Journal: The Journal of Clinical Investigation

Article Title: Disruption of PPAR?/?-catenin-mediated regulation of apelin impairs BMP-induced mouse and human pulmonary arterial EC survival

doi: 10.1172/JCI43382

Figure Lengend Snippet: (A) Caspase 3/7 activity was used to measure the level of apoptosis in PAECs exposed for 24 hours to SF medium in the presence of vehicle control, apelin (100 nM), or VEGFA (50 ng/ml). (B) PAEC survival under these conditions was also determined by cell count and MTT analyses. (C) Survival in the presence of apelin was also assessed by cell counts in PMVECs isolated from control and IPAH patients. (D) Apoptosis level in control siRNA– and apelin siRNA–treated PAECs, as determined by caspase 3/7 assay under SF conditions. (E) Survival of apelin-deficient PAECs was determined by cell count after 24 hours of exposure to SF conditions. Baseline represents cell number before switching to SF medium. Bars represent mean ± SEM from 3 separate experiments with 6 replicates per condition in A and D and 3 replicates per condition in B, C, and E. *P < 0.05, **P < 0.01, ***P < 0.001 vs. respective control, 1-way ANOVA with Bonferroni multiple comparison test.

Article Snippet: PAECs (ScienCell) and human and mouse PMVECs were grown in commercial EC media (Sciencell).

Techniques: Activity Assay, Control, Cell Counting, Isolation, Comparison

Journal: Scientific Reports

Article Title: Mechanotransductive stabilization of HIF-1α is inhibited by mitochondrial antioxidant therapy in the setting of pulmonary overcirculation

doi: 10.1038/s41598-025-99062-0

Figure Lengend Snippet: Differentially expressed transcripts mapping to the canonical HIF-1α signaling pathway in shunt PAECs, as mapped by Ingenuity Pathway Analysis Canonical pathway analysis, p -value of .0054 and z-score of + 3.657 indicating significant overexpression of this pathway in the shunt PAECs compared to controls. Orange = Predicted activation, Blue = Predicted inhibition, Pink = Increased measurement. N = 4 control and N = 3 shunt primary PAEC lines.

Article Snippet: N = 3 shunt and N = 3 control cell lines. ( B ) MitoQ shunt PAECs (N = 4) have significantly lower HIF-1α protein levels than shunt cells (N = 5) by Western Blot. ( C ) Peripheral lung tissue from MitoQ shunt animals (N = 4) demonstrates lower superoxide levels than shunt animals (N = 4) and no different from lung tissue from physiologically normal control animals (N = 4) by EPR with representative waves included for each group.

Techniques: Over Expression, Activation Assay, Inhibition, Control

Journal: Scientific Reports

Article Title: Mechanotransductive stabilization of HIF-1α is inhibited by mitochondrial antioxidant therapy in the setting of pulmonary overcirculation

doi: 10.1038/s41598-025-99062-0

Figure Lengend Snippet: Primary PAECs and snap frozen peripheral lung tissues from shunt and control animals were compared. ( A ) Shunt PAECs exhibit higher mitochondrial ROS production based on fluorescence intensity with MitoSOX staining. Results were analyzed as control and shunt pairs, with each shunt normalized to paired control PAEC fluorescence level. N = 3 shunt and 3 control cell lines. Scale bars = 50 μM ( B ) Shunt PAECs have significantly higher levels of HIF-1α protein by western blot. N = 4 shunt and N = 5 control cell lines. ( C ) Peripheral lung tissue from shunt animals shows significantly higher levels of superoxide content by EPR analysis ( D ) Peripheral lung tissue from shunt animals also demonstrates significantly higher levels of HIF-1α protein. N = 5 shunt and N = 5 control animals for tissue studies. Western blots HIF-1α levels are normalized to β-actin as an internal loading control and expressed as band intensity ratios. All p -values calculated by unpaired, 2-tailed t-test with p < 0.05 considered significant. For bar graphs * denotes p -value ≤ 0.05, ** denotes p -value ≤ 0.01, *** denotes p -value ≤ 0.001 and **** denotes p -value ≤ 0.0001.

