Abstract P318: Dna Tension Probes Show That Cardiomyocyte Maturation Is Sensitive To The Pico-Newton Traction Forces Transmitted By Integrins

Cardiac muscle cells (CMCs) are the unit cells that comprise the heart. CMCs go through different stages of differentiation and maturation pathways to fully mature into beating cells. These cells can sense and respond to mechanical cues through receptors such as integrins which influence maturation pathways. For example, cell traction forces are important for the differentiation and development of functional CMCs, as CMCs cultured on varying substrate stiffness function differently. Most work in this area has focused on understanding the role of bulk ECM (extracellular matrix) stiffness in mediating the functional fate of CMC. Given that stiffness sensing mechanisms are mediated by individual integrin receptors, an important question in this area pertains to the specific magnitude of integrin piconewton (pN) forces that can trigger CMC functional maturation. To address this knowledge gap, we used DNA adhesion tethers that rupture at specific thresholds of force (~12, ~56, and ~160 pN) to test whether capping peak integrin tension to specific magnitudes affects CMC function. The work shows that adhesion tethers with greater force tolerance lead to functionally mature CMCs as determined by morphology, twitching frequency, transient calcium flux measurements and protein expression (F-actin, vinculin, α-actinin, YAP and SERCA2). Additionally, sacromeric actinin alignment and multinucleation were significantly enhanced as the mechanical tolerance of integrin tethers was increased. Taken together, the results show that CMCs harness defined pN integrin forces to influence early stage development. This study represents an important step toward biophysical characterization of the contribution of pN forces in early stage cardiac differentiation.

Acute Exhaustive Exercise under Normoxic and Normobaric Hypoxic Conditions Differentially Regulates Angiogenic Biomarkers in Humans

Background and Objectives: Angiogenesis describes the outgrowth of new capillaries from already existing ones. Different biomarkers regulate this process. Physical exercise and hypoxia are key stimuli for the activation of different angiogenic molecules, such as the vascular endothelial growth factor (VEGF). matrix metalloproteases (MMPs)-2 and -9 or the extracellular matrix cleavage fragment endostatin. The present study aimed to investigate influences of short-term, intensive cycling exercise under both normoxic and normobaric hypoxic conditions on the mentioned parameters. Materials and Methods: Twelve male subjects (age: 23.3 ± 2.0 years) participated in the study. All subjects conducted four intensive cycling tests until individual exhaustion in a randomized order under the following conditions: normoxia, 2000 m, 3000 m and 4000 m above sea level. Blood samples were taken before (pre) and 10 min, 30 min, 60 min and 240 min post exercise and were analyzed by ELISA. Results: VEGF showed a significantly reduced concentration compared to the pre-value solely under 4000 m at 10 min post exercise. MMP-2 showed significantly reduced concentrations at 240 min post exercise under 4000 m. MMP-9 increased at 240 min post exercise under both 2000 m and 4000 m conditions. Endostatin was significantly increased at 10 min post exercise independently of the applied stimulus. Conclusions: The presented data show that intensive short-term exercise bouts facilitate the bioavailability of angiogenic, ECM (extracellular matrix)-related biomarkers. This finding is interesting for both health- and performance-related research as it demonstrates the positive effects of intensive short exercise interventions.

A pulsatile release platform based on photo-induced imine-crosslinking hydrogel promotes scarless wound healing

Effective healing of skin wounds is essential for our survival. Although skin has strong regenerative potential, dysfunctional and disfiguring scars can result from aberrant wound repair. Skin scarring involves excessive deposition and misalignment of ECM (extracellular matrix), increased cellularity, and chronic inflammation. Transforming growth factor-β (TGFβ) signaling exerts pleiotropic effects on wound healing by regulating cell proliferation, migration, ECM production, and the immune response. Although blocking TGFβ signaling can reduce tissue fibrosis and scarring, systemic inhibition of TGFβ can lead to significant side effects and inhibit wound re-epithelization. In this study, we develop a wound dressing material based on an integrated photo-crosslinking strategy and a microcapsule platform with pulsatile release of TGF-β inhibitor to achieve spatiotemporal specificity for skin wounds. The material enhances skin wound closure while effectively suppressing scar formation in murine skin wounds and large animal preclinical models. Our study presents a strategy for scarless wound repair.

