Platelet-derived Growth Factor Binding Research Articles

Roles of DSCC1 and GINS1 in gastric cancer.

Gastric carcinoma is a common malignant tumor originating from gastric mucosal epithelium. However, role of DS-cell cycle-dependent protein 1 (DSCC1) and GINS1 in gastric carcinoma remains unclear. The gastric carcinoma datasets GSE79973 and GSE118916 were downloaded from gene expression omnibus. Multiple datasets were merged and batched. Differentially expressed genes (DEGs) were screened and weighted gene co-expression network analysis was performed. Functional enrichment analysis, gene set enrichment analysis and immune infiltration analysis were performed. Construction and analysis of protein-protein interaction Network. Survival analysis and comparative toxicogenomics database were performed. A heat map of gene expression was drawn. Target Scan screen miRNAs regulating DEGs. Two thousand forty-four DEGs were identified. According to gene ontology analysis, in biological process, they were mainly enriched in cell migration, transforming growth factor β receptor signaling pathway, angiogenesis, and steroid metabolism process. In cellular component, they were mainly enriched in extracellular vesicles, basement membrane, endoplasmic reticulum lumen, and extracellular space. In molecular function, they focused on extracellular matrix structural components, protein binding, platelet-derived growth factor binding, and catalytic activity. In Kyoto encyclopedia of genes and genomes, they were mainly enriched in protein digestion and absorption, metabolic pathways, fatty acid degradation, Glycerophospholipid metabolism, ether lipid metabolism. Gene set enrichment analysis showed that DEGs were mainly enriched in transforming growth factor β receptor signaling pathway, steroid metabolism process, basement membrane, endoplasmic reticulum lumen, structural components of extracellular matrix, platelet-derived growth factor binding, Glycerophospholipid metabolism, ether lipid metabolism. The results of immune infiltration analysis showed that expression of T cell CD4 memory resting was lower in the samples of gastric cancer. The core genes (TRIP13, CHEK1, DSCC1, GINS1) are protective factors, their expression shows a downward trend with increase of risk score. Comparative toxicogenomics database analysis showed that TRIP13, CHEK1, DSCC1, GINS1 were related to gastric tumors, gastric diseases, tumors, inflammation, and necrosis. DSCC1 and GINS1 are highly expressed in gastric cancer. Higher expression levels of DSCC1 and GINS1, worse the prognosis.

Transcriptomic analysis of the cerebral hippocampal tissue in spontaneously hypertensive rats exposed to acute hypobaric hypoxia: associations with inflammation and energy metabolism

We evaluated the effect of acute hypobaric hypoxia (AHH) on the hippocampal region of the brain in early-stage spontaneously hypertensive male rats. The rats were classified into a control (ground level; ~ 400 m altitude) group and an AHH experimental group placed in an animal hypobaric chamber at a simulated altitude of 5500 m for 24 h. RNA-Seq analysis of the brains and hippocampi showed that differentially expressed genes (DEGs) were primarily associated with ossification, fibrillar collagen trimer, and platelet-derived growth factor binding. The DEGs were classified into functional categories including general function prediction, translation, ribosomal structure and biogenesis, replication, recombination, and repair. Pathway enrichment analysis revealed that the DEGs were primarily associated with relaxin signaling, PI3K-Akt signaling, and amoebiasis pathways. Protein–protein interaction network analysis indicated that 48 DEGs were involved in both inflammation and energy metabolism. Further, we performed validation experiments to show that nine DEGs were closely associated with inflammation and energy metabolism, of which two (Vegfa and Angpt2) and seven (Acta2, Nfkbia, Col1a1, Edn1, Itga1, Ngfr, and Sgk1) genes showed up and downregulated expression, respectively. Collectively, these results indicated that inflammation and energy metabolism-associated gene expression in the hippocampus was altered in early-stage hypertension upon AHH exposure.

Identification of hub genes for early detection of bone metastasis in breast cancer.

