Tail Vein Administration Research Articles

Galuminox: Preclinical validation of a novel PET tracer for non-invasive imaging of oxidative stress in vivo

Overproduction of reactive oxygen species (ROS) is a well-established indicator of ongoing tissue inflammation. However, there is a scarcity of molecular imaging probes capable of providing noninvasive sensitive detection of ROS for allowing longitudinal studies of disease pathology and/or monitoring therapeutic efficacy of ROS scavengers. Herein, we report synthesis and chemical characterization of a novel metalloprobe, Galuminox, a moderately fluorescent agent that detects superoxide and hydrogen peroxide generation. Using live-cell fluorescence imaging analysis, Galuminox demonstrates ability to detect superoxide and monitor effects of ROS-attenuating agents, such as Carvedilol, Dexrazoxane, and mitoTempo in lung epithelial A549 cells. Furthermore, LPS stimulation of A549 cells that either express the mitochondria targeted fluorescent protein Keima or are stained with MitoSOX, a mitochondria-specific superoxide probe, indicates preferential co-localization of Galuminox with mitochondria producing elevated amounts of superoxide. Dynamic PET/CT scans 45 min post tail-vein administration of 68Ga-Galuminox show 4-fold higher uptake and stable retention in lungs of LPS treated mice compared to their saline-only treated counterparts. Post preclinical PET imaging, quantitative biodistribution studies also correlate with 4-fold higher retention of the radiotracer in lungs of LPS treated mice compared with their saline-only treated control counterparts. Consistent with these observations, lung cells isolated from LPS-treated mice demonstrated elevated ROS production deploying CellROX, the ROS probe. Finally, Galuminox uptake correlates with histological and physiological evidence of acute lung injury as evident by polynuclear infiltration, thickening of the alveolar epithelial membranes and increased bronchioalveolar lavage protein content. Taken collectively, these data indicate that 68Ga-Galuminox tracer uptake is a measure of ROS activity in acutely injured lungs and suggests its potential utility in monitoring oxidative stress in other diseases.

Design and Validation of Liposomal ApoE2 Gene Delivery System to Evade Blood-Brain Barrier for Effective Treatment of Alzheimer's Disease.

Targeting gene-based therapeutics to the brain is a strategy actively sought to treat Alzheimer's disease (AD). Recent findings discovered the role of apolipoprotein E (ApoE) isoforms in the clearance of toxic amyloid beta proteins from the brain. ApoE2 isoform is beneficial for preventing AD development, whereas ApoE4 is a major contributing factor to the disease. In this paper, we demonstrated efficient brain-targeted delivery of ApoE2 encoding plasmid DNA (pApoE2) using glucose transporter-1 (glut-1) targeted liposomes. Liposomes were surface-functionalized with a glut-1 targeting ligand mannose (MAN) and a cell-penetrating peptide (CPP) to enhance brain-targeting and cellular internalization, respectively. Among various CPPs, rabies virus glycoprotein peptide (RVG) or penetratin (Pen) was selected as a cell-penetration enhancer. Dual (RVGMAN and PenMAN)-functionalized liposomes were cytocompatible at 100 nM phospholipid concentration and demonstrated significantly higher expression of ApoE2 in bEnd.3 cells, primary neurons, and astrocytes compared to monofunctionalized and unmodified (plain) liposomes. Dual-modified liposomes also showed ∼2 times higher protein expression than other formulation controls in neurons cultured below the in vitro BBB model. These results translated well to in vivo efficacy study with significantly higher transfection of pApoE2 in the C57BL/6 mice brain following single tail vein administration of RVGMAN and PenMAN functionalized liposomes without any noticeable signs of toxicity. These results illustrate the potential of surface-modified liposomes for safe and brain-targeted delivery of the pApoE2 gene for effective AD therapy.

Hepcidin deficiency causes bone loss through interfering with the canonical Wnt/β-catenin pathway via Forkhead box O3a

