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Oncotarget. 2017 Jan 3;8(1):1481-1494. doi: 10.18632/oncotarget.13644.

Angiogenesis. 2017 Feb;20(1):85-96. doi: 10.1007/s10456-016-9530-9.

Tissue factor is an angiogenic specific receptor, a novel oncotarget in cancer stem cells and the target for factor VII-targeted immunotherapy and photodynamic therapy 

Zhiwei Hu

The Ohio State University College of Medicine, The James Comprehensive Cancer Center, Department of Surgery and Division of Surgical Oncology, Columbus OH 43210.

* Correspondence should be addressed to: Dr. Zhiwei Hu, The Ohio State University College of Medicine, Department of Surgery, OSUCCC, Columbus, OH 43210. USA. Phone: 614-685-4606; Fax: 614-685-4606; Email: zhiwei.hu@osumc.edu


Tumor microenvironment is composed of several tumor compartments, importantly the cancer cells, tumor neovascular endothelial cells and cancer stem cells. The cancer cells represent the majority of a tumor mass. Tumor neovascular endothelial cells are the inner layer of tumor neovasculature that provides not only nutrients and oxygen for cancer cells to proliferate, but also serve as conduct for cancer cells to metastasize into distant organs and consequently form metastases. Cancer stem cells (CSC) are a small subpopulation of neoplastic cells within a tumor that theoretically possess the capacity to self-renew and develop into the heterogeneous lineages of cancer cells that comprise the tumor (1) and may contribute to tumor angiogenesis, tumor heterogeneity, resistance to multiple therapies (2, 3), recurrence and metastasis (2, 4, 5). Thus, it would be ideal to identify common yet specific surface target molecules and to development of corresponding targeted therapies so that the same targeted therapeutic agent can simultaneously target all three important tumor compartments.


Tissue factor (TF) is previously known for overexpression on many types of slid cancer cells and leukemic cancer cells (6) and for selective expression on tumor neovascular endothelial cells (TVEC) in patients’ tumors (7) and in tumor xenografts in mice (8-12), but not on normal resting vascular endothelial cells (VECs). But it was unknown whether cancer stem cells express TF. In the January 2017 issue of Oncotarget, Hu and colleagues (13) reported for the first time that TF is constitutively expressed on CD133 positive (CD133+) or CD24-CD44+ CSCs isolated from human cancer cell lines, tumor xenografts from mice and breast tumor tissues from patients (Figure 1). TF-targeted agents, i.e., a factor VII (fVII)-conjugated photosensitizer (fVII-PS for targeted photodynamic therapy, fVII-tPDT) and fVII-IgG1Fc (Immunoconjugate or called an ICON for immunotherapy), can eradicate CSCs without drug resistance (Figure 2) via the induction of apoptosis and necrosis and via antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity, respectively.



Figure 1. TF expression in CD133+ cancer stem cells (CSCs) isolated from in vitro cultured human lung cancer H460 cell line (a), human triple-negative breast cancer MDA-MB-231 line (b) and from patients’ breast tumor tissues (c). a: CD133+ CSCs from H460 lung cancer cell line were immunofluorescently stained for expression of CD133 (red) and TF (green). Their nuclei were stained by DAPI (blue) and the cells were photographed under confocal microscopy (Zeiss). Scale bar: 20 μm. b. Immunoblotting for TF expression on CD133+ CSCs and CD133- non-CSC MDA-MB-231 cells. CD133 expression was confirmed on CD133+ CSCs and GAPDH was for assessing sample loading. c. Representative imaging of TF expression on breast cancer patients’ CD133+ CSCs and CD133- non-CSCs, CD133 expression was confirmed only on CD133+ CSCs, not on CD133- cells (Original magnification: 25 μm under ZEO Fluorescent Cell Imager, Bio-Rad). Picture from “Hu et al. Oncotarget. 2017”



Figure 2. Tissue factor-targeted ICON and fVII-tPDT are effective in eradicating CSCs and non-CSC cancer cells without drug resistance via ADCC and CDC (a-b) or via by inducing apoptosis and necrosis (c-d), respectively. a-b: ICON can kill CD24-CD44+ CSCs (a) and CD133+ CSCs (b), as well as non-CSCs (CD24-CD44- in a and CD133- in b), isolated from MDA-MB-231 line separately using CD24- and CD44-based breast cancer stem cell kit and CD133-PE positive selection protocol. Data presented as Mean ± SEM. Each experiment was repeated 2-3 times. c-d: After fVII-tPDT, the CD133+ H460 CSC cells (c) were stained with Annexin V-FITC and then stained with propidium iodide (PI). Untreated CD133+ H460 CSCs were the control cells (d). The cells were photographed under a fluorescent microscope using green (FITC), red (PI), and phase channels. Original magnification: 200 X. e-f: Representative imaging of CD133+ CSCs from OVCAR5 line were stained with crystal violet dye after fVII-tPDT (showing that cancer cells were completed eradicated, only residual membrane remained) (e), and the control CSCs (showing that untreated cancer cells were intact) were not treated (f). Original magnification: 400 X. Picture from “Hu et al. Oncotarget. 2017”



Figure 3. TF is an angiogenic specific receptor on angiogenic endothelial cells. Representative confocal imaging of TF (green) and endothelial marker CD31 (red) expression on HMVEC before (0 hr) and 4 hrs after VEGF stimulation (4-6 hours reaching peak expression). Cell nuclei were counterstained by DAPI (blue). Scale bars: 20 μm. Picture from “Hu et al. Angiogenesis. 2017”


In addition, identification of target molecules specific for angiogenic vascular endothelial cells (VEC), the inner layer of pathological neovasculature, is critical for discovery and development of neovascular-targeting therapy for angiogenesis-dependent human diseases, notably cancer, macular degeneration and endometriosis, in which vascular endothelial growth factor (VEGF) plays a central pathophysiological role. Using VEGF-stimulated VECs isolated from micro-, venous and arterial blood vessels as in vitro angiogenic models and unstimulated VECs as a quiescent VEC model, Hu and colleagues (14) examined the expression of tissue factor (TF), a membrane-bound receptor on the angiogenic VEC models compared with quiescent VEC controls. Hu and colleagues reported in the February 2017 issue of Angiogenesis (14) that TF is specifically expressed on angiogenic VECs in a time-dependent manner in micro-, vein and artery vessels. TF-targeted therapeutic agents, including ICON and fVII-conjugated photosensitizer can selectively bind angiogenic VECs, but not the quiescent VECs. Moreover, fVII-targeted photodynamic therapy can selectively and completely eradicate angiogenic VECs. We conclude that TF is an angiogenic-specific receptor (Figure 3) and the target molecule for fVII-targeted therapeutics (Figure 4). This study supports clinical trials of TF-targeted therapeutics for the treatment of angiogenesis-dependent diseases such as cancer, macular degeneration and endometriosis.



Figure 4. TF is the target for fVII-targeted ICON. Representative Western blots using mouse ICON (mfVII/hIgG1Fc) and human ICON (hfVII/hIgG1Fc) to immune-precipitate their cognate receptor TF that was detected by monoclonal antibody against human TF (HTF) (clone HTF1). The negative control was the untransfected CHO-K1 cells. Human IgG was an isotype control. Cell lysates were derived from CHO-K1 cells expressing tissue factor (TF), endothelial protein C receptor (EPCR) or both (TF+EPCR). Picture from “Hu et al. Angiogenesis. 2017”



Figure 5. fVII-tPDT is selective and effective in eradicating angiogenic VEC. a. The fVII-tPDT is effective and selective in killing angiogenic VEC (angiogenic HMVEC) with an EC50 of 0.031 μM SnCe6 in fVII/NLS-SnCe6, whereas it has no side effects to quiescent VEC (HMVEC). The fVII-tPDT conditions were as follows: 635 nm laser light at 36 J/cm2 and the SnCe6 concentrations in the fVII/NLS-SnCe6 conjugate (x axis) were 0.0 (buffer only), 0.5, 1 and 2 μM, respectively. Note that the VEC cells without fVII/NLS-SnCe6 (0.0 μM) also served as the light only control as they were also irradiated with 635 nm laser light (36 J/cm2). b-c. Representative imaging of crystal violet-stained VEGF-stimulated and unstimulated HMVECs right after being treated with fVII-tPDT or ntPDT (2 μM and 635 nm laser light at 36 J/cm2) (b). Control HMVECs include an untreated control and a maximal killing control (completely lysed by 1% Triton X-100) (c). Original magnification: 400 x phase contrast. Picture from “Hu et al. Angiogenesis. 2017”


In conclusion, these two recent studies (13, 14) demonstrate that TF is a novel surface therapeutic oncotarget for CSC (Figure 1), an angiogenic-specific receptor (Figure 3) and the target for fVII-targeted immunotherapy and photodynamic therapy (Figures 2, 3 and 5). Moreover, these research highlights that TF-targeting therapeutics can effectively and selectively eradicate angiogenic VECs without harming normal quiescent VECs (Figure 5) and CSCs without drug resistance (Figure 2) (hopefully to overcome CSC’s drug resistance and prevent recurrence and metastasis), in addition to targeting TF-expressing cancer cells. Since Vascular endothelial growth factor (VEGF) can induce TF expression on angiogenic VECs (Figure 3) and plays a central role in angiogenesis-dependent cancer and non-malignant human diseases (15), such as age-related macular degeneration (AMD) (16), rheumatoid arthritis (RA) (17) and endometriosis (18), the findings reported in the February 2017 issue of Angiogenesis may be applied not only to cancer, but also to other angiogenesis-dependent human diseases. Thus, we anticipate that TF-targeted immunotherapy and fVII-tPDT could have broad potential to achieve optimal therapeutic efficacy not only for the treatment of cancer, including solid cancers, acute myeloid leukemia and acute lymphocytic leukemia, in which their cancer cells, tumor neovascular endothelial cells and/or cancer stem cells express TF (6, 7, 13, 14), but also for the treatment of angiogenesis-dependent non-cancerous diseases, notably AMD, RA and endometriosis, etc, in which TF is selectively expressed on in vitro VEGF-stimulated angiogenic VEC models (14) and on their angiogenic VECs in vivo in animal models of AMD (19, 20) and endometriosis (21).