BackgroundGlobally, among all women, the most frequently detected and diagnosed and the most lethal type of cancer is breast cancer (BC). In particular, bone is one of the most frequent distant metastases 24in breast cancer patients and bone metastasis arises in approximately 80% of advanced patients. Thus, we need to identify and validate early detection markers that can differentiate metastasis from non-metastasis breast cancers.MethodsGSE55715, GSE103357, and GSE146661 gene expression profiling data were downloaded from the GEO database. There was 14 breast cancer with bone metastasis samples and 8 breast cancer tissue samples. GEO2R was used to screen for differentially expressed genes (DEGs). The volcano plots, Venn diagrams, and annular heatmap were generated by using the ggplot2 package. By using the cluster Profiler R package, KEGG and GO enrichment analyses of DEGs were conducted. Through PPI network construction using the STRING database, key hub genes were identified by cytoHubba. Finally, K-M survival and ROC curves were generated to validate hub gene expression.ResultsBy GO enrichment analysis, 143 DEGs were enriched in the following GO terms: extracellular structure organization, extracellular matrix organization, leukocyte migration class II protein complex, collagen tridermic protein complex, extracellular matrix structural constituent, growth factor binding, and platelet-derived growth factor binding. In the KEGG pathway enrichment analysis, DEGs were enriched in Staphylococcus aureus infection, Complement and coagulation cascades, and Asthma. By PPI network analysis, we selected the top 10 genes, including SLCO2B1, STAB1, SERPING1, HLA-DOA, AIF1, GIMAP4, C1orf162, HLA-DMB, ADAP2, and HAVCR2. By using TCGA and THPA databases, we validated 2 genes, SERPING1 and GIMAP4, that were related to the early detection of bone metastasis in BC.Conclusions2 abnormally expressed hub genes could play a pivotal role in the breast cancer with bone metastasis by affecting bone homeostasis imbalance in the bone microenvironment.

Single Nucleus RNA-sequencing Reveals Altered Intercellular Communication and Dendritic Cell Activation in Nonobstructive Hypertrophic Cardiomyopathy.

End stage, nonobstructive hypertrophic cardiomyopathy (HCM) is an intractable condition with no disease-specific therapies. To gain insights into the pathogenesis of nonobstructive HCM, we performed single nucleus RNA-sequencing (snRNA-seq) on human HCM hearts explanted at the time of cardiac transplantation and organ donor hearts serving as controls. Differential gene expression analysis revealed 64 differentially expressed genes linked to specific cell types and molecular functions. Analysis of ligand-receptor pair gene expression to delineate potential intercellular communication revealed significant reductions in expressed ligand-receptor pairs likely affecting the extracellular matrix, growth factor binding, peptidase regulator activity, platelet-derived growth factor binding and protease binding in the HCM tissue. Changes in Integrin-β1 receptor expression were responsible for many observed changes related to extracellular matrix interactions, by increasing in dendritic, smooth muscle and pericyte cells while decreasing in endothelial and fibroblast cells, suggesting potential mechanisms for fibrosis and microvascular disease in HCM and a potential role for dendritic cells. In contrast, there was an increase in ligand-receptor pair expression associated with adenylate cyclase binding, calcium channel molecular functions, channel inhibitor activity, ion channel inhibitor activity, phosphatase activator activity, protein kinase activator activity and titin binding, suggesting important shifts in various signaling cascades in nonobstructive, end stage HCM.

