Haptoglobin and hemopexin inhibit vaso-occlusion and inflammation in murine sickle cell disease: Role of heme oxygenase-1 induction.

During hemolysis, hemoglobin and heme released from red blood cells promote oxidative stress, inflammation and thrombosis. Plasma haptoglobin and hemopexin scavenge free hemoglobin and heme, respectively, but can be depleted in hemolytic states. Haptoglobin and hemopexin supplementation protect tissues, including the vasculature, liver and kidneys. It is widely assumed that these protective effects are due primarily to hemoglobin and heme clearance from the vasculature. However, this simple assumption does not account for the consequent cytoprotective adaptation seen in cells and organs. To further address the mechanism, we used a hyperhemolytic murine model (Townes-SS) of sickle cell disease to examine cellular responses to haptoglobin and hemopexin supplementation. A single infusion of haptoglobin or hemopexin (± equimolar hemoglobin) in SS-mice increased heme oxygenase-1 (HO-1) in the liver, kidney and skin several fold within 1 hour and decreased nuclear NF-ĸB phospho-p65, and vaso-occlusion for 48 hours after infusion. Plasma hemoglobin and heme levels were not significantly changed 1 hour after infusion of haptoglobin or hemopexin. Haptoglobin and hemopexin also inhibited hypoxia/reoxygenation and lipopolysaccharide-induced vaso-occlusion in SS-mice. Inhibition of HO-1 activity with tin protoporphyrin blocked the protections afforded by haptoglobin and hemopexin in SS-mice. The HO-1 reaction product carbon monoxide, fully restored the protection, in part by inhibiting Weibel-Palade body mobilization of P-selectin and von Willebrand factor to endothelial cell surfaces. Thus, the mechanism by which haptoglobin and hemopexin supplementation in hyperhemolytic SS-mice induces cytoprotective cellular responses is linked to increased HO-1 activity.

Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease.

Treatment of sickle cell disease (SCD) is hampered by incomplete understanding of pathways linking hemolysis to vaso-occlusion. We investigated these pathways in transgenic sickle mice. Infusion of hemoglobin or heme triggered vaso-occlusion in sickle, but not normal, mice. Methemoglobin, but not heme-stabilized cyanomethemoglobin, induced vaso-occlusion, indicating heme liberation is necessary. In corroboration, hemoglobin-induced vaso-occlusion was blocked by the methemoglobin reducing agent methylene blue, haptoglobin, or the heme-binding protein hemopexin. Untreated HbSS mice, but not HbAA mice, exhibited ∼10% vaso-occlusion in steady state that was inhibited by haptoglobin or hemopexin infusion. Antibody blockade of adhesion molecules P-selectin, von Willebrand factor (VWF), E-selectin, vascular cell adhesion molecule 1, intercellular adhesion molecule 1, platelet endothelial cell (EC) adhesion molecule 1, α4ß1, or αVß3 integrin prevented vaso-occlusion. Heme rapidly (5 minutes) mobilized Weibel-Palade body (WPB) P-selectin and VWF onto EC and vessel wall surfaces and activated EC nuclear factor κB (NF-κB). This was mediated by TLR4 as TAK-242 blocked WPB degranulation, NF-κB activation, vaso-occlusion, leukocyte rolling/adhesion, and heme lethality. TLR4(-/-) mice transplanted with TLR4(+/+) sickle bone marrow exhibited no heme-induced vaso-occlusion. The TLR4 agonist lipopolysaccharide (LPS) activated ECs and triggered vaso-occlusion that was inhibited by TAK-242, linking hemolysis- and infection-induced vaso-occlusive crises to TLR4 signaling. Heme and LPS failed to activate VWF and NF-κB in TLR4(-/-) ECs. Anti-LPS immunoglobulin G blocked LPS-induced, but not heme-induced, vaso-occlusion, illustrating LPS-independent TLR4 signaling by heme. Inhibition of protein kinase C, NADPH oxidase, or antioxidant treatment blocked heme-mediated stasis, WPB degranulation, and oxidant production. We conclude that intravascular hemolysis in SCD releases heme that activates endothelial TLR4 signaling leading to WPB degranulation, NF-κB activation, and vaso-occlusion.

The risk factors of CVA in Jordan.

