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1.  Diagnostic Performance of Magnetic Resonance Elastography in Staging Liver Fibrosis: A Systematic Review and Meta-analysis of Individual Participant Data 
Background & Aims
Magnetic resonance elastography (MRE) is a non-invasive tool for staging liver fibrosis. We conducted a meta-analysis of individual participant data collected from published studies to assess the diagnostic accuracy of MRE and for staging liver fibrosis in patients with chronic liver diseases (CLD).
Through a systematic literature search of multiple databases (2003–2013), we identified studies on diagnostic performance of MRE for staging liver fibrosis in patients with CLD with native anatomy, using liver biopsy as the standard. We contacted study authors to collect data on each participant’s age, sex, body mass index (BMI), liver stiffness (measured by MRE), fibrosis stage, staging system used, degree of inflammation, etiology of CLD, and interval between MRE and biopsy. Through pooled analysis, we calculated the cluster-adjusted area under receiver-operating curve (AUROC), sensitivity, and specificity of MRE for any fibrosis (≥stage 1), significant fibrosis (≥stage 2), advanced fibrosis (≥stage 3), and cirrhosis (stage 4)
We analyzed data from 12 retrospective studies, comprising 697 patients (mean age, 55±13 years; 59.4% male; mean BMI, 26.9±6.7 kg/m2; 92.1% with <1 year interval between MRE and biopsy; hepatitis C in 47.1%). Participants had fibrosis stages 0, 1, 2, 3, or 4 (19.5%, 19.4%, 15.5%, 15.9% and 29.7%, respectively). Mean AUROC values (and 95% confidence intervals) for diagnosis of any (≥stage 1), significant (≥stage 2), or advanced fibrosis (≥stage 3), and cirrhosis, were 0.84 (0.76–0.92), 0.88 (0.84–0.91), 0.93 (0.90–0.95), and 0.92 (0.90–0.94), respectively. Similar diagnostic performance was observed in stratified analysis based on sex, obesity, and etiology of CLD. The overall rate of failure of MRE was 4.3%.
Based on pooled analysis of data from individual participants, MRE has high accuracy for diagnosis of significant or advanced fibrosis and cirrhosis, independent of BMI and etiology of CLD. Prospective studies are warranted to better understand the diagnostic performance of MRE.
PMCID: PMC4333001  PMID: 25305349
IPD; non-invasive; elastography; diagnostic performance; pooled analysis
2.  Non-invasive detection of liver fibrosis: MR imaging features vs. MR elastography 
Abdominal imaging  2015;40(4):766-775.
To compare accuracy of morphological features of liver on MRI and liver stiffness with MR elastography (MRE) for detection of significant liver fibrosis and cirrhosis.
Materials and Methods
In this retrospective study, we evaluated 62 patients who underwent liver MRI with MRE and histological confirmation of liver fibrosis within 6 months. Two radiologists, blinded to histology results, independently evaluated liver parenchyma texture, surface nodularity, signs of volumetric changes and portal hypertension for presence of significant fibrosis and cirrhosis. Two more readers independently calculated mean liver stiffness values with MRE. Interobserver agreement was evaluated with kappa and intra-class correlation coefficient (ICC) analysis. Diagnostic accuracy was assessed with area under receiver operating characteristic (AUROC) analysis. Comparison of AUROCs of MRI and MRE was performed.
Liver fibrosis was present in 37 patients. The interobserver agreement was poor to good (kappa= 0.12 - 0.74) for MRI features and excellent for MRE (ICC, 0.97, 95% CI, 0.95-0.98). MRI features had 48.5-87.9%sensitivity, 55.2%-100%specificity and 71.5-81.6% accuracy //for detection of significant fibrosis. MRE performed better with 100% sensitivity, 96.5% specificity and 98.9% accuracy .For the detection of cirrhosis, MRE performed better than MRI features with 88.2% sensitivity (vs.41.2-82.3%), 91.1% specificity (vs. 64.4-95.6%) and 93.5% accuracy (vs. 60.6%-80.5%) Among the MRI features, surface nodularity and overall impression had the best accuracies of 80.3% and 81.6% for detection of significant fibrosis respectively. For cirrhosis, parenchyma texture and overall impression had the best accuracies of 80.5% and 79.7% respectively . Overall, MRE had significantly greater AUROC than MRI features for detection of both significant fibrosis (0.98.9 vs 0.71-0.82, p<0.001) and cirrhosis (0.93.5-vs. 0.61 -0.80.5, p<0.01).
MRE is superior to MRI for the non-invasive diagnosis of significant liver fibrosis and cirrhosis.
PMCID: PMC4739358  PMID: 25805619
3.  Chronic Passive Venous Congestion drives Hepatic Fibrogenesis via Sinusoidal Thrombosis and Mechanical Forces 
Hepatology (Baltimore, Md.)  2015;61(2):648-659.