Acknowledgments: This work was supported by the Dr. Ralph and Marian Falk Medical Research Trust Awards Programs. The project described was also partly supported by Award Number UL1TR001070 from the National Center for Advancing Translational Sciences through a Phase 1 L-Pilot Award and a voucher award from the Ohio State University Center for Clinical and Translational Science.


Conflicts of Interest: Z.H. is co-inventor of the first generation tissue factor-targeting “neovascular-targeted immunoconjugates” (ICON) and is the inventor of a second and a third generation tissue factor-targeting ICONs, named L-ICON1 and L-ICON3 (Patents Pending).



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6. Z. Hu, Factor VII-Targeted Photodynamic Therapy for Breast Cancer and Its Therapeutic Potential for Other Solid Cancers and Leukemia, Breast Cancer – Current and Alternative Therapeutic Modalities, Esra Gunduz and Mehmet Gunduz (Ed.), ISBN: 978-953-307-776-5, InTech, Available from: http://www.intechopen.com/articles/show/title/factor-vii-targeted-photodynamic-therapy-for-breast-cancer-and-its-therapeutic-potential-for-other-s. E. Gunduz, Gunduz, M., Ed., Breast Cancer – Current and Alternative Therapeutic Modalities (InTech, 2011), pp. 175-196.
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J Cell Physiol. 2017 Jun;232(6):1368-1386.

Identification of a novel human E-cadherin splice variant and assessment of its effects upon EMT-related events



María Laura Matos 1, §; Lara Lapyckyj 1, §; Marina Rosso 1 ; María José Besso 1; María Victoria Mencucci 1; Clara Isabel Marín Briggiler 1; Silvina Giustina 1; Laura Inés Furlong 1; Mónica Hebe Vazquez-Levin 1,*

§ both authors equally contributed to this work

1 Instituto de Biología y Medicina Experimental (IBYME) National Research Council (CONICET)-FIBYME. Vuelta de Obligado 2490. Buenos Aires. 1428DN. Argentina.


*Corresponding author 

Mónica Vazquez-Levin, PhD.

Instituto de Biología y Medicina Experimental (IBYME)





Epithelial Cadherin (E-cadherin) is involved in calcium-dependent cell-cell adhesion and signal transduction. The E-cadherin decrease/loss is a hallmark of Epithelial to Mesenchymal Transition (EMT), a key event in tumor progression. The underlying molecular mechanisms that trigger E-cadherin loss and consequent EMT have not been completely elucidated. This study reports the identification of a novel human E-cadherin variant mRNA produced by alternative splicing. A bioinformatics evaluation of the novel mRNA sequence and biochemical verifications suggest its regulation by Nonsense-Mediated mRNA Decay (NMD). The novel E-cadherin variant was detected in 29/42 (69%) of human tumor cell lines, expressed at variable levels (E-cadherin variant expression relative to the wild type mRNA = 0.05%-11.6%). Stable transfection of the novel E-cadherin variant in MCF-7 cells (MCF7Ecadvar) resulted in downregulation of wild type E-cadherin expression (transcript/protein) and EMT-related changes, among them cell acquisition of fibroblastic-like phenotype, increased expression of Twist, Snail, Zeb1 and Slug transcriptional repressors and decreased expression of ESRP1 and ESRP2 RNA binding proteins. Moreover, loss of cytokeratins and gain of vimentin, N-cadherin and Dysadherin/FXYD5 proteins was observed. Dramatic changes in cell behavior were found in MCF7Ecadvar, as judged by the decreased cell-cell adhesion (Hanging-drop assay), increased cell motility (Wound Healing) and increased cell migration (Transwell) and invasion (Transwell w/Matrigel). Some changes were found in MCF-7 cells incubated with culture medium supplemented with conditioned medium from HEK-293 cells transfected with the E-cadherin variant mRNA. Further characterization of the novel E-cadherin variant will help understanding molecular basis of tumor progression and improve cancer diagnosis.



Identification of a Novel Human E-Cadherin Splice Variant and Assessment of Its Effects Upon EMT-Related Events. 

Matos ML, Lapyckyj L, Rosso M, Besso MJ, Mencucci MV, Marín-Briggiler CI, Giustina S, Furlong LI,Vazquez-Levin MH.

J Cell Physiol. 2017 Jun;232(6):1368-1386. doi: 10.1002/jcp.25622. Epub 2016 Dec 29.


Cover Image, Volume 232, Number 6, June 2017.

Matos ML, Lapyckyj L, Rosso M, Besso MJ, Mencucci MV, Marín-Briggiler CI, Giustina S, Furlong LI,Vazquez-Levin MH.

J Cell Physiol. 2017 Jun;232(6):i. doi: 10.1002/jcp.25866.



Identification of a human novel E-cadherin variant mRNA

A novel E-cadherin transcript was identified during the screening of a unidirectional lambda ZAP Express (Stratagene, USA) vector expression library prepared with RNA from human epididymis. Alignment with the reference nucleotide E-cadherin wild type sequence (Ecadwt; NM_004360) led to the identification of a 34 nt-deletion in Exon 14. With the NetGene2 server (http://www.cbs.dtu.dk/services/NetGene2/), the site used to produce the novel E-cadherin mRNA sequence was identified as a putative alternative splicing site (Figure 1.A., 1.B.).

The expression of the protein encoded by the variant E-cadherin (Ecadvar) mRNA was analyzed using a set of studies that combined bioinformatics tools and transfection assays. Firstly, the amino acidic sequence of the protein encoded by the Ecadvar mRNA was deduced using the ExPASy Translate software (http://web.expasy.org/translate/). Contrasting with the 882 amino acids of the wild type form, the novel Ecad protein spans a total of 757 residues (Figure 1.C.). Both sequences showed 100% homology up to the residue in position 721. The transcript 34 nt-deletion results in a frame-shift, giving rise to a new peptide sequence of 36 amino acids and a premature termination codon. A sequence analysis run through the TMHMM server (http://www.cbs.dtu.dk/services/TMHMM/) predicted a secretory nature of the novel protein (Figure 1.D.). Moreover, the transient expression of the novel Ecadvar mRNA in COS-7 cells identified a 94 KDa protein isoform in the cellular conditioned medium (CM), confirming the bioinformatics predictions.


Effects of Ecadvar expression in a human breast cancer cell model

MCF-7 human breast cancer cells stably transfected with a pcDNA3 expression plasmid containing the Ecadvar mRNA (MCF7Ecadvar) showed dramatic changes in cell phenotype, depicting a fibroblast-like morphology and a decrease in cell-cell contacts (Figure 2.A.). In line with these findings, a significant decrease in Ecadwt transcript levels (Figure 2.B.), as well as in protein expression (Figure 2.C.) and localization (Figure 2.D.), was observed. Also, filamentous actin (F-actin) was organized in non-junctional stress fibers oriented towards the boundary between cells (Figure 2.D.), a typical distribution found in invasive cells. In contrast, control cells (MCF7pcDNA3) showed E-cadherin and F-actin localization mainly in cell borders. Concomitant with the decrease in Ecadwt, an increased expression of transcriptional repressors (Twist, Snail, Zeb1 and Slug), loss of cytokeratin 19 and gain of vimentin, and expression of the mesenchymal N-cadherin mRNA, was found in MCF7Ecadvar cells (Figure 2.B.). All these changes have been reported in the Epithelial to Mesenchymal Transition (EMT), a key event in tumor progression. In addition, low levels of transcripts encoding ESRP1 and ESRP2 RNA binding proteins, responsible for maintaining an epithelial cellular phenotype, were observed (Figure 2.B.). Moreover, the expression of Dysadherin, a cell membrane glycoprotein highly expressed in several tumors that depict decreased E-cadherin expression, was increased in MCF7Ecadvar cells (Figure 2.B.). In agreement with these molecular changes, MCF7Ecadvar cells depicted reduced adhesiveness and increased motility, migration and invasion properties, all indicative of a more aggressive cellular behavior.

Next, a set of experiments were done to assess whether changes in cell morphology and E-cadherin expression could be attributed, at least in part, to an autocrine/paracrine effect of the secreted novel Ecadvar protein. Firstly, HEK-293 cells were stably transfected with Ecadvar (HEK293Ecadvar) and pcDNA3 empty vector (HEK293pcDNA3). Once the expression of the novel Ecadvar mRNA was verified, the CM of both cell lines was collected and used to challenge MCF-7 cells during 7 days. Cells exposed to the HEK293Ecadvar CM displayed a fibroblastic-like morphology and showed a significant decrease in Ecadwt transcript levels. No changes were observed in cells incubated with HEK293pcDNA3 CM.


