Hendrik Bergert

ANRIL Expression Is Associated With Atherosclerosis Risk at Chromosome 9p21

Objective—We tested the hypothesis that expression of transcripts adjacent to the chromosome 9p21 (Chr9p21) locus of coronary artery disease was affected by the genotype at this locus and associated with atherosclerosis risk. Methods and Results—We replicated the locus for coronary artery disease (P=0.007; OR=1.28) and other manifestations of atherosclerosis such as carotid plaque (P=0.003; OR=1.31) in the Leipzig Heart Study, a cohort of 1134 patients with varying degree of angiographically assessed coronary artery disease. Expression analysis in peripheral blood mononuclear cells (n=1098) revealed that transcripts EU741058 and NR_003529 of antisense noncoding RNA in the INK4 locus (ANRIL) were significantly increased in carriers of the risk haplotype (P=2.1×10−12 and P=1.6×10−5, respectively). In contrast, transcript DQ485454 remained unaffected, suggesting differential expression of ANRIL transcripts at Chr9p21. Results were replicated in whole blood (n=769) and atherosclerotic plaque tissue (n=41). Moreover, expression of ANRIL transcripts was directly correlated with severity of atherosclerosis (EU741058 and NR_003529; P=0.02 and P=0.001, respectively). No consistent association of Chr9p21 or atherosclerosis was found with expression of other genes such as CDKN2A, CDKN2B, C9orf53, and MTAP. Conclusion—Our data provide robust evidence for an association of ANRIL but not CDKN2A, CDKN2B, C9orf53, and MTAP, with atherosclerosis and Chr9p21 genotype in a large cohort.

Alu Elements in ANRIL Non-Coding RNA at Chromosome 9p21 Modulate Atherogenic Cell Functions through Trans -Regulation of Gene Networks

The chromosome 9p21 (Chr9p21) locus of coronary artery disease has been identified in the first surge of genome-wide association and is the strongest genetic factor of atherosclerosis known today. Chr9p21 encodes the long non-coding RNA (ncRNA) antisense non-coding RNA in the INK4 locus (ANRIL). ANRIL expression is associated with the Chr9p21 genotype and correlated with atherosclerosis severity. Here, we report on the molecular mechanisms through which ANRIL regulates target-genes in trans, leading to increased cell proliferation, increased cell adhesion and decreased apoptosis, which are all essential mechanisms of atherogenesis. Importantly, trans-regulation was dependent on Alu motifs, which marked the promoters of ANRIL target genes and were mirrored in ANRIL RNA transcripts. ANRIL bound Polycomb group proteins that were highly enriched in the proximity of Alu motifs across the genome and were recruited to promoters of target genes upon ANRIL over-expression. The functional relevance of Alu motifs in ANRIL was confirmed by deletion and mutagenesis, reversing trans-regulation and atherogenic cell functions. ANRIL-regulated networks were confirmed in 2280 individuals with and without coronary artery disease and functionally validated in primary cells from patients carrying the Chr9p21 risk allele. Our study provides a molecular mechanism for pro-atherogenic effects of ANRIL at Chr9p21 and suggests a novel role for Alu elements in epigenetic gene regulation by long ncRNAs.

Expression of Chr9p21 genes CDKN2B (p15INK4b), CDKN2A (p16INK4a, p14ARF) and MTAP in human atherosclerotic plaque

OBJECTIVE The pathophysiology underlying the chromosome (Chr) 9p21 locus of atherosclerosis susceptibility is presently unknown. Here, we sought to determine whether protein coding genes in the Chr9p21 region, i.e. cyclin-dependent kinase inhibitors CDKN2B (p15(INK4b)), CDKN2A (p16(INK4a), p14(ARF)) and methylthioadenosine phosphorylase (MTAP) were expressed in human atherosclerotic lesions and whether expression was correlated with lesion composition. METHODS AND RESULTS Protein expression of p15(INK4b), p16(INK4a), p14(ARF) and MTAP was demonstrated by immunostaining in normal and atherosclerotic coronary arteries and co-localized with CD68 and smooth muscle alpha-actin positive cells. Quantitative RT-PCR in human endarteryectomy specimens (n = 57) revealed increased p16(INK4a) and decreased MTAP expression in macrophage-rich lesions (P<0.001 and P = 0.007, respectively). Functional studies suggest that decreased MTAP expression in macrophage-rich lesions might be mediated through down-regulation by TNF-alpha. No clear association of p15(INK4b), p16(INK4a), p14(ARF), and MTAP expression in plaque tissue with Chr9p21 haplotypes was found. The latter finding was corroborated by the lack of correlation of RNA expression of 9p21-regulated transcripts EU741058 and NR_003529 of antisense non-coding RNA in the INK4 locus (ANRIL) with mRNA expression of these genes. In contrast, ANRIL DQ485454 which is not genetically determined by the 9p21 genotype was significantly correlated with MTAP expression (P = 0.01). CONCLUSION CDKN2B (p15(INK4b)), CDKN2A (p16(INK4a), p14(ARF)), and MTAP are abundantly expressed in atherosclerotic lesions. While expression levels showed no clear association with Chr9p21 genotype, association of high p16(INK4a) and low MTAP expression with a less stable plaque phenotype suggests a more general role of these proteins in atherogenesis.

