osteoimmunology,” has resulted in the development of clinical therapeutics for seemingly disparate diseases linked by the common themes of inflammation and bone remodeling. Here, the important concepts and discoveries in osteoimmunology are discussed in the context of the diseases bridging these two organ systems, including arthritis, osteoporosis, cancer, and infection, and the targeted treatments used by clinicians to combat them.
Dallas Jones, Laurie H. Glimcher, Antonios O. Aliprantis
The many challenges associated with lung transplantation provide a strong rationale for the development of cell- and tissue-based therapies for patients with respiratory failure caused by the loss of lung tissue that is associated with chronic pulmonary disease, injury, or resection. In this issue of the JCI, Chapman et al. take an important step forward in the development of regenerative medicine for the treatment of lung disease by identifying a novel integrin α6β4–expressing alveolar epithelial cell that serves as a multipotent progenitor during repair of the severely injured lung.
Jeffrey A. Whitsett, Vladimir V. Kalinichenko
HDL cholesterol activates endothelial cell production of the atheroprotective signaling molecule NO, and it promotes endothelial repair. In this issue of the JCI, Besler et al. provide new data indicating that HDL from stable coronary artery disease (CAD) or acute coronary syndrome patients inhibits rather than stimulates endothelial NO synthesis and endothelial repair. This may be related to decreased HDL-associated paraoxonase 1 (PON1) activity. These observations support the concept that the cardiovascular impact of HDL is not simply related to its abundance, and the translation of the present findings to prospective studies of CAD risk and to evaluations of HDL-targeted therapeutics is a logical future goal.
Chieko Mineo, Philip W. Shaul
Many individuals with epilepsy benefit from consuming a ketogenic diet, which is similar to the more commonly known Atkins diet. The underlying molecular reason for this has not been determined. However, in this issue of the JCI, Masino et al. have elucidated the mechanism responsible for the antiepileptic effects of the ketogenic diet in mice. The diet is shown to decrease expression of the enzyme adenosine kinase (Adk), which is responsible for clearing the endogenous antiepileptic agent adenosine (Ado) from the extracellular CNS space. Decreased expression of Adk results in increased extracellular Ado, activation of inhibitory Ado A1 receptors, and decreased seizure generation, the desired therapeutic effect. The authors’ work serves to emphasize the importance of controlling Adk expression, not only as the mechanism of action of the ketogenic diet, but also as a potential target of future therapies.
Robert W. Greene
Chronic inflammation has long been suspected to support tumorigenesis in a variety of cancers. The IκB kinase (IKK)/NF-κB pathway is the critical signal transduction pathway regulating inflammation, and loss-of-function studies have demonstrated its involvement in tumorigenesis. In this issue of the JCI, Vlantis et al. present evidence that persistent genetic activation of IKK/NF-κB signaling in intestinal epithelial cells not only accelerates tumorigenesis in models of both carcinogen- and mutation-induced colorectal cancer, but also is sufficient to induce intestinal tumors.
Yoshiaki Sunami, Thomas Wirth
STAT1 is the main signal transducer for type I and II IFNs and plays a central role in the regulation of innate and adaptive immune responses. We used Stat1-deficient mice to test the role of donor Stat1 in MHC-matched minor histocompatibility antigen–mismatched (mHA-mismatched) and fully MHC-mismatched models of bone marrow transplantation. Lack of Stat1 in donor splenocytes reduced graft-versus-host disease (GVHD) in both immunogenetic disparities, leading to substantially attenuated morbidity and mortality. Donor Stat1 deficiency resulted in reduced alloantigen-induced activation and expansion of donor T cells and correlated with the expansion of CD4+CD25+Foxp3+ Tregs in vivo. This expansion of Tregs was further confirmed by studies showing that Stat1 deficiency promoted the proliferation, while inhibiting the apoptosis, of natural Tregs, and that absence of Stat1 enhanced the induction of inducible Tregs both in vitro and in vivo. Ex vivo expanded Stat1–/– Tregs were superior to wild-type Tregs in suppressing alloantigen-driven expansion of T cells in vitro and in inhibiting the development of GVHD. These observations demonstrate that Stat1 is a regulator of Tregs and that targeting Stat1 in CD4+ T cells may facilitate in vitro and in vivo expansion of Tregs for therapeutic use.
Huihui Ma ... Suzanne Lentzsch, Markus Y. Mapara
Vaccination using DCs pulsed with tumor lysates or specific tumor-associated peptides has so far yielded limited clinical success for cancer treatment, due mainly to the low immunogenicity of tumor-associated antigens. In this study, we have identified intratumoral virus-induced inflammation as a precondition for effective antitumor DC vaccination in mice. Administration of a tumor-targeted DC vaccine during ongoing virus-induced tumor inflammation, a regimen referred to as oncolysis-assisted DC vaccination (ODC), elicited potent antitumoral CD8+ T cell responses. This potent effect was not replicated by TLR activation outside the context of viral infection. ODC-elicited immune responses mediated marked tumor regression and successful eradication of preestablished lung colonies, an essential prerequisite for potentially treating metastatic cancers. Unexpectedly, depletion of Tregs during ODC did not enhance therapeutic efficacy; rather, it abrogated antitumor cytotoxicity. This phenomenon could be attributed to a compensatory induction of myeloid-derived suppressor cells in Treg-depleted and thus vigorously inflamed tumors, which prevented ODC-mediated immune responses. Consequently, Tregs are not only general suppressors of immune responses, but are essential for the therapeutic success of multimodal and temporally fine-adjusted vaccination strategies. Our results highlight tumor-targeting, replication-competent viruses as attractive tools for eliciting effective antitumor responses upon DC vaccination.
