Sb204741

The collagen gel contractility assay was adapted from previously published work [32 (link)]. A collagen gel solution consisting of 8:1:1 parts bovine collagen (Advanced BioMatrix PureCol), 10x Dulbecco’s phosphate buffered saline (DPBS, Gibco), and 0.1 M NaOH was prepared and the pH adjusted to 7.4. 200 μL of gel solution was dispensed to 1.27 cm diameter Teflon rings (Seastrom Manufacturing Company) and the gel allowed to crosslink for 1.5 hours at 37°C. The top of the gel was seeded with a 200 μL of a cell suspension containing 40,000 immortalized microvascular endothelial and smooth muscle cells with or without a doxycycline inducible BMPR2 receptor mutation and allowed to settle for 30 minutes. The Teflon rings were removed and media added containing 300 ng/mL of doxycycline and the cells treated with either 1 ng/mL TGF-β1 (porcine, R&D Systems Inc.), 1 μM SB204741 (Tocris), both, or neither. Gels were imaged using a dissection microscope (Olympus) at 30 minutes and 72 hours after seeding, and the treatment media was changed every 24 hours. Gel size was determined using ImageJ (National Institutes of Health).

Ritanserin, SB204741 and mesulergine were from Tocris Bioscience. All other ligands were from Sigma-Aldrich. Cell culture reagents were from Invitrogen. PZQ enantiomers ((R)-[-]PZQ and (S)-[+]PZQ) were resolved (Supplementary Fig. 2) using methods published by Woelfle et al.^{13 (link)}.

Standard cell culture media was provided by the Molecular and Cellular core of the Pharmacology Department at Louisiana State University Health Sciences Center; media supplements were purchased from Invitrogen (Carlsbad, CA, USA). The 5-HT_2A receptor antagonist ketanserin, 5-HT_2C receptor antagonist RS102221, and 5HT_2B receptor antagonist SB204741 were purchased from Tocris Bioscience (Ellisville, MO, USA). (R)-DOI and the selective 5-HT_2A receptor antagonist MDL100907 were gifts from Dr. David Nichols (Purdue University, West Lafayette, IN, USA). Isobutyl-methylxanthine (IBMX), dexamethasone, and insulin were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Ketanserin (Sigma), an antagonist for 5HTR2A, was dissolved in 0.1 M HCl, diluted with PBS, and administered in a dosing volume of 0.1 mg/mouse. SB-204741 (Tocris Bioscience, Bristol, UK), an antagonist for 5HTR2B, was dissolved in DMSO, diluted with PBS such that the final concentration of DMSO was 0.1%, and administered in a dosing volume of 0.08 mg/mouse. SB-269970 (Sigma), 5HTR7 antagonist, and methysergide (Sigma), a 5HTR1, 2 and 7 antagonist, were dissolved in PBS and administered in a dosing volume of 0.6 and 0.1 mg/mouse, respectively. All antagonists were i.p. injected at 30 min before the injection of 1 mg 5-HT. After 120 min, samples were collected from skeletal muscle.

All animal procedures were approved and carried out in accordance with relevant guidelines and regulations established by the Institutional Animal Care and Use Committee at Vanderbilt University. Briefly, Notch1^+/- and Htr2b^-/- mice were crossed to create Notch1; Htr2b double mutant animals [26 (link), 27 (link)]. In a separate cohort, Alzet minipumps (model 1004) delivering either the high-affinity 5-HT_2B antagonist SB204741 (Tocris Biosciences; 1 mg kg^-1d^-1) or vehicle (50% dimethyl sulfoxide [Sigma-Aldrich] and polyethylene glycol-400 [Fisher Chemical]) were implanted subcutaneously in Notch1^+/- mice at 4 months of age (Fig 1). SB204741 is a well-validated and specific 5-HT_2B receptor antagonist commonly used in pharmacological studies targeting this ligand [28 (link), 29 (link)]. SB204741 dosing varies widely across the literature, but we use 1 mg kg^-1d^-1 to allow for longer time course administration with good results [30 ].
All mice were fed a 1% cholesterol Western diet (TestDiet 5TJT) for a total of 3.5 months beginning at 2.5 months of age. Food and water were provided ad libitum. Mice were sacrificed at 6 months of age using carbon dioxide inhalation followed by cervical dislocation before harvesting biological samples for processing and analysis.