29 research outputs found

    ROLE OF TOPOGRAPHIC CUES ON CANCER CELL PROLIFERATION

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    Ph.DPH.D. IN MECHANOBIOLOGY (FOS

    A NOVEL ROLE OF THE MECHANO-SENSING PROTEIN P130CAS IN SUPPORTING BONE HOMEOSTASIS

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    Ph.DDOCTOR OF PHILOSOPH

    The effect of matrix stiffness on vascular smooth muscle cell contractility

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    Vascular smooth muscle cells (VSMCs) typically line the medial layer of the arterial wall, and exist in a quiescent contractile phenotype to regulate vessel tone. However, during ageing and early cardiovascular disease (CVD) development, the arterial wall becomes more rigid, and under these conditions, VSMC de-differentiate into the synthetic proliferative phenotype where they instead contribute to vessel repair. Arterial stiffness is a key predicative biomarker of CVD and our work focuses on the response of the VSMCs to the less compliant extracellular matrix (ECM). We hypothesise aberrations in VSMC structure and function in response to matrix stiffness, and speculate that this may contribute to the pathological vessel wall remodelling typically observed within CVD. To test this, we fabricated polyacrylamide gels with rigidities representative of both physiological (12kPa) and pathological (72kPa) stiffness. Our work presents an increase in VSMC force generation in response to matrix stiffness via traction force microscopy (TFM). We show this to occur via novel mechanisms and, importantly, highlight the key mechanosensors mediating this. When seeded on that 72kPa hydrogel, quiescent VSMCs were shown to undergo hypertrophy causing increased DNA damage. Our study identifies stretch activated channels (SACs) and N-acetyltransferase 10 (NAT10) as critical mechanosensors that facilitate this, as inhibition of both restored healthy morphology. Importantly, we highlight Piezo1 as a novel SAC within stiffness-induced VSMC dysregulation. Using qPCR, we show Piezo1 gene expression to increase in response to matrix rigidity, and also reveal that its knockdown, via siRNA-mediated methods, can reduce DNA damage accumulation. Due to this, we predict there to be an intricate crosstalk between the cytoskeletal networks and key mechanosensors present at the cell membrane, and introduce Piezo1 and NAT10 as therapeutic targets for stiffness-induced quiescent VSMC dysregulation

    Vascular smooth muscle cells and arterial stiffening : relevance in development, aging, and disease

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    The cushioning function of large arteries encompasses distension during systole and recoil during diastole which transforms pulsatile flow into a steady flow in the microcirculation. Arterial stiffness, the inverse of distensibility, has been implicated in various etiologies of chronic common and monogenic cardiovascular diseases and is a major cause of morbidity and mortality globally. The first components that contribute to arterial stiffening are extracellular matrix (ECM) proteins that support the mechanical load, while the second important components are vascular smooth muscle cells (VSMCs), which not only regulate actomyosin interactions for contraction but mediate also mechanotransduction in cell-ECM homeostasis. Eventually, VSMC plasticity and signaling in both conductance and resistance arteries are highly relevant to the physiology of normal and early vascular aging. This review summarizes current concepts of central pressure and tensile pulsatile circumferential stress as key mechanical determinants of arterial wall remodeling, cell-ECM interactions depending mainly on the architecture of cytoskeletal proteins and focal adhesion, the large/small arteries cross-talk that gives rise to target organ damage, and inflammatory pathways leading to calcification or atherosclerosis. We further speculate on the contribution of cellular stiffness along the arterial tree to vascular wall stiffness. In addition, this review provides the latest advances in the identification of gene variants affecting arterial stiffening. Now that important hemodynamic and molecular mechanisms of arterial stiffness have been elucidated, and the complex interplay between ECM, cells, and sensors identified, further research should study their potential to halt or to reverse the development of arterial stiffness

    Regulation Of The Human Vascular Smooth Muscle Cell Transcriptome By Extracellular Matrix Stiffness