Chronic passive hepatic congestion (congestive hepatopathy) leads to hepatic fibrosis; however the mechanisms involved in this process are not well understood. We developed a murine experimental model of congestive hepatopathy through partial ligation of the inferior vena cava (pIVCL). C57BL/6 and transgenic mice overexpressing tissue factor pathway inhibitor (SM22α -TFPI) were subjected to pIVCL or SHAM. Liver and blood samples were collected and analyzed in immunohistochemical, morphometric, real-time polymerase chain reaction and western blot assays. Hepatic fibrosis and portal pressure were significantly increased after pIVCL concurrent with hepatic stellate cell (HSC) activation. Liver stiffness, as assessed by magnetic resonance elastography, correlated with portal pressure and preceded fibrosis in our model. Hepatic sinusoidal thrombosis as evidenced by fibrin deposition was demonstrated both in mice after pIVCL as well as in humans with congestive hepatopathy. Warfarin treatment and TFPI overexpression both had a protective effect on fibrosis development and HSC activation after pIVCL. In vitro studies show that congestion stimulates HSC fibronectin (FN) fibril assembly through direct effects of thrombi as well as by virtue of mechanical strain. Pretreatment with either Mab13 or Cytochalasin-D, to inhibit β-integrin or actin polymerization, respectively, significantly reduced fibrin and stretch induced FN fibril assembly.
Chronic hepatic congestion leads to sinusoidal thrombosis and strain, which in turn promote hepatic fibrosis. These studies mechanistically link congestive hepatopathy to hepatic fibrosis.
PMCID: PMC4303520  PMID: 25142214
Congestive hepatopathy; Extracellular Matrix; mechanical stretch; hepatic stellate cell; fibronectin fibril assembly
4.  Involvement of the Hippo pathway in regeneration and fibrogenesis after ischaemic acute kidney injury: YAP is the key effector 
The Hippo pathway plays a stage-specific role in regeneration and fibrogenesis after ischaemia/reperfusion-induced acute kidney injury. The proper modulation of this pathway might be the key point of transition from acute kidney injury to chronic kidney disease.
Renal tubule cells can recover after they undergo AKI (acute kidney injury). An incomplete repair of renal tubules can result in progressive fibrotic CKD (chronic kidney disease). Studies have revealed the relationship between tubular epithelial cells and kidney fibrogenesis. However, the underlying mechanism remains unclear. Hippo pathway components were evaluated in complete/incomplete repair of I/R (ischaemia/reperfusion) AKI rat models, HK-2 cells and AKI human renal biopsy samples. We found that the expression levels of the Hippo pathway components changed dynamically during kidney regeneration and fibrogenesis in rat models of I/R-induced AKI and human renal biopsy samples. The transcription cofactor YAP (Yes-associated protein) might be a key effector of renal regeneration and fibrogenesis. Our results showed further that YAP might elicit both beneficial and detrimental effects on I/R AKI. After I/R injury occurred, YAP could promote the repair of the injured epithelia. The constant YAP increase and activation might be related to interstitial fibrosis and abnormal renal tubule differentiation. These results indicate that the proper modulation of the Hippo pathway, specifically the transcription cofactor YAP, during repair might be a potent therapeutic target in AKI–CKD transition after I/R injury.
PMCID: PMC4727597  PMID: 26574480
acute kidney injury; chronic kidney disease; fibrogenesis; Hippo pathway; repair; Yes-associated protein (YAP)
5.  Hepatic Magnetic Resonance Elastography: Clinical Performance in a Series of 1,377 Consecutive Examinations 
Radiology  2015;278(1):114-124.
To assess the technical success rate and diagnostic performance of liver MR elastography (MRE) in a retrospective study of clinical patients.
Materials and Methods
This retrospective study was IRB approved with informed consent. A total of 1377 consecutive MRE examinations in 1287 patients for clinical indications were included from 2007 to 2010. Medical records were used to retrieve MRE-assessed liver stiffness, histological analysis, blood work and other liver disease related information. Nonparametric Kruskal-Wallis tests and analysis of covariance methods were used to evaluate the diagnostic values and relationships of the collected data.
Hepatic MRE had a success rate of 94.4% (1300/1377) and reproducible measurements (r=0.9716, p<0.0001) in our study cohort with a complex patient profile and multiple interpreters. Body mass index had no significant effect on the success rate (p=0.2). In 289 patients with liver biopsy performed within 1 year of the MRE exam date, MRE-assessed liver stiffness is significantly higher in advanced fibrosis stages (F3-4) than mild to moderate fibrosis stages (F0-1-2) (5.93±2.31 versus 3.35±1.44 kPa, p<0.0001). Liver stiffness is associated with many factors other than fibrosis extent, including etiology of fibrosis (viral hepatitis C versus nonalcoholic fatty liver disease, p=0.025), inflammation (severe versus mild to moderate, p=0.03), and hepatic metabolic and synthetic function (no versus intermediate fibrosis, p≤0.01).
In our general clinical practice environment, hepatic MRE is a very robust imaging method with a high success rate for a broad spectrum of patients. It also demonstrates the complex association between liver stiffness and hepatic pathophysiology.