Assessment of Ecadvar mRNA expression levels in human cancer cell lines

The expression levels of the Ecadvar mRNA were determined in a panel of 42 established human cancer cell lines of diverse tissue origin (urological: prostate, bladder; gynecological: breast, ovary, endometrium; digestive: gastric, liver, pancreas, colon; other: skin, lung) by applying a primer-specific quantitative Real Time PCR strategy. Detectable levels of the Ecadvar transcript were found in 29 cell lines evaluated (69%). When Ecadvar mRNA levels were calculated relative to the wild type transcript, 23 cell lines depicted a 0.05 to 1.2% expression of the novel form, while the remaining showed levels between 2 and 11.6%. A survey done on the COSMIC and CCLE databases on the available cell lines (n=22) showed absence of mutations around the CDH1 Exon 13-Exon 14 constitutive splicing donor and/or acceptor sites. In silico and experimental approaches revealed that differences in Ecadvar mRNA levels may correspond to the Nonsense-Mediated mRNA Decay, a regulatory mechanism usually triggered by premature termination codons which may be altered during tumorigenesis.

Alterations in the expression and function(s) of E-cadherin are a key event in the deregulation of cell-cell adhesion during tumor progression and metastasis. Despite the vast literature in this field, the underlying mechanisms of E-cadherin changes in cancer are not fully understood. The present study reports, for the first time, the identification and characterization of a novel human Ecad alternative spliced-transcript that acts as a dominant negative modulator of Ecadwt and induces alterations in gene expression, cell morphology and behavior characteristic of the EMT process.

Considering the relevance of E-cadherin and EMT changes in progression and aggressiveness of numerous tumors of epithelial origin, understanding the mechanisms that control Ecadvar mRNA expression will contribute to the management of cancer diagnosis and treatment.

The short video summarizes the main findings of the present report.





Figure 1. Identification of a human novel E-cadherin variant mRNA

A. Sequence alignment of the novel transcript (Ecadvar) with the reference Ecadwt mRNA showing a 34-bp deletion in the variant form. B. Graphical representation of the constitutive and the alternative splicing mechanisms involved in the generation of Ecadwt (top) and Ecadvar (bottom) mRNA. C. Amino acidic sequence of Ecadwt (amino acids 1-882) and Ecadvar (amino acids 1-757) proteins (Grey box: signal peptide and propeptide sequences; Light blue box: extracellular domain sequence; Orange box: transmembrane domain sequence; Violet box: intracellular domain sequence; in Ecadvar Dark Green box: novel peptide sequence). D. Prediction of the secretory nature of the novel protein encoded by Ecadvar mRNA; analysis done with the TMHMM server.



Figure 2. Effects of Ecadvar expression in MCF7 human breast cancer cells

A. Representative phase-contrast images of MCF7pcDNA3 and MCF7Ecadvar cells. B. Immunodetection of E-cadherin protein (120 KDa full length form) after SDS-PAGE and Western immunoblotting analysis in both cell lines. Immunodetection of Tubulin (55 KDa) was used as loading control. C. Fluorescence immunocytochemistry and confocal laser microscopy analysis of E-cadherin and F-actin in both cell lines. D. E-cadherin, Twist, Snail, Zeb1, Slug, cytokeratin 19, vimentin, N-cadherin, ESRP1, ESRP1 and Dysadherin mRNA expression levels in MCF7EpcDNA3 and MCF7Ecadvar cells assessed by Real Time PCR. The mRNA expression fold change is also shown.




PLoS One. 2016 Aug 2;11(8):e0160530. doi: 10.1371/journal.pone.0160530.

Oxidized Lipoprotein as a Major Vessel Cell Proliferator in Oxidized Human Serum 

Authors: Yoshiro Saito and Noriko Noguchi 

Department of Medical Life Systems, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Kyoto 610–0321, Japan


Address for correspondence:

Yoshiro Saito, PhD

Department of Medical Life Systems, Faculty of Life and Medical Sciences, Doshisha University, 1-3 Miyakodani, Tatara, Kyotanabe, Kyoto 610–0394, Japan

Tel: +81-774-65-6258. E-mail: ysaito@mail.doshisha.ac.jp



Backgrounds: Oxidative stress is correlated with the incidence of several diseases such as atherosclerosis and cancer, and oxidized biomolecules have been determined as biomarkers of oxidative stress; however, the detailed molecular relationship between generated oxidation products and the promotion of diseases has not been fully elucidated.  To clarify the role of serum oxidation products in vessel cell proliferation, the major vessel cell proliferator in oxidized human serum was investigated.

Methods and Results: Oxidized human serum was prepared by free radical exposure, separated using gel chromatography, and then each fraction was added to several kinds of vessel cells including endothelial cells and smooth muscle cells.  A high molecular weight fraction in oxidized human serum specifically induced vessel cell proliferation.  Oxidized lipids were contained in this high molecular weight fraction, while cell proliferation activity was not observed in oxidized lipoprotein-deficient serum.

Conclusions: Oxidized lipoproteins containing lipid oxidation products function as a major vessel cell proliferator in oxidized human serum.

PMID: 27483438



The relationship between oxidative stress biomarkers and their biological action

The oxidation of biological molecules by free radicals yields a variety of oxidation products [1].  Oxidized products are measured as biomarkers of oxidative stress to assess the oxidative injury in the pathologic processes of free radical-related diseases [2].  However, the relationship between oxidative stress biomarkers and their biological action has not been well investigated.  The proliferation of vascular cells is related to the onset as well as the progress of several diseases such as atherosclerosis and cancer.  To clarify the role of serum oxidation products in vessel cell proliferation, the major vessel cell proliferator in oxidized human serum was investigated.

Effects of oxidized human serum components on the viability of vessel cells

To examine the effects of oxidized products in serum, a water-soluble radical initiator was added to human serum, and the serum component was oxidized [3].  The increase in cholesteryl ester hydroperoxide (CE-OOH), a major lipid peroxidation product in serum, and protein oxidation products (protein carbonyl) was confirmed [4].  Oxidized human serum was applied to gel chromatography, and then each fraction was added to the culture media of vessel cells such as endothelial cells (ECs) and smooth muscle cells (SMCs). After 48 h, cell viability was determined.  No statistically significant change was observed in the component of control human serum (Fig 1A).  In contrast, a significant increase in human aortic endothelial cells (HAEC) viability was observed in the case of oxidized human serum, in which two peaks are observed: one is a macromolecule of more than 200 kDa at fraction number 12 (Fr. 12) and the other is of low molecular weight at Fr. 36 (Fig 1B).  In the other types of vessel cells such as human aortic smooth muscle cells (AoSMCs) and human umbilical vein endothelial cells (HUVECs), a statistically significant increase was observed in macromolecule peaks at Fr. 12 (Fig 1C).  A significant increase in vessel cell viability with a single peak at around 200 kDa was observed in gel chromatography with a larger pore size (Fig 1E).  These results suggest that the macromolecule proliferator for vessel cells is generated in oxidized human serum.

Distribution of oxidized products in gel chromatography

The distribution of oxidized products, such as CE-OOH and protein carbonyls, in gel chromatography of human oxidized serum was analyzed. The peak of CE-OOH was consistent with that of the macromolecular proliferator for vessel cells in oxidized human serum (Fig 2A and 2B).  CE-OOH mainly exists in lipoproteins. Thus, these results suggest that oxidized lipoproteins containing CE-OOH act as the macromolecular proliferator for vessel cells in oxidized human serum.

Effects of oxidized lipoprotein-deficient serum on the viability of vessel cells

To investigate the involvement of oxidized lipoproteins, lipoprotein-deficient serum (LPDS) was prepared and oxidized by peroxyl radical initiator.  It was found that the significant increase in cell viability of ECs and SMCs disappeared in a high molecular weight fraction of oxidized LPDS (Fig 3). In contrast, HAEC viability increased at Fr. 12 when cultured with oxidized human serum components (Fig 3 inset). Collectively, these results suggest that the activity of the macromolecular proliferator for vessel cells in oxidized human serum is derived from oxidized lipoproteins.

Oxidized lipoprotein as a major vessel cell proliferator in oxidized human serum

In the present study using oxidized human serum, we could identify oxidized lipoproteins, containing CE-OOH, as a major proliferator of vascular cells (Fig. 4). This result clearly suggests the relationship between oxidative stress and the proliferation of vessel cells, which are related to the progression of arteriosclerosis and cancer, and suggests the significance of oxidized lipoproteins and lipids in the evaluation of the pathogenesis of atherosclerosis and cancer.



[1] Niki E, Yoshida Y, Saito Y, Noguchi N. Lipid peroxidation: mechanisms, inhibition, and biological effects. Biochem Biophys Res Commun. 2005;338(1):668–76. pmid:16126168

[2] Saito Y, Shichiri M, Hamajima T, Ishida N, Mita Y, Nakao S, et al. Enhancement of lipid peroxidation and its amelioration by vitamin E in a subject with mutations in the SBP2 gene. J Lipid Res. 2015;56(11):2172–82. doi: 10.1194/jlr.M059105. pmid:26411970

[3] Yoshida Y, Itoh N, Saito Y, Hayakawa M, Niki E. Application of water-soluble radical initiator, 2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride, to a study of oxidative stress. Free Rad Res. 2004;38(4):375–84.

