Norihito Inami

The effects of pitavastatin, eicosapentaenoic acid and combined therapy on platelet-derived microparticles and adiponectin in hyperlipidemic, diabetic patients.

Platelet-derived microparticles (PDMP) play an important role in the pathogenesis of diabetic vasculopathy, and statins or eicosapentaenoic acid (EPA) have been shown to have a beneficial effect on atherosclerosis in hyperlipidemic patients. However, the influence of EPA and statins on PDMP and adiponectin in atherosclerosis is poorly understood. We investigated the effect of pitavastatin and EPA on circulating levels of PDMP and adiponectin in hyperlipidemic patients with type II diabetes. A total of 191 hyperlipidemic patients with type II diabetes were divided into three groups: group A received pitavastatin 2 mg once daily (n = 64), group B received EPA 1800 mg daily (n = 55) and group C received both drugs (n = 72). PDMP and adiponectin were measured by ELISA at baseline and after 3 and 6 months of drug treatment. Thirty normolipidemic patients were recruited as healthy controls. PDMP levels prior to treatment in hyperlipidemic patients with diabetes were higher than levels in healthy controls (10.4 ± 1.9 vs. 3.1 ± 0.4 U/ml, p < 0.0001), and adiponectin levels were lower than controls (3.20 ± 0.49 vs. 5.98 ± 0.42 µg/ml, p < 0.0001). PDMP decreased significantly in group B (before vs. 6M, 10.6 ± 2.0 vs. 8.0 ± 1.7 U/ml, p < 0.01), but not in group A (before vs. 6M, 9.4 ± 1.9 vs. 9.6 ± 1.7 U/ml, not significant). In contrast, group A exhibited a significant increase in adiponectin levels after treatment (before vs. 6M, 3.29 ± 0.51 vs. 4.16 ± 0.60 µg/ml, p < 0.001). Furthermore, group C exhibited significant improvement in both PDMP and adiponectin levels after treatment (PDMP, before vs. 6M, 11.2 ± 2.0 vs. 4.5 ± 2.7 U/ml, p < 0.001; adiponectin, before vs. 6M, 3.24 ± 0.41 vs. 4.02 ± 0.70 µg/ml, p < 0.001). Reductions of PDMP in combined therapy were significantly greater than those observed with EPA alone (p < 0.05 by ANOVA). In addition, soluble CD40 ligand exhibited almost the same change as PDMP in all therapy groups. These results suggest that pitavastatin possesses an adiponectin-dependent antiatherosclerotic effect, and this drug is able to enhance the anti-platelet effect of EPA. The combination therapy of pitavastatin and EPA may be beneficial for the prevention of vascular complication in hyperlipidemic patients with type II diabetes.

Correlation and association between plasma platelet-, monocyte- and endothelial cell-derived microparticles in hypertensive patients with type 2 diabetes mellitus.

Elevated platelet-derived mircoparticles (MP) (PDMP), endothelial cell-derived MP (EDMP), and monocyte-derived MP (MDMP) concentrations are documented in almost all thrombotic diseases. However, the intricate interactions between PDMP, MDMP and EDMP in hypertensive patients with or without type 2 diabetes remains poorly understood. Therefore, to clarify the correlation and association of MPs, we measured and analysed the levels of MPs in 359 hypertensive patients. We compared the results of chemokines, cell adhesion molecules, platelet activation markers and microparticles in hypertensive patients with and without type 2 diabetes mellitus. The levels of all markers were significantly higher in the hypertensive patients with diabetes than in the non-diabetic patients. For hypertensive patients with diabetes, univariate analysis showed that age, body mass index, systolic blood pressure, high density lipoprotein cholesterol (HDL-CHO), creatinine (CRTN), soluble P-selectin (sP-selectin), soluble E-selectin (sE-selectin), soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble CD40 ligand (sCD40L), regulated on activation normally T-cell expressed and secreted (RANTES), monocyte chemotactic peptide-1 (MCP-1), MDMP and EDMP were significantly associated with PDMP. In addition, systolic blood pressure, HDL cholesterol, sP-selectin, sE-selectin, sVCAM-1, sCD40L, RANTES, MDMP and EDMP were significant factors in the multivariate model with PDMP. Furthermore, a correlation between plasma PDMP and MDMP or EDMP in hypertensive patients were observed both with and without diabetes. These results suggest that the existence of diabetes mellitus affects PDMP generation in hypertensive patients and that enhanced plasma levels of PDMP and an association between the plasma levels of PDMP, MDMP and EDMP may result in the development of atherothrombotic complications in hypertensive patients.

