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Type 1 And Type 2 Diabetic Patients Display Different Patterns of Cellular Microparticles

¹ INSERM EMI 0019, Laboratory of Immunology and Hematology, UFR de Pharmacie, Université de la Méditerranée, Marseille, France
² Federation of Autoimmunity and Thrombosis, AP-HM de la Conception, Marseille, France
³ Department of Endocrinology, Metabolic Diseases and Nutrition, AP-HM Nord, Marseille, France
⁴ Institute of Hematology and Immunology, Faculty of Medicine, Strasbourg, France
⁵ Department of Internal Medicine, AP-HM Nord, Marseille, France

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
The development of vasculopathies in diabetes involves multifactorial processes including pathological activation of vascular cells. Release of microparticles by activated cells has been reported in diseases associated with thrombotic risk, but few data are available in diabetes. The aim of the present work was to explore the number and the procoagulant activity of cell-derived microparticles in type 1 and 2 diabetic patients. Compared with age-matched control subjects, type 1 diabetic patients presented significantly higher numbers of platelet and endothelial microparticles (PMP and EMP), total annexin V-positive blood cell microparticles (TMP), and increased levels of TMP-associated procoagulant activity. In type 2 diabetic patients, only TMP levels were significantly higher without concomitant increase of their procoagulant activity. Interestingly, in type 1 diabetic patients, TMP procoagulant activity was correlated with HbA_1c, suggesting that procoagulant activity is associated with glucose imbalance. These results showed that a wide vesiculation process, resulting from activation or apoptosis of several cell types, occurs in diabetes. However, diabetic patients differ by the procoagulant activity and the cellular origin of microparticles. In type 1 diabetic patients, TMP-procoagulant activity could be involved in vascular complications. Moreover, its correlation with HbA_1c reinforces the importance of an optimal glycemic control in type 1 diabetes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

It is now well accepted that in response to activation or apoptosis, all eukariotic cells shed microparticles (12). These elements are produced from the plasma membrane after the flip-flop of the membrane phospholipids leading to a loss of membrane asymmetry. The blebs formed at the cell surface are then shed in the circulation under the form of vesicles ranging in size from 0.1 to 1 µm. Increased levels of microparticles, mainly derived from platelets and to a lesser extent from leukocytes and endothelial cells, have been described in several pathologies associated with prothrombotic and proinflammatory tendencies like heparin-induced thrombocytopenia, thrombotic thrombocytopenic purpura, paroxismal nocturnal hemoglobinuria, HIV infection, and acute coronary syndromes (13–17). Moreover, a recent work has shown that, in atherosclerotic plaques, procoagulant microparticles result from an apoptotic phenomenon involving monocytic and vascular cells (18).

The present study focused on microparticles originating from platelet and endothelial cells, defined as GPIIbIIIa and vß3-positive microparticles, respectively, and microparticles expressing phosphatidylserine defined as total annexin V-positive microparticles. Our initial aim was to investigate the level and procoagulant activity of circulating microparticles in type 1 and type 2 diabetic patients. These microparticles were then explored in relation with type of diabetes and with biological and clinical features of diabetic patients.

	RESEARCH DESIGN AND METHODS

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
Patients and control subjects.
All of the subjects gave informed consent in accordance with local ethics committee recommendations. A total of 47 healthy subjects aged 18–60 years were recruited. They had a normal biological check-up and BMI and presented no history of vascular complications (either micro- or macrovascular). Patients were distributed into two groups: type 1 diabetic individuals (n = 24; age range 16–55 years, mean 34) and type 2 diabetic individuals (n = 52; age range 36–69 years, mean 57).

Subjects with <16 years or >70 years and a previous history of neoplasm, auto-immune disease other than type 1 diabetes, liver disease, or acute infections were excluded from the study.

Retinal vascular morphology was evaluated by direct ophthalmoscopy and fluorescein angiography. Retinopathy was scored as absent, background, preproliferative, or proliferative. Nephropathy was diagnosed by confirmed microalbuminuria (30–300 mg/day) or macroalbuminuria (>300 mg/ day) on two 24-h samples of urine collected over a 6-month period in the absence of urinary infections or other renal diseases. Peripheral neuropathy was assessed for sensory and motor function by clinical examination and classified as normal or impaired.

Macroangiopathy was considered in subjects who met one of the following conditions: 1) history and/or absence of angina and/or permanent ischemic electrocardiogram abnormalities at rest or ischemic abnormalities in a stress, 2) claudication and/or abolished peripheral pulses and/or foot lesions due to vascular disease demonstrated by Doppler echography, and 3) history of stroke and/or significant carotid stenosis (>50%) as assessed by Doppler echography.

