Hana Totary-Jain

Vascular Smooth Muscle Cell Proliferation in Restenosis

Current therapeutic approaches to restore blood flow in stenotic blood vessels involve the use of percutaneous devices and coronary bypass surgery. In all procedures that disrupt the normal integrity of the blood vessels, there is an increased incidence of vessel luminal narrowing, termed restenosis. Restenosis, arbitrarily defined as greater than 50% narrowing of vessel diameter compared with the reference vessel, is modulated by genetic background and diseases that affect the cardiovascular system, including diabetes, hypertension, and hypercholesterolemia. In the 1970s, Andreas Gruntzig pioneered the use of transluminal dilatation of coronary arteries for symptomatic coronary artery disease and reported a 19% rate of restenosis (6 of 32 patients).1–3 Subsequent studies demonstrated a restenosis rate of approximately 33%.4 More than 25 years later, despite pharmacological and mechanical approaches to reduce the incidence of restenosis, it remains a significant problem, especially in high-risk patient groups, limiting overall success. Restenosis after percutaneous intervention is characterized by platelet aggregation, release of growth factors, inflammatory cell infiltration, medial smooth muscle cell proliferation and migration, and extracellular matrix remodeling. The vascular response to injury depends not only on the cells within the vessels but is also modulated by circulating bone marrow–derived cells. Understanding the molecular mechanisms underlying the physiological healing response and the pathological restenosis response has been the focus of extensive investigations, which have led to the development of novel approaches to control the pathological formation of the neointima. In this review, we will focus on some of the molecular mechanisms responsible for the abnormal neointimal hyperplasia, specifically focusing on cell cycle and microRNA (miRNA) in the vascular smooth muscle. Acute occlusion at the percutaneous intervention site within hours to days after the procedure is usually caused by an intimal flap, thrombus formation, subintimal hemorrhage extending into the media, or elastic recoil …

Comparative RNA-sequencing analysis of myocardial and circulating small RNAs in human heart failure and their utility as biomarkers

Significance Heart failure (HF) has a high morbidity and mortality and its incidence is increasing worldwide. While protein biomarkers have been established for diagnostic and prognostic evaluation of patients with HF, there is currently no systematic assessment of RNA biomarkers. We determined the composition of myocardial tissue and circulating microRNAs (miRNAs) in a large cohort of patients with stable and advanced HF and compared it to the composition of normal adult and fetal samples. The advanced HF patients underwent mechanical unloading with left ventricular assist devices and samples were collected at different postoperative time points. Our findings provide the underpinning for miRNA-based therapies and emphasize the usefulness of circulating miRNAs as biomarkers for heart injury performing similar to established diagnostic protein biomarkers. Heart failure (HF) is associated with high morbidity and mortality and its incidence is increasing worldwide. MicroRNAs (miRNAs) are potential markers and targets for diagnostic and therapeutic applications, respectively. We determined myocardial and circulating miRNA abundance and its changes in patients with stable and end-stage HF before and at different time points after mechanical unloading by a left ventricular assist device (LVAD) by small RNA sequencing. miRNA changes in failing heart tissues partially resembled that of fetal myocardium. Consistent with prototypical miRNA–target-mRNA interactions, target mRNA levels were negatively correlated with changes in abundance for highly expressed miRNAs in HF and fetal hearts. The circulating small RNA profile was dominated by miRNAs, and fragments of tRNAs and small cytoplasmic RNAs. Heart- and muscle-specific circulating miRNAs (myomirs) increased up to 140-fold in advanced HF, which coincided with a similar increase in cardiac troponin I (cTnI) protein, the established marker for heart injury. These extracellular changes nearly completely reversed 3 mo following initiation of LVAD support. In stable HF, circulating miRNAs showed less than fivefold differences compared with normal, and myomir and cTnI levels were only captured near the detection limit. These findings provide the underpinning for miRNA-based therapies and emphasize the usefulness of circulating miRNAs as biomarkers for heart injury performing similar to established diagnostic protein biomarkers.

Calreticulin Destabilizes Glucose Transporter-1 mRNA in Vascular Endothelial and Smooth Muscle Cells Under High-Glucose Conditions