CSIG-10. PLATELET-DERIVED GROWTH FACTOR RECEPTOR ALPHA ONCOGENE DEPENDENCY IN GLIOBLASTOMA

Abstract PDGFRA is the second most frequently amplified gene encoding receptor tyrosine kinase in adult glioblastoma (GBM), oftentimes as extrachromosomal elements (ecDNA). Our overall objective is to elucidate mechanisms underlying PDGFRα dependency in GBM tumor maintenance. We have isolated distinct subpopulations from a GBM model (HF3253), harboring two alterations in PDGFRA: constitutively active genomic rearrangement and extrachromosomal amplification, that differ in the frequency of PDGFRA ecDNA. HF3253 tumor growth rate correlates with the initial proportion of ecDNA+ population implanted. Furthermore, slower tumor growth is due to selection for initially low-frequency PDGFRA ecDNA amplified clones based on histology and TaqMan Copy Number assay. Further exploiting intra-tumoral heterogeneity, we have isolated single cell clones from bulk cells. Compared to bulk cells, single cell clones do not express PDGFRα, PDGFRA mRNA and exhibit diploid PDGFRA copy number. Tumor growth was reduced in 4 ecDNA(-) clones compared to parental ecDNA(+) (log-rank test p= 0.00772, 0.00379, 0.00076, 0.00379). In contrast to parental HF3253, ecDNA(-) tumors demonstrated diffuse tumor morphology and weak PDGFRα activation. HF3253 ecDNA(-) PDX tumors lack detectable PDGFRα. Correspondingly, HF3253 ecDNA(-) cell populations do not exhibit de novo PDGFRA copy number gains post-implant. We conducted paired, whole RNA-sequencing on 20 HF3253 populations (ecDNA+/-: 6 clones from 3 biological replicates PDXs and 4 clones from 4 in vitro technical replicates). Employing a false discovery rate of 0.05, we identified 785 differentially expressed genes. Platelet-derived growth factor binding (GO:0048407) and central carbon metabolism were down-regulated in ecDNA(-) while genes significantly associated with astrocytic processes were upregulated. We demonstrated the dependency on PDGFRα signaling in a patient-derived GBM model carrying ecDNA PDGFRA amplification. Our data validates PDGFRɑ as a therapeutic target in a subset of GBM patients and demonstrates that detection of ecDNA-amplified PDGFRA has the potential to be a predictive biomarker of future PDGFRɑ targeted therapies.

Distinct functional classes of PDGFRB pathogenic variants in primary familial brain calcification.

Platelet-derived growth factor receptor beta (PDGFRB) is one of the genes associated with primary familial brain calcification (PFBC), an inherited neurological disease (OMIM:173410). Genetic analysis of patients and families revealed at least 13 PDGFRB heterozygous missense variants, including two novel ones described in the present report. Limited experimental data published on five of these variants had suggested that they decrease the receptor activity. No functional information was available on the impact of variants located within the receptor extracellular domains. Here, we performed a comprehensive molecular analysis of PDGFRB variants linked to PFBC. Mutated receptors were transfected in various cell lines to monitor receptor expression, signaling, mitogenic activity and ligand binding. Four mutants caused a complete loss of tyrosine kinase activity in multiple assays. One of the novel variants, p.Pro154Ser, decreased the receptor expression and abolished binding of platelet-derived growth factor (PDGF-BB). Others showed a partial loss of function related to reduced expression or signaling. Combining clinical, genetic and molecular data, we consider nine variants as pathogenic or likely pathogenic, three as benign or likely benign and one as a variant of unknown significance. We discuss the possible relationship between the variant residual activity, incomplete penetrance, brain calcification and neurological symptoms. In conclusion, we identified distinct molecular mechanisms whereby PDGFRB variants may result in a receptor loss of function. This work will facilitate genetic counseling in PFBC.

A Transcriptional Signature of PDGF-DD Activated Natural Killer Cells Predicts More Favorable Prognosis in Low-Grade Glioma.

The binding of platelet-derived growth factor D (PDGF-DD) to the NKp44 receptor activates a distinct transcriptional program in primary IL-2 expanded human natural killer (NK) cells. We were interested in knowing if the PDGF-DD-NKp44 pathway of NK cell activation might play a clinically relevant role in anti-tumor immunity. In order to address this question, we determined transcriptional signatures unique to resting, IL-2 expanded, and PDGF-DD activated, NK cells, in addition to different T cell subsets, and established the abundance of these immune cell phenotypes in The Cancer Genome Atlas (TCGA) low-grade glioma (LGG) dataset using CIBERSORT. Our results show that LGG patient tumors enriched for either the PDGF-DD activated NK cell or memory CD8+ T cell phenotypes are associated with a more favorable prognosis. Combined cell phenotype analyses revealed that patients with LGG tumors enriched for the PDGF-DD activated NK cell phenotype and the CD4+ T helper cell phenotype had a more favorable prognosis. High expression of transcripts encoding members of the killer cell lectin-like receptor (KLR) family, such as KLRK1 and KLRC2, KLRC3 and KLRC4 in LGG tumors were associated with more favorable prognosis, suggesting that these NK cell family receptors may play a prominent role in LGG anti-tumor immunity. Finally, many of the TCGA findings were reciprocated in LGG patients from the Chinese Glioma Genome Atlas (CGGA) dataset. Our results provide transcriptomic evidence that PDGF-DD activated NK cells and KLR family receptors may play an important clinical role in immune surveillance of LGG.

