Publications by authors named "Polina Sysa-Shah"

22 Publications

  • Page 1 of 1

Osteopontin/secreted phosphoprotein-1 behaves as a molecular brake regulating the neuroinflammatory response to chronic viral infection.

J Neuroinflammation 2020 Sep 17;17(1):273. Epub 2020 Sep 17.

Department of Neurology and Neuroscience, Baltimore, USA.

Background: Osteopontin (OPN) as a secreted signaling protein is dramatically induced in response to cellular injury and neurodegeneration. Microglial inflammatory responses in the brain are tightly associated with the neuropathologic hallmarks of neurodegenerative disease, but understanding of the molecular mechanisms remains in several contexts poorly understood.

Methods: Micro-positron emission tomography (PET) neuroimaging using radioligands to detect increased expression of the translocator protein (TSPO) receptor in the brain is a non-invasive tool used to track neuroinflammation in living mammals.

Results: In humanized, chronically HIV-infected female mice in which OPN expression was knocked down with functional aptamers, uptake of TSPO radioligand DPA-713 was markedly upregulated in the cortex, olfactory bulb, basal forebrain, hypothalamus, and central grey matter compared to controls. Microglia immunoreactive for Iba-1 were more abundant in some HIV-infected mice, but overall, the differences were not significant between groups. TSPO microglia were readily detected by immunolabeling of post-mortem brain tissue and unexpectedly, two types of neurons also selectively stained positive for TSPO. The reactive cells were the specialized neurons of the cerebellum, Purkinje cells, and a subset of tyrosine hydroxylase-positive neurons of the substantia nigra.

Conclusions: In female mice with wild-type levels of osteopontin, increased levels of TSPO ligand uptake in the brain was seen in animals with the highest levels of persistent HIV replication. In contrast, in mice with lower levels of osteopontin, the highest levels of TSPO uptake was seen, in mice with relatively low levels of persistent infection. These findings suggest that osteopontin may act as a molecular brake regulating in the brain, the inflammatory response to HIV infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12974-020-01949-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7499959PMC
September 2020

Prenatal stress enhances NNK-induced lung tumors in A/J mice.

Carcinogenesis 2020 12;41(12):1713-1723

Sidney Kimmel Cancer Center, Department of Oncology, The Johns Hopkins University, School of Medicine, Baltimore, MD, USA.

Children born to women who experience stress during pregnancy have an increased risk of cancer in later life, but no previous animal studies have tested such a link. We questioned whether prenatal stress (PS) in A/J mice affected the development of lung tumors after postnatal response to tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Timed-bred A/J mice were randomly assigned on gestation day 12.5 to PS by restraint for 5 consecutive days or control (no restraint). Adult offspring of control and stressed pregnancies were all treated with three NNK injections (50 mg/kg every other day) and euthanized 16 weeks later to examine their lungs. Compared with controls, PS dams exhibited significantly increased levels of plasma corticosterone, increased adrenal weights and decreased fetus weights without fetal loss. Prenatally stressed litters had a significantly higher neonatal death rate within first week of life, and surviving male and female offspring developed lung epithelial proliferations with increase multiplicity, increased area and aggressive morphology. PS also induced more advanced atypical adenomatous hyperplasia lesions. We found no difference in lung NNK-derived methyl DNA adducts, but PS did significantly enhance CD3+ T cell and Foxp3+ T cell tumor infiltration. PS significantly increases multiplicity, area of NNK-induced lung tumors and advanced morphology. PS did not affect production of NNK-derived methyl DNA adducts but did increase lymphocytic infiltration of lung tumors. To our knowledge, this is the first animal model of PS with evaluation of cancer development in offspring.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/carcin/bgaa033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7947993PMC
December 2020

Imaging CAR T cell therapy with PSMA-targeted positron emission tomography.

Sci Adv 2019 07 3;5(7):eaaw5096. Epub 2019 Jul 3.

Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins Medical Institutions, Baltimore, MD 21287, USA.

Chimeric antigen receptor (CAR) T cell therapy for hematologic malignancies is fraught with several unknowns, including number of functional T cells that engage target tumor, durability and subsequent expansion and contraction of that engagement, and whether toxicity can be managed. Non-invasive, serial imaging of CAR T cell therapy using a reporter transgene can address those issues quantitatively. We have transduced anti-CD19 CAR T cells with the prostate-specific membrane antigen (PSMA) because it is a human protein with restricted normal tissue expression and has an expanding array of positron emission tomography (PET) and therapeutic radioligands. We demonstrate that CD19-tPSMA CAR T cells can be tracked with [F]DCFPyL PET in a Nalm6 model of acute lymphoblastic leukemia. Divergence between the number of CD19-tPSMA CAR T cells in peripheral blood and bone marrow and those in tumor was evident. These findings underscore the need for non-invasive repeatable monitoring of CAR T cell disposition clinically.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciadv.aaw5096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6609218PMC
July 2019

In Vivo Imaging With Confirmation by Histopathology for Increased Rigor and Reproducibility in Translational Research: A Review of Examples, Options, and Resources.