ObjectiveHepcidin deficiency is known to cause body iron accumulation and bone microarchitecture defects, but the exact underlying mechanisms of hepcidin deficiency-induced bone loss remain unclear. Our objective was to understand the molecular mechanism of hepcidin deficiency-induced bone loss. MethodsThe bone phenotypes of wild type (WT) and hepcidin knockout (Hepcidin-KO) mice were measured by microcomputed tomography. The osteoclastic marker of the bone was measured by tartrate-resistant acid phosphatase staining. The osteoblastic marker of the bone was measured by immunostaining of osteocalcin. Primary osteoblastic and osteoclastic differentiation was performed using bone marrow cells. The mature osteoclast was determined by tartrate-resistant acid phosphatase staining, pit formation assay and relative gene expression. The mature osteoblast was determined by alkaline phosphatase activity, alkaline phosphatase staining, Alizarin Red staining and relative gene expression. The protein expression of β-catenin, TCF4/TCF7L2 and Forkhead box O3a (FOXO3a) was measured by Western blot and their combination by co-immunoprecipitation. In vivo study was performed by tail vein administration of FOXO3a-RNAi using an adeno-associated virus in Hepcidin-KO mice. ResultsWe found that Hepcidin-KO mice exhibited iron accumulation and bone loss compared with WT mice. The osteoclastic differentiation of bone marrow-derived macrophages from Hepcidin-KO mice was not significantly different from that of bone marrow–derived macrophages from WT mice. However, the osteoblastic differentiation of bone marrow–derived mesenchymal stem cells from Hepcidin-KO mice was obviously decreased compared with that of bone marrow–derived mesenchymal stem cells from WT mice. Furthermore, it was confirmed in this study that upon hepcidin deficiency, β-catenin, TCF4/TCF7L2 and FOXO3a expression in bone tissues was not altered, but β-catenin combination with TCF4/TCF7L2 was strongly inhibited by β-catenin combination with FOXO3a, indicating that the canonical Wnt/β-catenin pathway was affected. Tail vein administration of FOXO3a-RNAi using an adeno-associated virus in Hepcidin-KO mice resulted in bone mass recovery. ConclusionThese findings suggested that hepcidin deficiency might cause bone loss by interfering with the canonical Wnt/β-catenin pathway via FOXO3a, and FOXO3a inhibition would be a possible approach to treat hepcidin deficiency-induced bone loss. The translational potential of this articleHepcidin deficiency, as well as iron accumulation, has been considered as a risk factor for osteoporosis. For this kind of osteoporosis, inhibition of FOXO3a either by neutralized antibody or AAV-mediated RNAi, represents an effective and promising method.

The effects of different doses of teriparatide on bisphosphonate‐related osteonecrosis of the jaw in mice

This study aimed to investigate the therapeutic effect of different doses of teriparatide (TPTD) on bisphosphonate-related osteonecrosis of the jaw (BRONJ). To establish the BRONJ model, 20 mice were randomly divided into two groups: a group that received tail vein administration of zoledronic acid with dexamethasone (ZA-125 µg/kg, DEX 5 mg/kg) and a group that received saline weekly. The mice subsequently underwent bilateral maxillary first molar extraction. After 8 weeks of modeling administration, the maxilla samples were examined by micro-computed tomography and histological staining (haematoxylin-eosin, Masson's trichrome, and tartrate-resistant acid phosphatase) and the cytokine level was measured (enzyme-linked immunosorbent assay and western blot). To determine the role of TPTD in BRONJ, the same protocol as previously described was applied in 100 mice (80 received ZA+DEX and 20 received saline). After 8 weeks of modeling administration, 80 ZA+DEX mice were randomly divided into four groups: three groups with subcutaneous administration of TPTD (i.e. T1-3, T2-10, and T3-30 µg/kg/d) and one group with saline daily for the next 8 weeks. The other 20 saline mice continued to receive saline daily. In PART ONE, the level of receptor activator of nuclear factor kappa-Β ligand and the numbers of osteoclasts differed between the model and control groups. In PART TWO, we found that TPTD had a positive effect on BRONJ in a mouse model based on clinical and histomorphological observations. Among the three treatment groups, the T1 and T2 groups significantly differed from the model group, whereas the T3 group showed no statistical differences. Subcutaneous administration of TPTD has a beneficial effect on BRONJ in mice. Nevertheless, further studies are needed to determine whether the therapeutic effect on BRONJ is dose-dependent.

Xenogeneic and Allogeneic Mesenchymal Stem Cells Effectively Protect the Lung Against Ischemia-reperfusion Injury Through Downregulating the Inflammatory, Oxidative Stress, and Autophagic Signaling Pathways in Rat

This study tested the hypothesis that both allogenic adipose-derived mesenchymal stem cells (ADMSCs) and human inducible pluripotent stem cell-derived MSCs (iPS-MSCs) offered a comparable effect for protecting the lung against ischemia-reperfusion (IR) injury in rodent through downregulating the inflammatory, oxidative stress, and autophagic signaling pathways. Adult male Sprague–Dawley rats (n = 32) were categorized into group 1 (sham-operated control), group 2 (IRI), group 3 [IRI + ADMSCs (1.0 × 106 cells)/tail-vein administration at 0.5/18/36 h after IR], and group 4 [IRI + iPS-MSCs (1.0 × 106 cells)/tail-vein administration at 0.5/18/36 h after IR], and lungs were harvested at 72 h after IR procedure. In vitro study demonstrated that protein expressions of three signaling pathways in inflammation (TLR4/MyD88/TAK1/IKK/I-κB/NF-κB/Cox-2/TNF-α/IL-1ß), mitochondrial damage/cell apoptosis (cytochrome C/cyclophilin D/DRP1/ASK1/APAF-1/mitochondrial-Bax/caspase3/8/9), and autophagy/cell death (ULK1/beclin-1/Atg5,7,12, ratio of LCB3-II/LC3B-I, p-AKT/m-TOR) were significantly higher in lung epithelial cells + 6h hypoxia as compared with the control, and those were significantly reversed by iPS-MSC treatment (all P < 0.001). Flow cytometric analysis revealed that percentages of the inflammatory cells in bronchioalveolar lavage fluid and circulation, and immune cells in circulation/spleen as well as circulatory early and late apoptotic cells were highest in group 2, lowest in group 1, and significantly higher in group 3 than in group 4 (all P < 0.0001). Microscopy showed the lung injury score and numbers of inflammatory cells and Western blot analysis showed the signaling pathways of inflammation, mitochondrial damage/cell apoptosis, autophagy, and oxidative stress exhibited an identical pattern of flow cytometric results among the four groups (all P < 0.0001). Both xenogeneic and allogenic MSCs protected the lung against IRI via suppressing the inflammatory, oxidative stress, and autophagic signaling.

