[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.

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.

[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.

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.

[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.

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.

[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.

Intracellular signal cascade in CD4+ T-lymphocyte migration stimulated by interferon-gamma-inducible protein-10.

The intracellular signal cascades involved in chemokine-stimulated migration of in vitro activated human peripheral blood CD4+ T-lymphocytes were investigated. IP-10-mediated chemotactic response of lymphocytes was decreased in the presence of selective inhibitors of Src-kinases (by 40-45%), PI3-kinases (35-40%), and MAP-kinases ERK1/2 (35-40%) and p38 (20%). Combined addition of specific inhibitors of Src-kinases and PI3-kinases and inhibitors of Src-kinases and ERK1/2 MAP-kinases did not result in the further increase of the inhibitory effect, while the combined addition of specific inhibitors of PI3-kinases and ERK1/2 MAP-kinases decreased migration of CD4+ T-lymphocytes more effectively (by 55-60%) than any individual inhibitor. Immunoblotting analysis of activation of MAP-kinases ERK1/2 and p38 revealed increased level of phosphorylation of ERK1/2 and p38 MAP-kinases in the presence IP-10. Selective inhibitors of Src-kinases and PI3-kinases significantly inhibited phosphorylation of p38 but did not influence phosphorylation of ERK1/2 MAP-kinases. Our results suggest that Src-kinases, PI3-kinases, and ERK1/2 MAP-kinases are involved in intracellular signal cascade activated during IP-10-stimulated migration of T-lymphocytes, whereas p38 MAP-kinases do not participate in the migration process, although its activation induced by IP-10 depends on Src-kinases and PI3-kinases.

[Peptide fragments and structural analogues of chemokine MPC-1: synthesis and effect on the MPC-1-induced migration of monocytes]. / Peptidnye fragmenty khemokina MCP-1, ikh strukturnye analogi i ikh vliianie na MCP-1-oposredovannuiu migratsiiu monotsitarnykh kletok.

Fourteen fragments and structural analogues of chemokine MCP-1 were synthesized using the Fmoc strategy of solid phase peptide synthesis. The effect of synthesized peptides on the MCP-1-stimulated migration of mononuclear cells was examined. Both in vitro stimulants and inhibitors of the monocyte migration were found among the peptides. A possible participation of the C-terminal part of the MCP-1 molecule in the inhibition of the MCP-1-stimulated cell migration was found for the first time. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.

[Effect of matrix composition on chemotaxis response of monocytes stimulated by monocyte chemoattractant protein-1]. / Zavisimost' khemotaksicheskogo otveta monotsitarnykh kletok, stimulirovannykh monotsitarnym khemotaksicheskim belkom-1, ot sostava podlozhki.

MCP-1-stimulated chemotaxic response of monocytic cell line THP-1 and peripheral blood monocytes were investigated through extra cellular matrix proteins fibronectin, fibrinogen and fibrinogen degradation products. Cellular migration was significantly decreased in the presence of fibrinogen as compared with fibronectin. Fibrinogen proteolysis with plasmin generating D and E degradation products, resulted in increase of the chemotaxic response.