Norman Woller ... Stefan Kubicka, Florian Kühnel
B cells play a central role in immune system function. Deregulation of normal B cell maturation can lead to the development of autoimmune syndromes as well as B cell malignancies. Elucidation of the molecular features of normal B cell development is important for the development of new target therapies for autoimmune diseases and B cell malignancies. Employing B cell–specific conditional knockout mice, we have demonstrated here that the transcription factor leukemia/lymphoma-related factor (LRF) forms an obligate dimer in B cells and regulates mature B cell lineage fate and humoral immune responses via distinctive mechanisms. Moreover, LRF inactivation in transformed B cells attenuated their growth rate. These studies identify what we believe to be a new key factor for mature B cell development and provide a rationale for targeting LRF dimers for the treatment of autoimmune diseases and B cell malignancies.
Nagisa Sakurai ... Ravi Bhatia, Takahiro Maeda
Macrophages are critical players in both innate and adaptive immunity. While the exogenous signaling events leading to the terminal differentiation of macrophages from monocytes have been studied extensively, the underlying intracellular transcriptional mechanisms remain poorly understood. Here we report that the homeobox transcription factor VentX plays a pivotal role in human macrophage terminal differentiation and proinflammatory function. Our study showed that VentX expression was upregulated upon human primary monocyte-to-macrophage differentiation induced by cytokines such as M-CSF, GM-CSF, and IL-3. Moreover, ablation of VentX expression in primary monocytes profoundly impaired their differentiation to macrophages, and ectopic expression of VentX in a myeloid progenitor cell line triggered its differentiation with prominent macrophage features. Further analysis revealed that VentX was pivotal for the proinflammatory response of terminally differentiated macrophages. Mechanistically, VentX was found to control expression of proteins key to macrophage differentiation and activation, including M-CSF receptor. Importantly, preliminary analysis of gene expression in leukocytes from patients with autoimmune diseases revealed a strong correlation between levels of VentX and those of proinflammatory cytokines. Our results provide mechanistic insight into the crucial roles of VentX in macrophage differentiation and proinflammatory activation and suggest that dysregulation of VentX may play a role in the pathogenesis of autoimmune diseases.
Xiaoming Wu, Hong Gao, Weixiong Ke, Roger W. Giese, Zhenglun Zhu
Chronic immune activation in HIV-infected individuals leads to accumulation of exhausted tissue-like memory B cells. Exhausted lymphocytes display increased expression of multiple inhibitory receptors, which may contribute to the inefficiency of HIV-specific antibody responses. Here, we show that downregulation of B cell inhibitory receptors in primary human B cells led to increased tissue-like memory B cell proliferation and responsiveness against HIV. In human B cells, siRNA knockdown of 9 known and putative B cell inhibitory receptors led to enhanced B cell receptor–mediated (BCR-mediated) proliferation of tissue-like memory but not other B cell subpopulations. The strongest effects were observed with the putative inhibitory receptors Fc receptor–like–4 (FCRL4) and sialic acid–binding Ig-like lectin 6 (Siglec-6). Inhibitory receptor downregulation also led to increased levels of HIV-specific antibody-secreting cells and B cell–associated chemokines and cytokines. The absence of known ligands for FCRL4 and Siglec-6 suggests these receptors may regulate BCR signaling through their own constitutive or tonic signaling. Furthermore, the extent of FCLR4 knockdown effects on BCR-mediated proliferation varied depending on the costimulatory ligand, suggesting that inhibitory receptors may engage specific pathways in inhibiting B cell proliferation. These findings on HIV-associated B cell exhaustion define potential targets for reversing the deleterious effect of inhibitory receptors on immune responses against persistent viral infections.
Lela Kardava ... Susan K. Pierce, Anthony S. Fauci
Impaired cardiac function is associated with myocardial triglyceride accumulation, but it is not clear how the lipids accumulate or whether this accumulation is detrimental. Here we show that hypoxia/ischemia-induced accumulation of lipids in HL-1 cardiomyocytes and mouse hearts is dependent on expression of the VLDL receptor (VLDLR). Hypoxia-induced VLDLR expression in HL-1 cells was dependent on HIF-1α through its interaction with a hypoxia-responsive element in the Vldlr promoter, and VLDLR promoted the endocytosis of lipoproteins. Furthermore, VLDLR expression was higher in ischemic compared with nonischemic left ventricles from human hearts and was correlated with the total lipid droplet area in the cardiomyocytes. Importantly, Vldlr–/– mice showed improved survival and decreased infarct area following an induced myocardial infarction. ER stress, which leads to apoptosis, is known to be involved in ischemic heart disease. We found that ischemia-induced ER stress and apoptosis in mouse hearts were reduced in Vldlr–/– mice and in mice treated with antibodies specific for VLDLR. These findings suggest that VLDLR-induced lipid accumulation in the ischemic heart worsens survival by increasing ER stress and apoptosis.
