Monocyte Chemotactic Protein-1 Protein Research Articles

Modulation of Monocyte Chemotactic Protein-1 Expression During Lipopolysaccharide-Induced Preterm Delivery in the Pregnant Mouse

Preterm delivery is often associated with increased cytokine and chemokine production. These studies characterize the expression of the chemokine monocyte chemotactic protein-1 (MCP-1) in mice during lipopolysaccharide (LPS)-induced preterm delivery. Uterine and other tissues were harvested from CD-1 mice on gestational day 15 after intrauterine LPS injection. Quantitative real-time reverse-transcriptase polymerase chain reactions determined MCP-1 and toll-like receptor 4 (TLR4) mRNA expression during the 24 hours after LPS. MCP-1 protein expression was determined using a cytokine/chemokine protein array, enzyme-linked immunosorbant assay, and immunohistochemistry. Intrauterine LPS injection caused preterm delivery in CD-1 mice between 12 and 24 hours. Expression of MCP-1 mRNA significantly increased at 2 and 6 hours, while TLR4 expression did not significantly change over 24 hours. The MCP-1 protein levels peaked by 2 to 6 hours in maternal serum, liver, lung, kidney, and uterus. Immunohistochemistry confirmed MCP-1 in the myometrium and endometrium. These studies provide evidence suggesting that MCP-1 potentially plays an important role during the proinflammatory immune response, leading to preterm labor in the mouse.

(−)-Epigallocatechin-3-gallate inhibits monocyte chemotactic protein-1 expression in endothelial cells via blocking NF-κB signaling

Monocyte chemotactic protein-1 (MCP-1) is a potent chemoattractant for monocytes and plays a key role in various inflammatory responses, including atherosclerosis. In this study, we examined the effect of (−)-epigallocatechin-3-gallate (EGCG), a major green tea catechin, on the expression of MCP-1 in human endothelial ECV304 cells. EGCG markedly inhibited the phorbol 12-myristate 13-acetate (PMA)-induced MCP-1 mRNA and protein levels in a dose-dependent manner. EGCG was also found to reduce the MCP-1 transcriptional activity. The upregulation of MCP-1 by PMA was significantly inhibited by blockade of P38 mitogen-activated protein kinase (MAPK) and NF-κB, but not by blockade of extracellular-signal-regulated kinase and c-Jun N-terminal kinase pathway. Furthermore, The PMA-induced p38 MAPK and NF-κB activation were obviously attenuated after pretreating ECV304 cells with EGCG. The conditioned media from the endothelial ECV304 cells treated with PMA could remarkably stimulate the migration of THP-1 monocytes and this effect was partially abrogated by MCP-1 neutralizing antibodies. Moreover, the media from the EGCG-pretreated ECV304 cells lost the stimulatory activity for THP-1 migration. These results suggest that EGCG may exert an anti-inflammatory effect in endothelial cells by controlling MCP-1 expression, at least in part, mediated through the suppression of p38 MAPK and NF-κB activation.

Resveratrol, a Polyphenolic Phytostilbene, Inhibits Endothelial Monocyte Chemotactic Protein-1 Synthesis and Secretion

Background/Aims: Resveratrol is a naturally occurring polyphenol phytoestrogen and one of several constituents of red wine thought to be cardioprotective. We investigated the effect of resveratrol on the expression of the atherogenic chemokine, monocyte chemotactic protein-1 (MCP-1). Methods: Human umbilical vein endothelial cells were stimulated with interleukin-1β (IL-1β) in the absence or presence of resveratrol. MCP-1 levels were determined by ELISA and MCP-1 mRNA was measured. Results: Resveratrol (1–100 µM) dose-dependently inhibited IL-1β-stimulated MCP-1 secretion, with ∼45% inhibition at 50 µM resveratrol. This was a Gi-protein- and NO-dependent effect. Resveratrol also significantly inhibited MCP-1 gene expression in a Gi-protein-dependent but NO-independent manner. While resveratrol had no effect on MCP-1 mRNA degradation, it inhibited MCP-1 promoter activity and reduced nuclear factor ĸB and activator protein-1 binding activity induced by IL-1β. Moreover, while hemoxygenase-1 (HO-1) expression was induced by resveratrol in human umbilical vein endothelial cells, neither treatment with the HO-1 inhibitor tin-protoporphyrin IX nor siRNA-directed knockdown of HO-1 had any effect on the inhibition of MCP-1 mRNA or protein secretion by resveratrol. Conclusion: These data demonstrate an inhibitory effect of resveratrol on MCP-1 synthesis and secretion, mediated via distinct signaling pathways. The inhibition of MCP-1 may represent a novel cardioprotective mechanism of resveratrol.

