AP-1 signaling modulates cardiac fibroblast stress responses.

Matrix remodeling outcomes largely dictate patient survival post myocardial infarction. Moreover, human-restricted noncoding regulatory elements have been shown to worsen fibrosis, but their mechanism of action remains elusive. Here, we demonstrate, using induced pluripotent stem cell-derived cardiac fibroblasts (iCFs), that inflammatory ligands abundant in the remodeling heart after infarction activate AP-1 transcription factor signaling pathways resulting in fibrotic responses. This observed signaling induces deposition of fibronectin matrix and is further capable of supporting immune cell adhesion; pathway inhibition blocks iCF matrix production and cell adhesion. Polymorphisms in the noncoding regulatory elements within the 9p21 locus (also referred to as ANRIL) redirect stress programs, and in iCFs, they transcriptionally silence the AP-1 inducible transcription factor GATA5. The presence of these polymorphisms modulate iCF matrix production and assembly and reduce cell-cell signaling. These data suggest that this signaling axis is a critical modulator of cardiac disease models and might be influenced by noncoding regulatory elements.

Conductive electrospun polymer improves stem cell-derived cardiomyocyte function and maturation.

Despite numerous efforts to generate mature human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), cells often remain immature, electrically isolated, and may not reflect adult biology. Conductive polymers are attractive candidates to facilitate electrical communication between hPSC-CMs, especially at sub-confluent cell densities or diseased cells lacking cell-cell junctions. Here we electrospun conductive polymers to create a conductive fiber mesh and assess if electrical signal propagation is improved in hPSC-CMs seeded on the mesh network. Matrix characterization indicated fiber structure remained stable over weeks in buffer, scaffold stiffness remained near in vivo cardiac stiffness, and electrical conductivity scaled with conductive polymer concentration. Cells remained adherent and viable on the scaffolds for at least 5 days. Transcriptomic profiling of hPSC-CMs cultured on conductive substrates for 3 days showed upregulation of cardiac and muscle-related genes versus non-conductive fibers. Structural proteins were more organized and calcium handling was improved on conductive substrates, even at sub-confluent cell densities; prolonged culture on conductive scaffolds improved membrane depolarization compared to non-conductive substrates. Taken together, these data suggest that blended, conductive scaffolds are stable, supportive of electrical coupling in hPSC-CMs, and promote maturation, which may improve our ability to model cardiac diseases and develop targeted therapies.

Age-dependent Lamin changes induce cardiac dysfunction via dysregulation of cardiac transcriptional programs.

As we age, structural changes contribute to progressive decline in organ function, which in the heart act through poorly characterized mechanisms. Taking advantage of the short lifespan and conserved cardiac proteome of the fruit fly, we found that cardiomyocytes exhibit progressive loss of Lamin C (mammalian Lamin A/C homologue) with age, coincident with decreasing nuclear size and increasing nuclear stiffness. Premature genetic reduction of Lamin C phenocopies aging's effects on the nucleus, and subsequently decreases heart contractility and sarcomere organization. Surprisingly, Lamin C reduction downregulates myogenic transcription factors and cytoskeletal regulators, possibly via reduced chromatin accessibility. Subsequently, we find a role for cardiac transcription factors in regulating adult heart contractility and show that maintenance of Lamin C, and cardiac transcription factor expression, prevents age-dependent cardiac decline. Our findings are conserved in aged non-human primates and mice, demonstrating that age-dependent nuclear remodeling is a major mechanism contributing to cardiac dysfunction.

Improved epicardial cardiac fibroblast generation from iPSCs.

Since the initial isolation of human embryonic stem cells and subsequent discovery of reprogramming methods for somatic cells, thousands of protocols have been developed to create each of the hundreds of cell types found in-vivo with significant focus on disease-prone systems, e.g., cardiovascular. Robust protocols exist for many of these cell types, except for cardiac fibroblasts (CF). Very recently, several competing methods have been developed to generate these cells through a developmentally conserved epicardial pathway. Such methods generate epicardial cells, but here we report that prolonged exposure to growth factors such as bFGF induces fibroblast spindle-like morphology and similar chromatin architecture to primary CFs. Media conditions for growth and assays are provided, as well as suggestions for seeding densities and timepoints for protein harvest of extracellular matrix. We demonstrate marker expression and matrix competency of resultant cells as shown next to primary human cardiac fibroblasts. These methods provide additional guidance to the original protocol and result in an increasingly stable phenotype.

E-cigarette Aerosol Mixtures Inhibit Biomaterial-Induced Osseointegrative Cell Phenotypes.

OBJECTIVES: Smoking is a known contributor to the failure of dental implants. Despite a decline in cigarette use, the popularity of e-cigarettes has exploded. However, little is known about how e-cigarettes affect the biologic response to implants. This study examines the effect of e-cigarette aerosol mixtures (ecig-AM) on macrophage activation and osteoblastogenesis of mesenchymal stem cells (MSCs) in response to titanium (Ti) implant surfaces. METHODS: Ecig-AMs were prepared by bubbling aerosol through PBS. Human-derived MSCs or murine-derived macrophages were plated on smooth, rough-hydrophobic, or rough-hydrophilic Ti surfaces in media supplemented with ecig-AM. In macrophages, expression of inflammatory markers was measured by qPCR and macrophage immunophenotype characterized by flow cytometry after 24 hours of exposure. In MSCs, expression of osteogenic markers and inflammatory cytokines was measured by qPCR and ELISA, while alkaline phosphatase activity (ALP) was determined by colorimetric assay. RESULTS: Ecig-AM polarized primary macrophages into a pro-inflammatory state with higher effect on ecig-AM with flavorants and nicotine. Metabolic activity of MSCs decreased in a concentration dependent fashion and was stronger in ecig-AM containing nicotine. MSCs reduced expression of osteogenic markers in response to ecig-AM, but increased RANKL secretion, particularly at the highest ecig-AM concentrations. The effect of ecig-AM exposure was lessened when macrophages or MSCs were cultured on rough-hydrophilic substrates. SIGNIFICANCE: Ecig-AM activated macrophages into a pro-inflammatory phenotype and impaired MSC-to-osteoblast differentiation in response to Ti implant surfaces. These effects were potentiated by flavorants and nicotine, suggesting that e-cigarette use may compromise the osseointegration of dental implants.

