Growth Factor Binding Research Articles

Enzyme responsive GAG-based natural-synthetic hybrid hydrogel for tunable growth factor delivery and stem cell differentiation

We describe an enzymatically formed chondroitin sulfate (CS) and poly(ethylene glycol) (PEG) based hybrid hydrogel system, which by tuning the architecture and composition of modular building blocks, allows the application-specific tailoring of growth factor delivery and cellular responses. CS, a negatively charged sulfate-rich glycosaminoglycan of the extracellular matrix (ECM), known for its growth factor binding and stem cell regulatory functions, is used as a starting material for the engineering of this biomimetic materials platform. The functionalization of CS with transglutaminase factor XIII specific substrate sequences is utilized to allow cross-linking of CS with previously described fibrin-mimetic TG-PEG hydrogel precursors. We show that the hydrogel network properties can be tuned by varying the degree of functionalization of CS as well as the ratio and concentrations of PEG and CS precursors. Taking advantage of TG-PEG hydrogel, compatible tagged bio-functional building blocks, including RGD peptides or matrix metalloproteinase sensitive domains, can be incorporated on demand allowing the three-dimensional culture and expansion of human bone marrow mesenchymal stem cells (BM-MSCs). The binding of bone morphogenetic protein-2 (BMP-2) in a CS concentration dependent manner and the BMP-2 release mediated osteogenic differentiation of BM-MSCs indicate the potential of CS-PEG hybrid hydrogels to promote regeneration of bone tissue. Their modular design allows facile incorporation of additional signaling elements, rendering CS-PEG hydrogels a highly flexible platform with potential for multiple biomedical applications.

Improved Pancreatic Adenocarcinoma Diagnosis in Jaundiced and Non-Jaundiced Pancreatic Adenocarcinoma Patients through the Combination of Routine Clinical Markers Associated to Pancreatic Adenocarcinoma Pathophysiology.

BackgroundThere is still no reliable biomarker for the diagnosis of pancreatic adenocarcinoma. Carbohydrate antigen 19–9 (CA 19–9) is a tumor marker only recommended for pancreatic adenocarcinoma follow-up. One of the clinical problems lies in distinguishing between this cancer and other benign pancreatic diseases such as chronic pancreatitis. In this study we will assess the value of panels of serum molecules related to pancreatic cancer physiopathology to determine whether alone or in combination could help to discriminate between these two pathologies.MethodsCA 19–9, carcinoembryonic antigen (CEA), C-reactive protein, albumin, insulin growth factor-1 (IGF-1) and IGF binding protein-3 were measured using routine clinical analyzers in a cohort of 47 pancreatic adenocarcinoma, 20 chronic pancreatitis and 15 healthy controls.ResultsThe combination of CA 19–9, IGF-1 and albumin resulted in a combined area under the curve (AUC) of 0.959 with 93.6% sensitivity and 95% specificity, much higher than CA 19–9 alone. An algorithm was defined to classify the patients as chronic pancreatitis or pancreatic cancer with the above specificity and sensitivity. In an independent validation group of 20 pancreatic adenocarcinoma and 13 chronic pancreatitis patients, the combination of the four molecules classified correctly all pancreatic adenocarcinoma and 12 out of 13 chronic pancreatitis patients.ConclusionsAlthough this panel of markers should be validated in larger cohorts, the high sensitivity and specificity values and the convenience to measure these parameters in clinical laboratories shows great promise for improving pancreatic adenocarcinoma diagnosis.

Microdomain forming proteins in oncogenesis

Lipid rafts are lateral assembles of cholesterol, sphingomyelin, glicosphingolipids and specific proteins within cell plasma membrane. These microdomains are involved into a number of important cellular processes including membrane rearrangement, protein internalization, signal transduction, entry of viruses into the cell. Some of lipid rafts are stabilized by special microdomain-forming proteins such as caveolins, SPFH domain containing superfamily, tetraspanins, galectins, which maintain integrity of rafts and regulate signal transduction via forming of “signalosomes”. Involvement of the different lipid rafts is necessary in many situations such as binding of growth factors with their receptors, integrin regulation, cytoskeleton and extracellular matrix rearrangements, vesicular transport, etc. However, such classes of microdomain-forming proteins are still considered separately from each other. In this review we tried to perform complex analysis of microdomain-forming proteins in regulation of cancer assotiated processes.

