Cysteine-Containing Peptides Stimulate Monocyte Migration through NADPH-Oxidase Activation.

We analyzed migration of monocytes under the effect of apocinin (NADPH inhibitor) and PD98059 (blocker of extracellular MEK/ERK kinase involved in Nox4 oxidase-mediated migration of monocytes). Migration of monocytes stimulated by cysteine-containing peptides (fragments of chemokines with free thiol group MCP-1 and fractalkine) was completely inhibited by apocinin and MEK/ERK blocker. It is assumed that the stimulating effect of cysteine-containing peptides on monocyte migration is mediated by the NADPH-oxidase system, in particular, Nox4.

Ingramon, a Peptide Inhibitor of MCP-1 Chemokine, Reduces Migration of Blood Monocytes Stimulated by Glioma-Conditioned Medium.

Malignant gliomas are most common and fatal primary brain tumors. In addition to neoplastic cells, the tumor tissue contains microglial cells and monocyte-derived macrophages. It is an established fact that monocyte recruiting promotes the tumor growth and dissemination. Monocyte chemotactic protein-1 (MCP-1) is the major attractant for monocytes. We have previously synthesized an MCP-1 antagonist ingramon, a synthetic peptide fragment (65-76) of this chemokine. In the present study, we demonstrated that glioma-conditioned medium contains MCP-1 and stimulates migration of blood monocytes. Ingramon inhibited the effect of glioma-conditioned medium on monocyte migration.

[Lipoprotein(a), its autoantibodies, and circulating T lymphocyte subpopulations as independent risk factors for coronary artery atherosclerosis]. / Lipoproteid(a), autoantitela k nemu i tsirkuliruyushchie subpopulyatsii T-limfotsitov kak nezavisimye faktory riska ateroskleroza koronarnykh arterii.

AIM: To study the role of lipoprotein(a) [Lp(a)] as a potential autoantigen causing the activation of immunocompetent cells in atherosclerosis. SUBJECTS AND METHODS: A total of 104 men with stable coronary artery (CA) disease and different degrees of progressive coronary atherosclerosis were examined. Clinical blood analysis was carried out and lymphocyte subpopulations (CD4+, Th1, Th17, and Treg) were determined using immunofluorescence and flow cytometry. In addition, the indicators of blood lipid composition, Lp(a), autoantibody (autoAb) titer to Lp(a), and low-density lipoproteins (LDL), and the lymphocyte activation marker sCD25 were also measured. RESULTS: The Lp(a) level was shown to predict the severity of CA lesions (ß=0.28, p<0.05), regardless of age, the level of cholesterol, different T-lymphocyte subpopulations, sCD25, and autoAb. A combination of the concentration of Lp(a) above 11.8 mg/dl, that of Th17 over 11.4∙103 cells/ml and the reduced levels of regulatory T cells and IL-10-producing CD4+ T cells showed a manifold increase in the risk of severe and progressive CA atherosclerosis. There was a direct correlation of the blood level of Th1 with that of IgG autoAb specific to all atherogenic apoB-containing lipoproteins, including Lp(a). There was an inverse correlations of the lymphocyte activation marker sCD25 with IgM anti-Lp(a) autoAb titers (r=-0.36; p<0.005), but this was less significant with autoAbs to native and oxidized LDL (r=-0.21 and r=-0.24; p<0.05, respectively). CONCLUSION: The slightly elevated Lp(a) concentration along with changes in the level of T lymphocyte subpopulations was first shown to significantly potentiate the risk of progressive and multiple CA lesion in the examinees. The correlation of IgM anti-Lp(a) autoAb with the lymphocyte activation marker sCD25 and that of IgG anti-Lp(a) autoAb with Th1 have demonstrated that Lp(a) is involved in the autoimmune inflammatory processes in atherosclerosis.

[Cys-containing peptides cause migration of monocytes].

