PCSK9 deficiency unmasks a sex- and tissue-specific subcellular distribution of the LDL and VLDL receptors in mice

Are PCSK9 antibodies (Praluent, Repatha) for hypercholesterolemia treatment more efficient in men than women?

Significance Statement

 In 2015, monoclonal antibodies (mAbs) directed against PCSK9, and preventing its interaction with the LDLR, were made available as Praluent (alirocumab, Sanofi & Regeneron) or Repatha (evolocumab, Amgen). They are subcutaneously injected every two weeks and lower LDLc to levels never achieved before (by ~60%), making them the biggest weapon against cardiovascular disease since the advent of statins. In 2003, we identified the proprotein convertase subtilisin-kexin 9 (PCSK9) and established that its gene is the 3rd one known to be involved in FH after those encoding the LDL receptor and apoB. Indeed, PCSK9 enhanced degradation of the LDLR in endosomes/lysosomes, with ensuing high LDLc. In agreement, healthy individuals lacking functional PCSK9 exhibited a spectacular ~80% drop in their LDLc (~0.4 mM), thus reinforcing the validity of PCSK9 as an excellent target to treat hypercholesterolemia.

The absence of PCSK9 leads to a sex- and tissue-specific subcellular distribution of the LDLR Like humans, mice lacking PCSK9 exhibit very low levels of cholesterol. In these mice, the fraction of the LDL receptor present at the cell surface of hepatocytes seems to be regulated in a sex-dependent manner. In the liver of male and female mice lacking PCSK9, we observed a similar increase in the total amount of LDL receptor. However, its subcellular distribution differed in a sex-dependent manner: cell surface levels of the LDL receptor were dramatically increased in males, but not females.

Estrogens are responsible for the sex-dependent subcellular distribution of the LDLR Ovariectomized PCSK9-deficient females treated with placebo exhibited typical male patterns, with an accumulation of the LDLR at the hepatocyte cell surface. In contrast, those receiving 17β-estradiol (E2) maintained female patterns. Our working model is that the “eraser” effect of PCSK9 on cell surface LDL receptor is dominant, and that its presence masks the mechanism by which E2 regulates the surface levels of these two receptors.
Does a similar mechanism exist in women? If this E2-mediated regulation has its counterpart in humans and leads to a lower LDLR activity, the LDL uptake by the liver may differ in hypercholesterolemic men and women who receive therapeutic PCSK9 mAbs. Could this treatment have a lower efficacy in pre-menopausal women (with high E2 levels) than in men? Although >100 publications reported on clinical trials that led to the approval of PCSK9 mAbs to treat hypercholesterolemia, none of them compared the sex-dependent efficacy of these mAbs. However, the Clinical Briefing Document for alirocumab (FDA) revealed that, in a long term study, 1,438 men and 872 women (predominantly post-menopausal) responded by 65.5% and 53.4% reductions in LDLc, respectively, thus showing a 22% higher efficacy in men. Moreover, a subgroup analysis revealed that post-menopausal women responded with a 16% higher efficacy than pre-menopausal women (54.8% versus 47.3%). Thus, men seem to respond with a 38.5% higher efficacy than pre-menopausal (65.5% versus 47.3%). Independently, in the Amgen Clinical Briefing Document, a graph comparing the efficacy of evolocumab injection every two weeks versus that of ezetimibe in 46 women and 52 men also indicates a higher efficacy in men.

A thorough analysis of the clinical data accumulated in phase II and III trials concerning the possible sex-dependent efficacy of PCSK9 monoclonal antibodies will hopefully be soon available. If confirmed, the elucidation of the mechanism implicated will certainly be valuable.

About The Author

Anna Stepanova Roubtsova, M.D., M.S. Anna, did her medical studies in Yaroslavl State Madical Academy in Russia in 1986. She completed a Master in Experimental Medicine (McGill University, Canada) in 2008, and a Clinical Research Professional Development Program in 2009. She is now a Research Assistant at the Institut de Recherches Cliniques de Montréal (IRCM).

About The Author

Dr. Annik Prat received her PhD in molecular biology in 1988 from the University Pierre et Marie Curie in Paris, France. After 3 years at the Biozentrum in Basel, Switzerland, as a post-doctoral fellow, she became Associate Professor in Molecular Biology at the University Pierre et Marie Curie. Since 1997, she is a staff scientist at the Clinical Research Institute of Montreal (IRCM) and works in Nabil G. Seidah’s laboratory, in which PCSK9 was discovered.

Model for the E2 regulation of LDLR subcellular trafficking

PCSK9 exerts a dominant “eraser” effect on surface LDLR (left green panel). PCSK9 deficiency (KO) leads to ~3-fold higher LDLR levels in the liver (right red panel). In KO males, both homogenates and plasma membrane (PM) fractions show a ~ 3-fold increase of LDLR levels. In KO females, the increase is seen in homogenates only, not in PM fractions. E2 reduces the access of the LDLR to the plasma membrane or its residence time therein. Ovariectomy (Ovx) leads to a male pattern with high levels of cell surface LDLR. E2 treatment reverts this phenotype to a female one with LDLR being mostly intracellular.

Journal Reference

J Lipid Res. 2015 Nov;56(11):2133-42

Roubtsova A¹, Chamberland A¹, Marcinkiewicz J¹, Essalmani R¹, Fazel A², Bergeron JJ², Seidah NG¹, Prat A¹.

¹Laboratory of Biochemical Neuroendocrinology, Institut de Recherches Cliniques de Montréal (affiliated with the University of Montreal), Montreal, Quebec, Canada.