Effects of pitavastatin on adiponectin in patients with hyperlipidemia.

The effects of treatment with pitavastatin on inflammatory and platelet activation markers and adiponectin in 117 patients with hyperlipidemia were investigated to determine whether pitavastatin may prevent the progression of atherosclerotic changes in hyperlipidemic patients. Adiponectin levels prior to pitavastatin treatment in hyperlipidemic patients with and without diabetes were lower than levels in normolipidemic controls. Both total cholesterol and the low-density lipoprotein cholesterol decreased significantly after pitavastatin administration. Additionally, hyperlipidemic patients with or without type 2 diabetes exhibited a significant increase in adiponectin levels 6 months after pitavastatin treatment (diabetes: 3.52 ± 0.80 vs. 4.52 ± 0.71 µg/ml, p < 0.001; no diabetes: 3.48 ± 0.71 vs. 4.23 ± 0.82 µg/ml, p < 0.05). However, high-sensitivity C-reactive protein, platelet-derived microparticle and soluble P-selectin did not exhibit any differences before or after pitavastatin administration. Levels of adiponectin significantly increased after pitavastatin administration in the group of lower soluble P-selectin (soluble P-selectin before pitavastatin treatment <200 ng/ml). These results suggest that pitavastatin possesses an adiponectin-increasing effect in patients with hyperlipidemia and this effect is influenced by intensive platelet activation.

Evaluation of Angiopoietins and Cell-Derived Microparticles after Stem Cell Transplantation

Although stem cell transplantation (SCT) is being used for hematopoietic reconstitution following high-dose chemotherapy for malignancy, it involves certain serious transplant-related complications such as graft-versus-host disease (GVHD). Angiopoietins play important roles in angiogenesis. However, the role of angiopoietins after SCT is poorly understood. In this study, 52 patients underwent SCT; 26 patients received allogeneic SCT, while the remaining 26 received autologous SCT. In 48 of 52 patients, levels of angiopoietins, cytokines, and soluble factors were measured by enzyme-linked immunosorbent assay. Soluble Fas ligand (sFasL) and endothelial cell-derived microparticle (EDMP) exhibited significant elevation in the early phase (2-3 weeks) after SCT. In addition, the elevation of interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, and sIL-2 receptor (sIL-2R), which are GVHD markers after allogeneic SCT was observed. The level of angiopoietin (Ang)-2 in allogeneic SCT continued to increase for up to 4 weeks, although the level of Ang-1 did not show significant changes. The patients with high Ang-2 exhibited significant increase of sFasL and EDMP compared with those with low Ang-2. In addition, the patients with high-grade GVHD exhibited a significant increase in Ang-2 compared to patients with low-grade GVHD. In the in vitro experiment using endothelial cells, the suppressive effect of Ang-1 on EDMP generation by TNF-alpha was partially inhibited by the addition of Ang-2. Furthermore, multivariate regression analysis showed that EDMP and sFasL were significant factors in Ang-2 elevation. Our results suggest that Ang-2 generation after allogeneic SCT relates to GVHD.

Platelet activation markers, microparticles and soluble adhesion molecules are elevated in patients with arteriosclerosis obliterans: therapeutic effects by cilostazol and potentiation by dipyridamole.

We evaluated the plasma concentrations of platelet activation markers, microparticles and soluble adhesion molecules in patients with arteriosclerosis obliterans (ASO) and compared the beneficial effects of cilostazol alone and combination therapy of cilostazol and dipyridamole in these patients. There was a significant elevation of CD62P, CD63, PAC-1, annexin V, platelet-derived microparticles (PDMPs), sP-selectin, sE-selectin, sICAM-1 and sVCAM-1 in the ASO patients compared with the controls. Platelet aggregation was decreased by 2 weeks of cilostazol monotherapy in the ASO patients. Adding dipyridamole to the cilostazol therapy for 2 weeks further reduced platelet aggregation. While treatment with cilostazol alone reduced levels of CD62P, CD63, PAC-1, annexin V, PDMP, and sP-selectin, the combination therapy reduced these parameters further. While sE-selectin and cell adhesion molecules did not change significantly after 2 weeks of combination therapy, they exhibited a remarkable decrease after 16 weeks of combination treatment. These findings suggest that platelets are activated in ASO patients, and cilostazol is effective to reduce platelet activation. Furthermore, dipyridamole may potentiate the beneficial effect of cilostazol in ASO patients. Combination use of both drugs may help to prevent the onset of cardiovascular complications in patients with ASO by activated platelets and PDMP.