S-Nitrosylation of Plastin-3 Exacerbates Thoracic Aortic Dissection Formation via Endothelial Barrier Dysfunction.

Thoracic aortic dissection (TAD) is a fatal disease that leads to aortic rupture and sudden death. However, little is known about the effect and molecular mechanism of S-nitrosylation (SNO) modifications in TAD formation. Approach and Results: SNO levels were higher in aortic tissues from TAD patients than in those from healthy controls, and PLS3 (plastin-3) SNO was identified by liquid chromatography-tandem mass spectrometry analysis. Furthermore, tail vein administration of endothelial-specific adeno-associated viruses of mutant PLS3-C566A (denitrosylated form) suppressed the development of TAD in mice, but the wild-type PLS3 (S-nitrosylated form) virus did not. Mechanistically, Ang II (angiotensin II)-induced PLS3 SNO enhanced the association of PLS3 with both plectin and cofilin via an iNOS (inducible nitric oxide synthase)-dependent pathway in endothelial cells. The formation of PLS3/plectin/cofilin complex promoted cell migration and tube formation but weakened adherens junction formation in Ang II-treated endothelial cells. Interestingly, denitrosylated form of PLS3 partially mitigated Ang II-induced PLS3/plectin/cofilin complex formation and cell junction disruption. Additionally, the inhibition of iNOS attenuated PLS3 SNO and the association of PLS3 with plectin and cofilin, thereby modulating endothelial barrier function. Our data indicate that protein SNO modification in endothelial cells modulates the progression of aortic aneurysm and dissection. The iNOS-mediated SNO of PLS3 at the Cys566 site promoted its interaction with cofilin and plectin, thus contributing to endothelial barrier disruption and pathological angiogenesis in TAD.

Abstract 1442: Chimeric antigen receptor T cell therapy targeting ICAM-1 in gastric cancer

Abstract Introduction: Despite advances in treatment, gastric cancer (GC) remains among the most fatal malignancies. Intercellular adhesion molecule-1 (ICAM-1) is overexpressed and associated with various cancers, including GC. We developed a third-generation chimeric antigen receptor (CAR) targeting ICAM-1 (ICAM-1-CAR) and investigated ICAM-1 as a immunotherapeutic target for CAR T therapy in GC. Methods: We investigated ICAM-1 expression in tissues of curatively resected GC patients (n=134) by immunohistochemical staining to determine its prognostic value. Effector to target assays were performed with ICAM-1-CAR T cells co-cultured with 8 GC cell lines with varying levels of ICAM-1 expression to investigate specific target cell death. We created a firefly luciferase-expressing human GC model in mice to measure tumor growth and killing by whole body bioluminescence imaging. To find the best treatment route and optimal dose of T cells in GC mice model, ICAM-1-CAR was injected via tail vein and intraperitoneal administration (IP) at two different doses, i.e., low dose (1 million CAR T) and high dose (10 million live T cells). Results: The ICAM-1 expression was higher in advanced stages (22.2% in stage II vs. 48.8% in stage III, p=0.002) and the patients with high ICAM-1 expression showed significantly poor survival (disease free survival, hazard ration [HR], 4.55, p&amp;lt;0.001; overall survival, HR, 3.89, p&amp;lt;0.001). The efficacy of ICAM-1-CAR T cells in vitro showed a strong correlation with the level of ICAM-1 expression in target cells, i.e., faster killing of GC with higher ICAM-1 expression. ICAM-1-CART facilitated regression of tumor in MKN-28 xenograft IP model. Additionally, the efficacy of ICAM-1-CAR was more prominent in mice treated with high dose of T cell and IP route. Compared to no treatment, ICAM-1CAR via IP led to tumor reduction that persisted for over 80 days and significantly improved survival without toxicity (p=0.049). Conclusion: ICAM-1 specific CAR T cells demonstrated significant therapeutic efficacy in vitro and in vivo against preclinical GC models. ICAM-1-CAR T cells may be developed into a promising treatment strategy for patients with ICAM-1 positive GC cancers. Citation Format: Minkyu Jung, Marjan Zaman, Ygindra Vedvyas, Xianglan Zhang, Jaclyn E. McCloskey, Yanping Yang, Irene M. Min, Raza Zamegar, Yoon Young Choi, Jae-Ho Cheong, Sung Hoon Noh, Sun Young Rha, Hyun Cheol Chung, Moonsoo M. Jin. Chimeric antigen receptor T cell therapy targeting ICAM-1 in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1442.

