Journal of Thrombosis and Haemostasis, 7: 1566–1575
DOI: 10.1111/j.1538-7836.2009.03525.x
ORIGINAL ARTICLE
Chronic venous insufficiency is associated with elevated level of circulating microparticles A. GEORGESCU,* N. ALEXANDRU,* D. POPOV,* M. AMUZESCU, E. ANDREI,* C. ZAMFIR,à H . M A N I U * and A . B A D I L A § *Institute of Cellular Biology and Pathology, ÔNicolae SimionescuÕ; Medical Centrum ÔSTSÕ; àÔEliasÕ University Emergency Hospital; and §University Emergency Hospital, Bucharest, Romania
To cite this article: Georgescu A, Alexandru N, Popov D, Amuzescu M, Andrei E, Zamfir C, Maniu H, Badila A. Chronic venous insufficiency is associated with elevated level of circulating microparticles. J Thromb Haemost 2009; 7: 1566–75.
Summary. Background: Chronic venous insufficiency (CVI) results when the veins in the legs no longer pump blood back to the heart effectively. Microparticles (MPs) are small membrane vesicles released by several circulating and vascular cells upon activation or apoptosis. Objectives: The purpose of this study was to assess the subpopulations of circulating endothelial (EMPs) and platelet microparticles (PMPs) in CVI, and to disclose their contribution in mediating dysfunction of human peripheral venules. Patients and methods: Human peripheral venules were explanted during leg surgery on patients with CVI and on control subjects (C); concurrently, blood samples were collected and circulating MPs isolated. The techniques used were: flow cytometry, fluorescence and electron microscopy, myograph technique and western-blotting technique. Results: The results showed that compared with controls, patients with CVI had: (i) a marked elevation of circulating EMPs and PMPs; (ii) a structural modification of the venous wall consisting of activation of endothelial and smooth muscle cells, an abundance of intermediary filaments and synthesis of hyperplasic-multilayered basal lamina; (iii) a significantly altered reactivity of the venous wall, closely associated with EMPs and PMPs adherence; (iv) altered contractile response to noradrenaline, acetylcholine, 5-hydroxytryptamine and KCl, and an impeded relaxation in response to sodium nitroprusside; and (iv) a substantially increased protein expression of tissue factor (TF) and of P-Selectin both in the venular vascular wall and on the surface of EMPs and PMPs. Conclusions: The findings indicate that CVI is accompanied by an enhanced release of EMPs and PMPs that contribute to altered dysfunctional response of the venous wall. Correspondence: Adriana Georgescu, Institute of Cellular Biology and Pathology ÔNicolae SimionescuÕ, 8, BP Hasdeu Street, PO Box 35-14, 050568 Bucharest, Romania. Tel.: +401 319 4518; fax: +401 319 4519. E-mail:
[email protected] Received 26 July 2008, accepted 2 June 2009
Keywords: endothelial dysfunction, endothelial microparticles, platelets microparticles, venous wall reactivity.
Introduction Microparticles (MPs) are membrane vesicle fragments endowed with procoagulant and proinflamatory properties released in biological fluids from the plasma membrane of stimulated or apoptotic cells [1,2]. MPs are rather heterogeneous in size (0.05–1 lm), in protein and in lipid composition [1]. Endothelial microparticles (EMPs) are shed from the surface of endothelial cells, whereas platelet microparticles (PMPs) are the result of shedding of activated platelets. Despite being previously considered as inert non-functional debris, recent data demonstrate important pathophysiologic mechanisms orchestrated by MPs in vascular diseases associated with endothelial dysfunction. Thus, MPs can now be viewed not only as a hallmark of cell damage, but also as a fine biological tool [3]. Although the increased numbers of circulating MPs have been implicated in many cardiovascular diseases, their pathophysiological role has not been fully investigated yet. Thus, the participation of MPs in chronic venous insufficiency (CVI) is still obscure. There are just a few data regarding the involvement of MPs in CVI and regarding the response of the venous wall to thrombosis. Because elevation of EMPs, PMPs and leukocyte MPs has been reported in patients with venous thromboembolism [4] we hypothesize that the accumulation of MPs in the venous wall may contribute to the CVI. Chronic venous disease is a common problem that has a significant impact on afflicted individuals and on the healthcare system. Normal venous function requires the axial veins with a series of venous valves, the perforating veins to allow communication of the superficial with the deep venous system, and the venous muscle pumps. Dysfunction of any of the normal structures may lead to venous hypertension and the development of CVI [5]. Other contributors to the development of CVI are the proinflammatory properties of 2009 International Society on Thrombosis and Haemostasis
Microparticles in venous insufficiency 1567