Effect of Berberine on Monocyte Chemotactic Protein-1 and Cytokine-Induced Neutrophil Chemoattractant-1 Expression in Rat Lipopolysaccharide-Induced Uveitis

Purpose: The aims of this study were to examine the in vivo effects of berberine, an alkaloid isolated from some medicinal herbs, on monocyte chemotactic protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant-1 (CINC-1) expression in rat lipopolysaccharide (LPS)-induced uveitis. Methods: LPS was injected intraperitoneally. Berberine was orally administered. MCP-1 mRNA and CINC-1 mRNA were measured by semiquantitative reverse-transcription polymerase chain reaction and real-time polymerase chain reaction. MCP-1 and CINC-1 protein concentration in the aqueous humor were measured by enzyme-linked immunosorbent assay. Histopathologic study was performed in the anterior ocular segments. Results: Berberine dose-dependently inhibited LPS-induced MCP-1 mRNA and CINC-1 mRNA expression of the iris-ciliary body. The alkaloid inhibited chemokines, protein and cell levels in the aqueous humor in rats stimulated with LPS. On histopathologic study, the inflammatory cell infiltration was diminished by the berberine treatment. Conclusions: These findings indicate that berberine dose-dependently inhibited the expression of MCP-1 and CINC-1 induced by LPS and diminished the anterior uveitis.

Role of monocyte chemotactic protein-1 and nuclear factor kappa B in the pathogenesis of proliferative diabetic retinopathy

Intraocular concentrations of monocyte chemotactic protein-1 (MCP-1) are increased in proliferative diabetic retinopathy (PDR). Nuclear factor kappa B (NF-κB) is a transcription factor, and NF-κB binding site is located in gene promoter of MCP-1. This study was conducted to investigate the potential role of MCP-1 and NF-κB in the pathogenesis of PDR. Epiretinal membrane (ERM) samples were obtained during vitrectomy from 19 eyes with PDR and 16 eyes with idiopathic ERM. They were processed for RT–PCR analysis. Four PDR ERMs were processed for immunohistochemical analysis. In addition, cultured Müller glial cells were stimulated with glycated albumin or high glucose. After the stimulation, we examined nuclear localization of NF-κB p50, MCP-1 promoter activity, and MCP-1 concentration in culture media. MCP-1 mRNA expression was significantly higher in PDR (74%) than in idiopathic ERMs (38%) ( P < 0.05). Immunohistochemical analysis revealed that MCP-1 protein is colocalized with active form of NF-κB p50. In vitro studies demonstrated that glycated albumin or high glucose induces NF-κB activation followed by up-regulation of MCP-1 promoter activity and protein production in glial cells. These results suggest that MCP-1, under the regulation of NF-κB, is involved in the pathogenesis of PDR.

Effect of berberrubine on interleukin-8 and monocyte chemotactic protein-1 expression in human retinal pigment epithelial cell line

