Mechanisms of pain in sickle cell disease - Sickle cell disease (SCD) affects approximately 6 million people. The hallmark of SCD is severe painful vaso-occlusive crises (VOCs), which often requires hospitalization. The mechanisms underlying pain during VOC are not understood. Using transgenic mice with SCD, we developed a clinically-relevant model in which cold exposure causes VOCs characterized by hypoxia, vaso-occlusion, red blood cell (RBC) sickling, hemolysis, acidosis, acute pain, and elevated level of blood lysophosphatidic acid (LPA), a known pain mediator. LPA metabolism is regulated by peroxisome proliferator-activated receptor-γ (PPARγ), which, in turn, is regulated by hypoxia-induced factors (HIF-1α/2α). Hypoxia inhibits PPARγ transcription, and activation of PPARγ decreases expression of autotaxin (ATX), the enzyme that synthesizes LPA, and increases expression of lipid phosphate phosphatases (LPP), the enzymes that hydrolyze LPA. Since bone marrow adipocytes (BMAs) express ATX, LPP1 and PPARγ, a close relationship is predicted between LPA metabolism in BMAs and acute painful VOC. Also, LPA signaling is closely related to extracellular vesicles (EVs) that deliver LPA to various targets, including LPA receptor 1 (LPA1) on dorsal root ganglia (DRG) neurons. We will test the general hypothesis that acute pain associated with VOC in SCD results from hypoxia-induced aberrant LPA metabolism in BM, which leads to an increase in LPA and sensitization of nociceptors in an LPA1-dependent manner. Preliminary data suggest that 1) cold-induced acute spontaneous nocifensive behavior and hyperalgesia in sickle mice (HbSS) were associated with elevated levels of LPA and EVs in the blood. Inhibition of LPA synthesis decreased hyperalgesia in HbSS mice. EVs isolated from HbSS mice produced hyperalgesia in control (HbAA) mice. Inhibition of LPA1 receptor by siRNA prevented painful VOC. 2) Hyperalgesia in HbSS mice was accompanied by increased expression of ATX and a reduction in activity of PPARγ in BM. Pioglitazone, a PPARγ agonist, prevented cold-induced painful VOC in HbSS mice. 3) Electrophysiological studies showed that hyperalgesia in HbSS mice is associated with sensitization of C-fiber nociceptors and small DRG neurons in an LPA1-dependent manner. Sensitization occurred via potassium, ASIC and TRPV1 channels and was potentiated by low pH. Parallel behavioral, electrophysiological, cellular and molecular studies will test our hypothesis through the following specific aims: 1) Determine if LPA-LPA1 signaling contributes to cold-induced painful VOC in HbSS mice; 2) Determine the role of PPARγ in the regulation of LPA metabolism in BM adipocytes; and 3) Determine the mechanisms underlying LPA-LPA1-induced sensitization of primary afferent nociceptors during VOC. The proposed studies are innovative in that they will investigate novel mechanisms underlying painful VOC and may identify new therapeutic targets, highlighting the significance and translational potential of our study.
