6 research outputs found

    Increased SK3 expression in DM1 lens cells leads to impaired growth through a greater calcium-induced fragility

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    Although cataract is a characteristic feature of myotonic dystrophy type 1 (DM1), little is known of the underlying mechanisms. We generated four lens epithelial cell lines derived from DM1 cataracts and two from age-matched, non-DM cataracts. Small-pool PCR revealed typical large triplet repeat expansions in the DM1 cells. Furthermore, real-time PCR analysis showed reduced SIX5 expression and increased expression of the Ca2+-activated K+ channel SK3 in the DM1 cells. These cells also exhibited longer population doubling times which did not arise through reduced proliferation, but rather increased cell death as shown by increased release of lactate dehydrogenase (LDH). Using 86Rb+ as a tracer for K+, we found no difference in the resting K+ influx or efflux kinetics. In all cases, the ouabain sensitive component of the influx contributed ~50% of the total. However, stimulating internal Ca2+ by exposure to ionomycin not only caused greater stimulation of K+ (86Rb) efflux in the DM1 cells but also induced a higher rate of cell death (LDH assay). Since both the hyper-stimulation of K+ efflux and cell death were reduced by the highly specific SK inhibitor apamin, we suggest that increased expression of SK3 has a critical role in the increased Ca2+-induced fragility in DM1 cells. The present data, therefore, both help explain the lower epithelial cell density previously observed in DM1 cataracts and provide general insights into mechanisms underlying the fragility of other DM1-affected tissues

    Vascular mechanisms of post-COVID-19 conditions: rho-kinase is a novel target for therapy

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    BackgroundIn post-COVID-19 conditions (Long COVID), systemic vascular dysfunction is implicated but the mechanisms are uncertain, and treatment is imprecise.MethodsPatients convalescing after hospitalisation for COVID-19 and risk-factor matched controls underwent multisystem phenotyping using blood biomarkers, cardiorenal and pulmonary imaging, and gluteal subcutaneous biopsy (NCT04403607). Small resistance arteries were isolated and examined using wire myography, histopathology, immunohistochemistry, and spatial transcriptomics. Endothelium-independent (sodium nitroprusside) and -dependent (acetylcholine) vasorelaxation and vasoconstriction to the thromboxane A2 receptor agonist, U46619, and endothelin-1 (ET-1) in the presence or absence of a RhoA/Rho-kinase inhibitor (fasudil), were investigated.ResultsThirty-seven patients, including 27 (mean age 57 years, 48% women, 41% cardiovascular disease) three months post-COVID-19 and 10 controls (mean age 57 years, 20% women, 30% cardiovascular disease), were included. Compared with control responses, U46619-induced constriction was increased (p = 0.002) and endothelium-independent vasorelaxation was reduced in arteries from COVID-19 patients (p < 0.001). This difference was abolished by fasudil. Histopathology revealed greater collagen abundance in COVID-19 arteries (Masson's Trichrome (MT) 69.7% [95%CI: 67.8, 71.7]; picrosirius red 68.6% [95% CI: 64.4, 72.8]) versus controls (MT 64.9% [95%CI:59.4, 70.3] [p = 0.028]; picrosirius red 60.1% [95% CI: 55.4, 64.8], [p = 0.029]). Greater phosphorylated myosin light chain antibody-positive staining in vascular smooth muscle cells was observed in COVID-19 arteries (40.1%; 95% CI: 30.9, 49.3) vs. controls (10.0%; 95% CI: 4.4, 15.6) (p < 0.001). In proof-of-concept studies, gene pathways associated with extracellular matrix alteration, proteoglycan synthesis, and viral mRNA replication appeared to be upregulated.ConclusionPatients with post-COVID-19 conditions have enhanced vascular fibrosis and myosin light change phosphorylation. Rho-kinase activation represents a novel therapeutic target for clinical trials

    Osteoprotegerin regulates vascular function through syndecan-1 and NADPH oxidase-derived reactive oxygen species