Abstract 5016: STACT-TREX1: A systemically-administered STING pathway agonist targets tumor-resident myeloid cells and induces adaptive anti-tumor immunity in multiple preclinical models

Abstract Systemic delivery of STING agonists to tumor-resident myeloid cells will be required to elicit optimal type I interferon-mediated anti-tumor immunity in a metastatic disease setting. To this end, we have generated a microbial-based immunotherapy (STACT- Salmonella Typhimurium (Attenuated) Checkpoint Therapy) that utilizes a highly attenuated, clinically developed strain that is specifically enriched in tumors after intravenous administration. The strain has been modified to reduce pro-inflammatory TLR signaling in order to limit its immunosuppressive microbial profile and significantly improve its therapeutic index. An inhibitory microRNA to TREX1 was designed and introduced into the STACT strain. TREX1 is a 3′ exonuclease immune checkpoint that degrades cytosolic DNA, thereby preventing it from binding cGAS and activating the STING pathway. Mutations in human TREX1 cause a type I interferonopathy known as Aicardi-Goutières syndrome and autoimmune chilblain lupus. Systemic delivery of small-molecule inhibitors targeting TREX1 are intractable due to its ubiquitous expression in healthy tissue. We have engineered our systemically administered therapy to specifically deliver RNAi against TREX1 to the myeloid compartment of the tumor microenvironment, rather than to the inflammatory TNFα- and IL-6-producing stromal compartment. This enables targeted cGAS/STING induction of type I interferon, thereby promoting optimal T cell priming against tumor neoantigens. STACT-TREX1 was evaluated for therapeutic efficacy in several tumor models, including CT26 and MC38 colon carcinoma models, and B16.F10 melanoma. A high degree of tumor-specific colonization of STACT-TREX1 therapy was observed after a single intravenous tail vein administration, with 100,000-fold enrichment detected in tumor tissue relative to spleen and liver. The therapy was well tolerated, with very low TNFα and IL-6 serum cytokines detected. In multiple flank tumor models, potent tumor growth inhibition and complete tumor regressions were observed as a monotherapy. Efficacy was CD8-dependent, and cured mice were protected from tumor re-challenge. Importantly, immune correlates post-treatment demonstrated a significant shift away from the recruitment of microbe-induced innate myeloid populations, and towards type I interferon-induced adaptive immunity. This novel therapeutic approach will allow for systemic delivery of a STING pathway agonist that induces type I interferon specifically in tumor-resident myeloid cells, inducing potent adaptive anti-tumor immunity in solid tumors that are refractory to existing immunotherapies. Citation Format: Anastasia M. Makarova, Alexandre Iannello, Chris S. Rae, Beverly King, Marina Besprozvannaya, John Faulhaber, Justin Skoble, Christopher D. Thanos, Laura Hix Glickman. STACT-TREX1: A systemically-administered STING pathway agonist targets tumor-resident myeloid cells and induces adaptive anti-tumor immunity in multiple preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5016.

Imaging Cancer Immunology: Tracking Immune Cells in vivo with Magnetic Particle Imaging

Abstract The rapid growth of research into immuno-oncology has fueled a need to be able to determine the location of a variety of immune cells in solid tumors. However, existing methods for that have been insufficient. Magnetic Particle Imaging (MPI) is a novel pre-clinical cellular imaging technique that can be used to non-invasively track iron-oxide tagged immune cells in vivo. In this study, we imaged the differences in biodistribution and migration of macrophages in naïve and tumor bearing mice. Imaging was performed with the MOMENTUM MPI system (Magnetic Insight Inc., CA). Macrophages were pre-loaded with tracers in vitro for tracking specific populations or loaded in situ for detection of localized inflammation. Initial macrophage biodistribution varied depending on the route of administration. Intravenous murine tail vein administration provided the best systemic delivery, but also resulted in macrophage accumulation within the liver and spleen. Direct intracranial injection increased local retention but resulted in the most tissue disruption. In the tumor bearing animals, macrophages were predominately detected in the expanding margins of the tumor. By combining the accurate quantification and specificity of MPI, information can be obtained on macrophage function and biodistribution. Other cells can also be labeled (dendritic cells, MDSCs, etc) and this information can be utilized to better understand the factors influencing immune cell migration in and around tumors.

A Liquid Chromatography with Tandem Mass Spectrometry-Based Proteomic Analysis of the Proteins Secreted by Human Adipose-Derived Mesenchymal Stem Cells

