Shosaku Nomura

Measuring circulating cell-derived microparticles

Cell-derived microparticles (MPs) are receiving increasing attention in recent years, both as a diagnostic aid and investigative tool [1–4]. Because they carry markers of the parent cell, including those induced by activation or apoptosis, endothelial MPs (EMPs) can provide valuable information on the status of the parent cell, obtainable in no other way. In addition, there is a growing belief that MPs can function as important diffusible vectors of specific adhesins and cytokines promoting cellular interactions and signal transmission [2]. ThusMP analysis constitutes a new avenue for investigation of pathologies in various diseases. Although still considered investigational [1–4], recent results from several laboratories suggest that MP analysis may be poised to enter the mainstream of clinical testing. However, a major impediment to that end is the wide variety ofmethodologies used by different laboratories in this field, few of which can be directly compared to the others, and results from which are sometimes inconsistent or conflicting. As a first step in addressing that problem, the Editor has organized this Forum article, consisting of a brief description of the preferred methods and rationality from each of six active laboratories in the field, including our own [5–10]. Table 1 lists some key features of the six methodological approaches. It is seen that major differences exist in the preparation of the MP samples (such as centrifugation), whether or not they are first sedimented and resuspended, means of generic MP detection (4 of 6 use annexin V), and cell lineage-specific antigenic markers. These differences probably account for some of the different findings among the groups.

High-shear-stress-induced activation of platelets and microparticles enhances expression of cell adhesion molecules in THP-1 and endothelial cells.

Interaction between leukocyte and endothelial cells (ECs) is essential for vascular homeostasis and competent immune-inflammatory responses in vivo. Platelet-derived microparticles (PMPs) are generated by high shear stress and may appear in diseased small arteries and arterioles in various clinical settings. In this study, we used flow cytometry and confocal laser scanning microscopy to investigate the effects of high-shear-induced platelet and microparticle activation in adhesion molecules of THP-1 and ECs. We also measured the production of some cytokines and studied cytokine mRNA from THP-1 and ECs after PMP stimulation. PMP stimulation of THP-1 cells increased CD11b, CD32, and CD33 but not CD29, CD31, and CD36. PMP stimulation of ECs increased CD54 and CD63 but not CD9, CD29, and CD31. PMPs induced interleukin-8 (IL-8), interleukin-1 beta (IL-1 beta), and tumor necrosis factor alpha (TNF alpha) production by THP-1. PMPs also induced IL-8, IL-1 beta, and interleukin-6 (IL-6) production by ECs. Production was time-dependent. With RT-PCR, some cytokine mRNAs were detected in THP-1 and ECs after PMP stimulation. In relation to adhesiveness after PMP stimulation, we could clearly observe a shift in distribution not only of CD11b in THP-1 cells but also of CD54 in ECs. In addition, anti-P-selectin glycoprotein ligand-1 antibody reduced the expression of CD11b, CD32, and CD33 in THP-1 after PMP stimulation. These results suggest that high-shear-induced microparticles may contribute to the development of atherosclerosis and participate in vascular damage in inflammatory disorders.

Platelet-derived microparticles may influence the development of atherosclerosis in diabetes mellitus.

We investigated the association between low-density lipoprotein (LDL), triglycerides, and platelet activation in 18 patients with hypertension age 41-64 years and 18 with diabetes mellitus aged 43-70 years. Platelet P-selectin positivity and the microparticle level (indicators of activation) were both significantly higher in the diabetics than in healthy controls (P-selectin: 28.0% +/- 7.5% vs. 7.3% +/- 4.2%, P < 0.001; microparticles: 1900 +/- 966 vs. 526 +/- 158/10(4) platelets, P < 0.01). In contrast, there was no significant increase of either parameter in the patients with hypertension. Plasma microparticle levels were also significantly greater in the diabetics with high LDL levels than in those with low LDL levels (2375 +/- 949 vs. 1519 +/- 796/10(4) platelets, P < 0.05), and in those with high rather than low triglyceride levels (2188 +/- 845 vs. 1492 +/- 783/10(4) platelets, P < 0.05). However, platelet positivity for P-selectin was not significantly different between these two subgroups. Microparticle and P-selectin levels both showed no significant difference between the hypertensive patients with high and low LDL or triglyceride levels. These results suggest that platelet-derived microparticles may participate in the development or progression of atherosclerosis in patients with diabetes mellitus.