the venous endothelium [6]. Understanding the CVI is important because relatively small changes in venous function may have a substantial effect on the entire cardiovascular system. In addition, understanding the modulation of MPsÕ generation and their role in the pathogenesis of thrombosis, inflammation and mediating vascular dysfunction may lead to novel therapeutic avenues. This study emphasizes the significance of circulating MPs of endothelial and platelet origin as both a marker and a trigger of endothelial dysfunction in chronic venous insufficiency. Materials and methods Reagents
Noradrenaline, KCl, 5-hydroxytryptamine, acetylcholine and sodium nitroprusside were purchased from Sigma Chemical Co. (St Louis, MO, USA). The specific antibodies to VEcadherin (C-19) PE (for CD144), Annexin V FITC (for phosphatidylserine, PS), Integrin aIIb (M 148) PE (for CD41), TF (FL-295) rabbit polyclonal IgG, P-Selectin (C-20) goat polyclonal IgGb and mouse monoclonal IgG were purchased from Santa Cruz Inc. (Santa Cruz, CA, USA), the mouse IgG1-PE was from Dako (Carpinteria, CA, USA), anti-goat IgG (whole molecule)-FITC, antibody produced in rabbit and goat anti-rabbit IgG coupled with peroxidase were from Sigma (USA), rabbit polyclonal to beta Actin was from Abcam (USA) and PKH26 from Zynaxis (Cell Science, Malvern, PA, USA). All other reagents used were of analytical grade.
University Hospital of Emergency, both from Bucharest, Romania. Preparation of platelet-free plasma, the source for circulating microparticles
Plasma EMPs and PMPs were separated according to the method reported by Boulanger et al. [7]. Briefly, the procedure consisted of collection of venous blood in 0.138 M tri–sodium citrate 9/1 (vol/vol), centrifugation at 1000 g for 15 min at 15 C and separation of platelet-rich plasma (PRP). The latter was further centrifuged at 2500 g for 15 min at 15 C and the platelet-free plasma (PFP) was obtained. Centrifugation of PFP at 13 000 g for 5 min at 15 C allowed collection of MPs in the supernatant. Sorting of EMPs and PMPs by flow cytometry
The experiments were conducted using the Flow cytometer MoFlo (Dako, USA) equipped with high-speed cell sorter. MP numeration
At 20 lL PFP, 20 lL Flowcount beads (diameter 10 lm) and 200 lL freshly filtered PBS buffer were added, mixed and counted for 60 s. The concentration of MPs was calculated using the formula: MP as events=lL MP counts * volume Flowcount beads * beads concentration ¼ Beads count * PFP volume
or, Study groups and samples
In collaboration with the surgery departments of ÔEliasÕ University Emergency Hospital, and of the University Emergency Hospital, both from Bucharest, Romania, and upon certified consent of the patients, we performed the study on 90 subjects. These subjects were divided into two experimental groups: healthy control subjects (C, n = 20) and patients with chronic venous insufficiency (CVI, n = 70). Human peripheral venules (superficial veins with internal diameter £ 350 lm) were explanted during leg surgery on patients with CVI (n = 70), and during routine orthopedic surgery on control subjects (n = 20). Patients in the CVI group (41 women and 29 men) were 50.21 ± 2.89 years old and had been for the last 22.44 ± 1.38 years under treatment with Detralex 500 (phlebotonic drug, micronized purified flavonoid fraction and vascular protecting agent increasing venous tone, produced by Servier Laboratories, Orle´ans, France). Patients in the C group (12 women and 8 men) were 44.77 ± 2.08 years old and were clinically healthy. The blood was collected by venipuncture from every subject considered in this study. The experiments were approved by the Ethics Committees of the ÔEliasÕ University Emergency Hospital and of the 2009 International Society on Thrombosis and Haemostasis
Concentration MP=lL ¼
NMP 1000 100%; NB
where NMP = MP counts, NB = Beads counts. MPs were characterized as EMPs using specific antibodies to VE-cadherin (C-19) PE (for CD144), and Annexin V FITC (for phosphatidylserine, PS). As negative control, mouse IgG1-PE (Dako, Glostrup, Denmark) was used. MPs were characterized as PMPs using specific antibodies to Integrin aIIb (M 148) PE (for CD41) and Annexin V FITC (for PS). The number of experiments was 20 for C subjects and 70 for CVI patients. Flow cytometry analysis was followed by cell sorting involving physical separation (purification) of EMPs and PMPs from the remaining MPs in the suspension stained with specific fluorescent dyes (as above). Sorting speed was around 10 000–12 000 MPs per second. Characterization of circulating microparticles by electron and fluorescence microscopy
MPs sorted by flow cytometry were examined by electron microscopy. Briefly, the protocol applied was: fixation of MP