ILAR J 2018 12;59(1):80-98

Department of Radiology, Miller Research Building Molecular Imaging Service Center, Johns Hopkins University, Baltimore, Maryland.

Preclinical noninvasive imaging can be an indispensable tool for studying animal models of disease. In vivo imaging to assess anatomical, functional, and molecular features requires verification by a comparison to the macroscopic and microscopic morphological features, since all noninvasive in vivo imaging methods have much lower resolution than standard histopathology. Comprehensive pathological evaluation of the animal model is underutilized; yet, many institutions have veterinary or human pathologists with necessary comparative pathology expertise. By performing a rigorous comparison to gross or histopathology for image interpretation, these trained individuals can assist scientists with the development of the animal model, experimental design, and evaluation of the in vivo imaging data. These imaging and pathology corroboration studies undoubtedly increase scientific rigor and reproducibility in descriptive and hypothesis-driven research. A review of case examples including ultrasound, nuclear, optical, and MRI is provided to illustrate how a wide range of imaging modalities data can be confirmed by gross or microscopic pathology. This image confirmation and authentication will improve characterization of the model and may contribute to decreasing costs and number of animals used and to more rapid translation from preclinical animal model to the clinic.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/ilar/ily010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6645176PMC
December 2018

Peptide-based PET quantifies target engagement of PD-L1 therapeutics.

J Clin Invest 2019 02 7;129(2):616-630. Epub 2019 Jan 7.

The Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

Immune checkpoint therapies have shown tremendous promise in cancer therapy. However, tools to assess their target engagement, and hence the ability to predict their efficacy, have been lacking. Here, we show that target engagement and tumor-residence kinetics of antibody therapeutics targeting programmed death ligand-1 (PD-L1) can be quantified noninvasively. In computational docking studies, we observed that PD-L1-targeted monoclonal antibodies (atezolizumab, avelumab, and durvalumab) and a high-affinity PD-L1-binding peptide, WL12, have common interaction sites on PD-L1. Using the peptide radiotracer [64Cu]WL12 in vivo, we employed positron emission tomography (PET) imaging and biodistribution studies in multiple xenograft models and demonstrated that variable PD-L1 expression and its saturation by atezolizumab, avelumab, and durvalumab can be quantified independently of biophysical properties and pharmacokinetics of antibodies. Next, we used [64Cu]WL12 to evaluate the impact of time and dose on the unoccupied fraction of tumor PD-L1 during treatment. These quantitative measures enabled, by mathematical modeling, prediction of antibody doses needed to achieve therapeutically effective occupancy (defined as >90%). Thus, we show that peptide-based PET is a promising tool for optimizing dose and therapeutic regimens employing PD-L1 checkpoint antibodies, and can be used for improving therapeutic efficacy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI122216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6355241PMC
February 2019

Chemogenetics revealed: DREADD occupancy and activation via converted clozapine.

Science 2017 08;357(6350):503-507

Biobehavioral Imaging and Molecular Neuropsychopharmacology Unit, National Institute on Drug Abuse (NIDA) Intramural Research Program, Baltimore, MD 21224, USA.

The chemogenetic technology DREADD (designer receptors exclusively activated by designer drugs) is widely used for remote manipulation of neuronal activity in freely moving animals. DREADD technology posits the use of "designer receptors," which are exclusively activated by the "designer drug" clozapine N-oxide (CNO). Nevertheless, the in vivo mechanism of action of CNO at DREADDs has never been confirmed. CNO does not enter the brain after systemic drug injections and shows low affinity for DREADDs. Clozapine, to which CNO rapidly converts in vivo, shows high DREADD affinity and potency. Upon systemic CNO injections, converted clozapine readily enters the brain and occupies central nervous system-expressed DREADDs, whereas systemic subthreshold clozapine injections induce preferential DREADD-mediated behaviors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.aan2475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7309169PMC
August 2017

Regulatory Forum Opinion Piece*: Imaging Applications in Toxicologic Pathology-Recommendations for Use in Regulated Nonclinical Toxicity Studies.