Automated Fmoc solid-phase technique was used to synthesize Cys-containing linear peptide fragments of monocyte chemoattractant protein-1 and chemokine domain of fractalkine along with their analogues with Cys residue being either modified or replaced with Ser. Chimeric symmetric and asymmetric disulfides were also prepared from the former linear precursors. A SAR study on a set of the newly synthesized peptides revealed that capacity to stimulate migration of monocytes and to influence cell motility in vitro, in general, critically depends on the presence of Cys free thiol group in the molecule. Notably, all analogs lacking this feature, including chimeric disulfides, demonstrated lack of effect on monocyte migration.

[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.

Synthetic peptide fragment (65-76) of monocyte chemotactic protein-1 (MCP-1) inhibits MCP-1 binding to heparin and possesses anti-inflammatory activity in stable angina patients after coronary stenting.

OBJECTIVE AND DESIGN: The peptide from C-terminal domain of MCP-1 (Ingramon) has been shown to inhibit monocyte migration and possess anti-inflammatory activity in animal models of inflammation and post-angioplasty restenosis. Here, we investigate the effect of Ingramon treatment on blood levels of acute-phase reactants and chemokines in patients after coronary stenting and the mechanisms of Ingramon anti-inflammatory activity. SUBJECTS: Eighty-seven patients with ischemic heart disease (IHD) who faced the necessity of coronary angiography (CA) were enrolled. In 67 patients, one-stage coronary stenting was performed; 33 of them were treated with Ingramon in addition to standard therapy. Twenty patients underwent CA only. METHODS: High-sensitivity C-reactive protein (hsCRP) and fibrinogen blood levels were detected routinely. The chemokine concentration in plasma was measured by enzyme-linked immunosorbent assay (ELISA) or cytometric bead array-based immunoassay. Intracellular Ca(2+) levels and cell surface integrin exposure were assayed by flow cytometry. MCP-1 dimerization was studied by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). MCP-1-heparin binding was assessed with a biosensor and ELISA. RESULTS AND CONCLUSIONS: Ingramon treatment was accompanied by less pronounced elevation of hsCRP and fibrinogen levels and decreased MCP-1 concentration in plasma in patients after coronary stenting. Ingramon had no effect on MCP-1 interaction with cell receptors or MCP-1 dimerization, but inhibited MCP-1 binding to heparin. The anti-inflammatory activity of the peptide may be mediated by an impaired chemokine interaction with glycosaminoglycans.

Changes in the concentration of monocytic chemotaxic protein-1 in patients with unstable angina treated with arixtra.

The time course of inflammatory reaction markers in the blood of patients with unstable angina was studied during therapy including arixtra. Plasma concentration of monocytic chemotaxic protein-1 (MCP-1) decreased on days 2 and 3 in patients receiving arixtra and a trend to an increase in MCP-1 concentration was observed on day 7 after the drug was discontinued. After 1 month, MCP-1 level decreased in all patients. The concentration of highly sensitive C-reactive protein also decreased 1 month after the disease onset; no changes in the concentrations of IL-8 and IL-2 receptor α-subunit were detected during these periods. It seems that arixtra is characterized by an anti-inflammatory effect manifesting by reduction of plasma chemokine MCP-1 concentration.

[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.

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 markers of regulatory CD4+CD25+foxp3+ cells in atherosclerotic plaques of human coronary arteries.

The content of marker foxp3 of regulatory T cells and chemokines in atherosclerotic plaques of human coronary arteries was measured by the polymerase chain reaction. In vitro migration of regulatory CD4(+)CD25(+)foxp3(+) cells in the CD4(+) lymphocyte population from healthy donors was studied after treatment with chemokines I-309, IP-10, and SDF-1. mRNA for the factor foxp3 and chemokines SDF-1, I-309, and MIP-1beta were found in the majority of samples from atherosclerotic plaques. SDF-1 induced maximum migratory response of CD4(+)CD25(+)foxp3(+) cells.

[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.

[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.

[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.

[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.

