Content uploaded by Zufar A Gabbasov
Author content
All content in this area was uploaded by Zufar A Gabbasov on Oct 06, 2017
Content may be subject to copyright.
1
Can. J. Physiol. Pharmacol. 2007; 85(3-4), 295-300
Circulating stromal osteonectin-positive progenitor cells and stenotic coronary atherosclerosis
Zufar A Gabbasov, PhD, Alexander A Agapov, MD, Olga S Saburova, PhD,
Alexei E Komlev, Emma L Soboleva, PhD, Renat S Akchurin, MD
and Vladimir N Smirnov, PhD
Cardiology Research Center, Moscow, Russia
Address for correspondence:
Zufar A Gabbasov, Laboratory of Stem Cells, Institute of Experimental Cardiology, Cardiology Research Center,
3
rd
Cherepkovskaya st., 15A, 121 552, Moscow, Russia.
Telephone/fax +7 (495) 414 69 23, e-mail
gabbasov@cardio.ru
KEY WORDS. Atherosclerosis, coronary artery disease, osteonectin, circulating stromal progenitor cell.
2
ABSTRACT
The level of circulating stromal progenitor cell carrying osteonectin (ON), a marker of osteogenic differentiation, was evaluated by flow cytometry
in blood of patients with coronary artery disease (CAD).
Ninety nine patients with CAD were included into the study. Coronary angiography of all patients showed critical stenosis of at least two coronary
arteries or their major branches. The control groups included 8 patients without CAD and 19 healthy volunteers. In control patients no lesions of
coronary bed were found by angiography. The absence of CAD in the volunteers was confirmed by bicycle stress test.
The content of ON-positive cells in blood was examined in various populations of lymphocyte-like cells. It was found that the number of ON+
lymphocyte-like cells with CD41 positivity in blood of patients without coronary stenosis (0.27% ± 0.11, mean±SD) did not differ significantly from
corresponding value in healthy volunteers (0.26% ± 0.07, p=0.94). In CAD patients the percentage of these ON+ cells was 1.01% ± 0.49 and was
significantly higher than in blood of healthy volunteers (p<0.0001) and patients without CAD (p<0.0001). High content of ON+ lymphocyte-like cells
with CD41 positivity in blood may serve as non-invasive marker of arterial atherosclerosis.
3
INTRODUCTION
Recently, significant progress has been made in understanding the role of bone marrow stem/progenitor cells in atherogenesis (Caplice et al.
2003, Sata 2003, Soboleva et al. 2005). Earlier, using colony-forming tests, the presence of bone marrow colony-forming units (CFU’s) of hemopoietic
and stromal lineages in atheromatous human aorta were demonstrated, in addition to terminally differentiated cells (Soboleva and Popkova 1989,
Soboleva et al. 1994a). Using a clonal technique stromal CFU’s were also found in blood of patients with primary hyperlipidemia and coronary artery
disease (CAD) (Soboleva et al. 1994b). In stromal colonies cells were shown to synthesize fibrillar collagen and osteoid matrices. In the colonies
where bone matrix was formed cells expressed osteonectin (Romanov et al. 1995) which is known to be a marker of osteoid differentiation. Taken
together, these data suggested that 1) proliferating intimal cells are likely to originate from bone marrow and 2) an important factor in the progression
of atherosclerosis is transport of circulating hemopoietic and stromal colony-forming progenitor cells into vascular zones loaded with lipids. The
appearance in the circulation of progenitor cells with a certain stromal phenotype and variation in their numbers might serve as an informative indicator
of the presence of atherosclerosis in coronary patients. In this investigation the proportion of circulating stromal progenitor cells carrying the marker of
osteogenic differentiation, osteonectin (ON), was evaluated by flow cytometry in patients with documented stenosis of coronary arteries.
4
MATERIALS AND METHODS
Patients
Ninety nine patients with CAD were included into the study. Clinical examination of all the patients was as follows: ECG at rest and during
physical exercise, 24-hours ECG monitoring, echocardiography, coronary angiography. In some patients myocardial scintiography was done at rest and
after a standard bicycle exercise test. The history of the disease (case report), the results of the bicycle stress test and 24-hours ECG monitoring showed
that CAD of various severity was present in all cases. Coronary angiography of these patients showed stenosis of coronary arteries or their branches.