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pellets in 2% glutaraldehyde; washing in 0.14 M Na cacodylate buffer; postfixation in 1% OsO4; washing and mordanting; dehydration in graded ethanol; exposure to propylene and to a mixture of propylene oxide and Epon 812, 1:1 (vol/vol); embedding in Epon 812; and finally examination with the electron microscope (FEI Tecnai F20 field emission gun TEM, Philips Electron Optics, Eindhoven, the Netherlands) [8]. To identify MPsÕ presence in samples sorted by flow cytometry, the particles were labeled with the lipophilic dye PKH26 (2 · 10)6 M) followed by examination by fluorescence microscopy (Nikon E800 Fluorescence Microscope, Nikon, El Segundo, CA, USA). In addition, fluorescently labeled MPs were employed to investigate the adherence of MPs to peripheral venule walls. Identification of proteins on the surface of EMPs and PMPs
EMPs (1 · 104 mL)1) and PMPs (1 · 104 mL)1), from control subjects and patients with CVI, sorted by flow cytometry in 1.5 mL PBS, were examined again by flow cytometry in order to identify and quantify the membrane receptors TF and P-Selectin on the surface of EMPs and PMPs . The structural investigation of the venous wall
The structure of the venular wall and of the venous valves was prepared for electron microscopy examination as previously reported by Georgescu et al., 2006 [9].
exposed to agonists, as above. The incubation time was chosen to conform with experiments on thoracic aortas obtained from male Sprague-Dawley rats, described by Sergey et al., 2003 [14]. For all the experiments (n = 20 for C subjects and n = 35 for CVI patients) the recordings of the force (F, in mN) developed by the venular wall were carried out for each concentration added, at 2-min time intervals. Western-blotting technique
The classical Western-blotting technique was applied for the mechanistic insights of EMPs and PMPs. Data analysis
For flow cytometry experiments a software based on auto and manual compensation was used (SUMMIT 4.0 b2060 Software, DakoCytomation, USA). The tension developed by the NA, ACh, 5-HT and K+ on venules was expressed as active wall tension (mN mm)1 venular length) and the relaxation induced by SNP was considered as percentage of NA-induced contraction. Half-maximal concentrations (EC50) were calculated from the dose–response curves. All values were expressed as mean ± SEM. One-way analysis of variance (ANOVA) was employed to quantify the results. The data were considered significant when P < 0.05. Results
The myograph technique
Several characteristics of patientsÕ plasma
One mm long segments of venules were dissected out, two stainless steel wires (u: 40 lm) were threaded through their lumen and then the preparation was mounted in a small vessel myograph (Model 410A; J.P. Trading, Aarhus, Denmark) as described by Mulvany and Halpern [10]. The myograph chamber was filled with HEPES, maintained at 37 C and continuously gassed with O2 [11,12]. Following an equilibration period of 20 min, the venules were set to a normalized internal circumference at which they gave the maximum of the isometric response. The mean internal diameter of the venules used in these experiments was in the range of 300 ± 50 lm.
Patients in the C group (44.77 ± 2.08 years old) and in the CVI group (50.21 ± 2.89 years) were clinically evaluated. The status of the coagulation and the standard parameters of
Functional investigation of the venous wall
The venous wall reactivity, namely the contractile response to noradrenaline (NA, 10)8 to 10)4 M), the relaxation response to acetylcholine (ACh, 10)8 to 10)4 M), 5-hydroxytryptamine (5HT, 10)8 to 10)4 M) and KCl (K+, 24.4 mM, 42.46 mM, 64.1 mM, 83.93 mM, 123.7 mM) and the endothelium independent relaxation to sodium nitroprusside (SNP, 10)8 to 10)4 M), were investigated using the wire myograph technique [9,13]. In other experiments, the venules were incubated with isolated MPs (2 · 105 mL)1 EMP or PMP) at 37 C for 3 h in a 5% CO2 atmosphere in a cell culture incubator. Then venules were washed three times with HEPES buffer, pH 7.45, and
Table 1 Clinical characteristics of the control subjects and of the patients with chronic venous insufficiency (CVI) Clinical characteristics Prothrombin time (s) *P = 0,41 Activated partial thromboplastin time (%) *P = 0.03 Number of platelets/mm3 *P = 0.04 Medium platelet volume (fL) *P = 0,37 Fibrinogen concentration (mg dL)1) *P = 0.02 Sedimentation speed of red blood cells (mm) *P = 0.03
Control group (n = 20)
CVI group (n = 70)
11.6 ± 1.69
12.44 ± 0.27
79.21 ± 8.39
100.1 ± 2.91
210 520 ± 39.33
253 620 ± 50.84
8.72 ± 0.41
9.29 ± 0.41
296.83 ± 18.70
354.77 ± 18.88
8.77 ± 2.41
19.64 ± 4.17
Data are means ± SD. The control group was 12 women and 8 men and the CVI group was 41 women and 29 men. 2009 International Society on Thrombosis and Haemostasis
Microparticles in venous insufficiency 1569
the patients with and without CVI are presented in Table 1. The prothrombin time and the medium platelet volume were not significantly different between the CVI and control groups (P > 0.05), whereas the partial activated thromboplastin time, the number of platelets, the fibrinogen concentration and the sedimentation speed of red blood cells were significantly different (P < 0.05).