[4] Saito Y, Noguchi N. 7-Hydroxycholestrol as a possible biomarker of cellular lipid peroxidation: difference between cellular and plasma lipid peroxidation. Biochem Biophys Res Commun. 2014;446(3):741–4. doi: 10.1016/j.bbrc.2013.12.083. pmid:24380859



Figure legend

Fig 1. Effects of oxidized human serum components on vessel cell viability.

A-D. Control (A) and oxidized human serum (B, C) were fractionated by Sephacryl S-300 gel chromatography, and protein contents were determined by absorbance at 280 nm.  HAEC (A, B), AoSMC (C), and HUVEC (D) were treated with each fraction (10%) for 48h, and the viability was measured.  The maclomolecule proliferator for vessel cells is indicated by black arrow. D. Oxidized human serum were fractionated by Sephacryl S-500 gel chromatography, and HUVEC was treated each fraction for 48h. The maclomolecule proliferator for vessel cells at Fr.31 is indicated by black arrow. * P < 0.01, compared with vehicle control.




Fig 2. Distribution of oxidized products in Sephacryl S-300 gel chromatography.

A. Oxidized human serum were fractionated by Sephacryl S-300 gel chromatography, and contents of protein (A280), cholesterol ester hydroperoxide (CE-OOH), and protein carbonyl were determined, respectively. B. Both AoSMC viability and CE-OOH contents are plotted. The peak of maclomolecule proliferator for vessel cells and CE-OOH at Fr.12 is indicated by black arrow.



Fig 3. Effects of oxidized lipoprotein deficient serum on the viability of vessel cells.



A and B. Oxidized lipoprotein deficient serum (LPDS) were fractionated by Sephacryl S-300 gel chromatography, and protein contents were determined by absorbance at 280 nm. HAEC (A) and AoSMC (B) were treated with each fraction (10%) for 48h, and the viability was measured by WST assay. The cell viability in the case of oxidized human serum fractions are shown in inner figure. The maclomolecule proliferator for vessel cells at Fr.12 is indicated by black arrow. * P < 0.01, compared with vehicle control.

Fig 4. Oxidized lipoprotein as a major vessel cell proliferator in oxidized human serum.

This study could identify oxidized lipoproteins as a major proliferator of vascular cells in human serum treated with radical initiator, and indicates the significance for the evaluation of oxidized lipoproteins and lipids in atherosclerosis and cancer patients to assume disorder of vessel cells.







Biomed Res Int. 2017;2017:7023078.

Fluid-Structure Interaction in Abdominal Aortic Aneurysm: Effect of Modeling Techniques.

Lin S1, Han X2, Bi Y2, Ju S3, Gu L4.
1 School of Civil Engineering and Architecture, Xiamen University of Technology, Xiamen, China; Department of Mechanical and Materials Engineering, University of Nebraska-Lincoln, Lincoln, NE 68588-0656, USA.
2 Department of Interventional Radiology, The First Affiliated Hospital, Zhengzhou University, Henan Province, China.
3 Department of Mechanical and Materials Engineering, University of Nebraska-Lincoln, Lincoln, NE 68588-0656, USA.
4 Department of Mechanical and Materials Engineering, University of Nebraska-Lincoln, Lincoln, NE 68588-0656, USA; Nebraska Center for Materials and Nanoscience, Lincoln, NE 68588-0656, USA.


In this work, the impact of modeling techniques on predicting the mechanical behaviors of abdominal aortic aneurysm (AAA) is systematically investigated. The fluid-structure interaction (FSI) model for simultaneously capturing the transient interaction between blood flow dynamics and wall mechanics was compared with its simplified techniques, that is, computational fluid dynamics (CFD) or computational solid stress (CSS) model. Results demonstrated that CFD exhibited relatively smaller vortexes and tends to overestimate the fluid wall shear stress, compared to FSI. On the contrary, the minimal differences in wall stresses and deformation were observed between FSI and CSS models. Furthermore, it was found that the accuracy of CSS prediction depends on the applied pressure profile for the aneurysm sac. A large pressure drop across AAA usually led to the underestimation of wall stresses and thus the AAA rupture. Moreover, the assumed isotropic AAA wall properties, compared to the anisotropic one, will aggravate the difference between the simplified models with the FSI approach. The present work demonstrated the importance of modeling techniques on predicting the blood flow dynamics and wall mechanics of the AAA, which could guide the selection of appropriate modeling technique for significant clinical implications.

PMID: 28321413; DOI:10.1155/2017/7023078



Abdominal aortic aneurysms (AAA) are local dilations of the infrarenal aorta. If left untreated, it may continue to expand which eventually rupture. The rupture is a mechanical failure when the stress at the vessel wall exceeds its strength. To determine whether the aneurysm needs to be operated or not, the traditional approach is solely based on its maximum transversal diameter. When the maximum transversal diameter exceeds 55 mm the surgery is normally recommended as current clinical practice. However, small aneurysms can also rupture and has a high mortality rate [1]. Thus, the decision to repair an aneurysm should not be determined by the maximum transversal diameter alone but a more reliable criterion which taking more factors into account. Since aneurysm rupture is a mechanical failure, a criterion associated with the mechanical performance of the AAA such as peak wall stress and strength is proposed by Fillinger et al. [2].


Finite element analysis (FEA) is an effective tool for determining the stress distribution on the aneurysm wall [2]. Currently there are three types of models which have been widely used to model the mechanical behavior of the AAA, (i) computational solid stress (CSS) model [3] which only consider the aneurysm wall only and pressure is uniform along the inner surface; (ii) computational fluid dynamics (CFD) [4, 5] model used to estimate wall shear stress which assume the aneurysm wall as rigid; (iii) fluid-structure interaction (FSI) [6, 7]model which couples the flow the inside the aneurysm and the motion of the aneurysm wall together. However, each technique has its own advantages and disadvantages, a detailed comparison between different modeling techniques is made in current study to help people choosing when they have different goals.


Results show that the FSI approach is preferred for presurgical planning of the AAA. Compared with FSI, CFD predicted that flow dynamics within AAA might underestimate the development of vortices and overestimate the shear stress. A softer wall material will aggravate the differences between these two modeling approaches. In addition, the CSS model might underestimate the wall stresses unless the pressure profile within the aneurysm could be adopted. The present work demonstrates the differences of three popular modeling techniques on predicting the AAA behaviors, which can be used to provide a fundamental understanding of the AAA behavior, especially blood flow dynamics and wall mechanics, to guide the selection of appropriate modeling technique for preclinical planning and to illuminate the possibilities for exploiting their potential to prevent AAA rupture.



Figure 1: Aneurysm wall behavior at peak systolic pressure in FSI model. 


Figure 2: Flow streamlines colored by velocity magnitude (top) and wall shear stress distribution (bottom) at four different time points for FSI model with anisotropic material property



1. Valentine, R.J., et al., Watchful waiting in cases of small abdominal aortic aneurysms—appropriate for all patients? Journal of vascular surgery, 2000. 32(3): p. 441-450.
2. Fillinger, M.F., et al., In vivo analysis of mechanical wall stress and abdominal aortic aneurysm rupture risk. Journal of vascular surgery: official publication, the Society for Vascular Surgery [and] International Society for Cardiovascular Surgery, North American Chapter, 2002. 36(3): p. 589.
3. Fillinger, M.F., et al., In vivo analysis of mechanical wall stress and abdominal aortic aneurysm rupture risk. Journal of Vascular Surgery, 2002. 36(3): p. 589-597.
4. Morris, L., et al., Effects of flat, parabolic and realistic steady flow inlet profiles on idealised and realistic stent graft fits through Abdominal Aortic Aneurysms (AAA). Medical engineering & physics, 2006. 28(1): p. 19-26.
5. Lasheras, J.C., The biomechanics of arterial aneurysms. Annu. Rev. Fluid Mech., 2007. 39: p. 293-319.
6. Scotti, C.M., et al., Fluid-structure interaction in abdominal aortic aneurysms: effects of asymmetry and wall thickness. BioMedical Engineering OnLine, 2005. 4(1): p. 64.
7. Einav, S., J. Ricotta, and D. Bluestein, Abdominal aortic aneurysm risk of rupture: patient-specific FSI simulations using anisotropic model. Journal of biomechanical engineering, 2009. 131: p. 031001-1.





Br J Nutr. 2017 Jan;117(1):83-92. doi: 10.1017/S0007114516004268.

Maternal short-chain fructo-oligosaccharide supplementation increases intestinal cytokine secretion, goblet cell number, butyrate concentration and Lawsonia intracellularis humoral vaccine response in weaned pigs.

Le Bourgot C1, Le Normand L1, Formal M1, Respondek F2, Blat S1, Apper E2, Ferret-Bernard S1, Le Huërou-Luron I1.
1French National Institute for Agricultural Research (INRA),UR1341 Food and Digestive, Nervous and Behavioural Adaptations (ADNC),Saint-Gilles,F-35590,France.