Liquid chromatography using a tandem mass spectrometer (LC-MS/MS) is a method of proteomic analysis. A shotgun analysis by LC-MS/MS comprehensively identifies proteins from tissues and cells with high resolution. The hepatic function of mice with acute hepatitis following the intraperitoneal administration of CCL4 was improved by the tail vein administration of the culture conditional medium (CM) of human mesenchymal stem cells from adipose tissue (hMSC-AT). In this study, a secreted protein expression analysis of hMSC-AT was performed using LC-MS/MS; 128 proteins were identified. LC-MS/MS showed that 106 new functional proteins and 22 proteins (FINC, PAI1, POSTN, PGS2, TIMP1, AMPN, CFAH, VIME, PEDF, SPRC, LEG1, ITGBL, ENOA, CSPG2, CLUS, IBP4, IBP7, PGS1, IBP2, STC2, CTHR1, CD9) were previously reported in hMSC-AT-CMs. In addition, various proteins associated with growth (SAP, SEM7A, PTK7); immune system processes (CO1A2, CO1A1, CATB, TSP1, GAS6, PTX3, C1 S, SEM7A, G3P, PXDN, SRCRL, CD248, SPON2, ENPP2, CD109, CFAB, CATL1, MFAP5, MIF, CXCL5, ADAM9, CATK); and reproduction (MMP2, CATB, FBLN1, SAP, MFGM, GDN, CYTC) were identified in hMSC-AT-CMs. These results indicate that a comprehensive expression analysis of proteins by LC-MS/MS is useful for investigating new factors associated with cellular components, biological processes, and molecular functions.

Abstract 354: Accelerated Autophagy is Required for Doxorubicin-induced Oxidative Damage and Cardiomyopathy

Doxorubicin (DOX) is a highly effective chemotherapeutic agent used in the treatment of a broad spectrum of malignancies. However, clinical use of DOX is limited by the risk of severe cardiotoxicity. In patients, the acute onset of cardiomyopathy often precludes chemotherapy treatment, resulting in reduced quality of life and increased morbidity. While the mechanisms by which DOX promotes cardiac dysfunction are not well understood, our previous findings show that intracellular metabolism of DOX elicits mitochondrial reactive oxygen species (ROS) generation, and subsequent activation of the autophagy/lysosomal system. Equally, evidence suggests that elimination of vital cellular organelles and proteins within the cell as a result of pathologically elevated autophagy may in turn stimulate an increase in ROS generation. Therefore, we hypothesize that DOX-induced autophagy promotes cardiac dysfunction as a result of an autophagy-induced increase in oxidative stress in the heart. To prevent elevated activity of the autophagy/lysosomal system, we administered a dominant negative mutation of the autophagy protein ATG5 (rAAV-dnATG5) to female Sprague-Dawley rats via tail vein administration four weeks prior to either DOX (20 mg/kg ip) or saline treatment. Two days following treatment, assessment of cardiac function showed significant alterations to the myocardial performance index and fractional shortening percentage in control, DOX treated animals. These changes corresponded with deficits to mitochondrial respiration, increased mitochondrial ROS emission and increased appearance of autophagic vacuoles within the heart. In contrast, rAAV-dnATG5 administration in DOX treated animals prevented the conjugation of ATG12 to ATG5 thereby reducing autophagosome formation and attenuating cardiac dysfunction. Interestingly, protein expression of the antioxidant enzymes catalase and superoxide dismutase 2 (SOD2) was elevated in these animals, which may account for improved mitochondrial respiration and reduced mitochondrial free radical production. These findings indicate that accelerated autophagy promotes DOX-induced cardiac dysfunction and is an underlying cause of chemotherapy-induced oxidative damage to the heart.

Hepatocyte Early Growth Response 1 (EGR1) Regulates Lipid Metabolism in Nonalcoholic Fatty Liver Disease

ObjectiveNonalcoholic fatty liver disease (NAFLD) is a burgeoning health problem worldwide due to significant increases in the prevalence of obesity, insulin resistance diabetes, and hyperlipidemia. A risk factor for NAFLD is insulin resistance as it promotes the excessive accumulation of lipids into the hepatocytes, causing cellular stress and death. As a stress response gene, early growth response 1 (EGR1) plays critical roles in inflammation and tissue repair. The aim for this study is to explore a novel role of hepatocyte EGR1 in the regulation of lipid metabolism in NAFLD.MethodsHuman microarray data from NCBI GEO database (accession #GSE48452) was used to obtain relative EGR1 expression in human normal and NAFLD livers. To determine the expression of EGR1 during the development of NAFLD, C57Bl6/J mice were fed a high in fat, cholesterol, and fructose (HFCF) for up to 9 months and EGR1 levels were examined. An in vitro insulin stimulation experiment was performed in wild type and Egr1‐deficient primary hepatocytes to assess insulin response. In vivo hepatocyte‐specific Egr1 knockout (Egr1‐HKO) mice was accomplished in 2‐month‐old Egr1flox/flox male mice by tail vein administration of adeno‐associated viral vector 8 (AAV8) expressing cre recombinase driven by thyroxine binding globulin (TBG) promoter (AAV8‐Tbg‐Cre). An empty vector AAV8‐Tbg‐Null was included as a control. WT and Egr1‐HKO mice were fed either a normal chow or HFCF diet for 5 months and followed by the examination of body composition, adiposity, metabolic rates, insulin sensitivity, and liver histology.ResultsEGR1 was dramatically induced by insulin and abundantly expressed in the postprandial liver. The expression of EGR1 was significantly reduced in human and mouse NAFLD livers, suggesting a biological relevance of EGR1 downregulation during the pathogenesis of NAFLD. In vitro hepatocyte cultures showed that Egr1‐deficient hepatocytes have impaired activation of AKT but enhanced de novo lipogenesis in response to insulin. In vivo knockout of hepatocyte‐specific Egr1 led to liver steatosis and increased whole body adiposity under the chow diet. Additionally, in vivo EGR1 deficiency in hepatocytes exacerbated NAFLD progression by promoting liver inflammation and fibrosis in mice fed a HFCF diet.ConclusionsOur studies reveal a previously unrecognized role of hepatic EGR1 in the regulation of lipid metabolism. Our findings suggest that hepatocyte EGR1 is crucial to maintain hepatic insulin response and loss of EGR1 in hepatocytes leads to liver steatosis that exacerbates NAFLD development and progression.Support or Funding InformationNCI K22CA184146 and NIH P20 GM103549 to Yuxia Zhang.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Protective effects of geniposide and ginsenoside Rg1 combination treatment on rats following cerebral ischemia are mediated via microglial microRNA‑155‑5p inhibition.