²Department of Medicine, McGill University Hospital Research Institute, Montreal, Quebec, Canada.

ABSTRACT

Proprotein convertase subtilisin kexin type 9 (PCSK9), the last member of the family of Proprotein Convertases related to Subtilisin and Kexin, regulates LDL-cholesterol by promoting the endosomal/lysosomal degradation of the LDL receptor (LDLR). Herein, we show that the LDLR cell surface levels dramatically increase in the liver and pancreatic islets of PCSK9 KO male but not female mice. In contrast, in KO female mice, the LDLR is more abundant at the cell surface enterocytes, as is the VLDL receptor (VLDLR) at the cell surface of adipocytes. Ovariectomy of KO female mice led to a typical KO male pattern, whereas 17β-estradiol (E2) treatment restored the female pattern without concomitant changes in LDLR adaptor protein 1 (also known as ARH), disabled-2, or inducible degrader of the LDLR expression levels. We also show that this E2-mediated regulation, which is observed only in the absence of PCSK9, is abolished upon feeding the mice a high-cholesterol diet. The latter dramatically represses PCSK9 expression and leads to high surface levels of the LDLR in the hepatocytes of all sexes and genotypes. In conclusion, the absence of PCSK9 results in a sex- and tissue-specific subcellular distribution of the LDLR and VLDLR, which is determined by E2 levels.

Go To Journal of Lipid Research 

 

An inducible transgenic mouse breast cancer model for the analysis of tumor antigen specific CD8+ T-cell responses

Significance Statement

 Immune therapy is a promising approach for improving the treatment of cancer. However, the major obstacles in its successful application, the tumor-induced mechanisms that lead to immune-evasion, have not been satisfactorily resolved. Analysis of the immune status of a given tumor entity and identification of the obstructed immune checkpoints thus are crucial issues for the development of immune-therapeutic anti-cancer strategies. Due to the limited possibilities for analyzing the respective parameters in humans, suitable animal models should be of great value. However, animal models that allow the analysis of how the growth of naturally arising tumors can be controlled by inducing tumor antigen specific immune responses are scarce.

In this study we tested the suitability of our BALB/c mouse based transgenic WAP-T animal model for mammary carcinoma to study tumor antigen specific CD8⁺ T-cell responses during tumor growth and progression, as well as parameters which obstruct a successful immune response. In WAP-T mice, tumorigenesis is induced in the adult mammary gland by expression of viral oncogenes (the SV40 tumor antigens) in parity-induced epithelial mammary gland progenitor cells. Tumorigenesis and the ensuing invasive carcinomas have been well characterized in previous studies, and WAP-T mice have been validated by cross-species comparison as a suitable animal model for human triple-negative mammary carcinomas. For this study we extended our WAP-T mouse lines by WAP-T_NP mice, in which the transgene additionally codes for the NP_118-126-epitope contained within the nucleoprotein of lymphocytic choriomeningitis virus (LCMV), resulting in the expression of a chimeric T-Ag/NP protein (T-Ag_NP). We thus were able to compare immune responses against the “weak” (i.e. low affinity) T-cell epitopes of SV40 T-Ag expressed by WAP-T mice with those against the immune-dominant LCMV NP-epitope in T-Ag_NP expressed by WAP-T_NP mice.

While immunization of WAP-T mice with SV40 did not induce a measurable CTL immune response, immunization of WAP-T_NP mice with LCMV induced a strong response which led to transient tumor cell elimination. Most intriguingly, we found that WAP-T_NP mice mount an endogenous immune response (i.e. without immunization) against the LCMV NP-epitope, as elimination of CD8⁺ T-cells by anti-CD8⁺ antibodies or by irradiation promoted the outgrowth of tumors in WAP-T_NP mice.  WAP-T_NP tumor mice thus contain NP-epitope specific CD8⁺ T-cells, which, however, are only weakly active due to expression of the programmed death-1 protein (PD1), an important player in the PD1/PD-L1 axis of immune checkpoints obstructing CTL activity. Consequently, treatment of WAP-T_NP tumor with anti-PD1 antibodies largely restored their activity. This is demonstrated by the experiment shown in Fig. 1, where we exchanged endogenous lymphocytes of WAP-T_NP tumor mice by lymphocytes derived from different donor mice by adoptive transfer (see scheme in Fig. 1A). Fig. 1B, panel a shows the typical T-Ag_NP expression in nuclei of WAP-T_NP mammary carcinoma cells (red dots). No T-Ag_NP expressing cells can be seen in tumor areas of WAP-T_NP mice that had received lymphocytes from BALB/c mice infected with LCMV due to the presence of a highly active NP-epitope specific CTL population (Fig. 1B, panel b). Similarly, tumor areas from WAP-T_NP mice, which had received lymphocytes from anti-PD1 treated WAP-T_NP mice, were also devoid of T-Ag_NP expressing cells (Fig. 1B, panel c). Thus the anti-tumor activity of exhausted PD1 expressing CD8⁺ T-cells could be largely re-activated.

Comparative analysis of WAP-T and WAP-T_NP mice thus is suited to analyze the parameters leading to immune evasion of tumors expressing “weak” (WAP-T) or “strong” (WAP-T_NP) tumor antigen epitopes, and to test strategies for overcoming blockades in immune response. Furthermore, WAP-T_NP mice show that in the case of tumors expressing a “strong” T-cell epitope, immunization strategies might be developed which, together with strategies blocking immune checkpoints might lead to successful tumor elimination. 

