Synopsis of alpha-galactosidase gene mutations in 14 patients included in the study.

<p>Synopsis of alpha-galactosidase gene mutations in 14 patients included in the study.</p

Baseline data, medical history, biomarkers and cardiac work up in patients with FD in relation to cardiac troponin I elevation and normal values.

<p>*a small fibre dysfunction was proved by quantitative sensory testing or by skin biopsy.</p>†<p>measurement end-diastolic in the posterior wall of the left ventricle.</p>$<p>Arrhythmia was considered if one of the following conditions was detected: persistent or intermittent atrial fibrillation of flatter, sustained tachycardia (heart rate ≥100/minute for more than 30 seconds), non-sustained tachycardia (heart rate ≥100/minute for less than 30 seconds in at least 3 subsequent hear cycles), incomplete bundle branch block (QRS-duration: 100–119 ms) or complete bundle branch block (QRS-duration ≥120 ms).</p>§<p>lower level of quantification.</p

Cardiac work up in patient 2.

<p>A+B: Coronary angiography (A: right coronary artery; B: left coronary artery) demonstrating no relevant pathology. C+D: Cardiac MRI showing increase in myocardial wall thickness (C) and pathological late gadolinium enhancement (D, arrow). E: Myocardial biopsy revealing strong accumulation of Gb_3, as indicated by numerous vacuoles within the cardiomyocytes (arrow).</p

cTNI-values in 3 patients with Fabry disease.

<p>cTNI-values in 3 patients with Fabry disease.</p

Role of the Phosphatase PTEN in Early Vascular Remodeling

<div><p>Background</p><p>The phosphatase PTEN represents an important physiological inhibitor of phosphatidylinositol-3 kinase (PI3-K)/protein kinase B (Akt) signalling, however, the functional role of PTEN in the initial phase of angioplasty-induced vascular injury remains elusive. In the present study we sought to determine PTEN's effect on vascular smooth muscle cell (VSMC) apoptosis following acute injury <i>in vivo</i> and <i>in vitro</i>.</p> <p>Methods and Results</p><p>Immunohistochemistry indicated a faint basal expression and equal distribution of PTEN in uninjured rat carotid arteries. 12 h following balloon-injury, PTEN expression was strongly increased in apoptotic (TUNEL+) VSMC. In vitro, stimulation with serum or different growth factors or subjecting VSMC to cyclic stretch had no effect on PTEN expression, whereas stimulation with H₂O₂ robustly increased PTEN expression in a time- and dose-dependent manner. To evaluate the functional role of PTEN expression, human VSMC were transduced with WT-PTEN. Overexpression of PTEN increased the number of apoptotic VSMC (19.8%±4.4 vs. 5.6%±2.3; <i>P</i><0.001) as determined by TUNEL assay. In contrast, siRNA-mediated knock-down of PTEN attenuated the basal as well as H₂O₂-induced apoptosis of VSMC. Mechanistically, overexpression of PTEN prevented serum-induced Akt-phosphorylation, whereas siRNA-mediated knock down of PTEN augmented Akt-activation. Moreover, co-transfection of PTEN and a constitutive active Akt mutant prevented PTEN-dependent augmentation of VSMC apoptosis, indicating, that PTEN regulates VSMC apoptosis by inhibition of Akt phosphorylation/activation.</p> <p>Conclusion</p><p>By interfering with the PI3-K/Akt-dependent survival signalling, the oxidative stress-induced up regulation of PTEN in VSMC of injured arteries augments the sensitivity of VSMC to apoptotic stimuli in the early phase following vascular injury, augmenting the initial injury and cell loss of the injured vessel wall. Thus, these data add to our understanding of PTEN's role during vascular remodelling.</p> </div

PTEN expression in human coronary VSMC <i>in vitro</i>.