We examined the effects of berberrubine, a protoberberine alkaloid, on interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) expression in a human retinal pigment epithelial cell line (ARPE-19) stimulated with interleukin-1β (IL-1β) or tumor necrosis factor α (TNF-α). ARPE-19 cells were cultured to confluence. Berberrubine and IL-1β or TNF-α were added to the medium. IL-8 and MCP-1 protein concentrations were measured using enzyme-linked immunosorbent assay. IL-8 and MCP-1 mRNA were measured by real time polymerase chain reaction. Nuclear factor κB (NF-κB) translocation was examined by immunofluorescent staining/microscopy. Berberrubine dose-dependently inhibited IL-8 and MCP-1 protein levels in the media and mRNA expression of the cells stimulated with IL-1β or TNF-α. Immunofluorescent staining/microscopy of NF-κB in the nucleus of unstimulated cells was faint (51 ± 14 arbitrary units). Fluorescein was dense (215 ± 42 or 170 ± 24 arbitrary units, respectively) 30 min after stimulation with IL-1β or TNF-α and was decreased to 62 ± 18 or 47 ± 16 arbitrary units, respectively, by berberrubine. Berberrubine dose-dependently inhibited IL-8 and MCP-1 expression and protein secretion induced by IL-1β or TNF-α. Possibly, the effect on chemotactic factors may be via suppression of NF-κB translocation.

Expression of Monocyte Chemotactic Protein-1 in Human Endometrial Cancer Cells and the Effect of Treatment with Tamoxifen or Buserelin

Monocyte chemotactic protein-1 (MCP-1) is an important determinant of macrophage infiltration in tumours. This study investigated the effect of tamoxifen and the gonadotrophin-releasing hormone agonist buserelin on MCP-1 in the human endometrial cancer cell line EFE-184. Reverse transcription polymerase chain reaction and Western blot analysis were used to determine MCP-1 mRNA and protein expression, respectively. Immunoreactive MCP-1 in the cell culture media was quantified by enzyme-linked immunosorbent assay. Tamoxifen inhibited MCP-1 mRNA and protein expression in endometrial cancer cells and inhibited MCP-1 secretion in a time- and dose-dependent manner at concentrations of 10(-7) to 10(-5) M. Buserelin had no significant effect on MCP-1 mRNA and protein expression. These results suggest that tamoxifen directly inhibits the expression of MCP-1 in this cell line by blocking the MCP-1 signalling pathways. These findings may contribute to the understanding of the mechanisms underlying the different effects of tamoxifen and gonadotrophin-releasing hormone agonists in the treatment of endometrial cancer.

Differential response of vascular smooth muscle cells to oxidized LDL in mouse strains with different atherosclerosis susceptibility

Oxidized low-density lipoprotein (LDL) has numerous atherogenic properties, including induction of inflammatory genes, and vascular smooth muscle cells (VSMC) are involved in the development of atherosclerosis. In this study, we examined whether variations of VSMC in the capacity to oxidize LDL or in response to minimally modified LDL (MM-LDL) constitute a genetic component in atherosclerosis. VSMC were isolated from the aorta of two inbred mouse strains C57BL/6J (B6) and C3H, which differ markedly in susceptibility to atherosclerosis. LDL oxidation was assessed by measuring thiobarbituric acid-reactive substance (TBARS) production. Responses to MM-LDL were evaluated by examining the expression of inflammatory genes involved atherosclerosis, including monocyte chemotactic protein-1 (MCP-1) and vascular cell adhesion molecule-1 (VCAM-1), and an oxidant stress gene, heme oxygenase-1 (HO-1). VSMC from the two strains exhibited a comparable ability to transform native LDL to oxidized LDL, whereas their response to MM-LDL differed markedly. MM-LDL resulted in dramatic induction of MCP-1, VCAM-1, and HO-1 mRNAs in the cells from B6 mice but exerted little effect in cells from C3H mice. MCP-1 and soluble VCAM-1 protein levels in conditioned media were measured by ELISA. B6 cells produced significantly more MCP-1 and VCAM-1 proteins in response to MM-LDL than C3H cells. These data suggest that variation in the response of VSMC to oxidized LDL may contribute to the difference between B6 and C3H mice in atherosclerosis susceptibility.