PMCID: PMC4688072  PMID: 26162026
MR Elastography; liver; hepatic fibrosis; hepatic inflammation; clinical application; technical success
6.  Decreased Yes-Associated Protein-1 (YAP1) Expression in Pediatric Hearts with Ventricular Septal Defects 
PLoS ONE  2015;10(10):e0139712.
Ventricular septal defects (VSDs) are the most common and simplest type of congenital heart diseases (CHDs). Animal studies have suggested that the downregulation of Yes-associated protein 1 (YAP1) during embryonic development causes VSD-associated CHDs. However, how YAP1 contributes to isolated VSD (iVSD) is unclear.
Methods and Results
Twenty right atrial specimens were obtained from iVSD patients during routine congenital cardiac surgery and we assessed YAP1 expression in these specimens. For controls, six right atrial specimens were obtained from normal hearts of children without heart disease, three of whom died from cerebral palsy, and three who underwent heart transplants. YAP1 mRNA and protein levels and nuclear localization were significantly reduced in iVSD specimens compared to normal heart specimens. Concomitantly, mRNA levels of YAP1 downstream targets CTGF and AXL were also significantly decreased in iVSD specimens. Although Ki67-positive cardiomyocytes in iVSD specimens were comparable to normal heart specimens, Ki67-positive non-cardiomyocytes were significantly decreased.
YAP1 expression was markedly decreased in hearts of iVSD children. Given the important role of YAP1 during heart development, downregulation of YAP1 expression may contribute to iVSD and affect the proliferation of non-cardiomyocytes.
PMCID: PMC4591351  PMID: 26426694
7.  MR Elastography of Liver Disease: State of the Art 
Applied radiology  2013;42(4):5-12.
PMCID: PMC4564016  PMID: 26366024
Liver fibrosis; MR elastography; Hepatitis C Virus; Nonalcoholic Fatty Liver Disease; Methotrexate Toxicity
8.  Magnetic Resonance Elastography for Liver Fibrosis in Methotrexate Treatment 
Hepatic magnetic resonance elastography (MRE) allows for noninvasive assessment of liver fibrosis. The purpose of this study was to evaluate the usefulness of MRE in detecting and quantifying liver fibrosis in patients with rheumatoid arthritis (RA) who have received methotrexate (MTX).
The association between mean liver stiffness value as determined by MRE and variables of interest was determined. The decision for a liver biopsy in participants with an abnormal liver stiffness was made based on clinical judgment.
Sixty-five RA patients were enrolled. Mean liver stiffness value by MRE was abnormal in 7 patients, suggestive of hepatic injury. As a result of findings from the MRE, biopsies were performed in 5 patients and all correlated with elevated liver stiffness values. Elevated mean liver stiffness values were associated with body mass index (BMI) (OR= 1.18 per 1 kg/m2; 95% CI: 1.03, 1.36; p=0.017). Neither the total MTX dose nor the duration of MTX treatment was associated with mean liver stiffness value (p=0.51 and P=0.20, respectively).
MRE provides a reliable, non-invasive assessment of liver fibrosis in patients with RA receiving MTX. Patients with RA receiving MTX who have an elevated BMI may be at increased risk for chronic hepatic injury, regardless of MTX cumulative dose or duration of treatment.
PMCID: PMC4562680  PMID: 26361561
Methotrexate; Hepatic Magnetic Resonance Elastography; liver fibrosis; rheumatoid arthritis
9.  Stable Automated Segmentation of Liver MR Elastography Images for Clinical Stiffness Measurement 
Magnetic Resonance Elastography (MRE) is an MRI-based technique that is used for the clinical diagnosis and staging of liver fibrosis by quantitatively measuring the stiffness of the liver. Due to the complexity of the signal characteristics and the presence of artifacts both in the acquired images and in the resulting stiffness images, the selection of the ROI for the stiffness measurement is currently performed manually, which may lead to significant inter- and intrareader variability. An algorithm has been developed to fully automate this analysis for liver MRE images.
Automated segmentation of liver MRE images is challenging due to signal inhomogeneity, low contrast, and variability in patient anatomy. An initial liver contour is found by fitting Gaussian peaks to the image histogram and selecting the peak that comprises intensities in the expected range and produces a mask near the expected location of the liver. After correction to reduce intensity inhomogeneity, an active contour based on intensity, with morphology used to implicitly enforce smoothness, is used to segment liver tissue while avoiding blood vessels. The resulting mask is used to initialize another segmentation which splits the region of the elastogram belonging to the liver into homogeneous liver tissue and areas with inclusions, partial volume effects, and artifacts.
In a set of 88 cases the algorithm had a -6.0 ± 14.2% stiffness difference from an experienced reader, which was superior to the 6.8 ± 22.8% difference between two readers. The segmentation was run on an additional 200 cases and the final ROIs were subjectively rated by a radiologist. The ROIs in 98% of cases received an average rating of “good” or “acceptable.”