Modulation of Mammalian Cardiomyocyte Cytokinesis by the Extracellular Matrix

Rationale: After birth, cycling mammalian CMs (cardiomyocytes) progressively lose the ability to undergo cytokinesis and hence they become binucleated, which leads to cell cycle exit and loss of regenerative capacity. During late embryonic and early postnatal heart growth, CM development is accompanied by an expansion of the cardiac fibroblast (cFb) population and compositional changes in the ECM (extracellular matrix). Whether and how these changes influence cardiomyocyte cytokinesis is currently unknown. Objective: To elucidate the role of postnatal cFbs and the ECM in cardiomyocyte cytokinesis and identify ECM proteins that promote cardiomyocyte cytokinesis. Methods and Results: Using primary rat cardiomyocyte cultures, we found that a proportion of postnatal, but not embryonic, cycling cardiomyocytes fail to progress through cytokinesis and subsequently binucleate, consistent with published reports of in vitro and in vivo observations. Direct coculture with postnatal cFbs increased cardiomyocyte binucleation, which could be inhibited by RGD peptide treatment. In contrast, cFb-conditioned medium or transwell coculture did not significantly increase cardiomyocyte binucleation, suggesting that cFbs inhibit cardiomyocyte cytokinesis through ECM modulation rather than by secreting diffusible factors. Furthermore, we found that both embryonic and postnatal CMs binucleate at a significantly higher rate when cultured on postnatal cFb-derived ECM compared with embryonic cFb-derived ECM. These cytokinetic defects correlate with cardiomyocyte inefficiency in mitotic rounding, a process which is key to successful cytokinesis. To identify ECM proteins that modulate cardiomyocyte cytokinesis, we compared the composition of embryonic and postnatal cFb-derived ECM by mass spectrometry followed by functional assessment. We found that 2 embryonically enriched ECM proteins, SLIT2 and NPNT (nephronectin), promote cytokinesis of postnatal CMs in vitro and in vivo. Conclusions: We identified the postnatal cardiac ECM as a nonpermissive environment for cardiomyocyte cytokinesis and uncovered novel functions for the embryonic ECM proteins SLIT2 and NPNT (nephronectin) in promoting postnatal cardiomyocyte cytokinesis. Graphic Abstract: A graphic abstract is available for this article.

TGFβ (Transforming Growth Factor-Beta)–Activated Kinase 1 Regulates Arteriovenous Fistula Maturation

Objective: Arteriovenous fistulae (AVF) are the optimal conduit for hemodialysis access but have high rates of primary maturation failure. Successful AVF maturation requires wall thickening with deposition of ECM (extracellular matrix) including collagen and fibronectin, as well as lumen dilation. TAK1 (TGFβ [transforming growth factor-beta]–activated kinase 1) is a mediator of noncanonical TGFβ signaling and plays crucial roles in regulation of ECM production and deposition; therefore, we hypothesized that TAK1 regulates wall thickening and lumen dilation during AVF maturation. Approach and Results: In both human and mouse AVF, immunoreactivity of TAK1, JNK (c-Jun N-terminal kinase), p38, collagen 1, and fibronectin was significantly increased compared with control veins. Manipulation of TAK1 in vivo altered AVF wall thickening and luminal diameter; reduced TAK1 function was associated with reduced thickness and smaller diameter, whereas activation of TAK1 function was associated with increased thickness and larger diameter. Arterial magnitudes of laminar shear stress (20 dyne/cm 2 ) activated noncanonical TGFβ signaling including TAK1 phosphorylation in mouse endothelial cells. Conclusions: TAK1 is increased in AVF, and TAK1 manipulation in a mouse AVF model regulates AVF thickness and diameter. Targeting noncanonical TGFβ signaling such as TAK1 might be a novel therapeutic approach to improve AVF maturation.

Extracellular Matrix dari Vesica Urinaria Babi Mempercepat Kesembuhan Luka Sayat Tanpa Menimbulkan Anemia dan Leukositosis pada Tikus Putih

Luka terbuka memerlukan biomaterial untuk mempercepat proses kesembuhan. extracellular matrix (ECM) dari vesica urinaria babi merupakan salah satu biomaterial yang sering digunakan sebagai perancah sehingga pada resipien proses kesembuhan luka menjadi lebih cepat. Keamanan penggunaan extracellular matrix dapat dianalisa melalui gambaran darah. Tujuan penelitian ini untuk mengetahui biokompatibilitas bahan ECM yang berasal dari vesica urinaria babi dievaluasi dari hasil pemeriksaan darah khususnya total eritrosit, kadar hemoglobin, dan total leukosit. Penelitian ini menggunakan 32 ekor tikus putih jantan yang telah dianestesi kemudian diberikan luka insisi pada punggung sepanjang 2 cm dengan kedalaman 0,2 mm dan dibagi kedalam dua kelompok. KI sebagai kontrol, tidak diberi bahan ECM dan KII sebagai perlakuan, diberi bahan ECM. Extracellular matrix dari vesica urinaria babi dibuat menggunakan metode Freytes yang sedikit dimodifikasi. Extracellular matrix berbentuk serat halus diaplikasikan sampai menutupi daerah luka sayat kemudian dioleskan vaselin sebagai perekat ECM pada luka. Pengambilan sampel darah dilakukan empat kali yaitu pada hari ke-1, hari ke-5, hari ke-10, hari ke-15 pasca pembedahan, sampel darah diambil dari vena lateralis sebanyak 0,5 ml dan kemudian diperiksa menggunakan mesin Animal Blood Counter iCell-800Vet. Data yang diperoleh dianalisis secara statistik dengan analisis ragam. Hasil penelitian menunjukkan pemberian ECM dari vesica urinaria babi dapat mempercepat kesembuhan luka tanpa menimbulkan perubahan nilai total eritrosit, kadar hemoglobin, dan total leukosit dari nilai rujukan normal, sehingga dapat disimpulkan ECM dari vesica urinaria babi tidak memiliki efek negatif terhadap profil hematologi tikus putih jantan.