Effect of Berberine on Interleukin 8 and Monocyte Chemotactic Protein 1 Expression in a Human Retinal Pigment Epithelial Cell Line

Purpose: The aims of this study were to examine the effects of berberine, an alkaloid isolated from some medicinal herbs, on interleukin 8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) expression in a human retinal pigment epithelial cell line (ARPE-19) stimulated with interleukin 1β (IL-1β) or tumor necrosis factor α (TNF-α). Methods: ARPE-19 cells were cultured to confluence. Berberine and IL-1β or TNF-α were added to the medium. IL-8 mRNA and MCP-1 mRNA were measured by semiquantitative reverse-transcription polymerase chain reaction and real-time polymerase chain reaction. IL-8 and MCP-1 protein concentrations in the media were measured using enzyme-linked immunosorbent assay. Results: Berberine dose-dependently inhibited IL-8 mRNA and MCP-1 mRNA expression of the cells and protein levels in the media stimulated with IL-1β or TNF-α. Conclusion: These findings indicate that berberine dose-dependently inhibited the expression of IL-8 and MCP-1 induced by IL-1β or TNF-α.

Scoparone inhibits PMA-induced IL-8 and MCP-1 production through suppression of NF-κB activation in U937 cells

Scoparone is a major component of the shoot of Artemisia capillaris (Compositae), which has been used for the treatment of hepatitis and biliary tract infection in oriental countries. In this study, the effects of scoparone on the expression of interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) and activation of nuclear factor-kappaB (NF-κB) were examined in U937 human monocytes activated with phorbol 12-myristate 13-acetate (PMA). Scoparone (5–100 μM) had no cytotoxic effect in unstimulated cells and concentration-dependently reversed PMA-induced toxicity in the cells stimulated with PMA. Scoparone concentration-dependently reduced the release of IL-8 and MCP-1 protein and expression of IL-8 and MCP-1 mRNA levels induced by PMA. Moreover, scoparone inhibited the levels of NF-κB-DNA complex and NF-κB activity in the cells stimulated with PMA in a concentration-dependent manner. Scoparone dose-dependently inhibited the phosphorylation of IκBα and nuclear translocation of NF-κB 1 p50, RelA p65, and c-Rel p75. These data suggest that scoparone may inhibit the expression of chemokines (IL-8 and MCP-1) in PMA-stimulated U937 cells and a potential mechanism of scoparone may be inhibition of NF-κB activation, which is linked to inhibition of NF-κB subunits (NF-κB 1 p50, RelA p65, and c-Rel p75) translocation via suppression of IκBα phosphorylation.

Immortalized Mouse Inner Ear Cell Lines Demonstrate a Role for Chemokines in Promoting the Growth of Developing Statoacoustic Ganglion Neurons

The target-derived factors necessary for promoting initial outgrowth from the statoacoustic ganglion (SAG) to the inner ear have not been fully characterized. In the present study, conditioned medium from embryonic Immortomouse inner ear cell lines that maintain many characteristics of developing inner ear sensory epithelia were screened for neurite-promoting activity. Conditioned medium found to be positive for promoting SAG neurite outgrowth and neuronal survival was then tested for the presence of chemokines, molecules that have not previously been investigated for promoting SAG outgrowth. One candidate molecule, monocyte chemotactic protein 1 (MCP-1), was detected in the conditioned medium and subsequently localized to mouse hair cells by immunocytochemistry. In vitro studies demonstrated that function-blocking MCP-1 antibodies decreased the amount of SAG neurite outgrowth induced by the conditioned medium and that subsequent addition of MCP-1 protein was able to promote outgrowth when added to the antibody-treated conditioned medium. The use of the Immortomouse cell lines proved valuable in identifying this candidate cofactor that promotes outgrowth of early-stage SAG nerve fibers and is expressed in embryonic hair cells.

