A monocyte-TNF-endothelial activation axis in sickle transgenic mice: Therapeutic benefit from TNF blockade.

Elaboration of tumor necrosis factor (TNF) is a very early event in development of ischemia/reperfusion injury pathophysiology. Therefore, TNF may be a prominent mediator of endothelial cell and vascular wall dysfunction in sickle cell anemia, a hypothesis we addressed using NY1DD, S+SAntilles , and SS-BERK sickle transgenic mice. Transfusion experiments revealed participation of abnormally activated blood monocytes exerting an endothelial activating effect, dependent upon Egr-1 in both vessel wall and blood cells, and upon NFκB(p50) in a blood cell only. Involvement of TNF was identified by beneficial impact from TNF blockers, etanercept and infliximab, with less benefit from an IL-1 blocker, anakinra. In therapeutic studies, etanercept ameliorated multiple disturbances of the murine sickle condition: monocyte activation, blood biomarkers of inflammation, low platelet count and Hb, vascular stasis triggered by hypoxia/reoxygenation (but not if triggered by hemin infusion), tissue production of neuro-inflammatory mediators, endothelial activation (monitored by tissue factor and VCAM-1 expression), histopathologic liver injury, and three surrogate markers of pulmonary hypertension (perivascular inflammatory aggregates, arteriolar muscularization, and right ventricular mean systolic pressure). In aggregate, these studies identify a prominent-and possibly dominant-role for an abnormal monocyte-TNF-endothelial activation axis in the sickle context. Its presence, plus the many benefits of etanercept observed here, argue that pilot testing of TNF blockade should be considered for human sickle cell anemia, a challenging but achievable translational research goal.

Evidence for a profound remodeling of skeletal muscle and its microvasculature in sickle cell anemia.

Sickle cell anemia (SCA) is a hemoglobinopathy leading to major hematologic, hemorheologic, and hemodynamic disorders that induce various complications, including organ failure, and ultimately lead to death. Here, we assessed for the first time repercussions of SCA on skeletal muscle and its microvasculature. Twenty-seven sedentary Cameroonian volunteer men participated in the study. They were assigned to one of three groups according to their hemoglobin status (healthy control subjects, n = 10; sickle cell trait carriers, n = 10; and SCA patients, n = 7) and underwent muscle biopsy of the vastus lateralis. SCA was associated with microvessel rarefaction, decrease in capillary tortuosity, and widening of microvessel diameter. The absence of capillary wall reinforcement was shown by lack of wall thickening and lack of fibrous tissue or smooth muscle in their constitution. We also observed changes in fiber type distribution, muscle atrophy, an increase in satellite cell number, and a decrease in activity of creatine kinase and several oxidative enzymes. No signs of tissue necrosis, inflammatory stress, fibrosis, or segmented fibers were observed. The present study highlighted marked effects of SCA on microvascular, structural, and energetic characteristics of skeletal muscle.

Cannabinoid receptor-specific mechanisms to alleviate pain in sickle cell anemia via inhibition of mast cell activation and neurogenic inflammation.

Sickle cell anemia is a manifestation of a single point mutation in hemoglobin, but inflammation and pain are the insignia of this disease which can start in infancy and continue throughout life. Earlier studies showed that mast cell activation contributes to neurogenic inflammation and pain in sickle mice. Morphine is the common analgesic treatment but also remains a major challenge due to its side effects and ability to activate mast cells. We, therefore, examined cannabinoid receptor-specific mechanisms to mitigate mast cell activation, neurogenic inflammation and hyperalgesia, using HbSS-BERK sickle and cannabinoid receptor-2-deleted sickle mice. We show that cannabinoids mitigate mast cell activation, inflammation and neurogenic inflammation in sickle mice via both cannabinoid receptors 1 and 2. Thus, cannabinoids influence systemic and neural mechanisms, ameliorating the disease pathobiology and hyperalgesia in sickle mice. This study provides 'proof of principle' for the potential of cannabinoid/cannabinoid receptor-based therapeutics to treat several manifestations of sickle cell anemia.

Mast cell activation contributes to sickle cell pathobiology and pain in mice.

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.

Small-molecule nociceptin receptor agonist ameliorates mast cell activation and pain in sickle mice.