Function and role of microparticles in various clinical settings

Microparticles released from cells (MPs) may play a role in the normal hemostatic response to vascular injury and a role in clinical diseases because they express phospholipids, which function as procoagulants. Although flow cytometry is the most widely used method for studying MPs, some novel assays such as tissue factor-dependent procoagulant assay or the ELISA method have been reported. However, the use of MP quantification as a clinical tool is still a matter of debate. Elevated platelet-derived MP, endothelial cell-derived MP, and monocyte-derived MP concentrations are documented in almost all thrombotic diseases occurring in both venous and arterial beds. However, the clear significance of MPs in various clinical conditions remains controversial. For example, it is not known if MPs found in peripheral blood vessels cause thrombosis, or whether they are the result of thrombosis. On the other hand, numerous studies have shown that not only the quantity but also the cellular origin and composition of circulating MPs are dependent on the type of disease, the disease state and medical treatment. In addition, many different functions have also been attributed to MPs. Thus, the number and type of clinical disorders associated with elevated MPs is currently increasing.

Function and clinical significance of platelet-derived microparticles

Microparticles released from platelets (PMPs) may play a role in the normal hemostatic response to vascular injury because they demonstrate prothrombinase activity. PMPs were first observed as released vesicles from platelets following adhesion to vessel walls, and flow cytometry is now the most widely used method for studying PMPs. PMPs are thought to play a role in clinical disease because they express phospholipids that function as procoagulants. High shear stress can initiate both platelet aggregation and shedding of procoagulant-containing PMP, suggesting that PMP generation by high shear stress occurs in small diseased arteries and arterioles under various clinical conditions. In addition, the possibility that PMPs evoke cellular responses in their immediate microenvironments has recently been suggested. Despite many interesting findings, the significance of PMPs in various clinical conditions remains controversial. For example, it is not known whether PMPs found in peripheral blood vessels cause thrombosis, or if they are the results of thrombosis. There has been some question about whether the PMPs found in thromboses are consumed locally, meaning that PMPs circulating in the peripheral blood are not functionally important. Currently, the number of clinical disorders associated with elevated PMPs is increasing.

Is eradication therapy useful as the first line of treatment in Helicobacter pylori-positive idiopathic thrombocytopenic purpura? Analysis of 207 eradicated chronic ITP cases in Japan.

A retrospective study was performed to determine the prevalence of Helicobacter pylori (H pylori) infection, the effect of H pylori eradication on platelet counts, and the characteristic clinical features of chronic immune or idiopathic thrombocytopenic purpura (ITP) with H pylori infection. H pylori infection was found in 300 patients, a group that was significantly older (P < .005) and had more cases of hyperplastic megakaryocytes in the bone marrow (P = .01) than patients without H pylori infection.H pylori eradication therapy was performed in 207 H pylori-positive ITP cases, and the platelet count response was observed in 63% of the successful eradication group and in 33% of the unsuccessful eradication group (P < .005). In the successful group, the complete remission and partial remission rates were 23% and 42%, respectively, 12 months after eradication. In the majority of responders, the platelet count response occurred 1 month after eradication therapy, and the increased platelet count continued without ITP treatment for more than 12 months. H pylori eradication therapy was effective even in refractory cases, which were unresponsive to splenectomy. In conclusion, H pylori infection was involved in most ITP patients older than 40 years in Japan, and eradication therapy should be the first line of treatment in H pylori-positive ITP patients.

Detection of monocyte-derived microparticles in patients with Type II diabetes mellitus

