243 - PDE3 Isoforms Differentially Regulate Cell Growth in Murine Pulmonary Artery Smooth Muscle Cells

Poster Number: 243
Publication Number: 243.222

Paulina N. Krause, Nationwide Children's Hospital, Columbus, OH, United States; Gabrielle McGeorge, Nationwide Children's Hospital, Westerville, OH, United States; Xiaomei Meng, Ohio State University College of Medicine, Columbus, OH, United States; Leif D. Nelin, Nationwide Children's Hospital, Coumbus, OH, United States; Yusen Liu, Nationwide Children's Hospital, Columbus, OH, United States; Bernadette Chen, The Ohio State University College of Medicine, Columbus, OH, United States

Background: Pulmonary vascular cell proliferation and remodeling are hallmarks of pulmonary hypertension (PH). Despite the use of nonisoform-specific PDE3 inhibitors for PH treatment, it remains unclear whether PDE3A and 3B isoforms function similarly within pulmonary vasculature. PDE3 degrades cAMP and cGMP, second messengers important in vasodilatory and proliferative responses. We have shown that PDE3A can modulate AMPK in human pulmonary artery smooth muscle cells (PASMC) and pharmacological PDE3 inhibition decreases PASMC proliferation.

Objective: To test the hypothesis that deficiency in Pde3a and Pde3b may differentially effect AMPK activation and PASMC proliferation.

Design/Methods: PASMC were isolated from male (M) and female (F) C57bl/6J (WT), Pde3a-/- (3A-KO), and Pde3b-/- (3B-KO) mice. Equally seeded PASMC incubated in 21% O2 were grown to 90% confluence or seeded at 15x104 cells/well and grown for 5 d. Viable cells were counted using trypan blue exclusion. Protein was harvested. PDE3A, PDE3B, phosphorylated (p)- and total (T) AMPK, p- and T CREB, cleaved and T caspase-3, and b-actin expression were quantified by Western blot (n=3 per group/gender).

Results: Deficiency of PDE3A in 3A-KO and PDE3B in 3B-KO PASMC were confirmed with no compensatory increase in the non-deficient isoform. AMPK phosphorylation decreased by ~86% (p < 0.0001) in M and ~66% (p < 0.003) in F 3A-KO vs WT PASMC. Similarly, p-AMPK decreased by ~62% (p < 0.004) in M+F 3B-KO vs WT PASMC. CREB phosphorylation decreased by ~36% (p < 0.004) in M and ~80% (p < 0.02) in F 3A-KO vs WT PASMC. No difference was seen in p-CREB between WT and 3B-KO PASMC. Both AMPK and CREB phosphorylation were lower in M 3A-KO vs 3B-KO PASMC (p < 0.03). Cleaved caspase-3 increased ~6.2-fold (p=0.0002) in M and ~5.5-fold (p < 0.0001) in F 3A-KO vs WT PASMC. Similarly, cleaved caspase-3 increased ~2.9-fold (p < 0.03) in M and ~4.3-fold (p < 0.0001) in F 3B-KO vs WT PASMC. WT viable cell numbers increased by ~230% during the 5-d incubation period, whereas 3B-KO resulted in ~94% greater viable cell numbers and 3A-KO led to ~7% increase in viable cells relative to seeded cells (M+F, p<0.002).Conclusion(s): Deficiency of Pde3a or Pde3b inhibited PASMC growth, as indicated by lower viable cell numbers, at least in part due to caspase-3 activation and resultant apoptosis. 3A-KO inhibited PASMC growth and resulted in greater caspase-3 activation than 3B-KO. These data coincide with a decrease in AMPK and CREB activation in 3A-KO PASMC. Our results strongly support the idea that in PASMC, Pde3a and Pde3b differentially regulate AMPK and CREB pathways and cellular proliferation/survival.