Mast Cell Activation in Sickle Mice Stimulates Endothelial Dysfunction

We showed that mast cell activation/degranulation contributes to pain and neurogenic inflammation characterized by increased vascular permeability in sickle mice (Vincent et al., Blood 2013). Mast cells are tissue resident inflammatory cells, which are located in the vicinity of vasculature and nerve fibers. Neurogenic inflammation is mediated by activation of peripheral nerve fibers via the release of vasoactive and neurinflammatory peptide, substance P. However, the products of mast cell activation may have direct effects on the vasculature. Sickle pathobiology is characterized by endothelial dysfunction, inflammation and oxidative stress. We hypothesized that the neuropeptides, proteases, and cytokines released from activated mast cells lead to endothelial dysfunction by stimulating endoplasmic reticulum (ER) stress, and mitochondrial dysfunction, leading to oxidative stress. We examined the direct effect of mast cell activation on endothelium. Since morphine is used to treat pain in sickle cell disease (SCD) and also influences endothelial signaling (Gupta et al., Cancer Res 2002), we investigated if morphine contributes to endothelial dysfunction.

Methods. We isolated mast cells from the skin of HbSS-BERK sickle mice, which demonstrate severe mast cell activation and hyperalgesia (pain) and HbAA-BERK control mice. Mast cells from sickle mouse skin continue to degranulate in culture, but the mast cells from control mice do not. We collected the supernatant from mast cell cultures and used it to treat primary mouse brain microvascular endothelial cells (MBMEC) in vitro. ER stress was assayed using ER-Tracker Green (Glibenclamide BODIPY FL) dye (Molecular Probes) on live cells followed by laser scanning confocal microscopy (LSCM). ER stress markers, E74-like factor 2a (ELF2a), X-box binding protein 1 (XBP1), and glucose regulated protein 78 (GRP78), were analyzed with Western Immunoblotting. Mitochondrial function was analyzed by estimating mitochondrial membrane potential with MitoProbe JC-1 (Molecular Probes), which exhibits potential-dependent accumulation in mitochondria, causing a fluorescence emission shift from green (~529 nm) to red (~590 nm). Mitochondrial depolarization (dysfunction) was analyzed by a decrease in red/green ratio using LSCM. ROS was assayed using 2’7’-dichlorofluorescein diacetate and quantifying the fluorescence at the max excitation and emission spectra of 495 nm and 529 nm, respectively.

Results. Supernatant from sickle mast cells led to significant ER stress in MBMEC, as compared to the supernatant from control mast cells (p<0.05). Western blotting demonstrated an increase in ER stress markers, phosphor-elF2a, sXBP1 and GRP78, in MBMEC incubated with sickle mast cell supernatant as compared to control mast cell supernatant. Complementary to the sickle mast cell-induced ER stress, mitochondria potential decreased in MBMEC treated with sickle mast cell supernatant as compared to control mast cell supernatant (p < 0.05). We observed that supernatant from activated cutaneous mast cells stimulated a 10-fold increase in reactive oxygen species (ROS) in MBMEC (p < 0.05). This effect was further exacerbated in MBMEC treated with both sickle mast cell supernatant and morphine (p < 0.01). Morphine alone increased ROS production 4-fold in MBMEC. ER stress inhibitor, Salubrinal, inhibited ROS production in MBMEC induced by sickle mast cells. Together, these data suggest that mast cell activation stimulates ER stress in MBMEC, which may lead to mitochondrial dysfunction and generation of ROS. Thus, mast cell degranulation alone/and in addition to morphine, may contribute to endothelial dysfunction in SCD.