Long non-coding RNAs ENST00000429730.1 and MSTRG.93125.4 are associated with metabolic activity in tuberculosis lesions of sputum-negative tuberculosis patients.

Accurate diagnosis of complete inactivation of tuberculosis lesions is still a challenge with respect to sputum-negative tuberculosis. RNA-sequencing was conducted to uncover potential lncRNA indicators of metabolic activity in tuberculosis lesions. Lung tissues with high metabolic activity and low metabolic activity demonstrated by fluorine-18-fluorodeoxyglucose positron emission tomography/computed tomography were collected from five sputum-negative tuberculosis patients for RNA-sequencing. Differentially-expressed mRNAs and lncRNAs were identified. Their correlations were evaluated to construct lncRNA-mRNA co-expression network, in which lncRNAs and mRNAs with high degrees were confirmed by quantitative real-time PCR using samples collected from 11 patients. Prediction efficiencies of lncRNA indicators were assessed by receiver operating characteristic curve analysis. Bioinformatics analysis was performed for potential lncRNAs. 386 mRNAs and 44 lncRNAs were identified to be differentially expressed. Differentially-expressed mRNAs in lncRNA-mRNA co-expression network were significantly associated with fibrillar collagen, platelet-derived growth factor binding, and leukocyte migration involved in inflammatory response. Seven mRNAs (C1QB, CD68, CCL5, CCL19, MMP7, HLA-DMB, and CYBB) and two lncRNAs (ENST00000429730.1 and MSTRG.93125.4) were validated to be significantly up-regulated. The area under the curve of ENST00000429730.1 and MSTRG.93125.4 was 0.750 and 0.813, respectively. Two lncRNAs ENST00000429730.1 and MSTRG.93125.4 might be considered as potential indicators of metabolic activity in tuberculosis lesions for sputum-negative tuberculosis.

Engineered receptors for human cytomegalovirus that are orthogonal to normal human biology.

A trimeric glycoprotein complex on the surface of human cytomegalovirus (HCMV) binds to platelet-derived growth factor (PDGF) receptor α (PDGFRα) to mediate host cell recognition and fusion of the viral and cellular membranes. Soluble PDGFRα potently neutralizes HCMV in tissue culture, and its potential use as an antiviral therapeutic has the benefit that any escape mutants will likely be attenuated. However, PDGFRα binds multiple PDGF ligands in the human body as part of developmental programs in embryogenesis and continuing through adulthood. Any therapies with soluble receptor therefore come with serious efficacy and safety concerns, especially for the treatment of congenital HCMV. Soluble virus receptors that are orthogonal to human biology might resolve these concerns. This engineering problem is solved by deep mutational scanning on the D2-D3 domains of PDGFRα to identify variants that maintain interactions with the HCMV glycoprotein trimer in the presence of competing PDGF ligands. Competition by PDGFs is conformation-dependent, whereas HCMV trimer binding is independent of proper D2-D3 conformation, and many mutations at the receptor-PDGF interface are suitable for functionally separating trimer from PDGF interactions. Purified soluble PDGFRα carrying a targeted mutation succeeded in displaying wild type affinity for HCMV trimer with a simultaneous loss of PDGF binding, and neutralizes trimer-only and trimer/pentamer-expressing HCMV strains infecting fibroblasts or epithelial cells. Overall, this work makes important progress in the realization of soluble HCMV receptors for clinical application.

Retraction of "Design of Refolding DNA Aptamer on Single-Walled Carbon Nanotubes for Enhanced Optical Detection of Target Proteins".