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    Arterial stiffness is a risk factor for several cardiometabolic diseases and is caused by pathological remodeling of the vascular extracellular matrix (ECM). Vascular smooth muscle cells (VSMCs) respond to ECM stiffness by proliferating, migrating, and further remodeling the vascular ECM, thus contributing to vascular disease like atherosclerosis and hypertension. VSMCs along the vasculature are highly diverse as they arise from different embryologic origins, reside in ECMs of diverse compositions, and are exposed to various mechanical forces. This dissertation aims to understand how ECM stiffness regulates the transcriptional response of VSMCs from different origins, namely aortic (Ao) and coronary (Co) VSMCs. We conducted deep sequencing of RNA from Ao and Co VSMCs grown on engineered polyacrylamide hydrogel surfaces tuned to physiologic and pathologic stiffness. Using several bioinformatic approaches, we compared the transcriptional landscapes in Ao and Co VSMCs by looking at whole-gene level expression, splicing, and conservation properties, with a focus on long non-coding RNAs (lncRNAs), as they compose a significant portion of the unexplored VSMC transcriptome. We found evidence suggesting that the overall transcriptional response to stiffness may be conserved across species and cell types, and that stiffness significantly dictates VSMC transcriptional identity over contributions from embryologic origins. However, we also discovered instances of origin-specific stiffness responses in stiffness-mediated lncRNA expression and stiffness-mediated splicing. We identified a highly correlated network of adjacent stiffness-sensitive lncRNAs-protein coding gene pairs, which led us to experimentally interrogate the lncRNA PACER as a regulator of stiffness-mediate expression. We surprisingly found that PACER’s previously established regulatory pathway is absent in VSMCs. Using enrichment methods, we identified TBX5 and show that it is a stiffness-sensitive transcription factor specific to Co VSMCs. We also demonstrated a new role for MALAT1 as a lncRNA regulator of stiffness-dependent VSMC proliferation and migration. Thus, this dissertation reveals many novel characteristics of the VSMC stiffness-regulated transcriptome that may have clinical utility in understanding and managing the pathogenesis of arterial stiffening

    Noncanonical NF-KB Signaling Drives Glioma Invasion by Promoting MT1-MMP Activation, Pseudopodia Formation, and ITGA11 Expression

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    A hallmark of high grade glioma is highly aggressive, diffuse invasion into normal brain tissue, contributing to a 100% recurrence rate and resistance to current therapies. Recent efforts to determine molecular differences in high grade glioma and define tumor subtypes have revealed that the noncanonical NF-KB transcription factor RelB is upregulated in the highly aggressive mesenchymal subtype, as well as in recurrent tumors. The studies presented here seek to better understand how noncanonical NF-KB signaling drives glioma cell invasion in a three-dimensional (3D) environment. Stabilization of NF- KB-inducing kinase (NIK), a critical driver of noncanonical NF-KB signaling, promoted glioma cell adhesion, spreading, and pseudopodia formation on collagen, which is expressed at low levels in normal brain tissue but highly upregulated within the stroma and surrounding tissue of glioma. As NIK expression appeared to regulate glioma cell behavior on collagen, we investigated whether NIK controls the expression of integrins known to bind collagen. We found NIK expression upregulated the integrin alpha 11 subunit (ITGA11), while it did not significantly affect the expression of ITGA1, ITGA2, or ITGA10. Analysis of human tumor samples revealed that ITGA11 expression was increased in glioma tissue compared to normal brain tissue. Furthermore, when testing multiple glioma lines, ITGA11 expression positively correlated with invasiveness into 3D collagen matrices. Investigation of a key transmembrane metalloproteinase revealed that NIK expression enhanced the localization of active, phosphorylated membrane-type 1 matrix metalloproteinase (MT1-MMP) to pseudopodial structures. In a heterologous system, ITGA11 and MT1-MMP formed a complex, suggesting these transmembrane proteins could interact in glioma cells to facilitate coordinated recognition and degradation of collagen during invasion. Finally, silencing of ITGA11 in an invasive glioma line attenuated invasion into 3D collagen matrices. Collectively, these data reveal an ability of NIK to promote directed glioma cell invasion, pseudopodia formation, ITGA11 expression, and activated MT1-MMP localization to pseudopodia. These data suggest ITGA11 could serve as a novel marker for more invasive glioma and a potential therapeutic target in glioma

