Table 1.
Primer pairs of rat PDEs for quantitative realtime-PCR.
Figure 1.
Expression of PDE7-PDE11 isoforms in rat lung tissue and rat PASMCs.
A) mRNA expression of PDE7-PDE11 in lung homogenates from control rats (gray bars) and MCT-PH rats (black bars) as shown by qRT-PCR after normalization to PBGD. *P<0.05, **P<0.01, ***P<0.001 vs control lungs. n = 4 in each group. B) Relative mRNA levels of PDE7-PDE11 in control (gray bars) and MCT (black bars) PASMCs demonstrated by qRT-PCR after normalization to PBGD. **P<0.01 vs control PASMCs. n = 4 in each group. Values are expressed as the mean ± SEM.
Figure 2.
PDE10A expression and localization in rat pulmonary vasculature.
A) Immunohistochemistry staining of PDE10A in lung sections from control (a, b) and MCT (c, d) rats. Scale bar: 20 µm. B) PDE10A mRNA expression in the pulmonary artery (P.A.), aortic artery (A.A.) and femoral artery (F.A.) from control rats (gray bars) and 4-week MCT-PH rats (black bars) are shown as a percentage of control by qRT-PCR after normalization to PBGD. **P<0.01 vs control PASMCs. n = 4 in each group. Values are expressed as the mean ± SEM.
Figure 3.
PDE10A expression and localization in PASMCs.
A) A representative blot of PDE10A protein expression in MCT and control PASMCs, which was determined by immunoblots using GAPDH as the loading control. B) Densitometric quantification of PDE10A expression in PASMCs is shown as the ratio to GAPDH in the bar graph. *P<0.05 vs control PASMCs, n = 3 in each group. Values are expressed as the mean ± SEM. C) Cellular localization of PDE10A in both control and MCT PASMCs was determined by representative immunofluorescence: green (PDE10A, FITC-conjugated), Blue (DAPI). Staining is shown at 400× magnification.
Figure 4.
cAMP PDE activities of control and MCT PASMCs.
A) Total cAMP activity in control and MCT PASMCs. †P<0.05 vs control PASMCs, ***P<0.001 vs total activity, n = 3 in each group. cAMP activity of B) control PASMCs and C) MCT PASMCs mediated by PDE10A or other PDEs (PDE1, PDE2, PDE3 and PDE4). Total cAMP PDE activity was suppressed in varying degrees by a PDE10A inhibitor (papaverine, 10 µM) or by a combination of inhibitors against PDE1 (8 mm-IBMX, 30 µM), PDE2 (EHNA, 30 µM), PDE3 (milrinone, 5 µM) and PDE4 (rolipram, 10 µM). ***P<0.001 vs total activity, n = 3 in each group. Values are expressed as the mean ± SEM.
Figure 5.
Effects of PDE10A knockdown by siRNA on PASMC proliferation.
Representative blots showing PDE10A protein expression in A) control PASMCs and B) MCT PASMCs transiently transfected with 100 nM scramble siRNA or PDE10A siRNA for 48 h. PASMCs treated with transfection reagent alone served as a negative control (NTC), and GAPDH was used as a loading control. C) PASMC proliferation stimulated by 10% FBS with siRNA transfection was determined by the [3H]thymidine incorporation assay. †††P<0.001 vs 0.1% FBS, *P<0.05 vs 10% FBS, n = 4 in each group. Values are expressed as the mean ± SEM.
Figure 6.
Effects of the PDE10A inhibitor papaverine (Pap) on PASMC proliferation, intracellular cAMP accumulation and CREB activation.
A) FBS stimulated PASMC proliferation after Pap (10 µM, 25 µM) treatment for 24 h. [3H]thymidine incorporation was used to evaluate cell proliferation. †††P<0.001 vs 0.1% FBS, *P<0.05 vs 10% FBS, n = 4 in each group. B) Intracellular cAMP levels in PASMCs after Pap treatment measured by a cAMP enzyme immunoassay. The cAMP content of PASMC lysates is given as nmol/mg protein. *P<0.05, ***P<0.001 vs NTC (negative control), n = 4 in each group. C) CREB phosphorylation after Pap (10 µM, 25 µM) treatment. Representative immunoblots are shown in the figure, Values are expressed as the mean ± SEM.
Figure 7.
Effects of papaverine on MCT-PH rats in vivo.
Papaverine was applied by continuous intravenous infusion with osmotic minipumps from days 21 to 35. A) RVSP (mm Hg), B) SAP (mm Hg), C) PVRI (mm Hg min/ml 100 g body), and D) SVRI (mm Hg min/ml 100 g body) are given as the mean ± SEM. ***P<0.001 vs control; †P<0.05 vs. MCT [35 d]/saline. Control: n = 9; MCT [35 d]/saline: n = 8; MCT [35 d]/papaverine: n = 8. E) Right heart mass, which was measured by the ratio of RV/LV+S. F) Effect of papaverine on the extent of muscularization of peripheral pulmonary arteries. The percentage of nonmuscularized (N), partially muscularized (P), or fully (M) muscularized pulmonary arteries related to the total number of pulmonary arteries is given as the mean ± SEM. A total of 80 to 100 intra-acinar vessels were analyzed in the lung of each rat from each group. ***P<0.001 vs control; †P<0.05 vs MCT [35 d]/saline. Values are expressed as the mean ± SEM.
Figure 8.
Anti-proliferative effects of papaverine in vivo.
Proliferating cell nuclear antigen (PCNA) staining was performed to identify proliferating pulmonary vascular cells. A) Representative PCNA immunostaining microphotographs of the rat lung sections from control (left), MCT [35 day]/saline placebo (middle) and MCT [35 day]/papaverine (right), with black arrows indicating the PCNA-positive vascular cells in red. Scale bar: 20 µm. B) Effects of papaverine on pulmonary vascular cell proliferation are expressed as fold changes compared to control lungs, n = 5 in each group. ***P<0.001 vs control; †††P<0.001 vs MCT [35 d]/saline. Values are expressed as the mean ± SEM.
Figure 9.
Pulmonary vascular expression and localization of PDE10A in lung tissues from donor and IPAH patients.
Representative PDE10A immunostaining microphotographs of the human lung sections from donors (A–D) and IPAH patients (E–H). Scale bar: 20 µm.