Prebiotic supplementation modulates immune system development and function. However, less is known about the effects of maternal prebiotic consumption on offspring intestinal defences and immune system responsiveness. We investigated the effects of maternal short-chain fructo-oligosaccharide (scFOS) supplementation on mucin-secreting cells, ileal secretory IgA and cytokine secretion of weaned offspring and their humoral response to an oral vaccine against obligate intracellular Lawsonia intracellularis. Sows were fed a control diet (CTRL) or scFOS-supplemented diet during the last third of gestation and throughout lactation. At weaning, each litter was divided into two groups receiving a post-weaning CTRL or scFOS diet for a month. Pigs from the four groups were either non-vaccinated (n 16) or vaccinated (n 117) at day 33. Biomarkers related to intestinal defences and immune parameters were analysed 3 weeks later. SCFA production was assessed over time in suckling and weaned pigs. Maternal scFOS supplementation improved ileal cytokine secretions (interferon (IFN)-γ, P<0·05; IL-4, P=0·07) and tended to increase caecal goblet cell number (P=0·06). It increased IgA vaccine response in the serum (P<0·01) and ileal mucosa (P=0·08). Higher bacterial fermentative activity was observed during lactation (total faecal SCFA, P<0·001) and after weaning (colonic butyrate, P=0·10) in pigs from scFOS-supplemented mothers. No synergistic effect between maternal and post-weaning scFOS supplementation was observed. Therefore, maternal scFOS supplementation has long-lasting consequences by strengthening gut defences and immune response to a vaccine against an intestinal obligate intracellular pathogen. Prebiotic consumption by gestating and lactating mothers is decisive in modulating offspring intestinal immunity.


IFN interferon; PND postnatal day; sIgA secretory IgA; scFOS short-chain fructo-oligosaccharides; Intestinal immune system; Maternal nutrition; Prebiotics; Short-chain fructo-oligosaccharides; Vaccination

PMID: 28115029



Perinatal nutrition plays a decisive role in controlling gut bacterial primo-colonization and, thereby, is an important driver for the development and the maturation of intestinal immunity, particularly acquisition of mucosal immune tolerance as well as protection against pathogens. Since short-chain fructooligosaccharide (scFOS) prebiotic was known to positively modulate the microbiota composition and activity, it appeared as an interesting nutritional strategy during the perinatal life which is a critical window of development. We previously demonstrated, in a pig model, that scFOS supplementation of the maternal diet positively modulated the offspring gut microbiota and immune system maturation before weaning (Le Bourgot et al., 2014). Our recent study (Le Bourgot et al., 2017) showed that such improvement in intestinal immune system maturation induced beneficial effects on its functionality, later in life, by enhancing local defence mechanisms and responsiveness to a bacterial challenge. Interestingly, the maternal scFOS supplementation improved the immune response to a vaccine directed against the intestinal bacterium Lawsonia intracellularis, but not that to a vaccine directed against the Influenza respiratory virus (Le Bourgot et al., 2016). Yet, the direct scFOS supplementation of weaned piglets increased the response to Influenza virus challenge, but not that to Lawsonia intracellularis. No synergistic effect between maternal and post-weaning scFOS supplementation was observed for both types of vaccination. Thus, the window of nutritional exposure to scFOS (maternal versus post-weaning) conditioned the responses of the immune system.

Optimization of the maturation of intestinal immune system during the neonatal life, associated with a modulation of the intestinal microbiota by maternal scFOS supplementation, promoted the functionality of the mucosal immune system after weaning as evidenced by a local intestinal bacterial challenge. We speculate that such maternal supplementation programmed the mucosal immune system to respond appropriately to intestinal pathogens. Conversely, the direct intake of scFOS after weaning could have differently impacted the microbiota composition with different consequences on immunity. In fact, the abrupt process of weaning induces strong modifications of the composition and complexity of the intestinal microbiota and several weeks are needed to establish a stabilized bacterial balance. In this context, the consumption of prebiotic during post-weaning phase could have favoured the growth of specific bacteria species with consequences on humoral response.

Due to the complexity of the microbiota, it is difficult to pinpoint the exact bacterial determinants of the immune response to a vaccine challenge. Further studies are required to highlight the role of a specific microbiota composition to confer immuno-competence against different types of vaccination, depending on the timing of exposure to prebiotic supplementation. Our promising results confirm the role played by early prebiotic supplementation on the intestinal functionality.



Figure : Effects of scFOS supplementation on vaccine immune responses of the weaned pig.



Le Bourgot, C., Le Normand, L., Formal, M., Respondek, F., Blat, S., Apper, E., Ferret-Bernard, S., & Le Huërou-Luron, I. (2017). Maternal short-chain fructo-oligosaccharide supplementation increases intestinal cytokine secretion, goblet cell number, butyrate concentration and Lawsonia intracellularis humoral vaccine response in weaned pigs. Br. J. Nutr. 117(1):83-92.
Le Bourgot, C., Ferret-Bernard, S., Blat, S., Apper, E., & Le Huërou-Luron, I. (2016). Short-chain fructooligosaccharide supplementation during gestation and lactation or after weaning differentially impacts pig growth and IgA response to influenza vaccination. J.  Funct. Foods 24: 307-315.
Le Bourgot, C., Ferret-Bernard, S., Le Normand, L., Savary, G., Menendez-Aparicio, E., Blat, S., Apper-Bossard, E., Respondek, F., & Le Huërou-Luron, I. (2014). Maternal short-chain fructooligosaccharide supplementation influences intestinal immune system maturation in piglets. PloS one 9(9): e107508.




Arthritis Care & Research. 2016 June;68(6):729-37.

Can creatine supplementation improve body composition and objective physical function in rheumatoid arthritis patients? A randomized controlled trial

Thomas Wilkinson1, Andrew Lemmey2, Jeremy Jones1,2, Fazal Sheikh2, Yasmeen Ahmad2, Sarang Chitale2, Peter Maddison2, Thomas O’Brien3

1 School of Sport, Health & Exercise Sciences, Bangor University, George Building, Bangor, Gwynedd, UK.

2 Peter Maddison Rheumatology Centre, Llandudno Hospital, Llandudno, North Wales, UK.

3 Research Institute for Sports and Exercise Sciences, Liverpool John Moores University, Liverpool, UK.

Corresponding author

Prof. Andrew Lemmey

School of Sport, Health & Exercise Sciences, Bangor University, George Building, Bangor, Gwynedd, UK

Phone: +44-01248 383932

Email: a.b.lemmey@bangor.ac.uk



Objective: Muscle wasting (‘rheumatoid cachexia’) is evident in most rheumatoid arthritis (RA) patients, including those with well-controlled disease, and contributes substantially to the reductions in strength and physical function that are characteristic of this disease. The aim of this randomized controlled trial was to investigate the efficacy of oral creatine (Cr) supplementation on improving muscle mass, strength and function in stable RA patients.

Method: Forty RA patients were randomized to 12 weeks supplementation of Cr or placebo, in a double-blind fashion. Body composition (by whole-body dual-energy X-ray absorptiometry, DXA, and bioelectrical impedance spectroscopy, BIS), strength and objectively-assessed physical function measures were taken at baseline, week 12, and week 24 (i.e. after 12 weeks of treatment withdrawal).  Data was analyzed by ANCOVA.

Results: Cr supplementation increased appendicular lean mass (ALM; a surrogate DXA measure of muscle mass) by (mean±SE) 0.52±0.13kg (P=0.004 vs placebo), and total LM by 0.60±0.37kg (P=0.158 vs placebo). The increment in LM by DXA corresponded with the elevation in intracellular water (ICW) estimated by BIS (0.64±0.22 L, P=0.035 vs placebo).  However, the observed increases in ALM, total LM and ICW were not accompanied by improvements in isometric knee extensor strength (P=0.408), hand-grip strength (P=0.833), or objectively assessed function (30s sit-to-stand, 50’ walk, 8’ up-&-go, estimated VO2max; P’s=0.335-0.764)

Conclusion: Twelve weeks of Cr supplementation improved muscle mass, but not strength or objectively-measured physical function in RA patients. As no adverse treatment-related effects occurred, Cr supplementation appears to be a safe and acceptable adjunct treatment for attenuating muscle loss in RA patients.  This treatment may be especially suitable for patients with severe rheumatoid cachexia.

KEYWORDS: rheumatoid cachexia, body composition, function, rheumatoid arthritis

PMID: 26414681



Patients with RA usually experience substantial loss of lean mass (LM) (known as ‘rheumatoid cachexia’ (RC) (1). This loss of LM is a major contributor to the decreased strength and impaired physical function (2,3) that characterizes RA.

Unfortunately, current pharmacologic treatments for RA do not ameliorate this LM loss, nor fully restore physical function (3). Whilst exercise (specifically, progressive resistance training (PRT)) has been shown to be highly effective in restoring both LM and function in RA patients (2), the lack of adherence to sufficiently intense training means this form of therapy is unlikely to be widely adopted (4). Anabolic nutritional supplementation offers a potential adjunct treatment intervention for improving LM and function that should be widely acceptable. Indeed, our group (5) has previously demonstrated that daily oral protein supplementation for 12 weeks improved LM and some measures of objectively-assessed physical function in RA patients.

