Journal: Journal of Smooth Muscle Research

Article Title: Glucose-6-phosphate dehydrogenase and MEG3 controls hypoxia-induced expression of serum response factor (SRF) and SRF-dependent genes in pulmonary smooth muscle cell

doi: 10.1540/jsmr.58.34

Figure Lengend Snippet: G6PD inhibitor and MEG-3 siRNA regulates Srf expression in human pulmonary arterial SMCs. A) MEG3 expression level is decreased by MEG3-siRNA (50 nmol/l) in human pulmonary arterial SMCs exposed to normoxia (Nx; 21% O 2 ) and hypoxia (Hx; 3% O 2 ). Administration of 4091 (1 µmol/l) to human pulmonary arterial SMCs cultured under Nx or Hx upregulated MEG3 expression and this was blocked by MEG3-siRNA (50 nmol/l). There were no differences in expression of MEG3 between the application of control-siRNA with and without 4091. B) Srf expression in human SMCs exposed to Nx or Hx and treated with 4091 (1 µmol/l) and control (Ctrl)- or MEG3-siRNA (50 nmol/l). N=5–6. Statistical analysis was performed using the one-way ANOVA. Sidak’s test was used to compare multiple groups after ANOVA analysis.

Article Snippet: Human pulmonary artery smooth muscle cells (SMCs) were purchase from Lonza (MD, USA).

Techniques: Expressing, Cell Culture, Control

Journal:

Article Title: Hypoxia-induced mitogenic factor/FIZZ1 induces intracellular calcium release through the PLC-IP 3 pathway

doi: 10.1152/ajplung.90416.2008

Figure Lengend Snippet: Hypoxia-induced mitogenic factor (HIMF) increased intracellular Ca2+ levels in human pulmonary artery smooth muscle cells (SMC) maintained in Ca2+-containing buffer. Cells were preloaded with fluo 4-AM for 30 min at room temperature and washed twice before stimulation. Serial 2-dimensional fluorescence images show intracellular Ca2+ concentration ([Ca2+]i) at indicated times after cells were stimulated by 60 nmol/l HIMF (A), 60 nmol/l FLAG (B), and 10−7 mol/l ANG II (C). D–F: [Ca2+]i dynamics shown as changes in fluorescence density ratio (F/F0) over time during stimulation by 60 nmol/l HIMF, 60 nmol/l FLAG, and 10−7 mol/l ANG II. Values are means ± SE of 3 independent experiments.

Article Snippet: Primary human pulmonary artery SMC (Cambrex Biosciences, Walkersville, MD) were maintained in the growth medium SGM-2 (Cambrex Biosciences) containing 5% FCS, 0.5 ng/ml human epithelial growth factor, 2 ng/ml human fibroblast growth factor, and 5 μg/ml insulin under 5% CO 2 -21% O 2 in a humidified incubator at 37°C.

Techniques: Fluorescence, Concentration Assay

Journal:

Article Title: Hypoxia-induced mitogenic factor/FIZZ1 induces intracellular calcium release through the PLC-IP 3 pathway

doi: 10.1152/ajplung.90416.2008

Figure Lengend Snippet: HIMF increased [Ca2+]i in human pulmonary artery SMC maintained in Ca2+-free buffer. Cells were preloaded with fluo 4-AM in Ca2+-containing buffer and maintained in Ca2+-free buffer at the time of stimulation. Serial 2-dimensional fluorescence images show [Ca2+]i by fluorescence intensity at indicated times after cells were stimulated by 60 nmol/l HIMF (A), 60 nmol/l FLAG (B), and 10−7 mol/l ANG II (C). D–F: [Ca2+]i dynamics shown as changes in fluorescence intensity ratio over time during stimulation by 60 nmol/l HIMF, 60 nmol/l FLAG, and 10−7 mol/l ANG II. Results indicate that HIMF-induced [Ca2+]i transient is independent of extracellular Ca2+ and is released from intracellular stores. Values are means ± SE of 3 independent experiments.

Article Snippet: Primary human pulmonary artery SMC (Cambrex Biosciences, Walkersville, MD) were maintained in the growth medium SGM-2 (Cambrex Biosciences) containing 5% FCS, 0.5 ng/ml human epithelial growth factor, 2 ng/ml human fibroblast growth factor, and 5 μg/ml insulin under 5% CO 2 -21% O 2 in a humidified incubator at 37°C.