Figure Legend

Elimination of T-Ag_NP expressing WAP-T_NP tumor cells in mammary carcinomas via adoptive transfer of NP-specific CTLs from LCMV infected mice or of re-activated exhausted CTLs from WAP-T_NP tumor mice. (A) Schematic display of immune therapies and (B) immune histologic analysis of T-Ag_NP expression in WAP-T_NP tumor cells in mammary glands of acceptor mice, whose own immune cells were eliminated by sublethal irradiation with 4 Gy. Strong elimination of T-Ag_NP expressing cells is seen after transfer of immune cells either from LCMV infected BALB/c (b) as donors or from WAP-T_NP tumor mice after “therapy” with anti-PD1 antibodies (c) as donors; untreated WAP-T_NP tumor mice served as positive controls (a).

 

 

 

 

 

 

 

 

 

Journal Reference

Oncotarget. 2015 Nov 17;6(36):38487-503.

Bruns M¹, Wanger J¹, Utermöhlen O², Deppert W^1,3. 

Show Affiliations

1.Heinrich-Pette-Institute, Leibniz-Institute for Experimental Virology, Hamburg, Germany.

2. Institute for Medical Microbiology, Immunology and Hygiene, Medical Center and Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany.

3.Institute forTumor Biology, University Medical Center Hamburg-Eppendorf (UKE), University of Hamburg, Hamburg, Germany.

Abstract

In Simian virus 40 (SV40) transgenic BALB/c WAP-T mice tumor development and progression is driven by SV40 tumor antigens encoded byinducible transgenes. WAP-T mice constitute a well characterized mouse model for breast cancer with strong similarities to the corresponding human disease. BALB/c mice mount only a weak cellular immune response against SV40 T-antigen (T-Ag). For studying tumor antigen specific CD8+ T-cellresponses against transgene expressing cells, we created WAP-TNP mice, in which the transgene additionally codes for the NP118-126-epitope contained within the nucleoprotein of lymphocytic choriomeningitis virus (LCMV), the immune-dominant T-cell epitope in BALB/c mice. We then investigated in WAP-TNP mice the immune responses against SV40 tumor antigens and the NP-epitope within the chimeric T-Ag/NP protein (T-AgNP). Analysis of the immune-reactivity against T-Ag in WAP-T and of T-AgNP in WAP-TNP mice revealed that, in contrast to wild type (wt) BALB/c mice, WAP-T and WAP-TNP mice were non-reactive against T-Ag. However, like wtBALB/c mice, WAP-T as well as WAP-TNP mice were highly reactive against the immune-dominant LCMV NP-epitope, thereby allowing the analysis of NP-epitope specific cellular immune responses in WAP-TNP mice. LCMV infection of WAP-TNP mice induced a strong, LCMV NP-epitope specific CD8+ T-cell response, which was able to specifically eliminate T-AgNP expressing mammary epithelial cells both prior to tumor formation (i.e. in cells of lactating mammary glands), as well as in invasive tumors. Elimination of tumor cells, however, was only transient, even after repeated LCMV infections. Further studies showed that already non-infected WAP-TNP tumor mice contained LCMV NP-epitope specific CD8+ T-cells, albeit with strongly reduced, though measurable activity. Functional impairment of these ‘endogenous’ NP-epitope specific T-cells seems to be caused by expression of the programmed death-1 protein (PD1), as anti-PD1 treatment of splenocytes from WAP-TNP tumor mice restored their activity. These characteristics are similar to those found in many tumor patients and render WAP-TNP mice a suitable model for analyzing parameters to overcome the blockade of immune checkpoints in tumor patients.

=5750&pubmed-linkout=1″ target=”blank” ]Go To Oncotarget.

 

 

 

 

Ablation of the Ferroptosis Inhibitor Glutathione Peroxidase 4 in Neurons Results in Rapid Motor Neuron Degeneration and Paralysis

Significance Statement

Degeneration of spinal motor neurons leads to muscle weakness and pathogenesis of diseases such as Amyotrophic Lateral Sclerosis (ALS). Glutathione peroxidase 4 (Gpx4), an antioxidant defense enzyme in repairing oxidative damage to lipids, is a key regulator of ferroptosis, a non-apoptotic form of cell death involving lipid reactive oxygen species. In this study, we showed that, conditional ablation of Glutathione peroxidase 4 in neurons of adult mice resulted in rapid onset and progression of paralysis driven by a dramatic degeneration of motor neurons in spinal cord. We also showed that spinal motor neuron degeneration induced by Gpx4 ablation exhibited features of ferroptosis. Our results indicate that ferroptosis inhibition by Glutathione peroxidase 4 is essential for motor neuron health and survival in vivo, suggesting that increased activity/level of Glutathione peroxidase 4 may protect against degeneration of spinal motor neurons.

 

About The Author

Dr. Qitao Ran is an Associate Professor at Department of Cellular & Structural Biology of the University of Texas Health Science Center at San Antonio. Dr. Ran received his PhD degree from Peking Union Medical College (Beijing, China) in 1995. He conducted postdoctoral research at Baylor College of Medicine (Houston, Texas) from 1995 to 2000, and continued his research at the University of Texas Health Science Center at San Antonio afterwards. Dr. Ran was appointed Assistant Professor at Department of Cellular & Structural Biology and Barshop Institute for Longevity and Aging Research of the University of Texas Health Science Center at San Antonio in 2006. Dr. Ran was also appointed Research Health Scientist at South Texas Veterans Health Care System in 2007. The main research interest of Dr. Ran’s lab is to interrogate the mechanisms of neurodegenerative diseases such as Alzheimer’s disease and ALS as well as aging. 