The comparative examination of MPsÕ fine structure by electron microscopy showed that both EMPs and PMPs have larger dimensions in samples from controls [Fig. 2B(a,c)] and smaller dimensions in CVI patients [Fig. 2B(b,d)]. However, in both situations the MPs displayed a double-layered membrane surrounding an electrondense content.
Plasma level of EMPs and PMPs in control subjects and in CVI patients
Interaction of MPs with the vascular wall of human peripheral venules
The isolation and the specific quantification of EMPs and PMPs were performed by flow cytometry analysis, employing for double staining the specific antibodies. EMPs and PMPs in plasma were obtained in the blood collected from the control subjects and from patients with CVI, applying standard procedures (by centrifugation) to obtain the platelet-free plasma (PFP). Representative recordings in PFP are shown in Fig. 1. Quantification of EMP levels in CVI patients vs. controls showed that in patients the concentration of EMPs positive for both CD144 and PS [as revealed by VE-cadherin (C-19) PE and AnnexinV FITC binding, respectively] was 93 639 ± 2520 mL)1 [Fig. 1B(d)], while in the control group it was 10 783 ± 2112 mL)1 (P < 0.05) [Fig. 1A(d)]. No positive level of EMPs, for CD144 only, was found in PFP collected from the two groups [Fig. 1A(d),B(d)]. The plasma level of PMPs positive for both CD 41 and PS [as revealed by Integrin aIIb (M-148) and AnnexinV FITC binding, respectively] was 246 246 ± 2946 mL)1 for CVI patients [Fig. 1B(c)], while in the control group the concentration was 15 745 ± 2115 mL)1 (P < 0.05) [Fig. 1A(c)]. In addition, the concentration of PMPs positive for CD41 was 6961 ± 985 mL)1 in control subjects and 13 711 ± 1879 mL)1 in patients (P < 0.05) [Fig. 1A(c),B(c)]. MPs numeration using Flowcount beads [F = 10 lm, (103 mL)1)] [Fig. 1A(a),B(a)] and control experiments with negative control mouse IgG1-PE [Fig. 1A(b),B(b)] were taken into consideration to quantify EMP and PMP levels. Together, these results showed that plasma levels of EMPs (positive for both CD144 and PS), and of PMPs (positive for both CD41 and PS consi), were significantly augmented (8.68and 15.63-fold, respectively) in patients with CVI compared with control subjects (P < 0.05); moreover, the PMPs population was significantly increased compared with EMPs, namely by 2.62-fold for CVI patients and 1.46-fold for control subjects (P < 0.05).
Exposure of the lumen of the venules originating from CVI or control groups to either EMPs or PMPs indicates their adhesion to the endothelial surface, in a process that still persists after three washings for 1 min each. Representative examples of the adherence of C or CVI PMPs (2 · 105 mL)1) to peripheral venules of C subjects and of CVI patients are given in Fig. 3. Five experiments were performed by fluorescence microscopy. The quantification of PMP or EMP (2 · 105 mL)1) degree of adhesion to human peripheral venules showed that, compared with the adherence of PMPs from C subjects to the homologus venules (considered as 100%), the degree of adhesion of CVI PMPs to peripheral venules of C subjects increased by 8.75 ± 0.54% and that of CVI PMPs to homologus patients by 70.87 ± 1.28% (P < 0.05).