PI 3 kinase-dependent Akt kinase and PKCε independently regulate interferon-γ-induced STAT1α serine phosphorylation to induce monocyte chemotactic protein-1 expression

Monocyte chemotactic protein-1 (MCP-1) recruits activated phagocytes to the site of tissue injury. Interferon-γ (IFN-γ) present in the microenvironment of glomerulus acts on mesangial cells to induce local production of MCP-1. The mechanism by which IFN-γ stimulates expression of MCP-1 is not clear. We therefore examined the role of PI 3 kinase signaling in regulating the IFN-γ-induced MCP-1 expression in mesangial cells. Blocking PI 3 kinase activity with Ly294002 attenuated IFN-γ-induced MCP-1 protein and mRNA expression. IFN-γ increased Akt kinase activity in a PI 3 kinase-dependent manner. Expression of dominant negative Akt kinase inhibited serine phosphorylation of STAT1α, without any effect on its tyrosine phosphorylation, and decreased IFN-γ-induced expression of MCP-1. These data for the first time indicate a role for PI 3 kinase-dependent Akt kinase in MCP-1 expression. We have recently shown that along with Akt, PKCε is a downstream target of PI 3 kinase in IFN-γ signaling. Similar to dominant negative Akt kinase, dominant negative PKCε also inhibited serine phosphorylation of STAT1α without any effect on tyrosine phosphorylation. Dominant negative PKCε also abrogated MAPK activity, resulting in decrease in IFN-γ-induced MCP-1 expression. Furthermore, Akt and PKCε are present together in a signaling complex. IFN-γ had no effect on this complex formation, but did increase PKCε-associated Akt kinase activity. PKCε did not regulate IFN-γ-induced Akt kinase. Finally, expression of dominant negative Akt kinase blocked IFN-γ-stimulated MAPK activation. These data provide the first evidence that PI 3 kinase-dependent Akt and PKCε activation independently regulate MAPK activity and serine phosphorylation of STAT1α to increase expression of MCP-1.

Simian Human Immunodeficiency Virus-Associated Pneumonia Correlates with Increased Expression of MCP-1, CXCL10, and Viral RNA in the Lungs of Rhesus Macaques

Pulmonary disorders are the most frequent cause of death in HIV-1-infected individuals with AIDS and remain important even in the current era of potent antiretroviral therapy. Macaques infected with Simian/Human Immunodeficiency Virus (SHIV) develop pulmonary disease and concurrent opportunistic infections similar to those observed in HIV-infected individuals, thereby providing an excellent working model to elucidate the pathogenesis of the human lung disease. Since chemokines play a crucial role in the recruitment of inflammatory cells to tissues, we investigated the relationship between respiratory disease and the levels of chemokines, monocyte chemotactic protein-1 (MCP-1) and CXCL10, in the lungs of SHIV-infected rhesus macaques. We found that lung pathology in infected macaques was closely associated with overexpression of MCP-1 and CXCL10. In addition, these chemokines could, in part, be responsible for the recruitment of inflammatory cells infiltrating into the diseased lungs as demonstrated by chemotactic assays. Lung pathology and increased levels of MCP-1 and CXCL10 correlated with high viral loads in the lung parenchyma. Using confocal microscopy, we identified SHIV-infected macrophages as the major producers of MCP-1 and CXCL10 in the diseased lungs. These data suggest that chemokine overexpression plays an important role in the pathogenesis of SHIV-associated pulmonary disease in macaques.

Effect of Alpha-Melanocyte-Stimulating Hormone on Interleukin 8 and Monocyte Chemotactic Protein 1 Expression in a Human Retinal Pigment Epithelial Cell Line

Purpose: To examine melanocortin receptor (from MC-1 to MC-5) mRNA and the effect of α-melanocyte-stimulating hormone (α-MSH) on interleukin 8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) expression in a human retinal pigment epithelial cell line (ARPE-19) stimulated with IL-1β or tumor necrosis factor α (TNF-α). Methods: Expressions of MC-1 to MC-5 mRNA were examined by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). α-MSH and IL-1β or TNF-α were added to serum-free medium. IL-8 and MCP-1 mRNA were measured by real-time PCR. IL-8 and MCP-1 protein concentrations were measured using enzyme-linked immunosorbent assay. Nuclear factor ĸB (NF-ĸB) translocation was examined by immunofluorescent staining/microscopy. Results: MC-1 to MC-5 receptor mRNA was expressed in unstimulated cells. IL-1β stimulated IL-8 and MCP-1 mRNA at 6 h. TNF-α stimulated IL-8 and MCP-1 mRNA expression at 1.5 and 3 h. α-MSH (10^–14 to 10^–10M) inhibited IL-8 and MCP-1 mRNA expression in the cells stimulated with IL-1β or TNF-α. α-MSH inhibited IL-1β or TNF-α-stimulated IL-8 and MCP-1 protein levels in the media. Immunofluorescent staining/microscopy of NF-ĸB in the nucleus was dense 30 min after stimulation with IL-1β or TNF-α and was decreased by α-MSH. Conclusions: ARPE-19 cells had MC-1 mRNA. α-MSH inhibited IL-8 and MCP-1 expression and protein secretion. Possibly, the effect on chemotactic factors may be via suppression of NF-ĸB translocation.