DNA aptamer conjugated single-walled carbon nanotube (Aptamer-SWNT) hybrids have demonstrated effective optical biosensors because of their high selectivity and specificity to a target protein, however, the understanding of SWNT hybridization with an aptamer forming a secondary or tertiary structure is still lacking. We study wrapping methods dependent optical biosensing modulation by insulin and platelet-derived growth factor (PDGF) using the Aptamer-SWNT hybrids and the Aptamer-Anchor-SWNT hybrids with a periodically sequenced single-stranded DNA (ssDNA) as anchoring phases. We observe that the refolding nature of the aptamer and its combination with an anchoring phase are critical to the hybridization, where the remarkable optical sensing properties are attributed to the wrapping procedures including the direct wrapping with sonication and the indirect wrapping through dialysis. The tetrameric parallel-stranded G-quadrupole conformation of insulin binding aptamers (IBA) shows an enhanced fluorescence response quenching when using the directly wrapped Aptamer-Anchor-SWNT hybrids. In addition, helix-structural refolding of PDGF binding aptamers (PBA) indirectly wrapped on the SWNT vicinity is influenced by anchoring length for optical modulation. Furthermore, the consecutive centrifuging processes with the indirect wrapping demonstrate fluorescence response brightening, in which the diameter dependent brightening effect is observed by aptamer-protein interactions. This study provides an understanding the underlying conjugation nature of both the Aptamer-SWNT and the Aptamer-Anchor-SWNT hybrids formation, facilitating exceptional optical sensing modules with consideration of refolding feature of aptamers, selection of anchoring phases, wrapping methods and centrifuging process, and the hybridization voyage for DNA-SWNT platforms maneuvers their outcoming optical biosensing capabilities.

Design of Refolding DNA Aptamer on Single-Walled Carbon Nanotubes for Enhanced Optical Detection of Target Proteins.

DNA aptamer conjugated single-walled carbon nanotube (Aptamer-SWNT) hybrids have demonstrated effective optical biosensors because of their high selectivity and specificity to a target protein, however, the understanding of SWNT hybridization with an aptamer forming a secondary or tertiary structure is still lacking. We study wrapping methods dependent optical biosensing modulation by insulin and platelet-derived growth factor (PDGF) using the Aptamer-SWNT hybrids and the Aptamer-Anchor-SWNT hybrids with a periodically sequenced single-stranded DNA (ssDNA) as anchoring phases. We observe that the refolding nature of the aptamer and its combination with an anchoring phase are critical to the hybridization, where the remarkable optical sensing properties are attributed to the wrapping procedures including the direct wrapping with sonication and the indirect wrapping through dialysis. The tetrameric parallel-stranded G-quadrupole conformation of insulin binding aptamers (IBA) shows an enhanced fluorescence response quenching when using the directly wrapped Aptamer-Anchor-SWNT hybrids. In addition, helix-structural refolding of PDGF binding aptamers (PBA) indirectly wrapped on the SWNT vicinity is influenced by anchoring length for optical modulation. Furthermore, the consecutive centrifuging processes with the indirect wrapping demonstrate fluorescence response brightening, in which the diameter dependent brightening effect is observed by aptamer-protein interactions. This study provides an understanding the underlying conjugation nature of both the Aptamer-SWNT and the Aptamer-Anchor-SWNT hybrids formation, facilitating exceptional optical sensing modules with consideration of refolding feature of aptamers, selection of anchoring phases, wrapping methods and centrifuging process, and the hybridization voyage for DNA-SWNT platforms maneuvers their outcoming optical biosensing capabilities.

Identification of key genes, pathways and potential therapeutic agents for liver fibrosis using an integrated bioinformatics analysis.

BackgroundLiver fibrosis is often a consequence of chronic liver injury, and has the potential to progress to cirrhosis and liver cancer. Despite being an important human disease, there are currently no approved anti-fibrotic drugs. In this study, we aim to identify the key genes and pathways governing the pathophysiological processes of liver fibrosis, and to screen therapeutic anti-fibrotic agents.MethodsExpression profiles were downloaded from the Gene Expression Omnibus (GEO), and differentially expressed genes (DEGs) were identified by R packages (Affy and limma). Gene functional enrichments of each dataset were performed on the DAVID database. Protein–protein interaction (PPI) network was constructed by STRING database and visualized in Cytoscape software. The hub genes were explored by the CytoHubba plugin app and validated in another GEO dataset and in a liver fibrosis cell model by quantitative real-time PCR assay. The Connectivity Map L1000 platform was used to identify potential anti-fibrotic agents.ResultsWe integrated three fibrosis datasets of different disease etiologies, incorporating a total of 70 severe (F3–F4) and 116 mild (F0–F1) fibrotic tissue samples. Gene functional enrichment analyses revealed that cell cycle was a pathway uniquely enriched in a dataset from those patients infected by hepatitis B virus (HBV), while the immune-inflammatory response was enriched in both the HBV and hepatitis C virus (HCV) datasets, but not in the nonalcoholic fatty liver disease (NAFLD) dataset. There was overlap between these three datasets; 185 total shared DEGs that were enriched for pathways associated with extracellular matrix constitution, platelet-derived growth-factor binding, protein digestion and absorption, focal adhesion, and PI3K-Akt signaling. In the PPI network, 25 hub genes were extracted and deemed to be essential genes for fibrogenesis, and the expression trends were consistent with GSE14323 (an additional dataset) and liver fibrosis cell model, confirming the relevance of our findings. Among the 10 best matching anti-fibrotic agents, Zosuquidar and its corresponding gene target ABCB1 might be a novel anti-fibrotic agent or therapeutic target, but further work will be needed to verify its utility.ConclusionsThrough this bioinformatics analysis, we identified that cell cycle is a pathway uniquely enriched in HBV related dataset and immune-inflammatory response is clearly enriched in the virus-related datasets. Zosuquidar and ABCB1 might be a novel anti-fibrotic agent or target.

