Inhibitors of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase (Statins) Suppress Proliferation and Motility of Human CD4+ T Lymphocytes in Culture.

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) along with their blood lipid-lowering effect exhibit anti-inflammatory and immunomodulatory activity. We studied the effects of long-term (72-h or longer) exposure of human T lymphocytes in culture to atorvastatin and rosuvastatin (5-80 nM) on their functional activity. Treatment with statins inhibited PHA/IL-2-induced proliferation of CD4+ T lymphocytes isolated from the peripheral blood of healthy donors. This was accompanied by a decrease in the relative content of cells expressing active caspase-3. Addition of mevalonate or fetal bovine serum simultaneously with statins restored proliferative activity of cells. Culturing of CD4+ T lymphocytes with statins in the presence of IL-2 did not significantly affect the expression of chemokine receptors CCR4, CCR5, CXCR3, and CXCR4. Pretreatment with statins suppressed spontaneous and SDF-1-stimulated migration of CD4+ T lymphocytes, but little changed the content of intracellular phosphorylated protein kinases Akt, p38 and p42/44 (ERK1/2). The cellular effects of "lipophilic" atorvastatin were observed at lower concentrations compared to "hydrophilic" rosuvastatin.

Inhibitors of 3-Hydroxy-3-Methylglutaryl Coenzyme a Reductase (Statins) Suppress Differentiation and Reduce LPS/IFNγ-Induced Cytokine Production in Human Monocyte/Macrophage Culture.

We examined the effects of 72-h exposure to atorvastatin and rosuvastatin in concentrations of 2-10 nM on the cytokine expression in LPS/IFNγ-activated monocyte/macrophages derived from peripheral blood monocytes of healthy donors by culturing in the presence of GM-CSF. Pretreatment with statins was found to inhibit cytokine production in monocytes/macrophages after activation, while the level of cytokine mRNA in cells did not decrease. The number of cells containing active caspase-3 decreased in the culture. Culturing of monocytes/macrophages with statins was accompanied by changes in cell morphology and deceleration of cell growth. Cellular effects of "lipophilic" atorvastastin were observed at lower concentration compared to "hydrophilic" rosuvastatin.

A Low-Molecular-Weight Phenotype of Apolipoprotein(a) as a Factor Provoking Accumulation of Cholesterol by THP-1 Monocyte-Like Cells.

Increased concentration of lipoprotein(a) is a risk factor of coronary heart disease. lipoprotein(a) consists of LDL-like and highly polymorphic apolipoprotein(a). Here we studied the effect of lipoprotein(a)-containing sera with different apolipoprotein(a) phenotypes on lipid accumulation by THP-1 monocyte-like cells. Cholesterol concentration in lysates of THP-1 cells was significantly higher after their incubation with lipoprotein(a)-containing serum samples with low-molecular-weight phenotype of apolipoprotein(a) in comparison with samples with a high-molecular-weight apolipoprotein(a) phenotype irrespective of initial cholesterol level as well as serum concentrations of apoB-100, oxidized LDL, and circulating immune complexes. The presence of the most atherogenic small dense LDL subfractions in examined sera in addition to a low-molecular-weight apolipoprotein(a) phenotype resulted in significant elevation of cholesterol accumulation by THP-1 cells. The data obtained explain greater atherogenicity of lipoprotein(a) with low-molecular-weight apolipoprotein(a) phenotype.

Low Blood Content of IL-10-Producing CD4+ T Cells as a Risk Factor for Progression of Coronary Atherosclerosis.