Acetabular Bone Marrow Aspiration During Total Hip Arthroplasty.

Biologically augmented surgical treatments of orthopaedic conditions are increasingly popular. Bone marrow aspirate concentrate is a key orthobiologic tissue source, and the field is moving from the standard iliac crest marrow aspiration toward local aspirations of marrow depots that are accessible during the standard-of-care procedures in an attempt to reduce morbidity, surgery time, and cost. Here, we present the aspiration of the standard iliac marrow depot, but through a novel acetabular approach during total hip arthroplasty. This procedure markedly simplifies biologic augmentation with bone marrow aspirate concentrate in this large patient cohort.

Atomic Force Microscopy for Live-Cell and Hydrogel Measurement.

Atomic force microscopy (AFM) has emerged as a popular method for determining the mechanical properties of cells, their components, and biomaterials. Here, we describe AFM setup and application to obtain stiffness measurements from single indentations for hydrogels and myofibroblasts.

Regenerative cross talk between cardiac cells and macrophages.

Aside from the first week postnatal, murine heart regeneration is restricted and responses to damage follow classic fibrotic remodeling. Recent transcriptomic analyses have suggested that significant cross talk with the sterile immune response could maintain a more embryonic-like signaling network that promotes acute, transient responses. However, with age, this response-likely mediated by neonatal yolk sac macrophages-then transitions to classical macrophage-mediated, cardiac fibroblast (CF)-based remodeling of the extracellular matrix (ECM) after myocardial infarction (MI). The molecular mechanisms that govern the change with age and drive fibrosis via inflammation are poorly understood. Using multiple ribonucleic acid sequencing (RNA-Seq) datasets, we attempt to resolve the relative contributions of CFs and macrophages in the bulk-healing response of regenerative (postnatal day 1) and nonregenerative hearts (postnatal day 8+). We performed an analysis of bulk RNA-Seq datasets from myocardium and cardiac fibroblasts as well as a single-cell RNA-Seq dataset from cardiac macrophages. MI-specific pathway differences revealed that nonregenerative hearts generated more ECM and had larger matricellular responses correlating with inflammation, produced greater chemotactic gradients to recruit macrophages, and expressed receptors for danger-associated molecular patterns at higher levels than neonates. These changes could result in elevated stress-response pathways compared with neonates, converging at NF-κB and activator protein-1 (AP-1) signaling. Profibrotic gene programs, which greatly diverge on day 3 post MI, lay the foundation for chronic fibrosis, and thus postnatal hearts older than 7 days typically exhibit significantly less regeneration. Our analyses suggest that the macrophage ontogenetic shift in the heart postnatally could result in detrimental stress signaling that suppresses regeneration.NEW & NOTEWORTHY Immediately postnatal mammalian hearts are able to regenerate after infarction, but the cells, pathways, and molecules that regulate this behavior are unclear. By comparing RNA-Seq datasets from regenerative mouse hearts and older, nonregenerative hearts, we are able to identify biological processes that are hallmarks of regeneration. We find that sterile inflammatory processes are upregulated in nonregenerative hearts, initiating profibrotic gene programs 3 days after myocardial infarction that can cause myocardial disease.

Novel hydrophilic nanostructured microtexture on direct metal laser sintered Ti-6Al-4V surfaces enhances osteoblast response in vitro and osseointegration in a rabbit model.

The purpose of this study was to compare the biological effects in vivo of hierarchical surface roughness on laser sintered titanium-aluminum-vanadium (Ti-6Al-4V) implants to those of conventionally machined implants on osteoblast response in vitro and osseointegration. Laser sintered disks were fabricated to have micro-/nano-roughness and wettability. Control disks were computer numerical control (CNC) milled and then polished to be smooth (CNC-M). Laser sintered disks were polished smooth (LST-M), grit blasted (LST-B), or blasted and acid etched (LST-BE). LST-BE implants or implants manufactured by CNC milling and grit blasted (CNC-B) were implanted in the femurs of male New Zealand white rabbits. Most osteoblast differentiation markers and local factors were enhanced on rough LST-B and LST-BE surfaces in comparison to smooth CNC-M or LST-M surfaces for MG63 and normal human osteoblast cells. To determine if LST-BE implants were osteogenic in vivo, we compared them to implant surfaces used clinically. LST-BE implants had a unique surface with combined micro-/nano-roughness and higher wettability than conventional CNC-B implants. Histomorphometric analysis demonstrated a significant improvement in cortical bone-implant contact of LST-BE implants compared to CNC-B implants after 3 and 6 weeks. However, mechanical testing revealed no differences between implant pullout forces at those time points. LST surfaces enhanced osteoblast differentiation and production of local factors in vitro and improved the osseointegration process in vivo. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2086-2098, 2016.