Interleukin-2- and phytohemagglutinin-activated proliferation of human T-lymphocytes is accompanied by stimulation of phosphoinositide turnover.

Interleukin-2 (IL-2) was more effective than phytohemagglutinin (PHA) in increasing the proliferative activity of human T-lymphocytes. Unlike PHA, IL-2 stimulated phosphoinositide turnover (PI turnover) only in those T-lymphocytes which had been preactivated by PHA with IL-2 and expressed the IL-2 receptors. The effect of PHA on PI turnover was, in general, stronger than that of IL-2. These results indicate that IL-2 and PHA-activated proliferation of human T-lymphocytes is accompanied by stimulation of PI turnover. However, IL-2-induced proliferative response may not be a direct consequence of PI turnover stimulation in these cells.

[Primary pulmonary hypertension: activation of sympathico-adrenal system and free radical oxidation. Positive effects of carvedilol therapy].

AIM: To study the condition of the sympathico-adrenal system (SAS), synthesis of cAMP dependent on beta2-adrenoreceptors and parameters of free radical oxidation in patients with primary pulmonary hypertension (PPH); to examine efficacy of non-selective beta- and alpha1-adrenoblocker carvedilol in PPH patients. MATERIAL AND METHODS: Twenty patients with PPH had 6-minute walk test, ECG monitoring with assessment of heart rhythm variability (HRV). Tests for noradrenalin and adrenalin concentration in blood plasma, cAMP synthesis by blood lymphocytes in basal conditions and under stimulation with isoproterenol and forskolin, free radical oxidation were made initially, 1 and 6 months later. Ten patients received carvedilol in addition to standard therapy, 10 patients served control. RESULTS: PPH patients had higher NA in the blood, low cAMP synthesis, high malonic aldehyde, low activity of glutathionperoxidase, increased activity of superoxidedismutase and catalase of erythrocytes. The most pronounced changes in the above parameters were observed in patients with PPH FC III-IV. HRV declined in progression of cardiac failure. 6-months of combined treatment with carvedilol increased the distance of 6-min walk. Carvedilol had no effect on HRV, it reduced NA, stimulated cAMP synthesis, demonstrated no antioxidant activity. CONCLUSION: In PPH there is activation of SAS and desensitization of beta2-AR cells, oxidative stress develops. Addition of carvedilol to standard therapy with PPH improves clinical condition due to adrenoblocking properties of the drug.

[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.

Alteration of beta-adrenoceptor function in hypertensive patients with different degrees of left ventricular hypertrophy.

Previous assessment of beta-adrenoceptor function has shown alterations in essential hypertension (EH). In the present study, we compared lymphocyte beta-adrenoceptor density (Bmax) and adenylate cyclase (AC) activity stimulated by l-isoproterenol, Gpp(NH)p, Gpp(NH)p + l-isoproterenol, and forskolin in 46 patients with EH and in 17 normotensive subjects. The patients with EH were divided into two subgroups, one with left ventricular myocardial mass (LVMM) less than 200 g and the second with LVMM greater than 200 g (according to Teichholz' formula). There were no significant differences in Bmax or in AC activity [basal and stimulated by Gpp(NH)p and forskolin] between the patients and the normotensive subjects. Adenylate cyclase activity stimulated by l-isoproterenol was reduced (% from basal AC) in the patients (P less than .05), and Bmax was increased only in the patients with left ventricular hypertrophy (P less than .05). There were no differences in AC activity between the two patient subgroups, and Bmax and AC activity did not correlate with blood pressure in either the patients or the normotensive subjects. Correlations were found between Bmax and LVMM (r = 0.38, P less than .02) and between Bmax and interventricular septum thickness (r = 0.412, P less than .02) among the patients. Thus, beta-adrenergic-mediated AC sensitivity to catecholamines is reduced in patients with EH and may represent a generalized defect in beta-receptor function in EH. Increased Bmax is likely to characterize more pronounced involvement of the target organs in the pathologic process associated with EH than is a higher blood pressure level.