Effect of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) on IL-6, IL-8, and MCP-1 Expression in Human Retinal Pigment Epithelial Cell Line

Purpose: To examine pituitary adenylate cyclase–activating polypeptide (PACAP) receptors (PAC1, VPAC1, and VPAC2) mRNA and the effect of PACAP on interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1) expression in human retinal pigment epithelial cell line (ARPE-19) stimulated with interleukin-1β (IL-1β). Methods: Expression of PACAP receptor mRNA was examined by reverse transcription polymerase chain reaction (RT-PCR). PACAP and IL-1β were added to serum-free medium. IL-6, IL-8, and MCP-1 mRNA were measured by real-time PCR. IL-6, IL-8, and MCP-1 protein concentrations were measured using enzyme-linked immunosorbent assay. Nuclear factor κB (NF-κB) translocation was examined by immunofluorescence. Results: PAC1 and VCAP1 receptors mRNA were expressed in unstimulated cells. VCAP2 mRNA was expressed in cells stimulated with IL-1β. IL-1β stimulated IL-6, IL-8, and MCP-1 mRNA expression and protein levels. PACAP (10− 7 to 10− 6 M) inhibited IL-1β –stimulated IL-6, IL-8, and MCP-1 mRNA and protein levels. Immunofluorescence of NF-κB in the nucleus was dense 30 min after stimulation with IL-1β, and it was decreased by PACAP. Conclusions: ARPE-19 cells had PACAP receptors mRNA. PACAP inhibited IL-6, IL-8, and MCP-1 expression and protein secretion. Possibly, the effect on cytokines may be via suppression of NF-κB translocation.

The -2518 Promotor Polymorphism in the MCP-1 Gene Is Associated with Systemic Sclerosis

Factors influencing the initiation or progression of sclerosis in patients with systemic sclerosis (SSc) are poorly understood. Monocyte chemotactic protein-1 (MCP-1) is a potent chemokine, which is upregulated in fibroblasts during development of sclerosis. In this study, we investigated the frequency of the functional -2518G MCP-1 promoter polymorphism in 18 patients with SSc and 139 healthy controls. In the lesional skin of the same SSc patients, expression of MCP-1 protein was examined by immunohistochemistry. To investigate a genotype/phenotype correlation, basal as well as tumor necrosis factor (TNF)-induced MCP-1 expression was analyzed in fibroblasts isolated from the skin of SSc patients with different MCP-1 genotypes by quantitative RT-PCR and ELISA. Genotyping for the -2518 (A/G) MCP-1 promotor polymorphism showed that GG homozygotes were significantly more frequent in patients with SSc than in controls (28%vs 6%). Results of immunohistochemistry revealed that MCP-1 was expressed in keratinocytes, infiltrating inflammatory cells, fibroblasts, and endothelial cells in scleroderma skin, whereas normal control skin showed no MCP-1 expression. MCP-1 expression in fibroblasts from GG-homozygote individuals tended to be stronger as compared to AG or AA genotypes. Furthermore, basal as well as TNF-induced MCP-1 expression of fibroblasts isolated from a GG-homozygote SSc patient was significantly higher than MCP-1 expression of fibroblasts isolated from heterozygote or AA-homozygote donors. The A -2518G polymorphism of the MCP-1 gene appears to affect MCP-1 expression of skin fibroblasts of patients with SSc. In accordance, the G/G genotype may predispose patients to SSc.