Geniposide, an active component of Gardenia, has been reported to protect against cerebral ischemia in animals. Ginsenoside Rg1, a component of Panaxnotoginseng, is usually administered in combination with Gardenia for the treatment of acute ischemic stroke; however, there are unknown effects of ginsenoside Rg1 that require further investigation. In the present study, the effects of geniposide and ginsensoide Rg1 combination treatment on focal cerebral ischemic stroke were investigated. For invivo analysis, male rats were separated into three groups, including the (control), model and geniposide + ginsenoside Rg1 groups (n=8 per group). A middle cerebral artery occlusion model was established as the model group. The treatment group was treated with geniposide (30mg/kg, tail vein injection) + ginsenoside Rg1 (6mg/kg, tail vein injection), and the model group received saline instead. Neurobehavioral deficits, infarct volume, brain edema, and the expression of microRNA (miR)‑155‑5p and CD11b by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and immunohistochemistry, were assessed following 24h of ischemia. For invitro analysis, BV2 mouse microglial cells were cultured and exposed to geniposide (40µg/ml) + ginsenoside Rg1 (8µg/ml) during various durations of oxygen‑glucose deprivation (OGD). The expression levels of miR‑155‑5p, pri‑miR‑155 and pre‑miR‑155 were detected by RT‑qPCR. The results demonstrated that increases in brain infarct volume, edema volume, CD11b‑positive cells and miR‑155‑5p levels were alleviated following geniposide + ginsenoside administration in rats exposed to ischemia. Furthermore, geniposide + ginsenoside Rg1 treatment suppressed the miR‑155‑5p, pri‑miR‑155 and pre‑miR‑155 expression levels in OGD‑injured BV2 microglial cells. The results of the present study demonstrated that tail vein administration of geniposide in combination with ginsenoside Rg1 protected against focal cerebral ischemia in rats through inhibition of microglial miR‑155‑5p following ischemic injury, which may serve as a novel therapeutic agent for the treatment of strokes.

Development of a validated UPLC-MS/MS method for determination of humantenmine in rat plasma and its application in pharmacokinetics and bioavailability studies.

Humantenmine (HMT), the most toxic compound isolated from Gelsemium elegans Benth, is a well-known active herbal compound. A rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to estimate the absolute oral bioavailability of HMT in rats. Quantification was performed by multiple reaction monitoring using electrospray ionization operated in positive ion mode with transitions of m/z 327.14 → m/z 296.19 for HMT and m/z 323.20 → m/z 236.23 for gelsemine (internal standard, IS). The linear range of the calibration curve was 1-256 nmol/L, with a lower limit of quantification at 1 nmol/L. The accuracy of HMT ranged from 89.39 to 107.5%, and the precision was within 12.24% (RSD). Excellent recovery and negligible matrix effect were observed. HMT remained stable during storage, preparation and analytical procedures. The pharmacokinetics of HMT in rats showed that HMT reached the concentration peak at 12.50 ± 2.74 min with a peak concentration of 28.49 ± 6.65 nmol/L, and the corresponding area under the concentration-time curve (AUC0-t ) was 1142.42 ± 202.92 nmol/L min after 200 μg/kg HMT was orally administered to rats. The AUC0-t of HMT given at 20 μg/kg by tail vein administration was 1518.46 ± 192.24 nmol/L min. The calculated absolute bioavailability of HMT was 7.66%.

Effect of Androgen on Normal Biodistribution of [18F]-2'-Fluoro-5-methyl-1-beta-D-arabinofuranosyluracil (18F-FMAU) in Athymic Non-tumor-bearing Male Mice.