Toxicol Pathol 2017 06;45(4):444-471

8 Hadassah-Hebrew University Medical Center, Jerusalem, Israel.

Available imaging systems for use in preclinical toxicology studies increasingly show utility as important tools in the toxicologic pathologist's armamentarium, permit longitudinal evaluation of functional and morphological changes in tissues, and provide important information such as organ and lesion volume not obtained by conventional toxicology study parameters. Representative examples of practical imaging applications in toxicology research and preclinical studies are presented for ultrasound, positron emission tomography/single-photon emission computed tomography, optical, magnetic resonance imaging, and matrix-assisted laser desorption ionization-imaging mass spectrometry imaging. Some of the challenges for making imaging systems good laboratory practice-compliant for regulatory submission are presented. Use of imaging data on a case-by-case basis as part of safety evaluation in regulatory submissions is encouraged.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/0192623317710014DOI Listing
June 2017

An Fc-Small Molecule Conjugate for Targeted Inhibition of the Adenosine 2A Receptor.

Chembiochem 2016 10 26;17(20):1951-1960. Epub 2016 Aug 26.

Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 302 Wood Basic Science Building, 725 N Wolfe St., Baltimore, MD, 21205, USA.

The adenosine A receptor (A R) is expressed in immune cells, as well as brain and heart tissue, and has been intensively studied as a therapeutic target for multiple disease indications. Inhibitors of the A R have the potential for stimulating immune response, which could be valuable for cancer immune surveillance and mounting a response against pathogens. One well-established potent and selective small molecule A R antagonist, ZM-241385 (ZM), has a short pharmacokinetic half-life and the potential for systemic toxicity due to A R effects in the brain and the heart. In this study, we designed an analogue of ZM and tethered it to the Fc domain of the immunoglobulin IgG3 by using expressed protein ligation. The resulting protein-small molecule conjugate, Fc-ZM, retained high affinity for two Fc receptors: FcγRI and the neonatal Fc receptor, FcRn. In addition, Fc-ZM was a potent A R antagonist, as measured by a cell-based cAMP assay. Cell-based assays also revealed that Fc-ZM could stimulate interferon γ production in splenocytes in a fashion that was dependent on the presence of A R. We found that Fc-ZM, compared with the small molecule ZM, was a superior A R antagonist in mice, consistent with the possibility that Fc attachment can improve pharmacokinetic and/or pharmacodynamic properties of the small molecule.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cbic.201600337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5292873PMC
October 2016

Echocardiographic Characterization of a Murine Model of Hypertrophic Obstructive Cardiomyopathy Induced by Cardiac-specific Overexpression of Epidermal Growth Factor Receptor 2.

Comp Med 2016 ;66(4):268-77

Department of Molecular and Comparative Pathobiology, School of Medicine, Johns Hopkins University, Baltimore, Maryland, USA.

Although rare, hypertrophic cardiomyopathy (HCM) with midventricular obstruction is often associated with severe symptoms and complications. None of the existing HCM animal models display this particular phenotype. Our group developed a mouse line that overexpresses the ErbB2 receptor (ErbB2(tg)) in cardiomyocytes; we previously showed that the ErbB2 receptor induces cardiomyocyte hypertrophy, myocyte disarray, and fibrosis compatible with HCM. In the current study, we sought to further echocardiographically characterize the ErbB2(tg) mouse line as a model of HCM. Compared with their wild-type littermates, ErbB2(tg) mice show increased left ventricular (LV) mass, concentric LV hypertrophy, and papillary muscle hypertrophy. This hypertrophy was accompanied by diastolic dysfunction, expressed as reduced E:A ratio, prolonged deceleration time, and elevated E:e' ratio. In addition, ErbB2(tg) mice consistently showed midcavity obstruction with elevated LV gradients, and the flow profile revealed a prolonged pressure increase and a delayed peak, indicating dynamic obstruction. The ejection fraction was increased in ErbB2(tg) mice, due to reduced end-diastolic and end-systolic LV volumes. Furthermore, systolic radial strain and systolic radial strain rate but not systolic circumferential strain and longitudinal strain were decreased in ErbB2(tg) compared with wild-type mice. In conclusion, the phenotype of the ErbB2(tg) mouse model is consistent with midventricular HCM in many important aspects, including massive LV hypertrophy, diastolic dysfunction, and midcavity obstruction. This pattern is unique for a small animal model, suggesting that ErbB2(tg) mice may be well suited for research into the hemodynamics and treatment of this rare form of HCM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4983168PMC
October 2017

[64Cu]XYIMSR-06: A dual-motif CAIX ligand for PET imaging of clear cell renal cell carcinoma.