In a 2-year prospective study, prognostic significance of the blood content of IL-10-producing CD4+ T lymphocytes for progression of coronary artery atherosclerosis was assessed. Patients with verified stable angina (n=36) admitted for scheduled coronary angiography and coronary stenting were enrolled. The blood levels of CD4+FoxpP3+ Treg, CD4+IFNγ+ Th1, CD4+IL17+ Th17, CD4+IL10+ cells, sCD25, IL-10, IL-17, C-reactive protein, and lipoprotein (a) were assayed before endovascular interventions. The blood content of CD4+IL10+ T cells below 3.3% was associated with progression of coronary artery atherosclerosis (OR 12.0 (2.3, 61.0), sensitivity 77%, specificity 78%, p=0.003). No differences in other immunological parameters and common atherosclerosis risk factors in the groups were revealed. We hypothesize that the content of CD4+IL10+ T cells can be an important predictive marker for the progression of coronary atherosclerosis.

[INFLUENCE OF 29-40 AND 65-76 MCP-1 FRAGMENTS ON MYOCARDIUM MORPHOLOGY IN RATS AFTER T9CTHFMIA-R'FPFRFTTON].

Monocyte chemotactic protein-1 (MCP-1) is a chemokine that stimulates monocytes and macrophage migration into the sites of acute of chronic inflammation. Our study shows morphological changes in ischemic myocardium followed by the administration of two synthetic structural fragments of MCP-1 that are monocyte/macrophage migration inductor peptide IX and peptide X an inhibitor. Results show that peptides can change time points of the inflammatory response in myocardium. Peptide IX administration leads to increased and accelerated inflammatory response, i. e. attracts an additional number of monocytes and macrophages into the inflammatory focus. The introduction of the peptide X observed prolonged inflammatory process with the overall gain signs of myocardial damage.

[Effector and regulatory blood lymphocyte subpopulations in stable coronary artery disease].

AIM: To investigate a balance between circulating regulatory T lymphocytes (Treg) exerting antiatherogenic activity and T helper type 1 (Th1) and T helper type 17 (Th1 7) cells having proatherogenic activity in patients with stable coronary artery disease (CAD) and different degrees of coronary atherosclerosis. SUBJECTS AND METHODS: According to coronary angiography findings, 80 patients were allocated to 4 groups: 1) 18 patients with intact coronary arteries; 2) 21 with no progressive coronary atherosclerosis; 3) 16 with progressive coronary atherosclerosis (more than 50% stenosis) in the native coronary arteries; 4) 25 patients with three-vessel lesions. Groups 2 and 3 patients had undergone coronary stenting 23.8 ± 8.4 and 22.4 ± 8.7 months before their enrollment, respectively. Lymphocytes were typed by direct immunocytofluorometry: Treg was defined as CD4+CD25highCD127low and CD4+FoxP3+ lymphocytes. For CD4+IL-17a+ Th17 and CD4+INFgamma Th1 analysis, mononuclear cells were preactivated by culture. The serum levels of high-sensitivity C-reactive protein, IL-10, sCD25, and IL-17a were determined by nephelometry, chemiluminescence (Immulite) and ELISA, respectively. RESULTS: Group 4 was found to have lower Treg levels and higher Th17 levels than Group 1. The ratio of Th17/Treg proved to be higher in Groups 3 and 4 than in Group 1 and that of (Th1+Th17)/Treg was higher in Group 3 than in Group 2. The female patients had higher Tregs levels than the male ones. The Th17/Treg index turned out to be increased in patients with a history of myocardial infarction. CONCLUSION: The imbalance of pro- and anti-atherogenic lymphocyte subpopulations plays a role in the pathogenesis of CAD and is associated with progressive atherosclerosis.

[Peptide fragments of chemokine domain of fractalkine: effect on human monocyte migration].

