Hypoxia-stimulated cardiac fibroblast production of IL-6 promotes myocardial fibrosis via the TGF-ß1 signaling pathway.

Interlukin-6 (IL-6) is a multifunctional cytokine produced by several cell types that has a role in fibrosis. Fibroblasts (FBs) maintain this underlying pathogenic change through regulation of IL-6 production; however, its potential functional role in regulating surrounding cellular structural changes during ischemic myocardial remodeling remains unexplored. Here, we generated FBs, cardiomyocytes (CMs), and blood vascular endothelial cells (ECs) from the ventricles of neonatal rats. IL-6 was then overexpressed in FBs and the cells were treated with IL-6 receptor inhibitor (IL6RI), TGF-ß1 receptor inhibitor (TßRI), or MMP2/MMP9 inhibitor (MMPI) using monoculture or coculture models under hypoxic conditions. The results indicate that overexpression of IL-6 is sufficient to induce myofibroblastic proliferation, differentiation, and fibrosis, probably via increased TGF-ß1-mediated MMP2/MMP3 signaling. The use of IL6RI, TßRI, or MMPI diminished these effects. In addition, IL-6 activated the apoptosis-associated factors Caspase3 and Smad3, and decreased the expression of anti-apoptotic factor Bcl2, resulting in apoptosis of CMs under hypoxic coculture: IL6RI or TßRI inhibited these effects. Unexpectedly, IL-6-overexpressing FBs significantly increased the angiogenesis of ECs, which involved significant increases in the expression of proangiogenic growth factors. Treatment of FBs with IL6RI or TßRI in coculture with ECs reduced the levels of secreted proangiogenic growth factors, and the angiogenesis of ECs was significantly downregulated. Thus, IL-6 functions in ischemic myocardial remodeling through multifunctional reprogramming of hypoxia-associated FBs towards fibrosis via upregulation of the TGF-ß1 signaling pathway.

CXCR6 deficiency attenuates pressure overload-induced monocytes migration and cardiac fibrosis through downregulating TNF-α-dependent MMP9 pathway.

An immerging role of TNF-α in collagen synthesis and cardiac fibrosis implies the significance of TNF-α production in the development of myocardial remodeling. Our previous study showed a reduction of TNF-α and attenuated cardiac remodeling in CXCR6 knockout (KO) mice after ischemia/reperfusion injury. However, the potential mechanism of TNF-α-mediated cardiac fibrosis with pressure overload has not been well elucidated. In the present study, we aim to investigate the role of CXCR6 in TNF-α release and myocardial remodeling in response to pressure overload. Pressure overload was performed by constriction of transverse aorta (TAC) surgery on CXCR6 KO mice and C57 wild-type (WT) counterparts. At 6 weeks after TAC, cardiac remodeling was assessed by echocardiography, cardiac TNF-α release and its type I receptor (TNFRI), were detected by ELISA and western blot, collagen genes Col1a1 (type I) and Col3a1 (type III) were examined by real-time PCR. Compared with CXCR6 WT mice, CXCR6 KO mice exhibited less cardiac dysfunction, reduced expression of TNFRI, Col1a1 and Col3a. In vitro, we confirmed that CXCR6 deficiency led to reduced homing and infiltration of CD11b(+) monocytes, which contributed to attenuated TNF-α release in myocardium. Furthermore, TNFRI antagonist pretreatment blocked AT1 receptor signaling and NOX4 expression, reduced collagen synthesis, and blunted the activity of MMP9 in CXCR6 WT mice after TAC, but these were not observed in CXCR6 KO mice. In the present work, we propose a mechanism that CXCR6 is essential for pressure overload-mediated myocardial recruitment of monocytes, which contributes to cardiac fibrosis through TNF-α-dependent MMP9 activation and collagen synthesis.

[Construction and identification of mouse wildtype-syndecan-1 and unshedding-syndecan-1 expressing vector].

AIM: To construct the expressing vector pcDNA3.0- wildtype-syndecan-1(WT-sdc1) and pcDNA3.0-unshedding-syndecan-1(uS-sdc1), and to explore the expression in vitro. METHODS: (1)The mouse WT-sdc1 DNA was successfully amplified by PCR and then cloned into pcDNA3.0. uS-sdc1 was construct by Gene splicing by overlap extension- PCR on the basis of WT-sdc1. The two vectors confirmed by DNA sequencing. (2)there are 4 groups in our research: control group, pcDNA3.0 transfected group, WT-sdc1 transfected group and uS-sdc1 transfected group. each vecter was transfected into IEC-6 cells by Lipofectamine(TM);2000. RT-PCR, Western blot and Dot blot were performed to detect the expression of syndecan-1 before and after stimulation of phorbol 12-myristate 13-acetate (PMA) for 15 min. RESULTS: The vector WT-sdc1 and uS-sdc1 were successfully constructed although an non-sense mutation was in uS-sdc1. Compared to control and pcDNA3.0 transfected groups, WT-sdc1 and uS-sdc1 groups showed a significant increase in the expression of syndecan-1 in both mRNA and protein levels. In response to the stimulation of PMA, the expression of syndecan-1 was down-regulated at the protein levels but not mRNA levels. CONCLUSION: The WT-sdc1 and uS-sdc1 are successfully constructed, which lays the foundation for further studying of syndecan-1 in gastrointestinal inflammation.

