Медицина / Клиническая медицина

PhD Inzhutova Alyona Ivanovna, PhD Lopatina Olga Leonidovna

Krasnoyarsk State Medical University named after Prof. Vojno-Yasenetsky, Russia

Easy used biomarkers for detection of endothelial dysfunction

Endothelial dysfunction is one of the most important factors determining the severity of condition in patients with cardiovascular or cerebrovascular pathology. Under arterial hypertension endothelial cells have surface shear stress by flow blood pressure and the tension of vascular wall. The shear stress activates endothelial cells and increases expression of intercellular adhesion molecules. It attracts the white blood cells and activates them. Interaction between lymphocytes and endothelial cells dramatically changes endothelial function and could result in apoptosis of endothelial cells [4]. As well cell activation as apoptosis produces phosphatidylserine externalization and actin-myosin premembrane dissociation. Membrane-derived microparticles are formed by changing of cytoplasm membrane and cytoskeleton interaction. Microparticles consist from cell membrane and carry antigens of parental cells. Cytoplasmic space of membrane microparticles has not nucleus but includes some cell proteins. Phosphatidylserine has high antigenic and procoagulation potential. Therefore, membrane-derived microparticles are not only a marker of cardiovascular pathology. Their formation can lead to endothelial dysfunction and thromboses formation. Membrane blebbing proceeds to the shedding of membrane microparticles [3]. As cell activation as apoptosis leads to plasma cell membrane blebbing. It looks like cell membrane protrusion which can result to membrane-derived microparticles, apoptotic microparticles or spontaneously disappears. Membrane blebbing facilitates the contact between cells, for example lymphocytes and endothelial cells [1]. Membrane microparticles potentiate and increase the effect of lymphocytes on endothelial cells. Moreover membrane microparticles play a role in atherosclerotic plaque formation [3]. For clinical diagnosis, we need to get the fastest result and it is impossible to use experimental methods [2].

The purpose of this study was to evaluate the reliability of phase-contrast microscopy in detecting the membrane-derived microparticles in the blood and cell plasma membrane blebbing for diagnosis a severity of condition in the patients with cardiovascular diseases.

Methods. We examined 200 patients with arterial hypertension; 150 patients with arterial hypertension and stable angina, 80 patients with arterial hypertension and progressive angina, 60 patients with arterial hypertension and stroke. Membrane microparticles were isolated from blood with method which previously was described by Boulanger et al. (2005) [5]. We assessed the number of lymphocytes with membrane blebbing by Lympholight (Cedalane, Hornby, Canada) gradient after centrifugation at 3000 rpm, 15 min. LUMAM-8 microscope (St. Petersburg, Russia) was used for phase-contrast and luminescent microscopy, magnification 900 times. sPECAM-1 levels were measured in the peripheral blood plasma with RIA using sPECAM-1 kit (Bender Med Systems GmbH, Vienna, Austria). Statistical analysis was performed with Kolmogorov-Smirnov test in Liliefors modification. Continuous variables were compared by unpaired Student’s t-test or Mann-Whitney U statistic test. Categorical variables were compared by the chi-square analysis. For all analyses, a P-value < .05 was defined as statistically significant.

Results. Patients with arterial hypertension and progressive angina or stroke have higher content of membrane-derived microparticles, percentage of lymphocytes with membrane blebbing, and levels of sPECAM-1 on 1^st day of investigation in comparison with other groups of the study (Table 1).

	Arterial hypertension	Arterial hypertension & stabile angina	Arterial hypertension & progressive angina	Arterial hypertension & stroke	Control group
MP	1280.6±5.3 **	1400.7±6.2**	2670.3±3.7**	5800.1±5.8***	200.0±1.4
MP CD62E	671.8±7.2*	531.0±2.4*	1342.1±1.8**	3899.2±3.8**	100.6±1.9
MP PS+	438.3±3.9*	442.9±4.1*	1884.7±1.8***	5698.9±6.2***	50.8±2.7
Blebbing	48.9±2.3%	49.2±2.3%	68.7±1.2%	94.9±1.9%	2.5±2.0%
sPECAM-1	6.6±1.9*	7.8±2.1*	9.7±1.9*	12.5±2.8***	0.23±0.03

Table 1: Baseline peripheral blood characteristics in patients with cardiovascular pathology. Data are presented as Mean ± SE. Amount of microparticles is presented as units in 1 µl, lymphocyte with membrane blebbing are presented as relative number (%), sPECAM-1 levels are presented as ng per ml. MP: membrane microparticles, PS: phosphatidylserine, Blebbing: protrusion of lymphocyte membrane. P-value is presented as *p< .05, **p< .01, ***p< .001 in comparison with control group.

We found strong correlation between the blebbing of lymphocytes and amount of microparticles in the blood (r = .76, p= .001).

Discussion. The using of phase-contrast microscopy is informative and reliable method for assessing the intensity of peripheral blood leukocytes plasma membrane blebbing and membrane-derived microparticels in the patients with cardiovascular pathology. Strong correlation between the levels of blebbing lymphocytes and the number of membrane-derived microparticels in the peripheral blood suggests the possibility to use such a simple method for express diagnostics in patients with cardiovascular pathology.

1. A.I. Inzhutova, AB Salmina, MM Petrova, AA. Larionov. “Role of CD38 in cell-cell interactions under conditions of endothelial dysfunction”. Bull Exp Biol Med. 145(6), pp 703-6, 2008

2. J.J. Jimenez, W Jy, LM Mauro, LL Horstman et al. Endothelial microparticles (EMP) as vascular disease markers. Adv Clin Chem, 39, pp 131–157, 2005

3. P-E. Rautou, AS. Leroyer, B Ramkhelawon et al. “Microparticles from human atherosclerotic plaques promote endothelial ICAM-1dependent monocyte adhesion and transendothelial migration”. Circ Res., 108, pp e2-e10, 2011.

4. S. Verma, T.J.Anderson. “Fundamentals of endothelial function for the clinical cardiologist”. Circulation. 105, pp 46-49, 2002;

5. Yu. Yuana, R.M. Bertina, S. Osanto “Pre-analytical and analytical issues in the analysis of blood microparticles” Thrombosis and Haemostasis, vol. 105, no 3, pp 1-13, 2011