Key Points• Inhibition of mast cells with cromolyn or imatinib results in reduced systemic inflammation and neurogenic inflammation in sickle mice.• Pharmacological inhibition or genetic depletion of mast cells in sickle mice ameliorates chronic and hypoxia/reoxygenationinduced pain.Sickle cell anemia (SCA) is an inherited disorder associated with severe lifelong pain and significant morbidity. The mechanisms of pain in SCA remain poorly understood.We show that mast cell activation/degranulation contributes to sickle pain pathophysiology by promoting neurogenic inflammation and nociceptor activation via the release of substance P in the skin and dorsal root ganglion. Mast cell inhibition with imatinib ameliorated cytokine release from skin biopsies and led to a correlative decrease in granulocyte-macrophage colony-stimulating factor and white blood cells in transgenic sickle mice. Targeting mast cells by genetic mutation or pharmacologic inhibition with imatinib ameliorates tonic hyperalgesia and prevents hypoxia/reoxygenation-induced hyperalgesia in sickle mice. Pretreatment with the mast cell stabilizer cromolyn sodium improved analgesia following low doses of morphine that were otherwise ineffective. Mast cell activation therefore underlies sickle pathophysiology leading to inflammation, vascular dysfunction, pain, and requirement for high doses of morphine. Pharmacological targeting of mast cells with imatinib may be a suitable approach to address pain and perhaps treat SCA. (Blood. 2013;122(11):1853-1862

Morphine does not affect the onset of tumour development, but it promotes growth of existing tumours, and reduces overall survival in mice. MOR may be associated with morphine-induced cancer progression, resulting in shorter survival. Mast cell activation by morphine may contribute to increased cytokine and SP levels, leading to cancer progression and refractory pain.

Morphine stimulates tumor angiogenesis and cancer progression in mice. We examined if morphine influences endothelial-pericyte interaction via platelet-derived growth factor-BB (PDGF-BB) and PDGF receptor-β (PDGFR-β). Clinically relevant doses of morphine stimulated PDGF-BB secretion from human umbilical vein endothelial cells and activated PDGFR-β and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) phosphorylation in human pericytes. These in vitro effects of morphine were translated into promotion of tumor angiogenesis in a transgenic mice model of breast cancer when treated with clinically used dose of morphine. Increased vessel-associated immunoreactivity of desmin and PDGFR-β was observed on pericytes in tumors of morphine-treated mice. These data suggest that morphine potentiates endothelial-pericyte interaction via PDGF-BB/PDGFR-β signaling and promotes tumor angiogenesis, pericyte recruitment, and coverage of tumor vessels. We speculate that morphine may impair the effectiveness of antiangiogenic therapy by influencing vascular pericyte coverage.

Endothelial dysfunction underlies the pathobiology of cerebrovascular disease. Mast cells are located in close proximity to the vasculature, and vasoactive mediators released upon their activation can promote endothelial activation leading to blood brain barrier (BBB) dysfunction. We examined the mechanism of mast cell-induced endothelial activation via endoplasmic reticulum (ER) stress mediated P-selectin expression in a transgenic mouse model of sickle cell disease (SCD), which shows BBB dysfunction. We used mouse brain endothelial cells (mBECs) and mast cells-derived from skin of control and sickle mice to examine the mechanisms involved. Compared to control mouse mast cell conditioned medium (MCCM), mBECs incubated with sickle mouse MCCM showed increased, structural disorganization and swelling of the ER and Golgi, aggregation of ribosomes, ER stress marker proteins, accumulation of galactose-1-phosphate uridyl transferase, mitochondrial dysfunction, reactive oxygen species (ROS) production, P-selectin expression and mBEC permeability. These effects of sickle-MCCM on mBEC were inhibited by Salubrinal, a reducer of ER stress. Histamine levels in the plasma, skin releasate and in mast cells of sickle mice were higher compared to control mice. Compared to control BBB permeability was increased in sickle mice. Treatment of mice with imatinib, Salubrinal, or P-selectin blocking antibody reduced BBB permeability in sickle mice. Mast cells induce endothelial dysfunction via ER stress-mediated P-selectin expression. Mast cell activation contributes to ER stress mediated endothelial P-selectin expression leading to increased endothelial permeability and impairment of BBB. Targeting mast cells and/or ER stress has the potential to ameliorate endothelial dysfunction in SCD and other pathobiologies.