[Value of deep small-bowel endoscopy in the diagnosis of Crohn's disease].

OBJECTIVE: To evaluate the value of deep small-bowel endoscopy (DSBE) in the diagnosis of Crohns disease (CD). METHODS: The endoscopic and clinical data of 54 patients with CD receiving capsule endoscopy (CE) and double-balloon enteroscopy (DBE) between January, 2004 and December, 2008 were summarized and analyzed retrospectively. RESULTS: The main indications for DSBE in our series were suspected CD (42.6%) and obscure gastrointestinal bleeding (25.9%). DSBE was obviously superior to barium imaging. The detection rate of CD was significantly higher with DSBE (92.6%) than with ileocolonoscopy (75.9%, P=0.017), and DSBE provides much more detailed descriptions of specific endoscopic features such as segmental distribution and lumen changes. DSBE significantly improve the diagnostic efficiency, giving priority to offer a guide and raise suspected diagnosis for CD. CONCLUSION: DSBE is a valuable modality for detecting CD lesions in the jejunum and ileum and for evaluating lesion involvement and severity. The combination with a comprehensive analysis of routine imaging findings, gastro endoscopy, and clinical data can further enhance the diagnostic efficiency of DSBE.

[Changes in the biological behaviors of colon cancer cells under simulated hypoxia in vitro].

OBJECTIVE: To investigate the reactivity of colon cancer cell line SW480 and CD133(+) SW480 subsets to hypoxia in vitro and the changes in the expressions of anti-apoptosis and angiogenesis genes. METHODS: SW480 cells was subjected to CoCl(2) exposure at varying concentrations and for different time lengths to induce hypoxia, and the protein expression of hypoxia induced factor 1α (HIF-1α) was detected by Western blotting. The CD133(+) SW480 cells were sorted by magnetic activated cell sorting (MACS) and their proportion was assayed by flow cytometry (FCM). The CD133(+) SW480 subsets were exposed to CoCl(2) at the optimal concentration with exposure time selected in terms of HIF-1α level, and their tumor stem cell sphere formation ability was evaluated. Real-time PCR was used to compare the mRNA expression levels of the surface markers of colon cancer stem cells (CD133 and PROM1), survivin, and vascular endothelial growth factor (VEGF). RESULTS: Exposure to 200 µmol/L CoCl(2) for 8 h resulted in the highest HIF-1α expression in SW480 cells, but the same exposure failed to induce HIF-1α expression in CD133(+) SW480 subsets. The CD133(+) SW480 subsets, after CoCl(2)-induced hypoxia, showed significantly enhanced ability of cell sphere formation. Hypoxia of SW480 cells caused significant increases in CD133, survivin and VEGF mRNA levels by 1.607∓0.103, 2.745∓0.370 and 3.798∓0.091 folds, respectively (P<0.05). CONCLUSION: CoCl(2) can simulate hypoxia in colon cancer cells in vitro to induce stable HIF-1α expression, which is concentration- and time-dependent. The hypoxia-stimulated tumor stem sells show an enhanced sphere formation and anti-apoptotic and anti-angiogenic abilities.

[In vivo cardiac magnetic resonance imaging of superparamagnetic iron oxides-labeled mesenchymal stem cells in swines].

