A preliminary study of phosphodiesterases and adenylyl cyclase signaling pathway on red blood cell deformability of sickle cell patients

Sickle cell disease (SCD) is an inherited hemoglobinopathy characterized by chronic anemia, intravascular hemolysis, and the occurrence of vaso-occlusive crises due to the mechanical obstruction of the microcirculation by poorly deformable red blood cells (RBCs). RBC deformability is a key factor in the pathogenesis of SCD, and is affected by various factors. In this study, we investigated the effects of adenylyl cyclase (AC) signaling pathway modulation and different phosphodiesterase (PDE) modulatory molecules on the deformability and mechanical stress responses of RBC from SCD patients (HbSS genotype) by applying 5 Pa shear stress with an ektacytometer (LORRCA). We evaluated RBC deformability before and after the application of shear stress. AC stimulation with Forskolin had distinct effects on RBC deformability depending on the application of 5 Pa shear stress. RBC deformability was increased by Forskolin before shear stress application but decreased after 5 Pa shear stress. AC inhibition with SQ22536 and protein kinase A (PKA) inhibition with H89 increased RBC deformability before and after the shear stress application. Non-selective PDE inhibition with Pentoxifylline increased RBC deformability. However, modulation of the different PDE types had distinct effects on RBC deformability, with PDE1 inhibition by Vinpocetine increasing deformability while PDE4 inhibition by Rolipram decreased RBC deformability after the shear stress application. The effects of the drugs varied greatly between patients suggesting some could benefit from one drug while others not. Developing drugs targeting the AC signaling pathway could have clinical applications for SCD, but more researches with larger patient cohorts are needed to identify the differences in the responses of sickle RBCs

Proteomic Analysis of the Role of the Adenylyl Cyclase–cAMP Pathway in Red Blood Cell Mechanical Responses

Red blood cell (RBC) deformability is modulated by the phosphorylation status of the cytoskeletal proteins that regulate the interactions of integral transmembrane complexes. Proteomic studies have revealed that receptor-related signaling molecules and regulatory proteins involved in signaling cascades are present in RBCs. In this study, we investigated the roles of the cAMP signaling mechanism in modulating shear-induced RBC deformability and examined changes in the phosphorylation of the RBC proteome. We implemented the inhibitors of adenylyl cyclase (SQ22536), protein kinase A (H89), and phosphodiesterase (PDE) (pentoxifylline) to whole blood samples, applied 5 Pa shear stress (SS) for 300 s with a capillary tubing system, and evaluated RBC deformability using a LORRCA MaxSis. The inhibition of signaling molecules significantly deteriorated shear-induced RBC deformability (p < 0.05). Capillary SS slightly increased the phosphorylation of RBC cytoskeletal proteins. Tyrosine phosphorylation was significantly elevated by the modulation of the cAMP/PKA pathway (p < 0.05), while serine phosphorylation significantly decreased as a result of the inhibition of PDE (p < 0.05). AC is the core element of this signaling pathway, and PDE works as a negative feedback mechanism that could have potential roles in SS-induced RBC deformability. The cAMP/PKA pathway could regulate RBC deformability during capillary transit by triggering significant alterations in the phosphorylation state of RBCs

Proteomic Analysis of the Role of the Adenylyl Cyclase–cAMP Pathway in Red Blood Cell Mechanical Responses

Red blood cell (RBC) deformability is modulated by the phosphorylation status of the cytoskeletal proteins that regulate the interactions of integral transmembrane complexes. Proteomic studies have revealed that receptor-related signaling molecules and regulatory proteins involved in signaling cascades are present in RBCs. In this study, we investigated the roles of the cAMP signaling mechanism in modulating shear-induced RBC deformability and examined changes in the phosphorylation of the RBC proteome. We implemented the inhibitors of adenylyl cyclase (SQ22536), protein kinase A (H89), and phosphodiesterase (PDE) (pentoxifylline) to whole blood samples, applied 5 Pa shear stress (SS) for 300 s with a capillary tubing system, and evaluated RBC deformability using a LORRCA MaxSis. The inhibition of signaling molecules significantly deteriorated shear-induced RBC deformability (p p p < 0.05). AC is the core element of this signaling pathway, and PDE works as a negative feedback mechanism that could have potential roles in SS-induced RBC deformability. The cAMP/PKA pathway could regulate RBC deformability during capillary transit by triggering significant alterations in the phosphorylation state of RBCs

Alterations of erythrocyte rheology and cellular susceptibility in end stage renal disease: Effects of peritoneal dialysis.