Oncotarget 2016 Aug;7(35):56471-56479

Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Carbonic anhydrase IX (CAIX) is a cell surface enzyme that is over-expressed in approximately 95% of cases of clear cell renal cell carcinoma (ccRCC), the most common renal cancer. We synthesized and performed in vitro and in vivo evaluation of a dual-motif ligand, [64Cu]XYIMSR-06, for imaging CAIX expression on ccRCC tumors using positron emission tomography (PET). [64Cu]XYIMSR-06 was generated in yields of 51.0 ± 4.5% (n=5) and specific activities of 4.1 - 8.9 GBq/μmol (110-240 Ci/mmol). Tumor was visualized on PET images by 1 h post-injection with high tumor-to-background levels (>100 tumor-to-blood and -muscle) achieved within 24 h. Biodistribution studies demonstrated a maximum tumor uptake of 19.3% injected dose per gram of radioactivity at 4 h. Tumor-to-blood, -muscle and -kidney ratios were 129.6 ± 18.8, 84.3 ± 21.0 and 2.1 ± 0.3, respectively, at 8 h post-injection. At 24 h a tumor-to-kidney ratio of 7.1 ± 2.5 was achieved. These results indicate pharmacokinetics superior to those of previously reported imaging agents binding to CAIX. [64Cu]XYIMSR-06 is a new low-molecular-weight PET ligand targeting CAIX, which can image localized and metastatic ccRCC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.10602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5302928PMC
August 2016

A humanized antibody for imaging immune checkpoint ligand PD-L1 expression in tumors.

Oncotarget 2016 Mar;7(9):10215-27

Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University, Baltimore, MD, USA.

Antibodies targeting the PD-1/PD-L1 immune checkpoint lead to tumor regression and improved survival in several cancers. PD-L1 expression in tumors may be predictive of response to checkpoint blockade therapy. Because tissue samples might not always be available to guide therapy, we developed and evaluated a humanized antibody for non-invasive imaging of PD-L1 expression in tumors. Radiolabeled [111In]PD-L1-mAb and near-infrared dye conjugated NIR-PD-L1-mAb imaging agents were developed using the mouse and human cross-reactive PD-L1 antibody MPDL3280A. We tested specificity of [111In]PD-L1-mAb and NIR-PD-L1-mAb in cell lines and in tumors with varying levels of PD-L1 expression. We performed SPECT/CT imaging, biodistribution and blocking studies in NSG mice bearing tumors with constitutive PD-L1 expression (CHO-PDL1) and in controls (CHO). Results were confirmed in triple negative breast cancer (TNBC) (MDAMB231 and SUM149) and non-small cell lung cancer (NSCLC) (H2444 and H1155) xenografts with varying levels of PD-L1 expression. There was specific binding of [111In]PD-L1-mAb and NIR-PD-L1-mAb to tumor cells in vitro, correlating with PD-L1 expression levels. In mice bearing subcutaneous and orthotopic tumors, there was specific and persistent high accumulation of signal intensity in PD-L1 positive tumors (CHO-PDL1, MDAMB231, H2444) but not in controls. These results demonstrate that [111In]PD-L1-mAb and NIR-PD-L1-mAb can detect graded levels of PD-L1 expression in human tumor xenografts in vivo. As a humanized antibody, these findings suggest clinical translation of radiolabeled versions of MPDL3280A for imaging. Specificity of NIR-PD-L1-mAb indicates the potential for optical imaging of PD-L1 expression in tumors in relevant pre-clinical as well as clinical settings.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.7143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4891115PMC
March 2016

Bidirectional cross-regulation between ErbB2 and β-adrenergic signalling pathways.

Cardiovasc Res 2016 Mar 21;109(3):358-73. Epub 2015 Dec 21.

Department of Molecular and Comparative Pathobiology, Johns Hopkins Medical Institutions, MRB 807, 733 N. Broadway, Baltimore, MD 21205, USA

Aims: Despite the observation that ErbB2 regulates sensitivity of the heart to doxorubicin or ErbB2-targeted cancer therapies, mechanisms that regulate ErbB2 expression and activity have not been studied. Since isoproterenol up-regulates ErbB2 in kidney and salivary glands and β2AR and ErbB2 complex in brain and heart, we hypothesized that β-adrenergic receptors (AR) modulate ErbB2 signalling status.