OBJECTIVE: To explore the feasibility of magnetic resonance imaging (MR) on detecting transplanted nanometer small superparamagnetic iron oxides (SPIO) labeled mesenchymal stem cells (MSCs) in swine model with acute myocardial infarction (MI). METHODS: MSCs isolated from swine were incubated with nanometer SPIO for 24 hours and the third-passage MSCs were labeled with DNA dye 4'-6-diamidino-2-phenylindole (DAPI) and aliphatic red fluorescent dye PKH(26)-GL. Presence of small particles of SPIO in MSCs was assessed by Prussian Blue staining and electron microscopy. Three animals in each group received SPIO-labeled MSCs (5 x 10(5); 1 x 10(6); 2 x 10(6)) and MSCs without SPIO (1 x 10(6)) injections into the infarcted myocardium approximately 1 hour following left anterior descending coronary artery. MRI (1.5-T) was performed 20 to 24 hours post infarction in all animals and the animals were subsequently sacrificed for histology 1 hour post MRI. RESULTS: In vitro Prussian Blue staining and electron microscopy examination revealed numerous iron particles in the cytoplasm of MSCs. Low signal intensity spots with the scanning T(2)(*)WI-Flash 2d sequence were detected in all SPIO-MSCs but not in SPIO-negative-MSCs injected myocardial sites in vivo with the clinical 1.5 T scanner. Prussian blue, DAPI and PKH(26) positive cells were detected histologically in sections corresponding to low signal intensity spots area shown on MRI. CONCLUSION: Magnetically labeled MSCs transplanted in myocardial ischemia area of swine can be visualized in vivo with a clinical 1.5-T MRI and could be used for tracking SPIO-MSCs clinically.

[Observation on the safety: clinical trail on intracoronary autologous bone marrow mononuclear cells transplantation for acute myocardial infarction].

OBJECTIVE: To investigate the safety of autologous bone marrow mononuclear cell (BM-MNCs) transplantation by intracoronary infusion in patients with acute myocardial infarction (AMI). METHODS: One hundred and eighty-four patients with AMI treated with percutaneous coronary intervention (PCI) were randomized in a 1:1 way to either intracoronary transplantation of autologous BM-MNCs (n = 92) right after PCI or to sodium chloride concluding heparin (controlled, n = 92) via a micro infusion catheter. In the process of the intracoronary infusion of BM-MNCs, the complications should be recorded, which were aberration reflect (including of pale, syncope, nausea, hypotension and shock), deterioration of angina or heart failure, arrhythmias (including of bradycardia, sinus arrest or atrial ventricular block or ventricular fibrillation), embolism etc. Body temperature, blood pressure and heart rates should be monitored during the first week after transplantation. Holter, coronary angiography and ultrasonic cardiography were performed at the designed time points. Main heart accidents, restenosis and tumor were recorded during 2-years follow up. RESULTS: During the period of bone marrow puncture and intracoronary infusion of BM-MNCs, few patients occurred pale, dizziness, bradycardia and hypotension, which were transient and due to vagus reflect. No stem cell-related arrhythmias, deterioration of angina were noted. In BM-MNCs group one patient developed in-stent reocclusion in one week after transplantation, five developed in-stent restenosis during further follow-up 30 months, which were similar with control group. There were no deaths, major adverse cardiac events, tumor and other late adverse events during follow-up period in both groups. CONCLUSION: Intracoronary transplantation of autologous BM-MNCs in the acute phase after AMI is feasible and seems safe in the 30 months of follow-up.

[Long term follow-up on emergent intracoronary autologous bone marrow mononuclear cell transplantation for acute inferior-wall myocardial infarction].

OBJECTIVE: To investigate the effects of emergent intracoronary autologous bone marrow mononuclear cell (BM-MNC) transplantation on left ventricular function and myocardium lesion area in patients with first acute inferior-wall myocardial infarction. METHODS: Forty patients with first onset of acute inferior-wall myocardial infarction, 28 males and 12 females, aged < or = 75, treated with emergent percutaneous coronary intervention (PCI) were randomly divided into 2 equal groups: group undergoing intracoronary transplantation of autologous BM-MNC via a micro-catheter right after PCI (BM-MNC group), and control group receiving normal saline and heparin. Blood routine examination, myocardium zymogram, and serum high sensitive C reactive protein (hsCRP) were detected, and 24-hour dynamic electrocardiography, delayed-enhancement myocardial magnetic resonance imaging (CMR), and angiography of the coronary artery and left ventricle were conducted before the transplantation and immediately, 1 week, and 6 months after transplantation. RESULTS: CMR showed that 6 months later the left ventricular ejection fraction (LVEF) of the control group was 47.9% +/- 6.7%, significantly higher than that 1 week later (43.4% +/- 6.7%, P = 0.001), and the LVEF of the BM-MNC group 6 months later was 51.5% +/- 5.2%, significantly higher than that 1 week later (44.5% +/- 7.1%, P = 0.001; however, the absolute change of LVEF (DeltaLVEF) of the BM-MNC group was 6.95% +/- 3.33%, significantly higher than that of the control group (4.05% +/- 1.68%, P = 0.047). Six months later the myocardial lesion area of the BM-MNC group decreased more significantly in comparison with the control group. Nevertheless, there was no difference in change of left ventricular end diastolic volume (LVEDV) between these two groups. The serum hsCRP 48 h after transplantation of the BM-MNC group was 2.8 g/L +/- 0.8 g/L, significantly lower than that before transplantation (13.4 g/L +/- 3.6 g/L, P < 0.001). No severe clinical events, such death, recurrent cardiac infarction, malignant arrhythmia, occur in these 2 groups. CONCLUSION: Emergent intracoronary transplantation of autologous BM-MNC in patients with acute inferior-wall myocardial infarction improves the left ventricular function and myocardial infusion, minimizes the myocardial lesion area significantly.

