Arlo Radtke

Locally Different Endothelial Nitric Oxide Synthase Protein Levels in Ascending Aortic Aneurysms of Bicuspid and Tricuspid Aortic Valve

Aims. Dysregulated expression of the endothelial nitric oxide synthase (eNOS) is observed in aortic aneurysms associated with bicuspid aortic valve (BAV). We determined eNOS protein levels in various areas in ascending aortic aneurysms. Methods and Results. Aneurysmal specimens were collected from 19 patients, 14 with BAV and 5 with tricuspid aortic valve (TAV). ENOS protein levels were measured in the outer curve (convexity), the opposite side (concavity), the distal and above the sinotubular junction (proximal) aneurysm. Cultured aortic cells were treated with NO synthesis inhibitor L-NAME and the amounts of 35 apoptosis-related proteins were determined. In patients with BAV, eNOS levels were significantly lower in the proximal aorta than in the concavity and distal aorta. ENOS protein levels were also lower in the convexity than in the concavity. While the convexity and distal aorta showed similar eNOS protein levels in BAV and TAV patients, levels were higher in TAV proximal aorta. Inhibition of NO synthesis in aneurysmal aortic cells by L-NAME led to a cytosolic increase in the levels of mitochondrial serine protease HTRA2/Omi. Conclusion. ENOS protein levels were varied at different areas of the aneurysmal aorta. The dysregulation of nitric oxide can lead to an increase in proapoptotic HTRA2/Omi.

Serum Levels of MicroRNA miR-371a-3p: A Sensitive and Specific New Biomarker for Germ Cell Tumours

BACKGROUND Clinical management of germ cell tumours (GCTs) relies on monitoring of serum tumour markers. However, the markers α-fetoprotein (AFP), the β-subunit of human chorionic gonadotropin (bHCG), and lactate dehydrogenase (LDH) are expressed in <60% of GCT cases. OBJECTIVE To test the utility of the microRNAs (miRNAs) miR-371a-3p, miR-372-3p, miR-373-3p, and miR-367-3p as sensitive and specific GCT serum biomarkers. DESIGN, SETTING, AND PARTICIPANTS Serum levels of miRNAs were measured in 166 consecutive patients with GCT before and after treatment and in 106 male controls. In the first 50 consecutive patients, all four miRNAs were measured. In the main study, only the most sensitive miRNA was further analysed. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS The specificity and sensitivity of the four miRNAs were studied using receiver operating characteristic curves. miRNA sensitivities were compared to those of classical markers. Statistical cross-comparisons of miRNA levels for GCT subgroups and controls were performed at various time points during treatment. RESULTS AND LIMITATIONS Overall, miR-371a-3p performed best, with 88.7% sensitivity (95% confidence interval [CI] 82.5-93.3%) and 93.4% specificity (95% CI 86.9-97.3%) and an area under the curve of 0.94, outperforming AFP, bHCG, and LDH (combined sensitivity 50%). According to Kernel density estimation, the sensitivity and specificity were 86.3% and 92.5%, respectively. miR-371a-3p levels dropped to normal after completion of treatment. The miRNA levels correlated with treatment failure and relapse. Teratoma did not express miR-371a-3p. CONCLUSIONS The miRNA miR-371a-3p is a specific and sensitive novel serum GCT biomarker that accurately correlates with disease activity. Validation of this test in a large-scale prospective study is needed. PATIENT SUMMARY: miR-371a-3p is a novel serum marker for germ cell tumours that is expressed by 88.7% of patients and thus is far more sensitive and specific than classical serum markers. It correlates with tumour burden and treatment results. Validation in a large patient cohort is needed.

Elevation of Matrix Metalloproteinases in Different Areas of Ascending Aortic Aneurysms in Patients with Bicuspid and Tricuspid Aortic Valves

Our aim is to investigate the elevation of matrix proteins in tissues obtained from distal, above the sinotubular junction (proximal), concave, and convex sites of aneurysms in the ascending aorta using a simultaneous multiplex protein detection system. Tissues were collected from 41 patients with ascending aortic aneurysms. A total of 31 patients had a bicuspid aortic valve (BAV), whereas 10 had a tricuspid aortic valve (TAV). Concave and convex aortic site samples were collected from all patients, whereas proximal and distal convexity samples were obtained from 19 patients with BAV and 7 patients with TAV. Simultaneous detection of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) was performed at each of the four aortic sites. MMP-2 levels were higher in the concave aortic sites than in the convex aortic sites. In contrast, MMP-8 levels were higher in the convex sites than in the concave sites, as were MMP-9 levels. In both BAV and TAV patients, TIMP-3 levels were higher in the concave sites than in the convex sites. However, TIMP-2 and TIMP-4 levels were significantly elevated in the sinotubular proximal aorta of BAV patients. Simultaneous detection of MMPs and TIMPs revealed different levels at different aortic sites in the same patient.