Figure Legend: Spinal motor neuron loss induced by Glutathione peroxidase 4 ablation in neurons.

A. Fluorescence microscopy images of spinal cord section from a control Gpx4NIKO mouse without tamoxifen treatment to ablate Glutathione peroxidase 4.

B. Fluorescence microscopy images of spinal cord section from a tamoxifen-treated Gpx4NIKO mouse at day 6, showing the absence of ChAT positive motor neurons.

 

 

 

 

 

 

 

 

 

 

 

Journal Reference

J Biol Chem. 2015 Nov 20;290(47):28097-106.

Chen L¹, Hambright WS¹, Na R¹, Ran Q². 

Show Affiliations

1. From the Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229 and.
2. From the Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229 and the Research Service, South Texas Veterans Health Care System, San Antonio, Texas 78229 ran@uthscsa.edu.

Abstract

Glutathione peroxidase 4 (GPX4), an antioxidant defense enzyme active in repairing oxidative damage to lipids, is a key inhibitor of ferroptosis, a non-apoptotic form of cell death involving lipid reactive oxygen species. Here we show that Glutathione peroxidase 4 is essential for motor neuron health and survival in vivo. Conditional ablation of Gpx4 in neurons of adult mice resulted in rapid onset and progression of paralysis and death. Pathological inspection revealed that the paralyzed mice had a dramatic  degeneration of motor neurons in the spinal cord but had no overt neuron degeneration in the cerebral cortex. Consistent with the role of Glutathione peroxidase 4 as a ferroptosis inhibitor, spinal motor neuron degeneration induced by Glutathione peroxidase 4 ablation exhibited features of ferroptosis, including no caspase-3 activation, no TUNEL staining, activation of ERKs, and elevated spinal inflammation. Supplementation with vitamin E, another inhibitor of ferroptosis, delayed the onset of paralysis and death induced by Glutathione peroxidase 4 ablation. Also, lipid peroxidation and mitochondrial dysfunction appeared to be involved in  ferroptosis  of  motor neurons induced by Glutathione peroxidase 4 ablation. Taken together, the dramatic motor neuron degeneration  and paralysis induced by Glutathione peroxidase 4 ablation suggest that ferroptosis inhibition by Glutathione peroxidase 4 is essential for  motor neuron health and survival in vivo.

© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Go To J Biol Chem

 

Self-Guided Supramolecular Cargo-Loaded Nanomotors with Chemotactic Behavior towards Cells

Significance Statement

Tumor cells and wounded tissues produce an excessive amount of hydrogen peroxide. Drug delivery agents that could actively search and locate the diseased areas based on gradient of disease signal molecules between disease areas and blood stream/normal tissues would be an improvement to current targeting drug delivery systems. Although vesicular drug delivery systems such as polymersomes or liposomes are already used routinely in the clinic, the efficiency of targeted drug delivery is still limited mainly because these structures rely almost exclusively on passive accumulations a result of the leaky vasculature formed by rapid tissue growth found in tumors. Wilson approach is to use self-assembly as a tool to construct catalytic nanomotors that are able to carry cargo and direct their movement via chemotaxis. We are aiming to solve one of the most fundamental unresolved challenges in nanomotor research: “The Fantastic Voyage” – application of nanomotors for targeted drug delivery and diagnostics where nanomotors are able to actively seek and precisely locate tumour tissues by following a concentration gradient provided by the tumour signalling molecules.

Here we report a self-assembled nanomotor system able to self-propel and self-guide in a gradient of hydrogen peroxide produced by neutrophil cells. This is the first proof of concept experiment of nanosize motors able to carry a drug cargo and sense sick cells.  The nanomotor is formed through the assembly of smart amphiphilic block polymers into bowl shape structures incorporating an active catalyst in their inner compartment. Of nanometer scale, these motors are ideal for drug delivery in blood vessels/interstitial tissues and particularly attractive in biomedical applications. 

About The Author

Daniela A. Wilson (neé Apreutesei) received her B.Sc. degree (Hon) in chemistry from “A. I. Cuza” University of Iasi, Romania, in 2001 and an M.S. degree (Hon) in environmental chemistry from the same university in 2003. In 2006 she received her PhD with distinction “summa cum laudae” from “Gh. Asachi” Technical University of Iasi, Romania. During her PhD she obtained two fellowships in Japan and UK as exchange PhD student and Marie Curie fellow. She then worked as postdoctoral researcher at University of Pennsylvania, Philadelphia, USA and Radboud University Nijmegen, Netherlands in the groups of Prof. Virgil Percec and Prof. Roeland Nolte on several topics in synthetic organic chemistry, organometallic chemistry, and supramolecular and polymer chemistry. In 2012 she was awarded an ERC starting grant to investigate nanomotors with autonomous propulsion in biological systems. In 2015 she was awarded the first Athena Award from the Netherlands Organisation for Scientific Research for her excellent scientific research. She is currently assistant professor at the Institute for Molecules and Materials, Radboud University. Her research interests span a broad range of topics at the interface of supramolecular chemistry, macromolecular chemistry, and nanotechnology. 

Journal Reference

Angew Chem Int Ed Engl. 2015;54(40):11662-5.

Peng F¹, Tu Y¹, van Hest JC², Wilson DA³.

