Macrophage-dependent IL-1β production induces cardiac arrhythmias in diabetic mice

Diabetes mellitus (DM) encompasses a multitude of secondary disorders, including heart disease. One of the most frequent and potentially life threatening disorders of DM-induced heart disease is ventricular tachycardia (VT). Here we show that toll-like receptor 2 (TLR2) and NLRP3 inflammasome activation in cardiac macrophages mediate the production of IL-1β in DM mice. IL-1β causes prolongation of the action potential duration, induces a decrease in potassium current and an increase in calcium sparks in cardiomyocytes, which are changes that underlie arrhythmia propensity. IL-1β-induced spontaneous contractile events are associated with CaMKII oxidation and phosphorylation. We further show that DM-induced arrhythmias can be successfully treated by inhibiting the IL-1β axis with either IL-1 receptor antagonist or by inhibiting the NLRP3 inflammasome. Our results establish IL-1β as an inflammatory connection between metabolic dysfunction and arrhythmias in DM.Facultad de Ciencias MédicasCentro de Investigaciones Cardiovasculare

Macrophage-dependent IL-1β production induces cardiac arrhythmias in diabetic mice

Diabetes mellitus (DM) encompasses a multitude of secondary disorders, including heart disease. One of the most frequent and potentially life threatening disorders of DM-induced heart disease is ventricular tachycardia (VT). Here we show that toll-like receptor 2 (TLR2) and NLRP3 inflammasome activation in cardiac macrophages mediate the production of IL-1β in DM mice. IL-1β causes prolongation of the action potential duration, induces a decrease in potassium current and an increase in calcium sparks in cardiomyocytes, which are changes that underlie arrhythmia propensity. IL-1β-induced spontaneous contractile events are associated with CaMKII oxidation and phosphorylation. We further show that DM-induced arrhythmias can be successfully treated by inhibiting the IL-1β axis with either IL-1 receptor antagonist or by inhibiting the NLRP3 inflammasome. Our results establish IL-1β as an inflammatory connection between metabolic dysfunction and arrhythmias in DM.Facultad de Ciencias MédicasCentro de Investigaciones Cardiovasculare

A fractal nature for polymerized laminin

Polylaminin (polyLM) is a non-covalent acid-induced nano- and micro-structured polymer of the protein laminin displaying distinguished biological properties. Polylaminin stimulates neuritogenesis beyond the levels achieved by ordinary laminin and has been shown to promote axonal regeneration in animal models of spinal cord injury. Here we used confocal fluorescence microscopy (CFM), scanning electron microscopy (SEM) and atomic force microscopy (AFM) to characterize its three-dimensional structure. Renderization of confocal optical slices of immunostained polyLM revealed the aspect of a loose flocculated meshwork, which was homogeneously stained by the antibody. On the other hand, an ordinary matrix obtained upon adsorption of laminin in neutral pH (LM) was constituted of bulky protein aggregates whose interior was not accessible to the same anti-laminin antibody. SEM and AFM analyses revealed that the seed unit of polyLM was a flat polygon formed in solution whereas the seed structure of LM was highly heterogeneous, intercalating rod-like, spherical and thin spread lamellar deposits. As polyLM was visualized at progressively increasing magnifications, we observed that the morphology of the polymer was alike independently of the magnification used for the observation. A search for the Hausdorff dimension in images of the two matrices showed that polyLM, but not LM, presented fractal dimensions of 1.55, 1.62 and 1.70 after 1, 8 and 12 hours of adsorption, respectively. Data in the present work suggest that the intrinsic fractal nature of polymerized laminin can be the structural basis for the fractal-like organization of basement membranes in the neurogenic niches of the central nervous system.This work was supported by a grant from the Brazilian National Research Council (CNPq; 476772/2008-7) to TCS. MSS acknowledges support from the European Research Council through ERC - 306990. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Hochman Méndez, C.; Cantini ., M.; Moratal Pérez, D.; Salmerón Sánchez, M.; Coelho-Sampaio, T. (2014). A fractal nature for polymerized laminin. PLoS ONE. 9(10):109388-1-109388-11. https://doi.org/10.1371/journal.pone.0109388S109388-1109388-11910Durbeej, M. (2009). Laminins. Cell and Tissue Research, 339(1), 259-268. doi:10.1007/s00441-009-0838-2Miner, J. H., & Yurchenco, P. D. (2004). LAMININ FUNCTIONS IN TISSUE MORPHOGENESIS. Annual Review of Cell and Developmental Biology, 20(1), 255-284. doi:10.1146/annurev.cellbio.20.010403.094555Yurchenco, P. D. (2010). Basement Membranes: Cell Scaffoldings and Signaling Platforms. Cold Spring Harbor Perspectives in Biology, 3(2), a004911-a004911. doi:10.1101/cshperspect.a004911Hohenester, E., & Yurchenco, P. D. (2013). 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Change the Laminin, Change the Cardiomyocyte: Improve Untreatable Heart Failure

No effective medical treatment exists for heart failure with preserved ejection fraction (HFpEF), accounting for approximately half of all heart failure cases. The elevated passive myocardial stiffness in HFpEF is attributed to a combination of alterations in the extracellular matrix (ECM) collagen content and modifications in the sarcomeric protein titin. Here, we propose polylaminin, a biomimetic polymer of laminin, as a promising approach for manipulating the titin isoform shift and phosphorylation in cardiomyocytes. Exploring the pleiotropic effects of polylaminin may be a novel strategy for alleviating symptoms in HFpEF’s multifactorial pathophysiology

Mining the Mesenchymal Stromal Cell Secretome in Patients with Chronic Left Ventricular Dysfunction

