Macrophage Migration Inhibitory Factor Expression Levels Research Articles

Main Session: Gap junctions in myocardial disease

# 435 The role of macrophage migration inhibitory factor in the regulation of connexin 43 in atrial myocytes {#article-title-2} Introduction: Atrial fibrillation (AF) is the most common arrhythmia encountered in clinical practice. Recent findings have demonstrated a mechanistic link between inflammatory and the development of AF. Macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, controls the inflammatory ‘set point’ by regulating the release of other pro-inflammatory cytokines. Recent studies have revealed that Connexin 43 (Cx 43) not only is involved in the electrical conductivity between the cells, but also forms a complex with PKP2 and Na+ channel to affect the sodium current. The purpose of this study is to observe the role of MIF in the expression of Cx43, and the role of Cx43 in the regulation of calcium channel current in atrial myocytes. Methods: Cx 43 expression in human atrial tissues and HL-1 cells were determined by Western blot and Real time PCR. Whole-cell voltage-clamp recordings and Real time PCR were used to study the regulation and expression of ICa,L and ICa,T in HL-1 cells before and after Cx 43 siRNA knocking down. Results: Expression levels of Cx 43 mRNA and protein are significantly reduced while MIF expression levels were increased in patients with AF. In HL-1 cells, the depression of Cx 43 expression levels induced by rMIF was prevented by ERK 1/2 inhibitor U0126, but not Src inhibitor Genistein and PP1. Both ICa,L and ICa,T are reduced after Cx 43 siRNA knocking down, probably by down-regulation of L type calcium channel a1C subunit and T type calcium channel a1G subunit. Conclusion: These results implicate MIF in the pathological mechanism of AF, probably by decreasing Cx43 expression through the activation of ERK1/2 kinases in atrial myocytes. And ICa,L and ICa,T are regulated directly by Cx43, implicate involvement of Cx 43 in the electrical remodeling. # 436 Up-regulation of myocardial connexin-43 by melatonin: novel mechanism involved in its antiarrhythmic effects in spontaneously hypertensive rats. {#article-title-3} Background and Purpose: It has been reported that melatonin can regulate blood pressure in humans as well as animals but its antiarrhythmic effects are less known. Circulating levels of melatonin are decreased in patients with CVD as well as in spontaneously hypertensive rats (SHR). We examined whether treatment with melatonin will affect both propensity of SHR heart to malignant arrhythmias and electrical coupling protein, connexin-43 (Cx43). Design and Methods: SHR and healthy rats fed a standard rat chow received melatonin (40 mikrog/ml/day) for 4 weeks and were compared with untreated rats. Myocardial Cx43 gene and protein expression as well as its functional ( phosphorylated) forms were examined. PKC-epsilon (involved in Cx43 phosphorylation) and PKC-delta (involved in myocardial remodeling) as well as myocardial distribution of Cx43 were determined. Isolated Langendorff-perfused heart was used to test its susceptibility to electrically inducible ventricular fibrillation (VF). Key Results: Melatonin significantly reduced blood pressure but did not affect body, heart and left ventricular weights of SHR. Compared to healthy rats the threshold to induce sustained VF was lower in SHR (18.7+2 vs 29.0+5 mA, p<0.05) and significantly increased in melatonin-treated SHR to 33.0+4 mA. Myocardial Cx43 mRNA levels did not differ from healthy rats but total Cx43 protein and its phosphorylated forms were decreased in SHR heart left ventricle. In contrast, melatonin treatment resulted in up-regulation of Cx43 and attenuation of its abnormal distribution in SHR. Moreover, melatonin normalized myocardial PKC-delta expression and enhanced PKC-epsilon in SHR hearts. Conclusions: Results indicate antiarrhythmic effects of melatonin in hypertensive rats that can be, in part, attributed to up-regulation of myocardial Cx43 and PKC signaling. Findings support monitoring of melatonin levels and its supplementation in patients with CVD. This work was supported by VEGA 2/0046/12, SK-CZ-0027-11 and SKS grants.