Analysis of EMPs and PMPs in flow-cytometry-sorted samples
To verify their presence in flow-cytometry sorted samples, MPs collected from control subjects were labelled with the lipophilic dye PKH26, and examined by fluorescence microscopy (Fig. 2A). Circulating EMPs [Fig. 2A(a)] and PMPs [Fig. 2A(b)], stained in red and very heterogeneous in size, were easily observed. 2009 International Society on Thrombosis and Haemostasis
Structural and functional changes of the venous wall and valves in CVI
Electron microscopic examination of the venular wall and of the venous valves in CVI patients showed activation of the endothelial cells (containing numerous Weibel-Palade bodies, plasmalemmal vesicles and biosynthetic organelles) and of the smooth muscle cells (endowed with numerous sarcolemmal vesicles), a hyperplasic-multilayered basal lamina, and an unusually thick extracellular matrix. We also observed smooth muscle cell proliferation, foam cell formation in the microenvironment of the vascular intima and abundance of collagen fibers (figure is not shown). Isometric force measurements using the wire myograph technique revealed that, in control subjects, the maximal contractile force developed by venules was 2.28 ± 0.3 mN mm)1 for 10)4 M NA, 0.97 ± 0.09 mN mm)1 for 10)4 M ACh, 6.76 ± 0.5 mN mm)1 for 10)4 M 5-HT and 5.28 ± 0.3 mN mm)1 for 42.46 mM K+ (Fig. 4A). These values were significantly increased (P < 0.05) compared with those obtained for patients with CVI, where the values were 0.14 ± 0.006 mN mm)1 for 10)4 M NA, 0.11 ± 0.006 mN mm)1 for 10)4 M ACh, 0.12 ± 0.004 mN mm)1 for 10)4 M 5-HT and 0.16 ± 0.004 mN mm)1 for 42.46 mM K+ (Fig. 4C). Also, the venules collected from patients with CVI relaxed in 10)4 M SNP to 53.43 ± 4.9% of NA-induced contraction and those from control subjects to 43.69 ± 4.5%. The values were not significantly different (data not shown).
1570 A Georgescu et al A (a)
(b)
(c)
(d)
B (a)
(b)
(c)
(d)
Fig. 1. (A) Specific identification and quantification of FMPs and PMPs in control subjects. (B) Specific identification and quantification of EMPs and PMPs in CVI patients. (a) MPs numeration and plasma concentration using Flowcount beads; (b) negative controls experiments; (c) PMPs quantification; (d) EMPs quantification.
The effect of the MPs on the vascular reactivity of human peripheral venules
To investigate the contribution of the MPs in mediating vascular dysfunction, the venous vascular wall reactivity for the two studied groups after incubation with a concentration of
2 · 105 mL)1, for either EMPs or PMPs, was recorded by the myograph technique. When venules collected from healthy subjects were exposed to homologous EMPs and PMPs, the responses to vasoconstrictor (10)4 M NA, 10)4 M ACh, 10)4 M 5-HT, 42.46 mM K+) (Fig. 4A) and to vasodilator (10)4 M SNP) (Figure is not 2009 International Society on Thrombosis and Haemostasis
Microparticles in venous insufficiency 1571 A (a)
(b)
3.75 µm
B (a)
3.75 µm
(b)
0.27 µm
0.27 µm (d)
(c)
0.27 µm
0.27 µm
Fig. 2. (A) Fluorescence minuscopy of MPs collected from control subjects. Analysis of EMPs (a) and of PMPs (b). (B) Electron microscopy of MPs. Fine structure of EMPs collected from C subjects (a) and from patients with CVI (b), and of PMPs collected from C subjects (c) and from patients with CVI (d).
shown) agents were unchanged. The similar values for EC50 were: 10)7 M for NA, 3 · 10)7 M for ACh, 3 · 10)6 M for 5-HT and 24.4 mM for K+ (dose–response curves are not shown). When control venules were exposed to EMPs and PMPs sorted from CVI patients, significantly diminished contractile responses to NA ( 1.66 fold for EMPs, and 2.27 fold for PMPs), ACh ( 1.66 fold for EMPs, and 2 fold for PMPs), 5HT ( 1.36 fold for EMPs, and 1.53 fold for PMPs), and K+ ( 1.42fold for EMPs, and 1.66fold for PMPs)were recorded (Fig. 4B) (P < 0.05). In this case, the values for EC50 were modified in the presence of EMPs and PMPs and were around 10)6 M for NA, 3 · 10)6 M for ACh, 3 · 10)5 M for 5-HT and 34.52 mM for K+ (dose–response curves are not shown). When exposed to EMPs and PMPs sorted from CVI patients, the homologous venules showed deeply reduced responses to 10)4 M NA ( 7 fold for EMPs, and 2009 International Society on Thrombosis and Haemostasis
14 fold for PMPs), 10)4 M ACh ( 11 fold for EMPs, and 11 fold for PMPs), 10)4 M 5HT ( 12 fold for EMPs, and 12 fold for PMPs), and 42.46 mM K+ ( 15 fold for EMPs, and 15 fold for PMPs) (Fig. 4C) (P < 0.05). In addition, the incubation of control MPs (EMPs or PMPs) with CVI venules did not induced significant changes in the venular wall responses to studied agonists. These values were around 0.13 ± 0.007 mN mm)1 for 10)4 M NA, 0.10 ± 0.004 mN mm)1 for 10)4 M ACh, 0.11 ± 0.005 mN mm)1 for 10)4 M 5-HT and 0.15 ± 0.008 mN mm)1 for 42.46 mM K+ (Fig. 4D). Dose–response curves (data not shown) for venules from CVI patients presented the following values for EC50: 10)5 M for NA, 3 · 10)5 M for ACh, 3 · 10)4 M for 5HT and 40.29 mM for K+. EMPs and PMPs did not induce significant changes in EC50 values. Incubation with EMPs and PMPs sorted from CVI patients did not affect endothelium-independent relaxation
1572 A Georgescu et al A
3.75 µm B
3.75 µm C
3.75 µm Fig. 3. Representative examples of the adherence of PMPs to the vascular wall of human peripheral venules. (A) Peripheral venules of C subjects intersected with homologus PMPs; (B) peripheral venules of C subjects intersected with CVI PMPs; (C) CVI venules intersected with homologus PMPs.