Article Snippet: N = 3 shunt and N = 3 control cell lines. ( B ) MitoQ shunt PAECs (N = 4) have significantly lower HIF-1α protein levels than shunt cells (N = 5) by Western Blot. ( C ) Peripheral lung tissue from MitoQ shunt animals (N = 4) demonstrates lower superoxide levels than shunt animals (N = 4) and no different from lung tissue from physiologically normal control animals (N = 4) by EPR with representative waves included for each group.

Techniques: Control, Fluorescence, Staining, Western Blot

Journal: Scientific Reports

Article Title: Mechanotransductive stabilization of HIF-1α is inhibited by mitochondrial antioxidant therapy in the setting of pulmonary overcirculation

doi: 10.1038/s41598-025-99062-0

Figure Lengend Snippet: Control PAECs were variably exposed to 4 h of shear (20 dynes) or stretch (18% amplitude at 1 Hz) compared to unexposed static controls. ( A ) Shear exposure did not result in a significant increase in mitochondrial ROS by fluorescence intensity with MitoSOX staining ( B ) Stretch exposure led to a significant increase in mitochondrial ROS production compared to static PAECs. Scale bars = 50 μM ( C ) Shear exposure reduced HIF-1α protein levels, while ( D ) Stretch exposure increased HIF-1α protein levels by Western Blot relative to static controls. N = 6 exposed and unexposed groups for MitoSOX staining in shear and stretch groups, N = 4 exposed and unexposed groups for HIF-1α blots. Western blots HIF-1α levels are normalized to β-actin as an internal loading control and expressed as band intensity ratios. All p -values calculated by unpaired, 2-tailed t-test with p < 0.05 considered significant.

Article Snippet: N = 3 shunt and N = 3 control cell lines. ( B ) MitoQ shunt PAECs (N = 4) have significantly lower HIF-1α protein levels than shunt cells (N = 5) by Western Blot. ( C ) Peripheral lung tissue from MitoQ shunt animals (N = 4) demonstrates lower superoxide levels than shunt animals (N = 4) and no different from lung tissue from physiologically normal control animals (N = 4) by EPR with representative waves included for each group.

Techniques: Control, Shear, Fluorescence, Staining, Western Blot

Journal: Scientific Reports

Article Title: Mechanotransductive stabilization of HIF-1α is inhibited by mitochondrial antioxidant therapy in the setting of pulmonary overcirculation

doi: 10.1038/s41598-025-99062-0

Figure Lengend Snippet: Primary PAECs derived from shunt animals treated in vitro with MitoQ (designated as Shunt + MitoQ) for 24 h exhibit: ( A ) significantly decreased mitochondrial ROS production based on fluorescence intensity with MitoSOX staining (N = 6) and ( B ) significantly decreased levels of HIF-1α protein by western blot (N = 4) compared to untreated cells. Treatment with TPP + alone, the mitochondrial targeting moiety of MitoQ, does not significantly alter mitochondrial ROS or HIF-1α. ( C ) Control PAECs were cultured and treated with MitoQ and media was collected for analysis of NO x . MitoQ did not impact NO x levels of treated compared to untreated controls. ( D ) Similarly, shunt PAECs were cultured and treated with MitoQ and TPP + and media was collected for analysis. NO x levels were significantly increased in media from shunt PAECs treated with MitoQ, while those treated with TPP + showed no significant difference, compared to untreated shunt cells. (N = 5 for all groups). 2-way ANOVA performed with α of .05 as the significance threshold. ( E ) Control PAECs (N = 6) were treated with the prolyl-hydroxylase inhibitor FG-4592/roxadustat (100 μM for 24 h) and exhibited significantly decreased NO production by DAF FM fluorescence intensity compared to untreated control cells. ( F ) Control PAECs (N = 6) were transfected with an adenoviral vector expressing HIF-1α at a multiplicity of infection (MOI) of 100 resulting in increased cellular HIF-1α protein levels. These AdHIF1a cells also demonstrated significantly decreased NO production by DAF FM fluorescence compared to untreated control cells. ( G ) Shunt PAECs (N = 6) were treated with the HIF-1α inhibitor CAY 10,585 (10 μM for 24 h), which significantly increased NO production by DAF FM fluorescence compared to untreated shunt cells. All p -values calculated by unpaired, 2-tailed t-test with p < 0.05 considered significant.

