Midkine drives cardiac inflammation by promoting neutrophil trafficking and NETosis in myocarditis

Heart failure due to dilated cardiomyopathy is frequently caused by myocarditis. However, the pathogenesis of myocarditis remains incompletely understood. Here, we report the presence of neutrophil extracellular traps (NETs) in cardiac tissue of patients and mice with myocarditis. Inhibition of NET formation in experimental autoimmune myocarditis (EAM) of mice substantially reduces inflammation in the acute phase of the disease. Targeting the cytokine midkine (MK), which mediates NET formation in vitro, not only attenuates NET formation in vivo and the infiltration of polymorphonuclear neutrophils (PMNs) but also reduces fibrosis and preserves systolic function during EAM. Low-density lipoprotein receptor-related protein 1 (LRP1) acts as the functionally relevant receptor for MK-induced PMN recruitment as well as NET formation. In summary, NETosis substantially contributes to the pathogenesis of myocarditis and drives cardiac inflammation, probably via MK, which promotes PMN trafficking and NETosis. Thus, MK as well as NETs may represent novel therapeutic targets for the treatment of cardiac inflammation

Strict regulation of CAIX(G250/MN) by HIF-1alpha in clear cell renal cell carcinoma.

Contains fulltext : 58051.pdf (publisher's version ) (Closed access)Renal cell carcinoma of the clear cell type (ccRCC) is associated with loss of functional von Hippel-Lindau (VHL) protein and high, homogeneous expression of the G250MN protein, an isoenzyme of the carbonic anhydrase family. High expression of G250MN is found in all ccRCCs, but not in most normal tissues, including normal human kidney. We specifically studied the mechanism of transcriptional regulation of the CAIXG250 gene in RCC. Previous studies identified Sp1 and hypoxia-inducible factor (HIF) as main regulatory transcription factors of G250MN in various non-RCC backgrounds. However, G250MN regulation in RCC has not been studied and may be differently regulated in view of the HIF accumulation under normoxic conditions due to VHL mutations. Transient transfection of different G250MN promoter constructs revealed strong promoter activity in G250MN -positive RCC cell lines, but no activity in G250MN -negative cell lines. DNase-I footprint and band-shift analysis demonstrated that Sp1 and HIF-1alpha proteins in nuclear extracts of RCC cells bind to the CAIX promoter and mutations in the most proximal Sp1 binding element or HIF binding element completely abolished CAIX promoter activity, indicating their critical importance for the activation of G250 expression in RCC. A close correlation between HIF-1alpha expression and G250MN expression was observed. In contrast, no relationship between HIF-2alpha expression and G250MN was seen. The participation of cofactor CBP/p300 in the regulation of G250 transcription was shown. In conclusion, HIF-1alpha and Sp1, in combination with CBP/p300, are crucial elements for G250MN expression in ccRCC, and CAIXG250 can be regarded as a unique HIF-1alpha target gene in ccRCC

The Role of 1.5 Tesla MRI and Anesthetic Regimen Concerning Cardiac Analysis in Mice with Cardiomyopathy

Accurate assessment of left ventricular function in rodent models is essential for the evaluation of new therapeutic approaches for cardiac diseases. In our study, we provide new insights regarding the role of a 1.5 Tesla (T) magnetic resonance imaging (MRI) device and different anesthetic regimens on data validity. As dedicated small animal MRI and echocardiographic devices are not broadly available, we evaluated whether monitoring cardiac function in small rodents with a clinical 1.5 T MRI device is feasible. On a clinical electrocardiogram (ECG) synchronized 1.5 T MRI scanner we therefore studied cardiac function parameters of mice with chronic virus-induced cardiomyopathy. Thus, reduced left ventricular ejection fraction (LVEF) could be verified compared to healthy controls. However, our results showed a high variability. First, anesthesia with medetomidine, midazolam and fentanyl (MMF) led to depressed cardiac function parameters and more variability than isoflurane gas inhalation anesthesia, especially at high concentrations. Furthermore, calculation of an average ejection fraction value from sequenced scans significantly reduced the variance of the results. To sum up, we introduce the clinical 1.5 T MRI device as a new tool for effective analysis of left ventricular function in mice with cardiomyopathy. Besides, we suggest isoflurane gas inhalation anesthesia at high concentrations for variance reduction and recommend calculation of an average ejection fraction value from multiple sequenced MRI scans to provide valid data and a solid basis for further clinical testing