PMCID: PMC4560351  PMID: 26346196
MR elastography; liver; automation; segmentation; hepatic fibrosis
10.  Cross-vendor validation of liver magnetic resonance elastography 
Abdominal imaging  2015;40(4):789-794.
To evaluate and validate the reproducibility of MR Elastography (MRE) derived liver stiffness values on two different MR vendor platforms performed on the same subject on the same day.
This investigation was approved by the hospital IRB. MRE exams were performed twice in identical fashion in eight volunteers and in five clinical patients on two different 1.5T MR scanners – once on a Philips MR scanner and immediately afterward in back-to-back fashion on a General Electric MR scanner, or vice versa. All scan parameters were kept identical on the two platforms to the best extent possible. After the MRE magnitude and phase images were obtained, the data was converted into quantitative images displaying the stiffness of the liver parenchyma. Mean liver stiffness values between the two platforms were compared using interclass correlation with a p-value < 0.05 considered statistically significant.
Interclass correlation coefficient (ICC) value of 0.994 was obtained for 13 subjects with p-value <0.001 indicating a significantly positive correlation.
As MRE gains in acceptance and as its availability becomes more widespread, it is important to ascertain and confirm that liver stiffness values obtained on different MRE vendor platforms are consistent and reproducible. In this small pilot investigation, we demonstrate that liver stiffness measurement with MRE is reproducible and has very good consistency across two vendor platforms.
PMCID: PMC4523216  PMID: 25476489
MRE; Liver Elastography; MRE validation
11.  Plasmodium falciparum coronin organizes arrays of parallel actin filaments potentially guiding directional motility in invasive malaria parasites 
Malaria Journal  2015;14:280.
Gliding motility in Plasmodium parasites, the aetiological agents of malaria disease, is mediated by an actomyosin motor anchored in the outer pellicle of the motile cell. Effective motility is dependent on a parasite myosin motor and turnover of dynamic parasite actin filaments. To date, however, the basis for directional motility is not known. Whilst myosin is very likely orientated as a result of its anchorage within the parasite, how actin filaments are orientated to facilitate directional force generation remains unexplained. In addition, recent evidence has questioned the linkage between actin filaments and secreted surface antigens leaving the way by which motor force is transmitted to the extracellular milieu unknown. Malaria parasites possess a markedly reduced repertoire of actin regulators, among which few are predicted to interact with filamentous (F)-actin directly. One of these, PF3D7_1251200, shows strong homology to the coronin family of actin-filament binding proteins, herein referred to as PfCoronin.
Here the N terminal beta propeller domain of PfCoronin (PfCor-N) was expressed to assess its ability to bind and bundle pre-formed actin filaments by sedimentation assay, total internal reflection fluorescence (TIRF) microscopy and confocal imaging as well as to explore its ability to bind phospholipids. In parallel a tagged PfCoronin line in Plasmodium falciparum was generated to determine the cellular localization of the protein during asexual parasite development and blood-stage merozoite invasion.
A combination of biochemical approaches demonstrated that the N-terminal beta-propeller domain of PfCoronin is capable of binding F-actin and facilitating formation of parallel filament bundles. In parasites, PfCoronin is expressed late in the asexual lifecycle and localizes to the pellicle region of invasive merozoites before and during erythrocyte entry. PfCoronin also associates strongly with membranes within the cell, likely mediated by interactions with phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) at the plasma membrane.
These data suggest PfCoronin may fulfil a key role as the critical determinant of actin filament organization in the Plasmodium cell. This raises the possibility that macro-molecular organization of actin mediates directional motility in gliding parasites.
Electronic supplementary material
The online version of this article (doi:10.1186/s12936-015-0801-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4506582  PMID: 26187846
Gliding motility; Coronin; Actin; Plasmodium; Tight junction; Merozoite
12.  Ezetimibe for the Treatment of Nonalcoholic Steatohepatitis: Assessment by Novel Magnetic Resonance Imaging and Magnetic Resonance Elastography in a Randomized Trial (MOZART Trial) 
Hepatology (Baltimore, Md.)  2015;61(4):1239-1250.