Chlorogenic Acid Alleviates Thiram-Induced Tibial Dyschondroplasia by Modulating Caspases, BECN1 Expression and ECM Degradation

Chlorogenic acid (CGA) is a widely applied traditional Chinese medicine ingredient which can be used for the treatment of osteoporosis. In this experiment, we investigated the potential therapeutic effect of chlorogenic acid on thiram-induced tibial dyschondroplasia (TD) and explored the underlying mechanisms that have been rarely mentioned by others yet. Performance indicator analysis and tibial parameter analysis showed that CGA exhibited a definite positive effect on thiram-induced TD chickens. In order to further explore the mechanisms underlying the positive actions of CGA, apoptotic, autophagic genes and MMPs involved in matrix mineralization of growth plate were evaluated in this study. The results showed that CGA decreased the expression of pro-apoptotic genes caspases-3 and caspases-9, leading to the reduction of apoptotic cells accumulated in growth plate. In addition, CGA also increased the level of BECN1, an important gene involved in autophagy, which benefits the survival of abnormal cells. Furthermore, CGA also increased the expression of MMP-9, MMP-10, and MMP-13, which can directly affect the ossification of bones. Altogether, these results demonstrate that CGA possesses a positive therapeutic effect on thiram-induced TD via modulating the expression of caspases and BECN1 and regulating the degradation of ECM (extracellular matrix).

Role of miRNAs in Melanoma Metastasis

Tumour metastasis is a multistep process. Melanoma is a highly aggressive cancer and metastasis accounts for the majority of patient deaths. microRNAs (miRNAs) are non-coding RNAs that affect the expression of their target genes. When aberrantly expressed they contribute to the development of melanoma. While miRNAs can act locally in the cell where they are synthesized, they can also influence the phenotype of neighboring melanoma cells or execute their function in the direct tumour microenvironment by modulating ECM (extracellular matrix) and the activity of fibroblasts, endothelial cells, and immune cells. miRNAs are involved in all stages of melanoma metastasis, including intravasation into the lumina of vessels, survival during circulation in cardiovascular or lymphatic systems, extravasation, and formation of the pre-metastatic niche in distant organs. miRNAs contribute to metabolic alterations that provide a selective advantage during melanoma progression. They play an important role in the development of drug resistance, including resistance to targeted therapies and immunotherapies. Distinct profiles of miRNA expression are detected at each step of melanoma development. Since miRNAs can be detected in liquid biopsies, they are considered biomarkers of early disease stages or response to treatment. This review summarizes recent findings regarding the role of miRNAs in melanoma metastasis.

Decellularised Human Liver is too Heterogeneous for Designing a Generic ECM-mimic Hepatic Scaffold

Decellularised human livers are considered the perfect ECM (extracellular matrix) surrogate because both 3-dimensional architecture and biological features of the hepatic microenvironment are thought to be preserved. However, donor human livers are in chronically short supply, both for transplantation or as decellularised scaffolds, and will become even scarcer as life expectancy increases. It is hence of interest to determine the structural and biochemical properties of human hepatic ECM to derive design criteria for engineering bio-mimetic scaffolds. The intention of this work was to obtain quantitative design specifications for fabricating scaffolds for hepatic tissue engineering using human livers as a template. To this end, hepatic samples from 5 human donors were decellularised using a protocol shown to reproducibly conserve matrix composition and micro-structure in porcine livers. The decellularisation outcome was evaluated through histological and quantitative image analyses to evaluate cell removal, protein and glycosaminoglycan content per unit area. Applying the same decellularisation protocol to human liver samples obtained from 5 different donors yielded 5 different outcomes. Only 1 liver out of 5 was completely decellularised, while the other 4 showed different levels of remaining cells. Moreover, protein and glycosaminoglycan content per unit area after decellularisation were also found to be donor-dependent. The donor-to-donor variability of human livers thus precludes their use as templates for engineering a generic 'one-size fits all' ECM-mimic hepatic scaffold.

Pathogenic aspects and therapeutic avenues of intestinal fibrosis in Crohn's disease

In Crohn's disease, one of the two major forms of inflammatory bowel diseases in human beings, persistent and chronic inflammation promotes fibrotic processes thereby facilitating formation of strictures, the most common indication for surgical intervention in this disorder. The pathogenesis of Crohn's disease-associated fibrosis is not fully understood, but variants of genes involved in the recognition of microbial components/products [e.g. CARD15 (caspase-activating recruitment domain 15) and ATG16L1 (autophagy-related 16-like 1)] are associated with this phenotype, and experimental evidence suggests that intestinal fibrosis results from an altered balance between deposition of ECM (extracellular matrix) and degradation of ECM by proteases. Studies have also contributed to identify the main phenotypic and functional alterations of cells involved in the fibrogenic process, as well as molecules that stimulate such cells to produce elevated amounts of collagen and other ECM-related proteins. In the present review, we assess the current knowledge about cellular and molecular mediators of intestinal fibrosis and describe results of recent studies aimed at testing the preventive/therapeutic effect of compounds in experimental models of intestinal fibrosis.