This study aimed to identify the main risk factors of cerebrovascular accident (CVA) in Jordan. Identification of risk factors may help to reduce the incidence of CVA. A form was prepared for data collection which consisted of two parts to gather biodata and the incidence of risk factors. A sample of 200 patients with CVA (60% men) were randomly selected from various areas of Jordan. An age, region distribution, and gender-matched sample were selected to serve as control. Hypertension in the experimental group (66%) was significantly higher than the control group (32%) p  <  0.001. Half of the subjects with stroke had diabetes compared to 22% of the control group (p  <  0.001). Cardiovascular diseases were found in 29% of subjects with CVA compared to 14% in the control group (p  <  0.001). About 27% of the CVA group had hyperlipidemia in comparison to 13% in the controls (p <  0.002). Smokers represented 54% of the experimental group compared to 30% of the control group (p <  0.05). An important finding in the present study was that about half of the selected subjects with strokes were under the age of 60 years. In conclusion, hypertension, diabetes, cardiovascular diseases, hyperlipidemia, and smoking are risk factors for CVA in Jordan.

The antinociceptive and anti-inflammatory effects of Salvia officinalis leaf aqueous and butanol extracts.

CONTEXT: The leaf of sage Salvia officinalis L. (Lamiaceae) is reputed in the folk medicine of Arabia, and Jordan in particular, to relieve pain associated with gastrointestinal disturbance. OBJECTIVES: Evaluation of the antinociceptive and anti-inflammatory activities of aqueous and butanol extracts of S. officinalis leaf. MATERIALS AND METHODS: The analgesic effects of the aqueous extract (10, 31.6, 100, 316, 1000 mg/kg) and butanol extract (10, 31.6, 100, 316 mg/kg) were studied using the hot-plate test for mice and the formalin-induced paw licking in rats. The effects were compared to those of morphine and the influence of naloxone on these effects was also evaluated. The same concentrations of both extracts were used to evaluate their anti-inflammatory effects using the cotton pellet granuloma and carrageenan-induced paw edema in rats. RESULTS: The aqueous extract (10, 31.6, 100, 316, 1000 mg/kg) and butanol extract (10, 31.6, 100, 316 mg/kg) caused analgesic effect in the hot-plate latency assay as well as in early and late phases of formalin-induced paw licking in rats. These effects were reduced by the opioid receptor antagonist, naloxone (5 mg/kg). The same range of doses of both extracts caused dose-dependent inhibition of carrageenan-induced paw edema in rats as well as inhibition of cotton pellet granuloma. DISCUSSION AND CONCLUSION: These observations suggest that the sage leaf aqueous and butanol extracts have analgesic and anti-inflammatory effects, confirming the traditional use of this plant for pain alleviation.

Nuclear factor-kappa B (NFkappaB) component p50 in blood mononuclear cells regulates endothelial tissue factor expression in sickle transgenic mice: implications for the coagulopathy of sickle cell disease.

Sickle cell anemia is accompanied by the activation of coagulation and thrombosis. We have studied the abnormal expression of tissue factor (TF) by the pulmonary vein endothelium of the mild-phenotype NY1DD sickle transgenic. As detected by immunofluorescence microscopy, this occurs only after the NY1DD mouse is exposed to hypoxia/reoxygenation (H/R), which actually causes ischemia/reperfusion in the sickle cell disease-but not the normal-mouse model. We tested the hypothesis that the nuclear factor-kappa B (NFkappaB)-activating inflammation that develops in post-H/R NY1DD mice is responsible for this phenotype switch. Various NFkappaB inhibitors (including p50-specific andrographolide) demonstrated that endothelial TF positivity is NFkappaB dependent. Several systemic inflammatory stimulators (tumor necrosis factor [TNFalpha], lipopolysaccharide, thioglycollate, and carageenan) given to control mice showed that the inflammatory promotion of TF expression by only pulmonary vein endothelium is not specific to the sickle cell disease model. We bred the NFkappaB(p50)-/- state into the NY1DD mouse. Combined with marrow transplantation, this allowed the creation of NY1DD mice that were NFkappaB(p50)-/- only in peripheral blood cells (and marrow) versus only in vessel walls (and tissues). This process revealed that endothelial TF expression in the NY1DD mouse is highly dependent on NFkappaB(p50) in peripheral blood mononuclear cells-but not in the vessel wall. In confirmation, the infusion of post-H/R sickle mouse blood mononuclear cells into naïve NY1DD mice stimulated endothelial TF expression; the infusion of such cells from unstimulated sickle cell disease mice at ambient air did not stimulate TF expression. We conclude that peripheral blood mononuclear cells indirectly promote endothelial TF expression via a NFkappaB(p50)-dependent mechanism. This approach may be relevant to the role of coagulopathy in clinical sickle cell disease.