Article Snippet: N = 3 shunt and N = 3 control cell lines. ( B ) MitoQ shunt PAECs (N = 4) have significantly lower HIF-1α protein levels than shunt cells (N = 5) by Western Blot. ( C ) Peripheral lung tissue from MitoQ shunt animals (N = 4) demonstrates lower superoxide levels than shunt animals (N = 4) and no different from lung tissue from physiologically normal control animals (N = 4) by EPR with representative waves included for each group.

Techniques: Derivative Assay, In Vitro, Fluorescence, Staining, Western Blot, Control, Cell Culture, Transfection, Plasmid Preparation, Expressing, Infection

Journal: Scientific Reports

Article Title: Mechanotransductive stabilization of HIF-1α is inhibited by mitochondrial antioxidant therapy in the setting of pulmonary overcirculation

doi: 10.1038/s41598-025-99062-0

Figure Lengend Snippet: Shunt animals were treated daily with oral MitoQ. Primary PAEC cell lines were derived, and peripheral lung tissues collected and snap frozen from treated (note that all tissues and PAECs derived from these treated animals are labelled “MitoQ Shunt”) and untreated shunt animals for analysis. ( A ) MitoQ shunt PAECs exhibit lower mitochondrial ROS production than shunt PAECs based on fluorescence intensity with MitoSOX staining. Results were analyzed as control and shunt pairs, with each shunt normalized to paired control PAEC fluorescence level. N = 3 shunt and N = 3 control cell lines. ( B ) MitoQ shunt PAECs (N = 4) have significantly lower HIF-1α protein levels than shunt cells (N = 5) by Western Blot. ( C ) Peripheral lung tissue from MitoQ shunt animals (N = 4) demonstrates lower superoxide levels than shunt animals (N = 4) and no different from lung tissue from physiologically normal control animals (N = 4) by EPR with representative waves included for each group. Analysis was performed by 2-way ANOVA with α of .05 as the significance threshold. ( D ) Peripheral lung tissue from MitoQ shunt animals (N = 4) has significantly lower levels of HIF-1α protein than untreated shunts. Western blots HIF-1α levels are normalized to β-actin as an internal loading control and expressed as ratios of band intensity. All p -values calculated by unpaired, 2-tailed t-test with p < 0.05 considered significant.

Article Snippet: N = 3 shunt and N = 3 control cell lines. ( B ) MitoQ shunt PAECs (N = 4) have significantly lower HIF-1α protein levels than shunt cells (N = 5) by Western Blot. ( C ) Peripheral lung tissue from MitoQ shunt animals (N = 4) demonstrates lower superoxide levels than shunt animals (N = 4) and no different from lung tissue from physiologically normal control animals (N = 4) by EPR with representative waves included for each group.