Techniques: Fluorescence

Journal:

Article Title: Hypoxia-induced mitogenic factor/FIZZ1 induces intracellular calcium release through the PLC-IP 3 pathway

doi: 10.1152/ajplung.90416.2008

Figure Lengend Snippet: HIMF dose dependently induced intracellular Ca2+ release. Human pulmonary artery SMC preloaded with fluo 4-AM were maintained in Ca2+-free buffer at the time of HIMF application. A: increasing HIMF concentration led to an increase in percentage of cells that responded with an elevation in [Ca2+]i. B: as HIMF concentration increased, fluorescence intensity also increased, indicating that Ca2+ release correlated positively with HIMF concentration. Values are means ± SE of 3 independent experiments.

Article Snippet: Primary human pulmonary artery SMC (Cambrex Biosciences, Walkersville, MD) were maintained in the growth medium SGM-2 (Cambrex Biosciences) containing 5% FCS, 0.5 ng/ml human epithelial growth factor, 2 ng/ml human fibroblast growth factor, and 5 μg/ml insulin under 5% CO 2 -21% O 2 in a humidified incubator at 37°C.

Techniques: Concentration Assay, Fluorescence

Journal:

Article Title: Hypoxia-induced mitogenic factor/FIZZ1 induces intracellular calcium release through the PLC-IP 3 pathway

doi: 10.1152/ajplung.90416.2008

Figure Lengend Snippet: Representative intracellular Ca2+ dynamics in individual cells in response to stimulation with HIMF and ANG II in Ca2+-free buffer. In contrast to a rapid biphasic [Ca2+]i transient in response to 10−7 mol/l ANG II (B), human pulmonary artery SMC show an oscillatory, long-lasting [Ca2+]i response to 60 nmol/l HIMF (A). Each trace represents Ca2+ transient of an individual cell.

Article Snippet: Primary human pulmonary artery SMC (Cambrex Biosciences, Walkersville, MD) were maintained in the growth medium SGM-2 (Cambrex Biosciences) containing 5% FCS, 0.5 ng/ml human epithelial growth factor, 2 ng/ml human fibroblast growth factor, and 5 μg/ml insulin under 5% CO 2 -21% O 2 in a humidified incubator at 37°C.

Techniques:

Journal:

Article Title: Hypoxia-induced mitogenic factor/FIZZ1 induces intracellular calcium release through the PLC-IP 3 pathway

doi: 10.1152/ajplung.90416.2008

Figure Lengend Snippet: Effects of 2-aminoethoxydiphenyl borate (2-APB) and ryanodine on HIMF-induced intracellular Ca2+ release and induction of intracellular inositol 1,4,5-trisphosphate (IP3). Fluo 4-loaded human pulmonary artery SMC were pretreated with 50 μmol/l 2-APB (IP3 receptor antagonist) or 100 μmol/l ryanodine (ryanodine receptor antagonist) for 30 min at room temperature and maintained in Ca2+-free buffer at the time of HIMF stimulation. Complete blockade of [Ca2+]i response to HIMF by 2-APB (A), but not by ryanodine (B), indicates that HIMF initiates Ca2+ release via IP3 receptors. Although ryanodine did not prevent HIMF-induced Ca2+ release, it did reduce the resulting fluorescence intensity (P < 0.05), suggesting that Ca2+ release via ryanodine receptor might also be attributable to HIMF-induced increases in [Ca2+]i. Values are means ± SE of 3 independent experiments. C: IP3 levels in human pulmonary artery SMC were determined by Amersham IP3 assay kit at 0, 30, 60, 90, 120, and 300 s after HIMF stimulation. HIMF induced a rapid IP3 increase that peaked at 60 s (P < 0.05) and returned to baseline after 120 s. Values are means ± SE of 3 independent determinations.

Article Snippet: Primary human pulmonary artery SMC (Cambrex Biosciences, Walkersville, MD) were maintained in the growth medium SGM-2 (Cambrex Biosciences) containing 5% FCS, 0.5 ng/ml human epithelial growth factor, 2 ng/ml human fibroblast growth factor, and 5 μg/ml insulin under 5% CO 2 -21% O 2 in a humidified incubator at 37°C.

Techniques: Fluorescence

Journal:

Article Title: Hypoxia-induced mitogenic factor/FIZZ1 induces intracellular calcium release through the PLC-IP 3 pathway

doi: 10.1152/ajplung.90416.2008

Figure Lengend Snippet: Effects of the phosopholipase C (PLC) inhibitor U-73122 on HIMF-induced intracellular Ca2+ release. Fluo 4-loaded human pulmonary artery SMC were pretreated with 20 μmol/l U-73122 for 30 min at room temperature and maintained in Ca2+-free buffer at the time of HIMF stimulation. Complete blockade of [Ca2+]i increase by U-73122 indicates that PLC activity is required for HIMF to trigger the [Ca2+]i transient. Values are means ± SE of 3 independent experiments.