Design and synthesis of new binding growth factors

Design and synthesis of new binding growth factors

Heparin microparticles loaded with bone morphogenetic protein-2 (BMP-2) induce bone regeneration in a critically sized rat femoral defect model

Event Abstract Back to Event Heparin microparticles loaded with bone morphogenetic protein-2 (BMP-2) induce bone regeneration in a critically sized rat femoral defect model Marian Hettiaratchi1*, Catherine Chou1, Laxminarayanan Krishnan2, Mon-Tzu A. Li1, Johnna S. Temenoff1, 2, Todd C. Mcdevitt3, 4 and Robert E. Guldberg2, 5 1 Georgia Institute of Technology & Emory University, Wallace H. Coulter Department of Biomedical Engineering, United States 2 Georgia Institute of Technology, Parker H. Petit Institute for Bioengineering and Bioscience, United States 3 Gladstone Institute of Cardiovascular Disease, United States 4 University of California - San Francisco, Department of Bioengineering & Therapeutic Sciences, United States 5 Georgia Institute of Technology, George W. Woodruff School of Mechanical Engineering, United States Introduction: Bone morphogenetic protein-2 (BMP-2), an osteoinductive growth factor, is delivered clinically to stimulate endogenous healing after severe bone loss. However, existing biomaterials exhibit limited affinity for BMP-2, resulting in rapid diffusion of growth factor away from the injury site. Heparin, a negatively charged glycosaminoglycan, binds growth factors electrostatically and has high affinity for BMP-2. We have previously fabricated heparin microparticles (HMPs) that can retain large amounts of bioactive BMP-2 (>300 μg/mg HMPs)[1]. Thus, here we hypothesize that HMPs can improve BMP-2 delivery in vivo and enhance healing in a critically sized bone defect. Methods: HMPs were fabricated from heparin methacrylamide as previously described [1]. HMPs were loaded with BMP-2 overnight at 4°C, and bioactivity was evaluated via C2C12 myoblast growth and alkaline phosphatase (ALP) activity. For in vivo delivery, 2% RGD-alginate gels within polycaprolactone mesh tubes were cast with or without HMPs and/or BMP-2. BMP-2 release from constructs was evaluated in vitro at 37°C. Constructs with 1 mg of fluorescent HMPs were implanted subcutaneously in rats for 6 weeks to evaluate HMP retention in vivo. Constructs with 2.5 μg of BMP-2 loaded onto 1 mg of HMPs or mixed directly into the alginate were implanted in rat femoral defects to evaluate bone healing (Fig 2A). X-ray and micro-CT were performed at 4, 8, and 12 weeks post-surgery, histology was completed on 2, 4, and 12-week femurs, and biomechanical testing was conducted at 12 weeks. Results: C2C12 cells (20,000 cells/well) exposed to 50 ng of total BMP-2 demonstrated increased ALP activity and cell proliferation when ≥10 ng of BMP-2 were loaded onto the HMPs (Fig 1A-B). Alginate constructs with HMP-bound BMP-2 exhibited lower BMP-2 release over 21 days in vitro compared to alginate-bound BMP-2 (Fig 1C). Fluorescent imaging of subcutaneously implanted constructs revealed persistence of ~55% of the HMPs after 6 weeks in vivo (Fig 1D-E). Bone volume within femoral defects increased over 12 weeks in response to both HMP-bound and alginate-bound BMP-2 (Fig 2B). Bony bridging occurred in all alginate-bound BMP-2 samples and 60% of HMP-bound BMP-2 samples (Fig 2D). Functional restoration of maximum torque and stiffness was comparable (Fig 2C). New bone formation (Fig 2E-F, blue) was observed in close proximity to HMPs and alginate (red) at 4 and 12 weeks. Proliferating Ki-67+ cells (Fig 2G, brown) were localized with HMPs (red) at 2 weeks, suggesting HMP-mediated proliferation similar to that observed in vitro. Conclusions: BMP-2-loaded HMPs induce functional regeneration of large bone defects; however, optimization is required to achieve consistent bridging of newly formed bone. Since bioactivity assays have demonstrated the benefits of HMP-bound BMP-2, ongoing experiments are being performed to deliver a combination of alginate-bound and HMP-bound BMP-2 in vivo, in order to provide both rapid release and sustained presentation of growth factor. Overall, HMPs may provide a more effective method of spatially controlling the delivery of BMP-2 for in vivo tissue regeneration applications. Funding is provided by the National Institutes of Health (R01 AR062006) and Armed Forces Institute of Regenerative Medicine (AFIRM-II).; MHH is supported by a Natural Sciences and Engineering Research Council (NSERC) of Canada Postgraduate Scholarship and Philanthropic Education Organization (PEO) Scholar Award.; CC is supported by the Petit Undergraduate Research Scholars Program of the Georgia Institute of Technology.

Sulfatase 2 promotes breast cancer progression through regulating some tumor-related factors.