All type 1 diabetic patients were insulin-treated, whereas 13 of 52 type 2 diabetic patients (25%) received insulin, alone or in combination with oral hypoglycemic agents (metformin and/or sulfonylureas and/or acarbose). Moreover, the proportion of subjects who were undergoing treatment with aspirin, ACE inhibitors, or statins was greater in type 2 than in type 1 diabetic patients (19.2 vs. 8.3, 26.9 vs. 12.5, and 21.2 vs. 8.3%, respectively), but those differences were not statistically significant.

Reagents.
Fluorescein isothyocyanate (FITC)-conjugated monoclonal antibody (mAb) against vß3 (FITC-CD51, clone AMF7) and phycoerythrine (PE)-conjugated mAb against platelet glycoprotein GPIIbIIIa (PE-CD41, clone P2) were used to identify endothelial microparticle (EMP) and platelet microparticle (PMP), respectively. FITC-conjugated mAbs against CD45 and CD66b were used to identify microparticles from leukocytes and neutrophils, respectively. PE-conjugated mAb against CD14 was used to identify microparticles from monocytes. PE- and FITC-conjugated isotype controls (PE-IgG1, FITC- IgG1) were used to define the background noise of the labeling. FITC-conjugated annexin V was used to label total microparticles. All labeling reagents were purchased at Immunotech Beckman Coulter (Marseille, France). Microparticle absolute values were determined using Flowcount beads (Beckman Coulter, Margency, France).

Blood sampling for microparticle quantitation and procoagulant activity measurements.
Venipuncture was performed on 0.129 mol/l sodium citrate tubes (Vacutainer; Becton Dickinson) on informed healthy donors and patients. Within 2 h, whole blood was treated as previously described (19). Briefly, samples were centrifuged 15 min at 1,500g, and the plasma was then harvested and spun down at 13,000g during 2 min. Supernatants were then immediately frozen at -80°C until used.

Microparticle quantitation.
Numeration of platelet- and endothelial-derived microparticles was performed, as previously described (19), using anti-CD41 and anti-CD51 labeling, respectively. After thawing, 30 µl of plasma was incubated with either FITC-CD51 or PE-CD41PE and their corresponding isotype control for EMP and PMP quantitation, respectively. For total annexin V-positive blood cell microparticle (TMP) quantitation, 30 µl of plasma was incubated with FITC-annexin V. After a 30-min incubation at room temperature, samples were diluted in 1.5 ml of PBS (Dulbecco’s; Life Technologies, Paisley, U.K.) or binding buffer (Beckman Coulter Immunotech) for mAbs and annexin V labeling, respectively. Then, 30 µl of Flowcount beads was added to each sample for calculation of microparticle absolute value.

Flow cytometric analysis.
After labeling and dilution, samples were analyzed by flow cytometry on an EPICS XL (Beckman Coulter). Microparticles present in plasma were analyzed according to their parameters of size and fluorescence. Briefly, on a LogFS-LogSS dotplot, the microparticle upper size limit was defined using 0.8-µm beads, and a gate was drawn around the population. The lower limit of the gate excludes the first channels that contain the electronic background noise of the machine. Only the events included in this gate were further analyzed for their fluorescence on a LogSS-LogFL dotplot. The Flowcount beads were counted in a third dotplot LogSS-LogFL3. Using the absolute value of this calibrant, microparticle count was expressed as microparticles per microliter of plasma.

Microparticle-linked procoagulant activity measurement.
The microparticle procoagulant activity was determined, as previously described (16), using a prothrombinase assay after capture on a microtitration plate coated with annexin V. The blood clotting factor concentrations were determined to ensure that phosphatidylserine is the rate-limiting parameter of the reaction, and results were expressed as nanomolar phosphatidylserine equivalent by reference to a standard curve constructed by the use of liposomes of defined composition.

Statistical analysis.
Differences between groups were evaluated by the nonparametric Mann-Whitney U test; P < 0.05 was considered significant.

	RESULTS

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Demographic description of patients.
Biological parameters and clinical features of diabetic patients and healthy donors are presented in Table 1. Patients suffering from type 1 or type 2 diabetes differed by their age, BMI, and levels of HDL cholesterol, triglycerides, and HbA_1c.

View larger version (28K): [in this window] [in a new window]   FIG. 1. Flow cytometric enumeration of microparticles. Flow cytometric analysis of microparticles in the platelet-free plasma from healthy donors (C: n = 47, ) and all diabetic patients (type 1 + 2, n = 76, ) was performed using mAb directed against either vß3 (FITC-CD51) or GPIIbIIIa (PE-CD41) for labeling of EMP (A) and PMP (B), respectively. Microparticles were quantitated in plasma as described in RESEARCH DESIGN AND METHODS. Then, young and elderly healthy subjects (C1: n = 19, , and C2: n = 28, ) were compared with their corresponding patient groups (type 1: n = 24, , and type 2: n = 52 ). Results were expressed as number of microparticles per microliter of platelet-free plasma. The difference between groups was analyzed using a Mann-Whitney U test (*P < 0.05; **P < 0.005).