Creatine (Cr), a combination of essential amino acids, is a popular dietary supplement generally shown to have greater benefits on both LM and physical function than generic protein. To date, only one study (6) has investigated the efficacy of oral Cr supplementation in RA patients. In this uncontrolled trial, twelve patients underwent three weeks of supplementation, and although strength increased, body composition changes were not investigated.

To further investigate the efficacy of Cr supplementation in improving LM, strength and function in RA patients, we recruited 40 patients with stable RA disease (i.e. no change in medications in the preceding 3 months) from outpatient clinics. Participants were randomised to receive either supplementary Cr or placebo drinks for 12 weeks, with the groups matched for age and sex. Both the principle researcher (TW) and participants were blinded to supplement assignment.

In accordance with manufacture recommendations, and previous strategies (e.g. 6,7), the Cr group received 20 g of Cr monohydrate (4 x 5 g/day) for a 5-day ‘loading phase’ followed by 3 g/day for the remainder of the 12 week supplementation period (‘maintenance dose’). The Cr was mixed with a mango-flavoured drink powder to improve taste. The placebo group received only the mango-flavoured drink powder. The appearance of the different treatment packets were indistinguishable, as were the flavouring and colouring of the drinks.



Table 1. Baseline demographics of rheumatoid arthritis patients who underwent 12 weeks of oral creatine or placebo supplementation


BM = body mass; BMI = body mass index; ALM = appendicular lean mass; FM = fat mass; DAS28 = disease activity score in 28 joints; NSAIDS = non-steroidal anti-inflammatory drugs; DMARDs = disease modifying anti-rheumatic drugs; IKES = isometric knee extensor strength; HGS = handgrip strength; STS-30 = sit-to- stand in 30 second test; 8’UG = 8-foot up and go; 50’W = 50-foot walk; VO2max = estimated V02max from Siconolfi step test; MDHAQ = Multi-dimensional Health Assessment Questionnaire. a = current corticosteroid use, range 2.5–5.0 mg. Unless stated, data presented as mean (±SD). * P < .05; #P = .05–.10.



The flow of patients through the study is shown in Figure 1. Subjects’ baseline demographics are presented in Table 1, and the effects of Cr supplementation on body composition in Table 2. Twelve weeks of Cr supplementation resulted in a significant increase in ALM of 0.52 (±0.13) kg in the Cr group, with no change in the placebo group (0.05 (±0.13) kg; between-group P = .004, effect size (η2) = .23 (medium)). Similarly, total LM increased by 0.60 (±0.37) kg) following Cr supplementation, with no change in the placebo group over the same period (-0.06 (±0.29) kg), albeit the between-group change was not significant (P = .158, η2 = .06 (small)). In the Cr group there was an increase in intra-cellular water (ICW) from baseline to week 12 (0.64 ±0.22 L, P = .035, η2 = .13 (medium)).


Table 2. Changes in body composition in rheumatoid arthritis patients following 12 weeks oral creatine supplementation and 12 weeks withdrawal from supplementation.


ALM = appendicular lean mass; BM = body mass (scales); FM = fat mass; TBW = total body water; ICW = intracellular water; ECW = extracellular water. Changes (Δ) between time points (B = baseline, 12 = week 12 (immediately post-supplementation); 24 = week 24 (12 weeks post-supplementation)) are presented as the adjusted mean (±SE) from ANCOVA. The between-group difference for each Δ is displayed with 95% confidence interval (CI) along and effect size, eta squared (η2): small = .01; medium = .08; large = .26; very large = .50. * P < .05.


After 12 weeks of cessation of Cr supplementation (week 24), there was a regression towards baseline for ALM and total LM, which further supports a Cr treatment effect. No changes in FM or body fat % were observed at any time point for either group.

In contrast to the effects on muscle mass, Cr supplementation had no effect on objectively-assessed physical function (Table 3).


Table 3. Changes in strength and objective physical function measures in rheumatoid arthritis patients following 12 weeks oral creatine supplementation and 12 weeks withdrawal from supplementation.


IKES = isometric knee extensor strength; HGS = handgrip strength; STS-30 = sit-to- stand in 30 second test; 8’UG = 8-foot up and go; 50’W = 50-foot walk; VO2max = estimated V02max from Siconolfi step test. Changes (Δ) between time points (B = baseline, 12 = week 12 (immediately post-supplementation); 24 = week 24 (12 weeks post-supplementation)) are presented as the adjusted mean (±SE) from ANCOVA. The between-group difference for each Δ is displayed with 95% confidence interval (CI) and effect size, eta squared (η2): small = .01; medium = .08; large = .26; very large = .50. * P < .05; #P = .05–.10.


The magnitude of LM increase we observed is comparable to that seen previously in older men and women following Cr supplementation. The body composition changes are also similar to those we previously observed following 12 weeks of protein supplementation in RA patients (i.e. increases of 0.40 kg in ALM and 0.73 kg in total LM, whilst FM remained unchanged (5)). These results, together with the response to PRT (2), and the finding that muscle quality (i.e. maximal force exerted per unit muscle) is not impaired in RA patients (8), further emphasise that RA patients are not, as once believed (9), resistant to muscle anabolic stimuli.

Interestingly, at week 24, despite the losses due to withdrawal of Cr, ALM and total LM were still 0.40 kg and 0.21 kg, respectively, above baseline values, suggesting some longer term retention of muscle mass following Cr supplementation.

The lack of a Cr-induced improvement in either strength or function that we observed in this study contrasts with the 14% gain in composite strength reported by Willer et al. (6) following short-term Cr supplementation in RA patients. However, the reported effects of Cr supplementation on measures of strength and function in older individuals are equivocal. Additionally, responsiveness to Cr supplementation is reported to vary, with only ~70–75% of individuals, irrespective of age, deemed to be ‘responders’ (7). Consistent with this estimation, 80% of our participants ‘responded’, when ‘response’ is defined by increased ALM (≥0.24 kg).

Although the lack of effects on strength and physical function are disappointing, the increase in LM we demonstrated suggests that Cr supplementation may be beneficial in patients with severe RC, since a marked loss of LM both impairs the body’s ability to fight infection due to limited expendable protein reserve for immune cell production, and increases the risk of mortality. The lack of efficacy demonstrated on physical function in this study further emphasises that sustained PRT (2) should be performed by RA patients wishing to substantially increase LM, and, subsequently, restore their strength and physical functioning.

The importance of this study: In patients with RA, 12 weeks of oral Cr supplementation had beneficial effects on muscle mass, but not on strength or objectively-assessed physical function. Given compliance to Cr was high, and no adverse treatment-related effects were observed, Cr may offer an acceptable, safe, low-cost, and reasonably effective means for RA patients with severe RC to help restore muscle mass.




Figure 1. CONSORT diagram showing recruitment and path of patients through the study

GFR = (estimated) glomerular filtration rate; Cr = Creatine supplementation group; DNC = randomised but did not commence treatment (i.e. did not attend baseline and were subsequently withdrawn); * = due to missing data, final analysis for body composition data included values using Expectation-Maximization imputed data; # = missed sessions (placebo) at day 6, week 12 and week 24 were not the same participant.




  1. Summers G, Deighton C, Rennie M, Booth A. Rheumatoid cachexia: a clinical perspective. Rheumatology 2008; 47: 1124-31.
  2. Lemmey AB, Marcora SM, Chester K, Wilson S, Casanova F, Maddison PJ. Effects of high‐intensity resistance training in patients with rheumatoid arthritis: A randomized controlled trial. Arthritis Care Res 2009; 61: 1726-34.
  3. Lemmey AB, Clayton R, Wilkinson T, Sheikh F, Ahmad Y, Whale J, Jones HSJ, Ahmad YA, Chitale S, Jones JG, Maddison PJ, O’Brien TD. Tight control of disease activity fails to improve body composition or physical function in rheumatoid arthritis patients. Rheumatology 2016; (in press) Advance Access June 10, 2016.
  4. Lemmey AB, Williams SL, Marcora SM, Jones J, Maddison PJ. Are the benefits of a high‐intensity progressive resistance training program sustained in rheumatoid arthritis patients? A 3‐year followup study. Arthritis Care Res 2012; 64: 71-75.
  5. Marcora S, Lemmey A, Maddison P. Dietary treatment of rheumatoid cachexia with β-hydroxy-β-methylbutyrate, glutamine and arginine: A randomised controlled trial. Clin Nutr 2005; 24: 442-54.
  6. Willer B, Stucki G, Hoppeler H, Brühlmann P, Krähenbühl S. Effects of creatine supplementation on muscle weakness in patients with rheumatoid arthritis. Rheumatology 2000; 39: 293-98.
  7. Greenhaff P, Bodin K, Soderlund K, Hultman E. Effect of oral creatine supplementation on skeletal muscle phosphocreatine resynthesis. Am J Physiol Endocrinol Metab 1994; 266: E725-E30.
  8. Matschke V, Murphy P, Lemmey AB, Maddison PJ, Thom JM. Muscle quality, architecture, and activation in cachectic patients with rheumatoid arthritis. J Rheumatol 2010; 37: 282-84.
  9. Rall LC, Meydani SN, Kehayias JJ, Dawson‐Hughes B, Roubenoff R. The effect of progressive resistance training in rheumatoid arthritis. Increased strength without changes in energy balance or body composition. Arthritis Rheum 1996; 39: 415-26.