    Dichotomic role of NAADP/two-pore channel 2/Ca2+ signaling in regulating neural differentiation of mouse embryonic stem cells

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    Poster Presentation - Stem Cells and Pluripotency: abstract no. 1866The mobilization of intracellular Ca2+stores is involved in diverse cellular functions, including cell proliferation and differentiation. At least three endogenous Ca2+mobilizing messengers have been identified, including inositol trisphosphate (IP3), cyclic adenosine diphosphoribose (cADPR), and nicotinic adenine acid dinucleotide phosphate (NAADP). Similar to IP3, NAADP can mobilize calcium release in a wide variety of cell types and species, from plants to animals. Moreover, it has been previously shown that NAADP but not IP3-mediated Ca2+increases can potently induce neuronal differentiation in PC12 cells. Recently, two pore channels (TPCs) have been identified as a novel family of NAADP-gated calcium release channels in endolysosome. Therefore, it is of great interest to examine the role of TPC2 in the neural differentiation of mouse ES cells. We found that the expression of TPC2 is markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebound during the late stages of neurogenesis. Correspondingly, perturbing the NAADP signaling by TPC2 knockdown accelerates mouse ES cell differentiation into neural progenitors but inhibits these neural progenitors from committing to the final neural lineage. Interestingly, TPC2 knockdown has no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Overexpression of TPC2, on the other hand, inhibits mouse ES cell from entering the neural lineage. Taken together, our data indicate that the NAADP/TPC2-mediated Ca2+signaling pathway plays a temporal and dichotomic role in modulating the neural lineage entry of ES cells; in that NAADP signaling antagonizes ES cell entry to early neural progenitors, but promotes late neural differentiation.postprin

    Immune and stress factors in the pathophysiology of the mdx mouse model of Duchenne Muscular Dystrophy

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    Duchenne Muscular Dystrophy (DMD) is a fatal multi-system neuromuscular disease caused by loss of dystrophin. The loss of dystrophin from membranes of contractile muscle cells and the dysregulation of the DAPC, induces chronic inflammation due to tissue necrosis and eventual replacement with collagen which weakens muscular force and strength. Dystrophin deficiency may cause under-diagnosed features of DMD include mood disorders such as depression and anxiety and dysfunction of the gastrointestinal tract. The first study in the thesis examined mood in the dystrophin-deficient mdx mouse model of DMD and examined the effects of the tri-cyclic antidepressant, amitriptyline on behaviours. Amitriptyline had anti-depressant and anxiolytic effects in the mdx mice possibly through effects on stress factors such as corticotrophin-releasing factor (CRF). This antidepressant also reduced skeletal muscle inflammation and caused a reduction in circulating interleukin (IL)-6 levels. In the second and third studies, we specifically blocked IL-6 signalling and used Urocortin 2, CRFR2 agonist to investigate their potential as therapeutic targets in mdx mice pathophysiology. Isometric and isotonic contractile properties of the diaphragm, were compared in mdx mice treated with anti IL-6 receptor antibodies (anti IL-6R) and/or Urocortin 2. Deficits in force production, work and power detected in mdx mice were improved with treatment. In study three I investigated contractile properties in gastrointestinal smooth muscle. As compared to wild type mice, mdx mice had slower faecal transit times, shorter colons with thickened muscle layers and increased contractile activity in response to recombinant IL-6. Blocking IL-6 signalling resulted in an increase in colon length, normalised faecal output times and a reduction in IL-6-evoked contractile activity. The findings from these studies indicate that for both diaphragm and gastrointestinal function in a dystrophin-deficient model, targeting of IL-6 and CRFR2 signalling has beneficial therapeutic effects
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