Close examination of the initial results of cardiovascular cell therapy clinical trials indicates the importance of patient-specific differences on outcomes and the need to optimize or customize cell therapies. The fields of regenerative medicine and cell therapy have transitioned from using heterogeneous bone marrow mononuclear cells (BMMNCs) to mesenchymal stromal cells (MSCs), which are believed to elicit benefits through paracrine activity. Here, we examined MSCs from the BMMNCs of heart failure patients enrolled in the FOCUS-CCTRN trial. We sought to identify differences in MSCs between patients who improved and those who declined in heart function, regardless of treatment received. Although we did not observe differences in the cell profile of MSCs between groups, we did find significant differences in the MSC secretome profile between patients who improved or declined. We conclude that “mining” the MSC secretome may provide clues to better understand the impact of patient characteristics on outcomes after cell therapy and this knowledge can inform future cell therapy trials

Laminin as a Potent Substrate for Large-Scale Expansion of Human Induced Pluripotent Stem Cells in a Closed Cell Expansion System

The number of high-quality cells required for engineering an adult human-sized bioartificial organ is greater than one billion. Until the emergence of induced pluripotent stem cells (iPSCs), autologous cell sources of this magnitude and with the required complexity were not available. Growing this number of cells in a traditional 2D cell culture system requires extensive time, resources, and effort and does not always meet clinical requirements. The use of a closed cell culture system is an efficient and clinically applicable method that can be used to expand cells under controlled conditions. We aimed to use the Quantum Cell Expansion System (QES) as an iPSC monolayer-based expansion system. Human iPSCs were expanded (up to 14-fold) using the QES on two different coatings (laminin 521 (LN521) and vitronectin (VN)), and a karyotype analysis was performed. The cells were characterized for spontaneous differentiation and pluripotency by RT-PCR and flow cytometry. Our results demonstrated that the QES provides the necessary environment for exponential iPSC growth, reaching 689.75 × 106 ± 86.88 × 106 in less than 7 days using the LN521 coating with a population doubling level of 3.80 ± 0.19. The same result was not observed when VN was used as a coating. The cells maintained normal karyotype (46-XX), expressed pluripotency markers (OCT4, NANOG, LIN28, SOX2, REX1, DPPA4, NODAL, TDGFb, TERT3, and GDF), and expressed high levels of OCT4, SOX2, NANOG, SSEA4, TRA1-60, and TRA1-81. Spontaneous differentiation into ectoderm (NESTIN, TUBB3, and NEFH), mesoderm (MSX1, BMP4, and T), and endoderm (GATA6, AFP, and SOX17) lineages was detected by RT-PCR with both coating systems. We conclude that the QES maintains the stemness of iPSCs and is a promising platform to provide the number of cells necessary to recellularize small human-sized organ scaffolds for clinical purposes

Polymerized Laminin-521: A Feasible Substrate for Expanding Induced Pluripotent Stem Cells at a Low Protein Concentration

Laminins (LNs) play a central role in the self-assembly and maintenance of basement membranes and are involved in critical interactions between cells and other extracellular matrix (ECM) proteins. Among the defined, xeno-free ECM culture matrices, LNs—namely LN521—have emerged as promising coating systems for the large-scale expansion of induced pluripotent stem cells (iPSCs). The biologic activity of LNs is enhanced by their acidification-induced self-polymerization into a cell-associated network called polylaminin (polyLN), which can recapitulate the native-like polymeric array in a cell-free system. Here, we show for the first time to our knowledge that polyLN521 displays a native-like hexagonal-like structure and that, at basal and low concentrations, it permits the large-scale expansion of human iPSCs. Human iPSCs expanded with polyLN521 maintained the pluripotent state and showed no impairment of karyotype stability or telomere length. These results suggest that low-concentration polyLN521 is a stable and cost-effective coating for large-scale iPSC expansion

Characterization of polymer units in polyLM and LM.

<p>Laminin polymers were analyzed at high magnification in order to characterize the morphologies of the seed units of each polymer. (A–C) At 1 hour polyLM forms star-like 2D structures as exemplified in the three panels. (D) The sizes of the longer axes in these structures were quantified and shown to average at 20.84±5.449 µm. (E, F) High magnification images of polyLM at 8 and 12 hours show a meshwork pattern compatible with the deposition of the star-like structures. (G–I) LM observed at high magnification reveals three types of seed structures: rods (pseudocolored yellow), spheres (pseudocolored pink) and lamellas (pesudocolored green). The scale bar in I applies to all panels and represents 10 µm.</p

Atomic force microscopy reveals the occurrence of hexagonal-like figures in polyLM.

<p>AFM was performed on polyLM matrices obtained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109388#pone-0109388-g004" target="_blank">Figure 4</a> and areas of 1 (A, B) or 0.25 µm² (C) were scanned in height mode. Hexagons-like figures similar to those occurring in natural laminin polymers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109388#pone.0109388-Yurchenco3" target="_blank">[12]</a> were identified. These hexagons were visible at different magnifications (A–C) and presented variable side lengths (sketched with white dashed lines), but they were never as short as 30 nm as they should be to correspond to the short arm of a laminin molecule. The smallest distinguishable structures contained within the sides of the hexagons were little globules (D) whose size and spacing was measured in images of 0.02 µm² (D). Panel E shows the distribution of spacing values, which are compatible with the characteristic length (∼30 nm) of laminin polymerized via the short arms. Panel F depicts a three-dimensional reconstruction of the same area shown in panel D, with superposition of compatible locations of laminin molecules. Schemes of one individual laminin molecule (long arm dashed and short arms colored blue, green and orange), with indication of its characteristic dimensions (G) and of the hexagonal polymer generated by the interaction between individual laminin molecules (H) are also shown.</p