Compared with healthy donors, numbers of TMP were significantly elevated in the whole diabetic group (Fig. 2A) (P = 0.0021). In reference with their respective control subjects, TMP was also significantly elevated in both type 1 and type 2 diabetic patients (Fig. 2A) (P = 0.0028 and P = 0.04, respectively), with levels significantly higher in type 1 than in type 2 diabetic patients (1,238 ± 556 and 988 ± 690 TMP/µl, respectively, P = 0.024). Interestingly, the procoagulant activity of TMP was significantly increased in the whole diabetic group (Fig. 2B, P = 0.009) and in type 1 but not in type 2 diabetic patients (P = 0.0013). Among diabetic patients, we found a higher procoagulant activity in type 1 than in type 2 diabetic patients (P < 0.0001). No correlation was observed between number and procoagulant activity of TMP. In summary, analysis of the pattern of cellular microparticles showed that EMP, PMP, and TMP numbers and procoagulant activity were elevated in type 1 diabetic patients, whereas only TMP numbers were increased in type 2 diabetic patients.

View larger version (29K): [in this window] [in a new window]   FIG. 2. Total microparticle number and procoagulant activity in diabetic patients. Annexin V was used for both TMP quantitation by flow cytometry (A) and TMP capture for determination of the procoagulant activity (B). Number and TMP procoagulant activity were measured in healthy donors (C: n = 47, ) and all diabetic patients (type 1 and 2: n = 76, ). Then, young and elderly healthy subjects (C1: n = 19, , and C2: n = 28, ) were compared with their corresponding patient groups (type 1: n = 24, , and type 2: n = 52, , respectively). TMP count was expressed as TMP per microliter of plasma and TMP procoagulant activity as nanomolar of equivalent phosphatidylserine. The difference between groups was analyzed using a Mann-Whitney U test (*P < 0.05; **P < 0.005; ***P < 0.0005).

View larger version (15K): [in this window] [in a new window]   FIG. 3. Numeration of leukocyte microparticles. Flow cytometry analysis of microparticles derived from leukocytes, neutrophils, and monocytes was performed using CD45, CD66b, and CD14 labeling, respectively. Microparticles were numerated in plasma from healthy donors (C: ), type 1 diabetic patients () and type 2 diabetic patients () as described in RESEARCH DESIGN AND METHODS. Results are expressed as number of microparticles per microliter of platelet-free plasma. The difference between groups was analyzed using a Mann-Whitney U test (* P < 0.05).

Although the proportion of patients with vascular complications was not sufficient to perform extensive correlations, type 1 diabetic patients suffering from one or more microvascular complications (retinopathy, nephropathy, or neuropathy) displayed higher EMP mean levels than those who did not (data not shown). It is important to note that the highest amounts of EMP were found in patients suffering from microvascular complications.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
The present study showed that both type 1 and type 2 diabetes are associated with increased levels of circulating microparticles. However, diabetic patients differ by the procoagulant activity and the cellular origin of microparticles. Indeed, only TMP numbers were increased in type 2 diabetes, whereas EMP, PMP, and TMP levels and procoagulant activity were elevated in type 1 diabetes. Interestingly, this procoagulant activity was correlated with levels of HbA_1c.

A wide vesiculation process, probably involving several cell types, is associated with diabetes, as shown by the elevated numbers of TMP in both type 1 and type 2 diabetes. The mechanisms of vesiculation are not completely elucidated. A large body of evidence obtained from in vitro studies and animal models has shown that this process can be triggered by activating factors such as the complement membrane attack complex (MAC) (20) or by cytokines like tumor necrosis factor (TNF) and interleukin-1ß (21). Vesiculation can also be induced in conditions like apoptosis or shear stress disturbance (18,21). All of these parameters are potentially implicated in the development of diabetic vascular complications and may thus account for a part of the vesiculation process (22–25). Previous studies have documented an increased number of PMP in diabetic patients, but none investigated vesiculation according to the type of diabetes (26–28). Our results showed that a significant elevation of PMP occurs in type 1 diabetes, whereas no significant increase was observed between type 2 diabetic patients and their age-matched control subjects. This observation differs from data reported by Nomura et al. (29) showing that PMP levels are increased in type 2 diabetic patients and therefore may be used as a marker of vascular disease. Differences in both patients and control population recruitment and/or in microparticle analysis may account for this discrepancy. Indeed, our population included patients with or without microvascular complications, whereas the study of Nomura et al. has focused on complicated forms of diabetes. Moreover, our detection of PMP was performed on platelet-free plasma in which PMP were labeled with anti-GPIIbIIIa mAb, whereas Nomura et al. used PMP extracted from a platelet suspension and labeled together with additional washed platelets using anti-GPIb mAb. In our study, we can rule out an effect of aspirin treatment on PMP levels since no significant difference in PMP number was noted between patients who were or were not undergoing this treatment. In addition, we have previously reported the lack of effect of aspirin on PMP levels in healthy donors and in patients undergoing coronary angioplasty (30).