<p><b>A</b>, PTEN-expression is not upregulated by mechanical stress in VSMC. Lysates of cells exposed to mechanical forces using a stretching device were analyzed by western blotting using specific antibodies. <b>B</b>, Lysates of SMC exposed to growth medium (FCS). PTEN expression was not upregulated after 24 h. Protein expression was determined using specific antibodies. Detection of Cdk4 served as loading control. <b>C</b>, Lysates of SMC exposed to oxidative stress using 500 µM H₂O₂. PTEN upregulation was triggered by oxidative stress within 24 h. Detection of p53 and Cdk4 served as apoptotic marker and loading control, respectively. <b>D</b>, The upregulation of PTEN protein levels was quantified by densitometric analysis of immunoblots (n = 3; *<i>P</i><0.05). <b>E</b>, The upregulation of PTEN activity is mediated by H₂O₂-induction. Shown is a phosphatase activity assay of immunoprecipitated protein from lysates of HcASMC with and without 24 h H₂O₂–treatment. Immunoprecipitations from lysates employing an IgG iso-antibody without H₂O₂-treatment with and without bpV supplementation, an anti-PTEN-antibody without H₂O₂ and bpV treatment and an anti-PTEN-antibody with H₂O₂– and bpV-treatment served as controls. Results are expressed as mean OD650 ± SD using an ELISA plate reader (^#<i>P</i><0.001, *<i>P</i><0.001; n = 4).</p

PTEN overexpression augments SMC apoptosis under basal conditions as well as following exposure to oxidative stress.

<p>HcASMC transfected with a plasmid carrying WT-PTEN or a control (empty) vector and PTEN protein-expression levels were determined by immunoblotting (<b>A</b>). SMC were incubated in basal medium (<b>B</b>) and in basal medium supplemented with H₂O₂ (<b>C</b>) and the relative number of apoptotic cells was evaluated following TUNEL-staining (expressed as % of total cells; *<i>P</i><0.001; n = 6). HcASMC were transfected as follows: non transfected (NT); transfected with an empty plasmid without PTEN-cDNA (pControl); transfected with a plasmid coding for PTEN (pPTEN).</p

PTEN overexpression prevents serum-induced Akt phosphorylation after 15 min (A) and 30 min (B) serum induction using 10% FCS as determined by western blotting using an anti-pAkt-antibody.

<p>An antibody against total Akt was used as control. The potent PI3-K-inhibitor Ly294002 (50 nM) was supplemented to the medium for not-transfected cells. HcASMC were transfected as follows: non transfected (NT); transfected with a plasmid coding for GFP (pControl); transfected with an empty plasmid without PTEN-cDNA (pControl); transfected with a plasmid coding for PTEN (pPTEN); mock transfected (without plasmid, but with transfection reagent). PTEN- and Akt co-overexpression reverses PTENs pro-apoptotic effect (<b>C</b>). HcASMC were co-transfected with a WT form of PTEN and a constitutively active form of Akt. An empty plasmid was used as control. Following the magnetic separation of positively transfected SMC, apoptosis was determined after cell growth for 24 h and expressed as mean OD405 ± SD using a cell death detection ELISA (^#<i>P</i><0.001, *<i>P</i><0.001; n = 4).</p

PTEN overexpression attenuates serum induced HcASMC proliferation (A). HcASMC transfected with a plasmid carrying WT-PTEN or a control vector carrying GFP alone were incubated either in basal medium or growth medium in the presence of BrdU.

<p>PTEN knock down enhances serum induced HcASMC proliferation (B). Cells were transfected with siRNA targeting PTEN or a scrambled control. Proliferation is expressed as mean OD450 ± SD as determined by anti-BrdU ELISA (^#<i>P</i><0.001, *<i>P</i><0.001; n = 4). HcASMC were transfected as following: mock transfected (without plasmid, but with transfection reagent); transfected with a plasmid coding for GFP (pGFP); transfected with a plasmid coding for PTEN (pPTEN); transfected with a non-targeting (scrambled) siRNA (Control siRNA); transfected with a targeting siRNA against PTEN (PTEN siRNA).</p

PTEN knock down attenuates SMC apoptosis under basal conditions or oxidative stress.

<p><b>A</b>, PTEN expression following siRNA-mediated knock down. Protein expression was determined by western blotting using specific antibodies. Detection of Cdk4 served as a loading control. SMC were transfected as follows: non transfected (NT); mock transfected (without siRNA, but with lipid carrier); transfected with a non-targeting (scrambled) siRNA (Control siRNA); transfected with a targeting siRNA against PTEN (PTEN siRNA). SMC transfected with siRNA targeting PTEN or a scrambled control were incubated in basal medium in the absence (<b>B</b>) or presence of H₂O₂ (<b>C</b>). SMC apoptosis is expressed as mean OD405 ± SD using a cell death detection ELISA (*<i>P</i><0.05; n = 3).</p