Methods And Results: ErbB2 transfection of HEK293 cells up-regulates β2AR, and β2AR transfection of HEK293 up-regulates ErbB2. Interestingly, cardiomyocytes isolated from myocyte-specific ErbB2-overexpressing (ErbB2(tg)) mice have amplified response to selective β2-agonist zinterol, and right ventricular trabeculae baseline force generation is markedly reduced with β2-antagonist ICI-118 551. Consistently, receptor binding assays and western blotting demonstrate that β2ARs levels are markedly increased in ErbB2(tg) myocardium and reduced by EGFR/ErbB2 inhibitor, lapatinib. Intriguingly, acute treatment of mice with β1- and β2-AR agonist isoproterenol resulted in myocardial ErbB2 increase, while inhibition with either β1- or β2-AR antagonist did not completely prevent isoproterenol-induced ErbB2 expression. Furthermore, inhibition of ErbB2 kinase predisposed mice hearts to injury from chronic isoproterenol treatment while significantly reducing isoproterenol-induced pAKT and pERK levels, suggesting ErbB2's role in transactivation in the heart.

Conclusion: Our studies show that myocardial ErbB2 and βAR signalling are linked in a feedback loop with βAR activation leading to increased ErbB2 expression and activity, and increased ErbB2 activity regulating β2AR expression. Most importantly, ErbB2 kinase activity is crucial for cardioprotection in the setting of β-adrenergic stress, suggesting that this mechanism is important in the pathophysiology and treatment of cardiomyopathy induced by ErbB2-targeting antineoplastic drugs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/cvr/cvv274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4752042PMC
March 2016

Imaging of carbonic anhydrase IX with an 111In-labeled dual-motif inhibitor.

Oncotarget 2015 Oct;6(32):33733-42

Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

We developed a new scaffold for radionuclide-based imaging and therapy of clear cell renal cell carcinoma (ccRCC) targeting carbonic anhydrase IX (CAIX). Compound XYIMSR-01, a DOTA-conjugated, bivalent, low-molecular-weight ligand, has two moieties that target two separate sites on CAIX, imparting high affinity. We synthesized [111In]XYIMSR-01 in 73.8-75.8% (n = 3) yield with specific radioactivities ranging from 118 - 1,021 GBq/μmol (3,200-27,600 Ci/mmol). Single photon emission computed tomography of [111In]XYIMSR-01 in immunocompromised mice bearing CAIX-expressing SK-RC-52 tumors revealed radiotracer uptake in tumor as early as 1 h post-injection. Biodistribution studies demonstrated 26% injected dose per gram of radioactivity within tumor at 1 h. Tumor-to-blood, muscle and kidney ratios were 178.1 ± 145.4, 68.4 ± 29.0 and 1.7 ± 1.2, respectively, at 24 h post-injection. Retention of radioactivity was exclusively observed in tumors by 48 h, the latest time point evaluated. The dual targeting strategy to engage CAIX enabled specific detection of ccRCC in this xenograft model, with pharmacokinetics surpassing those of previously described radionuclide-based probes against CAIX.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.5254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4741798PMC
October 2015

Electrocardiographic Characterization of Cardiac Hypertrophy in Mice that Overexpress the ErbB2 Receptor Tyrosine Kinase.

Comp Med 2015 Aug;65(4):295-307

Department of Molecular and Comparative Pathobiology, Johns Hopkins Medical Institutions, Baltimore, Maryland, USA.

Electrocardiography is an important method for evaluation and risk stratification of patients with cardiac hypertrophy. We hypothesized that the recently developed transgenic mouse model of cardiac hypertrophy (ErbB2(tg)) will display distinct ECG features, enabling WT (wild type) mice to be distinguished from transgenic mice without using conventional PCR genotyping. We evaluated more than 2000 mice and developed specific criteria for genotype determination by using cageside ECG, during which unanesthetized mice were manually restrained for less than 1 min. Compared with those from WT counterparts, the ECG recordings of ErbB2(tg) mice were characterized by higher P- and R-wave amplitudes, broader QRS complexes, inverted T waves, and ST interval depression. Pearson's correlation matrix analysis of combined WT and ErbB2(tg) data revealed significant correlation between heart weight and the ECG parameters of QT interval (corrected for heart rate), QRS interval, ST height, R amplitude, P amplitude, and PR interval. In addition, the left ventricular posterior wall thickness as determined by echocardiography correlated with ECG-determined ST height, R amplitude, QRS interval; echocardiographic left ventricular mass correlated with ECG-determined ST height and PR interval. In summary, we have determined phenotypic ECG criteria to differentiate ErbB2(tg) from WT genotypes in 98.8% of mice. This inexpensive and time-efficient ECG-based phenotypic method might be applied to differentiate between genotypes in other rodent models of cardiac hypertrophy. Furthermore, with appropriate modifications, this method might be translated for use in other species.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4549675PMC
August 2015

ErbB2 overexpression upregulates antioxidant enzymes, reduces basal levels of reactive oxygen species, and protects against doxorubicin cardiotoxicity.

