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Results: 8

1.
Figure 6

Figure 6. DNA methyltransferase expression is increased in FHR lung and PASMCs. From: Epigenetic Attenuation of Mitochondrial Superoxide Dismutase 2 (SOD2) in Pulmonary Arterial Hypertension.

A)DNA MT1 and 3B mRNA is increased in FHR versus SDR lungs (n=12 each). *, **p<0.05 and 0.01, respectively. In low passage (3-4) PASMCs (n=8 in each group), FHR had higher DNA MT3B expression and a trend toward increased DNA MT1.

Stephen L. Archer, et al. Circulation. ;121(24):2661-2671.
2.
Figure 7

Figure 7. SOD2 supplementation inactivates HIF-1α and increases Kv1.5 expression in FHR PASMC. From: Epigenetic Attenuation of Mitochondrial Superoxide Dismutase 2 (SOD2) in Pulmonary Arterial Hypertension.

A) The SOD2 virus and a control GFP virus are effective, increasing expression of SOD2 and GFP mRNA, respectively, in FHR PASMC.
B) Immunofluorescence using an Alexa647 secondary antibody shows that the SOD2 adenovirus increases human SOD2 expression (green, left lower panel). This restores Kv1.5 expression (green) and inactivates HIF-1α (red nuclei). Nuclei are counterstained blue (DAPI).
C-D) 48-hours incubation with MnTBAP decreases nuclear localization of HIF-1α and restores Kv1.5 expression.

Stephen L. Archer, et al. Circulation. ;121(24):2661-2671.
3.
Figure 2

Figure 2. Inhibition of SOD2 expression in normal Sprague-Dawley PASMC recapitulates the PAH phenotype. From: Epigenetic Attenuation of Mitochondrial Superoxide Dismutase 2 (SOD2) in Pulmonary Arterial Hypertension.

A) siRNAs reduce SOD2 mRNA and H2O2 production.
B) siSOD2 hyperpolarizes ΔΨm (increased red TMRM intensity).
C) SOD2 siRNA activates HIF-1α (note translocation to the nucleus-red in middle panel) and decreases Kv1.5 expression (decreased green, left panel).
D) Immunoblotting confirmed that siSOD2 decreases Kv1.5 expression (left panel). In line with loss of Kv channels, siSOD2 increased intracellular K+ concentration (middle panel). siSOD2 also reduced caspase activity (right panel).

Stephen L. Archer, et al. Circulation. ;121(24):2661-2671.
4.
Figure 4

Figure 4. 5-aza-2′-deoxycytidine restores SOD2 expression, reduces proliferation and enhances apoptosis in FHR PASMC. From: Epigenetic Attenuation of Mitochondrial Superoxide Dismutase 2 (SOD2) in Pulmonary Arterial Hypertension.

A-B) Representative and mean data showing that FHR PASMC express less SOD2 than SDR PASMC and this is partially reversible with 5-AZA treatment, n=4 per group. *p<0.05.
C) 5-aza-2′-deoxycytidine-induced, dose-dependent increase in Kv1.5 mRNA in FHR PASMC, *p<0.05.
D-E) 5-aza-2′-deoxycytidine (black bars) increases apoptosis and decreases proliferation in FHR PASMC and lung cancer cells (A549), but not SDR PASMC, n=4-6/group. *p<0.05 between baseline and 5-aza-2′-deoxycytidine, † significant difference from SDR PASMC.
F) 5-aza-2′-deoxycytidine increases O2-consumption in FHR and SDR PASMC, but not A549 cells (n=3-8/group).

Stephen L. Archer, et al. Circulation. ;121(24):2661-2671.
5.
Figure 1

Figure 1. Decreased SOD2 expression in FHR and patients with WHO Category-1 PAH. From: Epigenetic Attenuation of Mitochondrial Superoxide Dismutase 2 (SOD2) in Pulmonary Arterial Hypertension.

A) Confocal microscopy shows decreased PASMC SOD2 expression and increased mitochondrial fragmentation in FHR PASMC. Note the loss of SOD2 (green) in FHR as evidenced by the reduced intensity and peak height.
B) Confocal immunofluorescence images of 3 control patients (who died from non lung-related conditions, upper 3 rows) and 3 patients who died of PAH (lower rows). Note the medial hypertrophy and plexiform lesions in the PAH patients. SOD2 expression (red) is decreased in the media and adventitia of small PAs and in the plexiform lesions. α-smooth muscle actin (green) is increased in PAH.
C) mean±SEM of red fluorescence intensity showing decreased SOD2 expression in the media of small PAs. * p<0.05.

Stephen L. Archer, et al. Circulation. ;121(24):2661-2671.
6.
Figure 8

Figure 8. MnTBAP regresses PAH in FHR. From: Epigenetic Attenuation of Mitochondrial Superoxide Dismutase 2 (SOD2) in Pulmonary Arterial Hypertension.

A) MnTBAP reduces mean PA pressure measured by Doppler (lengthens PAAT) and decreases right ventricular thickness in FHR treated for 4 weeks, n=5/group. * p<0.05.
B) MnTBAP therapy reduces mean PAP and total pulmonary resistance (TPR).
C) FHR treated with MnTBAP exercise longer on a graded treadmill, n=15/group.
D) Lung sections were stained for von Willebrand factor (red), alpha smooth muscle cell actin (green) and DAPI (blue). Note the fully muscularized (white arrows), partially muscularized (yellow arrows) and non-muscularized blood vessels (red arrows). Lower panel: A representative fully muscularized PA in a vehicle-treated FHR (left) versus MnTBAP (right). The % medial thickness of precapillary resistance PAs was reduced and the number of non-muscularized resistance PAs was increased by MnTBAP. ** p<0.01 versus control.

Stephen L. Archer, et al. Circulation. ;121(24):2661-2671.
7.
Figure 5

Figure 5. Lower ROS levels and reduced cellular environment in FHR PASMC. From: Epigenetic Attenuation of Mitochondrial Superoxide Dismutase 2 (SOD2) in Pulmonary Arterial Hypertension.

A) Decreased ROS levels in freshly isolated resistance PAs from FHR versus consomic rats, measured using L-012 chemiluminescence at 37°C.
B) PASMCs were transfected with redox-sensitive GFP constructs targeted to the cytoplasm (upper panel) and mitochondria (lower panel). FHR PASMC had a reduced cytoplasm and mitochondria, relative to Sprague Dawley PASMC. Dithiothreitol and tert-butyl hydroperoxide were used to completely reduce and oxidize the cells, respectively.
C) ROS mean data and representative trace (inset) showing the CMH triplet signal. Peak height (EPR amplitude units) is proportional to ROS levels and is normalized to wet weight. PAs from FHR treated with 5-aza-2′-deoxycytidine (black bars) have increased production of ROS (left) and H2O2 (right) versus untreated FHR. Control rats show no induction of ROS or H2O2, n=5/group.

Stephen L. Archer, et al. Circulation. ;121(24):2661-2671.
8.
Figure 3

Figure 3. Methylation of SOD2 in FHR PASMC is reversible by 5-aza-2′-deoxycytidine. From: Epigenetic Attenuation of Mitochondrial Superoxide Dismutase 2 (SOD2) in Pulmonary Arterial Hypertension.

A) Schematic of the CG dinucleotide percentage and CpG islands within the SOD2 promoter and first 2 KB after the transcriptional start site. 7 amplicons were surveyed within the SOD2 gene. Their approximate locations are represented by solid, horizontal lines. The positions of individual CpG dinucleotides are shown as vertical tick marks in the panel below the amplicon map. * denotes the differentially methylated CpG dinucleotides in FHR within amplicon 7 compared to BN1 tissue. No methylated CpG pairs were identified in amplicons 3-6.
B) Corresponding methylation percentage of the differentially methylated CpG in intron 2. Results are expressed as a frequency of cytosine methylation in PAs from consomic control BN1 rats (n=2), FHR (n=3) and FHR treated with 5-aza-2′-deoxycytidine (n=3).
C) Representative sequencing traces of genomic DNA from cultured PASMCs. Only methylated cytidines are protected against bisulfite-mediated deamination of cytidine into uridine (which is recognized as thymidine when the PCR product is amplified). As indicated by the arrow, the cytidine in FHR PASMCs was methylated (and therefore remains a cytidine, upper left) and this is reversed by 5-Azacytidine. The site is not methylated in FHR aortic SMC or in SDR PASMCs. The bar graph shows the mean data indicating the reversibility and tissue specificity of this SOD2 methylation in intron 2 in cultured PASMCs.
D) FHR PASMC have lower SOD2 mRNA levels versus consomic PASMC. 5-aza-2′-deoxycytidine (5-AZA) causes a dose-dependent increase in SOD2 expression.

Stephen L. Archer, et al. Circulation. ;121(24):2661-2671.

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