In previous studies Sulf2 has been evidenced to play an important role in tumor progression through editing sulfate moieties on heparan sulfate proteoglycans (HSPGs) and modulating heparin binding growth factors. However, the role of Sulf2 in breast cancer progression is still poorly understood. In the present study, we hypothesized that Sulf2 promoted breast cancer progression. Two different breast cancer cell lines, MCF-7 and MDA-MB-231, were chosen for this study because of high and low Sulf2 expression levels. We also altered their Sulf2 expression by establishing Sulf2 knockdown and overexpressing breast cancer cell lines MCF-7 shSulf2 and MDA-MB-231 Sulf2. To evaluate the functions of Sulf2, cell proliferation, apoptosis, cell cycle, invasion, mobility and adhesion of these cell lines were measured in vitro, and xenograft formation, invasion and metastasis ability were examined in vivo. Furthermore, expression of related genes were screened and were certified in these cell lines. We found that Sulf2 increased breast cancer proliferation, invasion, mobility and adhesion both in vitro and in vivo. Sulf2 also decreased cisplatin inducing breast cancer apoptosis without affecting the cell cycle. Sulf2 upregulated c-fos induced growth factor (FIGF) and nuclear receptor subfamily 4 group A member 3 (NR4A3) expression and downregulated the cluster of differentiation 82 (CD82) and platelet-derived growth factor C (PDGFC) expression in breast cancer. Our data confirmed that Sulf2 promoted breast cancer progression and regulated the expression of tumor-related genes in breast cancer.

Bioinformatics approach to predict target genes for dysregulated microRNAs in hepatocellular carcinoma: study on a chemically-induced HCC mouse model.

BackgroundHepatocellular carcinoma (HCC) is an aggressive epithelial tumor which shows very poor prognosis and high rate of recurrence, representing an urgent problem for public healthcare. MicroRNAs (miRNAs/miRs) are a class of small, non-coding RNAs that attract great attention because of their role in regulation of processes such as cellular growth, proliferation, apoptosis. Because of the thousands of potential interactions between a single miR and target mRNAs, bioinformatics prediction tools are very useful to facilitate the task for individuating and selecting putative target genes. In this study, we present a chemically-induced HCC mouse model to identify differential expression of miRNAs during the progression of the hepatic injury up to HCC onset. In addition, we describe an established bioinformatics approach to highlight putative target genes and protein interaction networks where they are involved.ResultsWe describe four miRs (miR-125a-5p, miR-27a, miR-182, miR-193b) which showed to be differentially expressed in the chemically-induced HCC mouse model. The miRs were subjected to four of the most used predictions tools and 15 predicted target genes were identified. The expression of one (ANK3) among the 15 predicted targets was further validated by immunoblotting. Then, enrichment annotation analysis was performed revealing significant clusters, including some playing a role in ion transporter activity, regulation of receptor protein serine/threonine kinase signaling pathway, protein import into nucleus, regulation of intracellular protein transport, regulation of cell adhesion, growth factor binding, and regulation of TGF-beta/SMAD signaling pathway. A network construction was created and links between the selected miRs, the predicted targets as well as the possible interactions among them and other proteins were built up.ConclusionsIn this study, we combined miRNA expression analysis, obtained by an in vivo HCC mouse model, with a bioinformatics-based workflow. New genes, pathways and protein interactions, putatively involved in HCC initiation and progression, were identified and explored.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-015-0836-1) contains supplementary material, which is available to authorized users.

Identification of Genes Related to Growth and Lipid Deposition from Transcriptome Profiles of Pig Muscle Tissue.

Transcriptome profiles established using high-throughput sequencing can be effectively used for screening genome-wide differentially expressed genes (DEGs). RNA sequences (from RNA-seq) and microRNA sequences (from miRNA-seq) from the tissues of longissimus dorsi muscle of two indigenous Chinese pig breeds (Diannan Small-ear pig [DSP] and Tibetan pig [TP]) and two introduced pig breeds (Landrace [LL] and Yorkshire [YY]) were examined using HiSeq 2000 to identify and compare the differential expression of functional genes related to muscle growth and lipid deposition. We obtained 27.18 G clean data through the RNA-seq and detected that 18,208 genes were positively expressed and 14,633 of them were co-expressed in the muscle tissues of the four samples. In all, 315 DEGs were found between the Chinese pig group and the introduced pig group, 240 of which were enriched with functional annotations from the David database and significantly enriched in 27 Gene Ontology (GO) terms that were mainly associated with muscle fiber contraction, cadmium ion binding, response to organic substance and contractile fiber part. Based on functional annotation, we identified 85 DEGs related to growth traits that were mainly involved in muscle tissue development, muscle system process, regulation of cell development, and growth factor binding, and 27 DEGs related to lipid deposition that were mainly involved in lipid metabolic process and fatty acid biosynthetic process. With miRNA-seq, we obtained 23.78 M reads and 320 positively expressed miRNAs from muscle tissues, including 271 known pig miRNAs and 49 novel miRNAs. In those 271 known miRNAs, 20 were higher and 10 lower expressed in DSP-TP than in LL-YY. The target genes of the 30 miRNAs were mainly participated in MAPK, GnRH, insulin and Calcium signaling pathway and others involved cell development, growth and proliferation, etc. Combining the DEGs and the differentially expressed (DE) miRNAs, we drafted a network of 46 genes and 18 miRNAs for regulating muscle growth and a network of 15 genes and 16 miRNAs for regulating lipid deposition. We identified that CAV2, MYOZ2, FRZB, miR-29b, miR-122, miR-145-5p and miR-let-7c, etc, were key genes or miRNAs regulating muscle growth, and FASN, SCD, ADORA1, miR-4332, miR-182, miR-92b-3p, miR-let-7a and miR-let-7e, etc, were key genes or miRNAs regulating lipid deposition. The quantitative expressions of eight DEGs and seven DE miRNAs measured with real-time PCR certified that the results of differential expression genes or miRNAs were reliable. Thus, 18,208 genes and 320 miRNAs were positively expressed in porcine longissimus dorsi muscle. We obtained 85 genes and 18 miRNAs related to muscle growth and 27 genes and 16 miRNAs related to lipid deposition, which provided new insights into molecular mechanism of the economical traits in pig.