Overexpression of macrophage migration inhibitory factor in adenoid cystic carcinoma: correlation with enhanced metastatic potential

Adenoid cystic carcinoma (ACC) is a malignant tumor frequently arising in salivary glands with poor long-term prognosis due to high rates of local recurrences and distant metastases. Macrophage migration inhibitory factor (MIF) is a multi-functional cytokine and has recently emerged as a pro-tumorigenic factor in various cancers. This study is designed to investigate the expression status and functional significance of MIF in ACC. Immunohistochemical staining was performed to evaluate the expression levels of MIF, HIF-1α, MMP-9, p53, and p-JNK in ACC tissues. In vitro, ACC-2 cells were exposed to recombinant human MIF (rMIF) or ISO-1 (an inhibitor of MIF) at different concentrations and times, followed by the detection of cell growth, viability, migration, and invasion, as well as the expression levels of several cellular signals. The immunohistochemical results demonstrated the overexpression of MIF in ACC tissues as well as its association with the distant metastasis. Further analyses showed a significant correlation between the staining of MIF and p-JNK. Moreover, the in vitro studies revealed that the treatment for ACC cells with ISO-1 significantly attenuated cell migratory and invasive capacity, as opposed to the limited promotive effects of rMIF. More importantly, MIF inhibition could cause the activation of JNK, correlating with the immunohistochemical findings on ACC tissues. The results suggest that MIF is likely to be an important player in the pathogenesis of ACC and may promote cancer metastasis, which possibly involves JNK inactivation. Further investigation of MIF-mediated molecular events may provide novel insights into the treatment for ACC.

Macrophage migration inhibitory factor, Toll-like receptor 4, and CD14 polymorphisms with altered expression levels in patients with ulcerative colitis

Ulcerative colitis is a multifactorial disease in which genetic factors play a major role. Functional mutations in the genes related to innate immune response exacerbate mucosal damage coupled with persistent inflammation. The cytokine macrophage migration inhibitory factor (MIF), CD14, and Toll-like receptor 4 (TLR4) are the central players with clearly defined roles in inflammation. The aim of this study was to investigate the association between MIF-173G > C, CD14-159C > T, and TLR4-299A > G polymorphisms and mononuclear cell expression in patients with ulcerative colitis (UC). Genotyping of MIF-173G > C, CD14-159C > T, and TLR4-299A > G polymorphisms was performed by amplification refractory mutation system-polymerase chain reaction and allele-specific amplification in 139 and 176 patients with UC and controls, respectively. Simultaneously, the expression levels of intracellular MIF, mCD14, and mTLR4 were determined in mononuclear cells using a flow cytometer. Polymorphisms in CD14-159C > T and TLR4-299A > G significantly affected mCD14 and mTLR4 expression levels and also increased susceptibility to UC. Although intracellular MIF expression levels differed among patient and control groups, the polymorphism in MIF 173G > C was not observed to be associated with a risk of UC.

Involvement of Src in L-type Ca2+ channel depression induced by macrophage migration inhibitory factor in atrial myocytes

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that controls inflammatory processes, and inflammation is known to play an important role in the pathogenesis of atrial fibrillation (AF). The present study sought to investigate whether MIF expression is responsible for the changes in L-type Ca2+ currents (ICa,L) seen in AF. Whole-cell voltage-clamp recordings and biochemical assays were used to study the regulation and expression of ICa,L in human atrial myocytes and in HL-1 cells. Basal ICa,L was reduced in AF compared to sinus rhythm (SR) controls, mRNA and protein levels of the pore-forming α1C subunit of L-type Ca2+ channel (LCC α1C) were also decreased, while MIF expression levels were increased in AF. Levels of Src and activated Src (p-Src Y416) were higher in AF than in SR. Treatment of atrial myocytes from a patient with SR with human recombinant MIF (rMIF) (40 nM, 1 h) was found to depress ICa,L amplitudes, while mouse rMIF (20 or 40 nM, 24 h) suppressed peak ICa,L in HL-1 cells by ∼69% and ∼83% in a concentration-dependent manner. Mouse rMIF impaired the time-dependent recovery from inactivation of ICa,L and down-regulated LCC α1C subunit levels. The depression of ICa,L and decrease of LCC protein levels induced by rMIF were prevented by the Src inhibitors genistein and PP1. These results implicate MIF in the electrical remodeling that accompanies AF, probably by decreasing ICa,L amplitudes through impairment of channel function, down-regulation of LCC α1C subunit levels, and the activation of c-Src kinases in atrial myocytes.