Jeanna C. Perman ... Sven-Olof Olofsson, Jan Borén
Histone H3 lysine 4 (H3K4me) methyltransferases and their cofactors are essential for embryonic development and the establishment of gene expression patterns in a cell-specific and heritable manner. However, the importance of such epigenetic marks in maintaining gene expression in adults and in initiating human disease is unclear. Here, we addressed this question using a mouse model in which we could inducibly ablate PAX interacting (with transcription-activation domain) protein 1 (PTIP), a key component of the H3K4me complex, in cardiac cells. Reducing H3K4me3 marks in differentiated cardiomyocytes was sufficient to alter gene expression profiles. One gene regulated by H3K4me3 was Kv channel-interacting protein 2 (Kcnip2), which regulates a cardiac repolarization current that is downregulated in heart failure and functions in arrhythmogenesis. This regulation led to a decreased sodium current and action potential upstroke velocity and significantly prolonged action potential duration (APD). The prolonged APD augmented intracellular calcium and in vivo systolic heart function. Treatment with isoproterenol and caffeine in this mouse model resulted in the generation of premature ventricular beats, a harbinger of lethal ventricular arrhythmias. These results suggest that the maintenance of H3K4me3 marks is necessary for the stability of a transcriptional program in differentiated cells and point to an essential function for H3K4me3 epigenetic marks in cellular homeostasis.
Adam B. Stein ... José Jalife, Gregory R. Dressler
β-Adrenergic receptors (β-ARs) enhance cardiac contractility by increasing cAMP levels and activating PKA. PKA increases Ca2+-induced Ca2+ release via phosphorylation of L-type Ca2+ channels (LTCCs) and ryanodine receptor 2. Multiple cyclic nucleotide phosphodiesterases (PDEs) regulate local cAMP concentration in cardiomyocytes, with PDE4 being predominant for the control of β-AR–dependent cAMP signals. Three genes encoding PDE4 are expressed in mouse heart: Pde4a, Pde4b, and Pde4d. Here we show that both PDE4B and PDE4D are tethered to the LTCC in the mouse heart but that β-AR stimulation of the L-type Ca2+ current (ICa,L) is increased only in Pde4b–/– mice. A fraction of PDE4B colocalized with the LTCC along T-tubules in the mouse heart. Under β-AR stimulation, Ca2+ transients, cell contraction, and spontaneous Ca2+ release events were increased in Pde4b–/– and Pde4d–/– myocytes compared with those in WT myocytes. In vivo, after intraperitoneal injection of isoprenaline, catheter-mediated burst pacing triggered ventricular tachycardia in Pde4b–/– mice but not in WT mice. These results identify PDE4B in the CaV1.2 complex as a critical regulator of ICa,L during β-AR stimulation and suggest that distinct PDE4 subtypes are important for normal regulation of Ca2+-induced Ca2+ release in cardiomyocytes.
Jérôme Leroy ... Rodolphe Fischmeister, Grégoire Vandecasteele
Joubert syndrome (JBTS) is characterized by a specific brain malformation with various additional pathologies. It results from mutations in any one of at least 10 different genes, including NPHP1, which encodes nephrocystin-1. JBTS has been linked to dysfunction of primary cilia, since the gene products known to be associated with the disorder localize to this evolutionarily ancient organelle. Here we report the identification of a disease locus, JBTS12, with mutations in the KIF7 gene, an ortholog of the Drosophila kinesin Costal2, in a consanguineous JBTS family and subsequently in other JBTS patients. Interestingly, KIF7 is a known regulator of Hedgehog signaling and a putative ciliary motor protein. We found that KIF7 co-precipitated with nephrocystin-1. Further, knockdown of KIF7 expression in cell lines caused defects in cilia formation and induced abnormal centrosomal duplication and fragmentation of the Golgi network. These cellular phenotypes likely resulted from abnormal tubulin acetylation and microtubular dynamics. Thus, we suggest that modified microtubule stability and growth direction caused by loss of KIF7 function may be an underlying disease mechanism contributing to JBTS.
Claudia Dafinger ... Bernhard Schermer, Hanno Jörn Bolz
Numerous studies have suggested a link between the angiogenic FGF and VEGF signaling pathways; however, the nature of this link has not been established. To evaluate this relationship, we investigated VEGF signaling in ECs with disrupted FGF signaling in vitro and in vivo. ECs lacking FGF signaling became unresponsive to VEGF, caused by downregulation of VEGF receptor 2 (VEGFR2) expression after reduced Vegfr2 enhancer activation. FGF mediated VEGFR2 expression via activation of Erk1/2. Transcriptional analysis revealed that Ets transcription factors controlled VEGFR2 expression in an FGF- and Erk1/2-dependent manner. Mice with defective FGF signaling exhibited loss of vascular integrity and reduced vascular morphogenesis. Thus, basal FGF stimulation of the endothelium is required for maintenance of VEGFR2 expression and the ability to respond to VEGF stimulation and accounts for the hierarchic control of vascular formation by FGFs and VEGF.
Masahiro Murakami ... Brian L. Black, Michael Simons
A ketogenic diet (KD) is a high-fat, low-carbohydrate metabolic regimen; its effectiveness in the treatment of refractory epilepsy suggests that the mechanisms underlying its anticonvulsive effects differ from those targeted by conventional antiepileptic drugs. Recently, KD and analogous metabolic strategies have shown therapeutic promise in other neurologic disorders, such as reducing brain injury, pain, and inflammation. Here, we have shown that KD can reduce seizures in mice by increasing activation of adenosine A1 receptors (A1Rs). When transgenic mice with spontaneous seizures caused by deficiency in adenosine metabolism or signaling were fed KD, seizures were nearly abolished if mice had intact A1Rs, were reduced if mice expressed reduced A1Rs, and were unaltered if mice lacked A1Rs. Seizures were restored by injecting either glucose (metabolic reversal) or an A1R antagonist (pharmacologic reversal). Western blot analysis demonstrated that the KD reduced adenosine kinase, the major adenosine-metabolizing enzyme. Importantly, hippocampal tissue resected from patients with medically intractable epilepsy demonstrated increased adenosine kinase. We therefore conclude that adenosine deficiency may be relevant to human epilepsy and that KD can reduce seizures by increasing A1R-mediated inhibition.