Controlled release of fibrin matrix-conjugated platelet derived growth factor improves ischemic tissue regeneration by functional angiogenesis

Sustained, local, low dose growth factor stimulus of target tissues/cells is believed to be of imminent importance in tissue regeneration and engineering. Recently, a technology was developed to bind growth factors to a fibrin matrix using the transglutaminase (TG) activity of factor XIIIa, thus allowing prolonged release through enzymatic cleavage. In this study we aimed to determine whether TG-PDGF.AB in fibrin could improve tissue regeneration in a standard ischemic flap model. In vitro determination of binding and release kinetics of TG-PDGF.AB allowed proof of concept of the developed binding technology. A single spray application of TG-PDGF.AB in fibrin matrix at a concentration of 10 and 100ng/ml significantly reduced ischemia-induced flap tissue necrosis in vivo on day 7 after ischemic impact compared to controls. TG-PDGF.AB at a concentration of 100ng/ml fibrin induced distinct angiogenesis as reflected by significantly improved tissue perfusion assessed by laser Doppler imaging as well as enhanced von Willebrand factor (vWF) protein expression determined by immunohistochemical means. In addition, significantly more mature microvessels were observed with 100ng/ml TG-PDGF.AB in fibrin compared to control and vehicle groups as evidenced by an improved smooth muscle actin (sma)/vWF protein ratio. In conclusion, PDGF.AB in a conjugated fibrin matrix effectively reduced ischemia-induced tissue necrosis, increased tissue perfusion and induced the growth of a mature and functional neovasculature. The sealing properties of the fibrin matrix in conjunction with the prolonged growth factor stimulus enabled by the TG-hook binding technology may present an innovative and suitable tool in tissue regeneration. Statement of significanceIn our experimental study we elucidated recombinant platelet derived growth factor (PDGF) as a potential candidate in inducing angiogenesis. To avoid preterm growth factor degradation in vivo PDGF.AB was covalently linked to a fibrin scaffold using a bi-domain functionalized peptide (FXIII substrate site and plasmin cleavage site). This allowed PDGF binding to fibrin during spray application to the donor site and subsequent prolonged release via endogenous plasmin. This resulted in a mature vascular network thus enhancing tissue perfusion and consequently improved clinical outcome. With our present work we could certainly provide researchers and clinicians with an innovative versatile and reproducible technology not only to induce functional vascularity but also to improve attempts in tissue engineering in general by e.g. using different growth factors. Hence, we believe that this approach studied in the present work may provide a valuable input in an effort to drive the aim forward bringing experimental work in tissue engineering to clinic by using a clinically well characterized and used fibrin scaffold in combination with a human recombinant growth factor (fibrin scaffold linked with the specific binding technology).

Osteogenic Differentiation Evaluation of an Engineered Extracellular Matrix Based Tissue Sheet for Potential Periosteum Replacement.

Due to the indispensable role of periosteum in bone defect healing and regeneration, a promising method to enhance osteogenesis of bone grafts by using an engineered biomimetic periosteum would be highly beneficial. The stromal microenvironment of periosteum is composed of various highly organized extracellular matrix (ECM) fibers, so an aligned natural ECM sheet, derived from the human dermal fibroblast cell sheet, may be advantageous when applied for artificial periosteum fabrication. Human mesenchymal stem cells (hMSCs) have been used to replace the osteoprogenitor cell population in native periosteum due to hMSCs' great osteogenic potential and fast in vitro expansion capacity. The objective of this work is to investigate if the natural ECM sheet and the substrate alignment can promote in vitro osteogenesis of hMSCs. The conventional cell culture substrates collagen I-coated polydimethylsiloxane (PDMS) and tissue culture plastic (TCP) were used as controls. It was found that the ECM sheet significantly increased alkaline phosphatase activity and calcium deposition. The enhanced osteogenic potential was further confirmed by increased bone-specific gene expression. The ECM sheet can bind significantly higher amounts of growth factors including ANG-1, TGF-β1, bFGF, and VEGF, as well as calcium phosphate nanoparticles, which contributed to high osteogenesis of the hMSCs on ECM sheet. However, the alignment of the substrates did not show significant influence on osteogenic activity and growth factor binding. These results demonstrated the great potential of hMSC-seeded ECM sheet as a biomimetic periosteum to improve critical sized bone regeneration.