Techniques: Derivative Assay, Fluorescence, Staining, Control, Western Blot

Journal: Scientific Reports

Article Title: Mechanotransductive stabilization of HIF-1α is inhibited by mitochondrial antioxidant therapy in the setting of pulmonary overcirculation

doi: 10.1038/s41598-025-99062-0

Figure Lengend Snippet: Primary PAEC cell lines, serum and peripheral lung tissue derived from treated MitoQ shunt and untreated shunt animals were evaluated for NO levels and production. ( A ) MitoQ shunt PAECs exhibit increased NO production compared to untreated shunt cells by DAF FM fluorescence intensity. Results analyzed as control and shunt pairs, with each shunt normalized to paired control PAEC fluorescence level. N = 3 shunt and N = 3 control cell lines. ( B ) Culture media from MitoQ shunt PAECs contains higher levels of NO x than media from untreated shunt cells. (N = 3 MitoQ shunt and N = shunt cell lines) ( C ) Plasma samples and ( D ) peripheral lung tissue from MitoQ shunt animals have significantly higher NO x levels than corresponding samples from untreated shunt animals, though not as high as physiologically normal control animals. (N = 3 MitoQ shunt, shunt, and normal control animals per group performed on samples in triplicate). p -values for A&B calculated by unpaired, 2-tailed t-test with p < 0.05 considered significant. Analysis for C&D was performed by 2-way ANOVA with α of .05 as the significance threshold.

Article Snippet: N = 3 shunt and N = 3 control cell lines. ( B ) MitoQ shunt PAECs (N = 4) have significantly lower HIF-1α protein levels than shunt cells (N = 5) by Western Blot. ( C ) Peripheral lung tissue from MitoQ shunt animals (N = 4) demonstrates lower superoxide levels than shunt animals (N = 4) and no different from lung tissue from physiologically normal control animals (N = 4) by EPR with representative waves included for each group.

Techniques: Derivative Assay, Fluorescence, Control, Clinical Proteomics

Journal: Scientific Reports

Article Title: Mechanotransductive stabilization of HIF-1α is inhibited by mitochondrial antioxidant therapy in the setting of pulmonary overcirculation

doi: 10.1038/s41598-025-99062-0

Figure Lengend Snippet: Primary PAECs derived from shunt animals treated in vitro with MitoQ (designated as Shunt + MitoQ) for 24 h exhibit: ( A ) significantly decreased mitochondrial ROS production based on fluorescence intensity with MitoSOX staining (N = 6) and ( B ) significantly decreased levels of HIF-1α protein by western blot (N = 4) compared to untreated cells. Treatment with TPP + alone, the mitochondrial targeting moiety of MitoQ, does not significantly alter mitochondrial ROS or HIF-1α. ( C ) Control PAECs were cultured and treated with MitoQ and media was collected for analysis of NO x . MitoQ did not impact NO x levels of treated compared to untreated controls. ( D ) Similarly, shunt PAECs were cultured and treated with MitoQ and TPP + and media was collected for analysis. NO x levels were significantly increased in media from shunt PAECs treated with MitoQ, while those treated with TPP + showed no significant difference, compared to untreated shunt cells. (N = 5 for all groups). 2-way ANOVA performed with α of .05 as the significance threshold. ( E ) Control PAECs (N = 6) were treated with the prolyl-hydroxylase inhibitor FG-4592/roxadustat (100 μM for 24 h) and exhibited significantly decreased NO production by DAF FM fluorescence intensity compared to untreated control cells. ( F ) Control PAECs (N = 6) were transfected with an adenoviral vector expressing HIF-1α at a multiplicity of infection (MOI) of 100 resulting in increased cellular HIF-1α protein levels. These AdHIF1a cells also demonstrated significantly decreased NO production by DAF FM fluorescence compared to untreated control cells. ( G ) Shunt PAECs (N = 6) were treated with the HIF-1α inhibitor CAY 10,585 (10 μM for 24 h), which significantly increased NO production by DAF FM fluorescence compared to untreated shunt cells. All p -values calculated by unpaired, 2-tailed t-test with p < 0.05 considered significant.

Article Snippet: Evaluating primary PAECs from the MitoQ-treated shunt animals compared to untreated; we demonstrated increased levels of basal NO production by DAF fluorescence staining of the cells (Fig. A), as well as by measurement of bioavailable NO (NO x ) released into the cell culture media (Fig. B).