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    Osteogenic factors, such as osteoprotegerin (OPG), are protective against vascular calcification. However, OPG is also positively associated with cardiovascular damage, particularly in pulmonary hypertension, possibly through processes beyond effects on calcification. In the present study, we focused on calcification-independent vascular effects of OPG through activation of syndecan-1 and NADPH oxidases (Noxs) 1 and 4. Isolated resistance arteries from Wistar–Kyoto (WKY) rats, exposed to exogenous OPG, studied by myography exhibited endothelial and smooth muscle dysfunction. OPG decreased nitric oxide (NO) production, eNOS activation and increased reactive oxygen species (ROS) production in endothelial cells. In VSMCs, OPG increased ROS production, H2O2/peroxynitrite levels and activation of Rho kinase and myosin light chain. OPG vascular and redox effects were also inhibited by the syndecan-1 inhibitor synstatin (SSNT). Additionally, heparinase and chondroitinase abolished OPG effects on VSMCs-ROS production, confirming syndecan-1 as OPG molecular partner and suggesting that OPG binds to heparan/chondroitin sulphate chains of syndecan-1. OPG-induced ROS production was abrogated by NoxA1ds (Nox1 inhibitor) and GKT137831 (dual Nox1/Nox4 inhibitor). Tempol (SOD mimetic) inhibited vascular dysfunction induced by OPG. In addition, we studied arteries from Nox1 and Nox4 knockout (KO) mice. Nox1 and Nox4 KO abrogated OPG-induced vascular dysfunction. Vascular dysfunction elicited by OPG is mediated by a complex signalling cascade involving syndecan-1, Nox1 and Nox4. Our data identify novel molecular mechanisms beyond calcification for OPG, which may underlie vascular injurious effects of osteogenic factors in conditions such as hypertension and/or diabetes

    Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) blocks differentiation and maintains the expression of pluripotency markers in human embryonic stem cells

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    Human embryonic stem cells (hESCs) have enormous potential for use in pharmaceutical development and therapeutics, however to realise this potential there is a requirement for simple and reproducible cell culture methods that provide adequate numbers of cells of suitable quality. We have discovered a novel way of blocking the spontaneous differentiation of hESCs in the absence of exogenous cytokines by supplementing feeder-free conditions with erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), an established inhibitor of adenosine deaminase (ADA) and cyclic nucleotide phosphodiesterase- 2 (PDE2). hESCs maintained in feeder-free conditions with EHNA for more than 10 passages showed no reduction in hESC associated markers including NANOG, POU5F1 and SSEA 4 compared to cells maintained in feeder-free conditions containing basic fibroblast growth factor (bFGF). Spontaneous differentiation was reversibly suppressed by the addition of EHNA, but upon removing EHNA hESC populations underwent efficient spontaneous, multi-lineage and directed differentiation. EHNA also acts as a strong blocker of directed neuronal differentiation. Chemically distinct inhibitors of ADA and PDE2 lacked the capacity of EHNA to suppress hESC differentiation, suggesting that the effect is not driven by inhibition of either ADA or PDE2. Preliminary structure activity relationship analysis found the differentiation blocking properties of EHNA to reside in a pharmacophore comprised of a close adenine mimetic with an extended hydrophobic substituent in the 8- or 9-position. We conclude that EHNA and simple 9-alkyladenines can block directed neuronal and spontaneous differentiation in the absence of exogenous cytokine addition, and may provide a useful replacement for bFGF in large scale or cGMP compliant processes

    Some lessons from history: Morphomes in diachrony

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    What follows informally recounts some ‘lessons I have learned’ from several years spent exploring and describing in close detail the history of the inflectional morphology of the Romance verb.1 This programme of research, which has considered the entire range of Romance languages and dialects, and in as much historical depth and comparative breadth as possible, has been concerned in particular to identify, and describe the development of, a number of phenomena for which it can be argued that they are clear-cut,2 unambiguous cases of ‘morphomes’ in the sense of Aronoff; phenomena, that is, for which extramorphological (phonological or morphosyntactic) synchronic conditioning can be ruled out. Reproduction of all the relevant arguments would require far more space than I have here, and they have been published elsewhere. This is not to say, of course, that these arguments are selfevidently correct or that all morphologists would necessarily be persuaded by them (far from it), but I am interested here in standing back and developing some general reflections on what I believe the unusual comparative breadth and historical depth offered by the descriptive literature3 on Romance languages has to offer for the study of morphomes. The principal lessons that have emerged from the diachronic observation of Romance verb morphology seem to me to be the following: i. Diachrony can provide evidence for the psychological reality of putative morphomes. ii. Diachrony can be used as a diagnostic of the synchronically morphomic nature of some pattern of alternation. iii. Typological comparison can serve to falsify the putatively morphomic status of some pattern of alternation iv. Speakers do not especially prefer ‘non-morphomic’ over ‘morphomic’ patterns. v. An alternation pattern can be morphomic even when it appears to be phonologically conditioned. Each of these findings will be explained and illustrated at least in outline4 in the following sections. In conclusion, I shall briefly present some thoughts on why morphomes persist diachronically, which I shall relate to the observation that all morphomes seem to originate in allomorphy affecting lexical roots
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