PLoS One. 2016 Mar 15;11(3):e0151245.

Rheumatoid Arthritis and Incidence of Twelve Initial Presentations of Cardiovascular Disease: A Population Record-Linkage Cohort Study in England.

Pujades-Rodriguez M1,2, Duyx B1, Thomas SL3, Stogiannis D1, Rahman A4, Smeeth L1,3, Hemingway H1.

  • 1Farr Institute of Health Informatics Research, University College London, 222 Euston Road, London NW1 2DA, United Kingdom.
  • 2Leeds Institute of Biomedical and Clinical Sciences, MRC Medical Bioinformatics Centre, Worsley Building, University of Leeds, Leeds LS2 9JT, United Kingdom.
  • 3Department of Non-communicable Disease Epidemiology, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom.
  • 4Centre of Rheumatology Research, Division of Medicine, Faculty of Medical Sciences, University College London, London WC1E 6JF, United Kingdom.




While rheumatoid arthritis is an established risk factor for cardiovascular disease (CVD), our knowledge of how the pattern of risk varies for different cardiovascular phenotypes is incomplete. The association between rheumatoid arthritis and the initial presentation of 12 types of CVDs were examined in a contemporary population of men and women of a wide age range.


CALIBER data, which links primary care, hospital and mortality data in England, was analysed. A cohort of people aged ≥18 years and without history of CVD was assembled and included all patients with prospectively recorded rheumatoid arthritis from January 1997, until March 2010, matched with up to ten people without rheumatoid arthritis by age, sex and general practice. The associations between rheumatoid arthritis and the initial presentation of 12 types of CVDs were estimated using multivariable random effects Poisson regression models.


The analysis included 12,120 individuals with rheumatoid arthritis and 121,191 comparators. Of these, 2,525 patients with and 18,146 without rheumatoid arthritis developed CVDs during a median of 4.2 years of follow-up. Patients with rheumatoid arthritis had higher rates of myocardial infarction (adjusted incidence ratio [IRR] = 1.43, 95%CI 1.21-1.70), unheralded coronary death (IRR = 1.60, 95%CI 1.18-2.18), heart failure (IRR = 1.61, 95%CI 1.43-1.83), cardiac arrest (HR = 2.26, 95%CI 1.69-3.02) and peripheral arterial disease (HR = 1.36, 95%CI 1.14-1.62); and lower rates of stable angina (HR = 0.83, 95%CI 0.73-0.95). There was no evidence of association with cerebrovascular diseases, abdominal aortic aneurysm or unstable angina, or of interactions with sex or age.


The observed associations with some but not all types of CVDs inform both clinical practice and the selection of cardiovascular endpoints for trials and for the development of prognostic models for patients with rheumatoid arthritis.

PMID: 26978266



Rheumatoid arthritis is a common long-lasting disease that causes pain, swelling and stiffness in the joints. Previous studies have shown that people with rheumatoid arthritis might be more likely to suffer from cardiovascular diseases and die than people without this disease. The increased of cardiovascular risk might be partly related to the widespread inflammation that is present in people with this rheumatoid arthritis and to the use of certain types of medication used for its treatment.

The aim of the study was to compare the risk of developing different types of cardiovascular diseases in people with or without rheumatoid arthritis who had not been previously diagnosed with any cardiovascular disease and to examine whether a higher risk is found for all or only for some types of cardiovascular diseases. To do this work we analysed clinical information contained in electronic medical records routinely collected by general practitioners and during hospitalisation in England. We first classified people according to whether they had been diagnosed or not with rheumatoid arthritis and excluded those with prior history of cardiovascular disease in the records. We then identified people who were diagnosed with any of the most common types of cardiovascular diseases during the following months and years, for example angina, heart attack, cardiac arrest, heart failure and stroke. We finally compared the risk of each of these different types of cardiovascular diseases in people with and without rheumatoid arthritis.

The results of the study showed that people with rheumatoid arthritis had a higher risk of suffering from heart attack, coronary death, heart failure, cardiac arrest and peripheral arterial disease. This higher risk was found in both patients recently diagnosed with rheumatoid arthritis and in those who had the disease for ten years or more, in men and women and in all age groups. In our study population, people with rheumatoid arthritis did not have a higher risk of stroke and had a lower risk of being diagnosed with stable angina. The last finding suggests that people with rheumatoid arthritis and clinicians might not always easily recognise the symptoms of coronary diseases such as chest pain related angina.

Because we wanted to understand how important was the observed increase in risk of cardiovascular diseases related to rheumatoid arthritis, we compared this to the magnitude of risk found in patients with diabetes, and our data showed that the size of risk related to these two diseases was similar.

The importance of this study is two-fold: first, our findings suggest that people with rheumatoid arthritis are more likely to experience cardiovascular diseases than people without this disease, highlighting the importance of regularly assessing, monitoring and adequately managing cardiovascular risk in patients with rheumatoid arthritis. Second, the results suggest that some deaths could be prevented if patients with rheumatoid arthritis are adequately informed about the increased cardiovascular risk conferred by this disease and they are educated on how to recognise cardiovascular symptoms.





Endocr J. 2017 Feb 27;64(2):221-227. doi: 10.1507/endocrj.EJ16-0170.

Gradually increasing ethinyl estradiol for Turner syndrome may produce good final height but not ideal BMD

Yukihiro Hasegawa

Division of Endocrinology and Metabolism, Tokyo Metropolitan Children’s Medical Center.

Corresponding Author:

Yukihiro Hasegawa, M.D. and Ph.D.

2-8-29 Musashi-dai, Fuchu, Tokyo, Japan 183-8561

Division of Endocrinology and Metabolism

Tokyo Metropolitan Children€™s Medical Center


Disclosure statement: Nothing to declare



Estrogen replacement therapy in Turner syndrome should theoretically mimic the physiology of healthy girls. The objective of this study was to describe final height and bone mineral density (BMD) in a group of 17 Turner syndrome patients (group E) who started their ethinyl estradiol therapy with an ultra-low dosage (1-5 ng/kg/day) from 9.8~13.7 years. The subjects in group E had been treated with GH 0.35 mg/kg/week since the average age of 7.4 years. The 30 subjects in group L, one of the historical groups, were given comparable doses of GH, and conjugated estrogen 0.3125 mg/week ~0.3125 mg/day was initiated at 12.2-18.7 years. The subjects in group S, the other historical group, were 21 patients who experienced breast development and menarche spontaneously. Final height (height gain < 2 cm/year) in group E was 152.4 +/- 3.4 cm and the standard deviation (SD) was 2.02 +/- 0.62 for Turner syndrome. The final height in group L was 148.5 +/- 3.0 cm with a SD of 1.30 +/- 0.55, which was significantly different from the values for group E. The volumetric BMD of group S (0.290 +/- 0.026 g/cm3) was significantly different from that of group L or E (0.262 or 0.262 g/cm3 as a mean, respectively). This is the first study of patients with Turner syndrome where estrogen was administered initially in an ultra-low dose and then increased gradually. Our estrogen therapy in group E produced good final height but not ideal BMD.

PMID: 27916781



Turner syndrome (TS) is one of the most common sex chromosome aneuploidies and one of the main treatable symptoms is delayed puberty. Estrogen replacement for TS should ideally mimic a physiology of healthy girls; hence treatment should begin with an ultra-low dosage and gradually increase. However, no studies to date have examined the merits of this kind of therapy. We administered EE2 to 17 patients with TS (Early-group; group E) to imitate physiological pubertal development.

This is the first study of patients with TS where estrogen was administered initially in an ultra-low dose and then increased gradually. The initial dose of EE2, 1-5 ng/kg in group E, was extremely low (cf. adult dose is roughly 200~400 ng/kg). Additionally, this is the first study of TS, where volumetric BMD (vBMD) was also taken into consideration in terms of the timing and method of exogenous estrogen administration. One of the representative cases is shown in Figure 1.

In this study, the higher vBMD values contrasting with the lower final height in group S (Spontaneous puberty-group), compared with the corresponding values for group E, should be noted. Estrogen administration in group E should have been started earlier than done in this study in order to achieve adequate BMD in patients with TS who are not expected to experience regular menstruation. One of our previous studies reported a decrease in actual BMD as measured by quantitative computed tomography in TS patients not only during, but also before puberty. The decline started as early as 7-10 years of age [1]. It is classically speculated that less estrogen is needed to maintain bone mineral density than to maintain and develop pubertal/adult physical change [2].

Since it is commonly believed that transdermal estradiol could be more physiological treatment than others [3-5], the future study should be ideally done with transdermal estradiol, titrated starting at an ultra-low dose and at earlier ages. The endpoints should consist of not only height and bone parameters but also uterus size, lipid and coagulation profiles, and QOL evaluations.



Figure 1. One of the representative cases who started ultra-low EE2 at an early age. Her final height was 159.2 cm (0.1 SD for Japanese girls) and DXA value at lumbar 2-4 at the final height was -0.1 SD.