Abstract 1237: PDGF induces cell growth and changes in glucose metabolism in colon cancer in the absence of PDGF receptors

Abstract Background: The Platelet derived growth factor (PDGF) and its receptors play a major role in inducing proliferation, migration, and angiogenesis by activating intracellular PI3K/Akt/mTOR signaling events in different solid tumors and therefore represent attractive targets in tumor therapy. Recently we showed a PDGF-induced activation of metabolism and proliferation in HT29 colon cancer cells in the absence of specific PDGF receptors. The aim of this study was to analyze PDGFR and VEGFR expression and possible alternative PDGF binding partners in colorectal cancer (CRC). Methods: Human HT29 colon cancer cells were cultured and stimulated with PDGF in a time-dependent manner. Additionally, VEGFR2 and EGFR inhibition was performed to analyze alternative PDGF signaling events. Whole cell or RNA extracts were analyzed by Western Blot and RT-q-PCR for receptors and PI3K/Akt/mTOR signaling. To investigate the effects of specific receptor inhibition on proliferation, MTS proliferation assays were performed. Moreover, mRNA levels of PDGFR and VEGFR in tumors from patients with CRC were analyzed by RT-qPCR. Results: Human UICC stage I-IV tumors exhibited a significantly increased PDGFRβ and VEGFR1,2 gene expression. As observed previously, HT29 colon cancer cells showed only positivity for VEGFR1,2, but no PDGFR protein expression. Despite that, stimulation with PDGF resulted in increased proliferation and metabolic changes. In contrast, Caco-2 and SW480 colon cancer cells showed PDGFR and VEGFR expression on protein level. Interestingly, PDGF stimulation increased VEGFR1 and 2 gene expression in HT29 colon cancer cells and secondly inhibition of VEGFR2 and EGFR showed alterations in proliferation and Akt signaling in PDGF stimulated cancer cells. Conclusion: Despite the absence of PDGF receptors in HT29 colon cancer cells, PDGF induced proliferating and metabolic effects in the tumor cells, suggesting an alternative binding partner on the tumor cell surface. Further investigation of the alternative PDGF binding partners could be of clinical relevance in CRC. Note: This abstract was not presented at the meeting. Citation Format: Romana Moench, Tanja Grimmig, Christoph-Thomas Germer, Ana Maria Waaga-Gasser, Martin Gasser. PDGF induces cell growth and changes in glucose metabolism in colon cancer in the absence of PDGF receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1237. doi:10.1158/1538-7445.AM2017-1237

Comparative analysis of gene expression profiles in normal hip human cartilage and cartilage from patients with necrosis of the femoral head

