Cardiomyopathy associated with the Ala143Thr variant of the α-galactosidase A gene

Objective To investigate whether the Ala143Thr variant of the α-galactosidase A gene (A143T/GLA), with conflicting interpretations of pathogenicity, is associated with Fabry cardiomyopathy.Methods The index patient, a woman in her 60s with cardiomyopathy, was screened for variants in 59 cardiomyopathy-related genes. A143T/GLA, the only rare variant found, was screened in 10 relatives. GLA activity and lyso-Gb3 levels were measured and echocardiography was performed in 8 of 9 subjects carrying A143T/GLA. Cardiac magnetic resonance (CMR) imaging and 18F-fluorodeoxyglucose (FDG) positron emission tomography/CT (PET/CT) were performed in four adult A143T/GLA carriers. Endomyocardial biopsy was obtained from two adult A143T/GLA carrying sons of the index patient.Results The index patient and her elder son had a pacemaker implantation because of sick sinus syndrome and atrioventricular block. GLA activities were decreased to 25%–40% of normal in both sons and one granddaughter. Lyso-Gb3 levels were elevated in both sons. In CMR, the index patient and her two sons had left ventricular (LV) hypertrophy and/or dilatation. The elder son had late gadolinium enhancement, high CMR-derived T1 time and positive FDG signal in PET/CT in the basal inferolateral LV wall. The younger son had low T1 time and the mother had positive FDG signal in PET/CT in the basal inferolateral LV wall. Endomyocardial biopsy of both sons showed myocardial accumulation compatible with glycolipids in light and electron microscopy, staining with anti-Gb3 antibody available for the younger son. Five female relatives with A143T/GLA had no cardiomyopathy in cardiac imaging.Conclusions A143T/GLA is likely a late-onset Fabry cardiomyopathy causing variant with incomplete penetrance.</p

Nuclear factor E2-related factor 2 deficiency impairs atherosclerotic lesion development but promotes features of plaque instability in hypercholesterolaemic mice

Aims Oxidative stress and inflammation play an important role in the progression of atherosclerosis. Transcription factor NF-E2-related factor 2 (Nrf2) has antioxidant and anti-inflammatory effects in the vessel wall, but paradoxically, global loss of Nrf2 in apoE deficient mice alleviates atherosclerosis. In this study, we investigated the effect of global Nrf2 deficiency on early and advanced atherogenesis in alternative models of atherosclerosis, LDL receptor deficient mice (LDLR-/-), and LDLR-/- mice expressing apoB-100 only (LDLR-/- ApoB(100/100)) having a humanized lipoprotein profile. Methods and results LDLR-/- mice were fed a high-fat diet (HFD) for 6 or 12weeks and LDLR(-/-)ApoB(100/100) mice a regular chow diet for 6 or 12months. Nrf2 deficiency significantly reduced early and more advanced atherosclerosis assessed by lesion size and coverage in the aorta in both models. Nrf2 deficiency in LDLR-/- mice reduced total plasma cholesterol after 6weeks of HFD and triglycerides in LDLR(-/-)ApoB(100/100) mice on a chow diet. Nrf2 deficiency aggravated aortic plaque maturation in aged LDLR(-/-)ApoB(100/100) mice as it increased plaque calcification. Moreover, approximate to 36% of Nrf2(-/-)LDLR(-/-)ApoB(100/100) females developed spontaneous myocardial infarction (MI) or sudden death at 5 to 12months of age. Interestingly, Nrf2 deficiency increased plaque instability index, enhanced plaque inflammation and calcification, and reduced fibrous cap thickness in brachiocephalic arteries of LDLR(-/-)ApoB(100/100) female mice at age of 12months. Conclusions Absence of Nrf2 reduced atherosclerotic lesion size in both atherosclerosis models, likely via systemic effects on lipid metabolism. However, Nrf2 deficiency in aged LDLR(-/-)ApoB(100/100) mice led to an enhanced atherosclerotic plaque instability likely via increased plaque inflammation and oxidative stress, which possibly predisposed to MI and sudden death.Peer reviewe

VEGF-A knockdown via RNAi in mouse endothelial cells and cardiomyocytes in a doxycycline-regulatable fashion.