Aims/hypothesis. The role of plasma monocyte-derived microparticles (MDMPs) and platelet-activation markers (platelet-derived microparticle [PDMP], platelet-bound CD62P [plt-CD62P], and platelet-bound CD63 [plt-CD63]) in diabetic vascular complications is not clear. We measured and compared plasma concentrations of MDMPs and the platelet-activation markers to investigate their possible contribution to diabetic vascular complications. Methods. Activated platelets and microparticles (PDMP and MDMP) were analysed by flow cytometry. Concentrations of serum sE-selectin were measured with enzyme-linked immunosorbent assay. Results. The concentration of MDMPs in diabetic patients was higher than in normal subjects. We found no differences in the binding of anti-GPIIb/IIIa and anti-GPIb monoclonal antibodies between groups. There were differences, however, in the concentrations of PDMPs, plt-CD62P, and plt-CD63 between Type II (non-insulin-dependent) diabetes mellitus patients and control subjects (PDMPs: 585 ± 25 vs 263 ± 9, p < 0.01; plt-CD62P: 28.1 % ± 1.4 % vs 9.4 % ± 0.6 %, p < 0.001; plt-CD63: 28.1 % ± 1.4 % vs 8.6 % ± 0.5 %, p < 0.001). Amounts of MDMPs correlated positively with these platelet activation markers, and the relation between PDMP and MDMP was the most significant. The concentration of MDMP in patients who had diabetes complicated with nephropathy, retinopathy, or neuropathy was higher than in those without diabetes-related complications. The increase in MDMP was particularly significant in patients with nephropathy. Concentrations of sE-selectin were higher in Type II diabetes patients than in control subjects, and correlated with MDMP, PDMP, plt-CD62P, and plt-CD63 levels in nephropathy patients. Conclusion/interpretation. In Type II diabetes patients, we detected increased activation of monocytes, which could have been stimulated by activated platelets and PDMPs. Because the activation of monocytes is associated with vascular endothelial damage, high concentrations of MDMPs could indicate vascular complications in diabetes patients, especially those who have diabetes-related nephropathy. [Diabetologia (2002) 45: ▪–▪]

Effects of efonidipine on platelet and monocyte activation markers in hypertensive patients with and without type 2 diabetes mellitus

We compared the levels of microparticles, platelet activation markers, soluble cell adhesion molecules, and soluble selectins between hypertensive patients with and without type 2 diabetes and control subjects. Binding of anti-glycoprotein IIb/IIIa and anti-glycoprotein Ib monoclonal antibodies to platelets did not differ significantly between the hypertensive patients and controls, but platelet expression of activation markers (CD62P, CD63, PAC-1, and annexin V) was higher in the hypertensive patients. Platelet-derived microparticle (PDMP) and monocyte-derived microparticle (MDMP) levels were significantly higher in the hypertensive patients than in the controls. Soluble ICAM-1, VCAM-1, P-selectin, and E-selectin levels were also higher in the hypertensive patients, and they were significantly higher in the hypertensive patients with diabetes. After treatment with efonidipine, the levels of PDMPs, CD62P-, CD63-, PAC-1-, and annexin V-positive platelets, sICAM-1, sVCAM-1, sP-selectin, and sE-selectin all decreased significantly. The MDMP levels decreased, and the decrease was significant in the hypertensive patients with diabetes. These findings suggest that administration of efonidipine to hypertension patients with diabetes may prevent the development of cardiovascular complications caused by cell adhesion molecules or activated platelets and monocytes.

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.

Effects of Losartan and Simvastatin on Monocyte-Derived Microparticles in Hypertensive Patients With and Without Type 2 Diabetes Mellitus

Monocyte-derived microparticles play an important role in the pathogenesis of diabetic vasculopathy, and angiotensin II receptor blocker and statin have been shown to have a beneficial effect on the angiopathies of hypertension and hyperglycemia in patients with type 2 diabetes mellitus. However, the interaction between angiotensin II receptor blocker and statin, and monocyte-derived microparticles in atherosclerosis is poorly understood. The effects of losartan and simvastatin on circulating concentrations of monocyte-derived microparticles, chemokines, and soluble adhesion markers were studied in hypertensive patients with or without type 2 diabetes mellitus. Monocyte-derived microparticles were measured by flow cytometry, and levels of serum chemokines (MCP-1 and RANTES) and soluble adhesion markers (sP-selectin and sVCAM-1) were measured by enzyme-linked immunosorbent assay. Losartan decreased both the systolic and diastolic blood pressure in hypertensive patients with and without type 2 diabetes mellitus. The concentrations of monocyte-derived microparticles, chemokines, and soluble adhesion molecules were higher in hypertensive patients who also had type 2 diabetes mellitus vs. those who did not. The administration of angiotensin II receptor blocker decreased the circulating concentration of all these markers. In addition, all markers were decreased by combination therapy, and monocyte-derived microparticles were decreased more with combination therapy with losartan and simvastatin than monotherapy with losartan. The administration of angiotensin II receptor blocker inhibited monocyte-derived microparticle generation and suggests that angiotensin II is intimately related to vascular changes that occur in type 2 diabetes mellitus. Combination therapy with a statin and angiotensin II receptor blocker might be valuable as anti-atherosclerotic therapy in patients with type 2 diabetes mellitus and nephropathy.