The effects of Helicobacter pylori eradication on chemokine production in patients with immune thrombocytopenic purpura.

To the Editor: Immune thrombocytopenic purpura (ITP) is an autoimmune disease caused by circulating antibodies that react with platelet membranes (1). Emilia et al. (2) reported a high incidence of Helicobacter pylori (HP) infection in patients with ITP as well as a significant increase in platelet count after bacterium eradication. Although the pathogenetic mechanisms of HP-induced thrombocytopenia remain unknown, HP causes an immunological response resulting in the production of high concentrations of pro-inflammatory cytokines and chemokines in non-ITP patients (3). On the contrary, we recently reported on the significance of chemokines in patients with ITP (4). Monocyte chemoattractant protein-1 (MCP-1) and regulated on activation normally T-cell expressed and secreted (RANTES) were significantly increased in ITP. This elevation was thought to be related to the CD40/CD40 ligand system. We thus investigated the concentrations of different chemokines before and after HP eradication in ITP patients. Serum samples from 42 patients (15 men and 27 women) with chronic ITP were studied. Plateletassociated immunoglobulin G (IgG) was measured by a competitive enzyme-linked immunosorbent assay. Platelet counts and platelet-associated IgG were 31 ± 6 (·10/L) and 134 ± 12 (ng/10 plt), respectively. The patients had not received any transfusions, but 21 had previously been treated for ITP (splenectomy in six patients and prednisolone therapy in 17 patients). The urea breath test was used to determine whether or not patients were infected with HP, and if found to be infected, treatment to eradicate HP included amoxicillin (1000 mg twice daily), clarithromycin (400 mg twice daily), and omeprazole (20 mg twice daily) for 1 wk. The prednisolone treatment was not changed during the eradication therapy, which was evaluated at 8 wk following treatment. We measured human 3 CC chemokines (MCP-1, RANTES, and Eotaxin) and 3 CXC chemokines [interleukin-8 (IL-8), epithelial cell-derived neutrophil attractant78; ENA-78, and stromal cell-derived factor-1; SDF-1] because HP influences both CC and CXC chemokine (5, 6) levels, which were measured with a monoclonal antibody-based ELISA kit from BioSourse International Inc. (Camarillo, CA, USA) and R & D Systems (Minneapolis, MN, USA) according to the manufacturer’s instructions. HP infection was found in 28 of 42 chronic ITP patients (66.7%). After eradication, 15 of the 28 patients (53.6%) had a significant increase in platelet count (increase of greater than 50 · 10/L vs. pretreatment platelet count), and the eradication rate was 42.9% (12/28). This eradication rate was low compared with the average rate usually obtained after a standard eradication regimen. This reason is unknown. However, one of them could be an ethnic difference. There were no significant differences in concentrations of IL-8, SDF-1 and Eotaxin between HP-positive and HP-negative ITP patients (Table 1). However, significant increases in

P-selectin and platelet-derived microparticles associated with monocyte activation markers in patients with pulmonary embolism.

Platelet activation markers (platelet-derived microparticles and P-selectin on activated platelets), chemokines (monocyte chemotactic peptide and regulated on activation normally T-cell expressed and secreted), and soluble markers (sP-selectin, sE-selectin, sVCAM-1, and sCD14) were measured and compared in patients with pulmonary embolism (PE). These substances are thought to participate in the pathogenesis of PE. Levels of all of the platelet activation markers, chemokines, and soluble markers were higher in the patients with PE than in normal controls. Levels of platelet activation markers were also significantly increased postoperatively after total knee arthroplasty. Anti-platelet therapy significantly inhibited the elevation of platelet activation markers after total knee arthroplasty. These findings suggest that antiplatelet therapy may be useful for PE-related interaction of platelets, leukocytes, and endothelial cells.

Elevation of Platelet Activation Markers and Chemokines during Peripheral Blood Stem Cell Harvest with G-CSF