Exclusion criteria were chronic hepatic or renal disease, endocrine pathology, renal hypertension. Patients with gastrointestinal tract and respiratory
system damage who required constant drug therapy were also excluded from the study.
The control groups included 8 patients with suspected CAD and 19 healthy volunteers 21-52 years old. The absence of CAD in these 8 patients
was documented by coronary angiography and a radionuclide study; chest pain was due to vertebral pathology and esophagitis or gastralgia. The
absence of myocardial ischemia in 19 healthy volunteers was confirmed by a bicycle stress test and 24-hours ECG monitoring. Stenotic atherosclerosis
of aorta and/or its branches in the control groups was excluded by ultrasound examination.
All the patients received standard therapy (beta-blockers, aspirin, nitrates). Patients with concomitant hypertension occasionally took diuretics,
Ca-blockers, ACF-inhibitors. No other medication was prescribed.
Ethical Committee approval of this project and informed consent from each patient and volunteer was duly provided.
5
Fluorescence activated cell sorter (FACS) analysis
Blood was taken from the cubital vein of patients or healthy donors after 14 hours of fasting, and anticoagulated with EDTA. Staining of
various antigens on blood cells was carried out within 2 hours after blood collection using fluorescein-isothiocyanate-labelled polyclonal rabbit
antibodies to human osteonectin (ON-FITC, IMTEK, Russia); phycoerythrin-labelled monoclonal antibodies to CD41 (CD41-PE, Becton Dickinson,
USA); CY5-PE-labelled monoclonal antibodies to human CD45 (CD45-TC, Becton Dickinson, USA). As controls, corresponding isotypic antibodies
were used (rabbit Ig(G+A+M)-FITC, IMTEK, Russia; mouse IgG1-PE and mouse IgG1-TC, Becton Dickinson, USA). Cell aliquots were incubated
with antibodies for 30 minutes at room temperature in the dark. The reaction was stopped by addition of lysing buffer (FACS Lysing Solution, Becton
Dickinson, USA). 15 min later cells were centrifuged at 500g for 15 min, washed with phosphate buffer (0.1 M, pH 7.4), fixed with 1%
paraformaldehyde and analyzed by FACS Calibur (Becton Dickinson, USA). Data collection and analysis were carried out using CELL Quest software
(Becton Dickinson, USA). In each sample 100,000 leukocytes were analyzed.
Determination of circulating ON+ cells
In blood samples the number of ON+ cells was determined using 3-color flow cytometry. The combination of antibodies was as follows: ON-
FITC/CD41-PE/CD45-TC. A typical analysis of ON+ cells in peripheral blood of CAD patients is presented in Fig. 1. A lymphocyte gate was
determined based on light side-scatter and forward-scatter (gate R2, Fig. 1A). In this gate cells were then separated which expressed both CD41 and
CD45 (gate R3, Fig. 1B). The number of these CD41+/CD45+ cells reflects the presence of lymphocytes/platelets conjugates/aggregates. On the plot
of light side-scatter versus ON-FITC fluorescence intensity of gated cells (gate R2&R3) the number of ON+ cells was determined (Fig. 1C). The
6
content of these ON+ lymphocyte-like cells with CD41 positivity was expressed as percentages (
o
/
o
) of total number of lymphocyte-like
CD41+/CD45+ cells. For each blood sample control binding of isotypic immunoglobulins with CD41+/CD45+ cells was carried out.
Statistical analysis
Data are reported as median (lower quartile, upper quartile). Patients and controls were compared using the non-parametric 2-tailed Fisher exact
test or Mann-Whitney U-test for comparing two unmatched samples and Kruskal-Wallis ANOVA by Ranks test for comparing three or more samples.
Differences were considered to be statistically significant if the null hypothesis could be rejected with >95% confidence. Statistical analysis was
carried out using Statistica software (StatSoft, USA).
RESULTS
The characteristics of the patients are presented in Table 1. No significant differences were observed in age, gender, lipid profile, the presence
of hypertension and diabetes between the groups of CAD patients and patients with stenosis-free arteries. Coronary angiography of all patients with
CAD showed critical coronary stenosis of at least two coronary arteries or their major branches. Degree of stenosis showed that bypass surgery was
obligatory. In the control group of 8 patients no lesions of coronary bed were found by angiography. No lesions of aorta and/or its branches were found
in patients or volunteers of the control group by ultrasound examination.