Techniques: Derivative Assay, In Vitro, Fluorescence, Staining, Western Blot, Control, Cell Culture, Transfection, Plasmid Preparation, Expressing, Infection

Journal: Scientific Reports

Article Title: Mechanotransductive stabilization of HIF-1α is inhibited by mitochondrial antioxidant therapy in the setting of pulmonary overcirculation

doi: 10.1038/s41598-025-99062-0

Figure Lengend Snippet: Shunt animals were treated daily with oral MitoQ. Primary PAEC cell lines were derived, and peripheral lung tissues collected and snap frozen from treated (note that all tissues and PAECs derived from these treated animals are labelled “MitoQ Shunt”) and untreated shunt animals for analysis. ( A ) MitoQ shunt PAECs exhibit lower mitochondrial ROS production than shunt PAECs based on fluorescence intensity with MitoSOX staining. Results were analyzed as control and shunt pairs, with each shunt normalized to paired control PAEC fluorescence level. N = 3 shunt and N = 3 control cell lines. ( B ) MitoQ shunt PAECs (N = 4) have significantly lower HIF-1α protein levels than shunt cells (N = 5) by Western Blot. ( C ) Peripheral lung tissue from MitoQ shunt animals (N = 4) demonstrates lower superoxide levels than shunt animals (N = 4) and no different from lung tissue from physiologically normal control animals (N = 4) by EPR with representative waves included for each group. Analysis was performed by 2-way ANOVA with α of .05 as the significance threshold. ( D ) Peripheral lung tissue from MitoQ shunt animals (N = 4) has significantly lower levels of HIF-1α protein than untreated shunts. Western blots HIF-1α levels are normalized to β-actin as an internal loading control and expressed as ratios of band intensity. All p -values calculated by unpaired, 2-tailed t-test with p < 0.05 considered significant.

Article Snippet: Evaluating primary PAECs from the MitoQ-treated shunt animals compared to untreated; we demonstrated increased levels of basal NO production by DAF fluorescence staining of the cells (Fig. A), as well as by measurement of bioavailable NO (NO x ) released into the cell culture media (Fig. B).

Techniques: Derivative Assay, Fluorescence, Staining, Control, Western Blot

Journal: Scientific Reports

Article Title: Mechanotransductive stabilization of HIF-1α is inhibited by mitochondrial antioxidant therapy in the setting of pulmonary overcirculation

doi: 10.1038/s41598-025-99062-0

Figure Lengend Snippet: Primary PAEC cell lines, serum and peripheral lung tissue derived from treated MitoQ shunt and untreated shunt animals were evaluated for NO levels and production. ( A ) MitoQ shunt PAECs exhibit increased NO production compared to untreated shunt cells by DAF FM fluorescence intensity. Results analyzed as control and shunt pairs, with each shunt normalized to paired control PAEC fluorescence level. N = 3 shunt and N = 3 control cell lines. ( B ) Culture media from MitoQ shunt PAECs contains higher levels of NO x than media from untreated shunt cells. (N = 3 MitoQ shunt and N = shunt cell lines) ( C ) Plasma samples and ( D ) peripheral lung tissue from MitoQ shunt animals have significantly higher NO x levels than corresponding samples from untreated shunt animals, though not as high as physiologically normal control animals. (N = 3 MitoQ shunt, shunt, and normal control animals per group performed on samples in triplicate). p -values for A&B calculated by unpaired, 2-tailed t-test with p < 0.05 considered significant. Analysis for C&D was performed by 2-way ANOVA with α of .05 as the significance threshold.

Article Snippet: Evaluating primary PAECs from the MitoQ-treated shunt animals compared to untreated; we demonstrated increased levels of basal NO production by DAF fluorescence staining of the cells (Fig. A), as well as by measurement of bioavailable NO (NO x ) released into the cell culture media (Fig. B).

Techniques: Derivative Assay, Fluorescence, Control, Clinical Proteomics