in response to SNP of the control venules and of the venules collected from CVI patients (data not shown). Mechanistic insights of MPs
Using the Western blot technique, we compared the TF and P-Selectin expression (Fig. 5) from vascular walls of control subjects with that of venules explanted from CVI patients. The densitometry of TF and P-Selectin protein (in eight separate experiments each) gave mean values of 123.29 ± 0.32 and 125.35 ± 0.29 (considered in controls as 100%); thus, CVI resulted in a 23% increase in TF (Fig. 5A) and a 25% increase in P-Selectin (Fig. 5B). The Western blotting experiments (n = 8) for b actin showed similar results in both control and CVI venules (Fig. 5A,B). Also, no bands were detected in lysates from C and CVI venules with non-immune IgG antibody (Fig. 5A). By flow cytometry analysis, in eight separate experiments, we identified and quantified the membrane receptors (TF and P-Selectin) on the surface of sorted EMPs (1 · 104 mL)1) and PMPs (1 · 104 mL)1), from control subjects compared with patients with CVI (Table 2).
In CVI patients, EMPs and PMPs were positive at TF, and the percentage was 64.59 ± 3.12 (%) and 52.26 ± 2.92 (%), respectively, while in the control group it was 23.18 ± 2.11 (%) and 35.91 ± 2.22 (%), respectively (P < 0.05) (Table 2). Also, it was found that EMPs and PMPs were positive at P-Selectin in CVI patients and the percentage was 68.62 ± 2.94 (%) and 82.56 ± 2.81 (%), respectively, while in the control group this percentage was 39.77 ± 2.31 (%) and 20.72 ± 2.12 (%), respectively (P < 0.05) (Table 2). These results showed that the number of the membrane receptors, TF and P-Selectin, on the surface of EMPs and PMPs, was significantly augmented in patients with CVI compared with control subjects (P < 0.05). Discussion Circulating MPs constitute a reservoir of bioactive vascular effectors involved in thrombotic responses, vascular wall inflammation and remodeling, enabling the assessment of the individual atherothrombotic risk [2]. 2009 International Society on Thrombosis and Haemostasis
Microparticles in venous insufficiency 1573 A
B
C
D
Fig. 4. The effect of EMPs and PMPs on the contractile function of human peripheral venules. (A) The venules collected from C subjects were exposed to EMPs and PMPs sorted from C subjects; (B) the control venules were exposed to EMPs and PMPs sorted from CVI patients; (C) the venules collected fromCVI patients were exposed to EMPs and PMPs sorted from CVI patients; (D) CVI venules were incubated with control EMPs and PMPs.
A 200 kDa 116 kDa 97 kDa 66 kDa 55 kDa
Tissue factor (TF-47 kDa)
45 kDa 36 kDa 29 kDa
Beta actin (42 kDa)
24 kDa 20 kDa
C CVI Non-immune IgG
B
C CVI
C CVI
P-Selectin (140 kDa)
Beta actin (42 kDa) C
CVI
Fig. 5. Representative Western blot of the venules explanted from CVI patients compared with control subjects. The expression of tissue factor (TF) (A) and of P-Selectin. (B) Beta Actin and non-immune IgG on the same transfers were used as internal controls.