Show Affiliations

1. Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ, Nijmegen (The Netherlands).
2. Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ, Nijmegen (The Netherlands). j.vanhest@science.ru.nl.
3. Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ, Nijmegen (The Netherlands). d.wilson@science.ru.nl.

Abstract

Delivery vehicles that are able to actively seek and precisely locate targeted tissues using concentration gradients of signaling molecules have hardly been explored. The directed movement toward specific cell types of cargo-loaded polymeric nanomotors along a hydrogen peroxide concentration gradient (chemotaxis) is reported. Through self-assembly, bowl-shaped poly(ethylene glycol)-b-polystyrene nanomotors, or stomatocytes, were formed with platinum nanoparticles entrapped in the cavity while a model drug was encapsulated in the inner compartment. Directional movement of the stomatocytes in the presence of a fuel gradient (chemotaxis) was first demonstrated in both static and dynamic systems using glass channels and a microfluidic flow. The highly efficient response of these motors was subsequently shown by their directional and autonomous movement towards hydrogen peroxide secreting neutrophil cells.

© 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Go To Angew Chem Int Ed Engl

 

 

 

 

 

Nuclear EGFR characterize still controlled proliferation retained in better differentiated clear cell RCC

 

 

 

 

 

 

 

Figure Legend

Nuclear EGFR is visible in the low grade part of clear cell RCC (arrow), while large, irregular nuclei are negative (asterix), 200x.

Acknowledgement: We thank our statistician Prof. Ivan Dražić (Department of Mathematics, Faculty of Engineering, University of Rijeka)  for expert assistance in the statistical analysis of this study.

About The Author

Gordana Đorđević-pathologist and associated professor  at the Department of Pathology, School of Medicine, University of Rijeka and Clinical Hospital Centre of Rijeka, Croatia. Main activities and responsibilities in my daily practice are  Uropathology and  Nephropathology. Special interest in renal cell carcinoma research has led to collaboration with colleagues of different specialties as follows:

Ahel ,Urologist, Dr. med. Department of Urology, Clinical Hospital Centre Rijeka.
Markic, ass. prof., urologist, Department of Urology, Clinical Hospital Centre Rijeka
Mozetic , ass. prof., urologist, Medico Polyclinic, Rijeka.
Spanjol , ass. prof., urologist, Department of Urology, Clinical Hospital Centre Rijeka
Grahovac, prof, Department of molecular biology, Department of Pathology, School of Medicine, University of Rijeka
Stifter, assoc. prof. ; Department of Pathology, School of Medicine, University of Rijeka [/box]

 

 

Journal Reference

Med Hypotheses. 2015;85(2):183-5. 

Ahel ¹, G. Dordevic ² D. Markic¹, V. Mozetic ³, J. Spanjol ¹, B. Grahovac ², S. Stifter ² 

Show Affiliations

1. Department of Urology, Clinical Hospital Centre Rijeka, Krešimirova 42, 51000 Rijeka, Croatia
2. Department of Pathology, School of Medicine, University of Rijeka, Braće Branchetta 20, 51000 Rijeka, Croatia
3. Medico Polyclinic, Agatićeva 8, 51000 Rijeka, Croatia

 

Abstract

Renal cell carcinoma (RCC) is the most common solid kidney tumor representing 2–3% of all cancers, with the highest frequency occurring in Western countries. There was a worldwide and European annual increase in incidence of approximately 2% although incidence has been stabilized in last few years. One third of the patients already have metastases in the time of the diagnosis with poor prognosis because renal cell carcinoma are radio and chemoresistant. The prognostic value of EGFR over-expression in renal cell carcinoma is a controversial issue that could be explained by different histological types of study tumors and non-standardized criteria for evaluation of expression. Recent evidences points to a new mode of EGFR signaling pathway in which activated EGFR undergoes nuclear translocalization and then, as transcription factor, mediates gene expression and other cellular events required for highly proliferating activities. According to our observations, the membranous expression of EGFR associates with high nuclear grade and poor differentiated tumors. On the other hand, nuclear EGFR expression was high in low nuclear graded and well differentiated tumors with good prognosis. We hypothesize that this mode of EGFR signaling characterizes still controlled proliferation retained in well differentiated renal cell carcinoma. From a practical point of view, EGFR immunohistochemical localization can be useful in preselection of patients who could potentially benefit of EGFR targeting therapy.

Go To Med Hypotheses.

 

 

 

 

 

Blood transfusion is associated with increased risk of perioperative complications and prolonged hospital duration of stay among patients undergoing amputation

Journal Reference

Surgery. 2015 Dec;158(6):1609-16.

Tan TW¹, Eslami M², Rybin D², Doros G², Zhang WW³, Farber A².

Show Affiliations

1. Louisiana State University Health Shreveport, Shreveport, LA. Electronic address: ttan1@lsuhsc.edu.
2. Boston University Medical Center, Boston, MA.
3. Louisiana State University Health Shreveport, Shreveport, LA.

Abstract

OBJECTIVE:

We evaluated the outcomes of patients undergoing major lower-extremity amputation who received packed red blood cell transfusion.

METHODS:

Using the dataset of the National Surgical Quality Improvement Program (2005-2011), we examined 5,739 above-knee and 6,725 below-knee amputations. Patients were stratified by perioperative (preoperative, intraoperative, or postoperative) blood transfusion. Outcomes included perioperative mortality, myocardial infarction (MI), thromboembolism, and duration of stay (DOS) at the hospital. Adjusted comparisons of outcomes between transfused and not-transfused patients were performed by matching the 2 groups for age, smoking, diabetes, renal failure, coronary artery disease, classification of the American Society of Anesthesiologists, functional status, and procedure type. Multivariable logistic and gamma regression were used to examine associations between transfusion and outcomes.