Susan A. Masino ... Eleonora Aronica, Detlev Boison
The popular media and personal anecdotes are rich with examples of stress-induced eating of calorically dense “comfort foods.” Such behavioral reactions likely contribute to the increased prevalence of obesity in humans experiencing chronic stress or atypical depression. However, the molecular substrates and neurocircuits controlling the complex behaviors responsible for stress-based eating remain mostly unknown, and few animal models have been described for probing the mechanisms orchestrating this response. Here, we describe a system in which food-reward behavior, assessed using a conditioned place preference (CPP) task, is monitored in mice after exposure to chronic social defeat stress (CSDS), a model of prolonged psychosocial stress, featuring aspects of major depression and posttraumatic stress disorder. Under this regime, CSDS increased both CPP for and intake of high-fat diet, and stress-induced food-reward behavior was dependent on signaling by the peptide hormone ghrelin. Also, signaling specifically in catecholaminergic neurons mediated not only ghrelin’s orexigenic, antidepressant-like, and food-reward behavioral effects, but also was sufficient to mediate stress-induced food-reward behavior. Thus, this mouse model has allowed us to ascribe a role for ghrelin-engaged catecholaminergic neurons in stress-induced eating.
Jen-Chieh Chuang ... Michael Lutter, Jeffrey M. Zigman
Therapies that raise levels of HDL, which is thought to exert atheroprotective effects via effects on endothelium, are being examined for the treatment or prevention of coronary artery disease (CAD). However, the endothelial effects of HDL are highly heterogeneous, and the impact of HDL of patients with CAD on the activation of endothelial eNOS and eNOS-dependent pathways is unknown. Here we have demonstrated that, in contrast to HDL from healthy subjects, HDL from patients with stable CAD or an acute coronary syndrome (HDLCAD) does not have endothelial antiinflammatory effects and does not stimulate endothelial repair because it fails to induce endothelial NO production. Mechanistically, this was because HDLCAD activated endothelial lectin-like oxidized LDL receptor 1 (LOX-1), triggering endothelial PKCβII activation, which in turn inhibited eNOS-activating pathways and eNOS-dependent NO production. We then identified reduced HDL-associated paraoxonase 1 (PON1) activity as one molecular mechanism leading to the generation of HDL with endothelial PKCβII-activating properties, at least in part due to increased formation of malondialdehyde in HDL. Taken together, our data indicate that in patients with CAD, HDL gains endothelial LOX-1– and thereby PKCβII-activating properties due to reduced HDL-associated PON1 activity, and that this leads to inhibition of eNOS-activation and the subsequent loss of the endothelial antiinflammatory and endothelial repair–stimulating effects of HDL.
Christian Besler ... Thomas F. Lüscher, Ulf Landmesser
Cisplatin is a widely used cancer therapy drug that unfortunately has major side effects in normal tissues, notably nephrotoxicity in kidneys. Despite intensive research, the mechanism of cisplatin-induced nephrotoxicity remains unclear, and renoprotective approaches during cisplatin-based chemotherapy are lacking. Here we have identified PKCδ as a critical regulator of cisplatin nephrotoxicity, which can be effectively targeted for renoprotection during chemotherapy. We showed that early during cisplatin nephrotoxicity, Src interacted with, phosphorylated, and activated PKCδ in mouse kidney lysates. After activation, PKCδ regulated MAPKs, but not p53, to induce renal cell apoptosis. Thus, inhibition of PKCδ pharmacologically or genetically attenuated kidney cell apoptosis and tissue damage, preserving renal function during cisplatin treatment. Conversely, inhibition of PKCδ enhanced cisplatin-induced cell death in multiple cancer cell lines and, remarkably, enhanced the chemotherapeutic effects of cisplatin in several xenograft and syngeneic mouse tumor models while protecting kidneys from nephrotoxicity. Together these results demonstrate a role of PKCδ in cisplatin nephrotoxicity and support targeting PKCδ as an effective strategy for renoprotection during cisplatin-based cancer therapy.
Navjotsingh Pabla ... Robert O. Messing, Zheng Dong
Intratumor heterogeneity is a major clinical problem because tumor cell subtypes display variable sensitivity to therapeutics and may play different roles in progression. We previously characterized 2 cell populations in human breast tumors with distinct properties: CD44+CD24– cells that have stem cell-like characteristics, and CD44–CD24+ cells that resemble more differentiated breast cancer cells. Here we identified 15 genes required for cell growth or proliferation in CD44+CD24– human breast cancer cells in a large-scale loss-of-function screen and found that inhibition of several of these (IL6, PTGIS, HAS1, CXCL3, and PFKFB3) reduced Stat3 activation. We found that the IL-6/JAK2/Stat3 pathway was preferentially active in CD44+CD24– breast cancer cells compared with other tumor cell types, and inhibition of JAK2 decreased their number and blocked growth of xenografts. Our results highlight the differences between distinct breast cancer cell types and identify targets such as JAK2 and Stat3 that may lead to more specific and effective breast cancer therapies.