A 24-Hour Study of the Hypothalamo-Pituitary Axes in Huntington's Disease.

BackgroundHuntington’s disease is an inherited neurodegenerative disorder characterised by motor, cognitive and psychiatric disturbances. Patients exhibit other symptoms including sleep and mood disturbances, muscle atrophy and weight loss which may be linked to hypothalamic pathology and dysfunction of hypothalamo-pituitary axes.MethodsWe studied neuroendocrine profiles of corticotropic, somatotropic and gonadotropic hypothalamo-pituitary axes hormones over a 24-hour period in controlled environment in 15 healthy controls, 14 premanifest and 13 stage II/III Huntington’s disease subjects. We also quantified fasting levels of vasopressin, oestradiol, testosterone, dehydroepiandrosterone sulphate, thyroid stimulating hormone, free triiodothyronine, free total thyroxine, prolactin, adrenaline and noradrenaline. Somatotropic axis hormones, growth hormone releasing hormone, insulin-like growth factor-1 and insulin-like factor binding protein-3 were quantified at 06:00 (fasting), 15:00 and 23:00. A battery of clinical tests, including neurological rating and function scales were performed.Results24-hour concentrations of adrenocorticotropic hormone, cortisol, luteinizing hormone and follicle-stimulating hormone did not differ significantly between the Huntington’s disease group and controls. Daytime growth hormone secretion was similar in control and Huntington’s disease subjects. Stage II/III Huntington’s disease subjects had lower concentration of post-sleep growth hormone pulse and higher insulin-like growth factor-1:growth hormone ratio which did not reach significance. In Huntington’s disease subjects, baseline levels of hypothalamo-pituitary axis hormones measured did not significantly differ from those of healthy controls.ConclusionsThe relatively small subject group means that the study may not detect subtle perturbations in hormone concentrations. A targeted study of the somatotropic axis in larger cohorts may be warranted. However, the lack of significant results despite many variables being tested does imply that the majority of them do not differ substantially between HD and controls.

HSulf-1 deficiency dictates a metabolic reprograming of glycolysis and TCA cycle in ovarian cancer.

Warburg effect has emerged as a potential hallmark of many cancers. However, the molecular mechanisms that led to this metabolic state of aerobic glycolysis, particularly in ovarian cancer (OVCA) have not been completely elucidated. HSulf-1 predominantly functions by limiting the bioavailability of heparan binding growth factors and hence their downstream signaling. Here we report that HSulf-1, a known putative tumor suppressor, is a negative regulator of glycolysis. Silencing of HSulf-1 expression in OV202 cell line increased glucose uptake and lactate production by upregulating glycolytic genes such as Glut1, HKII, LDHA, as well as metabolites. Conversely, HSulf-1 overexpression in TOV21G cells resulted in the down regulation of glycolytic enzymes and reduced glycolytic phenotype, supporting the role of HSulf-1 loss in enhanced aerobic glycolysis. HSulf-1 deficiency mediated glycolytic enhancement also resulted in increased inhibitory phosphorylation of pyruvate dehydrogenase (PDH) thus blocking the entry of glucose flux into TCA cycle. Consistent with this, metabolomic and isotope tracer analysis showed reduced glucose flux into TCA cycle. Moreover, HSulf-1 loss is associated with lower oxygen consumption rate (OCR) and impaired mitochondrial function. Mechanistically, lack of HSulf-1 promotes c-Myc induction through HB-EGF-mediated p-ERK activation. Pharmacological inhibition of c-Myc reduced HB-EGF induced glycolytic enzymes implicating a major role of c-Myc in loss of HSulf-1 mediated altered glycolytic pathway in OVCA. Similarly, PG545 treatment, an agent that binds to heparan binding growth factors and sequesters growth factors away from their ligand also blocked HB-EGF signaling and reduced glucose uptake in vivo in HSulf-1 deficient cells.

A fine structural modification of glycosaminoglycans is correlated with the progression of muscle regeneration after ischaemia: towards a matrix-based therapy?

Critical limb ischaemia often leads to amputation of the limb and potential mortality. Moreover, there are still significant problems with current therapeutic treatments, according to poor revascularisation of degenerated tissue probably due to modifications within the microenvironment. This study is focused on the changes of structure and bioactivity of glycosaminoglycans (GAGs), especially heparan sulphate (HS) and chondroitin sulphate (CS) in rat Extensor Digitorum Longus (EDL) muscle after ischaemia. Male Wistar rats were subjected to ischaemic-injury by ligation of the neurovascular trunk accompanying EDL-tendon. After 4, 8, 15, 21, 60 and 90 d, the rats were sacrificed and the muscles were collected and submitted to histological, biochemical and gene expression assays. We demonstrated that ischaemia induced modification of expression of enzymes involved in GAG biosynthesis which correlated with significant changes in HS and CS structural features such as size and sulphation pattern. These major structural changes are associated to modifications of GAG abilities to bind growth factors and to modulate cell activity. Moreover, a CS hallmark of injury is maintained as well after the regeneration process. Finally, we showed the relevance of the role of this glycanic matrix remodelling, since a GAG mimetic treatment accelerated muscle repair after ischaemia.