The content of ON+ cells in blood from CAD patients and control subjects was examined in various populations of lymphocyte-like cells. It
was found that the number of ON+ lymphocyte-like cells with CD41 positivity was many times higher in CAD patients compared to the control
groups. These ON+ cells were determined in the population of CD41+/CD45+ cells within the lymphocyte gate. The number of the CD41+/CD45+
7
cells reflects the presence in blood of lymphocyte/platelet conjugates. The number of lymphocyte/platelet conjugates in blood from the CAD patients
[3800 (2350, 5800)] did not statistically differ from their number in the groups of patients without coronary stenosis [4315 (3570, 5200), p=0.78] and
healthy volunteers [3650 (2500, 5500), p=0.61]. However, the number of ON+ cells in this pool (lymphocyte-like ON+/CD41+/CD45+ cells) was
significantly higher in CAD patients compared to the control groups.
In blood from patients without CAD and healthy volunteers only a low level of ON+ lymphocyte-like cells with CD41 positivity was observed.
The number of these ON+ cells in blood of patients without coronary stenosis [0.25% (0.20, 0.30)] did not differ significantly from the number in
healthy volunteers [0.27% (0.17, 0.36), p=0.94]. Non-specific binding by cells of isotypic immunoglobulins was 2-4 cells per 100,000 cells which
corresponds to 0.08 (0.06, 0.11) percents of lymphocyte/platelet conjugates (Table 2). In some cases (volunteers) the percentage of ON+ cells was
extremely low comparable to binding level of isotypic immunoglobulins. In these cases it was not possible to detect ON+ cells reliably. In blood from
CAD patients the percentage of ON+ lymphocyte-like cells with CD41 positivity was 0.86% (0.62, 1.26) and was significantly higher than in blood of
volunteers (p<0.0001) and patients with stenosis-free arteries (p<0.0001, Fig.2). The content of these ON+ cells in blood of CAD patients without
hypertension and diabetes did not differ significantly from the content of cells in blood of CAD patients with hypertension and CAD patients with
diabetes and hypertension (p=0.91, Fig. 3).
DISCUSSION
Arterial stenotic disease is a complex phenomenon. As understanding of the pathogenesis of vascular atherosclerosis progresses, new stenosis
markers are being found that are useful in diagnosis of atherosclerosis of arteries and the risk of developing cardiovascular complications. Factors such
8
as the level of total blood cholesterol, dyslipidemia, hypertension, diabetes and smoking are involved in progression of atherosclerosis. Starting from
1990s, it became evident that the number of other factors, for example, inflammatory events, thrombogenesis or genetic variables are also relevant.
Recent studies of patients with various cardiovascular diseases revealed the presence of unexpected populations of cells in blood. A shortage of
endothelial progenitor cells may result in disturbances of angiogenesis (Vasa et al. 2001). The presence in blood of circulating endothelial cells may
reflect the existence of a vascular pathology and may serve as a biomarker (Blann et al. 2005). In the present study it is shown that 1) a pool of
circulating lymphocyte-like ON+ cells exists and 2) the level of these cells is elevated manyfold in patients with stenotic atherosclerosis of coronary
arteries compared to volunteers and stenosis-free patients.
Non-collagen protein, osteonectin, was first isolated from bone tissue in 1981 (Termine et al. 1981). Later it was demonstrated in platelets
(Stenner et al. 1986) and in vascular cells (Hirota et al. 1993). At present, osteonectin, often named as SPARC (secreted protein acidic and rich in
cystein), is known to participate in the regulation of a large number of cellular and humoral reactions. Osteonectin is involved in bone mineralization
(Hoshi et al. 2001), regulation of cell migration/proliferation (Sage et al. 1989, Rempel et al. 2001), and remodeling of extracellular matrix (Engel et al.
1987, Dobaczewski et al. 2006). Osteonectin participates in wound healing (Bradshaw et al. 2000, Basu et al. 2001), modulation of fibrinolysis
(Hasselaar et al. 1991) and formation of cataracts (Norose et al. 1998). In spite of its involvement in many physiological responses osteonectin is
certainly the marker of differentiation of corresponding progenitor cells into osteoblasts. Localization of osteonectin in osteoprogenitor cells, active
osteoblasts and in young osteocytes was demonstrated by antibody binding, while in adult senescent osteocytes osteonectin is absent (Jundt et al.
1987).