<p>Secreted VEGF-A protein amount was most efficiently decreased with sh1 -knockdown vector (T-1 and TT-1) in mouse endothelial cells in comparison to sh2 (T-2 and TT-2) and sh3 (T-3 and TT-3) vectors. With the sh1 construct the normal TRE promoter (T-1) seemed to be slightly more efficient and less leaky than the tight TRE promoter (TT-1) (a). The magnitude of VEGF-A knockdown with the selected T-1 vector increased with increasing dox doses in endothelial cells (b). With the increasing dox doses also the amount of Venus expressing cells (%) was increased as follows: 40 ± 7% (–dox), 46 ± 8% (dox 10 ng/ml), 85 ± 3% (dox 100 ng/ml) and 78 ± 2% (dox 1000 ng/ml) (c). In cardiomyocytes the knockdown of VEGF-A with the T-1 vector was shown to be dependent of the amount of virus (MOI) and dox exposure time and the decrease was larger at the cellular level in comparison to the secreted protein (d-e). Results are shown as mean ± S.D., n = 3/group. The percentage of VEGF-A protein concentration compared to control +dox group (a) or non-transduced (NT) group (b, d, e) are shown above bars. *p<0.05, **p<0.01 and ***p<0.001 compared to control +dox group (a) or non-transduced (NT) group (b, d, e), 1-way ANOVA with Dunnett´s post hoc test. NT = non-transduced cells, MOI = multiplicity of infection, Contr = Control lentivirus vector targeting Luciferase. Scale bar 200 μm (c).</p

Doxycycline modulates VEGF-A expression: Failure of doxycycline-inducible lentivirus shRNA vector to knockdown VEGF-A expression in transgenic mice - Fig 3

<p><b>Plasma VEGF-A concentration in response to dox treatment (a). Tissue VEGF-A mRNA and protein levels after two weeks of 1 mg/ml dox treatment and five weeks of 2 mg/ml dox dose treatment (b-k).</b> Dox treatment decreased plasma VEGF-A levels in TG mice after 1 week, after which the plasma VEGF-A increased. In WT mice a decreasing effect on plasma VEGF-A was seen (a). *p<0.05, **p<0.01 and ***p<0.001 compared to baseline within each group, 1-way ANOVA with Dunnett´s post hoc test, n = 9-10/group. In the selected tissues, aorta, heart and kidney, the dox treatment with the 1 mg/ml dox dose for 2 weeks showed decreasing trend in VEGF-A mRNA expression in TG mice in comparison to WT mice (b, d, h), which was associated with increased cardiac VEGF-A protein (e) and decreased kidney VEGF-A protein levels (i). When the dox dose was doubled and the treatment time increased to 5 weeks, a trend towards increasing VEGF-A expression was seen in all three tissues in both WT and TG mice (c, f, j). However, no changes were detected in protein levels (g, k). **p<0.01 and ***p<0.001 compared to WT+dox group in 2 weeks experiment (b, d, e, h, i) or to no dox group (-dox) in 5 weeks experiment (c, f, g, j, k), <i>t-test</i>, n = 6-24/group.</p

Schematic drawing of the dox-regulatable VEGF-A shRNA lentivirus vector.

<p>Lentivirus vector for inducible knockdown permits constitutive expression of a Tet-transactivating component (rtTA3) with a Venus selection marker (green fluorescent protein -like protein) and tetracycline-regulated expression of VEGF-A-shRNAs. The shRNA transcripts were designed as primary microRNA mimics i.e. they were embedded in the primary transcript of human miRNA30. The lentiviruses were self-inactivating (SIN) third generation vectors, in which part of the viral 3´LTR has been deleted preventing the viral replication. The vectors contain a central polypurine tract (indicated as FLAP) for enhancement of viral titers and a woodchuck hepatitis virus posttranscriptional regulatory element (WRE) for better transgene expression [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190981#pone.0190981.ref024" target="_blank">24</a>].</p