BackgroundThe pathogenesis of necrosis of the femoral head (NFH) remains elusive. Limited studies were conducted to investigate the molecular mechanism of hip articular cartilage damage in NFH. We conducted genome-wide gene expression profiling of hip articular cartilage with NFH.MethodsHip articular cartilage specimens were collected from 18 NFH patients and 18 healthy controls. Gene expression profiling of NFH articular cartilage was carried out by Agilent Human 4x44K Gene Expression Microarray chip. Differently expressed genes were identified using the significance analysis of microarrays (SAM) software. Gene Ontology (GO) enrichment analysis of differently expressed genes was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Significantly differently expressed genes in the microarray experiment were selected for quantitative real-time PCR (qRT-PCR) and immunohistochemical validation.ResultsSAM identified 27 differently expressed genes in NFH articular cartilage, functionally involved in extracellular matrix, cytokines, growth factors, cell cycle and apoptosis. The expression patterns of the nine validation genes in qRT-PCR were consistent with that in proteinaceous extracellular matrix (false discovery rate (FDR) = 3.22 × 10-5), extracellular matrix (FDR = 5.78 × 10-5), extracellular region part (FDR = 1.28 × 10-4), collagen (FDR = 3.22 × 10-4), extracellular region (FDR = 4.78 × 10-4) and platelet-derived growth factor binding (FDR = 5.23 × 10-4).ConclusionsThis study identified a set of differently expressed genes, implicated in articular cartilage damage in NFH. Our study results may provide novel insight into the pathogenesis and rationale of therapies for NFH.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-0991-4) contains supplementary material, which is available to authorized users.

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).

Pathway Analysis of Metabolic Syndrome Using a Genome-Wide Association Study of Korea Associated Resource (KARE) Cohorts.

Metabolic syndrome (MetS) is a complex disorder related to insulin resistance, obesity, and inflammation. Genetic and environmental factors also contribute to the development of MetS, and through genome-wide association studies (GWASs), important susceptibility loci have been identified. However, GWASs focus more on individual single-nucleotide polymorphisms (SNPs), explaining only a small portion of genetic heritability. To overcome this limitation, pathway analyses are being applied to GWAS datasets. The aim of this study is to elucidate the biological pathways involved in the pathogenesis of MetS through pathway analysis. Cohort data from the Korea Associated Resource (KARE) was used for analysis, which include 8,842 individuals (age, 52.2 ± 8.9 years; body mass index, 24.6 ± 3.2 kg/m2). A total of 312,121 autosomal SNPs were obtained after quality control. Pathway analysis was conducted using Meta-analysis Gene-Set Enrichment of Variant Associations (MAGENTA) to discover the biological pathways associated with MetS. In the discovery phase, SNPs from chromosome 12, including rs11066280, rs2074356, and rs12229654, were associated with MetS (p < 5 × 10-6), and rs11066280 satisfied the Bonferroni-corrected cutoff (unadjusted p < 1.38 × 10-7, Bonferroni-adjusted p < 0.05). Through pathway analysis, biological pathways, including electron carrier activity, signaling by platelet-derived growth factor (PDGF), the mitogen-activated protein kinase kinase kinase cascade, PDGF binding, peroxisome proliferator-activated receptor (PPAR) signaling, and DNA repair, were associated with MetS. Through pathway analysis of MetS, pathways related with PDGF, mitogen-activated protein kinase, and PPAR signaling, as well as nucleic acid binding, protein secretion, and DNA repair, were identified. Further studies will be needed to clarify the genetic pathogenesis leading to MetS.

Aptamer-based label-free detection of PDGF using ruthenium(II) complex as luminescent probe

We report a simple, cost-effective, and label-free detection method, consisting of a platelet-derived growth factor (PDGF) binding aptamer and hydrophobic Ru(II) complex as a sensor system for PDGF. The binding of PDGF with the aptamer results in the weakening of the aptamer-Ru(II) complex, monitored by luminescence signal. A substantial enhancement in the luminescence intensity of Ru(II) complex is observed in the presence of aptamer due to the hydrophobic interaction. Upon addition of PDGF, the luminescence intensity is decreased, due to the stronger interaction between the aptamer and PDGF resulting in the displacement of Ru(II) complex to the aqueous solution. Our assay can detect a target specifically in a complex medium such as the mixture of proteins, at a concentration of 0.8pM.

An aptamer-based biosensing platform for highly sensitive detection of platelet-derived growth factor via enzyme-mediated direct electrochemistry

In this work, a new label-free electrochemical aptamer-based sensor (aptasensor) was constructed for detection of platelet-derived growth factor (PDGF) based on the direct electrochemistry of glucose oxidase (GOD). For this proposed aptasensor, poly(diallyldimethylammonium chloride) (PDDA)-protected graphene-gold nanoparticles (P-Gra-GNPs) composite was firstly coated on electrode surface to form the interface with biocompatibility and huge surface area for the adsorption of GOD layer. Subsequently, gold nanoclusters (GNCs) were deposited on the surface of GOD to capture PDGF binding aptamer (PBA). Finally, GOD as a blocking reagent was employed to block the remaining active sites of the GNCs and avoid the nonspecific adsorption. With the direct electron transfer of double layer GOD membranes, the aptasensor showed excellent electrochemical response and the peak current decreased linearly with increasing logarithm of PDGF concentration from 0.005nM to 60nM with a relatively low limit of detection of 1.7pM. The proposed aptasensor exhibited high specificity, good reproducibility and long-term stability, which provided a new promising technique for aptamer-based protein detection.