RESULTS:

Of the 12,464 amputations in the study cohort 2,133 (17%) required transfusion. The majority of the cases were performed for critical limb ischemia (8,205 amputations; 66%) and the overall 30-days mortality was 9%. In both crude and matched cohorts, although perioperative mortality and cardiac complication rates were similar, transfusion was associated with a greater incidence of pneumonia (crude: 6.1% vs 3%, P < .001; matched: 5.9% vs 3.7%, P < .001), thromboembolism (2.5% vs 1.6%, P = .003; 2.5% vs 1.4%, P = .002) and longer DOS (18 ± 19 vs 13.6 ± 14.3 days, P < .001; 17.8 ± 18.4 vs 14.2 ± 14.5 days, P < .001). Multivariable adjustment for confounding variables in the crude cohort demonstrated that transfusion was independently associated with a greater odds of perioperative pneumonia (odds ratio [OR]:1.6; 95% confidence interval [CI]:1.3-2; P < .001), thromboembolism (OR 1.3, 95% CI 1.0-1.9, P = .09) and longer DOS (mean ratio: 1.1; 95% CI 1.1-1.6; P = .006).

CONCLUSION:

Among patients who had major lower-extremity amputation, perioperative transfusion independently predicted greater risks for perioperative pneumonia, thromboembolism, and prolonged hospital DOS.

Copyright © 2015 Elsevier Inc. All rights reserved.

Go To Surgery

 

 

Independent effect of polymeric nanoparticle zeta potential /surface charge, on their cytotoxicity and affinity to cells

Significance Statement

Nanoparticles have shown great potentials in drug delivery, diagnosis and therapeutics. However, comprehensive understanding of the effect of nanoparticle zeta potential on its interaction with cells is still lack although such interaction has substantial impacts on nanoparticles efficacy. In this present work, we aim to investigate how nanoparticle zeta potential affects its cytotoxicity and disclose the underlying mechanisms by quantifying the interaction force between cells and nanoparticles via atom force microscopy (AFM). Four kinds of nanoparticles that possessed similar sizes around 230 nm and gradient zeta potentials ranging from -30 mv to +40 mv were fabricated. The cytotoxicity of these nanoparticles was found to be zeta potential-dependent. The nanoparticles  possessing positive surface charges were more toxic than negative nanoparticles, and the nanoparticles possessing higher like charges led to higher cytotoxicity. AFM showed that the positively charged nanoparticles or the nanoparticles with higher like charges had significantly higher interaction force to cells. The interaction force indicated the affinity to cells and could be the main reason of nanoparticles to induce cytotoxicity. This is the first report investigating the independent effect of nanoparticles zeta potential on its cytotoxicity and cellular affinity. Our findings suggest that in practical application, negative nanoparticles would be better for delivering general agents for diagnosis and therapy. On the other hand, positive nanoparticles would be better for delivering anti-tumor agents for chemotherapy. This work proposes a novel approach to investigate the cellular affinity of nanoparticles and demonstrates the importance of controlling zeta potential in practical use of nanoparticles.

About The Author

Yunfeng Lin, D.D.S., M.D., Ph.D., is Professor of State Key Laboratory of Oral Diseases, Sichuan University. He serves as the Vice-director of State Key Laboratory of Oral Diseases and Assistant Dean of West China School of Stomatology. He received his Ph.D. from Sichuan University in 2006. Dr. Lin’s research focus on adipose stem cells, biomaterials and craniofacial regeneration, such as bone, cartilage, tooth, fat et al. He has published over 100 papers, reviews and book chapters, and made several seminal contributions to the stem cells fields. He received Young scientific and technological innovation leader of China, Chinese Youth Science and Technology Award, Ministry of Education Science and Technology Progress Award, Henry Fok prize for young teachers in Colleges and Universities, New Century Excellent Talents of Chinese Ministry of Education and National Excellent Doctoral Dissertation of China.

About The Author

Dr. Peng received his PhD from Sichuan University (Chengdu, China) in 2012 and was appointed as a lecturer in Sichuan University in the same year and promoted as an associate professor in 2014. He studied in Keele University (Stoke-On-Trent, UK) as an exchange PhD student from 2010 to 2011 and researched in University of Copenhagen (Copenhagen, Denmark) as a visiting scholar in 2015. His research interests are biomaterials and drug delivery. He currently focuses on the interactions between nanoparticles and proteins/cells. He has received the Sichuan Provincial Award of Outstanding PhD Dissertation in 2013 and the First Prize of Chinese Outstanding Young Investigator Award (IADR-China Division) in 2014.  

Journal Reference

Cell Prolif. 2015 ;48(4):465-74. 

Shao XR¹, Wei XQ¹, Song X², Hao LY¹, Cai XX¹, Zhang ZR², Peng Q¹, Lin YF¹.

Show Affiliations

1. State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, China.
2. Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, 610041, China.

Abstract

OBJECTIVES:

Up to now, little research has been focussed on discovering how zeta potential independently affects polymeric nanoparticle (NP) cytotoxicity.

METHODS:

Polymeric nanoparticles of gradient zeta potential ranging from -30 mv to +40 mv were fabricated using the same poly-3-hydroxybutyrate-co-3-hydroxyhexanoate (PHBHHx) biopolymer. Interaction forces between nanoparticles and cells were measured by atomic force microscopy (AFM). Cytotoxicity of the nanoparticles to cells was investigated by using MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay.