In this study, we investigated the effects of peritoneal dialysis on hemorheological and hematological parameters and their relations with oxidant and antioxidant status of uremic patients. Hemorheological parameters (erythrocyte deformability, erythrocyte aggregation, osmotic deformability, blood and plasma viscosity) were measured in patients with renal insufficiency undergoing peritoneal dialysis (PD) and volunteers. Erythrocyte deformability, osmotic deformability and aggregation in both autologous plasma and 3% dextran 70 were measured by laser diffraction ektacytometry. Enzyme activities of glutathione peroxidase, superoxide dismutase and catalase were studied in erythrocytes; lipid peroxidation was studied by measuring the amount of malondialdehyde in both erythrocytes and plasma samples. Blood viscosity at native hematocrit was significantly lower in PD patients at all measured shear rates compared to controls, but it was high in PD patients at corrected (45%) hematocrit. Erythrocyte deformability did not show any difference between the two groups. Osmotic deformability was significantly lower in PD patients compared to controls. Aggregation index values were significantly high in PD patients in plasma Catalase and glutathione peroxidase activities in erythrocytes were decreased in PD patients whereas superoxide dismutase activity was increased compared to controls. Malondialdehyde was significantly increased in erythrocytes and plasma samples of PD patients which also shows correlations with aggregation parameters. It has been concluded that erythrocytes in PD patients are more prone to aggregation and this tendency could be influenced by lipid peroxidation activity in patient's plasma. These results imply that uremic conditions, loss of plasma proteins and an increased risk of oxidative stress because of decreasing levels of antioxidant enzymes affect erythrocyte rheology during peritoneal dialysis. This level of distortion may have crucial effects, impairing the blood flow dynamics and causing inadequate microcirculatory perfusion

Increased complexin-1 and decreased miR-185 expression levels in Beh double dagger et's disease with and without neurological involvement

Although complexin 1 (CPLX1) is not known as an inflammation factor, recent identification of a complexin 1 (CPLX1) polymorphism in Beh double dagger et's disease (BD) has sparked an interest in the role of this molecule in autoinflammation. DNA samples were isolated from peripheral blood mononuclear cells (PBMC) of BD and neuro-Beh double dagger et's disease (NBD) patients and expression levels of CPLX1 and miR-185, a predicted target miRNA for CPLX1 and an inflammation-related miRNA, were investigated by real time PCR assays. PBMC expression levels of CPLX1 were significantly increased in BD and NBD patients. By contrast, levels of miR-185 were reduced in both patient groups. A moderate inverse correlation was found between levels of CPLX1 and miR-185. No correlation could be found between expression levels and clinical features of patients. Significant expression alterations of CPLX1 in BD and NBD patients suggest that this molecule has a proinflammatory action. The putative role of CPLX1 in BD pathogenesis remains to be further studied

Novel Anti-Neuronal Antibodies in Neuromyelitis Optica Patients with or without Aquaporin-4 Antibodies

65th Annual Meeting of the American-Academy-of-Neurology (AAN) -- MAR 16-23, 2013 -- San Diego, CAWOS: 000332068601199…Amer Acad Neuro

Elevated Switch-Associated Protein 70 (SWAP70) Antibody Levels during Attacks in Multiple Sclerosis Patients

64th Annual Meeting of the American-Academy-of-Neurology (AAN) -- APR 21-28, 2012 -- New Orleans, LAWOS: 000303204801195…Amer Acad Neurol (AAN

Switch-associated protein 70 (SWAP 70) antibody levels are associated with clinical relapse in multiple sclerosis

11th International Congress of Neuroimmunology (ISNI) -- NOV 04-08, 2012 -- Boston, MAWOS: 000312764800046…European Sch Neuroimmunol, Int Soc Neuroimmunol (ISNI