Sphingosine 1 phosphate (s1p)

In order to load the selected reporter molecule, 2 × 10⁸ MSVs were suspended in 500 µL of fluorescein isothiocyanate (FITC) solution (Sigma–Aldrich) in distilled water (10 mg mL⁻¹), followed by 2 h of incubation in physiological-like conditions (37°C, under mild agitation). Centrifugation at 4500 r/min for 10 min allowed for particle isolation. The collected supernatant was used to estimate the amount of absorbed reporter molecules by mass difference, using a spectrophotometer (SpectraMax M2; Molecular Devices, Sunnyvale, CA, USA), at λ = 495 nm/555 nm. The FITC-loaded particles were then lyophilized overnight and subsequently encapsulated in a 5% 50:50 PLGA shell, as described above. Following the same procedure, S1P (TOCRIS, Bristol, UK) was loaded into PLGA-MSV microspheres at a concentration of 300 µg mL⁻¹. An enzyme-linked immunosorbent assay (ELISA) kit (ECHELON, Salt Lake City, UT, USA) was used to quantify S1P concentration in the microspheres and samples. We measured the absorbance at 450 nm and determined the S1P concentration in the collected supernatants using a standard curve.

The intracellular calcium (Ca²⁺) levels were measured in human immortalized (hHM and HM6) and primary hMSMCs as described previously (40 (link)). In brief, 10⁴ cells were seeded in a 96-well black-wall, clear-bottom polystyrene microplate (Corning, NY, USA). The next day, cells were serum starved overnight in 1% charcoal-stripped FBS. The following day, media were replaced with 80 μL media containing the respective S1PR antagonist, as indicated in each figure legend and incubated for 30 minutes at 37 ^oC. Then 20 μL of Brilliant Calcium indicator solution (Ion Biosciences, TX, USA; working solution prepared using DrySolv and TRS reagent in provided assay buffer) was added to each well. After incubation for 1 hour, a Synergy2 plate reader (BioTek, VT, USA) was used to add 100 μL/well of either 1 μM S1P or oxytocin (OXT; Tocris, Bristol, UK) and the fluorescence intensities (excitation filter 485/20 nm, emission filter 528/20 nm) were recorded every 0.14 seconds for 20 seconds/well. Fluorescence increase (indicating an increase in Ca²⁺) was calculated as the average of fluorescence intensity readings from 10 to 20 seconds after S1P or OXT addition minus the minimum fluorescence intensity averaged over 5 points from 0 to 10 seconds.

S1P (Tocris Bioscience, Bio-Techne Corp, MN, USA) was dissolved in methanol:water 95:5 (0.5 mg/mL) and heated to 50 to 60 ^oC to dissolve. The methanol:water was then evaporated and S1P was stored at −20 ^oC until use. Working stock solution (1 mM S1P) was diluted in 4 mg/mL bovine serum albumin (BSA). All agonists and antagonists, Ex26, JTE03, TY52156, CYM50358, TY52156, SEW2871, and CYM5541 (Tocris Bioscience, Bio-Techne Corp, MN, USA) were dissolved in DMSO and stored at −20 ^oC until use. S1PR1-4 antagonists were used at a concentration of 200 nM, as this is above the IC50 dose for all, according to the manufacturer (Ex26 IC₅₀ = 0.93 nM, JTE03 IC₅₀ = 17.6 nM, TY52156 Ki = 110 nM, CYM50358 IC₅₀ = 25 nM). ERK1/2 inhibitor, U0126 (Cell Signaling, MA, USA), was dissolved in DMSO and used at 10 µM concentration based on previously published dose response data (28 (link)) and stored at −20 ^oC until use.