Overexpression of macrophage migration inhibitory factor induces angiogenesis and deteriorates prognosis after radical resection for hepatocellular carcinoma

Macrophage migration inhibitory factor (MIF) is a pivotal cytokine that regulates inflammatory and immune responses. Recently, many investigators reported that MIF is expressed highly in several tumors, including hepatocellular carcinoma (HCC). However, the role of MIF in tumor angiogenesis and patient prognosis has not been examined in patients with HCC. The authors evaluated MIF expression in 56 samples of HCC by Western blot analysis, and the results were correlated with clinicopathologic factors and patient prognosis. MIF localization was determined by immunohistochemical methods, and the results were compared with tumor microvessel density (MVD), as assessed by anti-CD34 antibody. Furthermore, to validate the role of MIF in angiogenesis, both MIF expression during culture of HCC cells (using the Hep3B, HepG2, and Huh7 cell lines) under hypoxic condition and the angiogenic potential of recombinant MIF in an in vitro angiogenic model were examined. Tumors with high MIF expression had high alpha-fetoprotein levels (P = 0.049) and frequent intrahepatic recurrence (P = 0.043). Immunohistochemical MIF scores had a significant correlation with MVD (P = 0.007). Patients who had tumors with high MIF expression levels had a significantly worse (P = 0.025) disease-free survival, and this finding remained significant as an independent prognostic factor in the multivariate analysis. Hep3B cells had high expression of MIF at 6 hours and 12 hours after hypoxic stress and exogenous MIF stimulated endothelial tube formation in in vitro angiogenesis. The current findings suggest that MIF expression may play a pivotal role in the dismal prognosis of patients with HCC that may be attributable to the modulation of angiogenesis.

Upregulation of macrophage migration inhibitory factor contributes to induced N-Myc expression by the activation of ERK signaling pathway and increased expression of interleukin-8 and VEGF in neuroblastoma.

Macrophage migration inhibitory factor (MIF) has been linked to fundamental processes such as control of cell proliferation, cell survival, angiogenesis, and tumor progression. The expression of MIF has been reported in several tumors. However, the precise role of MIF in tumor cells remains unclear. In the present study, we investigated the expression pattern and the function of MIF in neuroblastoma. Our results showed that intracellular MIF was upregulated in neuroblastoma tumor tissues and cell lines. MIF protein expression significantly correlated with the grade of tumor differentiation. In addition, we found that MIF induced a significant dose-dependent increase of vascular endothelial growth factor and interleukin-8 secretion. We also observed that an increased MIF expression level correlated with N-Myc protein (the N-myc oncogene product) expression in neuroblastoma tissues. MIF increased the expression of N-myc mRNA and N-Myc protein and induced N-Myc translocation from the cytoplasm to nucleus in neuroblastoma cell lines. MIF-induced N-Myc expression was found to be dependent on ERK signaling pathways. The inhibition of ERK activation reduced MIF-mediated N-Myc expression. These results suggest that MIF may contribute to the progression of neuroblastoma by (a) inducing N-Myc expression and (b) upregulating the expression of angiogenic factors.

Intracellular distribution of macrophage migration inhibitory factor predicts the prognosis of patients with adenocarcinoma of the lung

Macrophage migration inhibitory factor (MIF) is known to be a proinflammatory cytokine and glucocorticoid-induced immunomodulator as well as a regulator of tumor growth. Although positive and negative effects of MIF on tumor cell growth have been reported, to the authors' knowledge the precise role of MIF in tumorigenesis remains unclear. In the current study the authors assessed expression of MIF protein and mRNA in lung adenocarcinomas with regard to patient prognosis. Immunohistochemical analysis was performed on tissue specimens surgically obtained from 74 patients with primary lung adenocarcinoma (American Joint Committee on Cancer pathologic Stages I, II, and IIIa). In addition, expression of MIF mRNA in the cancerous tissue was investigated using in situ hybridization. Patient prognosis was evaluated with regard to MIF expression levels and its distribution was analyzed with the Kaplan-Meier method. MIF mRNA and MIF protein were observed in the bronchial epithelium, alveolar epithelium, vascular smooth muscle, and alveolar macrophages in the normal lung tissue. In tumor tissue from lung adenocarcinoma specimens, both MIF mRNA and protein were observed at much higher levels than in the normal alveolar epithelium. MIF protein was observed diffusely in the cytoplasm of tumor cells in all tumor specimens examined. MIF protein also was observed in the nuclei of tumor cells from 59 patients (79.7%), whereas it was not observed in the nuclei of tumor cells from 15 patients (20.3%). The patients without nuclear MIF expression had a worse prognosis compared with those patients with MIF expression in the nuclei (P = 0.04). The results of the current study suggest that intracellular MIF distribution predicts patient prognosis in individuals with adenocarcinoma of the lung.