Lauren L.C. Marotta ... David A. Frank, Kornelia Polyak
Current paradigms suggest that two macrophage subsets, termed M1 and M2, are involved in inflammation and host defense. While the distinct functions of M1 and M2 macrophages have been intensively studied — the former are considered proinflammatory and the latter antiinflammatory — the determinants of their speciation are incompletely understood. Here we report our studies that identify Krüppel-like factor 4 (KLF4) as a critical regulator of macrophage polarization. Macrophage KLF4 expression was robustly induced in M2 macrophages and strongly reduced in M1 macrophages, observations that were recapitulated in human inflammatory paradigms in vivo. Mechanistically, KLF4 was found to cooperate with Stat6 to induce an M2 genetic program and inhibit M1 targets via sequestration of coactivators required for NF-κB activation. KLF4-deficient macrophages demonstrated increased proinflammatory gene expression, enhanced bactericidal activity, and altered metabolism. Furthermore, mice bearing myeloid-specific deletion of KLF4 exhibited delayed wound healing and were predisposed to developing diet-induced obesity, glucose intolerance, and insulin resistance. Collectively, these data identify KLF4 as what we believe to be a novel regulator of macrophage polarization.
Xudong Liao ... Karine Clément, Mukesh K. Jain
Triple-negative breast cancer (TNBC) is a highly diverse group of cancers, and subtyping is necessary to better identify molecular-based therapies. In this study, we analyzed gene expression (GE) profiles from 21 breast cancer data sets and identified 587 TNBC cases. Cluster analysis identified 6 TNBC subtypes displaying unique GE and ontologies, including 2 basal-like (BL1 and BL2), an immunomodulatory (IM), a mesenchymal (M), a mesenchymal stem–like (MSL), and a luminal androgen receptor (LAR) subtype. Further, GE analysis allowed us to identify TNBC cell line models representative of these subtypes. Predicted “driver” signaling pathways were pharmacologically targeted in these cell line models as proof of concept that analysis of distinct GE signatures can inform therapy selection. BL1 and BL2 subtypes had higher expression of cell cycle and DNA damage response genes, and representative cell lines preferentially responded to cisplatin. M and MSL subtypes were enriched in GE for epithelial-mesenchymal transition, and growth factor pathways and cell models responded to NVP-BEZ235 (a PI3K/mTOR inhibitor) and dasatinib (an abl/src inhibitor). The LAR subtype includes patients with decreased relapse-free survival and was characterized by androgen receptor (AR) signaling. LAR cell lines were uniquely sensitive to bicalutamide (an AR antagonist). These data may be useful in biomarker selection, drug discovery, and clinical trial design that will enable alignment of TNBC patients to appropriate targeted therapies.
Brian D. Lehmann ... Yu Shyr, Jennifer A. Pietenpol
Nanoparticle-based materials, such as drug delivery vehicles and diagnostic probes, currently under evaluation in oncology clinical trials are largely not tumor selective. To be clinically successful, the next generation of nanoparticle agents should be tumor selective, nontoxic, and exhibit favorable targeting and clearance profiles. Developing probes meeting these criteria is challenging, requiring comprehensive in vivo evaluations. Here, we describe our full characterization of an approximately 7-nm diameter multimodal silica nanoparticle, exhibiting what we believe to be a unique combination of structural, optical, and biological properties. This ultrasmall cancer-selective silica particle was recently approved for a first-in-human clinical trial. Optimized for efficient renal clearance, it concurrently achieved specific tumor targeting. Dye-encapsulating particles, surface functionalized with cyclic arginine–glycine–aspartic acid peptide ligands and radioiodine, exhibited high-affinity/avidity binding, favorable tumor-to-blood residence time ratios, and enhanced tumor-selective accumulation in αvβ3 integrin–expressing melanoma xenografts in mice. Further, the sensitive, real-time detection and imaging of lymphatic drainage patterns, particle clearance rates, nodal metastases, and differential tumor burden in a large-animal model of melanoma highlighted the distinct potential advantage of this multimodal platform for staging metastatic disease in the clinical setting.
Miriam Benezra ... Ulrich Wiesner, Michelle S. Bradbury
Many cancers display increased NF-κB activity, and NF-κB inhibition is known to diminish tumor development in multiple mouse models, supporting an important role of NF-κB in carcinogenesis. NF-κB activation in premalignant or cancer cells is believed to promote tumor development mainly by protecting these cells from apoptosis. However, it remains unclear to what extent NF-κB activation exhibits additional protumorigenic functions in premalignant cells that could be sufficient to induce spontaneous tumor development. Here we show that expression of constitutively active IκB kinase 2 (IKK2ca) in mouse intestinal epithelial cells (IECs) induced spontaneous tumors in aged mice and also strongly enhanced chemical- and Apc mutation–mediated carcinogenesis. IECs expressing IKK2ca displayed altered Wnt signaling and increased proliferation and elevated expression of genes encoding intestinal stem cell–associated factors including Ascl2, Olfm4, DLK1, and Bmi-1, indicating that increased IKK2/NF-κB activation synergized with Wnt signaling to drive intestinal tumorigenesis. Moreover, IECs expressing IKK2ca produced cytokines and chemokines that induced the recruitment of myeloid cells and activated stromal fibroblasts to become myofibroblasts, thus creating a tumor-promoting microenvironment. Taken together, our results show that constitutively increased activation of IKK2/NF-κB signaling in the intestinal epithelium is sufficient to induce the full spectrum of cell-intrinsic and stromal alterations required for intestinal tumorigenesis.
