106 - Deficiency of PDE3A and 3B Differentially Regulates Murine Pulmonary Artery Smooth Muscle Cell Growth and Metabolism

Monday, May 1, 2023

9:30 AM – 11:30 AM ET

Poster Number: 106
Publication Number: 106.426

Paulina Krause, Nationwide Children's Hospital, Columbus, OH, United States; Gabrielle McGeorge, Nationwide Children's Hospital, Westerville, OH, United States; Xiaomei Meng, Nationwide Children's Hospital, Columbus, OH, United States; Jenna L. McPeek, Nationwide Children's Hospital, Westerville, OH, United States; Leif D. Nelin, Nationwide Children's Hospital, Columbus, OH, United States; Yusen Liu, Abigail Wexner Research Institute at Nationwide Children's Hospital, Columbus, OH, United States; Bernadette Chen, Nationwide Children's Hospital, Columbus, OH, United States

Presenting Author(s)

Paulina Krause, MD

NICU Fellow
Nationwide Children's Hospital
Columbus, Ohio, United States

Background: Alterations in cell metabolism are emerging as triggers of pulmonary vascular dysfunction associated with pulmonary hypertension (PH). PDE3 degrades cAMP/cGMP, key second messengers in cellular functions. Nonisoform-specific PDE3 inhibitors are used to treat PH, but the mechanism by which PDE3A and 3B isoforms alter metabolic function in the pulmonary vasculature is not clear. We have shown that PDE3A modulates AMPK in human pulmonary artery smooth muscle cells (PASMC). Preliminary data also demonstrate that Pde3a knockout mice have increased serum free fatty acid (FFA) levels.

Objective:

To test the hypothesis that Pde3a and 3b deficiencies have differential effects on metabolic pathways and cell growth in PASMCs that may be triggered by FFA.

Design/Methods: PASMC isolated from wild-type (WT) mice (n=3 per gender) were transfected with scramble, PDE3A (siPDE3A), or PDE3B (siPDE3B) siRNA. Conditioned media (CM) experiment was also performed using SMC growth medium (SmGM) 1:1 with media from harvested from adipocytes (Figure 1). FFA was measured in the siPDE3A adipocyte media. PASMC were incubated in 21%O² for 48h after transfection and protein was harvested. PDE3A, PDE3B, p- and total (T) AMPK, p- and T CREB, cleaved and T caspase 3, and β-actin proteins were quantified by Western blotting. PASMC were seeded (12.5x10⁴ cells/well) and grown for 7d in respective media. Viable cells were counted using trypan blue exclusion.

Results: No differences were observed between genders. PDE3A levels were decreased in siPDE3B PASMC, and PDE3B levels increased in siPDE3A PASMC. AMPK and CREB phosphorylation were decreased in siPDE3A (p< 0.0001) > > siPDE3B (p< 0.001) vs scramble siRNA PASMC. Cleaved caspase-3 was increased in siPDE3A (p< 0.0001) > > siPDE3B (p< 0.001) vs scramble PASMC. In the CM experiment, FFA levels were higher in media harvested from siPDE3A-adipocytes relative to scramble siRNA adipocytes. Exposure to CM also decreased AMPK and CREB phosphorylation and increased caspase 3 activity in the siPDE3A-CM PASMC. A greated increase in viable PASMC numbers was observed for siPDE3A-PASMC in WT CM > > scramble PASMC in siPDE3A-CM vs scramble PASMC in WT CM.

Conclusion(s): siRNA knockdown of Pde3a or Pde3b resulted in differential decreases in AMPK and CREB phosphorylation, and increases in caspase 3 activity compared to scramble siRNA, with a greater effect with PDE3A deficiency. Media from siPDE3A-transfected adipocytes with higher FFA levels also led to similar significant, but less robust changes in both protein expression and increased viable cell numbers when compared to siPDE3A-transfected PASMC.