9
At the beginning of 1980s experimental data showing circulation of bone marrow stromal progenitor cells in blood was not yet available. It was
commonly accepted that stem colony-forming units for fibroblasts in the postnatal period are localized only in tissues. From 1982 a few publications
appeared showing the presence of stromal progenitor cells in the circulation. Thus, in animal models (Piersma et al. 1985) and in humans (Keating et
al. 1982), after bone marrow transplantation, circulation of stromal progenitors was indirectly demonstrated. The finding of hemopoietic and stromal
progenitor cells in the intima of atheromatous human aorta and the presence in the peripheral blood of CAD patients of stromal progenitor cells also
supported the idea of their presence in circulation and pointed to the bone marrow as their putative source (Soboleva and Popkova 1989, Soboleva et al.
1994a, Soboleva et al. 1994b). Later, other authors directly demonstrated the presence in circulation of bone marrow clonogenic CFU for fibroblasts
capable to target inflammatory regions, injury sites and to remodel various tissues. It was shown that it is the circulating endothelial progenitors that
form the endothelial layer on vascular prostheses (Shi et al. 1998). It was also found that in mice bone marrow progenitors of muscle cells entering
circulation overcome vascular barriers and can be found in the sites of muscle injury where new muscle elements are formed (Ferrari and Mavilio
1998). Direct proof of the presence in circulation of “skeletal” stem cells with osteogenic and adipogenic potential was presented in 2001 (Kuznetsov
et al. 2001). Today, it is commonly accepted that bone marrow stromal
progenitor cells do circulate and are capable of entering various tissues and
organs under normal and pathological conditions.
In our study the population of circulating ON+ lymphocyte-like cells capable of binding to platelets was examined. Platelets play important
roles in connecting inflammation, thrombosis and atherogenesis. Inflammation is characterized by interactions between platelets, leukocytes and
endothelial cells. These interactions initiate autocrine and paracrine reactions of cellular activation resulting in recruitment of leukocytes into the
10
vascular wall. Appearance in the circulation of leukocyte/platelets aggregates promotes the reaction of acute inflammation via stimulation of rolling
and subsequent recruitment of leukocytes into the vascular wall. Circulating leukocyte/platelet aggregates are seen much earlier than routine
myocardial necrosis markers such as MD-isoform of creatine kinase and cardiac troponins (Furman et al. 2001).
The formation of platelet/leukocyte aggregates may accelerate transportation of blood cells to the sites of injury. It was found that endothelial
progenitor cells (EPC) via PSGL-1 and P-selectin interact with platelets and the latter provide adhesion and rolling of EPC on collagen-covered
surfaces. Besides, platelets serve in fact as the source for a number of cytokines and other biologically active compounds which facilitate cellular
proliferation and differentiation of EPC into adult endothelial cells (Langer et al. 2006).
Osteonectin expression is elevated in cells of the vascular wall when atherosclerosis progresses, namely, when the atherosclerotic plaque is
calcified (Dhore et al. 2001, Gadeau et al. 2001). Using clonal cultures of intimal cells from atherosclerotic lesions (autopsy samples) colony-forming
cells were identified form hemopoietic as well as stromal colonies in test systems. In stromal colonies cells synthesize fibrillar collagen and osteoid
matrices (Soboleva et al. 1994a, Romanov et al. 1995). When a mononuclear fraction of peripheral blood of patient with primary hyperlipidemia and
coronary atherosclerosis was seeded into culture, growth of stromal fibroblastoid-like cells which synthesized fibrillar and osteoid extracellular
matrices was seen. In colonies where an osteoid matrix was formed cells expressed osteonectin. In healthy volunteers no stromal colonies were formed
in vitro from a blood mononuclear fraction (Soboleva et al. 1994b). Taking together, these data suggest close relationships between progression of
stenotic atherosclerosis and appearance in peripheral blood of bone marrow stromal progenitor cells.
11
In this investigation, it was not possible to define precisely the nature and biological characteristics of lymphocyte-like ON+ cells present in
peripheral blood. Nevertheless, elevated levels of these cells may be a reflection of the productive stage of inflammation in the vascular wall. It is
believed that the content of ON+ cells in blood may serve as an indication of the presence of stenotic arterial atherosclerosis. Clinical interest lays in
the fact that this indicator can be considered as a possible diagnostic test for the “acute“ stage of atherosclerosis. Thus, determination of circulating
ON+ cells content may be useful in diagnosis, prognosis and treatment of atherosclerotic vascular lesions.