Article Snippet: Primary human pulmonary artery SMC (Cambrex Biosciences, Walkersville, MD) were maintained in the growth medium SGM-2 (Cambrex Biosciences) containing 5% FCS, 0.5 ng/ml human epithelial growth factor, 2 ng/ml human fibroblast growth factor, and 5 μg/ml insulin under 5% CO 2 -21% O 2 in a humidified incubator at 37°C.

Techniques: Activity Assay

Journal:

Article Title: Hypoxia-induced mitogenic factor/FIZZ1 induces intracellular calcium release through the PLC-IP 3 pathway

doi: 10.1152/ajplung.90416.2008

Figure Lengend Snippet: Effects of Gαi and Gαs protein inhibitors on HIMF-induced intracellular Ca2+ release. Fluo 4-loaded human pulmonary artery SMC were pretreated with 1 μg/ml pertussis toxin (PTX, a Gαi protein inhibitor) for 1 h or 10 μM NF-449 (a Gαs protein inhibitor) for 30 min at room temperature and maintained in Ca2+-free buffer at the time of HIMF stimulation. PTX and NF-449 failed to prevent HIMF-induced Ca2+ release (A and B). Values are means ± SE of 3 independent experiments.

Article Snippet: Primary human pulmonary artery SMC (Cambrex Biosciences, Walkersville, MD) were maintained in the growth medium SGM-2 (Cambrex Biosciences) containing 5% FCS, 0.5 ng/ml human epithelial growth factor, 2 ng/ml human fibroblast growth factor, and 5 μg/ml insulin under 5% CO 2 -21% O 2 in a humidified incubator at 37°C.

Techniques:

Journal:

Article Title: Hypoxia-induced mitogenic factor/FIZZ1 induces intracellular calcium release through the PLC-IP 3 pathway

doi: 10.1152/ajplung.90416.2008

Figure Lengend Snippet: Effects of Gαq/11 small interfering RNA (siRNA) knockdown on HIMF-induced intracellular Ca2+ release. A: in human pulmonary artery SMC transfected with siRNA specific for Gαq/11, expression of Gαq/11 was remarkably reduced 48 h after electroporation. Compared with scrambled siRNA-treated cells, which showed an oscillatory, sustained Ca2+ release in response to HIMF stimulation (B), Gαq/11 knockdown did not prevent HIMF-induced Ca2+ release from internal stores but caused intermittent Ca2+ spikes with return to baseline between spikes (C). B and C show Ca2+ transients of 5 representative individual cells responsive to HIMF application from experiments performed in triplicate.

Article Snippet: Primary human pulmonary artery SMC (Cambrex Biosciences, Walkersville, MD) were maintained in the growth medium SGM-2 (Cambrex Biosciences) containing 5% FCS, 0.5 ng/ml human epithelial growth factor, 2 ng/ml human fibroblast growth factor, and 5 μg/ml insulin under 5% CO 2 -21% O 2 in a humidified incubator at 37°C.

Techniques: Small Interfering RNA, Knockdown, Transfection, Expressing, Electroporation

Journal:

Article Title: Hypoxia-induced mitogenic factor/FIZZ1 induces intracellular calcium release through the PLC-IP 3 pathway

doi: 10.1152/ajplung.90416.2008

Figure Lengend Snippet: Effects of the protein tyrosine kinase inhibitor genistein on HIMF-induced Ca2+ release. Fluo 4-loaded human pulmonary artery SMC were pretreated with 10 μmol/l genistein or daidzein (an inactive analog of genistein) for 30 min at room temperature and maintained in Ca2+-free buffer at the time of HIMF stimulation. Genistein, but not daidzein, abolished the cellular response to HIMF. Values are means ± SE of 3 independent experiments.

Article Snippet: Primary human pulmonary artery SMC (Cambrex Biosciences, Walkersville, MD) were maintained in the growth medium SGM-2 (Cambrex Biosciences) containing 5% FCS, 0.5 ng/ml human epithelial growth factor, 2 ng/ml human fibroblast growth factor, and 5 μg/ml insulin under 5% CO 2 -21% O 2 in a humidified incubator at 37°C.

Techniques: