Fig 1: PDE4D is primarily expressed in smooth muscle cells of AAA tissues.a Immunofluorescence staining of PDE4D and a smooth muscle actin (a-SMA, smooth muscle cell marker) in human non-AAA sections and AAA tissues (6 µm). We obtained similar results from at least three different sets of human tissues in separate experiments. PDE4D: red, a-SMA: green. b Immunofluorescence staining of PDE4D and a-SMA in mouse control sections and AAA tissues (6 µm). We obtained similar results from at least three different sets of mouse samples in separate experiments. PDE4D: red, a-SMA: green. L: lumen. c RT-PCR analysis of Pde4d expression in rat aortic SMCs treated with Ang II (100 nM, 24 h). **p < 0.0001, Welch’s t test, mean ± SEM, n = 3 separate experiments. d Representative immunoblot analysis of PDE4D protein expression in SMCs treated with Ang II (100 nM, 24 h). e Quantification of PDE4D protein expression by immunoblotting in (d) normalized to GAPDH protein (fold change versus control). **p < 0.01, unpaired Student’s t test, mean ± SEM, n = 3 separate experiments.
Fig 2: PDE4D promotes apoptosis through Bad phosphorylation regulated by PKA.a Representative immunoblot analysis of caspase-3 and cleaved caspase-3 expression in the SMCs treated with PKI (PKA inhibitor, 10 μM, 60 min) and/or Ang II (100 nM, 24 h) and/or PDE4D siRNA (200 nM, 48 h) as indicated. b Quantification of cleaved caspase-3 protein expression by immunoblotting in (a) normalized to caspase-3 protein (fold change versus control). **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA with Holm-Sidak’s post hoc test, mean ± SEM, n = 3 separate experiments. c Immunoblot analysis of caspase-3 and cleaved caspase-3 expression in the SMCs treated with ESI-09 (Epac inhibitor, 100 μM, 60 min) and/or Ang II (100 nM, 24 h) and/or PDE4D siRNA (200 nM, 48 h) as indicated. d Quantification of cleaved caspase-3 protein expression by immunoblotting in (c) normalized to caspase-3 protein (fold change versus control). ****p < 0.0001, ns: no significant difference, one-way ANOVA with Holm–Sidak’s post hoc test, mean ± SEM, n = 3 separate experiments. e Representative immunoblot analysis of phospho-Bad (pBad) and Bad expression in the SMCs treated with PKI (PKA inhibitor, 10 μM, 60 min) and/or Ang II (100 nM, 24 h) and/or PDE4D siRNA (200 nM, 48 h) as indicated. f Quantification of pBad expression by immunoblotting in (e) normalized to Bad protein (fold change versus control). **p < 0.01, ****p < 0.0001, one-way ANOVA with Holm-Sidak’s post hoc test, mean ± SEM, n = 3 separate experiments. g Quantification of pBad expression by immunoblotting in (e) normalized to GAPDH protein (fold change versus control). **p < 0.01, ***p < 0.001, one-way ANOVA with Holm–Sidak’s post hoc test, mean ± SEM, n = 3 separate experiments. h Quantification of Bad expression by immunoblotting in (e) normalized to GAPDH protein (fold change versus control). **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA with Holm-Sidak’s post hoc test, mean ± SEM, n = 3 separate experiments. i PDE4D expression was significantly increased in Ang II-induced AAA, aggravating SMC apoptosis, which promoted pBad via the cAMP-PKA axis.
Fig 3: PDE4D regulates SMC apoptosis induced by Ang II.a The top 15 MetaCore pathways (based on their minimum FDR) enriched for both the upregulated genes in Ang II (100 nM, 24 h)-stimulated rat aortic SMCs and the downregulated genes in PDE4D siRNA (200 nM, 48 h)-treated SMCs. b Representative immunoblot analysis of caspase-3 and cleaved caspase-3 expression in the SMCs treated with Ang II (100 nM, 24 h) and/or PDE4D siRNA (200 nM, 48 h) as indicated. c Quantification of cleaved caspase-3 protein expression by immunoblotting in (b) normalized to caspase-3 protein (fold change versus control). **p < 0.01, ***p < 0.001, two-way ANOVA with Holm-Sidak’s post hoc test, mean ± SEM, n = 3 separate experiments. d Flow cytometric analysis of annexin V/propidium iodide (PI)-stained SMCs treated with or without Ang II (100 nM, 24 h)/PDE4D siRNA (200 nM, 48 h) as indicated. e Quantification of the total (early and late) apoptosis rates of annexin V/PI-stained SMCs treated with or without Ang II (100 nM, 24 h)/PDE4D siRNA (200 nM, 48 h) as indicated. ****p < 0.0001, two-way ANOVA with Holm–Sidak’s post hoc test, mean ± SEM, n = 3 separate experiments. f Immunoblot analysis of caspase-3 and cleaved caspase-3 expression in the SMCs treated with or without Ang II (100 nM, 24 h)/rolipram (500 nM, 24.5 h). g Quantification of cleaved caspase-3 protein expression by immunoblotting in (f) normalized to caspase-3 protein (fold change versus control). **p < 0.01, two-way ANOVA with Holm–Sidak’s post hoc test, mean ± SEM, n = 3 separate experiments. h Flow cytometric analysis of annexin V/PI-stained SMCs treated with or without Ang II (100 nM, 24 h)/rolipram (500 nM, 24.5 h) as indicated. i Quantification of the total (early and late) apoptosis rates of annexin V/PI-stained SMCs treated with or without Ang II (100 nM, 24 h)/rolipram (500 nM, 24.5 h) as indicated. ****p < 0.0001, two-way ANOVA with Holm-Sidak’s post hoc test, mean ± SEM, n = 3 separate experiments.