Katerina Vlantis ... Tania Roskams, Manolis Pasparakis
Metastasis depends on the ability of tumor cells to establish a relationship with the newly seeded tissue that is conducive to their survival and proliferation. However, the factors that render tissues permissive for metastatic tumor growth have yet to be fully elucidated. Breast tumors arising during pregnancy display early metastatic proclivity, raising the possibility that pregnancy may constitute a physiological condition of permissiveness for tumor dissemination. Here we have shown that during murine gestation, metastasis is enhanced regardless of tumor type, and that decreased NK cell activity is responsible for the observed increase in experimental metastasis. Gene expression changes in pregnant mouse lung and liver were shown to be similar to those detected in premetastatic sites and indicative of myeloid cell infiltration. Indeed, myeloid-derived suppressor cells (MDSCs) accumulated in pregnant mice and exerted an inhibitory effect on NK cell activity, providing a candidate mechanism for the enhanced metastatic tumor growth observed in gestant mice. Although the functions of MDSCs are not yet understood in the context of pregnancy, our observations suggest that they may represent a shared mechanism of immune suppression occurring during gestation and tumor growth.
Laetitia A. Mauti ... Paolo Provero, Ivan Stamenkovic
Stem cell–based therapy has been proposed as a potential means of treatment for a variety of brain disorders. Because ethical and technical issues have so far limited the clinical translation of research using embryonic/fetal cells and neural tissue, respectively, the search for alternative sources of therapeutic stem cells remains ongoing. Here, we report that upon transplantation into mice with chemically induced hippocampal lesions, human olfactory ecto–mesenchymal stem cells (OE-MSCs) — adult stem cells from human nasal olfactory lamina propria — migrated toward the sites of neural damage, where they differentiated into neurons. Additionally, transplanted OE-MSCs stimulated endogenous neurogenesis, restored synaptic transmission, and enhanced long-term potentiation. Mice that received transplanted OE-MSCs exhibited restoration of learning and memory on behavioral tests compared with lesioned, nontransplanted control mice. Similar results were obtained when OE-MSCs were injected into the cerebrospinal fluid. These data show that OE-MSCs can induce neurogenesis and contribute to restoration of hippocampal neuronal networks via trophic actions. They provide evidence that human olfactory tissue is a conceivable source of nervous system replacement cells. This stem cell subtype may be useful for a broad range of stem cell–related studies.
Emmanuel Nivet ... François Féron, François S. Roman
Rapamycin is an antibiotic inhibiting eukaryotic cell growth and proliferation by acting on target of rapamycin (TOR) kinase. Mammalian TOR (mTOR) is thought to work through 2 independent complexes to regulate cell size and cell replication, and these 2 complexes show differential sensitivity to rapamycin. Here we combine functional genetics and pharmacological treatments to analyze rapamycin-sensitive mTOR substrates that are involved in cell proliferation and tissue regeneration after partial hepatectomy in mice. After hepatectomy, hepatocytes proliferated rapidly, correlating with increased S6 kinase phosphorylation, while treatment with rapamycin derivatives impaired regeneration and blocked S6 kinase activation. In addition, genetic deletion of S6 kinase 1 (S6K1) caused a delay in S phase entry in hepatocytes after hepatectomy. The proliferative defect of S6K1-deficient hepatocytes was cell autonomous, as it was also observed in primary cultures and hepatic overexpression of S6K1-rescued proliferation. We found that S6K1 controlled steady-state levels of cyclin D1 (Ccnd1) mRNA in liver, and cyclin D1 expression was required to promote hepatocyte cell cycle. Notably, in vivo overexpression of cyclin D1 was sufficient to restore the proliferative capacity of S6K-null livers. The identification of an S6K1-dependent mechanism participating in cell proliferation in vivo may be relevant for cancer cells displaying high mTOR complex 1 activity and cyclin D1 accumulation.
Catherine Espeillac ... Chantal Desdouets, Mario Pende
Hutchinson-Gilford progeria syndrome (HGPS), a devastating premature aging disease, is caused by a point mutation in the lamin A gene (LMNA). This mutation constitutively activates a cryptic splice donor site, resulting in a mutant lamin A protein known as progerin. Recent studies have demonstrated that progerin is also produced at low levels in normal human cells and tissues. However, the cause-and-effect relationship between normal aging and progerin production in normal individuals has not yet been determined. In this study, we have shown in normal human fibroblasts that progressive telomere damage during cellular senescence plays a causative role in activating progerin production. Progressive telomere damage was also found to lead to extensive changes in alternative splicing in multiple other genes. Interestingly, elevated progerin production was not seen during cellular senescence that does not entail telomere shortening. Taken together, our results suggest a synergistic relationship between telomere dysfunction and progerin production during the induction of cell senescence, providing mechanistic insight into how progerin may participate in the normal aging process.
Kan Cao ... Elizabeth G. Nabel, Francis S. Collins
In addition to its role as an essential neurotransmitter, dopamine serves important physiologic functions in organs such as the kidney. Although the kidney synthesizes dopamine through the actions of aromatic amino acid decarboxylase (AADC) in the proximal tubule, previous studies have not discriminated between the roles of extrarenal and intrarenal dopamine in the overall regulation of renal function. To address this issue, we generated mice with selective deletion of AADC in the kidney proximal tubules (referred to herein as ptAadc–/– mice), which led to selective decreases in kidney and urinary dopamine. The ptAadc–/– mice exhibited increased expression of nephron sodium transporters, decreased natriuresis and diuresis in response to l-dihydroxyphenylalanine, and decreased medullary COX-2 expression and urinary prostaglandin E2 excretion and developed salt-sensitive hypertension. They had increased renin expression and altered renal Ang II receptor (AT) expression, with increased AT1b and decreased AT2 and Mas expression, associated with increased renal injury in response to Ang II. They also exhibited a substantially shorter life span compared with that of wild-type mice. These results demonstrate the importance of the intrarenal dopaminergic system in salt and water homeostasis and blood pressure control. Decreasing intrarenal dopamine subjects the kidney to unbuffered responses to Ang II and results in the development of hypertension and a dramatic decrease in longevity.