Leukocyte chemotaxis to the area of tissue damage is mediated by chemokines. According to the primary structure, chemokines are divided into four families, fractalkine (CX3CL1) is the only one member of CX3C family and the only membrane-bound chemokine. Fractalkine molecule includes the extracellular N-terminal chemokine domain, mucin-like rod, the transmembrane and the intracellular domains. In membrane-bound state fractalkine has the properties of an adhesion molecule. Chemokine domain of fractalkine (CDF) is released from cell membrane by proteolysis, and this soluble form acts as a chemoattractant for leukocytes expressing fractalkine receptor CX3CR1. Fractalkine is involved in development of a number of pathological processes caused by inflammation, and therefore a search for fractalkine inhibitors is very important. For this purpose we identified several antigenic determinants--the fragments of CDF, and the following peptides were synthesized--P41-52 H-Leu-Glu-Thr-Arg-Gln-His-Arg-Leu-Phe-Cys-Ala-Asp-NH2, P53-60 H-Pro-Lys-Glu-Gln-Trp-Val-Lys-Asp-NH2 and P60-71 H-Asp-Ala-Met-Gln-His-Leu-Asp-Arg-Gln-Ala-Ala-Ala-NH2. The peptide effects on adhesion and migration of human peripheral blood monocytes expressing fractalkine receptors were investigated. In the presence of CDF and P41-52 we observed the increased adhesion and migration of monocytes compared with spontaneous values. Peptides P53-60 and P60-71 significantly inhibited monocyte adhesion and migration stimulated by CDF. Since the chemotactic activity of chemokines was shown to be dependent on their binding to glycosaminoglycans of the cell surface and extracellular matrix, the effect ofpeptides on the interaction of CDF with heparin was analyzed by ELISA. Peptide P41-52 competed with CDF for heparin binding, while peptides P53-60 and P60-71 had no significant activity.

[Participation of beta2-integrins CD11b/CD18 and CD11c/CD18 in adhesion and migration of cells on fibrinogen].

The role of beta2-integrins CD11b/CD18 and CD 11c/CD 18 in adhesion and migration of leukocytes on fibrinogen was studied. The monoclonal antibodies against CD11b inhibited the spontaneous adhesion of monocytic THP-1 cells on fibrinogen, whereas antibodies to CD11c more effectively inhibited the adhesion stimulated by chemokine MCP-1. By the RNA-interference method the clones of THP-1 with reduced expression of CD11b and general beta2-subunit CD18 were obtained. MCP-I stimulated the adhesion to fibrinogen of THP-1 cells of wild-type and mutant cells with reduced expression of CD11b (THP-1-CD11b-low), but not of cells with low expression of CD18 (THP-1-CD18-low). THP-1-CD18-low cells were also characterized by the impaired chemotaxis in presence of MCP-1. The data obtained suggest that spontaneous cell adhesion to fibrinogen is mediated to a greater extent by CD11b/CD18 integrins, while chemokine-stimulated adhesion and migration is mostly dependent on CD11c/CD18 molecules.

[Changes of content of regulatory lymphocytes and concentration of soluble interleukine-2 receptor in blood of patients with ischemic heart disease after coronary artery angioplasty with implantation of stents with rapamycin covering].

We studied dynamics of content of subpopulation of lymphocytes including regulatory and effector T-lymphocytes as well as concentration of soluble form of interleukine-2 receptor (sCD25) in peripheral blood of patients after coronary stenting (CS) with implantation of stents with rapamycin covering (SRC). We included into the study 62 patients with stable effort II-III functional class angina. Coronary angiography (CA) was carried out in all, CS with implantation of 1 - 2 SRC - in 42 patients. Blood samples were taken before CA/CS, in 24, 48 hours, 7 days, 1 and 3 months after intervention. Content of T-, helper and cytotoxic T-cells, -, NK-, NKT-cells, activated effector T-lymphocytes (CD4+CD251owCD127high) and regulatory T-lymphocytes (CD4+CD25highCD1271ow) were measured by direct immunofluorescence and flow cytometry. CD4+ lymphocytes were isolated from mononuclear cell fraction of donor blood by magnetic separation. Content of regulatory T-lymphocytes in culture were determined by expression of a specific marker FOXP3+. Concentration of sCD25 was measured by chemiluminescent method. It was shown that content of main subpopulations of lymphocytes in blood changed after CS or CF. Blood content of regulatory T-lymphocytes and sCD25 significantly increased after 7 days and 1 month after CS but not after CA. Plasma sCD25 concentration correlated with content of regulatory T-lymphocytes in 1 month after SRC implantation. During cultivation of CD4+ lymphocytes in the presence of rapamycin we noted antiproliferative effect relative to FOXP3-cells and accumulation of regulatory +-lymphocytes. Thus implantation of SRC in coronary arteries leads to increase of number of circulating regulatory T-lymphocytes and blood concentration of sCD25. Changes of these parameters after CS can reflect peculiarities of local and systemic reaction arising in response to introduction of stent with drug covering and be significant for assessment of prognosis of the disease.