Abstract 2184: Gene expression analysis of human papillomavirus positive head and neck cancer primary tumor samples reveals two distinct subgroups

Abstract Introduction: Human Papillomavirus (HPV) infection is recognized as a major cause for an increasing percent of oropharyngeal squamous cell carcinomas (OPSCC). Patients with HPV-induced OPSCC have a better prognosis than those who are HPV-negative, which implies distinct molecular mechanisms. However, predicting prognosis with the HPV-positive population remains elusive. In this study, we aimed to characterize molecular aspects of HPV expression, HPV integration, and host gene expression to later correlate with survival and recurrence as those data become available for our study population. Materials and methods: Thirty-six primary head and neck SCC (HNSCC) tumor samples were collected between 2011-2013 at University of Michigan hospital, of which 18 were determined to be HPV+ (type16, 18 or 35). Paired-end RNA-Seq libraries were generated using Illumina HiSeq for all samples. The sequences were aligned to both the human and HPV genome. Gene expression levels were quantified for both human and HPV genes using TopHat2 and HTSeq, and HPV integration events were determined using Virus-seq. Results: Hierarchical clustering of the gene expression levels showed three distinct clusters among the samples. The HPV- samples were first separated from the HPV+ samples, and then there were two robust subgroups within the HPV+ samples. Subsequent analysis revealed one subgroup had significantly higher HPV expression levels than the other (p-value: 0.03; Wilcoxon test). The subgroups were also highly correlated with HPV-integration status (8 of the 16 tumors had at least one identified HPV integration event). Differentially expressed genes between HPV-integration positive and negative samples were found to be enriched for interesting pathways, including “keratinocyte differentiation”, “cell adhesion” and “growth factor binding”, suggesting possible differences in carcinogenesis We also examined the HPV integration sites and found the majority of them were transcription factors. These also include several cancer-related genes, such as “BIRC3”, “CD274” and “PBX1”. One gene, KLF12, contained an integration event in one of our tumors and in one of the 36 HPV+ tumors from The Cancer Genome Atlas (TCGA). Network analysis of the genes harboring an integration event in our population or the TCGA HNSCC population (38 total genes) revealed interactions around p63 and ETS. Conclusion: Our data is an important addition to TCGA HNSCC cohort data, because as opposed to TCGA, we did not enforce selection bias towards large tumor size. With our data, we identified two distinct HPV+ subgroups characterized by RNA-Seq expression levels and strongly correlated with HPV viral expression levels and integration status. A significant portion of the HPV+ tumors (50%) had integrated HPV genome, implying important oncogenic potentials for integration events. Citation Format: Yanxiao Zhang, Lada A. Koneva, Shama Virani, Alisha Virani, Katie M. Rentschler, Thomas E. Carey, Laura S. Rozek, Maureen A. Sartor, the University of Michigan Head and Neck SPORE. Gene expression analysis of human papillomavirus positive head and neck cancer primary tumor samples reveals two distinct subgroups. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2184. doi:10.1158/1538-7445.AM2015-2184

Methods for assessing pre-induction cervical ripening.