Molecular cloning and immunogenicity of renal cell carcinoma-associated antigen G250

The molecular cloning of the cDNA and gene encoding the renal cell carcinoma (RCC)-associated protein G250 is described. This protein is one of the best markers for clear cell RCC: all clear-cell RCC express this protein, whereas no expression can be detected in normal kidney and most other normal tissue. Antibody studies have indicated that this molecule might serve as a therapeutic target. In view of the induction/up-regulation of G250 antigen in RCC, its restricted tissue expression and its possible role in therapy, we set out to molecularly define the G250 antigen, which we identified as a transmembrane protein identical to the tumor-associated antigen MN/CAIX. We determined, by FISH analysis, that the G250/MN/CAIX gene is located on chromosome 9p12-13. In view of the relative immunogenicity of RCC, we investigated whether the G250 antigen can be recognized by TIL derived from RCC patients. The initial characterization of 18 different TIL cultures suggests that anti-G250 reactivity is rare. (C) 2000 Wiley-Liss, Inc

Anesthesia with isoflurane reduces variability of data.

<p>Compared to MMF, isoflurane is able to reduce the variability of the results. Additionally, isoflurane at higher concentrations than commonly used might further reduce the variability of heart rate and heart rate dependent values such as cardiac output and cardiac index. However, compared with lower isoflurane concentrations (1.75%), high isoflurane concentrations (3%) show no significant cardiodepressive effects. A) Shows percentual standard deviations of single cycle values, and B) of mean values (average of 3 consecutive cycles). (C) Shows percentual standard deviations for cardiac output and cardiac index obtained with either MMF, 1.75% isoflurane or 3% isoflurane. D) Shows heart rate, E) cardiac output and F) cardiac index under different anesthetic regimens. EF = LVEF, EDV = end diastolic volume, ESV = end systolic volume, SV = stroke volume, HR = heart rate, CO = cardiac output, CI = cardiac index. 100 µl MMF: n = 6, 1.75% isoflurane and 3% isoflurane: n = 7. ***: p<0.001.</p

Comparison of MRI devices and results on cardiac function in small animals and in humans.

#<p><i> = LVEF evaluation in human populations;</i></p><p><i>y = yes, n = no; SD = standard deviation;</i></p><p><i>* = mean relative SD in % of LVEF of the control group only.</i></p

Calculation of an average LVEF value reduces variability of data independently from the anesthetic regimen.

<p>For evaluation of cardiac function parameters, 3 consecutive cycles were measured and averaged. Mean values showed a reduced variability compared to values obtained by single cycle measurements. A) Shows single cycle and mean values for the group anesthetized with 1.75% isoflurane, B) for the group anesthetized with 3% isoflurane and C) for the group anesthetized with 100 µl MMF. EF = LVEF, EDV = end diastolic volume, ESV = end systolic volume, SV = stroke volume, HR = heart rate, CO = cardiac output, CI = cardiac index. 100 µl MMF: n = 6, 1.75% isoflurane and 3% isoflurane: n = 7.</p

Left ventricular function parameters of CVB3-infected mice compared to age matched healthy controls at baseline.

<p><i>Values are presented as mean and standard deviation, along with the corresponding p values.</i></p><p><i>(Student's t test).</i></p

Echocardiography confirms values obtained by MRI.

<p>A) Echocardiography was able to reproduce the low LVEF values we obtained from MMF anesthetized mice investigated by MRI. B) Heart rates (HR) and C) cardiac output (CO) measured by our Vevo2100 system did not significantly differ from those in the MRI study. D) Anesthesia with isoflurane revealed the cardiodepressive effects of MMF. n.s.: not significant; **: p<0.01; ***: p<0.001.</p

Left ventricular function parameters in healthy mice under different anesthetic regimen.

<p><i>1.75% = anesthesia with 1.75% isoflurane; 3% = 3% isoflurane. MMF = 100 µl MMF injected i.p. for maintenance of anesthesia. RR = relative reduction comparing MMF and 1.75% isoflurane and MMF and 3% isoflurane. Values are presented as mean, along with the corresponding p values (Student's t test).</i></p><p><i>*: p<0.05,</i></p><p><i>**: p<0.01,</i></p><p><i>***: p<0.001.</i></p><p><i>No statistical differences were observed between the 1.75% group and the 3% group.</i></p