[Transplantation of autologous bone marrow mononuclear cells on patients with idiopathic dilated cardiomyopathy: early results on effect and security].

OBJECTIVE: The aim of this study is to identify short-term result of cell transplantation in idiopathic dilated cardiomyopathy (IDC) patients who were treated by intracoronary transplantation of autologous mononuclear bone marrow cells (BMCs) in addition to standard therapy. METHODS: Based on given standard therapy, eighteen patients with idiopathic dilated cardiomyopathy were enrolled and divided into transplantation group and control group. The clinical characteristics of two groups were comparable. Among these patients, 10 patients were performed percutaneous coronary autologous BMCs transplantation. Blood routine test, hepatic function, renal function, glucose, triglyceride (TG), cholesterol, low density cholesterol (LDL), high density cholesterol (HDL), uric acid (UA) and high sensitive C-reactive protein (hsCRP) were measured at the time point of pre-operation and some time after transplantation. All patients were monitored under ultrasonic cardiography, Holter, six-minute-walk test and magnetic resonance imaging over a period of at least 6 months. Annual hospital days were recorded during two-year follow-up. RESULTS: Blood routine test, hepatic function, renal function, glucose, TG, cholesterol, LDL, HDL, UA and hsCRP had no significant differences among 48 hours, 3 months and 6 months after transplantation compared with control and pre-transplantation (P > 0.05). Six-minute-walk distance elevated significantly six months after BMCs transplantation compared with control and pre-transplantation [(494.3 +/- 62.8) m vs (307.2 +/- 75.0) m, (321.5 +/- 63.7) m, P < 0.05]. Left ventricular ejection fraction (LVEF) and the sizes of LVEDd had no significant changes compared with that of control and pre-transplantation (P > 0.05). Myocardium lesion area measured by (MRI) seemed decrease in transplantation group compared with that of control and pre-operation [(4.96 +/- 0.47) cm(2) vs (5.12 +/- 0.54) cm(2), (5.02 +/- 0.39) cm(2), P > 0.05], but there was no significance. None of proarrhythmias and side effects had been observed around transplantation and 2 years follow-up. There was no significant difference in survival between two groups in 2 years follow-up. Interestingly, annual hospital day in BMCs transplantation patients was significantly shorter than that in control group [(30.2 +/- 11.2) d vs (43.6 +/- 9.8) d, P < 0.05]. CONCLUSIONS: Autologous bone marrow mononuclear cells transplantation can prolong six-minute-walk, decrease re-hospitalization rate, elevate exercise ability and help to improve cardiac function in patients with IDC. In addition, it was demonstrated that cell transplantation is safe.

[The role of first pass and delayed contrast-enhancement magnetic resonance imaging in patients with myocardial infarction].

OBJECTIVE: To evaluate the role of contrast-enhancement magnetic resonance imaging (CeMRI) in patients with myocardial infarction (MI). METHODS: There were twenty-three patients enrolled in this study. After dynamic observation, there were 20 patients who were diagnosed as MI. All those patients underwent coronary artery angiography and CeMRI. MRI was performed with a 1.5-T magnet (AVANTO, SIMENS). After tagged images were acquired, the patients received an intravenous bolus of 0.1 mmol/kg Gd-DTPA at a rate of 5 ml/s. A first-pass perfusion scan was acquired simultaneously with a bolus injection. A second bolus of 0.3 mmol/kg Gd-DTPA was given following the first-pass images. Delayed images were acquired 5 minutes after the second bolus by using an inversion-recovery prepared gated fast-gradient echo-pulse sequence. RESULTS: Hypoenhancement was seen in 20 patients at the first-pass perfusion at the myocardial infarction site, while hyperenhancement was seen at delayed CeMRI. Myocardial infarction area in delayed CeMRI was 16.58% +/- 9.73%, which was correlated positively with peak CK and cTnT (r = 0.821, P < 0.01 and r = 0.565, P < 0.05), respectively. The ejection fraction (EF) detected by MRI was 0.46 +/- 0.13, while the left ventricular EF (LVEF) detected by left ventriculography was 0.49 +/- 0.16. There was no difference between two parameters. CONCLUSIONS: CeMRI may play an important role in the diagnosis and prognosis of patients with MI.