Ming-Zhi Zhang ... Shilin Yang, Raymond C. Harris
Laminins and their integrin receptors are implicated in epithelial cell differentiation and progenitor cell maintenance. We report here that a previously unrecognized subpopulation of mouse alveolar epithelial cells (AECs) expressing the laminin receptor α6β4, but little or no pro–surfactant C (pro-SPC), is endowed with regenerative potential. Ex vivo, this subpopulation expanded clonally as progenitors but also differentiated toward mature cell types. Integrin β4 itself was not required for AEC proliferation or differentiation. An in vivo embryonic lung organoid assay, which we believe to be novel, was used to show that purified β4+ adult AECs admixed with E14.5 lung single-cell suspensions and implanted under kidney capsules self-organized into distinct Clara cell 10-kDa secretory protein (CC10+) airway-like and SPC+ saccular structures within 6 days. Using a bleomycin model of lung injury and an SPC-driven inducible cre to fate-map AECs, we found the majority of type II AECs in fibrotic areas were not derived from preexisting type II AECs, demonstrating that SPC– progenitor cells replenished type II AECs during repair. Our findings support the idea that there is a stable AEC progenitor population in the adult lung, provide in vivo evidence of AEC progenitor cell differentiation after parenchymal injury, and identify a strong candidate progenitor cell for maintenance of type II AECs during lung repair.
Harold A. Chapman ... Ying Wei, Thiennu H. Vu
The airway is a primary portal of entry for noxious environmental stimuli that can trigger airway remodeling, which contributes significantly to airway obstruction in chronic obstructive pulmonary disease (COPD) and chronic asthma. Important pathologic components of airway remodeling include fibrosis and abnormal innate and adaptive immune responses. The positioning of fibroblasts in interstitial spaces suggests that they could participate in both fibrosis and chemokine regulation of the trafficking of immune cells such as dendritic cells, which are crucial antigen-presenting cells. However, physiological evidence for this dual role for fibroblasts is lacking. Here, in two physiologically relevant models — conditional deletion in mouse fibroblasts of the TGF-β–activating integrin αvβ8 and neutralization of αvβ8 in human COPD fibroblasts — we have elucidated a mechanism whereby lung fibroblast chemokine secretion directs dendritic cell trafficking, in a manner that is critically dependent on αvβ8-mediated activation of TGF-β by fibroblasts. Our data therefore indicate that fibroblasts have a crucial role in regulating both fibrotic and immune responses in the lung.
Hideya Kitamura ... Jody Lynn Baron, Stephen L. Nishimura
Bicuspid aortic valve (BAV), the leading congenital heart disease, occurs in 1%–2% of the population. Genetic studies suggest that BAV is an autosomal-dominant disease with reduced penetrance. However, only 1 gene, NOTCH1, has been linked to cases of BAV. Here, we show that targeted deletion of Gata5 in mice leads to hypoplastic hearts and partially penetrant BAV formation. Endocardial cell–specific inactivation of Gata5 led to BAV, similar to that observed in Gata5–/– mice. In all cases, the observed BAVs resulted from fusion of the right-coronary and noncoronary leaflets, the subtype associated with the more severe valve dysfunction in humans. Neither endocardial cell proliferation nor cushion formation was altered in the absence of Gata5. Rather, defective endocardial cell differentiation, resulting from the deregulation of several components of the Notch pathway and other important endocardial cell regulators, may be the underlying mechanism of disease. The results unravel a critical cell-autonomous role for endocardial Gata5 in aortic valve formation and identify GATA5 as a potential gene responsible for congenital heart disease in humans. Mice with mutated Gata5 alleles represent unique models to dissect the mechanisms underlying degenerative aortic valve disease and to develop much-needed preventive and therapeutic interventions.
Brigitte Laforest, Gregor Andelfinger, Mona Nemer
Multidrug resistance–associated protein 4 (MRP4, also known as Abcc4) regulates intracellular levels of cAMP and cGMP in arterial SMCs. Here, we report our studies of the role of MRP4 in the development and progression of pulmonary arterial hypertension (PAH), a severe vascular disease characterized by chronically elevated pulmonary artery pressure and accompanied by remodeling of the small pulmonary arteries as a prelude to right heart failure and premature death. MRP4 expression was increased in pulmonary arteries from patients with idiopathic PAH as well as in WT mice exposed to hypoxic conditions. Consistent with a pathogenic role for MRP4 in PAH, WT mice exposed to hypoxia for 3 weeks showed reversal of hypoxic pulmonary hypertension (PH) following oral administration of the MRP4 inhibitor MK571, and Mrp4–/– mice were protected from hypoxic PH. Inhibition of MRP4 in vitro was accompanied by increased intracellular cAMP and cGMP levels and PKA and PKG activities, implicating cyclic nucleotide-related signaling pathways in the mechanism underlying the protective effects of MRP4 inhibition. Our data suggest that MRP4 could represent a potential target for therapeutic intervention in PAH.