We assessed the association between the presence and absence of androgen on the normal biodistribution of the positron emission tomography (PET) cellular proliferation imaging biomarker, [18F]-2'-Fluoro-5-methyl-1-beta-D-arabinofuranosyluracil (18F-FMAU), in mice. Non-castrated (n=4) and castrated (n=4) athymic non-tumor-bearing male mice served as models for presence and absence, respectively, of androgen. MicroPET-CT scans were performed 1 h following tail vein administration of 200 uCi of 18F-FMAU. Imaging was performed at baseline and then at 7-day intervals longitudinally for 35 days only in castrated mice following subcutaneous introduction of a 12.5 mg, 21-day release, dihydrotestosterone pellet. Mean standardized uptake values (SUVmean) were obtained for liver, heart, and muscle. Several two-group comparisons of average of SUVmean were performed. Pre-pellet baseline average SUVmean (±s.d.) values in castrated mice were significantly lower than baseline non-castrated values, increased on day 15 and reached peak values on day 28, at which time they were significantly higher than corresponding baseline levels in both non-castrated and pre-pellet castrated mice. The peak values decreased significantly following dihydrotestosterone withdrawal. There is a significant modulatory effect of androgen on normal 18F-FMAU uptake levels in mice liver, heart and muscle tissues.

Antinociceptive Effects of AGAP, a Recombinant Neurotoxic Polypeptide: Possible Involvement of the Tetrodotoxin-Resistant Sodium Channels in Small Dorsal Root Ganglia Neurons.

Antitumor-analgesic peptide (AGAP) is a novel recombinant polypeptide. The primary study showed that AGAP 1.0 mg/kg exhibited strong analgesic and antitumor effects. The tail vein administration of AGAP potently reduced pain behaviors in mice induced by intraplantar injection of formalin or intraperitoneal injection of acetic acid, without affecting basal pain perception. To further assess the mechanisms of AGAP, the effects of AGAP on sodium channels were assessed using the whole-cell patch clamp recordings in dorsal root ganglia (DRG) neurons. The results showed that AGAP (3–1000 nM) inhibited the sodium currents in small-diameter DRG neurons in a dose-dependent manner. 1000 nM AGAP could inhibit the current density-voltage relationship curve of sodium channels in a voltage-dependent manner and negatively shift the activation curves. 1000 nM AGAP could reduce the tetrodotoxin-resistant (TTX-R) sodium currents by 42.8% in small-diameter DRG neurons. Further analysis revealed that AGAP potently inhibited NaV1.8 currents by 59.4%, and negatively shifted the activation and inactivation kinetics. 1000 nM AGAP also reduced the NaV1.9 currents by 33.7%, but had no significant effect on activation and inactivation kinetics. Thus, our results demonstrated that submicromolar concentrations of AGAP inhibited TTX-R sodium channel in rat small-diameter DRG neurons. It is concluded that these new results may better explain, at least in part, the analgesic properties of this polypeptide.

CaMKII Phosphorylation in Primary Somatosensory Cortical Neurons is Involved in the Inhibition of Remifentanil-induced Hyperalgesia by Lidocaine in Male Sprague-Dawley Rats.

Previous clinical studies have shown that lidocaine can alleviate severe postoperative pain after remifentanil-based anesthesia. Experimental studies have also demonstrated that lidocaine can inhibit remifentanil-induced hyperalgesia, yet the mechanism remains unknown. The present study explored the role of the primary somatosensory (S1) cortex in remifentanil-induced hyperalgesia as well as its inhibition by lidocaine through evaluation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) phosphorylation and protein expression levels in S1 cortical neurons. Male Sprague-Dawley rats were randomly allocated to the following 3 groups: remifentanil only (R), lidocaine only (L), and remifentanil+lidocaine (RL). Experimentally naive animals were used as controls for immunoblotting and immunofluorescence evaluations. Via intravenous tail vein administration (24 G catheter), the animals received remifentanil at 2.4 μg/kg/min, lidocaine at 200 μg/kg/min, and remifentanil at 2.4 μg/kg/min plus lidocaine at 200 μg/kg/min for 2 hours. Paw withdrawal threshold (PWT) values for both mechanical and thermal hyperalgesia, along with immunoblotting and immunofluorescence, were used to measure remifentanil-induced hyperalgesia and changes in CaMKII phosphorylation (P-CaMKII) and total protein expression (T-CaMKII). There was a significant decrease in the PWT for mechanical stimulation at 0.5 and 2 hours after discontinuing infusion in groups R and RL (P<0.05, n=10 per group). However, there were no differences in thermal PWT in any group at any time period when compared with that of baseline. There was also a significant increase of P-CaMKII (not T-CaMKII) in S1 cortical neurons of group R (not L and RL groups) at 0 to 2 hours after discontinuing infusion when compared with that of the corresponding control group (P<0.05, n=6 per group) as determined by immunoblotting and immunofluorescence microscopy. These results suggested that the phosphorylation of CaMKII in S1 cortical neurons increases significantly during the process of remifentanil-induced hyperalgesia. The increase of CaMKII phosphorylation could be inhibited by systemic application of lidocaine. This inhibition may play a role in the antihyperalgesia effects of lidocaine.

Detection of an Abundant Plant-Based Small RNA in Healthy Consumers.