1. Nanao K, Tsuchiya Y, Kotoh S, Hasegawa Y (2002) Low vertebral cancellous bone density in peripubertal girls with Turner’s syndrome and boys with hypogonadism. J Pe-diatr Endocrinol Metab 15:1537-1542.
2. Barbieri RL. (1992) Hormone treatment of endometriosis: the estrogen threshold hy-pothesis. Am J Obsttet Gynecol 166:740-5.
3. Scarabin PY, Oger E, Plu-Bureau (2003) Differential association of oral and trans-dermal oestrogen-replacement therapy with venous thromboembolism risk. Lancet 362:428–432.
4. Canonico M, Oger E, Plu-Bureau G, Conard J, Meyer G, et al. (2007) Hormone ther-apy and venous thromboembolism among postmenopausal women: impact of the route of estrogen administration and progestogens: the ESTHER study. Circulation 115:840–845.
5. Torres-Santiago L, Mericq V, Taboada M, Unanue N, Klein KO, et al. (2013) Meta-bolic effects of oral vs. transdermal 17β etradiol: a randomized clinical trial in girls with Turner syndrome. J Clin Endocrinol Metab 98:2716-24.




Langmuir.2016 Oct;32(42):10957-10966

Insight into the Tribological Behavior of Liposomes in Artificial Joints

Yiqin Duan1, Yuhong Liu1,*, Caixia Zhang2, Zhe Chen1, Shizhu Wen1

1State Key Laboratory of Tribology, Tsinghua University, Beijing 100084, China

2 Beijing Key Laboratory of Advanced Manufacturing Technology, Beijing University of Technology, Beijing 100124, China

Correspondence should be addressed to Yuhong Liu, E-mail: liuyuhong@tsinghua.edu.cn



Liposomes are widely used in drug delivery and gene therapy, and their new role as boundary lubricant in natural/artificial joints has been found in recent years. In this study, the tribological properties of liposomes on titanium alloy (Ti6Al4V)/UHMWPE interface were studied by a ball-on-disc tribometer. The efficient reduction of friction coefficient and wear on both surfaces under various velocities and loads is found. A multilayer structure of physically adsorbed liposomes on Ti6Al4V surface was also observed by atomic force microscope (AFM). Except for the hydration mechanism by phosphatidylcholine (PC) groups, the well-performed tribological properties by liposomes is also attributed to the existence of adsorbed liposomes layers on both surfaces, which could reduce asperities contact and show great bearing capacity. This work enriches the research on liposomes for lubrication improvement on artificial surface and shows their value on clinical application.



The adsorption morphology of DPPC on Ti6Al4V was obtained by AFM, shown in Figure 1. When lipid concentration is 1mg/ml (Figure 1(a)), the coating is composed of a close-packed layer of liposomes, with a loose coating of aggregated liposomes on top of this layer. When scanning the same area by AFM tip for several times, the close-packed liposomes in lower layer appear (inset). The height of this flattened liposome is 50±10 nm, which is much lower than the unperturbed liposome height from DLS result (122±35 nm), due to attraction force from substrate and compression by AFM tip. By decreasing the lipid concentration (Figure 1(b-d)), there are fewer and flatter liposomes on top layer (mark a). Meanwhile, in lower layer (insets), due to the stronger attraction force from substrate, large liposomes rupture to bilayers1 with thickness of 5 nm and smaller liposomes are flattened with height of 10 nm, which is equal to a double value of one bilayer (mark b).

Based on the AFM results, the sequential adsorption process and mechanisms of liposome vesicles are proposed in Figure 1(e-g). For a single vesicle (Figure 1(e)), the closed structure (from strong chain-chain hydrophobic attractions2) and the lateral constraints arising from neighboring vesicles could keep vesicles intact and robust. However, the forces arising from the substrate, AFM tip1 tend to rupture the bilayer membrane. Thus, adsorbed liposomes are metastable, especially for these liposomes which are firstly adsorbed on Ti6Al4V substrate (They rupture to bilayers with thickness of 5-6 nm). For subsequent liposomes, they would retain their closed shape, due to decreased attraction forces (Figure 1(f)). As a result, a multilayer structure of liposomes adsorbed on Ti6Al4V substrate is proposed as Figure 1(g).



Figure 1. AFM images of liposomes absorbed onto Ti6Al4V with lipid concentrations of (a) 1 mg/ml, (b) 0.1 mg/ml, (c) 0.05 mg/ml, and (d) 0.01 mg/ml. Insets in the bottom left corner are images below top layers. (e) Mechanical analysis of liposome vesicles. (f) – (g) Schemes of continuous adsorption process of vesicles and final structure of liposomes adsorbed on Ti6Al4V substrate. Scale bars are 500 nm.


The adsorption morphology of DPPC on UHMWPE was obtained by AFM, shown in Figure 2. Similarly, with lipid concentration of 1mg/ml, there is a dense distribution of liposomes on UHMWPE3. So far, the liposomes are found on both sliding surfaces with a high lipid concentration. Notably, there is a difference on the adsorption strength between liposomes and two surfaces. Compared with Figure 2(b) and Figure 1(d), it is obvious that liposomes cannot stay on UHMWPE surface with low lipid concentration, since UHMWPE is known as a hydrophobic polymer4.



Figure 2. AFM images of liposomes absorbed onto UHMWPE foil with lipid concentrations of (a) 1 mg/ml and (b) 0.01 mg/ml. Insets in the bottom left corner are images below top layers. Scale bars are 500 nm.


To study the tribological behaviors of DPPC liposomes on Ti6Al4V against UHMWPE, four friction tests were conducted using ball-on-disc equipment. It is worth noting that the velocity and normal load are related to the friction coefficient according to Stribeck Curve5. From Figure 3(a), there is an excellent friction reduction by DPPC liposomes under all velocities. Besides, the average friction coefficient by DPPC is stable mostly due to the same wear scars on UHMWPE surfaces under different velocities. To the contrary, the wear scar is severer under higher velocity when lubricated by water, causing high friction. Meanwhile, there is also an obvious friction reduction by DPPC liposomes under all loads from Figure 3(b). Even under high load of 400g (ca.39MPa), the friction is low in the case of DPPC, which means that the adsorbed layer on both surface can prevent asperities contact.

Since the vesicle size and lipid concentration can directly influence the structure of boundary layers, it is considerable in our study. Figure 3(c) gives a clear sight that the lowest friction coefficient is achieved under size around 100 nm. Because the vesicles are well-distributed on adsorbed layer with uniform size, the energy dissipation is low when two surfaces sliding past each other. As for the impact of lipid concentration, the decrease in average friction coefficient indicates higher lipid concentration could form a thicker adsorbed layers, leading to better bearing capacity, shown in Figure 3(d).



Figure 3. (a) Effect of sliding velocity on average friction coefficient. The lipid concentration is 1mg/ml and normal load is 2N (P=ca. 30.87 MPa). (b) Effect of normal load on average friction coefficient. The lipid concentration is 1mg/ml and sliding velocity is 0.3 mm/s. (c) Average friction coefficient with three membrane pore sizes and their mixture (lipid concentration is 1 mg/ml). (d) Effect of lipid concentration on average friction coefficient (vesicle size is around 100 nm). Experiments in Fig(c) and (d) were carried out under load of 2 N (P=ca. 30.87 MPa), sliding velocity of 0.3 mm/s.


At last, a lubrication model is proposed in Figure 4. The rubbing of two surfaces can be considered as the mechanical effect of asperities from surfaces. Between these asperities, a mount of liposomes exist and form the adsorbed layers. Liposomes physically adsorb onto two surfaces, with stronger attraction interaction towards Ti6Al4V surface due to hydrophilicity and opposite charges. A multilayer structure is formed and shear slip happen within these layers. The low friction is achieved by the highly hydrated PC groups6 exposed in water, where liposome vesicles move easily between each other. Meanwhile, adsorbed liposomes layers show great bearing capacity and wear resistance due to elastic property1 of vesicle and softness of adsorbed films, which contribute to the low friction.



Figure 4. Schematic illustration of the lubrication model by liposomes between Ti6Al4V- UHMWPE surfaces.




(1) Sorkin, R.; Dror, Y.; Kampf, N.; Klein, J. Mechanical Stability and Lubrication by Phosphatidylcholine Boundary Layers in the Vesicular and in the Extended Lamellar Phases. Langmuir 2014, 30, 5005-5014.

(2) Sorkin, R.; Kampf, N.; Dror, Y.; Shimoni, E.; Klein, J. Origins of Extreme Boundary Lubrication by Phosphatidylcholine Liposomes. Biomaterials 2013, 34, 5465-5475.

(3) Bruck, A. L.; Kanaga Karuppiah, K. S.; Sundararajan, S.; Wang, J.; Lin, Z. Friction and Wear Behavior of Ultrahigh Molecular Weight Polyethylene as a Function of Crystallinity in the Presence of the Phospholipid Dipalmitoyl Phosphatidylcholine. J. Biomed. Mater. Res., Part B 2010, 93B, 351-358.

(4) Pawlak, Z.; Urbaniak, W.; Oloyede, A. The Relationship Between Friction and Wettability in Aqueous Environment. Wear 2011, 271, 1745-1749.

(5) Kalin, M.; Velkavrh, I.; Vizintin, J. The Stribeck Curve and Lubrication Design for Non-Fully Wetted Surfaces. Wear 2009, 267, 1232-1240.

(6) Klein, J. Hydration Lubrication. Friction 2013, 1, 1-23.