842 The mechanisms underlying non-healing leg ulcers in sickle cell disease (SCD) remain unknown and their treatment is difficult. We hypothesized that leg ulcers in SCD cause injury to nerve fibers leading to neurogenic inflammation, and peripheral and central sensitization resulting in chronic pain and impairment of wound healing. We examined our hypotheses by creating 4 mm punch biopsies on the lower thigh of the left leg (LL) of 12 wk old BERK and hemizygous BERK (hBERK) mice expressing sickle hemoglobin (HbS). Since BERK did not survive wounding, hBERK and control HbA-BERK, expressing normal human HbA and wild type C57/BL6, were used. hBERK appeared to be a suitable model for this study because we found that >5 mo old BERK and hBERK show deep tissue and cutaneous hyperalgesia similar to pain in human SCD (Blood 116:456-65, 2010). Wounds were treated with topical morphine (3 mg/g Eucerin base cream) or with Eucerin base cream twice a day. Behavioral measures of hyperalgesia were assessed by paw withdrawal frequency (PWF) per 10 applications of a 1.0 g von Frey monofilament and paw withdrawal latency (PWL) in response to a radiant heat stimulus, applied to the plantar surface of the hind paw of wounded left leg and unwounded right leg. No hyperalgesia was observed in 12 wk old hBERK (baseline), but wounding led to a sharp increase in PWF and decrease in PWL, in LL for 24h, suggestive of wound-induced pain. Topical application of morphine significantly decreased mechanical and thermal hyperalgesia after 1h and 24h as compared to the base cream treated wound (p<0.05) in LL. Pain measures returned to baseline 2 wks post-wounding in morphine- but base cream-treated hBERK mice exhibited hyperalgesia for 4 wks (last measurement). The right leg exhibited increased mechanical but not thermal hyperalgesia following the wounding of left leg in hBERK. HbA-BERK and C57/BL6 wounds treated with base cream showed an increase in pain which was significantly less than hBERK and reached baseline 2 wks post-wounding, suggesting that wounding results in chronic pain in hBERK mice. Increased pain was accompanied by decreased wound closure in hBERK mice. Morphine-treated mice showed 100% wound closure at 4d Vs 8d in base cream treated hBERK, indicative of morphine-induced augmentation of wound healing. Laser scanning confocal microscopy (LSCM) of whole dorsal root ganglion (DRG) showed that, activating transcription factor 3 (ATF3), a marker of neuronal injury, was highly expressed in left lumbar DRG of base cream Vs morphine treated wounded hBERK, 30d post-wounding (p<0.05), suggestive of ongoing wound-induced neuronal injury which is abrogated by topical morphine. LSCM of 100 micron thick sections of healed wounds showed that substance P (SP), a neuropeptide expressed in nociceptive primary afferents, was significantly increased in base cream treated Vs morphine treated healed wounds 30d post wounding (p<0.01). Increased SP was accompanied by reduced and disorganized protein gene product (PGP 9.5)-ir nerve fibers and vasculature in base cream treated wounds. In contrast, morphine treated wounds showed normally organized dense PGP 9.5-ir nerve fibers and blood vessels replete with nerve and vascular plexus similar to their presentation in normal skin. Together, these data show that wounds stimulate chronic pain accompanied by nerve injury, central sensitization and neurogenic inflammation; and that morphine ameliorates this noxious insult and promotes wound healing. In addition, morphine may directly influence wound healing by promoting angiogenesis. It is possible that chronic pain may contribute to impaired wound healing and that non-healing wounds contribute to chronic pain in SCD. We propose that wound-induced nerve signal conduction (ATF3 in DRG) and neurogenic inflammation/sensitization of primary afferents (SP in skin) may contribute to chronic pain and in turn interfere with wound healing. Our observations suggest that neurochemical and behavioral alterations orchestrated by wounding may contribute to the impairment of healing of leg ulcers in SCD. We speculate that topical application of morphine may ameliorate pain and promote healing of painful leg ulcers in SCD. Disclosures: No relevant conflicts of interest to declare.