Endothelial dysfunction is a common feature of diabetic patients who have early or advanced nephropathy. Recently, we and others have shown that the release of EMP reflects endothelial alterations in pathologies such as antiphospholipid syndrome (31), meningococcal sepsis (32), thrombotic thrombocytopenic purpura (33), and acute coronary syndromes (17). Moreover, we have demonstrated that cultured endothelial cells stimulated by TNF released microparticles that display the same procoagulant receptors and adhesion molecules as the cells of origin (31). A novel finding of the present study is that EMPs are found in higher amounts in type 1 diabetic patients, whereas the increase does not reach statistical significance in type 2 diabetic patients. The highest levels of EMP in type 1 diabetic patients with microvascular complications, together with the positive correlation between EMP levels and albuminuria, suggest that EMP could be a marker of the endothelial damage associated with type 1 diabetic nephropathy.

In type 2 diabetes, the TMP number was increased, whereas PMP and EMP numbers were not significantly modified. Enhanced vesiculation of leukocyte could account for the result, as shown by increased levels of CD45- and CD66-b- positive microparticles in these patients. Consistent with the involvement of neutrophil activation, several studies have reported upregulation of adhesion molecules on neutrophil from diabetic patients (34,35).

At present, no study has explored the procoagulant activity of the microparticles present in the circulation of diabetic patients. Numerous works reported that microparticles are rich in membrane receptors for coagulation factor Va and provide a catalytic surface for the assembly of the prothrombinase complex. Thus, microparticles produced by circulating blood cells could play a role in the dissemination of a procoagulant potential. Surprisingly, despite high numbers of TMP, type 2 diabetes was not associated with an elevation of microparticle procoagulant activity, whereas type 1 diabetes was characterized by a concomitant increase of numbers and procoagulant activity of TMP. The mechanism underlying the difference in this intrinsic procoagulant activity is actually unknown. In our functional assay, the amount of thrombin generated by microparticles strictly depends on phosphatidylserine exposure. Therefore, heterogeneity in aminophospholipid content could account for the differences observed in the procoagulant phenotype of microparticles released in the two diabetic populations. In addition, several studies have described changes of tissue factor pathway in diabetes and their involvement in the vascular complications (36). Accordingly, another procoagulant mechanism could result from tissue factor expression by microparticles. However, this hypothesis can be ruled out because no significant difference in tissue factor activity of the circulating microparticles has been observed between the two diabetic groups (data not shown).

Interestingly, a positive correlation was found between this total procoagulant activity and HbA_1c levels but not with fasting glycemia. The procoagulant activity of microparticles could be indicative of a chronic lack of balance of glycemia rather than an acute phenomenon and could enhance blood thrombogenicity in type 1 diabetic patients. This observation is sustained by the higher thrombotic risk of poorly controlled HbA_1c in diabetic patients (37).

In addition to coagulation receptors, microparticles carry adhesion molecules at their surface. They have the capacity to bind to circulating cells or endothelium and therefore to participate in a "long range" transmission of inflammatory information. Recently, we demonstrated that microparticles released by stimulated endothelial cells can activate tissue factor procoagulant pathway by adhering to monocytes (38). In the same way, leukocyte-derived microparticles interact with endothelial cells and induce tissue factor expression (39,40). Thus, we can speculate that microparticles released in both types of diabetes may adhere to circulating cells or endothelium, exacerbating cell activation and thus contributing to worsen vasculopathy and disease progress.

The different patterns of microparticle phenotype and procoagulant activity observed in both types of diabetes may have different causes and biological consequences. Among these consequences, the procoagulant activity carried by microparticles is potentially involved in type 1 diabetic vascular complications. Moreover, its relation with the impaired glucose balance suggests that it could be useful in monitoring disease equilibrium.

	ACKNOWLEDGMENTS

 
We thank Beckman Coulter Immunotech for providing antibodies and A. Boyer for secretarial support.

	FOOTNOTES

 
Address correspondence and reprint requests to Professor Françoise Dignat-George, INSERM EMI 0019, Laboratory of Immunology and Hematology, UFR de Pharmacie, Université de la Méditerranée, 27 Bd Baille, Marseille, France. E-mail: dignat{at}pharmacie.univ-mrs.fr.

Received for publication 31 October 2001 and accepted in revised form 4 June 2002.

EMP, endothelial microparticle; FITC, fluorescein isothyocyanate; mAb, monoclonal antibody; PE, phycoerythrine; PMP, platelet microparticle; TMP, total annexin V-positive blood cell microparticle; TNF, tumor necrosis factor.

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TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 

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