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The adsorbed protein layer on an implanted biomaterial surface is known to mediate downstream cell-material interactions that drive the host response. While the adsorption of plasma-derived proteins has been studied extensively, the adsorption of damage-associated molecular patterns (DAMPs) derived from damaged cells and matrix surrounding the implant remains poorly understood. Previously, our group developed a DAMP-adsorption model in which 3T3 fibroblast lysates were used as a complex source of cell-derived DAMPs and we demonstrated that biomaterials with adsorbed lysate potently activated RAWBlue macrophages via Toll-like receptor 2 (TLR2). In the present study, we characterized the response of mouse bone marrow derived macrophages (BMDM) from wildtype (WT), TLR2-/- and MyD88-/- mice on Teflon™ AF surfaces pre-adsorbed with 10% plasma or lysate-spiked plasma (10% w/w total protein from 3T3 fibroblast lysate) for 24 hours. WT BMDM cultured on adsorbates derived from 10% lysate in plasma had significantly higher gene and protein expression of IL-1β, IL-6, TNF-α, IL-10, RANTES/CCL5 and CXCL1/KC, compared to 10% plasma-adsorbed surfaces. Furthermore, the upregulation of pro-inflammatory cytokine and chemokine expression in the 10% lysate in plasma condition was attenuated in TLR2-/- and MyD88-/- BMDM. Proteomic analysis of the adsorbed protein layers showed that even this relatively small addition of lysate-derived proteins within plasma (10% w/w) caused a significant change to the adsorbed protein profile. The 10% plasma condition had fibrinogen, albumin, apolipoproteins, complement, and fibronectin among the top 25 most abundant proteins. While proteins layers generated from 10% lysate in plasma retained fibrinogen and fibronectin among the top 25 proteins, there was a disproportionate increase in intracellular proteins, including histones, tubulins, actins, and vimentin. Furthermore, we identified 7 DAMPs or DAMPrelated proteins enriched in the 10% plasma condition (fibrinogen, apolipoproteins), compared to 39 DAMPs enriched in the 10% lysate in plasma condition, including high mobility group box 1 and histones. Together, these findings indicate that DAMPs and other intracellular proteins readily adsorb to biomaterial surfaces in competition with plasma proteins, and that adsorbed DAMPs induce an inflammatory response in adherent macrophages that is mediated by the MyD88-dependent TLR2 signaling pathway. [ABSTRACT FROM AUTHOR]

Introduction: Continuity of care by family physicians in primary care settings may play a role in reducing health resource utilization and improving clinical outcomes and satisfaction of patients with chronic obstructive pulmonary disease. Clear evidence on the impact of continuity of care will support clinical programing and integration of services across health settings. Methods: The association between continuity of care and unplanned health service utilization in persons with a diagnosis of chronic obstructive pulmonary disease in a rural region in Ontario, Canada was evaluated. A retrospective cohort study was conducted using population-level health administrative data. The main exposure variable was continuity of care. Results: A continuity of care index was calculated for patients with at least five visits to a healthcare provider during the 5-year follow-up period (n = 40,033). Higher continuity of care (n = 20,008) and lower continuity of care (n = 20,025), based on the median continuity of care score were calculated. Patients with lower continuity of care had an increased adjusted relative risk of 2.12 (2.08, 2.33) of an emergency department visit, 2.81 (2.72, 2.9) risk of hospitalization, and 3.52 (3.24, 3.82) of being readmitted to hospital compared to those with higher continuity of care. Discussion: An association between continuity of care and unplanned health services utilization, where a lower use of unplanned health services was observed in the cohort of patients with chronic obstructive pulmonary disease experiencing higher continuity of care. Continuity of care makes philosophical and social sense in that care is provided by a known provider to a known patient and unnecessary investigations can be avoided. [ABSTRACT FROM AUTHOR]