Am J Physiol Heart Circ Physiol 2015 Oct 7;309(8):H1271-80. Epub 2015 Aug 7.

Program in Molecular and Translational Toxicology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland; Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland; Department of Molecular and Comparative Pathobiology, Johns Hopkins Medical Institutions, Baltimore, Maryland;

Levels of the HER2/ErbB2 protein in the heart are upregulated in some women during breast cancer therapy, and these women are at high risk for developing heart dysfunction after sequential treatment with anti-ErbB2/trastuzumab or doxorubicin. Doxorubicin is known to increase oxidative stress in the heart, and thus we considered the possibility that ErbB2 protein influences the status of cardiac antioxidant defenses in cardiomyocytes. In this study, we measured reactive oxygen species (ROS) in cardiac mitochondria and whole hearts from mice with cardiac-specific overexpression of ErbB2 (ErbB2(tg)) and found that, compared with control mice, high levels of ErbB2 in myocardium result in lower levels of ROS in mitochondria (P = 0.0075) and whole hearts (P = 0.0381). Neonatal cardiomyocytes isolated from ErbB2(tg) hearts have lower ROS levels and less cellular death (P < 0.0001) following doxorubicin treatment. Analyzing antioxidant enzyme levels and activities, we found that ErbB2(tg) hearts have increased levels of glutathione peroxidase 1 (GPx1) protein (P < 0.0001) and GPx activity (P = 0.0031) in addition to increased levels of two known GPx activators, c-Abl (P = 0.0284) and Arg (P < 0.0001). Interestingly, although mitochondrial ROS emission is reduced in the ErbB2(tg) hearts, oxygen consumption rates and complex I activity are similar to control littermates. Compared with these in vivo studies, H9c2 cells transfected with ErbB2 showed less cellular toxicity and produced less ROS (P < 0.0001) after doxorubicin treatment but upregulated GR activity (P = 0.0237) instead of GPx. Our study shows that ErbB2-dependent signaling contributes to antioxidant defenses and suggests a novel mechanism by which anticancer therapies involving ErbB2 antagonists can harm myocardial structure and function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajpheart.00517.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4666964PMC
October 2015

Parkin-independent mitophagy requires Drp1 and maintains the integrity of mammalian heart and brain.

EMBO J 2014 Dec 27;33(23):2798-813. Epub 2014 Oct 27.

Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

Mitochondrial dynamics and mitophagy have been linked to cardiovascular and neurodegenerative diseases. Here, we demonstrate that the mitochondrial division dynamin Drp1 and the Parkinson's disease-associated E3 ubiquitin ligase parkin synergistically maintain the integrity of mitochondrial structure and function in mouse heart and brain. Mice lacking cardiac Drp1 exhibited lethal heart defects. In Drp1KO cardiomyocytes, mitochondria increased their connectivity, accumulated ubiquitinated proteins, and decreased their respiration. In contrast to the current views of the role of parkin in ubiquitination of mitochondrial proteins, mitochondrial ubiquitination was independent of parkin in Drp1KO hearts, and simultaneous loss of Drp1 and parkin worsened cardiac defects. Drp1 and parkin also play synergistic roles in neuronal mitochondrial homeostasis and survival. Mitochondrial degradation was further decreased by combination of Drp1 and parkin deficiency, compared with their single loss. Thus, the physiological importance of parkin in mitochondrial homeostasis is revealed in the absence of mitochondrial division in mammals.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embj.201488658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4282557PMC
December 2014

Geranylgeranylacetone blocks doxorubicin-induced cardiac toxicity and reduces cancer cell growth and invasion through RHO pathway inhibition.

Mol Cancer Ther 2014 Jul 15;13(7):1717-28. Epub 2014 Apr 15.

