BMP-2 Up-Regulates PTEN Expression and Induces Apoptosis of Pulmonary Artery Smooth Muscle Cells under Hypoxia

To investigate the role of bone morphogenetic protein 2 (BMP-2) in regulation of phosphatase and tensin homologue deleted on chromosome ten (PTEN) and apoptosis of pulmonary artery smooth muscle cells (PASMCs) under hypoxia.Normal human PASMCs were cultured in growth medium (GM) and treated with BMP-2 from 5-80 ng/ml under hypoxia (5% CO(2)+94% N(2)+1% O(2)) for 72 hours. Gene expression of PTEN, AKT-1 and AKT-2 were determined by quantitative RT-PCR (QRT-PCR). Protein expression levels of PTEN, AKT and phosph-AKT (pAKT) were determined. Apoptosis of PASMCs were determined by measuring activities of caspases-3, -8 and -9. siRNA-smad-4, bpV(HOpic) (PTEN inhibitor) and GW9662 (PPARγ antagonist) were used to determine the signalling pathways.Proliferation of PASMCs showed dose dependence of BMP-2, the lowest proliferation rate was achieved at 60 ng/ml concentration under hypoxia (82.2±2.8%). BMP-2 increased PTEN gene expression level, while AKT-1 and AKT-2 did not change. Consistently, the PTEN protein expression also showed dose dependence of BMP-2. AKT activity significantly reduced in BMP-2 treated PASMCs. Increased activities of caspase-3, -8 and -9 of PASMCs were found after cultured with BMP-2. PTEN expression remained unchanged when Smad-4 expression was inhibited by siRNA-Smad-4. bpV(HOpic) and GW9662 (PPARγ inhibitor) inhibited PTEN protein expression and recovered PASMCs proliferation rate.BMP-2 increased PTEN expression under hypoxia in a dose dependent pattern. BMP-2 reduced AKT activity and increased caspase activity of PASMCs under hypoxia. The increased PTEN expression may be mediated through PPARγ signalling pathway, instead of BMP/Smad signalling pathway

Energy Saving Melting and Revert Reduction Technology: Improved Die Casting Process to Preserve the Life of the Inserts

The goal of this project was to study the combined effects of die design, proper internal cooling and efficient die lubricants on die life. The project targeted improvements in die casting insert life by: Optomized Die Design for Reduced Surface Temperature: The life of die casting dies is significantly shorter when the die is exposed to elevated temperature for significant periods of time. Any die operated under conditions leading to surface temperature in excess of 1050oF undergoes structural changes that reduce its strength. Optimized die design can improve die life significantly. This improvement can be accomplished by means of cooling lines, baffles and bubblers in the die. A key objective of the project was to establish criteria for the minimal distance of the cooling lines from the surface. This effort was supported with alloys and machining by BohlerUddeholm, Dunn Steel, HH Stark and Rex Buckeye. In plant testing and evaluation was conducted as in-kind cost share at St. Clair Die Casting. The Uddeholm Dievar steel evaluated in this program showed superior resistance to thermal fatigue resistance. Based on the experimental evidence, cooling lines could be placed as close as 0.5"Â from the surface. Die Life Extension by Optimized Die Lubrication: The life of die casting dies is affected by additions made to its surface with the proper lubricants. These lubricants will protect the surface from the considerable temperature peaks that occur when the molten melt enters the die. Dies will reach a significantly higher temperature without this lubricant being applied. The amount and type of the lubricant are critical variables in the die casting process. However, these lubricants must not corrode the die surface. This effort was supported with alloys and machining by BohlerUddeholm, Dunn Steel, HH Stark and Rex Buckeye. In plant testing and evaluation was conducted as in-kind cost share at St. Clair Die Casting. Chem- Trend participated in the program with die lubricants and technical support. Experiments conducted with these lubricants demonstrated good protection of the substrate steel. Graphite and boron nitride used as benchmarks are capable of completely eliminating soldering and washout. However, because of cost and environmental considerations these materials are not widely used in industry. The best water-based die lubricants evaluated in this program were capable of providing similar protection from soldering and washout. In addition to improved part quality and higher production rates, improving die casting processes to preserve the life of the inserts will result in energy savings and a reduction in environmental wastes. Improving die life by means of optimized cooling line placement, baffles and bubblers in the die will allow for reduced die temperatures during processing, saving energy associated with production. The utilization of optimized die lubricants will also reduce heat requirements in addition to reducing waste associated with soldering and washout. This new technology was predicted to result in an average energy savings of 1.1 trillion BTU's/year over a 10 year period. Current (2012) annual energy saving estimates, based on commercial introduction in 2010, a market penetration of 70% by 2020 is 1.26 trillion BTU's/year. Along with these energy savings, reduction of scrap and improvement in casting yield will result in a reduction of the environmental emissions associated with the melting and pouring of the metal which will be saved as a result of this technology. The average annual estimate of CO2 reduction per year through 2020 is 0.025 Million Metric Tons of Carbon Equivalent (MM TCE)