Abstract Background Transcriptomic studies in clinical research are essential tools for deciphering the functional elements of the genome and unraveling underlying disease mechanisms. Various technologies have been developed to deduce and quantify the transcriptome including hybridization and sequencing-based approaches. Recently, high density exon microarrays have been successfully employed for detecting differentially expressed genes and alternative splicing events for biomarker discovery and disease diagnostics. The field of transcriptomics is currently being revolutionized by high throughput DNA sequencing methodologies to map, characterize, and quantify the transcriptome. Methods In an effort to understand the merits and limitations of each of these tools, we undertook a study of the transcriptome in sickle cell disease, a monogenic disease comparing the Affymetrix Human Exon 1.0 ST microarray (Exon array) and Illumina’s deep sequencing technology (RNA-seq) on whole blood clinical specimens. Results Analysis indicated a strong concordance (R = 0.64) between Exon array and RNA-seq data at both gene level and exon level transcript expression. The magnitude of differential expression was found to be generally higher in RNA-seq than in the Exon microarrays. We also demonstrate for the first time the ability of RNA-seq technology to discover novel transcript variants and differential expression in previously unannotated genomic regions in sickle cell disease. In addition to detecting expression level changes, RNA-seq technology was also able to identify sequence variation in the expressed transcripts. Conclusions Our findings suggest that microarrays remain useful and accurate for transcriptomic analysis of clinical samples with low input requirements, while RNA-seq technology complements and extends microarray measurements for novel discoveries.

In this work, we expand on our understanding of the thrombogenicity of coatings prepared with three different recombinant elastin-like polypeptides (ELPs). The bulk platelet response of the ELP coatings was characterized following whole blood contact under physiological shear flow (300 s−1) using flow cytometry. Prolonged exposure to shear flow (1-h) indicated that materials coated with the longer ELP coatings (ELP2 and ELP4) had less bulk platelet activation and microparticle formation than materials coated with the shorter ELP1. Quartz crystal microbalance with dissipation (QCM-D) was used to monitor the binding of the platelet membrane receptor GPIIb/IIIa to ELP-adsorbed fibrinogen (Fg) surfaces. Compared to the shorter ELPs, a lower amount of Fg adsorbed to the ELP4 coated material and ELP4 appeared to form a softer, more structurally flexible coating layer. When Fg was adsorbed to the ELP coated surface it demonstrated an altered binding for GPIIb/IIIa that was inhibited in the presence of an AGDV-containing peptide but not an RGD-containing peptide. Conversely, on the shorter ELP coatings, binding of GPIIb/IIIa to an adsorbed Fg layer was partially inhibited in the presence of an RGD-containing peptide. These results indicate that both the quantity and conformational state of Fg varies when adsorbed to surfaces coated with ELPs of varying sequence length, which may be mediating their platelet response. Collectively, the findings reinforce the applicability of the ELPs as potential thromboresistant coatings, especially with the use of the longer polypeptide—ELP4. [ABSTRACT FROM AUTHOR]

Background: Increased platelet activation in sickle cell disease (SCD) contributes to a state of hypercoagulability and confers a risk of thromboembolic complications. The role for post-transcriptional regulation of the platelet transcriptome by microRNAs (miRNAs) in SCD has not been previously explored. This is the first study to determine whether platelets from SCD exhibit an altered miRNA expression profile. Methods and Findings: We analyzed the expression of miRNAs isolated from platelets from a primary cohort (SCD = 19, controls = 10) and a validation cohort (SCD = 7, controls = 7) by hybridizing to the Agilent miRNA microarrays. A dramatic difference in miRNA expression profiles between patients and controls was noted in both cohorts separately. A total of 40 differentially expressed platelet miRNAs were identified as common in both cohorts (p-value 0.05, fold change>2) with 24 miRNAs downregulated. Interestingly, 14 of the 24 downregulated miRNAs were members of three families - miR-329, miR-376 and miR-154 - which localized to the epigenetically regulated, maternally imprinted chromosome 14q32 region. We validated the downregulated miRNAs, miR-376a and miR-409-3p, and an upregulated miR-1225-3p using qRT-PCR. Over-expression of the miR-1225-3p in the Meg01 cells was followed by mRNA expression profiling to identify mRNA targets. This resulted in significant transcriptional repression of 1605 transcripts. A combinatorial approach using Meg01 mRNA expression profiles following miR-1225-3p overexpression, a computational prediction analysis of miRNA target sequences and a previously published set of differentially expressed platelet transcripts from SCD patients, identified three novel platelet mRNA targets: PBXIP1, PLAGL2 and PHF20L1. Conclusions: We have identified significant differences in functionally active platelet miRNAs in patients with SCD as compared to controls. These data provide an important inventory of differentially expressed miRNAs in SCD patients and an experimental framework for future studies of miRNAs as regulators of biological pathways in platelets. [ABSTRACT FROM AUTHOR]

Embryonic stem cells (ESCs) are an important source of cardiomyocytes for regenerating injured myocardium. The successful use of ESC-derived cardiomyocytes in cardiac tissue engineering requires an understanding of the important scaffold properties and culture conditions to promote cell attachment, differentiation, organization, and contractile function. The goal of this work was to investigate how scaffold architecture and coculture with fibroblasts influences the differentiated phenotype of murine ESC-derived cardiomyocytes (mESCDCs). Electrospinning was used to process an elastomeric biodegradable polyurethane (PU) into aligned or unaligned fibrous scaffolds. Bioreactor produced mESCDCs were seeded onto the PU scaffolds either on their own or after pre-seeding the scaffolds with mouse embryonic fibroblasts (MEFs). Viable mESCDCs attached to the PU scaffolds and were functionally contractile in all conditions tested. Importantly, the aligned scaffolds led to the anisotropic organization of rod-shaped cells, improved sarcomere organization, and increased mESCDC aspect ratio (length-to-diameter ratio) when compared to cells on the unaligned scaffolds. In addition, pre-seeding the scaffolds with MEFs improved mESCDC sarcomere formation compared to mESCDCs cultured alone. These results suggest that both fiber alignment and pre-treatment of scaffolds with fibroblasts improve the differentiation of mESCDCs and are important parameters for developing engineered myocardial tissue constructs using ESC-derived cardiac cells. Biotechnol. Bioeng. 2012; 109:813-822. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Despite a growing number of reports of gene expression analysis from blood-derived RNA sources, there have been few systematic comparisons of various RNA sources in transcriptomic analysis or for biomarker discovery in the context of cardiovascular disease (CVD). As a pilot study of the Systems Approach to Biomarker Research (SABRe) in CVD Initiative, this investigation used Affymetrix Exon arrays to characterize gene expression of three blood-derived RNA sources: lymphoblastoid cell lines (LCL), whole blood using PAXgene tubes (PAX), and peripheral blood mononuclear cells (PBMC). Their performance was compared in relation to identifying transcript associations with sex and CVD risk factors, such as age, high-density lipoprotein, and smoking status, and the differential blood cell count. We also identified a set of exons that vary substantially between participants, but consistently in each RNA source. Such exons are thus stable phenotypes of the participant and may potentially become useful fingerprinting biomarkers. In agreement with previous studies, we found that each of the RNA sources is distinct. Unlike PAX and PBMC, LCL gene expression showed little association with the differential blood count. LCL, however, was able to detect two genes related to smoking status. PAX and PBMC identified Y-chromosome probe sets similarly and slightly better than LCL. [ABSTRACT FROM AUTHOR]

Hematopoietic differentiation is strictly regulated by complex network of transcription factors that are controlled by ligands binding to cell surface receptors. Disruptions of the intricate sequences of transcriptional activation and suppression of multiple genes cause hematological diseases, such as leukemias, myelodysplastic syndromes, or myeloproliferative syndromes. From a clinical standpoint, deciphering the pattern of gene expression during hematopoiesis may help unravel disease-specific mechanisms in hematopoietic malignancies. Herein, we describe a human in vitro hematopoietic model system where lineage-specific differentiation of CD34+ cells was accomplished using specific cytokines. Microarray and RNAseq-based whole transcriptome and exome analysis was performed on the differentiated erythropoietic, granulopoietic, and megakaryopoietic cells to delineate changes in expression of whole transcripts and exons. Analysis on the Human 1.0 ST exon arrays indicated differential expression of 172 genes (P < 0.0000001) and significant alternate splicing of 86 genes during differentiation. Pathway analysis identified these genes to be involved in Rac/RhoA signaling, Wnt/B-catenin signaling and alanine/aspartate metabolism. Comparison of the microarray data to next generation RNAseq analysis during erythroid differentiation demonstrated a high degree of correlation in gene (R = 0.72) and exon (R = 0.62) expression. Our data provide a molecular portrait of events that regulate differentiation of hematopoietic cells. Knowledge of molecular processes by which the cells acquire their cell-specific fate would be beneficial in developing cell-based therapies for human diseases. [ABSTRACT FROM AUTHOR]