BackgroundInduction of labour is the artificial initiation of labour in a pregnant woman after the age of fetal viability but without any objective evidence of active phase labour and with intact fetal membranes. The need for induction of labour may arise due to a problem in the mother, her fetus or both, and the procedure may be carried out at or before term. Obstetricians have long known that for this to be successful, it is important that the uterine cervix (the neck of the womb) has favourable characteristics in terms of readiness to go into the labour state.ObjectivesTo compare Bishop score with any other method for assessing pre‐induction cervical ripening in women admitted for induction of labour.Search methodsWe searched the Cochrane Pregnancy and Childbirth Group's Trials Register (31 March 2015) and reference lists of retrieved studies to identify randomised controlled trials (RCTs).Selection criteriaAll RCTs comparing Bishop score with any other methods of pre‐induction cervical assessment in women admitted for induction of labour. Cluster‐RCTs were eligible for inclusion but none were identified. Quasi‐RCTs and studies using a cross‐over design were not eligible for inclusion. Studies published in abstract form were eligible for inclusion if they provided sufficient information.Comparisons could include the following. 1. Bishop score versus transvaginal ultrasound (TVUS). 2. Bishop score versus Insulin‐like growth factor binding protein‐1 (IGFBP‐1). 3. Bishop score versus vaginal fetal fibronectin (fFN). However, we only identified data for a comparison of Bishop score versus TVUS.Data collection and analysisTwo review authors independently assessed the trials for inclusion, extracted the data and assessed trial quality. Data were checked for accuracy.Main resultsWe included two trials that recruited a total of 234 women. The overall risk of bias was low for the two studies. Both studies compared Bishop score withTVUS.The two included studies did not show any clear difference between the Bishop score and TVUS groups for the following main outcomes: vaginal birth (RR 1.07, 95% CI 0.92 to 1.25, moderate quality evidence), caesarean delivery (RR 0.81, 95% CI 0.49 to 1.34, moderate quality evidence), neonatal admission into neonatal intensive care unit (RR 1.67, 95% CI 0.41 to 6.71, moderate quality evidence). Both studies only provided median data in relation to induction‐delivery interval and reported no clear difference between the Bishop and TVUS groups. Perinatal mortality was not reported in the included studies.For the review's secondary outcomes, the need for misoprostol for cervical ripening was more frequent in the TVUS group compared to the Bishop score group (RR 0.52, 95% CI 0.41 to 0.66, two studies, 234 women, moderate quality evidence). In contrast, there were no clear differences between the Bishop scope and TVUS groups in terms of meconium staining of the amniotic fluid, fetal heart rate abnormality in labour, and Apgar score less than seven. Only one trial reported median data on the induction‐delivery interval and induction to active phase interval, the trialist reported no difference between the Bishop group and the TVUS group for this outcome. Neither of the included studies reported on uterine rupture.Authors' conclusionsModerate quality evidence from two small RCTs involving 234 women that compared two different methods for assessing pre‐induction cervical ripening (Bishop score and TVUS) did not demonstrate superiority of one method over the other in terms of the main outcomes assessed in this review. We did not identify any data relating to perinatal mortality. Whilst use of TVUS was associated with an increased need for misoprostol for cervical ripening, both methods could be complementary.The choice of a particular method of assessing pre‐induction cervical ripening may differ depending on the environment and need where one is practicing since some methods (i.e. TVUS) may not be readily available and affordable in resource‐poor settings where the sequelae of labour and its management is prevalent.The evidence in this review is based on two studies that enrolled a small number of women and there is insufficient evidence to support the use of TVUS over the standard digital vaginal assessment in pre‐induction cervical ripening. Further adequately powered RCTs involving TVUS and the Bishop score and including other methods of pre‐induction cervical ripening assessment are warranted. Such studies need to address uterine rupture, perinatal mortality, optimal cut‐off value of the cervical length and Bishop score to classify women as having favourable or unfavourable cervices and cost should be included as an outcome.

Evaluation of insulin-like growth factor (IGF-1) and retinol binding protein (RBP-4) concentration levels in patients with newly diagnosed pancreatic adenocarcinoma

Evaluation of insulin-like growth factor (IGF-1) and retinol binding protein (RBP-4) concentration levels in patients with newly diagnosed pancreatic adenocarcinoma

Characterization of TCF21 Downstream Target Regions Identifies a Transcriptional Network Linking Multiple Independent Coronary Artery Disease Loci.

To functionally link coronary artery disease (CAD) causal genes identified by genome wide association studies (GWAS), and to investigate the cellular and molecular mechanisms of atherosclerosis, we have used chromatin immunoprecipitation sequencing (ChIP-Seq) with the CAD associated transcription factor TCF21 in human coronary artery smooth muscle cells (HCASMC). Analysis of identified TCF21 target genes for enrichment of molecular and cellular annotation terms identified processes relevant to CAD pathophysiology, including “growth factor binding,” “matrix interaction,” and “smooth muscle contraction.” We characterized the canonical binding sequence for TCF21 as CAGCTG, identified AP-1 binding sites in TCF21 peaks, and by conducting ChIP-Seq for JUN and JUND in HCASMC confirmed that there is significant overlap between TCF21 and AP-1 binding loci in this cell type. Expression quantitative trait variation mapped to target genes of TCF21 was significantly enriched among variants with low P-values in the GWAS analyses, suggesting a possible functional interaction between TCF21 binding and causal variants in other CAD disease loci. Separate enrichment analyses found over-representation of TCF21 target genes among CAD associated genes, and linkage disequilibrium between TCF21 peak variation and that found in GWAS loci, consistent with the hypothesis that TCF21 may affect disease risk through interaction with other disease associated loci. Interestingly, enrichment for TCF21 target genes was also found among other genome wide association phenotypes, including height and inflammatory bowel disease, suggesting a functional profile important for basic cellular processes in non-vascular tissues. Thus, data and analyses presented here suggest that study of GWAS transcription factors may be a highly useful approach to identifying disease gene interactions and thus pathways that may be relevant to complex disease etiology.

Important topics in the future of biomaterials and stem cells for bone tissue engineering: Comments from the participants of the International Symposium on Recent Trend of Biomaterials and Stem Cells for Bone Tissue Engineering at Changchun, China.