Protein Disulfide Isomerase Is Required for Platelet-derived Growth Factor-induced Vascular Smooth Muscle Cell Migration, Nox1 NADPH Oxidase Expression, and RhoGTPase Activation

Vascular Smooth Muscle Cell (VSMC) migration into vessel neointima is a therapeutic target for atherosclerosis and postinjury restenosis. Nox1 NADPH oxidase-derived oxidants synergize with growth factors to support VSMC migration. We previously described the interaction between NADPH oxidases and the endoplasmic reticulum redox chaperone protein disulfide isomerase (PDI) in many cell types. However, physiological implications, as well as mechanisms of such association, are yet unclear. We show here that platelet-derived growth factor (PDGF) promoted subcellular redistribution of PDI concomitant to Nox1-dependent reactive oxygen species production and that siRNA-mediated PDI silencing inhibited such reactive oxygen species production, while nearly totally suppressing the increase in Nox1 expression, with no change in Nox4. Furthermore, PDI silencing inhibited PDGF-induced VSMC migration assessed by distinct methods, whereas PDI overexpression increased spontaneous basal VSMC migration. To address possible mechanisms of PDI effects, we searched for PDI interactome by systems biology analysis of physical protein-protein interaction networks, which indicated convergence with small GTPases and their regulator RhoGDI. PDI silencing decreased PDGF-induced Rac1 and RhoA activities, without changing their expression. PDI co-immunoprecipitated with RhoGDI at base line, whereas such association was decreased after PDGF. Also, PDI co-immunoprecipitated with Rac1 and RhoA in a PDGF-independent way and displayed detectable spots of perinuclear co-localization with Rac1 and RhoGDI. Moreover, PDI silencing promoted strong cytoskeletal changes: disorganization of stress fibers, decreased number of focal adhesions, and reduced number of RhoGDI-containing vesicular recycling adhesion structures. Overall, these data suggest that PDI is required to support Nox1/redox and GTPase-dependent VSMC migration.

SPARC promotes the development of erythroid progenitors

Secreted protein acidic and rich in cysteine (SPARC) is a well-recognized regulator that affects cell proliferation, differentiation, and survival. Here, we investigated the impact of SPARC on erythropoiesis. Erythropoiesis in bone marrow (BM) from SPARC(-/-) mice was analyzed by flow cytometry and colony-forming assays. The mechanisms that affected erythropoiesis were investigated by BM transplantation experiments. CD34(+) cells from umbilical cord blood were isolated by MiniMACS, and the effect of SPARC on human erythropoiesis was assessed by colony-formation assay and erythroid differentiation culture. The hemoglobin level in peripheral blood (PB) was lower in SPARC(-/-) mice. Neither red blood cell count in PB nor the percentage of Ter119(+) erythrocytes in BM showed significant difference between SPARC(-/-) and WT mice. However, the ability of SPARC(-/-) bone marrow cells (BMCs) to form erythroid burst-forming units (BFU-E), as well as spleen colony-forming units (CFU-S8), was significantly decreased. The addition of exogenous SPARC could promote the formation of BFU-E from SPARC(-/-) BMCs in vitro. And the impaired ability of SPARC(-/-) BMCs to form BFU-E could be restored by transplanted into WT BM microenvironment, whereas the BFU-E formation of WT BMCs was impaired after transplanted into SPARC(-/-) recipients. Furthermore, exogenous SPARC promoted umbilical cord blood CD34(+) cells to form erythroid colonies and the hemoglobinization of erythroid, but did not affect the expression levels of glycophorin A and CD71. In conclusion, our results indicate that SPARC promotes the development of erythroid progenitors, but does not affect terminal erythroid differentiation.