Trypsin-activated PAR2 intracellular signaling in hASCs.

<p>(<b>A</b>) Schematic rendition of signal-transduction pathways linking PAR2 and <i>VEGF</i>. (<b>B</b>) The effect of specific kinase inhibitors on suppressing trypsin-induced <i>VEGF</i> activation after 5 min trypsin exposure was assessed by real-time RT-PCR (n = 6). Expression levels were normalized to the levels induced by trypsin (Ctrl). (<b>C</b>) The effect of PI3K and Mek inhibitors on phosphorylation of Akt and Erk1/2, respectively, as a result of 5 min trypsin exposure was determined by immunoblotting. PI3K and Mek inhibitors were added 2 hours prior to trypsin exposure. Cells after a 4-day culture at 20% oxygen were used as controls (Ctrl). Representative data obtained from ASC12 cells are presented. Values are represented as the mean and SEM. Abbreviations: PAR2, protease-activated receptor 2; VEGF, vascular endothelial growth factor; Ctrl, control.</p

Expression of PAR2 in hASCs and its association with transcriptional activation of <i>VEGF</i>.

<p>(<b>A</b>) Detection of PAR2 in hASC lines by immunoblotting. (<b>B</b>) Expression of PAR2 by indirect immunofluorescence using SAM11 antibody. Representative pattern as detected on the surface of ASC12 cells is presented. (<b>C</b>) SAM11 antibody blocked trypsin-induced PAR2 activation, measured using real-time RT-PCR to determine <i>VEGF</i> expression levels 12 hours after trypsin exposure (n = 15). Expression levels were corrected for basal <i>VEGF</i> activity in hASCs cultured at 20% oxygen and normalised to the levels induced by trypsin. Values are represented as the mean and SEM. Scale bar indicates 200 µm. Abbreviations: PAR2, protease-activated receptor 2; VEGF, vascular endothelial growth factor; Ctrl, control (NIH 3T3 cells).</p

Immunophenotypical analysis of hASC lines at passage 2.

<p>(<b>A</b>) Representative distributions of positive markers expressed on the ASC12 cells are presented. (<b>B</b>) Surface markers profile was obtained as an average from ASC12, 21, and 23 lines.</p

Stabilisation of HIF-1 in hASCs after trypsin and 1% oxygen exposure alone or in combination.

<p>(<b>A</b>) HIF-1 activation/stabilization during 6 hours culture after trypsin exposure in combination with 24 hours in hypoxic/normoxic conditions was analysed by ELISA. All cells were harvested <i>in situ</i>. Values are represented as the mean and SEM (n = 12). Asterisks denote statistical difference between this and all other groups (p<0.05). (<b>B</b>) Analysis of HIF-1α induction at 4 and 12 hours following 5 min trypsin exposure was done by immunoblotting. All cells were harvested <i>in situ</i>. HIF-1α positive controls are ASCs subjected to 48 hours of 1% oxygen.</p

Lung mRNA expression levels of A) eNOS, K_Ca2.1, K_Ca2.2, K_Ca2.3, K_Ca3.1, and K_Ca1.1; B) α-Smooth muscle actin (α-SMA), collagen-1, and TGFβ.

<p>Data are given as means ±SEM, n = 7–8. Data were analyzed by two-way ANOVA and differences were considered significant when *P<0.05 from wild type, ^# P<0.05 from normoxia. Statistical interaction (£) was observed in K_Ca2.3 expression (A). C–E) Genotyping: C:Gel electrophoresis shows that polymerase chain reacton (PCR) detected the K_Ca2.3-wild type allele (wild type (+)) and the tTA allele (T) in K_Ca2.3^T/+, and K_Ca2.3^T/T. D and E: PCR detected the targeted allele in K_Ca3.1^−/+ and in K_Ca3.1^−/− as well as the wild type allele in K_Ca3.1^+/− and K_Ca3.1^+/+. A DNA ladder was used to determine products sizes.</p

Right ventricular systolic blood pressure and hypertrophy.

<p>A) The effect of chronic hypoxia on right ventricular systolic blood pressure (RVSBP) in wild type and K_Ca3.1^−/−/K_Ca2.3^T/T(+Dox) mice. Values are means ±SEM, normoxic wild type (n = 8) and K_Ca3.1^−/−/K_Ca2.3^T/T(+Dox) mice (n = 7). B) Representative trace of right ventricular pressure measurements in normoxic wild type mice (top) and normoxic K_Ca3.1^−/−/K_Ca2.3^T/T(+Dox) mice (bottom). C) Hypoxia induced right ventricular hypertrophy as indicated by alterations of the weight ratio of right ventricle/left ventricle + septum, in wild type and K_Ca3.1^−/−/K_Ca2.3^T/T(+Dox) mice, n = 8. D) The effect of hypoxia on right ventricular wall thickness/heart weight (HW) in wild type and K_Ca3.1^−/−/K_Ca2.3^T/T(+Dox) mice. Values are mean ±SEM, n = 8. Data were analyzed by 2−way ANOVA and differences were considered significant when *P<0.05 vs. wild type, ^# P<0.05 vs. normoxia.</p

Characteristics of the animals.

<p>Selected morphological and functional characteristics and hematocrit of the normoxic and hypoxic strains. Values are means ±SEM, n = 7–8. BPM =  beats per minute. BW =  body weight. *P<0.05 vs. wild type;</p>#<p>P<0.05 vs. normoxia; 2-way ANOVA (n = 7–8 per group).</p

Morphometric measurements.

<p>A: Lumen diameter (µm) in vessels divided in groups of non-muscularized-, partially- and fully-muscularized vessels. B: Wall/lumen ratio divided in groups of non-muscularized-, partially- and fully-muscularized vessels. C: Number of non-muscularized-, partially- and muscularized vessels in each experimental group. D: Wall area of the vessels divided in groups of vessel diameter. * P<0.05 vs. wild type and ^# P<0.05 vs. normoxia. Statistical interaction was observed in Figure (B) for non-muscularized vessels.</p

Immunoblottings of right ventricle samples from normoxic and hypoxic rats.

<p>Proteins participating in metabolism A: ACAA2, B: HADHA and C: aquaporin 7 show tendency to downregulation by hypoxia. D: monoamine oxidase A, E: tissue transglutaminase and F: endothelin receptor B were all upregulated by hypoxia. ACAA2: acetyl-Coenzyme A acyltransferase 2, HADHA: hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A thiolase/enoyl-Coenzyme A hydratase (trifunctional protein), alpha subunit. Values are means ± SE and are calculated as percent of normoxia 1 week. n = 6 in both groups at all time points. *P<0.05 vs. normoxia at same time point.</p

Experimental design.

<p>*Three animals from each control and four animals from each hypoxic subgroup were used for GeneChip analysis. Four animals from each group either pulmonary trunk banded (PTB) or sham at week 5 were used for GeneChip analysis.</p