Cyclic AMP Prevents Hypoxia-Induced Arginase II Expression and Proliferation of Human Pulmonary Artery Smooth Muscle Cells; However, Mice Deficient in PDE3A have Evidence of Pulmonary Hypertension

Pulmonary hypertension (PH) associated with alveolar hypoxia is characterized by vascular remodeling and smooth muscle cell proliferation. Hypoxia induces the proliferation of human pulmonary artery smooth muscle cells (hPASMC) through induction of arginase II (arg II). The signal transduction pathways responsible for this induction are unknown. cAMP is involved in many intracellular processes including signal transduction. cAMP levels are regulated by a balance between production, via adenylate cyclases (AC), and degradation via phosphodiesterases (PDE). In the pulmonary vasculature, PDE3 is implicated as one of the predominant isoforms for cAMP degradation.

To test the hypothesis that 1) Hypoxia increases arg II expression and hPASMC proliferation through cAMP and 2) Mice deficient in Pde3a will have attenuated pulmonary hypertension.

hPASMC were treated with the cAMP analogs, 8-Br-cAMP or 8-pCPT-2'-O-Me-cAMP; the PDE3 inhibitor, cilostamide; or the AC activator, forskolin; then incubated in 21%O₂ or 1%O₂ for 48h. Cells were harvested for RNA and/or protein. Arg I and II mRNA and protein levels were measured by quantitative real-time PCR and Western blot and arginase activity measured. For proliferation studies, ˜10,000 cells were seeded/well in six-well plates, treated with pharmacologic agents as described, and exposed to 21%O₂ or 1%O₂ for 5d. Adherent cells were trypsinized, and viable cells counted using trypan blue exclusion. hPASMC were also treated with the PKA inhibitors, H-89 or Rp-cAMPS +/– 8-Br-cAMP, or transfected with siRNA targeted against PKA(α+β) +/– 8-Br-cAMP; arg II protein levels were measured and/or hPASMC proliferation assessed. Pde3a–/– (KO) and wild-type (WT) mice were exposed to room air for 28d. The mice were sedated and RV pressures were measured. The mice were then sacrificed using pentobarbital and the lungs were harvested for protein. Arg II protein expression was evaluated by Western blot analysis. The hearts were also harvested and the right ventricle (RV) and the left ventricle (LV)+septum (S) were weighed and the ratio of RV/(LV+S) weights was calculated. Some mice were also exposed to 12%O₂ for 28d.

There was no change in arg I mRNA or protein levels in vehicle-treated hPASMC or those treated with 8-Br-cAMP, cilostamide, or forskolin with exposure to 1%O₂ at 48h. Treatment with 8-Br-cAMP, cilostamide, or forskolin inhibited both hypoxia-induced arg II mRNA and protein levels at 48h to levels equivalent to vehicle-treated, 21%O₂ controls. Furthermore, these agents prevented hypoxia-induced hPASMC proliferation. The hypoxia-induced arginase activity in hPASMC was inhibited by the addition of 8-Br-cAMP. 8-pCPT-2'-O-Me-cAMP and the PKA inhibitors had little effect on hypoxia-induced arg II protein induction. siRNA against PKA(α+β) + 8-Br-cAMP resulted in a small, but statistically significant increase in hypoxia-induced hPASMC proliferation, relative to hPASMC treated with 8-Br-cAMP alone. KO mice had a greater RV/(LV+S) ratio and RV pressures compared to WT mice. There was no difference in lung arg II protein levels between WT and KO mice. Interestingly, KO mice exposed to hypoxia had similar RV/(LV+S) ratios and RV pressures as hypoxia-exposed WT mice.

Pharmacologic agents known to increase cAMP concentrations prevent the hypoxia-induced increase in arg II mRNA, protein and activity and the resultant pro-proliferative effects in hPASMC. The cAMP inhibitory effect may be explained, in part, by PKA, but other mechanisms may be involved. Due to the importance of vascular remodeling and smooth muscle proliferation in the pathogenesis of PH, agents acting to increase cAMP may represent an important therapeutic target for the prevention or treatment of PH; thus, we used mice deficient in Pde3a to determine whether they were protected against hypoxia-induced PH. To our surprise, the KO mice had evidence of PH, as seen by elevated RV/(LV+S) ratio and RV pressures. Interestingly, exposure to hypoxia did not result in any worsening of PH in the KO mice. Our results suggest that alterations in Pde3a gene function may underlie some forms of PH not associated with hypoxia, for example primary pulmonary arterial hypertension.