ACKNOWLEDGEMENTS
Authors would like to thank Dr. Helen Jarovaja for helpful suggestions in the statistical analysis of data.
REFERENCES
Basu, A., Kligman, L.H., Samulewicz, S.J., Howe, C.C. 2001. Impaired wound healing in mice deficient in a matricellular protein SPARC
(osteonectin, BM-40). BMC Cell Biol. 2: 15. Epub 2001 Aug 7.
Blann, A.D., Woywodt, A., Bertolini, F., Bull, T.M., Buyon, J.P., Clancy, R.M., Haubitz, M., Hebbel, R.P., Lip, G.Y., Mancuso, P., Sampol, J.,
Solovey, A., Dignat-George, F. 2005. Circulating endothelial cells. Biomarker of vascular disease. Thromb. Haemost. 93: 228-235.
Bradshaw, A.D., Sage, E.H. 2000. SPARC, a matricellular protein that functions in cellular differentiation and tissue response to injury. J. Clin.
Invest. 107: 1049-1054.
12
Caplice, N.M., Bunch, T.J., Stalboerger, P.G., Wang, S., Simper, D., Miller, D.V., Russell, S.J., Litzow, M.R., Edwards, W.D. 2003. Smooth
muscle cells in human coronary atherosclerosis can originate from cells administered at marrow transplantation. Proc. Nat. Acad. Sci. USA. 100: 4754-
4759.
Dhore, C.R., Cleutjens, J.P., Lutgens, E., Cleutjens, K.B., Geusens, P.P., Kitslaar, P.J., Tordoir, J.H., Spronk, H.M., Vermeer, C., Daemen, M.J.
2001. Differential expression of bone matrix regulatory proteins in human atherosclerotic plaques. Arterioscler. Thromb. Vasc. Biol. 21(12): 1998-
2003.
Dobaczewski, M., Bujak, M., Zymek, P., Ren, G., Entman, M.L., Frangogiannis, N.G. 2006. Extracellular matrix remodeling in canine and
mouse myocardial infarcts. Cell. Tissue. Res. 324(3): 475-488.
Engel, J., Taylor, W., Paulsson, M., Sage, H., Hogan, B. 1987. Calcium binding domains and calcium-induced conformational transition of
SPARC/BM-40/osteonectin, an extracellular glycoprotein expressed in mineralized and nonmineralized tissues. Biochemistry. 26: 6958–6965.
Ferrari, G., and Mavilio, F. 1998. Muscle regeneration by bone marrow-derived myogenic progenitors. Science. 279: 1528-1530.
Furman, M.I., Barnard, M.R., Krueger, L.A., Fox, M.L., Shilale, E.A., Lessard, D.M., Marchese, P., Frelinger, A.L. 3rd, Goldberg, R.J.,
Michelson, A.D. 2001. Circulating Monocyte-Platelet Aggregates Are an Early Marker of Acute Myocardial Infarction. J. Am. Coll. Cardiol. 38(4):
1002–1006.
Gadeau, A.P., Chaulet, H., Daret, D., Kockx, M., Daniel-Lamaziere, J.M., Desgranges, C. 2001. Time course of osteopontin, osteocalcin, and
osteonectin accumulation and calcification after acute vessel wall injury. J. Histochem. Cytochem. 49(1): 79-86.
13
Hasselaar, P., Loskutoff, D.J., Sawdey, M., Sage, E.H. 1991. SPARC induces the expression of type 1 plasminogen activator inhibitor in
cultured bovine aortic endothelial cells. J. Biol. Chem. 266: 13178–131784.
Hirota, S., Imakita, M., Kohri, K., Ito, A., Morii, E., Adachi, S., Kim, H.M., Kitamura, Y., Yutani, C., Nomura, S. 1993. Expression of
osteopontin messenger RNA by macrophages in atherosclerotic plaques: a possible association with calcification. Am. J. Pathol. 143: 1003–1008.
Hoshi, K., Ejiri, S., Ozawa, H. 2001. Ultrastructural analysis of bone calcification by using energy-filtering transmission electron microscopy.
Ital J Anat Embryol. 106 (2 Suppl 1): 141-150.