[Effect of the anti-inflammatory peptide preparation ingramon on the blood levels of acute-phase proteins and chemokines in patients after coronary stenting].

AIM: To study the effect of the anti-inflammatory peptide preparation ingramon on the peripheral blood levels of inflammatory markers in patients with exercise-induced stable angina after coronary stenting (CS). SUBJECTS AND METHODS: The investigation enrolled 64 patients with stable angina who had undergone coronary bypass surgery, of them 34 patients received ingramon in addition to standard therapy. The blood levels of high-sensitive C-reactive protein (hs-CRP), fibrinogen, the chemokines MCP-1, IL-8, IP-10, and MID were measured before and 1, 2, and 7 days and 1, 3, and 6 months after surgery. Twenty patients who had gone coronarography (CG) only were examined as a control group. RESULTS: In the post CS patients receiving only standard therapy, the levels of hs-CRP and fibrinogen were much higher on days 1, 2, and 7 after surgery than in the CG patients. On day 1 following CS, the increment in hs-CRP correlated with the length of implanted stents. During ingramon therapy, the content of hs-CRP and fibrinogen was considerably lower on days 1, 2, and 7 after CS than in the control group; this trend persisted a month after surgery; there was also a reduction in MCP-1 levels within the first 24 hours after initiation of therapy. The levels of the chemokines IP-10, MIG, and IL-8 were significantly unchanged. CONCLUSION. When added to standard therapy, ingramon exerts a positive effect against risk factors for coronary heart disease (CHD) and its events. Further investigations are required to define the impact of ingramon therapy on prognosis in patients with CHD.

[The influence of coronary stenting with rapamycin-eluting stents on the number of circulating CD4+CD25high+regulatory T-cells].

AIM: To investigate the effects of coronary stenting with rapamycin-eluting stents on parameters of cell immunity. METHODS: 26 patients (group 1) with stable coronary heart disease and angiographically proved coronary stenosis underwent stenting with rapamycin-eluting stents. The control group (group 2) consisted of 6 patients: 4 patients underwent diagnostic coronaroangiography, I patient got a bare metal stent and in 1 patient angioplasty was unsuccessful. Blood samples were obtained before and 1 month after the intervention. The quantity of activated (CD4+CD25low+) and regulatory (CD4+CD25high+) T cells was measured by direct immunofluorescence and flow cytometry. Plasma concentration of IL-10 was determined by ELISA. RESULTS: In group 1 the percentages of CD4+CD25high+ regulatory T-cells increased significantly one month after stenting, while in group 2 no difference in regulatory T-cell levels before and after the intervention was observed. No changes in total number of leukocytes, relative levels of lymphocytes, CD4+ T-cells, activated CD4+CD25+low T-cells and IL-10 plasma concentration before and after the procedure were detected in both groups. CONCLUSION: Rapamycin-eluting stent implantation is associated with a significant increase of circulating CD4+CD25high+ regulatory T-cell level.

[Development of a new anti-inflammatory peptide preparation--inhibitor of the monocytic chemotaxic protein-1 (MCP-1)].

A new anti-inflammatory peptide X (Ingramon)--a fragment of C-terminal sequence (65-76) of MCP-1, was elaborated. In Boyden chamber assay, Ingramon inhibited migration of human monocytic cells THP-1 and peripheral blood monocytes. In vitro in blood plasma, Ingramon was stable for at least 24 hours. Ingramon was accumulated in sites of inflammation in rats, and impaired monocyte and granulocyte accumulation occurred in different models of inflammation in rodents and primates. Ingramon exerted no effect on the MCP-1-dependent externalisation of p2 integrins CD lib CD18 (Mac-1). We propose that Ingramon might interfere with MCP-1-heparin/heparan sulphate binding on cell surface. Ingramon reception by volunteers showed its safety. The clinical trials of Ingramon in patients with acute coronary syndromes and percutaneous coronary interventions, have been launched.