The kinetics of peripheral blood stem cell mobilization in response to recombinant human granulocyte colony‐stimulating factor is well established. However, there have been few investigations of platelet activation markers during peripheral blood stem cell harvest. We measured the levels of the platelet activation markers, chemokines, and soluble factors in plasma obtained from patients undergoing peripheral blood stem cell harvest. The number of leukocytes, CD34+ cells, neutrophils, monocytes, and lymphocytes peaked on day 5 after granulocyte colony‐stimulating factor treatment, but the numbers of eosinophils and basophils showed no significant change. Regulated on activation normally T‐cell expressed and secreted (RANTES) level increased through day 10, and the monocyte chemotactic peptide‐1 (MCP‐1) level peaked on day 5. Platelet counts continued to increase through day 10. The level of thrombopoietin significantly increased on day 3, peaked on day 5, and decreased slightly by day 10. The levels of soluble CD40 ligand and soluble P‐selectin increased up to day 5. The platelet‐derived microparticle level peaked on day 5, and then began to decline. CD34+ cell numbers significantly correlated with those of leucocytes, neutrophils, monocytes, and lymphocytes, as well as levels of MCP‐1, and the CD34+ cells exhibited changes similar to platelet‐derived microparticles. The patterns of change in MCP‐1, platelet‐derived microparticles, and the CD34+ cell count are similar in that each peaks on day 5 and decreases thereafter. Further study is required to determine if a cause‐and‐effect relationship in their pattern of change exists among them.

Significance of microparticles in progressive systemic sclerosis with interstitial pneumonia.

We measured and compared the levels of microparticles, chemokines, cell adhesion molecules and platelet activation markers with a view to developing a better understanding of their potential contributions to the pathophysiology of progressive systemic sclerosis (PSS, scleroderma). The concentrations of all the factors in PSS patients were significantly higher than those in normal subjects. PSS patients were divided to two groups by whether they have interstitial pneumonia (IP) or not. There were no differences in the levels of soluble(s) VCAM-1, sICAM-1, sE-selectin and IL-8 between the two groups. However, there were significant between-group differences in the levels of sP-selectin, sCD40L, ENA-78, RANTES (regulated on activation normally T-cell expressed and secreted), platelet-derived microparticles (PDMPs), monocyte-derived microparticle (MDMPs) and KL-6. The level of tissue factor expression on monocytes by A23187 stimulation in PSS patients was found to be similar to that in healthy controls. Although PDMP did not induce the expression of tissue factor on monocytic cell line (THP-1) directly, the recombinant sCD40 ligand-induced expression of tissue factor on THP-1 and generation of MDMP from this cell line were enhanced by the addition of PDMPs. Our findings suggested that elevated levels of PDMPs and MDMPs may be interpreted as a sign of vascular complications in PSS patients, particularly those complicated with IP, offering a new treatment strategy in these patients.

Effects of pitavastatin on monocyte chemoattractant protein-1 in hyperlipidemic patients.

The effects of statins on platelet activation markers, chemokines and adiponectin, were investigated in 135 patients with hyperlipidemia. Of the 135 hyperlipidemic patients, 63 were allocated to the simvastatin group, treated with simvastatin at the dose of 10 mg daily, and the remaining 72 were allocated to the pitavastatin group, treated with pitavastatin at the dose of 2 mg daily. Plasma levels of platelet-derived microparticles (PDMP), cell adhesion molecules (sCD40L and sP-selectin), chemokines [monocyte chemoattractant protein-1 (MCP-1) and regulated on activation normally T-cell expressed and secreted] and adiponectin were measured at the baseline and after 6 months of treatment in both the groups. In addition, we carried out a basic study to investigate the MCP-1-dependent induction of tissue factor expression on a histiocytic cell line (U937 cells). The plasma levels of PDMP, sCD40L, sP-selectin, regulated on activation normally T-cell expressed and secreted and MCP-1 were higher, whereas those of adiponectin were lower, in the hyperlipidemic patients than in the normolipidemic controls. Plasma PDMP and sCD40L were positively correlated, whereas plasma adiponectin was negatively correlated, with the plasma levels of MCP-1. No significant differences in the plasma levels of PDMP, sCD40L, sP-selectin, regulated on activation normally T-cell expressed and secreted and MCP-1 measured before and after treatment were observed in either the simvastatin or pitavastatin group. A significant increase of the plasma adiponectin levels was observed after 6 months of treatment with pitavastatin but not after an equal duration of treatment with simvastatin. When pitavastatin-treated patients were divided into two groups according to the adiponectin response to pitavastatin treatment, significant decreases of the plasma MCP-1, PDMP and sCD40L levels were observed after pitavastatin treatment in the responder group. In the aforementioned basic study, MCP-1 by itself did not induce the expression of tissue factor on the U937 cells. However, the recombinant sCD40L-induced expression of tissue factor on U937 was enhanced by the addition of MCP-1. These findings suggest that PDMP, sCD40L and MCP-1 may participate in the development of atherothrombosis in patients with hyperlipidemia and that pitavastatin may exert an adiponectin-dependent antiatherothrombotic effect in hyperlipidemic patients.