Ezetimibe inhibits intestinal cholesterol absorption and lowers low-density lipoprotein cholesterol. Uncontrolled studies have suggested that it reduces liver fat as estimated by ultrasound in nonalcoholic steatohepatitis (NASH). Therefore, we aimed to examine the efficacy of ezetimibe versus placebo in reducing liver fat by the magnetic resonance imaging-derived proton density-fat fraction (MRI-PDFF) and liver histology in patients with biopsy-proven NASH. In this randomized, double-blind, placebo-controlled trial, 50 patients with biopsy-proven NASH were randomized to either ezetimibe 10 mg orally daily or placebo for 24 weeks. The primary outcome was a change in liver fat as measured by MRI-PDFF in colocalized regions of interest within each of the nine liver segments. Novel assessment by two-dimensional and three-dimensional magnetic resonance elastography was also performed. Ezetimibe was not significantly better than placebo at reducing liver fat as measured by MRI-PDFF (mean difference between the ezetimibe and placebo arms -1.3%, P = 0.4). Compared to baseline, however, end-of-treatment MRI-PDFF was significantly lower in the ezetimibe arm (15%-11.6%, P < 0.016) but not in the placebo arm (18.5%-16.4%, P = 0.15). There were no significant differences in histologic response rates, serum alanine aminotransferase and aspartate aminotransferase levels, or longitudinal changes in two-dimensional and three-dimensional magnetic resonance elastography-derived liver stiffness between the ezetimibe and placebo arms. Compared to histologic nonresponders (25/35), histologic responders (10/35) had a significantly greater reduction in MRI-PDFF (-4.35 ± 4.9% versus -0.30 ± 4.1%, P < 0.019). Conclusions: Ezetimibe did not significantly reduce liver fat in NASH. This trial demonstrates the application of colocalization of MRI-PDFF-derived fat maps and magnetic resonance elastography-derived stiffness maps of the liver before and after treatment to noninvasively assess treatment response in NASH. (Hepatology 2015;61:1239–1250)
PMCID: PMC4407930  PMID: 25482832
13.  Electrospun gelatin/polycaprolactone nanofibrous membranes combined with a coculture of bone marrow stromal cells and chondrocytes for cartilage engineering 
Electrospinning has recently received considerable attention, showing notable potential as a novel method of scaffold fabrication for cartilage engineering. The aim of this study was to use a coculture strategy of chondrocytes combined with electrospun gelatin/polycaprolactone (GT/PCL) membranes, instead of pure chondrocytes, to evaluate the formation of cartilaginous tissue. We prepared the GT/PCL membranes, seeded bone marrow stromal cell (BMSC)/chondrocyte cocultures (75% BMSCs and 25% chondrocytes) in a sandwich model in vitro, and then implanted the constructs subcutaneously into nude mice for 12 weeks. Gross observation, histological and immunohistological evaluation, glycosaminoglycan analyses, Young’s modulus measurement, and immunofluorescence staining were performed postimplantation. We found that the coculture group formed mature cartilage-like tissue, with no statistically significant difference from the chondrocyte group, and labeled BMSCs could differentiate into chondrocyte-like cells under the chondrogenic niche of chondrocytes. This entire strategy indicates that GT/PCL membranes are also a suitable scaffold for stem cell-based cartilage engineering and may provide a potentially clinically feasible approach for cartilage repairs.
PMCID: PMC4370944  PMID: 25834428
electrospinning; nanocomposite; cartilage tissue engineering; nanomaterials; stem cells
14.  Magnetic resonance elastography of the human abdominal aorta: a preliminary study 
Journal of magnetic resonance imaging : JMRI  2013;38(6):10.1002/jmri.24056.
To investigate the feasibility of using magnetic resonance elastography (MRE) for the evaluation of the stiffness of in vivo aortic wall.
Materials and Methods
To validate the experimental approach for imaging the aorta in vivo, a gel phantom with an embedded porcine aorta was imaged in the presence of fluid flow within the aorta. The potential changes in the elasticity of the vessel wall with changes in pressure were investigated. The feasibility of performing MRE of abdominal aorta was assessed in five volunteers (Age 22–40 years; BMI 21.5–25.2 kg/m2). The pulse-gated cine MRE technique was used to study the wave propagation along the aorta throughout the cardiac cycle and provide estimates of aortic stiffness in diastole.
In the phantom study, the wave propagation was well visualized within the porcine aorta embedded in the gel phantom. An increase of the Young's modulus-wall thickness (E*t) product with the increase in static pressure was observed. In the in vivo study, the waves were well visualized within the lumen of abdominal aorta in the five volunteers in diastolic phase, but they were not well visualized during systole.
MRE is feasible for noninvasively assessing the stiffness of the abdominal aorta and merits further investigation.
PMCID: PMC3652910  PMID: 23371244
MRI; elastography; aorta; stiffness
15.  Hepatic and Splenic Stiffness Augmentation Assessed with MR Elastography in an in vivo Porcine Portal Hypertension Model 
To investigate the influence of portal pressure on the shear stiffness of the liver and spleen in a well-controlled in vivo porcine model with MR Elastography (MRE). A significant correlation between portal pressure and tissue stiffness could be used to noninvasively assess increased portal venous pressure (portal hypertension), which is a frequent clinical condition caused by cirrhosis of the liver and is responsible for the development of many lethal complications.
Materials and Methods
During multiple intra-arterial infusions of Dextran-40 in three adult domestic pigs in vivo, 3-D abdominal MRE was performed with left ventricle and portal catheters measuring blood pressure simultaneously. Least-squares linear regressions were used to analyze the relationship between tissue stiffness and portal pressure.
Liver and spleen stiffness have a dynamic component that increases significantly following an increase in portal or left ventricular pressure. Correlation coefficients with the linear regressions between stiffness and pressure exceeded 0.8 in most cases.