[Circulating endothelial progenitor cells and vascular endothelial dysfunction].

Endothelial progenitor cells (EOCs) play a key role in reendothelization of injured blood vessels and in endogenous neovascularization of ischemic tissues. In the presented article, the mechanisms of endothelial function impairment and the role of EPCs in their correction are discussed. The objective of our study was to investigate the number of CD34+ cells in patients with different forms of ischemic heart disease (IHD) and association of this parameter with endothelial dysfunction. We quantified the numbers of CD34+ cells by flow cytometry, measured the brachial artery flow-mediated vasodilatation (FMD) and the common carotid artery intima-media thickness (IMT) by ultrasonography in 28 patients with ST-segment elevation acute coronary syndromes (ACS), in 33 patients with stable angina (SA) and in 17 subjects without IHD (control group). The level of CD34+ cells was lower in both groups of IHD, especially in patientes with ACS, and was associated with endothelial dysfunction. We suppose that CD34+ cells contribute to reparation of injured endothelium and the reduction of their numbers in peripheral blood indicates endothelial state.

Effects of synthetic monocyte chemotactic protein-1 fragment 65-76 on neointima formation after carotid artery balloon injury in rats.

The effects of the synthetic monocyte chemotactic protein-1 (MCP-1) peptide fragment 65-76 (peptide X) on the development of neointima after balloon injury to the carotid artery were studied. The agent was given i.m. at a dose of 33 microg/kg once daily for 28 days after balloon injury. Animals given peptide showed significant suppression of neointima growth 4 and 7 days after lesioning, as indicated by morphometric analysis of sections of lesioned arteries. On days 14 and 28, there were no significant differences in neointima formation in rats given and not given peptide. Peptide administration was not accompanied by any changes in C-reactive peptide concentrations, leukocyte counts, or the population composition of peripheral blood lymphocytes. Use of synthetic peptide X as an inhibitor of leukocyte migration during angioplasty may, along with traditional treatments, decrease the risk of restenosis.

[Expression of chemokines and cytokines in atherosclerotic plaques and internal membrane of the arteries in patients with coronary artery disease].

AIM: To analyse contents of leukocytes and chemokines expression cytokines and transforming growth factor beta (TGFB) in atherosclerotic plaques (AP) of coronary arteries and aortic intima of patients with coronary artery disease (CAD). MATERIAL AND METHODS: The samples of aortic intima and coronary artery tissues were obtained intraoperatively (coronary artery bypass grafting, endarterectomy). Leukocytes were typed immunohistochemically and cytometry in the flow. Gene expression was analysed using reverse transcription and polymerase chain reaction. RESULTS: AP of the coronary arteries and macroscopically unaffected fragments of aortic imtima contained leukocytes. All the samples contained mRNA of chemokines SDF- 1 and MCP-3. Two groups of the plaques were identified by chemokines expression. Group I AP had marked expression of TGFB, chemokines SDF-1, MCP-3, MIG, I-309, MCP-1, MIP-1beta, I-TAC, RANTES and IL-13. Group II AP had mRNA of the proteins only in single samples. Intima samples free of morphological signs of atherosclerotic lesion contained mRNA of proinflammatory chemokines MIG, I-309, IL-13, had no expression of TGFB. CONCLUSION: In IHD patients arterial intima free of macroscopic visual affection may be a site of developing inflammation. AP differ by chemokines expression, cytokines, TGFB. The differences may indicate different stages or mechanisms of AP formation.

[The effect of synthetic fragment 65-76 of monocyte chemiattractant protein-1 (MCP-1) on neointima growth after carotid artery balloon injury in rats].