Treatment of pain with morphine and its congeners in sickle cell anemia is suboptimal, warranting the need for analgesics devoid of side effects, addiction and tolerance liability. Small-molecule nociceptin opioid receptor ligands show analgesic efficacy in acute and chronic pain models. We show that AT-200, a high affinity nociceptin opioid receptor agonist with low efficacy at the mu opioid receptor, ameliorated chronic and hypoxia/reoxygenation-induced mechanical, thermal and deep tissue/musculoskeletal hyperalgesia in HbSS-BERK sickle mice. The antinociceptive effect of AT-200 was antagonized by SB-612111, a nociceptin opioid receptor antagonist, but not naloxone, a non-selective mu opioid receptor antagonist. Daily 7-day treatment with AT-200 did not develop tolerance and showed a sustained anti-nociceptive effect, which improved over time and led to reduced plasma serum amyloid protein, neuropeptides, inflammatory cytokines and mast cell activation in the periphery. These data suggest that AT-200 ameliorates pain in sickle mice via the nociceptin opioid receptor by reducing inflammation and mast cell activation without causing tolerance. Thus, nociceptin opioid receptor agonists are promising drugs for treating pain in sickle cell anemia.

Lower Muscle and Blood Lactate Accumulation in Sickle Cell Trait Carriers in Response to Short High-Intensity Exercise.

It remains unclear whether sickle cell trait (SCT) should be considered a risk factor during intense physical activity. By triggering the polymerization-sickling-vaso-occlusion cascade, lactate accumulation-associated acidosis in response to high-intensity exercise is believed to be one of the causes of complications. However, our understanding of lactate metabolism in response to high-intensity exercise in SCT carriers is incomplete. Thirty male SCT carriers (n = 15) and healthy subjects (n = 15) with and without α-thalassemia performed a 2-min high-intensity exercise. Blood and muscle lactate concentrations were measured at exercise completion. Time courses of blood lactate and glucose concentrations were followed during the subsequent recovery. Additional biochemical analyses were performed on biopsies of the vastus lateralis muscle. SCT was associated with lower blood and muscle lactate concentrations in response to the short high-intensity exercise. Compared to controls, the muscle content among SCT carriers of lactate transporter MCT4 and ß2-adrenergic receptor were higher and lower, respectively. During recovery, the lactate removal ability was higher in SCT carriers. In the present study, no effect of α-thalassemia was observed. The lower blood and muscle lactate accumulations in SCT carriers may, to some extent, act as protective mechanisms: (i) against exercise-related acidosis and subsequent sickling, that may explain the relatively rare complications observed in exercising SCT carriers; and (ii) against the deleterious effects of intracellular lactate and associated acidosis on muscle function, that might explain the elevated presence of SCT carriers among the best sprinters.

Remodeling of skeletal muscle microvasculature in sickle cell trait and alpha-thalassemia.

The influence of sickle cell trait and/or alpha-thalassemia on skeletal muscle microvascular network characteristics was assessed and compared with control subjects [hemoglobin (Hb) AA] in 30 Cameroonian residents [10 HbAA, 5 HbAA alpha-thalassemia (alpha-t), 6 HbAS, and 9 HbASalpha-t] matched for maximal work capacity and daily energy expenditure. Subjects performed an incremental exercise to exhaustion and underwent a muscle biopsy. Muscle fiber type and surface area were not different among groups. However, sickle cell trait (SCT) was associated with lower capillary density (P < 0.05), lower capillary tortuosity (P < 0.001), and enlarged microvessels (P < 0.01). SCT carriers had reduced counts of microvessels <5-microm diameter, but a higher percentage of broader microvessels, i.e., diameter >10 microm (P < 0.05). alpha-Thalassemia seemed to be characterized by a higher capillary tortuosity and unchanged capillary density and diameter. Thus, while SCT is a priori clinically benign, we demonstrate for the first time that significant remodeling of the microvasculature occurs in SCT carriers. These modifications may possibly reflect protective adaptations against hemorheological and microcirculatory dysfunction induced by the presence of HbS. The remodeling of the microvascular network occurs to a lesser extent in alpha-thalassemia. In alpha-thalassemic subjects, increased capillary tortuosity would promote oxygen supply to muscle tissues and might compensate for the lower Hb content often reported in those subjects.

Effects of regular physical activity on skeletal muscle structural, energetic, and microvascular properties in carriers of sickle cell trait.