RESULTS:

Four kinds of nanoparticle with similar sizes and gradient zeta potentials, were fabricated. Those with positive surface charges were found to be more toxic than those with negative surface charges. Positively charged nanoparticles or nanoparticles with higher ‘like’ charges, offered higher interaction force with cells.

CONCLUSION:

This work proposes a novel approach for investigating interaction between NPs and cells, and discloses the importance of controlling zeta potential in developing NPs-based formulations in the future.

© 2015 John Wiley & Sons Ltd.

Go To Cell Prolif

 

 

 

Nicotinic acetylcholine receptors mediate donepezil-induced oligodendrocyte differentiation

Journal Reference

J Neurochem. 2015 Aug 28.

Imamura O¹, Arai M¹, Dateki M¹, Ogata T², Uchida R³, Tomoda H³, Takishima K¹.

Show Affiliations

1. Department of Biochemistry, National Defense Medical College, Tokorozawa, Saitama, Japan.
2. Department of Rehabilitation for the Movement Functions, Research Institute, National Rehabilitation Center for Persons with Disabilities, Tokorozawa, Saitama, Japan.
3. Graduate School of Pharmaceutical Sciences, Kitasato University, Minato-ku, Tokyo, Japan.

Abstract

Oligodendrocytes are the myelin-forming cells of the central nervous system (CNS). Failure of myelin development and oligodendrocyte loss results in serious human disorders, including multiple sclerosis. Here, we show that donepezil, an acetlycholinesterase inhibitor developed for the treatment of Alzheimer’s disease, can stimulate oligodendrocyte differentiation and maturation of neural stem cell-derived oligodendrocyte progenitor cells without affecting proliferation or cell viability. Transcripts for essential myelin-associated genes, such as PLP, MAG, MBP, CNPase, and MOG, in addition to transcription factors that regulate oligodendrocyte differentiation and myelination, were rapidly increased after treatment with donepezil. Furthermore, luciferase assays confirmed that both MAG and MBP promoters display increased activity upon donepezil-induced oligodendrocytes differentiation, suggesting that donepezil increases myelin gene expression mainly through enhanced transcription. We also found that the increase in the number of oligodendrocytes observed following donepezil treatment was significantly inhibited by the nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine, but not by the muscarinic acetylcholine receptor antagonist scopolamine. Moreover, donepezil-induced myelin-related gene expression was suppressed by mecamylamine at both the mRNA and protein level. These results suggest that donepezil stimulates oligodendrocyte  differentiation and myelin-related gene expression via nAChRs in neural stem cell-derived oligodendrocyte progenitor cells. We show that donepezil, a drug for the treatment of Alzheimer disease, can stimulate oligodendrocyte differentiation and maturation of oligodendrocyte progenitor cells. Transcripts for essential myelin-associated genes, such as PLP, MAG, MBP, CNPase and MOG in addition to transcripton factors that regulate oligodendrocyte differentiation and myelination were rapidly increased after treatment with donepezil. These effects were partly dependent on nicotinic acetylcholine receptor (nAChR).

© 2015 International Society for Neurochemistry.

Go To J Neurochem

 

Development and validation of a HILIC- MS/MS multi-targeted method for metabolomics applications

Significance Statement

Metabolomics is a newly emerging field of “omics” research concerned with the comprehensive characterization of the small molecule basic polar metabolites in biological systems. The overall goal of metabolomics is to link the metabolite concentration information with clinical or physiology data and to assess the multi-parametric response of a living system to pathophysiological stimuli or genetic modification. Metabolites may describe phenomena which often are not successfully described or predicted by genomics or transcriptomics, such as the effects of xenobiotics or gut microbiome or the positive response or no response to pharmaceutical intervention.

The analysis of polar metabolites represents a challenging task for contemporary science. LC-MS presents a growing popularity as the platform for metabolomic studies due to its high throughput, robustness and wide coverage of metabolites. The success of a LC-MS-based metabolomic study often depends on multiple experimental steps. The comprehensive analysis of complex samples still requires meticulous method development due to lack of analyte free matrix and experience on both LC and MS methods.

To address this challenge we developed a HILIC-MS/MS profiling method for the quantification of ca 100 metabolites of high significance in central biosynthetic routes. The proposed method provides the identification and the generation of definite quantitative data. The method has been optimised and fully validated to accurately quantify polar metabolites in a single injection of 40 min. The use of a single analytical station enhances assay/practitioner control, reporting and inter-laboratory testing.

The developed method was applied first to the analysis of IVF extracellular culture medium of IVF procedure and subsequently to other biological matrices. The results from the analysis provided information on the metabolic activity reflecting from embryos with different growth potential. Differences in the profiles of media deriving from chromosomally normal embryos that resulted in blastocyst and abnormal that stayed in arrested stage could be detected.  Multivariate statistical analysis showed that alanine, glucose, lactic acid and creatine revealed to be the most significant to the differentiation of embryos with different growth potential.

Additional results showed that the developed method can sensitively detect the metabolic content of different matrices and could be used as a tool for detecting differences in metabolic signature and for different purposes in life sciences (diagnostic, nutritional assessment and other). Although the development of such an analytical tool may require time, hard work and dedication it is in our opinion definitely worth the effort.

Journal Reference

Electrophoresis. 2015:Volume 36: issue 18:2215-2225.