Fig 4: Phosphodiesterase (PDE) 4D expression is upregulated in human and mouse AAA tissues.a RT-PCR quantification of PDE4A, PDE4B, PDE4C, and PDE4D mRNA levels (fold change versus one of the non-AAA subjects) in human tissues. ***p < 0.001, Welch’s t test for PDE4A, Mann–Whitney test for PDE4B/PDE4C, unpaired Student’s t test for PDE4D, mean ± SEM, non-AAA (n = 6), AAA (n = 9). Human AAA tissues were collected from AAA patients during surgery, and non-AAA tissues were collected from corresponding tissues of deceased donors with no detectable vascular disease. b RT-PCR quantification of Pde4a, Pde4b, Pde4c, and Pde4d mRNA (fold change versus one of the control subjects) in mouse AAA tissues from the Apoe−/− mice treated with 1000 ng kg−1 min−1 angiotensin II (Ang II) and a high-fat diet (HFD) and control mouse abdominal aortas from the Apoe−/− mice treated with saline. *p < 0.05, unpaired Student’s t test, mean ± SEM, controls (n = 4), AAA (n = 8). Mouse AAA aortas were dissected at the maximal suprarenal outer aortic diameter, and controls were collected from the corresponding suprarenal abdominal aortas of control mice. (c, d). Representative western blots and quantification of PDE4D protein levels in human AAA and non-AAA tissues normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein (fold change versus non-AAA subjects). **p < 0.01, Mann-Whitney test, mean ± SEM, non-AAA (n = 6), AAA (n = 9). (e, f) Representative western blots and quantification of PDE4D protein levels in mouse AAA and control tissues normalized to GAPDH protein (fold change versus control subjects). ***p < 0.001, unpaired Student’s t test, mean ± SEM, controls (n = 4), AAA (n = 8). (g, h) Representative images of immunohistochemistry staining of PDE4D (g) and quantification of PDE4D staining intensity per medial area (h) in human tissues. A total of five random images per human in each group were selected for statistical analysis of the ratio of positive areas to vascular areas. L lumen. **p < 0.01, unpaired Student’s t test, mean ± SEM, non-AAA (n = 6), AAA (n = 9). (i, j) Representative images of immunohistochemistry analysis of PDE4D (i) and quantification of PDE4D staining intensity per medial area (j) in mouse tissues. A total of five random images per mouse in each group were selected for statistical analysis of the ratio of positive areas to vascular areas. L lumen. *p < 0.05, Welch’s t test, mean ± SEM, controls (n = 4), AAA (n = 8).
Fig 5: PDE4D knockout and rolipram alleviates apoptosis in vivo.a Representative immunoblot analysis of caspase-3 and cleaved caspase-3 expression in the Apoe-/-Pde4dflox/flox and Apoe-/-Pde4dSMC-/- mice with or without 1000 ng kg-1 min-1 Ang II treatment and a high-fat diet (HFD) for 28 days. b Quantification of cleaved caspase-3 protein expression by immunoblotting in (a) normalized to caspase-3 protein (fold change versus control). *p < 0.05, ****p < 0.0001, two-way ANOVA with Holm-Sidak’s post hoc test, mean ± SEM, Apoe-/-Pde4dflox/flox (n = 5) and Apoe-/-Pde4dSMC-/- (n = 5) with saline infusion, Apoe-/-Pde4dflox/flox (n = 5) and Apoe-/-Pde4dSMC-/- (n = 5) with Ang II infusion. c Quantification of cell apoptosis rates with TUNEL staining in Supplementary Fig. 13. A total of five random images per mouse were selected for TUNEL staining statistical analysis. Apoptotic cells examined by TUNEL staining were quantified as the ratio of apoptotic cells to total cells in five fields per mouse. *p < 0.05, **p < 0.01, Welch ANOVA with Dunnett’s T3 post hoc test, mean ± SEM, Apoe-/-Pde4dflox/flox (n = 5) and Apoe-/-Pde4dSMC-/- (n = 5) with saline infusion, Apoe-/-Pde4dflox/flox (n = 5) and Apoe-/-Pde4dSMC-/- (n = 5) with Ang II infusion. (d) Representative immunoblot analysis of caspase-3 and cleaved caspase-3 expression in the Apoe-/- mice with or without Ang II/rolipram treatment. e Quantification of cleaved caspase-3 protein expression by immunoblotting in (d) normalized to caspase-3 protein (fold change versus control). *p < 0.05, **p < 0.01, two-way ANOVA with Holm-Sidak’s post hoc test, mean ± SEM, vehicle (n = 5) and rolipram (n = 5) with saline infusion, vehicle (n = 5) and rolipram (n = 5) with Ang II infusion. f Quantification of cell apoptosis rates with TUNEL staining in Supplementary Fig. 14. A total of 5 random images per mouse were selected for TUNEL staining statistical analysis. Apoptotic cells examined by TUNEL staining were quantified as the ratio of apoptotic cells to total cells in five fields per mouse. **p < 0.01, Mann–Whitney test, mean ± SEM, vehicle (n = 5) and rolipram (n = 5) with saline infusion, vehicle (n = 5) and rolipram (n = 5) with Ang II infusion.
Supplier Page from Abcam for Anti-PDE4D antibody [EPR11823-31]