Endometriosis is a painful gynecological condition characterized by ectopic growth of endometrial cells. Little is known about its pathogenesis, which is partially due to a lack of suitable experimental models. Here, we use endometrial stromal (St-T1b), primary endometriotic stromal, epithelial endometriotic (12Z) and co-culture (1:1 St-T1b:12Z) spheroids to mimic the architecture of endometrium, and either collagen I or Matrigel to model ectopic locations. Stromal spheroids, but not single cells, assumed coordinated directional migration followed by matrix remodeling of collagen I on day 5 or 7, resembling ectopic lesions. While generally a higher area fold increase of spheroids occurred on collagen I compared to Matrigel, directional migration was not observed in co-culture or in 12Z cells. The fold increase in area on collagen I was significantly reduced by MMP inhibition in stromal but not 12Z cells. Inhibiting ROCK signalling responsible for actomyosin contraction increased the fold increase of area and metabolic activity compared to untreated controls on Matrigel. The number of protrusions emanating from 12Z spheroids on Matrigel was decreased by microRNA miR-200b and increased by miR-145. This study demonstrates that spheroid assay is a promising pre-clinical tool that can be used to evaluate small molecule drugs and microRNA-based therapeutics for endometriosis.

Altered plasma sphingosine-1-phosphate (S1P) concentrations are associated with clinical manifestations of atherosclerosis. However, whether long-term elevation of endogenous S1P is pro- or anti-atherogenic remains unclear. Here, we addressed the impact of permanently high S1P levels on atherosclerosis in cholesterol-fed apolipoprotein E-deficient (ApoE−/−) mice over 12 weeks. This was achieved by pharmacological inhibition of the S1P-degrading enzyme S1P lyase with 4-deoxypyridoxine (DOP). DOP treatment dramatically accelerated atherosclerosis development, propagated predominantly unstable plaque phenotypes, and resulted in frequent plaque rupture with atherothrombosis. Macrophages from S1P lyase-inhibited or genetically deficient mice had a defect in cholesterol efflux to apolipoprotein A-I that was accompanied by profoundly downregulated cholesterol transporters ATP-binding cassette transporters ABCA1 and ABCG1. This was dependent on S1P signaling through S1PR3 and resulted in dramatically enhanced atherosclerosis in ApoE−/−/S1PR3−/− mice, where DOP treatment had no additional effect. Thus, high endogenous S1P levels promote atherosclerosis, compromise cholesterol efflux, and cause genuine plaque rupture.

Membrane phospholipids affect GLUT function in RBC but the role of their large intercellular S1P pool is unknown. We show that altering S1P content by intervening in its precursor uptake, synthesis and efflux had major impacts on RBC glucose uptake and glycolysis. We identified the mechanism as direct activation of PP2A by S1P leading to cell-surface GLUT reduction. This mechanism was highly dynamic as it responded to metabolic cues from the environment by regulating intracellular S1P and thereby glucose uptake, hence counteracting pathophysiological conditions such as diabetes. Accordingly, RBC from hyperglycemic mice and humans with diabetes had higher S1P, Sphk1 and PP2A activities, and lower cell-surface GLUTs. Proof of concept is provided by the resistance of RBC lacking the main S1P exporter MFSD2B to pathological HbA1c increases in hyperglycemia. This mechanism may also be functional in other insulin-independent tissues and could be pharmacologically exploited by existing S1P homeostasis-modifying drugs.