The observed stiffness-pressure relationship of the liver and spleen could provide a promising noninvasive method for assessing portal pressure. Using MRE to study the tissue mechanics associated with portal pressure may provide new insights into the natural history and pathophysiology of hepatic diseases and may have significant diagnostic value in the future.
PMCID: PMC3661694  PMID: 23418135
MR Elastography; liver; spleen; portal pressure; shear stiffness
16.  A novel partner of Scalloped regulates Hippo signaling via antagonizing Scalloped-Yorkie activity 
Cell Research  2013;23(10):1201-1214.
The Hippo (Hpo) pathway controls tissue growth and organ size by regulating the activity of transcriptional co-activator Yorkie (Yki), which associates with transcription factor Scalloped (Sd) in the nucleus to promote downstream target gene expression. Here we identify a novel protein Sd-Binding-Protein (SdBP)/Tgi, which directly competes with Yki for binding to Sd through its TDU domains and inhibits the Sd-Yki transcriptional activity. We also find that SdBP retains Yki in the nucleus through the association with Yki WW domains via its PPXY motifs. Collectively, we identify SdBP as a novel component of the Hpo pathway, negatively regulating the transcriptional activity of Sd-Yki to restrict tissue growth.
PMCID: PMC3790236  PMID: 23999857
Hippo; Sd
17.  Isolation and characterization of a Sca-1+/CD31- progenitor cell lineage derived from mouse heart tissue 
BMC Biotechnology  2014;14:75.
Myocardial infarction remains the leading cause of mortality in developed countries despite recent advances in its prevention and treatment. Regenerative therapies based on resident cardiac progenitor cells (CPCs) are a promising alternative to conventional treatments. However, CPCs resident in the heart are quite rare. It is unclear how these CPCs can be isolated and cultured efficiently and what the effects of long-term culture in vitro are on their ‘stemness’ and differentiation potential, but this is critical knowledge for CPCs’ clinical application.
Here, we isolated stem cell antigen-1 positive cells from postnatal mouse heart by magnetic active cell sorting using an iron-labeled anti-mouse Sca-1 antibody, and cultured them long-term in vitro. We tested stemness marker expression and the proliferation ability of long-term cultured Sca-1+ cells at early, middle and late passages. Furthermore, we determined the differentiation potential of these three passages into cardiac cell lineages (cardiomyocytes, smooth muscle and endothelial cells) after induction in vitro. The expression of myocardial, smooth muscle and endothelial cell-specific genes and surface markers were analyzed by RT-PCR and IF staining. We also investigated the oncogenicity of the three passages by subcutaneously injecting cells in nude mice. Overall, heart-derived Sca-1+ cells showed CPC characteristics: long-term propagation ability in vitro, non-tumorigenic in vivo, persistent expression of stemness and cardiac-specific markers, and multipotent differentiation into cardiac cell lineages.
Our research may bring new insights to myocardium regeneration, for which even a small number of biopsy-derived CPCs could be enriched and propagated long term in vitro to obtain sufficient seed cells for cell injection or cardiac tissue engineering.
PMCID: PMC4133720  PMID: 25106452
Cardiac progenitor cell; Stem cell antigen-1; Differentiation; Multipotent; Self-renewal
18.  Automated Liver Stiffness Measurements with Magnetic Resonance Elastography 
To provide a fully-automated algorithm for obtaining stiffness measurements from hepatic MR Elastography images that are consistent with measurements performed by expert readers.
Materials and Methods
An initial liver contour was found using an adaptive threshold and expanded using an active contour to select a homogeneous area of the liver. The confidence map generated during the stiffness calculation was used to select a region of reliable wave propagation. The average stiffness within the automatically-generated ROI was compared to measurements by two trained readers in a set of 88 clinical test cases ranging from healthy to severely fibrotic.
The stiffness measurements reported by the readers differed by −6.76% ± 22.8 % (95% confidence) and had an ICC of 0.972 (p<0.05).The algorithm and the more experienced reader differed by 4.32% ± 14.9 with an ICC of 0.987.
The automated algorithm performed reliably, even though MRE acquisitions often have motion artifacts present. The correlation between the automated measurements and those from the trained readers was superior to the correlation between the readers.
PMCID: PMC3620931  PMID: 23281171
MR Elastography; Liver; Automation; Segmentation; Hepatic Fibrosis
19.  Magnetic Resonance Elastography of Liver: Clinical Applications 
Magnetic resonance elastography (MRE) has been successfully implemented in the assessment of diffuse liver diseases. Currently, MRE is the most accurate noninvasive technique for detection and staging of liver fibrosis with a potential to replace liver biopsy. Magnetic resonance elastography is able to differentiate isolated fatty liver disease from steatohepatitis with or without fibrosis. Potential clinical applications include the differentiation of benign and malignant focal liver masses and the assessment of treatment response in diffuse liver diseases.