Fibroid explants reveal a higher sensitivity against MDM2-inhibitor nutlin-3 than matching myometrium

BackgroundSpontaneous cessation of growth is a frequent finding in uterine fibroids. Increasing evidence suggests an important role of cellular senescence in this growth control. Deciphering the underlying mechanisms of growth control that can be expected not only to shed light on the biology of the tumors but also to identify novel therapeutic targets.MethodsWe have analyzed uterine leiomyomas and matching normal tissue for the expression of p14Arf and used explants to see if reducing the MDM2 activity using the small-molecule inhibitor nutlin-3 can induce p53 and activate genes involved in senescence and/or apoptosis. For these studies quantitative real-time RT-PCR, Western blots, and immunohistochemistry were used. Statistical analyses were performed using the students t test.ResultsAn in depth analysis of 52 fibroids along with matching myometrium from 31 patients revealed in almost all cases a higher expression of p14Arf in the tumors than in the matching normal tissue. In tissue explants, treatment with the MDM2 inhibitor nutlin-3 induced apoptosis as well as senescence as revealed by a dose-dependent increase of the expression of BAX as well as of p21, respectively. Simultaneously, the expression of the proliferation marker Ki-67 drastically decreased. Western-blot analysis identified an increase of the p53 level as the most likely reason for the increased activity of its downstream markers BAX and p21. Because as a rule fibroids express much higher levels of p14Arf, a major negative regulator of MDM2, than matching myometrium it was then analyzed if fibroids are more sensitive against nutlin-3 treatment than matching myometrium. We were able to show that in most fibroids analyzed a higher sensibility than that of matching myometrium was noted with a corresponding increase of the p53 immunopositivity of the fibroid samples compared to those from myometrium.ConclusionsThe results show that uterine fibroids represent a cell population of advanced cellular age compared to matching myometrium. Moreover, the data point to members of the p53-network as to potential novel therapeutic targets for the treatment of uterine fibroids.

Serum Levels of MicroRNA371a-3p: A Highly Sensitive Tool for Diagnosing and Staging Testicular Germ Cell Tumours: A Clinical Case Series

Introduction: MicroRNA (miR)371a-3p was suggested to be a sensitive and specific new serum biomarker of germ cell tumours (GCTs); however, its clinical usefulness remains unproven. Patients, Methods: In 312 consecutive cases with various testicular diseases, serum levels of miR371a-3p were measured. Measurement results became available only after completion of treatment. Five patients with testicular seminoma were selected for review because of unanticipated clinical courses. Results: In each two patients, elevated miR levels heralded undetected primary testicular GCT and metastases despite inconclusive radiological findings. In one case, a normal miR371a-3p level correctly pointed to the absence of metastases contrary to clinical assessment. In all cases, knowledge about the miR371a-3p levels would have altered the clinical management. Conclusions: These cases highlight the exceptional usefulness of the new GCT biomarker. In contrast to classical markers, miR371a-3p can identify primary testicular GCT. The marker can aid in clinical decision making in cases with ambiguous clinical findings.

Generation and Characterization of Vascular Smooth Muscle Cell Lines Derived from a Patient with a Bicuspid Aortic Valve

Thoracic aortic dilation is the most common malformation of the proximal aorta and is responsible for 1%–2% of all deaths in industrialized countries. In approximately 50% of patients with a bicuspid aortic valve (BAV), dilation of any or all segments of the aorta occurs. BAV patients with aortic dilation show an increased incidence of cultured vascular smooth muscle cell (VSMC) loss. In this study, VSMC, isolated from the ascending aorta of BAV, was treated with Simian virus 40 to generate a BAV-originated VSMC cell line. To exclude any genomic DNA or cross-contamination, highly polymorphic short tandem repeats of the cells were profiled. The cells were then characterized using flow cytometry and karyotyping. The WG-59 cell line created is the first reported VSMC cell line isolated from a BAV patient. Using an RT2 Profiler PCR Array, genes within the TGFβ/BMP family that are dependent on losartan treatment were identified. Endoglin was found to be among the regulated genes and was downregulated in WG-59 cells following treatment with different losartan concentrations, when compared to untreated WG-59 cells.

Can germ cell neoplasia in situ be diagnosed by measuring serum levels of microRNA371a-3p?