Substrate autoregulation of glucose transporter-1 (GLUT-1) mRNA and protein expression provides vascular endothelial and smooth muscle cells a sensitive mechanism to adapt their rate of glucose transport in response to changing glycemic conditions. Hyperglycemia-induced downregulation of glucose transport is particularly important in protecting these cells against an excessive influx of glucose and consequently increased intracellular protein glycation and generation of free radicals; both are detrimental in the development of vascular disease in diabetes. We aimed to investigate the molecular mechanism of high glucose–induced downregulation of GLUT-1 mRNA expression in primary bovine aortic vascular endothelial (VEC) and smooth muscle (VSMC) cell cultures. Using RNA mobility shift, UV cross-linking, and in vitro degradation assays, followed by mass-spectrometric analysis, we identified calreticulin as a specific destabilizing trans-acting factor that binds to a 10-nucleotide cis-acting element (CAE2181-2190) in the 3′-untranslated region of GLUT-1 mRNA. Pure calreticulin accelerated the rate of GLUT-1 mRNA-probe degradation in vitro, whereas overexpression of calreticulin in vascular cells decreased significantly the total cell content of GLUT-1 mRNA and protein. The expression of calreticulin was augmented in vascular cells exposed to high glucose in comparison with low-glucose conditions. Similarly, increased expression of calreticulin was observed in aortae of diabetic Psammomys obesus in comparison with normoglycemic controls. These data suggest that CAE2181-2190–calreticulin complex, which is formed in VSMC and VEC exposed to hyperglycemic conditions, renders GLUT-1 mRNA susceptible to degradation. This interaction underlies the process of downregulation of glucose transport in vascular cells under high-glucose conditions.

Tailoring mTOR-based therapy: molecular evidence and clinical challenges

The mTOR signaling pathway integrates inputs from a variety of upstream stimuli to regulate diverse cellular processes including proliferation, growth, survival, motility, autophagy, protein synthesis and metabolism. The mTOR pathway is dysregulated in a number of human pathologies including cancer, diabetes, obesity, autoimmune disorders, neurological disease and aging. Ongoing clinical trials testing mTOR-targeted treatments number in the hundreds and underscore its therapeutic potential. To date mTOR inhibitors are clinically approved to prevent organ rejection, to inhibit restenosis after angioplasty, and to treat several advanced cancers. In this review we discuss the continuously evolving field of mTOR pharmacogenomics, as well as highlight the emerging efforts in identifying diagnostic and prognostic markers, including miRNAs, in order to assess successful therapeutic responses.

Leptin-enhanced neointimal hyperplasia is reduced by mTOR and PI3K inhibitors

Despite the use of the sirolimus (rapamycin) drug-eluting coronary stent, diabetics are at increased risk of developing in-stent restenosis for unclear reasons. Hyperleptinemia, which often coexists with diabetes and metabolic syndrome, is an independent risk factor for progression of coronary artery disease. It has not been determined whether elevated circulating leptin decreases the efficacy of the sirolimus drug-eluting stent in inhibiting neointimal hyperplasia, the process underlying restenosis after stenting. Here we show that leptin activates the mammalian target of rapamycin (mTOR) signaling pathway in primary murine vascular smooth muscle cells (VSMC) and stimulates VSMC proliferation in a PI3K-dependent fashion. Exogenous leptin, administered at levels comparable to those found in obese humans, promotes neointimal VSMC hyperplasia in a murine femoral artery wire injury model. Leptin significantly increases the dose of the mTOR inhibitor sirolimus that is required for effective inhibition of neointimal formation. Combination therapy with LY294002, a PI3K inhibitor, and sirolimus effectively inhibits leptin-enhanced neointimal hyperplasia. These data show that, in the setting of hyperleptinemia, higher doses of an mTOR inhibitor, or combination therapy with mTOR and PI3K inhibitors, inhibits neointimal hyperplasia after arterial injury. These studies may explain the higher rates of restenosis observed in diabetics treated with a sirolimus-eluting coronary stent and suggest a potential novel therapeutic approach for inhibiting in-stent restenosis in such patients.

Reprogramming of the microRNA transcriptome mediates resistance to rapamycin.

Background: The role of miRNAs in the cellular response to mTOR inhibitors has never been tested. Results: Rapamycin resistance is associated with up-regulation of oncogenic miRNAs and down-regulation of tumor suppressor miRNAs. Conclusion: miRNAs influence the cellular response to mTOR inhibitors. Significance: miRNAs are potential markers and novel targets for cancer therapy involving mTOR inhibitors. The mammalian target of rapamycin (mTOR) is a central regulator of cell proliferation that is often deregulated in cancer. Inhibitors of mTOR, including rapamycin and its analogues, are being evaluated as antitumor agents. For their promise to be fulfilled, it is of paramount importance to identify the mechanisms of resistance and develop novel therapies to overcome it. Given the emerging role of microRNAs (miRNAs) in tumorigenesis, we hypothesized that miRNAs could play important roles in the response of tumors to mTOR inhibitors. Long-term rapamycin treatment showed extensive reprogramming of miRNA expression, characterized by up-regulation of miR-17–92 and related clusters and down-regulation of tumor suppressor miRNAs. Inhibition of members of the miR-17–92 clusters or delivery of tumor suppressor miRNAs restored sensitivity to rapamycin. This study identifies miRNAs as new downstream components of the mTOR-signaling pathway, which may determine the response of tumors to mTOR inhibitors. It also identifies potential markers to assess the efficacy of treatment and provides novel therapeutic targets to treat rapamycin-resistant tumors.