PMCID: PMC4075049  PMID: 24270110
magnetic resonance elastography; clinical applications; diffuse liver diseases; liver fibrosis; focal lesions; treatment response
20.  Electrospun gelatin/PCL and collagen/PLCL scaffolds for vascular tissue engineering 
Electrospun hybrid nanofibers prepared using combinations of natural and synthetic polymers have been widely investigated in cardiovascular tissue engineering. In this study, electrospun gelatin/polycaprolactone (PCL) and collagen/poly(l-lactic acid-co-ε-caprolactone) (PLCL) scaffolds were successfully produced. Scanning electron micrographs showed that fibers of both membranes were smooth and homogeneous. Water contact angle measurements further demonstrated that both scaffolds were hydrophilic. To determine cell attachment and migration on the scaffolds, both hybrid scaffolds were seeded with human umbilical arterial smooth muscle cells. Scanning electron micrographs and MTT assays showed that the cells grew and proliferated well on both hybrid scaffolds. Gross observation of the transplanted scaffolds revealed that the engineered collagen/PLCL scaffolds were smoother and brighter than the gelatin/PCL scaffolds. Hematoxylin and eosin staining showed that the engineered blood vessels constructed by collagen/PLCL electrospun membranes formed relatively homogenous vessel-like tissues. Interestingly, Young’s modulus for the engineered collagen/PLCL scaffolds was greater than for the gelatin/PCL scaffolds. Together, these results indicate that nanofibrous collagen/PLCL membranes with favorable mechanical and biological properties may be a desirable scaffold for vascular tissue engineering.
PMCID: PMC4026554  PMID: 24872696
electrospinning; gelatin; collagen; polycaprolactone; poly(l-lactic acid-co-ε-caprolactone)
21.  Magnetic Resonance Elastography of Liver: Technique, Analysis and Clinical Applications 
Many pathological processes cause marked changes in the mechanical properties of tissue. Magnetic Resonance Elastography (MRE) is a non-invasive MRI based technique for quantitatively assessing the mechanical properties of tissues in vivo. MRE is performed by using a vibration source to generate low frequency mechanical waves in tissue, imaging the propagating waves using a phase contrast MRI technique, and then processing the wave information to generate quantitative images showing mechanical properties such as tissue stiffness. Since its first description in 1995, published studies have explored many potential clinical applications including brain, thyroid, lung, heart, breast, and skeletal muscle imaging. However, the best-documented application to emerge has been the use of MRE to assess liver disease. Multiple studies have demonstrated that there is a strong correlation between MRE-measured hepatic stiffness and the stage of fibrosis at histology. The emerging literature indicates that MRE can serve as a safer, less expensive, and potentially more accurate alternative to invasive liver biopsy which is currently the gold standard for diagnosis and staging of liver fibrosis. This review describes the basic principles, technique of performing a liver MRE, analysis and calculation of stiffness, clinical applications, limitations, and potential future applications.
PMCID: PMC3579218  PMID: 23423795
Magnetic Resonance Elastography (MRE); Liver; Fibrosis; Technique; Analysis; Clinical applications
22.  Brahma is essential for Drosophila intestinal stem cell proliferation and regulated by Hippo signaling 
eLife  2013;2:e00999.
Chromatin remodeling processes are among the most important regulatory mechanisms in controlling cell proliferation and regeneration. Drosophila intestinal stem cells (ISCs) exhibit self-renewal potentials, maintain tissue homeostasis, and serve as an excellent model for studying cell growth and regeneration. In this study, we show that Brahma (Brm) chromatin-remodeling complex is required for ISC proliferation and damage-induced midgut regeneration in a lineage-specific manner. ISCs and enteroblasts exhibit high levels of Brm proteins; and without Brm, ISC proliferation and differentiation are impaired. Importantly, the Brm complex participates in ISC proliferation induced by the Scalloped–Yorkie transcriptional complex and that the Hippo (Hpo) signaling pathway directly restricted ISC proliferation by regulating Brm protein levels by inducing caspase-dependent cleavage of Brm. The cleavage resistant form of Brm protein promoted ISC proliferation. Our findings highlighted the importance of Hpo signaling in regulating epigenetic components such as Brm to control downstream transcription and hence ISC proliferation.
eLife digest
Most tissues can generate new cells to repair damage or replace worn-out cells. The new cells are often generated from stem cells—cells that can either reproduce themselves or mature into other types of cells. In the fruit-fly Drosophila, for example, intestinal stem cells in the midgut are capable of producing more stem cells or they can differentiate to produce immature cells called enteroblasts that go on to become either enterocytes (the cells that line the gut) or enteroendocrine cells (which secrete hormones).
Researchers have identified a number of signalling pathways that are involved in the proliferation and differentiation of intestinal stem cells in the midgut of fruit flies. These include the Hippo pathway, which is important for regulating both cell proliferation and programmed cell death (apoptosis). Activation of the Hippo protein triggers a cascade of signals that culminate in the regulation of many of the genes involved in cell proliferation, division and apoptosis.
Another process that is important for controlling the proliferation and differentiation of cells is chromatin remodelling. Chromatin is the ‘packaging’ that keeps DNA tightly wound within the cell nucleus, and remodelling refers to the structural changes that allow proteins called transcription factors to reach the genes and transcribe them into messenger RNA (which then leaves the nucleus to generate the protein).