Macrophage depletion reduces monocyte chemotactic protein-1 and transforming growth factor-β 1 in healing rat vein grafts

Objective We previously showed that treatment with liposomally encapsulated dichloromethylene bisphosphonate reduces intimal hyperplasia development and macrophage accumulation in a rat epigastric vein to femoral artery model of intimal hyperplasia. Our objective in this study was to determine the effect of liposomally encapsulated dichloromethylene bisphosphonate on the expression of two cytokines essential to neointimal development, monocyte chemotactic protein-1 (MCP-1) and transforming growth factor-β 1 (TGF-β). Methods We injected rats both 2 days preoperatively and 2 weeks postoperatively with liposomally encapsulated dichloromethylene bisphosphonate (Lip-Clod), liposomally encapsulated phosphate-buffered saline solution (Vector), or phosphate-buffered saline solution (PBS), and harvested the grafts at 1 and 4 weeks. In the perianastomotic region, MCP-1 and TGF-β protein expression in the total graft cross-section and in the neointima was determined with immunohistochemistry. In whole-graft lysates, MCP-1 and TGF-β protein were determined with an enzyme-linked immunosorbent assay, and messenger RNA expression was determined with reverse transcription quantitative polymerase chain reaction. Results Lip-Clod treatment reduced intimal hyperplasia when compared with Vector or PBS treatment. These reductions were significant ( P < .05) at both time points. When compared with the PBS treatment, at 1 week but not at 4 weeks Lip-Clod reduced both MCP-1 and TGF-β protein ( P ≤ .01 and P ≤ .006) in the perianastomotic region of vein grafts. In whole-graft lysates, no significant difference was seen in MCP-1 protein at either time point; however, TGF-β protein expression was significantly reduced at both 1 and 4 weeks ( P = .02 and P = .004). Message analysis in whole-graft lysates at 1 week showed that MCP-1 message expression increased in the Lip-Clod group compared with the PBS group ( P = .02), but no significant differences among groups for TGF-β message levels. Results with Vector were often intermediate to results with Lip-Clod and PBS. Conclusion The major effect of Lip-Clod treatment on TGF-β and MCP-1 protein levels in the perianastomotic region is observed at 1 week, and macrophage depletion with Lip-Clod inhibits graft neointimal hyperplasia and TGF-β protein expression in whole-graft lysates at 1 and 4 weeks. These results support the concept that the infiltrating macrophages contribute a significant portion of the cytokines that facilitate intimal hyperplasia and that reducing these cytokines early after grafting influences the development of intimal hyperplasia at later time points. Clinical relevance All vascular surgeons have patients who have undergone a technically satisfying vein graft, only to have the bypass fail during the first year due to perianastomotic intimal hyperplasia (IH). We hypothesize that vein graft IH is analogous to aberrant wound healing. Central to wound healing is the recruitment of macrophages with their cytokines. This work raises the question whether clinical strategies designed to either decrease macrophages or the cytokines released by macrophages at the time of vein graft placement will be efficacious for limiting the development of IH.

Effect of hypoxia on monocyte chemotactic protein-1 (MCP-1) gene expression induced by Interleukin-1� in human synovial fibroblasts

Rheumatoid arthritis (RA) synovial membrane is characterized by leucocyte infiltration and secretion of chemotactic and proinflammatory factors. Since hypoxia is an important pathogenic factor in inflamed synovium, we examined the effects of hypoxia on monocyte chemotactic protein-1 (MCP-1) expression in human rheumatoid arthritis synovial fibroblasts (RASF) under IL-1beta-stimulated and -unstimulated conditions.Synovial fibroblasts were isolated from RA, osteoarthritis (OA) and healthy knee joints and subjected to hypoxia or/and IL-1beta treatment. MCP-1 expression and protein secretion were measured by real-time PCR and ELISA, respectively.Hypoxia reduces MCP-1 expression and protein secretion in RASF. The same response to hypoxia was found in OA and healthy SF cultures. Treatment with actinomycin D showed that hypoxic down-regulation of MCP-1 expression was due to a decrease in transcription, since the half-life of MCP-1 mRNA was unchanged. A cycloheximide study demonstrated that de novo protein synthesis was not required for the hypoxic effect. The decrease in MCP-1 expression by hypoxia was mimicked by cobalt chloride in unstimulated RASF with no effect on IL-1beta-activated MCP-1, suggesting differences in the signaling mechanisms. The analysis of IkappaB degradation and NF-kappaB translocation revealed that hypoxia did not affect IL-1beta activation of NF-kappaB.Hypoxia regulates MCP-1 expression under both basal and cytokine-stimulated conditions, suggesting that reduced oxygen supply is an important factor that mediates chemotaxis of monocytes to the area of inflammation.