Jundt, G., Berghauser, K.H., Termine, J.D., Schulz, A. 1987. Osteonectin - a differentiation marker of bone cells. Cell Tissue Res. 248(2): 409-
415.
Keating, A., Singer, J.W., Killen, P.D., Striker, G.E., Salo, A.C., Sanders, J., Thomas, E.D., Thorning, D., Fialkow, P.J. 1982. Donor origin of
the in vitro haematopoietic microenvironment after marrow transplantation in man. Nature. 298(5871): 280-283.
Kuznetsov, S.A., Mankani, M.H., Gronthos, S., Satomura, K., Bianco, P., Robey, P.G. 2001. Circulating skeletal stem cells. J. Cell. Biol. 153:
1133-1140.
Langer, H., May, A.E., Daub, K., Heinzmann, U., Lang, P., Schumm, M., Vestweber, D., Massberg, S., Schonberger, T., Pfisterer, I.,
Hatzopoulos, A.K., Gawaz, M. 2006. Adherent Platelets Recruit and Induce Differentiation of Murine Embryonic Endothelial Progenitor Cells to
Mature Endothelial Cells In Vitro. Circ. Res. 98(2): e2-e10
14
Norose, K., Clark, J.I., Syed, N.A., Basu, A., Heber-Katz, E., Sage, E.H., Howe, C.C. 1998. SPARC deficiency leads to early onset
cataractogenesis. Invest. Ophthalmol. Vis. Sci. 39: 2674-2680.
Piersma, A.H., Ploemacher, R.E., Brockbank, K.G.M., Nikkels, P.J., Ottnheim, C.P.E. 1985. Migration of fibroblastoid stromal cells in murine
blood. Cell Tissue Kinet. 8: 589-595.
Rempel, S.A., Golembieski, W.A., Fisher, J.L., Maile, M., Nakeff, A. 2001. SPARC modulates cell growth, attachment and migration of U87
glioma cells on brain extracellular matrix proteins. J. Neurooncol. 53: 149-160.
Romanov, Y.A., Balyasnikova, I.V., Bystrevskaya, V.B., Byzova, T.V., Ilyinskaya, O.P., Krushinsky, A.V., Latsis, R.V., Soboleva, E.L.,
Tararak, E.M., Smirnov, V.N. 1995. Endothelial heterogeneity and intimal blood-borne cells. Relation to human atherosclerosis. Ann. N. Y. Acad. Sci.
748:12-37.
Sage, H., Vernon, R.B., Funk, S.E., Everitt, E.A., Agnello, J. 1989. SPARC, a secreted protein associated with cellular proliferation, inhibits
cell spreading in vitro and exhibits Ca12-dependent binding to the extracellular matrix. J. Cell. Biol. 109: 341–356.
Sata, M. 2003. Circulating vascular progenitor cells contribute to vascular repair, remodeling, and lesion formation. Trends Cardiovasc. Med.
13: 249-253.
Shi, Q., Rafii, S., Wu, M.H., Wijelath, E.S., Yu, C., Ishida, A., Fujita, Y., Kothari, S., Mohle, R., Sauvage, L.R., Moore, M.A., Storb, R.F.,
Hammond, W.P. 1998. Evidence for circulating bone marrow-derived endothelial cells. Blood. 92: 362-367.
15
Soboleva, E.L., and Popkova, V.M. 1989. Hemopoietic progenitor cells (CFU-GM) in the intima of human atheromatous aorta. Bull. Exp. Biol.
Med. 5: 600-604.
Soboleva, E.L., Popkova, V.M., Saburova, O.S., Tararak, E.M., Tvorogova, M.G., Smirnov, V.N. 1994a. Colony-forming units and
atherosclerosis. In Atherosclerosis X. Edited by F.P. Woodford, I. Davignon, and A. Sniderman. New York. Elsevier Science. pp. 919-925.
Soboleva, E.L., Shindler, E.M., Saburova, O.S., Tvorogova, M.G., and Smirnov, V.N. 1994b. Colony-forming units for fibroblasts (CFU-f) in
the peripheral blood of patients with primary hypercholesterolemia. In New Pathogenic Aspects of Atherosclerosis. Nordrhein-Westfalische Academie
der Wissenschaften, Westdeutscher Verlag. pp. 79-93.
