398 - Development of an Immortalized Ductus Arteriosus (DA) Smooth Muscle Cell (SMC) Platform for High Throughput Drug Screening to Identify Novel Therapeutics for PDA

Publication Number: 3385.398

Deanna Sekulich, Vanderbilt University School of Medicine, Charlottesville, VA, United States; Michael T. Yarboro, Robert Larner, M.D., College of Medicine at the University of Vermont, St Albans, VT, United States; Edith M. Charron, Monroe Carell Jr. Children's Hospital at Vanderbilt, Brentwood, TN, United States; Alexus J.. Brown, Monroe Carell Jr. Children's Hospital at Vanderbilt, Nashville, TN, United States; Charmaine R. Rock, Vanderbilt University Medical Center, Nashville, TN, United States; Naoko Boatwright, Vanderbilt University Medical Center, Franklin, TN, United States; Jennifer L. Herington, Vanderbilt University Medical Center, Nashville, TN, United States; Shajila Siricilla, Vanderbilt University Medical Center, Nashville, TN, United States; Elaine L.. Shelton, Vanderbilt University School of Medicine, Nashville, TN, United States; Jeff Reese, Vanderbilt University Medical Center, Nashville, TN, United States

Background: Persistent patency of the ductus arteriosus (PDA) is an ongoing dilemma in most NICUs. Limited therapeutic options, drug toxicity, and off-target effects hamper PDA decision-making. Identification of new candidate genes and molecules that regulate DA patency have not translated to new therapeutics. Alternatively, high throughput drug screens (HTS) offer the ability to find effective compounds, with subsequent identification of critical pathways or mechanisms for DA regulation.

Objective: Develop an innovative HTS drug screening platform using low passage DA SMCs to identify novel therapies for PDA.

Design/Methods: DA and aorta (AO) tissues were explanted from newborn CD-1 wild-type (WT) and C57Bl10/CBA “immortomice”, (harboring the H-2Kb-tsA58 SV-40 transgene which confers an immortal phenotype at 33C in the presence of IFgamma). SMC outgrowths were cultured in DMEM/FBS. Cell survival, motility (scratch assay), proliferation, SMC marker expression, and contractile response (Ca2+ mobilization) patterns were compared. DA and AO SMCs were plated in 384 well plates for HTS optimization assays. Male vs. female SMCs, high (10%) vs. low (1%) serum concentration, and normoxia vs. hypoxia conditions were compared. Data were analyzed by t-test or ANOVA; best-fit curves were plotted for analysis (GraphPad Prism 10).

Results: Immorto DA and AO SMCs had superior survival to native WT SMCs after passage 3 (P3). PCR and immunostaining showed stable expression of different SMC markers in Immorto SMCs up to P8-9. AO SMCs were more motile than DA SMCs. Hypoxic conditions (2% O2) improved time to confluence at P1 (p < 0.001), whereas normoxia and 10% FBS improved wound closure rates in both SMC types (p < 0.05). No consistent differences were noted between male and female cells. Standardization assays showed that 4000 SMCs/well and 4μM of the Fluo-4AM Ca2+ sensing dye were optimal for HTS. DA SMCs in 384-well plates had significantly more contraction than AO SMCs in response to the thromboxane mimetic U44619 (0.01 – 100uM) (p < 0.05).

Conclusion(s): SMCs from “Immortomice” provide a superior resource for phenotypic drug screens of DA SMC contraction. DA SMCs appear to retain a DA-like identity in culture and respond differently than AO SMCs. HTS in a 384-well format is a promising approach to identify novel therapeutics for PDA treatment. These studies represent the first essential steps to enable large-scale drug screening for PDA. Future studies will focus on application of this drug discovery platform to human DA SMCs.