Authors' Affiliations: Departments of Molecular and Comparative Pathobiology, and

Doxorubicin is a widely used chemotherapy for solid tumors and hematologic malignancies, but its use is limited due to cardiotoxicity. Geranylgeranylacetone (GGA), an antiulcer agent used in Japan for 30 years, has no significant adverse effects, and unexpectedly reduces ovarian cancer progression in mice. Because GGA reduces oxidative stress in brain and heart, we hypothesized that GGA would prevent oxidative stress of doxorubicin cardiac toxicity and improve doxorubicin's chemotherapeutic effects. Nude mice implanted with MDA-MB-231 breast cancer cells were studied after chronic treatment with doxorubicin, doxorubicin/GGA, GGA, or saline. Transthoracic echocardiography was used to monitor systolic heart function and xenografts evaluated. Mice were euthanized and cardiac tissue evaluated for reactive oxygen species generation, TUNEL assay, and RHO/ROCK pathway analysis. Tumor metastases were evaluated in lung sections. In vitro studies using Boyden chambers were performed to evaluate GGA effects on RHO pathway activator lysophosphatidic acid (LPA)-induced motility and invasion. We found that GGA reduced doxorubicin cardiac toxicity, preserved cardiac function, prevented TUNEL-positive cardiac cell death, and reduced doxorubicin-induced oxidant production in a nitric oxide synthase-dependent and independent manner. GGA also reduced heart doxorubicin-induced ROCK1 cleavage. Remarkably, in xenograft-implanted mice, combined GGA/doxorubicin treatment decreased tumor growth more effectively than doxorubicin treatment alone. As evidence of antitumor effect, GGA inhibited LPA-induced motility and invasion by MDA-MB-231 cells. These anti-invasive effects of GGA were suppressed by geranylgeraniol suggesting GGA inhibits RHO pathway through blocking geranylation. Thus, GGA protects the heart from doxorubicin chemotherapy-induced injury and improves anticancer efficacy of doxorubicin in breast cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1535-7163.MCT-13-0965DOI Listing
July 2014

Generation of a cre recombinase-conditional Nos1ap over-expression transgenic mouse.

Biotechnol Lett 2014 Jun 22;36(6):1179-85. Epub 2014 Feb 22.

McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, 733 N. Broadway, Baltimore, MD, 21205, USA,

Polymorphic non-coding variants at the NOS1AP locus have been associated with the common cardiac, metabolic and neurological traits and diseases. Although, in vitro gene targeting-based cellular and biochemical studies have shed some light on NOS1AP function in cardiac and neuronal tissue, to enhance our understanding of NOS1AP function in mammalian physiology and disease, we report the generation of cre recombinase-conditional Nos1ap over-expression transgenic mice (Nos1ap (Tg)). Conditional transgenic mice were generated by the pronuclear injection method and three independent, single-site, multiple copies integration event-based founder lines were selected. For heart-restricted over-expression, Nos1ap (Tg) mice were crossed with Mlc2v-cre and Nos1ap transcript over-expression was observed in left ventricles from Nos1ap (Tg); Mlc2v-cre F1 mice. We believe that with the potential of conditional over-expression, Nos1ap (Tg) mice will be a useful resource in studying NOS1AP function in various tissues under physiological and disease states.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10529-014-1473-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4850732PMC
June 2014

Loss of NAC1 expression is associated with defective bony patterning in the murine vertebral axis.

PLoS One 2013 26;8(7):e69099. Epub 2013 Jul 26.

Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, Maryland, United States of America.

NAC1 encoded by NACC1 is a member of the BTB/POZ family of proteins and participates in several pathobiological processes. However, its function during tissue development has not been elucidated. In this study, we compared homozygous null mutant Nacc1(-/-) and wild type Nacc1(+/+) mice to determine the consequences of diminished NAC1 expression. The most remarkable change in Nacc1(-/-) mice was a vertebral patterning defect in which most knockout animals exhibited a morphological transformation of the sixth lumbar vertebra (L6) into a sacral identity; thus, the total number of pre-sacral vertebrae was decreased by one (to 25) in Nacc1(-/-) mice. Heterozygous Nacc1(+/-) mice had an increased tendency to adopt an intermediate phenotype in which L6 underwent partial sacralization. Nacc1(-/-) mice also exhibited non-closure of the dorsal aspects of thoracic vertebrae T10-T12. Chondrocytes from Nacc1(+/+) mice expressed abundant NAC1 while Nacc1(-/-) chondrocytes had undetectable levels. Loss of NAC1 in Nacc1(-/-) mice was associated with significantly reduced chondrocyte migratory potential as well as decreased expression of matrilin-3 and matrilin-4, two cartilage-associated extracellular matrix proteins with roles in the development and homeostasis of cartilage and bone. These data suggest that NAC1 participates in the motility and differentiation of developing chondrocytes and cartilaginous tissues, and its expression is necessary to maintain normal axial patterning of murine skeleton.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0069099PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3724875PMC
April 2014

Cardiac-specific over-expression of epidermal growth factor receptor 2 (ErbB2) induces pro-survival pathways and hypertrophic cardiomyopathy in mice.