To assess the effects of regular physical activity on muscle functional characteristics of carriers of sickle cell trait (SCT), 39 untrained (U) and trained (T) hemoglobin (Hb)AA (CON) and SCT subjects (U-CON, n = 12; U-SCT, n = 8; T-CON, n = 10; and T-SCT, n = 9) performed a graded exercise and a time to exhaustion (T(ex)) test, and were subjected to a muscle biopsy. Maximal power, total work performed during T(ex), citrate synthase and cytochrome c oxidase (COX) activities, respiratory chain complexes I and IV content, and capillary density (CD), diameter (COD), and surface area (CSA) were upregulated by the same proportion in T-CON and T-SCT compared with their untrained counterparts. These proportionally similar differences imply that the observed discrepancies between U-SCT and U-CON remained in the trained subjects. Specifically, both CD and COX remained and tended to remain lower, and both COD and CSA remained and tended to remain higher in T-SCT than in T-CON. Besides, carriers of SCT displayed specific adaptations with regular physical activity: creatine kinase activity; complexes II, III, and V content; and type I fiber surface area and capillary tortuosity were lower or unchanged in T-SCT than in U-SCT. In summary, our results show that 1) carriers of SCT adapted almost similarly to CON to regular physical activity for most of the studied muscle characteristics, 2) oxidative potential remains altered in physically active carriers of SCT compared with HbAA counterparts, and 3) the specific remodeling of muscle microvascular network persists in the trained state.

Effects of carbohydrates-BCAAs-caffeine ingestion on performance and neuromuscular function during a 2-h treadmill run: a randomized, double-blind, cross-over placebo-controlled study.

BACKGROUND: Carbohydrates (CHOs), branched-chain amino acids (BCAAs) and caffeine are known to improve running performance. However, no information is available on the effects of a combination of these ingredients on performance and neuromuscular function during running. METHODS: The present study was designed as a randomized double-blind cross-over placebo-controlled trial. Thirteen trained adult males completed two protocols, each including two conditions: placebo (PLA) and Sports Drink (SPD: CHOs 68.6 g.L-1, BCAAs 4 g.L-1, caffeine 75 mg.L-1). Protocol 1 consisted of an all-out 2 h treadmill run. Total distance run and glycemia were measured. In protocol 2, subjects exercised for 2 h at 95% of their lowest average speeds recorded during protocol 1 (whatever the condition). Glycemia, blood lactate concentration and neuromuscular function were determined immediately before and after exercise. Oxygen consumption (V˙O2), heart rate (HR) and rate of perceived exertion (RPE) were recorded during the exercise. Total fluids ingested were 2 L whatever the protocols and conditions. RESULTS: Compared to PLA, ingestion of SPD increased running performance (p = 0.01), maintained glycemia and attenuated central fatigue (p = 0.04), an index of peripheral fatigue (p = 0.04) and RPE (p = 0.006). Maximal voluntary contraction, V˙O2, and HR did not differ between the two conditions. CONCLUSIONS: This study showed that ingestion of a combination of CHOs, BCAAs and caffeine increased performance by about 2% during a 2-h treadmill run. The results of neuromuscular function were contrasted: no clear cut effects of SPD were observed. TRIAL REGISTRATION: ClinicalTrials.gov, http://www.clinicaltrials.gov, NCT00799630.

Skeletal muscle structural and energetic characteristics in subjects with sickle cell trait, alpha-thalassemia, or dual hemoglobinopathy.

Previous studies have shown that subjects with sickle cell trait (SCT), alpha-thalassemia (alpha-t), and the dual hemoglobinopathy (SCT/alpha-t) manifest subtle, albeit significant, differences during exercise. To better understand such differences, we assessed skeletal muscle histomorphological and energetic characteristics in 10 control HbAA subjects (C), 5 subjects with alpha-t (alpha-t), 6 SCT carriers (SCT) and 9 SCT carriers with alpha-t (SCT/alpha-t). Subjects underwent a muscle biopsy and also performed an incremental maximal exercise and a time to exhaustion test. There were no observable differences in daily energy expenditure, maximal power output (Pmax), or time to exhaustion at 110% Pmax (Tex) among the groups. Blood lactate concentrations measured at the end of the Tex, muscle fiber type distribution, and mean phosphofructokinase (PFK), lactate dehydrogenase (LDH), beta-hydroxyacyl-CoA-dehydrogenase (HAD), and citrate synthase (CS) activities were all similar among the four groups. However, SCT was associated with a lower cytochrome-c oxidase (COx) activity in type IIa fibers (P<0.05), and similar trends were observed in fiber types I and IIx. Trends toward lower creatine kinase (CK) activity (P=0.0702) and higher surface area of type IIx fibers were observed in SCT (P=0.0925). In summary, these findings support most of the previous observations in SCT, such as 1) similar maximal power output and associated maximal oxygen consumption (VO2max) values and 2) lower exercise performances during prolonged submaximal exercise. Furthermore, performances during short supramaximal exercise were not different in SCT. Finally, the dual hemoglobinopathy condition does not seem to affect muscle characteristics.