The mechanisms of delivery of plant small RNAs to consumers must be investigated in order to harness this technology to positively impact biotechnology. Two groups have used honeysuckle (Lonicera japonica) feeding regimes to detect a plant-based small RNA, termed MIR2911, in sera. Meanwhile, numerous groups have failed to detect dietary plant-based small RNAs in consumers. Here we catalog levels of MIR2911 in different herbs, and suggest that in particular herb MIR2911 levels are elevated. Feeding these different herb-based diets to mice, we found MIR2911 levels in the sera and urine were associated with dietary intake levels. Abundance was not the sole determinate of apparent RNA bioavailability, as gavage-feeding large-doses of synthetic MIR2911 permitted only small transient increases in serum levels. Dietary MIR2911 were not modified in circulation by association with the host’s RNA-induced silencing complex, as the RNA did not co-immunoprecipitate with AGO2. The stability of dietary MIR2911 in circulation differed from synthesized small RNAs, as tail vein administration of various synthetic plant-based small RNAs resulted in rapid clearance. However, synthetic MIR2911 appeared to be more stable than the other plant miRNAs tested. Notably, this uptake of dietary MIR2911 was not related to perturbations in the host’s microbiome or gut permeability. We suggest dietary uptake of MIR2911 is commonplace in healthy consumers, and reproducible detection of plant-based small RNAs in consumers depends on dietary abundance, RNA stability and digestion from within the food-matrix.

Dissemination via the lymphatic or angiogenic route impacts the pathology, microenvironment and hypoxia-related drug response of lung metastases.

Complications associated with the development of lung metastases have a detrimental effect on the overall survival rate of many cancer patients. Preclinical models that mimic the clinical aspects of lung metastases are an important tool in developing new therapy options for these patients. The commonly used intravenous models only recapitulate dissemination of cancer cells to the lungs via the haematological route. Here we compared spontaneous and intravenous lung metastases of the highly metastatic KHT mouse fibrosarcoma cells after injecting KHT cells into the subcutaneous layer of the skin or directly into the tail vein. In contrast to the intravenous model, metastases spontaneously arising from the subcutaneous tumours disseminated most consistent with the lymph nodes/lymphatics route and were more hypoxic than the metastases observed following tail-vein administration and haematological spread. To ascertain whether this impacted on drug response, we tested the effectiveness of the hypoxia-sensitive cytotoxin AQ4N (Banoxantrone) in both models. AQ4N was more effective as an anti-metastatic drug in mice with subcutaneous KHT tumours, significantly reducing the metastatic score. Complementing the KHT studies, pathology studies in additional models of spontaneous lung metastases showed haematological (HCT116 intrasplenic implant) or mixed haematological/lymphatic (B16 intradermal implant) spread. These data suggest that preclinical models can demonstrate differing, clinically relevant dissemination patterns, and that careful selection of preclinical models is required when evaluating new strategies for targeting metastatic disease.

Abstract 184: Pulmonary Microvascular Thrombosis Enhances Extravasation via Myeloid Hypoxia-Inducible Factors

Introduction: Mechanisms that regulate the positive association between thrombosis and metastasis are incompletely understood. It was hypothesised that thrombus formation stimulates a hypoxic response, which in turn promotes extravasation. The primary aim was to determine whether thrombosis of the pulmonary microvasculature (T pm ) increases extravasation via myeloid (neutrophil and macrophage) hypoxia-inducible factor (HIF). Methods and Results: T pm was induced in wild type mice via tail vein administration of polystyrene microbeads (15μm diameter, 1000/mouse). T pm led to chronological increases in pulmonary HIF1α expression (P=0.01), HIF2α expression (P&lt;0.01), neutrophil infiltration (P&lt;0.05), and macrophage infiltration (P&lt;0.05; 1-5days post-T pm vs. non-thrombosed vehicle controls, n=8/group/time point); these increases were comparable with changes observed following vena cava thrombosis (assessed via image analysis of immunostained tissue throughout). In wild type mice with circulating Lewis lung cancer cells (LLCs, 1million/mouse i/v), T pm led to increases in pulmonary fibrin deposition (P&lt;0.0001), HIF1α expression (P&lt;0.05), HIF2α expression (P&lt;0.05), and LLC extravasation (P&lt;0.0001; 14days post-LLCs vs. non-thrombosed controls, n=15/group). Using conditional HIFα knockout mice (vs. wild type littermates), it was shown that T pm -induced increases in pulmonary fibrin deposition and LLC extravasation were dependent upon HIF1α or HIF2α in neutrophils and macrophages; myeloid HIFs were also responsible for T pm -induced increases in pulmonary tumour proliferation and vascularisation (n=15/group). In human breast tumour microarrays (n=221), fibrin deposition was positively correlated with HIF2α expression (RS=0.22, P&lt;0.001), while increases in HIF2α were associated with reductions in metastasis-free survival (HR=1.1, P&lt;0.05). Conclusions: Thrombus formation in mouse pulmonary microvasculature enhances cancer cell extravasation via neutrophil- and macrophage-specific HIF1α or HIF2α. In human breast tumours, HIF2α is associated with increased fibrin deposition, and reduced survival. Pulmonary microvascular thrombosis can enhance cancer cell dissemination via myeloid cell-specific HIFs.