Elastin-like polypeptide (ELP) surface modification represents a valuable approach for the development of biomaterials in a wide range of medical applications. In this study, ELP surface modification has been achieved through the use of elastin cross-linking peptide (ECP) bioactive fluorinated surface modifiers (ECP-BFSMs). The synthesis of low molecular weight fluorinated additives was described and their subsequent blending with a base polycarbonate urethane (PCNU) was shown to successfully enrich the surface to allow for ELP surface cross-linking via lysine moieties on the peptide segments of the ECP-BFSMs. The kinetics for the surface migration of fluorescent ECP-BFSMs was studied over a 2-week period by two-photon confocal microscopy. A decrease in advancing contact angle from 87.9° to 75.3° was observed for ECP-BFSM modified PCNU and was associated with the presence of ECP peptides on the surface. X-ray photoelectron spectroscopy demonstrated an increase in surface atomic percent of fluorine (from 0.2 to 7.2%) and nitrogen (from 1.0 to 3.0%) associated with the surface localization of fluoro groups and amide groups associated with the peptides in the ECP-BFSMs. A further increase in surface atomic percent of nitrogen (from 3.0 to 8.3%) was observed after ELP surface cross-linking. These ELP-modified surfaces were shown to promote increased smooth muscle cell adhesion, spreading and retention over a 7-day culture period relative to their non-ELP4 analogs. This novel surface modifying additive approach may be used for various biomimetic applications since it generates a stable ECM-like surface retained onto a relatively inert fluorinated background. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2011. [ABSTRACT FROM AUTHOR]

The inherent flexibility of polyurethane (PU) chemistry allows the incorporation of specific chemical moieties into the backbone structure conferring a unique biological function to these synthetic polymers. We describe here the synthesis and characterization of a PU containing a Gly–Leu linkage, the cleavage site of several matrix metalloproteinases. A Gly–Leu dipeptide was introduced into the chain extender of the polyurethane through the reaction with 1,4-cyclohexane dimethanol. PUs synthesized with the Gly–Leu-based chain extender had a high weight-average molecular weight (Mw > 125 × 103) and were phase segregated, semi-crystalline polymers with a low soft-segment glass-transition temperature (Tg < –50°C). Uniaxial tensile testing of PU films indicated that the polymer could withstand high ultimate tensile strengths (approx. 13 MPa) and were flexible with breaking strains of approx. 900%. The Gly–Leu PU had a significantly higher initial modulus, yield stress and ultimate stress compared to a PU previously developed in our laboratory containing a phenylalanine-based chain extender (Phe PU). The Gly–Leu-based chain extender allowed for better hard segment packing and hydrogen bonding leading to enhanced mechanical properties. Electrospinning was used to form scaffolds with randomly organized fibers and an average fiber diameter of approx. 3.6 μm for both the Gly–Leu and Phe PUs. Mouse embryonic fibroblasts were successfully cultured on the PU scaffolds out to 28 days. Further investigations into cell-mediated polymer degradation will help to identify the suitability of this new biomaterial as scaffolds for soft tissue applications. [ABSTRACT FROM AUTHOR]

To better understand endogenous parameters that influence pluripotent cell differentiation we used human embryonic stem cells (hESCs) as a model system. We demonstrate that differentiation trajectories in aggregate (embryoid body [EB])-induced differentiation, a common approach to mimic some of the spatial and temporal aspects of in vivo development, are affected by three factors: input hESC composition, input hESC colony size, and EB size. Using a microcontact printing approach, size-specified hESC colonies were formed by plating single-cell suspensions onto micropatterned (MP) extracellular matrix islands. Subsequently, size-controlled EBs were formed by transferring entire colonies into suspension culture enabling the independent investigation of colony and aggregate size effects on differentiation induction. Gene and protein expression analysis of MP-hESC populations revealed that the ratio of Gata6 (endoderm-associated marker) to Pax6 (neural-associated marker) expression increased with decreasing colony size. Moreover, upon forming EBs from these MP-hESCs, we observed that differentiation trajectories were affected by both colony and EB size-influenced parameters. In MPEBs generated from endoderm-biased (high Gata6/Pax6) input hESCs, higher mesoderm and cardiac induction was observed at larger EB sizes. Conversely, neural-biased (low Gata6/Pax6) input hESCs generated MP-EBs that exhibited higher cardiac induction in smaller EBs. Our analysis demonstrates that heterogeneity in hESC colony and aggregate size, typical in most differentiation strategies, produces subsets of appropriate conditions for differentiation into specific cell types. Moreover, our findings suggest that the local microenvironment modulates endogenous parameters that can be used to influence pluripotent cell differentiation trajectories. [ABSTRACT FROM AUTHOR]