Effect of hypoxia on monocyte chemotactic protein-1 (MCP-1) gene expression induced by Interleukin-1beta in human synovial fibroblasts.

Rheumatoid arthritis (RA) synovial membrane is characterized by leucocyte infiltration and secretion of chemotactic and proinflammatory factors. Since hypoxia is an important pathogenic factor in inflamed synovium, we examined the effects of hypoxia on monocyte chemotactic protein-1 (MCP-1) expression in human rheumatoid arthritis synovial fibroblasts (RASF) under IL-1beta-stimulated and -unstimulated conditions. Synovial fibroblasts were isolated from RA, osteoarthritis (OA) and healthy knee joints and subjected to hypoxia or/and IL-1beta treatment. MCP-1 expression and protein secretion were measured by real-time PCR and ELISA, respectively. Hypoxia reduces MCP-1 expression and protein secretion in RASF. The same response to hypoxia was found in OA and healthy SF cultures. Treatment with actinomycin D showed that hypoxic down-regulation of MCP-1 expression was due to a decrease in transcription, since the half-life of MCP-1 mRNA was unchanged. A cycloheximide study demonstrated that de novo protein synthesis was not required for the hypoxic effect. The decrease in MCP-1 expression by hypoxia was mimicked by cobalt chloride in unstimulated RASF with no effect on IL-1beta-activated MCP-1, suggesting differences in the signaling mechanisms. The analysis of IkappaB degradation and NF-kappaB translocation revealed that hypoxia did not affect IL-1beta activation of NF-kappaB. Hypoxia regulates MCP-1 expression under both basal and cytokine-stimulated conditions, suggesting that reduced oxygen supply is an important factor that mediates chemotaxis of monocytes to the area of inflammation.

Factor Xa and thrombin, but not factor VIIa, elicit specific cellular responses in dermal fibroblasts

Coagulation factors (F)VIIa, FXa and thrombin are implicated in cellular responses in vascular, mesenchymal and inflammatory cells. Fibroblasts are the most abundant cells in connective tissue, and damage to blood vessels places coagulation factors in contact with these and other cell types. To investigate cellular responses of primary dermal fibroblasts to FVIIa, FXa and thrombin by following changes in expression of candidate proteins: monocyte chemotactic protein-1 (MCP-1), interleukin-8 (IL-8), interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF), and to determine the expression of receptors implicated in signaling by these coagulation factors. Steady-state mRNA levels were quantified by RNase protection assay, and protein secretion by ELISA. PAR gene expression was assessed by ribonuclease protection assay and conventional and quantitative reverse-transcription-polymerase chain reaction. FVIIa did not induce the candidate genes. In contrast, FXa and thrombin induced MCP-1 mRNA and protein secretion strongly, IL-8 moderately, and IL-6 weakly. Neither FXa nor thrombin induced VEGF mRNA or protein secretion, although FXa induced VEGF protein secretion in lung fibroblasts. Comparison of the presence of candidate receptors in the two fibroblast subtypes demonstrated higher levels of PAR-1 and PAR-3 in lung fibroblasts relative to their dermal counterparts and the additional expression of PAR-2. FXa and thrombin induce expression of MCP-1, IL-8 and IL-6, and distribution and expression of PARs on dermal fibroblasts is reduced relative to their lung counterparts. Tissue origin may influence the cellular response of fibroblasts to coagulation proteases.