The present study demonstrates that patients with CVI have elevated levels of circulating EMPs and PMPs and pronounced structural alterations of the venous wall and of their valves. In
addition, it shows that, in vitro, circulating EMPs and PMPs cause alterations of venule wall reactivity, especially ones collected from CVI patients. Standard flow cytometry technology was used to quantify and partially characterize circulating MPs in control subjects and in patients with CVI. The increase of plasma levels, of EMPs (positive for both CD144 and PS) and of PMPs (positive for both CD41 and PS) in CVI patients, could be correlated with evidence of chronic systemic inflammation (based on increase in sedimentation speed of red blood cells, fibrinogen concentration, and number of platelets). The quantitative augmentation of PMPs compared with EMPs is not surprising because the subjects with CVI present an increase in platelet count. They also have an increase in aPTT (activated partial thromboplastin time), raising the possibility of underlying low grade DIC (disseminated intravascular coagulation). Compared with other previous studies, the number of EMPs and PMPs in control subjects is higher, maybe because most of our patients in the C group were women (12 women and 8 men) and were 44.77 ± 2.08 years old. Even though control subjects were clinically healthy there is the possibility for gender and age to induce increases in circulating MP levels.
Table 2 Pro-thrombotic activity of EMP and PMP
Patient group Control group P values
Total EMP data
Total PMP data
% TF + EMP
% P-Selectin + EMP
% TF + PMP
% P-Selectin + PMP
1 · 104 mL)1 1 · 104 mL)1
1 · 104 mL)1 1 · 104 mL)1
64.59 ± 3.12 23.18 ± 2.11 P = 0.02
68.62 ± 2.94 39.77 ± 2.31 P = 0.03
52.26 ± 2.92 35.91 ± 2.22 P = 0.04
82.56 ± 2.81 20.72 ± 2.12 P = 0.01
Identification of membrane receptors, TF and P-Selectin, on the surface of EMPs and PMPs. 2009 International Society on Thrombosis and Haemostasis
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Applying the electron and fluorescence microscopy techniques, we demonstrate here that both EMPs and PMPs display a double-layered membrane that expresses specific proteins on the external leaflet, such as CD144 (EMPs), CD41 (PMPs) and phosphatidyl serine residues. In addition, some electron-opaque material is enclosed by the MP membrane; this may originate from the cytoplasm of either endothelium or platelets and its chemical identification is now in progress. It is well established that changes in extracellular matrix proteins, such as collagen and elastin, may play a relevant role in the development of cardiovascular damage in hypertension [15]. Here we show that the venous wall and valves of patients with CVI display pronounced structural alterations such as activation of endothelial and smooth muscle cells, increased synthesis leading to a hyperplasic-multilayered basal lamina and abundance of intermediary filaments. All these may play an important role in explaining venular wall dysfunction in CVI. The morphological changes in the venous wall and valves could be correlated with the concomitant changes in vessels reactivity. Thus, the results demonstrated that the contractile force of the venous wall was significantly diminished for patients with CVI, compared with control subjects. No significant change was observed in endothelium-independent relaxation. Others have shown that the changes in structure and function of the vascular wall may have a relevant pathophysiological significance for the development of CVI [5]. The present study shows that EMPs and PMPs from patients with CVI reduced significantly the contractile function of the venous wall in both control subjects and CVI patients. Although the cellular mechanism of MP effects remains to be investigated, our results demonstrate that an effect of MPs on inductible NO in smooth muscle cells can be ruled out, because the SNP-induced relaxation was not modified by the presence of MPs. Moreover, our data reveal that protein expression of tissue factor (TF) and of P-Selectin are increased both in venular vascular wall and on the surface of EMPs and PMPs from CVI patients. Thus, shed membrane microparticles (EMPs and PMPs) existing in peripheral blood of patients with CVI may impair endothelial function, suggesting that increased microparticle levels (which can up-regulate TF and PSelectin) in plasma during inflammation predispose to venous thromboembolism and to CVI. Also, the results show that the effect of PMPs on inducing endothelial vascular dysfunction was more pronounced compared with the effect of EMPs. The reason for such difference could be found in our data, which revealed the increase in membrane receptor number, TF and P-Selectin, on the surface of PMPs in patients with CVI compared with control subjects, and also compared with EMP isolated from the same groups of studied patients. The origin and characteristics of TF and P-Selectin are targets of intense research as well as of an intense debate. Surprising observations now implicate the adhesion receptor P-Selectin, known for its role in inflammation, in MPsÕ generation. P-