Recombinantly-engineered elastin-like polypeptides (ELPs) possess many of the favourable attributes of the native elastin protein, making them an attractive option for designing biomaterials for tissue-engineering applications. The focus of this study was to synthesize and characterise the bulk material properties of two ELP sequences, ELP2 and ELP4, cross-linked with lysine diisocyanate (LDI). The two distinct ELPs consist of repeating hydrophobic and hydrophilic cross-linking domains in a block co-polymer structure, however, differ by the number of respective domains. Depending on the conditions sets for the cross-linking reactions, two different ELP-based materials were synthesized: a gel-like relatively non-porous material and a porous foam-like material, from both ELP sequences. The physical material properties were characterised by scanning electron microscopy, compression testing, differential calorimetry analysis and swelling analysis. The bulk material properties were found to vary depending on the ELP sequence investigated. ELP gels were also found to have a more dense solidified morphology, lower compressive moduli, higher melting temperature and greater swelling capacity than the porous ELP foams. These novel cross-linked bio-elastomeric materials show promising properties for soft tissue replacement, particularly in load-bearing applications. [ABSTRACT FROM AUTHOR]

A polyurethane designed to be biodegradable via hydrolysis and enzyme-mediated chain cleavage, has been investigated for its use as a temporary scaffold in tissue-engineering applications. The phase-segregated nature of the polyurethane imparts elastomeric properties that are attractive for soft tissue engineering. This polyurethane has been electrospun in order to create scaffolds that incorporate several biomimetic features including small fiber diameter, large void volume, and an interconnected porous network. Material properties were evaluated via gel-permeation chromatography, differential scanning calorimetry and Raman spectroscopy before and after processing. Analysis by gel-permeation chromatography showed that the molecular weights were similar, indicating that the bulk of the polymer chains were not degraded during processing. Thermal analysis revealed that the glass transition temperature did not shift and Raman spectra of the bulk polyurethane film compared to the electrospun mat were identical, confirming that the conformation of the polymer was unaffected by the shear and electric field used in the electrospinning process. In addition, field emission scanning electron microscopy revealed that the morphology of the electrospun mats had a broad fiber diameter distribution, and mechanical analysis showed that the mats had an ultimate tensile stress of 1.33 MPa and ultimate tensile strain of 78.6%. The degradation profile was investigated in the presence of chymotrypsin. These results were compared to a previous study of thin films of this polyurethane, and it was found that the increase of surface area aided the surface-mediated erosion of the material. It is believed that an electrospun matrix of this biodegradable polyurethane shows promise for use in soft tissue engineering and regenerative medicine applications. [ABSTRACT FROM AUTHOR]

Investigates the coacervation of elastin-like peptides (EP) by variable temperature in situ atomic force microscopy. Role of hydrophobic stabilization on coacervation; Discrete aggregates of EP on mica and highly oriented pyrolytic graphite; Increase in the rate of fibril formation.

Critical limb ischemia is a major clinical problem. Despite rigorous treatment regimes, there has been only modest success in reducing the rate of amputations in affected patients. Reduced level of blood flow and enhanced inflammation are the two major pathophysiological changes that occur in the ischemic tissue. The objective of this study was to develop a controlled dual gene delivery system capable of delivering therapeutic plasmid eNOS and IL-10 in a temporal manner. In order to deliver multiple therapeutic genes, an elastin-like polypeptide (ELP) based injectable system was designed. The injectable system was comprised of hollow spheres and an in situ -forming gel scaffold of elastin-like polypeptide capable of carrying gene complexes, with an extended manner release profile. In addition, the ELP based injectable system was used to deliver human eNOS and IL-10 therapeutic genes in vivo . A subcutaneous dose response study showed enhanced blood vessel density in the treatment groups of eNOS (20 μg) and IL-10 (10 μg)/eNOS (20 μg) and reduced inflammation with IL-10 (10 μg) alone. Next, we carried out a hind-limb ischemia model comparing the efficacy of the following interventions; Saline; IL-10, eNOS and IL-10/eNOS. The selected dose of eNOS, exhibited enhanced angiogenesis. IL-10 treatment groups showed reduction in the level of inflammatory cells. Furthermore, we demonstrated that eNOS up-regulated major proangiogenic growth factors such as vascular endothelial growth factors, platelet derived growth factor B, and fibroblast growth factor 1, which may explain the mechanism of this approach. These factors help in formation of a stable vascular network. Thus, ELP injectable system mediating non-viral delivery of human IL10-eNOS is a promising therapy towards treating limb ischemia. [ABSTRACT FROM AUTHOR]