Fundamental and clinical experts from Japan, Korea and China, warmly gathered on the beautiful ice and snow city—Changchun, the capital of Jilin Province, China—during 23-26 January 2015, to present their research findings and participated in discussion relating to progress in biomaterials, stem cells and bone tissue engineering. The International Symposium on Recent Trend of Biomaterials and Stem Cells for Bone Tissue Engineering (BTE 2015) was hosted by the Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, and co-organized by the Department of Orthopaedics, China-Japan Union Hospital, Jilin University. It provided a new platform of academic and technological communication for fundamental researchers and orthopedic surgeons to express their diverse ideas and inspiring new cooperation. The field of tissue engineering has emerged as an important approach to the clinical therapy of damaged bone due to trauma, tumor resections or congenital anomalies [1–3]. Besides bone forming cells and growth factors, we have profoundly recognized that the development of bone tissue engineering is directly related to changes in materials technology [4, 5]. However, after experiencing the rapid development of bone tissue engineering over the past several decades, we still currently face a tremendous challenge to design a new platform that can integrate bioscaffolds and growth factors or stem cells into biomimetic bone substitutes with new technologies for reconstruction of large-size orthopedic defects [6, 7]. The participants focused on discussing the recent development of synthetic or natural bioscaffolds that can provide 3D architecture [8, 9], appropriate topography and suitable surface chemistry [7, 10, 11], as well as enough mechanical strength [12, 13], which can encourage desired cellular activities and guide bone tissue regeneration. The introduction of bioactive molecules into 3D porous scaffolds physically or chemically to mimic the in vivo microenvironment is a promising strategy for tissue engineering and stem cell research [14]. A series of engineered binding growth factors [14–16] or peptides [17] have been designed and employed to study the biomaterial-stem cell interactions and to direct stem cell behaviors. Genetic tissue engineering was developed by bone marrow-derived mesenchymal stem cells (MSCs) reinforced with gene-transfecting for local delivery of bone morphogenetic proteins (BMPs) [18, 19]. The post-materials technology, or named offline materials technology, is also proposed in the symposium for researchers and surgeons to focus on the research of combination of current material products and clinical available resources, which will broaden and accelerate the clinical application of current material products. We made a concise report on this symposium, and some comments of the key speakers in the symposium were presented. We hope that their comments can arouse further interests of readers in discussing future strategies of bone tissue engineering and regenerative medicine. Moreover, we believed that the frequent appointment of researchers and orthopedic surgeons may inspire more and more ideas in the development of future clinical products of tissue engineering for orthopedic application.

Safety, pharmacokinetics, pharmacodynamics, and antitumor activity of necuparanib combined with nab-paclitaxel and gemcitabine in patients with metastatic pancreatic cancer: Phase 1 results.

4114 Background: Preclinical studies have shown necuparanib (necu)’s ability to bind growth factors, adhesion molecules, and chemokines to inhibit tumor progression, invasion, metastasis, and angio...

Perlecan heparan sulfate deficiency impairs pulmonary vascular development and attenuates hypoxic pulmonary hypertension.

Excessive vascular cell proliferation is an important component of pulmonary hypertension (PH). Perlecan is the major heparan sulfate (HS) proteoglycan in the vascular extracellular matrix. It binds growth factors, including FGF2, and either restricts or promotes cell proliferation. In this study, we have explored the effects of perlecan HS deficiency on pulmonary vascular development and in hypoxia-induced PH. In normoxia, Hspg2(Δ3/Δ3) mice, deficient in perlecan HS, had reduced pericytes and muscularization of intra-acinar vessels. Pulmonary angiography revealed a peripheral perfusion defect. Despite these abnormalities, right ventricular systolic pressure (RVSP) and myocardial mass remained normal. After 4 weeks of hypoxia, increases in the proportion of muscularized vessels, RVSP, and right ventricular hypertrophy were significantly less in Hspg2(Δ3/Δ3) compared with wild type. The early phase of hypoxia induced a significantly lower increase in fibroblast growth factor receptor-1 (FGFR1) protein level and receptor phosphorylation, and reduced pulmonary artery smooth muscle cell (PASMC) proliferation in Hspg2(Δ3/Δ3). At 4 weeks, FGF2 mRNA and protein were also significantly reduced in Hspg2(Δ3/Δ3) lungs. Ligand and carbohydrate engagement assay showed that perlecan HS is required for HS-FGF2-FGFR1 ternary complex formation. In vitro, proliferation assays showed that PASMC proliferation is reduced by selective FGFR1 inhibition. PASMC adhesion to fibronectin was higher in Hspg2(Δ3/Δ3) compared with wild type. Perlecan HS chains are important for normal vascular arborization and recruitment of pericytes to pulmonary vessels. Perlecan HS deficiency also attenuates hypoxia-induced PH, where the underlying mechanisms involve impaired FGF2/FGFR1 interaction, inhibition of PASMC growth, and altered cell-matrix interactions.