Selectin, translocated from granules to the cell surfaces of activated platelets and endothelial cells, was recently found to play multiple roles in hemostasis [16]. As mentioned by other authors, an increased level of platelet-derived TF-positive MPs may relate either to TF originally synthesized by other cells and then transferred to platelets or to a higher proportion of TFpositive platelets that release MPs into the blood [17]. On the other hand, platelets themselves have been demonstrated to store small amounts of TF in a-granules and to release TF-positive MPs, which can increase the blood TF activity [18]. There are other studies that showed that investigation into properties of MPs revealed a significant impact of these structures on vascular tone. Thus, EMPs can modulate NOinduced endothelial relaxation in rat models [19], while MP derived from T lymphocytes can promote endothelial dysfunction through effects on NO- and prostacyclin-dependent vasodilation [20]. All of these above-mentioned papers and also others, such as the article by VanWijk et al. (2002) [21], investigated the effect of MPs on arterial relaxation. In contrast, we were interested in studying venous function in CVI and in the contribution of MPs in mediating dysfunction of human peripheral venules. Thus, while it was demonstrated that MPs isolated from pre-eclampsia patients abolished endothelial-dependent vascular relaxation [21], we showed that EMPs or PMPs from CVI patients reduced the venous contractile responses induced by various agonists. Most current research focuses on these processes in arteries, leaving veins on the other side of vascular biology, in obscurity. Veins are different structurally and functionally from arteries. Equipped with a smaller smooth muscle layer compared with arteries, but being able to accommodate 70% of the circulating blood volume, veins can modulate cardiovascular homeostasis and contribute significantly to hypertension pathogenesis. Endothelium function is also different in veins compared with arteries. Venous endothelium produces less prostacyclin and NO than arterial endothelium and its overall response to atherogenic stimuli is different. Although the question of whether circulating levels of endothelial MPs cause or result from endothelial dysfunction is still much debated, in vitro evidence indicates that augmented plasma levels of EMPs, as observed in patients with end-stage renal diseases, could result from endothelial injury or apoptosis [22]. There are data that provide a rationale to explain the paracrine role of MPs as vectors of bioactive effectors promoting vascular dysfunction through transcellular exchanges during inflammatory disease [23]. All together, our results underline that the levels of circulating EMPs and PMPs are elevated in patients with CVI and are positively correlated with the changes in structure and function of the peripheral venous wall. The release of EMPs and PMPs, and their involvement in mediating vascular dysfunction, may be the result of venous insufficiency processes. Therefore, pharmacological control of MP release can be considered as the next promising challenge in restoration of vascular homeostasis. 2009 International Society on Thrombosis and Haemostasis
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Acknowledgements The authors gratefully acknowledge the advice, criticisms and permanent enthusiastic support of our mentor M. Simionescu. A. Georgescu gratefully acknowledges the hospitality of A. Tedgui and the kind help of C. Boulanger from INSERM U541, Paris, France, in training her in flow cytometry techniques in their laboratory. The authors appreciate also the dedicated work of M. Nemecz (Western blotting technique), M. Isachi and M. Toader (biochemistry), M. Misici (electron microscopy), and I. Alecsandra and A. Pascu (for explanation of human peripheral venules). This study was supported by the Excellence Research Projects for Young Researchers (Grant no. 15121/2006– 2008) financed by the Ministry of Education and Research, Romania. Disclosure of conflict of interests The authors state that they have no conflict of interest. Supporting Information Additional Supporting Information may be found in the online version of this article: Supplementary data. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. References 1 Freyssinet J-M. Cellular microparticles: what are they bad or good for? J Thromb Haemost 2003; 1: 1655–62. 2 Morel O, Toti F, Hugel B, Bakouboula B, Camoin-Jau L, DignatGeorge F, Freyssinet J-M. Procoagulant microparticles disrupting the vascular homeostasis equation? Arterioscler. Thromb Vasc Biol 2006; 26: 2594–604. 3 Morel O, Morel N, Hugel B, Jesel L, Vinzio S, Goichot B, Bakouboula B, Grunebaum L, Freyssinet J-M, Toti F. The semnificance of circulating microparticles in physiology, inflamatory and thrombotic deseases. Rev Med Interne 2005; 26: 791–801. 4 Chirinos JA, Heresi GA, Velasquez H, Jy W, Jimenez JJ, Ahn E, Horstman LL, Soriano AO, Zambrano JP, Ahn YS. Elevation of endothelial microparticles, platelets, and leukocyte in patients with venous thromboembolism. J Am Coll Cardiol 2005; 45: 1467–71. 5 Eberhardt RT, Raffetto JD. Chronic venous insufficiency. Circulation 2005; 111: 2398–409. 6 Eriksson EE, Karlof E, Ludmark K, Rotzius P, Hedin U, Xie X. Powerful inflammatory properties of large vein endothelium in vivo. Arterioscler Thromb Vasc Biol 2005; 25: 723–8.
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