PurposeDiagnosing germ cell neoplasia in situ (GCNis) can detect germ cell tumours (GCTs) at the pre-invasive stage. To date, testicular biopsy with the potential of surgical complications is the only way of safely diagnosing GCNis. Recently, microRNAs (miRs) 371-3, and miR 367 were shown to be valuable serum biomarkers of GCTs. We explored the usefulness of these candidate miRs as a marker for GCNis.Methods27 patients with GCNis and no concomitant GCT were enrolled. All patients underwent measuring serum levels of miR-371a-3p and miR-367-3p before treatment, 11 had repeat measurement after treatment, 2 also had testicular vein blood examinations. Serum levels were measured by quantitative PCR. In addition, four orchiectomy specimens of patients with GCT were examined immunohistochemically and by in situ hybridization (ISH) with a probe specific for miR-371a-3p to look for the presence of this miR in GCNis cells.ResultsThe median serum level of miR-371a-3p was significantly higher in patients with GCNis than in controls, miR-367 levels were not elevated. Overall, 14 patients (51.9%) had elevated serum levels of miR-371a-3p. The highest levels were found in patients with bilateral GCNis. Levels in testicular vein serum were elevated in both of the cases. After treatment, all elevated levels dropped to normal. In two orchiectomy specimens, miR-371a-3p was detected by ISH in GCNis cells.ConclusionsMeasuring miR-371a-3p serum levels can replace control biopsies after treatment of GCNis. In addition, the test can guide clinical decision making regarding the need of testicular biopsy in cases suspicious of GCNis.

MicroRNA 208 in Atrial Fibrillation

MicroRNAs (miRNAs) are critical regulators of most major cellular processes and seem to play a vital role in the pathogenesis of numerous diseases including atrial fibrillation, the most commonly encountered cardiac rhythm disorder. Among the several miRNAs that appear to be involved in pathogenesis of atrial fibrillation, miRNA 208a is linked to fibrosis and proper cardiac conduction. We quantified the expression levels of miRNA 208a in left atrial appendage tissue of patients with paroxysmal (n=2), persistent (n=10), and long-standing persistent (n=7) arrhythmia using quantitative PCR. In paroxysmal atrial fibrillation, miRNA 208a was expressed moderately, whereas the expression was enhanced in persistent atrial fibrillation and significantly reduced in long-standing persistent atrial fibrillation. The difference between persistent and long-standing persistent atrial fibrillation was significant at p=0.02. The findings from our study suggest a decline in miRNA 208a expression with ongoing arrhythmia, possibly preceded by a rise in expression from paroxysmal to persistent atrial fibrillation or even long-standing persistent. The significant changes in miRNA 208a expression over the course of the disease may be used as an additional diagnostic tool to monitor the progression of atrial fibrillation.

The Novel Biomarker of Germ Cell Tumours, Micro-RNA-371a-3p, Has a Very Rapid Decay in Patients with Clinical Stage 1

Background: Accumulating evidence suggests serum levels of microRNA (miR)-371a-3p to be a novel tumour marker of testicular germ cell tumours (GCTs). Presently, there is only limited information regarding the velocity of decline of serum levels in response to treatment. Patients and Methods: Twenty-four patients with testicular GCT (20 seminoma, 4 nonseminoma, median age 40 years) with clinical stage 1 had measurements of serum levels of miR-371a-3p preoperatively and repeatedly on the following 3 days. Three had additional tests done within 24 h after surgery. Measurement results were analysed using descriptive statistical methods. Results: Serum levels dropped to 2.62, 1.27, and 0.47% of the preoperative level within 1, 2, and 3 days, respectively. The computed half-life amounts to 3.7–7 h. The velocity of decay is significantly associated with tumour size. Conclusions: Serum-levels of miR-371a-3p have a short half-life of less than 12 h. The rapid decay after treatment represents a valuable feature confirming the usefulness of miR-371a-3p as a valuable serum biomarker of GCT.

Corrigendum re: “Serum Levels of MicroRNA miR-371a-3p: A Sensitive and Specific New Biomarker for Germ Cell Tumours” [Eur Urol 2017;71:213–20]

a Department of Urology, Albertinen Krankenhaus, Hamburg, Germany; b Centre for Human Genetics, University of Bremen, Bremen, Germany; c Department of Urology, Bundeswehr Krankenhaus, Hamburg, Germany; d Department of Medical Oncology, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany; e Department of Urology, Zentralklinikum Bremen, Bremen, Germany; f Institute of Statistics, University of Bremen, Bremen, Germany