Virgiliou C¹, Sampsonidis I¹, Gika HG², Raikos N³, Theodoridis GA¹.

Show Affiliations

1. Department of Chemistry, Aristotle University, Thessaloniki, 54124, Greece.
2. Department of Chemical Engineering, Aristotle University, Thessaloniki, 54124, Greece.
3. Department of Medicine, Aristotle University, Thessaloniki, 54124, Greece.

Abstract

The paper reports the development of a multi-analyte method and its application in metabolic profiling of biological fluids. The initial aim of the method was the quantification of metabolites existing in cell culture medium used in in-vitro fertilisation (IVF) and in other biological fluids related to embryo growth. Since most of these analytes are polar primary metabolites a hydrophilic interaction liquid chromatography (HILIC) system was selected. The analytical system comprised Ultra (High) performance liquid chromatography (UHPLC) with detection on a triple quadrupole mass spectrometer operating in both positive and negative modes. Mobile phase and gradient elution conditions were studied with the aim to achieve the highest coverage of metabolic space in a single injection namely the largest number of analytes that could be detected and quantified. The developed method provides absolute quantitation of ca. 100 metabolites belonging to key metabolite classes such as sugars, amino acids, nucleotides, organic acids and amines. Following validation, the method was applied for the metabolic profiling of hundreds of samples of spent culture medium originating from human IVF procedures and several hundreds of biological samples such as amniotic fluid, human urine and blood serum from pregnant women. The bioanalytical end-point was to provide assistance in the process of embryo transfer and improving IVF success rates but also to provide insight in complications related to the subsequent embryo growth during pregnancy. This article is protected by copyright. All rights reserved.

Go To Electrophoresis

 

 

 

Proteomics-level analysis of myelin formation and regeneration in a mouse model for Vanishing White Matter disease

Journal Reference

J Neurochem. 2015;134(3):513-26.

Gat-Viks I¹, Geiger T², Barbi M¹, Raini G¹, Elroy-Stein O^1,3.

Show Affiliations

1. Department of Cell Research & Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
2. Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
3. Sagol school of Neuroscience, Tel Aviv University, Tel Aviv, Israel.

Abstract

Vanishing white matter (VWM) is a recessive neurodegenerative disease caused by mutations in translation initiation factor eIF2B and leading to progressive brain myelin deterioration, secondary axonal damage, and death in early adolescence. Eif2b5(R132H/R132H) mice exhibit delayed developmental myelination, mild early neurodegeneration and a robust remyelination defect in response to cuprizone-induced demyelination. In the current study we used Eif2b5(R132H/R132H) mice for mass-spectrometry analyses, to follow the changes in brain protein abundance in normal- versus cuprizone-diet fed mice during the remyelination recovery phase. Analysis of proteome profiles suggested that dysregulation of mitochondrial functions, altered proteasomal activity and impaired balance between protein synthesis and degradation play a role in Vanishing white matter pathology. Consistent with these findings, we detected elevated levels of reactive oxygen species in mutant-derived primary fibroblasts and reduced 20S proteasome activity in mutant brain homogenates. These observations highlight the importance of tight translational control to precise coordination of processes involved in myelin formation  and  regeneration and  point at cellular functions that may contribute to Vanishing white matter pathology. Eif2b5(R132H/R132H)  mousemodel  for vanishing white matter disease was used for mass spectrometry of brain proteins at two time points under normal conditions and along recovery from cuprizone-induced demyelination. Comparisons of proteome profiles revealed the importance of mitochondrial function and tight coordination between protein synthesis and degradation to myelination formation and  regeneration, pointing at cellular functions that contribute to Vanishing white matter pathology.

© 2015 International Society for Neurochemistry.

Go To JOURNAL

 

 

 

 

Loss of p53 enhances the function of the endoplasmic reticulum through activation of the IRE1α/XBP1 pathway

Journal Reference

Oncotarget. 2015;6(24):19990-20001.

Namba T^1,2, Chu K², Kodama R¹, Byun S², Yoon KW², Hiraki M², Mandinova A², Lee SW².

Show Affiliations

1. Science Research Center, Kochi University, Kohasu Oko-cho Nankoku-shi, Kochi, Japan.
2. Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA.

Abstract

Altered regulation of ER stress response has been implicated in a variety of human diseases, such as cancer and metabolic diseases. Excessive ER function contributes to malignant phenotypes, such as chemoresistance and metastasis. Here we report that the tumor suppressor p53 regulates ER function in response to stress. We found that loss of p53 function activates the IRE1α/XBP1 pathway to enhance protein folding and secretion through upregulation of IRE1α and subsequent activation of its target XBP1. We also show that wild-type p53 interacts with synoviolin (SYVN1)/HRD1/DER3, a transmembrane E3 ubiquitin ligase localized to ER during ER stress and removes unfolded proteins by reversing transport to the cytosol from the ER, and its interaction stimulates IRE1α degradation. Moreover, IRE1α inhibitor suppressed protein secretion, induced cell death in p53-deficient cells, and strongly suppressed the formation of tumors by p53-deficient human tumor cells in vivo compared with those that expressed wild-type p53. Therefore, our data imply that the IRE1α/XBP1 pathway serves as a target for therapy of chemoresistant tumors that express mutant p53.

=4598&pubmed-linkout=1″ target=”blank” ]Go To Oncotarget

Figure legend. U2OS cells expressing calreticulin-RFP were transduced with the pDsRed-ER vector to visualize the amount of endoplasmic reticulum using confocal microscopy.