Growth profile of PASMCs cultured in GM supplemented with BMP-2, GW9662 (PPARγ antagonist) or bpV(HOpic) (PTEN inhibitor).

<p>PPARγ antagonist and PTEN inhibitor were added into respective cell culture medium 1 hour before adding BMP-2 (40 ng/ml). (The number of PASMCs after cultured in GM only was considered as 100%). (*: vs GM only) (Lipo = Lipofetamine −2000).</p

Addition of BMP-2 (40 ng/ml) in GM did not significantly change AKT-1 and AKT-2 gene expressions of PASMCs.

<p>No significant reduction or increment of AKT-1 gene expression was found when PASMCs were cultured in GM supplemented with BMP-2 (<b>A</b>). Similarly, no significant change of AKT-2 gene expression was found when PASMCs were cultured in GM supplemented with BMP-2 (<b>B</b>).</p

BMP-2 increased caspases 3, 8, and 9 activities.

<p>BMP-2 (40 ng/ml) significantly increased caspases 3 (<b>A</b>), 8 (<b>B</b>), and 9 (<b>C</b>) activities of PASMCs cultured in GM. However, pre-treat PASMCs with GW9662 and bpV(HOpic) reversed this effect (*: vs other samples, p<0.05).</p

Smad-4 gene and protein expressions of Lipo-siRNA-Smad-4 transfected PASMCs.

<p>(<b>A</b>) QRT-PCR demonstrated that the lowest Smad-4 gene expression was achieved when 120 nM siRNA-Smad-4 was used to transfect 1×10⁵ PASMCs. (<b>B</b>) Abolishment of Smad-4 protein expression did not affect PTEN protein expression. Qantification of PTEN (<b>C</b>) and pAKT (<b>D</b>) protein expression after normalized to GAPDH (consider as 100%). (<b>E</b>) A non-coding siRNA was used as a negative control to determine the specificity of siRNA-Smad-4 targeting. (Lipo = lipofetamine-2000. *: vs lipo only, p<0.05; &: vs GM only, p<0.05).</p

Reduced PASMCs proliferation rate as a function of BMP-2 concentration when cultured in GM under hypoxia (A).

<p>PASMCs cultured in GM supplemented with 0–80 ng/ml BMP-2. BMP-2 at the concentrations of 40 and 60 ng/ml significantly inhibited PASMC proliferation as compared with GM without BMP-2. Typical pictures of PASMCs cultured in GM only (<b>B</b>), or GM supplemented with BMP-2 at 40 ng/ml (<b>C</b>). (*: vs 0 ng/ml only, p<0.05) <b>(</b>Magnification = 100×).</p

RT-PCR primers and annealing temperature.

<p>RT-PCR primers and annealing temperature.</p

Typical pictures of EGFP expression from pEGFP lipoplexes transfected PASMC when the ratio between Lipofectamine-2000 and plasmid DNA was at (A) 1∶1, (B) 2∶1, (C) 3∶1 and (D) 4∶1, when 2 µg plasmid DNA was used per 1×10⁵ cells.

<p>(Magnification = 40×).</p

Addition of BMP-2 (40 ng/ml) in GM increased PTEN gene expression of PASMCs.

<p>BMP-2 significantly increased PTEN expression at 8 and 24 hours after treatment. (&: vs any other time point, p<0.05; *: vs 0, 1, and 4 hours, p<0.05).</p