Soboleva, E.L., Gabbasov, Z.A., Agapov, A.A., Akchurin, R.S., Saburova, O.S., Romanov, Y.A., and Smirnov, V.N. 2005. Circulating bone
marrow stem/progenitor cells in vascular atherogenesis and in non-invasive diagnosis of coronary stenosis. Experimental and Clinical Cardiology. 10:
184-188.
Stenner, D.D., Tracy, R.P., Riggs, B.L., Mann, K.G. 1986. Human platelets contain and secrete osteonectin, a major protein of mineralized
bone. Proc. Natl. Acad. Sci. USA. 83: 6892–6896.
Termine, J.D., Belcourt, A.B., Conn, K.M., Kleinman, H.K. 1981. Mineral and collagen-binding proteins of fetal calf bone. J. Biol. Chem.
256(20):10403-10408.
Vasa, M., Fichtlscherer, S., Aicher, A., Adler, K., Urbich, C., Martin, H., Zeiher, A.M., Dimmeler, S. 2001. Number and Migratory Activity of
Circulating Endothelial Progenitor Cells Inversely Correlate With Risk Factors for Coronary Artery Disease. Circ. Res. 89: E1-7.
16
Table 1. Baseline characteristics of patients
Variable
Patients with
coronary artery disease
(n=99)
Patients with stenosis-free
arteries
(n=8)
p
value
Mean age, years 58 (50, 65) 55 (42, 58) 0.12
Gender (male/female) 85/14 6/2 0.34
Cholesterol,
mmol/liter 5.1 (4.4, 5.9) 5.6 (5.2, 6.2) 0.17
Triglyceride s,
mmol/liter 1.58 (1.16, 2.50) 1.61 (0.82, 4.00) 0.86
LDL-cholesterol,
mmol/liter 3.05 (2.23, 4.38) 2.98 (2.61, 3.35) 0.85
HDL-cholesterol,
mmol/liter 1.23 (1.01, 1.45) 1.31 (1.16, 1.72) 0.35
Uric acid, µmol/liter 392 (331, 447) 307 (283, 397) 0.21
Hypertension 47 (47%) 5 (62%) 0.48
Diabetes 13 (13%) 1 (13%) 1.00
Number of diseased
vessels >50% 2.0 (2.0, 3.0) 0.0 (0.0, 0.0) 0.00
17
Table 2. The content of ON+ lymphocyte-like cells with CD41 positivity in blood of healthy volunteers, patients with stenosis-free arteries and
patients with coronary artery disease.
The number of ON+ cells (%)
Mean±SD Median Lower
quartile
Upper
quartile
Healthy volunteers (n=19) 0.27±0.11 0.27 0.17 0.36
Patients with stenosis-free
arteries
(n=8)
0.26±0.07 0.25 0.20 0.30
Patients with coronary
artery disease (n=99) 1.01±0.49 0.86 0.62 1.26
The number of cells labeled with isotype-matched
antibodies (%)
All subjects (n=126)
0.09±0.03 0.08 0.06 0.11
18
FIGURE CAPTIONS
Fig. 1. FACS-analysis of ON+-positive cells in the peripheral blood of a CAD patient. Isolation of lymphocyte-like cells (gate R2) on the plot of
light side-scatter versus forward-scatter (A). Isolation of platelet/lymphocyte conjugates (gate R3) on the plot of CD41-PE versus CD45-TC
fluorescence intensity of the gated lymphocyte-like cells (B). Binding of FITC-labeled antibodies to osteonectin by lymphocyte-like CD41/CD45-
positive cells (C).
Fig. 2. The content of ON+ lymphocyte-like cells with CD41 positivity in blood of volunteers, patients with stenosis-free arteries and patients
with coronary artery disease.
1 - volunteers (n=19), 2 - patients with stenosis-free arteries (n=8), 3 - patients with coronary artery disease (n=99).
p
1,2
=0.94, p
1,3
<0.0001, p
2,3
<0.0001, Mann-Whitney U-test.
Fig. 3. The content of ON+ lymphocyte-like cells with CD41 positivity in blood of CAD patients without hypertension and diabetes, CAD
patients with hypertension and without diabetes and CAD patients with hypertension and diabetes.
1 – CAD patients without hypertension and diabetes (n=52), 2 – CAD patients with hypertension and without diabetes (n=34), 3 – CAD patients with
diabetes and hypertension (n=13).
p=0.91, Kruskal-Wallis test.
19
Fig. 1.
20
Fig.2.
21
Fig.3.
