Journal: Cardiovascular Research

Article Title: Endothelium-specific SIRT7 targeting ameliorates pulmonary hypertension through Krüpple-like factor 4 deacetylation

doi: 10.1093/cvr/cvae011

Figure Lengend Snippet: Endothelial-specific loss of Sirt7 aggravates PH. ( A ) Immunofluorescence images of SIRT7 co-stained with CD31 in pulmonary arteries from lung tissues of IPAH patients ( n = 4) and control individuals ( n = 2). Scale bar = 20 µm. ( B ) Protein levels of SIRT7 in human PAECs isolated from IPAH patients ( n = 4) and control individuals (Ctrl, n = 4). ( C ) Western blotting analysis of Sirt7 protein expression in lung ECs isolated from Sirt7 f/f ; Cdh5 - Cre ERT mice intraperitoneally injected with TX (Tx, n = 6) or vehicle (Veh, n = 6). ( D–G ) Four-week-old Sirt7 f/f ; Cdh5 - Cre ERT mice were intraperitoneally injected with Tx or vehicle (Veh). Four weeks later, mice were exposed to normoxia (Nor) or SuHx for 4 weeks. RVSP ( D ) and Fulton Index ( E ) were measured. Vascularization of mice in various groups were shown by H&E staining ( F ) and angiography ( G ) of lung tissues. Scale bar = 20 µm ( F ) and 1 mm ( G ), respectively. Data are means ± SEM, and were analysed using Mann–Whitney U test ( B ), two-tailed Student t -test ( C ), and two-way ANOVA with Holm-Šídák's post-hoc test for multiple groups ( D–G ). * P < 0.05, ** P < 0.01, *** P < 0.001 with comparisons indicated by lines ( n = 6 mice per group).

Article Snippet: Human PAECs were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) and cultured with endothelial culture medium (ECM; ScienCell Research Laboratories).

Techniques: Immunofluorescence, Staining, Control, Isolation, Western Blot, Expressing, Injection, MANN-WHITNEY, Two Tailed Test

Journal: Cardiovascular Research

Article Title: Endothelium-specific SIRT7 targeting ameliorates pulmonary hypertension through Krüpple-like factor 4 deacetylation

doi: 10.1093/cvr/cvae011

Figure Lengend Snippet: SIRT7 maintains PAEC function. ( A–F ) PAECs transfected with siRNA against SIRT7 (si S7 ) or negative control (siNC), or infected with lentivirus- SIRT7 ( S7 ) or control virus (Ctrl), were cultured in either 0.1% or 5% FBS for 48 h. Proliferation ( A , D ) and migration ( B , E ) were measured by EdU and wound healing assays, respectively. Tube formation analysis using PAECs transfected with siNC or si SIRT7 , or infected with Ctrl and lentivirus- SIRT7 for 48 h. ( C , F ) Quantified data shows the number of tubes and branch points for each group. Scale bar = 20 µm. ( G , I ) qPCR measurement of SIRT7 , TGFB1 , CTGF , cadherin 1 (ECAD) , IL-6 , IL-1B , NLRP3 , EDN1 , platelet derived growth factor subunit B (PDGFB) and BMPR2 in PAECs transfected with siNC or si SIRT7 , or infected with indicated lentivirus for 48 h. ( H ) Western blotting analysis of protein levels of SIRT7, TGF-β, eNOS, and α-SMA in PAECs transfected with siNC or si S7 for 48 h, and ( J ) protein levels of SIRT7, eNOS TGF-β, and BMPR2 in PAECs with or without SIRT7 overexpression for 48 h. Data are means ± SEM from 5 or 10 independent experiments, and were analysed by Kruskal–Wallis test with Dunn post-hoc test ( A , B , D , and E ) and Student t -test or Mann–Whitney U test ( C , F , G – J ), * P < 0.05, ** P < 0.01, *** P < 0.001 with comparisons indicated by lines.

Article Snippet: Human PAECs were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) and cultured with endothelial culture medium (ECM; ScienCell Research Laboratories).

Techniques: Transfection, Negative Control, Infection, Control, Virus, Cell Culture, Migration, Derivative Assay, Western Blot, Over Expression, MANN-WHITNEY

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