Influence of synthetic fragment 65-76 of monocyte chemoattractant protein-1 (MCP-1) (peptide X) on development of neointima after balloon injury of carotid artery was investigated. Peptide X was introduced intramuscularly, 33 pg/kg, daily during 28 days after balloon injury. In days 4 and 7 after intervention, in animals receiving peptide X in comparison with control animals a substantial decrease of neointimal growth was observed. On 14 and 28 days there, was no significant difference in neointima development in rats with and without peptide treatment. Injections of peptide X did not after the C-reactive protein concentration, leukocyte number and lymphocyte subpopulations in peripheral blood. Peptide X treatment along with traditional therapy may be effective in preventing restenosis after angioplasty.

[Amount of circulating endothelium precursors in patients with stable angina of effort and patients with acute non-ST-elevation coronary syndrome].

AIM: To measure and compare the level of circulating precursors of endothelial cells in patients with acute coronary non-ST-elevation syndrome (NSTES), patients with stable effort angina (SEA) before coronary stenting and after it and persons with documented absence of coronary heart disease (CHD). MATERIAL AND METHODS: The trial included 19 patients with acute coronary NSTES, 20 patients with SEA and 14 patients free of CHD. Blood samples were stained with antibodies CD45-FITC (Becton Dickinson), CD34-PerCP (Becton Dickinson), KDR-PE (R&D systems). Count of cell populations was measured with flow cytofluorimetry (FACSCalibur (Becton Dickinson). RESULTS: SEA patients had the number of circulating cells CD34+ including hematopoietic cells and endothelial precursors (EP) two times lower versus that in the controls (0.650 +/- 0.086 and 1.216 +/- 0.242%, respectively, p = 0.037). No significant differences were found by the number of CD34+KDR+ cells between the groups. A trend was found to lower number of CD34-KDR+ cells in EA patients compared to control. The differences reached significance on day 1 and 3-5 after endovascular interventions (0.067 +/- 0.022, 0.060 +/- 0.017 and 0.188 +/- 0.052%, respectively, p = 0.024 and p = 0.021). CONCLUSION: The count of blood CD34+ cells in combination with other indicators can be used as an additional factor confirming the presence of chronic myocardial ischemia.

[Markers of inflammation--monocyte chemoattractant protein-1 (MCP-1) and C-reactive protein--in blood of patients with unstable angina pectoris and stable effort angina].

AIM: To estimate concentrations of C-reactive protein (CRP) and MCP-1 in blood plasma of patients with unstable angina (UA) and stable effort angina (SEA). MATERIAL AND METHODS: Multiprojection coronaroangiography was performed in 12 patients with UA and 11 patients with SEA. Hemodynamically significant stenosis (50% and more) at least in one major coronary artery was confirmed in all the patients. CRP and MCP-1 were measured with latex agglutination and enzyme immunoassay (Biosource kits), respectively. RESULTS: UA patients had significantly higher plasma levels of MCP-1 and CRP than those with SEA (107.25 +/- 16.19 vs. 63.0 +/- 16.16 pg/ml and 1.99 +/- 1.64 vs 0.44 +/- 0.28 mcg/ml, respectively). CONCLUSION: Estimation of MCP-1, as a marker of vascular wall inflammation, can be used, in line with other indices, for verification of UA.

[Peptide fragment 66-77 of monocyte chemoattractant protein 1 and its retro-enantio analogue inhibits the migration of cells in vitro and in vivo].

The retro-enantio analogue of peptide 66-77 of the chemokine MCP-1 and two hexapeptide fragments 66-71 and 72-77 of the C-terminal sequence of this protein were synthesized using the Fmoc strategy of solid phase peptide synthesis. The effect of the synthetic peptides upon the MCP-1-stimulated migration of THP-1 mononuclear cells was studied in vitro. The activity of the retro-enantio analogue was found to be comparable with that of the initial peptide 66-77: both peptides inhibit the migration of monocytes and granulocytes into inflammation zones of experimental animals.