Yannis Hara ... Marc Humbert, Jean-Sébastien Hulot
Immune mechanisms are known to control the pathogenesis of atherosclerosis. However, the exact role of DCs, which are essential for priming of immune responses, remains elusive. We have shown here that the DC-derived chemokine CCL17 is present in advanced human and mouse atherosclerosis and that CCL17+ DCs accumulate in atherosclerotic lesions. In atherosclerosis-prone mice, Ccl17 deficiency entailed a reduction of atherosclerosis, which was dependent on Tregs. Expression of CCL17 by DCs limited the expansion of Tregs by restricting their maintenance and precipitated atherosclerosis in a mechanism conferred by T cells. Conversely, a blocking antibody specific for CCL17 expanded Tregs and reduced atheroprogression. Our data identify DC-derived CCL17 as a central regulator of Treg homeostasis, implicate DCs and their effector functions in atherogenesis, and suggest that CCL17 might be a target for vascular therapy.
Christian Weber ... Tobias Junt, Alma Zernecke
Deep vein thrombosis (DVT) is a major cause of pulmonary thromboembolism, a leading cause of death in individuals with DVT. Several lines of evidence indicate proinflammatory cytokines such as TNF-α are involved in thrombus formation and resolution, but the roles of IFN-γ remain unclear. To address this issue, we performed ligation of the inferior vena cava to induce DVT in WT and IFN-γ–deficient (Ifng–/–) mice. In WT mice, intrathrombotic IFN-γ levels were elevated progressively as the postligation interval was extended. Thrombus size was substantially smaller at 10 and 14 days in Ifng–/– mice than in WT mice. Intrathrombotic collagen content was remarkably reduced at more than 10 days after the ligation in Ifng–/– mice compared with WT mice. The expression and activity of MMP-9, but not MMP-2, was higher at the late phase in Ifng–/– mice than in WT mice. Moreover, intrathrombotic recanalization was increased in Ifng–/– mice, with enhanced Vegf gene expression, compared with that in WT mice. Activation of the IFN-γ/Stat1 signal pathway suppressed PMA-induced Mmp9 and Vegf gene expression in peritoneal macrophages. Furthermore, administration of anti–IFN-γ mAbs accelerated thrombus resolution in WT mice. Collectively, these findings indicate that IFN-γ can have detrimental roles in thrombus resolution and may be a good molecular target for the acceleration of thrombus resolution in individuals with DVT.
Mizuho Nosaka ... Naofumi Mukaida, Toshikazu Kondo
Plasma HDL levels have a protective role in atherosclerosis, yet clinical therapies to raise HDL levels have remained elusive. Recent advances in the understanding of lipid metabolism have revealed that miR-33, an intronic microRNA located within the SREBF2 gene, suppresses expression of the cholesterol transporter ABC transporter A1 (ABCA1) and lowers HDL levels. Conversely, mechanisms that inhibit miR-33 increase ABCA1 and circulating HDL levels, suggesting that antagonism of miR-33 may be atheroprotective. As the regression of atherosclerosis is clinically desirable, we assessed the impact of miR-33 inhibition in mice deficient for the LDL receptor (Ldlr–/– mice), with established atherosclerotic plaques. Mice treated with anti-miR33 for 4 weeks showed an increase in circulating HDL levels and enhanced reverse cholesterol transport to the plasma, liver, and feces. Consistent with this, anti-miR33–treated mice showed reductions in plaque size and lipid content, increased markers of plaque stability, and decreased inflammatory gene expression. Notably, in addition to raising ABCA1 levels in the liver, anti-miR33 oligonucleotides directly targeted the plaque macrophages, in which they enhanced ABCA1 expression and cholesterol removal. These studies establish that raising HDL levels by anti-miR33 oligonucleotide treatment promotes reverse cholesterol transport and atherosclerosis regression and suggest that it may be a promising strategy to treat atherosclerotic vascular disease.
Katey J. Rayner ... Edward A. Fisher, Kathryn J. Moore
Thrombosis is initiated by tissue factor (TF), a coagulation cofactor/receptor expressed in the vessel wall, on myeloid cells, and on microparticles (MPs) with variable procoagulant activity. However, the molecular pathways that generate prothrombotic TF in vivo are poorly defined. The oxidoreductase protein disulfide isomerase (PDI) is thought to be involved in the activation of TF. Here, we found that in mouse myeloid cells, ATP-triggered signaling through purinergic receptor P2X, ligand-gated ion channel, 7 (P2X7 receptor; encoded by P2rx7) induced activation (decryption) of TF procoagulant activity and promoted release of TF+ MPs from macrophages and SMCs. The generation of prothrombotic MPs required P2X7 receptor–dependent production of ROS leading to increased availability of solvent-accessible extracellular thiols. An antibody to PDI with antithrombotic activity in vivo attenuated the release of procoagulant MPs. In addition, P2rx7–/– mice were protected from TF-dependent FeCl3-induced carotid artery thrombosis. BM chimeras revealed that P2X7 receptor prothrombotic function was present in both hematopoietic and vessel wall compartments. In contrast, an alternative anti-PDI antibody showed activities consistent with cellular activation typically induced by P2X7 receptor signaling. This anti-PDI antibody restored TF-dependent thrombosis in P2rx7–/– mice. These data suggest that PDI regulates a critical P2X7 receptor–dependent signaling pathway that generates prothrombotic TF, defining a link between inflammation and thrombosis with potential implications for antithrombotic therapy.
Christian Furlan-Freguia, Patrizia Marchese, András Gruber, Zaverio M. Ruggeri, Wolfram Ruf
Manzar Ashtari ... Kenneth S. Shindler, Jean Bennett
Andrea Casazza ... Paolo M. Comoglio, Luca Tamagnone
Copyright © 2014 American Society for Clinical Investigation