Now, Jin et al. have explored how the Hippo pathway and chromatin remodelling work together to regulate of stem cells. Using a technique called RNA interference to block the expression of various genes in intestinal stem cells and enteroblasts, Jin et al. found that a protein called Brahma—which is an essential part of a chromatin-remodelling complex—must be present for the stem cells to multiply normally.
Jin et al. also showed how the Hippo signalling pathway interacts with chromatin remodelling. Activation of the Hippo pathway inhibits gene expression by preventing two other proteins, Yorkie and Scalloped, from forming a complex in the nucleus. The new work shows that Brahma interacts physically with the Yorkie and Scalloped proteins to regulate the proliferation of the intestinal stem cells. It also shows that the Hippo protein regulates the activity of the Brahma protein by inducing a process called caspase-dependent cleavage. Because many of the proteins involved in these pathways are evolutionarily conserved and expressed in a variety of tissues, these findings may have implications for stem cell function and tissue repair in many species.
PMCID: PMC3796317  PMID: 24137538
Hippo signaling; brahma; midgut; D. melanogaster
23.  Neuropilin-1 stimulates tumor growth by increasing fibronectin fibril assembly in the tumor microenvironment 
Cancer research  2012;72(16):4047-4059.
The tumor microenvironment, including stromal myofibroblasts and associated matrix proteins, regulates cancer cell invasion and proliferation. Here we report that neuropilin-1 (NRP-1) orchestrates communications between myofibroblasts and soluble fibronectin (FN) that promote α5β1 integrin-dependent FN fibril assembly, matrix stiffness, and tumor growth. Tumor growth and FN fibril assembly was reduced by genetic depletion or antibody neutralization of NRP-1 from stromal myofibroblasts in vivo. Mechanistically, the increase in FN fibril assembly required glycosylation of serine 612 of the extracellular domain of NRP-1, an intact intracellular NRP-1 SEA domain, and intracellular associations between NRP-1, the scaffold protein GIPC, and the nonreceptor tyrosine kinase c-Abl, that augmented α5β1 FN fibril assembly activity. Analysis of human cancer specimens established an association between tumoral NRP-1 levels and clinical outcome. Our findings indicate that NRP-1 activates the tumor microenvironment, thereby promoting tumor growth. These results not only identify new molecular mechanisms of FN fibril assembly but also have important implications for therapeutic targeting of the myofibroblast in the tumor microenvironment.
PMCID: PMC3421041  PMID: 22738912
Fibronectin; Integrin; Neuropilin; Matrix; Myofibroblast
24.  Par-1 Regulates Tissue Growth by Influencing Hippo Phosphorylation Status and Hippo-Salvador Association 
PLoS Biology  2013;11(8):e1001620.
Par-1 regulates the Hippo signaling pathway in Drosophila melanogaster by modifying the phosphorylation status of Hippo and also by inhibiting the interaction of Hippo and Salvador.
The evolutionarily conserved Hippo (Hpo) signaling pathway plays a pivotal role in organ size control by balancing cell proliferation and cell death. Here, we reported the identification of Par-1 as a regulator of the Hpo signaling pathway using a gain-of-function EP screen in Drosophila melanogaster. Overexpression of Par-1 elevated Yorkie activity, resulting in increased Hpo target gene expression and tissue overgrowth, while loss of Par-1 diminished Hpo target gene expression and reduced organ size. We demonstrated that par-1 functioned downstream of fat and expanded and upstream of hpo and salvador (sav). In addition, we also found that Par-1 physically interacted with Hpo and Sav and regulated the phosphorylation of Hpo at Ser30 to restrict its activity. Par-1 also inhibited the association of Hpo and Sav, resulting in Sav dephosphorylation and destabilization. Furthermore, we provided evidence that Par-1-induced Hpo regulation is conserved in mammalian cells. Taken together, our findings identified Par-1 as a novel component of the Hpo signaling network.
Author Summary
An organism's organ size is determined by cell number, the size of each cell, and the distance between cells. All of these factors are controlled by the coordination of different cell signaling pathways and other mechanisms. The Hippo signaling pathway controls organ size by restricting the number of cells that make up the organ. Malfunction of this pathway leads to abnormal overgrowth, and is involved in a large number of human diseases and cancers. We identify here a component of the Hippo pathway, Par-1, which controls tissue growth by negatively regulating the Hippo pathway. We show that overexpression or depletion of Par-1 influences tissue growth in fruit flies via Hippo signaling. Then, by genetic and biochemical experiments, we show that Par-1 interacts with Hippo, regulating the Hippo Ser30 phosphorylation status to alter Hippo activity. In addition, we found that Par-1 regulates Hippo signaling via inhibition of the Hippo-Salvador association in a kinase-dependent fashion. We predict that Par-1 is a potential oncogene and that its regulatory role in Hippo signaling could be conserved.
PMCID: PMC3735459  PMID: 23940457

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