PLoS One 2012 9;7(8):e42805. Epub 2012 Aug 9.

Department of Molecular and Comparative Pathobiology, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America.

Background: Emerging evidence shows that ErbB2 signaling has a critical role in cardiomyocyte physiology, based mainly on findings that blocking ErbB2 for cancer therapy is toxic to cardiac cells. However, consequences of high levels of ErbB2 activity in the heart have not been previously explored.

Methodology/principal Findings: We investigated consequences of cardiac-restricted over-expression of ErbB2 in two novel lines of transgenic mice. Both lines develop striking concentric cardiac hypertrophy, without heart failure or decreased life span. ErbB2 transgenic mice display electrocardiographic characteristics similar to those found in patients with Hypertrophic Cardiomyopathy, with susceptibility to adrenergic-induced arrhythmias. The hypertrophic hearts, which are 2-3 times larger than those of control littermates, express increased atrial natriuretic peptide and β-myosin heavy chain mRNA, consistent with a hypertrophic phenotype. Cardiomyocytes in these hearts are significantly larger than wild type cardiomyocytes, with enlarged nuclei and distinctive myocardial disarray. Interestingly, the over-expression of ErbB2 induces a concurrent up-regulation of multiple proteins associated with this signaling pathway, including EGFR, ErbB3, ErbB4, PI3K subunits p110 and p85, bcl-2 and multiple protective heat shock proteins. Additionally, ErbB2 up-regulation leads to an anti-apoptotic shift in the ratio of bcl-xS/xL in the heart. Finally, ErbB2 over-expression results in increased activation of the translation machinery involving S6, 4E-BP1 and eIF4E. The dependence of this hypertrophic phenotype on ErbB family signaling is confirmed by reduction in heart mass and cardiomyocyte size, and inactivation of pro-hypertrophic signaling in transgenic animals treated with the ErbB1/2 inhibitor, lapatinib.

Conclusions/significance: These studies are the first to demonstrate that increased ErbB2 over-expression in the heart can activate protective signaling pathways and induce a phenotype consistent with Hypertrophic Cardiomyopathy. Furthermore, our work suggests that in the situation where ErbB2 signaling contributes to cardiac hypertrophy, inhibition of this pathway may reverse this process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0042805PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3415416PMC
April 2013

A composite polymer nanoparticle overcomes multidrug resistance and ameliorates doxorubicin-associated cardiomyopathy.

Oncotarget 2012 Jun;3(6):640-50

The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

Acquired chemotherapy resistance is a major contributor to treatment failure in oncology. For example, the efficacy of the common anticancer agent doxorubicin (DOX) is limited by the emergence of multidrug resistance (MDR) phenotype in cancer cells. While dose escalation of DOX can circumvent such resistance to a degree, this is precluded by the appearance of cardiotoxicity, a particularly debilitating condition in children. In vitro studies have established the ability of the natural phytochemical curcumin to overcome MDR; however, its widespread clinical application is restricted by poor solubility and low bioavailability. Building upon our recently developed polymer nanoparticle of curcumin (NanoCurc or NC) that significantly enhances the systemic bioavailability of curcumin, we synthesized a doxorubicin-curcumin composite nanoparticle formulation called NanoDoxCurc (NDC) for overcoming DOX resistance. Compared to DOX alone, NDC inhibited the MDR phenotype and caused striking growth inhibition both in vitro and in vivo in several models of DOX-resistant cancers (multiple myeloma, acute leukemia, prostate and ovarian cancers, respectively). Notably, NDC-treated mice also demonstrated complete absence of cardiac toxicity, as assessed by echocardiography, or any bone marrow suppression, even at cumulative dosages where free DOX and pegylated liposomal DOX (Doxil®) resulted in demonstrable attenuation of cardiac function and hematological toxicities. This improvement in safety profile was achieved through a reduction of DOX-induced intracellular oxidative stress, as indicated by total glutathione levels and glutathione peroxidase activity in cardiac tissue. A composite DOX-curcumin nanoparticle that overcomes both MDR-based DOX chemoresistance and DOX-induced cardiotoxicity holds promise for providing lasting and safe anticancer therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3442295PMC
http://dx.doi.org/10.18632/oncotarget.543DOI Listing
June 2012