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

Abstract P063: BMP-2 Upregulates PTEN Expression and Induces Apoptosis of Pulmonary Artery Smooth Muscle Cells Under Hypoxia

Background: Primary pulmonary hypertension is mainly caused by increased proliferation and decreased apoptosis in pulmonary artery smooth muscle cells (PASMCs). Aim: To investigate the role of BMP-2 in regulation of PTEN and apoptosis of PASMCs under hypoxia. Methods: Normal human PASMCs were cultured in basal medium (BM) or 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, caspase−3, −8, and −9, AKT-1 and -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 was used to determine whether Smad signaling pathway was involved in the regulation of PTEN expression by BMP-2. bpV(HOpic) (PTEN inhibitor) and GW9662 (PPAR-r antagonist) were also used. Results: Proliferation of PASMCs showed dose dependence of BMP-2, the lowest proliferative rate was achieved at 40–60ng/ml under hypoxia (BM=77.2±4%, GM=80±2.8%). Increased gene expression levels of PTEN, caspases−3, −8 and −9 were found, while AKT-1, and -2 did not change. Consistently, the PTEN protein expression also showed dose dependence of BMP-2. Though AKT was unchanged in all treated samples, reduced pAKT was found in BMP-2 treated PASMCs. Increased activities of caspase-3, -8 and -9 of PASMCs were found after cultured with BMP-2 in both mediums. PTEN expression was unchanged when Smad-4 expression was inhibited. However pre-treat PASMCs with bpV(HOpic) and GW9662 (PPAR-r inhibitor) inhibited PTEN protein expression and recovered PASMCs proliferation rate. Conclusion: BMP-2 can increase PTEN expression under hypoxia in a dose dependent pattern. BMP-2 can increase apoptosis of PASMCs under hypoxia. The increased PTEN expression may be mediated through PPAR-r signalling pathway, instead of BMP/Smad signalling pathway. </jats:p

The reconstructed surface water area time series (2000-2019) dataset for lakes >1 km2 in China

&lt;p&gt;This repository contains the &lt;strong&gt;revised version&lt;/strong&gt; of the supplementary data for the paper: &lt;strong&gt;Reconstruction of long-term high-resolution lake variability: Algorithm improvement and applications in China&nbsp; &lt;/strong&gt;(https://www.sciencedirect.com/science/article/pii/S0034425723003267?via%3Dihub).&nbsp;&lt;/p&gt; &lt;p&gt;Specifically, this dataset documents the reconstructed surface water area time series for all studied lakes in China during the period of 2000-2019. In the prior version of the dataset, there was an erroneous assignment of IDs to each lake. This issue has been rectified in the revised version, ensuring that the updated IDs now accurately correspond to the actual GLAKES_ID for each of the GLAKES lake polygons.&lt;/p&gt; &lt;p&gt;For more detailed information of the dataset, please refer to the README file.&lt;/p&gt

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

Western blot analysis of PASMCs cultured in GM supplemented with BMP-2 under hypoxia.

<p>(<b>A</b>) A dose dependent effect of BMP-2 on PTEN protein expression when BMP-2 was supplemented up to 80 ng/ml in GM. (<b>B</b>) Quantification of PTEN protein expression when BMP-2 was increased from 0–80 ng/ml after normalized to GAPDH (consider as 100%). (<b>C</b>) Typical pictures of PTEN and pAKT protein expressions as a function of time when BMP-2 was supplemented at 40 ng/ml in GM. Quantification of PTEN (<b>D</b>) and pAKT (<b>E</b>) protein expressions after normalized to GAPDH (consider as 100%). Reduced PTEN protein expression was found when GW9662 was added alone (<b>F</b>) or combined with BMP-2 (<b>G</b>). Reduced PTEN protein expression was also found when bpV (HOpic) was added alone (<b>H</b>) or combined with BMP-2 (<b>I</b>). (*: vs 0 ng/ml BMP-2, p<0.05; &: vs 0 hour, p<0.05).</p

Toxicity of BMP-2 towards PASMCs when PASMCs were cultured in GM supplemented with 0−80 ng/ml BMP-2 under normoxia.

<p>It appears that only at 80 ng/ml concentration of BMP-2 resulted in significantly increased LDH leakage as compared with GM with 0 ng/ml BMP-2. The percentage of LDH leakage was normalized to fresh GM (consider as 0%). (*: vs 0 ng/ml, 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