Rapamycin Resistance Is Linked to Defective Regulation of Skp2

The mammalian target of rapamycin (mTOR) plays a role in controlling malignant cellular growth. mTOR inhibitors, including rapamycin (sirolimus), are currently being evaluated in cancer trials. However, a significant number of tumors are rapamycin resistant. In this study, we report that the ability of rapamycin to downregulate Skp2, a subunit of the ubiquitin protein ligase complex, identifies tumors that are sensitive to rapamycin. RNA interference (RNAi)-mediated silencing of Skp2 in human tumor cells increased their sensitivity to rapamycin in vitro and inhibited the growth of tumor xenografts in vivo. Our findings suggest that Skp2 levels are a key determinant of antitumor responses to mTOR inhibitors, highlighting a potentially important pharmacogenomic marker to predict sensitivity to rapamycin as well as Skp2 silencing strategies for therapeutic purposes.

MicroRNAs and the cellular response to rapamycin: Potential role in diagnosis and therapy

The mammalian target of rapamycin (mTOR) is a major regulator of cell growth, motility and angiogenesis that is often deregulated in malignancies. mTOR inhibitors have been approved for the treatment of renal-cell carcinoma and mantle-cell lymphoma. Currently, second generation mTOR inhibitors are under clinical evaluation.1 Major challenges remain the identification of patients who will respond to mTOR inhibitors, and the development of therapeutics that can overcome intrinsic resistance or acquired resistance. There are currently no biomarkers that predict tumor response to mTOR inhibitors.

Freeze Drying Method with Gaseous Nitrogen to Preserve Fine Ultrastructure of Biological Organizations for Scanning Electron Microscopy, Helium Ion Beam Microscopy and Fluorescence Microscopy

Electron Microscopy Resource Center, Laboratory of Bacterial Pathogenesis and Immunology, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York, USA; Center for Biomedical Research, Population Council, New York, New York, USA, Department of Physiology and Cellular Biophysics, The Clyde and Helen Wu Center for Molecular Cardiology, Columbia University Medical Center, New York, New York, USA., Department of Molecular Pharmacology & Physiology, University of South Florida Morsani College of Medicine, Tampa, Florida, USA., Ion Microscopy Innovation Center, Zeiss Microscopy LLC, Peabody, Massachusetts, USA., Department of Infectious Diseases, Kings College London School of Medicine, Guys Hospital, Great Maze Pond, London SE1 9RT, UK

Decreased LIN28B in preeclampsia impairs human trophoblast differentiation and migration

Preeclampsia (PE) is a common cause of maternal morbidity, characterized by impaired trophoblast invasion and spiral artery transformation resulting in progressive uteroplacental hypoxia. Given the primary role of LIN28A and LIN28B in modulating cell metabolism, differentiation, and invasion, we hypothesized that LIN28A and/or LIN28B regulates trophoblast differentiation and invasion, and that its dysregulation may contribute to PE. Here we show that LIN28B is expressed ∼1300‐fold higher than LIN28A in human term placenta and is the predominant paralog expressed in primary human trophoblast cultures. The expression of LIN28B mRNA and protein levels are significantly reduced in gestational age–matched preeclamptic vs. normal placentas, whereas LIN28A expression is not different. First trimester human placental sections displayed stronger LIN28B immunoreactivity in extravillous (invasive) cytotrophoblasts and syncytial sprouts vs. villous trophoblasts. LIN28B overexpression increased HTR8 cell proliferation, migration, and invasion, whereas LIN28B knockdown in JEG3 cells reduced cell proliferation. Moreover, LIN28B knockdown in JEG3 cells suppressed syncytin 1 (SYN‐1), apelin receptor early endogenous ligand (ELABELA), and the chromosome 19 microRNA cluster, and increased mRNA expression of ITGp4and TNF‐α. Incubation of BeWo and JEG3 cells under hypoxia significantly decreased expression of LIN28B and LIN28A, SYN‐1, and ELABELA, whereas TNF‐α is increased. These results provide the first evidence that LIN28B is the predominant paralog in human placenta and that decreased LIN28B may play a role in PE by reducing trophoblast invasion and syncytialization, and by promoting inflammation.—Canfield, J., Arlier, S., Mong, E. F., Lockhart, J., Vanwye, J., Guzeloglu‐Kayisli, O., Schatz, F., Magness, R. R., Lockwood, C. J., Tsibris, J. C. M., Kayisli, U. A., Totary‐Jain, H. Decreased LIN28B in preeclampsia impairs human trophoblast differentiation and migration. FASEB J. 33, 2759–2769 (2019). www.fasebj.org