Quantitative Perfusion Analysis of Transabdominal Contrast-Enhanced Ultrasonography of Pancreatic Masses and Carcinomas

BACKGROUND & AIMS Preoperative differential diagnosis of pancreatic ductal adenocarcinoma (PDAC) and focal masses in patients with chronic pancreatitis (CP) can be challenging. There are fine differences in the vascularization of these lesions; ultrasound contrast agents can aid in their differentiation. We evaluated the value of software-aided quantitative analysis of transabdominal contrast-enhanced ultrasonography for differential diagnosis of PDAC vs focal masses. METHODS Sixty patients for whom it was not possible to differentiate between an inflammatory focal lesion of the pancreas and a pancreatic carcinoma underwent contrast-enhanced ultrasonography with a second-generation contrast agent. Time-intensity curves were obtained for all exams in 2 regions of interest within the lesion and within the normal pancreatic tissue. Images were processed using Axius ACQ software; the following parameters were obtained: maximum intensity, arrival time, time-to-peak, and area under the curve. Absolute values and differences between the lesion and the normal tissue were evaluated. RESULTS Histology analysis revealed 45 PDACs and 15 inflammatory masses in patients with CP. Time-dependent parameters (arrival time and time to peak) were significantly longer in PDACs compared to focal masses. Although markedly lower than in healthy pancreata, the maximum intensity and area under the curve parameters were not significantly different between PDACs and focal lesions in patients with CP. CONCLUSIONS In cases of CP, PDAC and focal masses exhibit different perfusion patterns at a capillary level that can be visualized using the small microbubbles of ultrasound contrast agents. Contrast quantification software supplements a subjective visual assessment with objective criteria to facilitate the differential diagnosis of focal lesions in pancreatic cancer and chronic pancreatitis.

Prospective evaluation of the retrograde percutaneous translaryngeal tracheostomy (Fantoni procedure) in a surgical intensive care unit: Technique and results of the Fantoni tracheostomy

Controversy surrounds the safety and practicality of the retrograde percutaneous translaryngeal tracheostomy (Fantoni procedure) compared with other percutaneous methods.

Analysis of Tissue Inhibitor of Metalloproteinase-2 Gene Polymorphisms in a Caucasian Population with Abdominal Aortic Aneurysms

BACKGROUND The formation of sporadic abdominal aortic aneurysm (AAA) is explained by a remodelling of the extracellular matrix (ECM) and breakdown of structural components of the vascular wall. Matrix metalloproteinases are the principle matrix-degrading proteases and are known to play a major role in the remodelling of the extracellular matrix in arterial vessels. Their activity is controlled by tissue inhibitors of metalloproteinases (TIMPs). Decreased TIMP-1 and TIMP-2 expression in the extracellular matrix of the walls of AAAs has been demonstrated in several studies. This case-control study was designed to investigate the possible impact of genetic variants of the TIMP-2 gene in the aetiology of AAA and to reproduce a recently described significant difference in allele frequency of the SNP 303G>A in a German population. METHODS TIMP-2 single nucleotide polymorphisms (SNPs) were analysed in a study sample of 50 patients with AAA and 41 controls. Differences in genotype and allele frequencies of the identified polymorphisms were determined after sequencing the entire coding region and selected parts of the promoter using the automated laser fluorescence technique. RESULTS Six polymorphisms were identified, one of which is described for the first time, located in the intron, (231+23C>T). An association of the SNP 303G>A with the phenotype was not confirmed in our study (p=0.648). However, the CT genotype of the SNP -479C>T was more frequent in patients with AAA than in the control group (p=0.054). CONCLUSIONS In our analysis of the TIMP-2 gene, we identified one new SNP. A previously published association of the SNP 303G>A with the phenotype could not be validated in our population. However, we detected an association for the CT genotype of one polymorphism in the promoter region (g-479C>T) and AAA. This result has to be proved in a second study sample.

Epithelioid hemangiosarcoma of the rectum

Dear Editor: Hemangiosarcoma is an uncommon malignant tumor, accounting for only 1–2% of all soft tissue sarcomas [1, 2]. Commonly involved sites include skin, liver, spleen, lungs, bones, deep soft tissue, and chest wall after mastectomy [3]. Primary colorectal angiosarcomas are exceedingly rare. In the literature, only 23 cases of primary colorectal hemangiosarcoma [4–17] have been reported, and only eight were localized in the rectum [4, 18– 23]. Histopathological recognition may be difficult, and immunocytochemical techniques are frequently required to confirm the diagnosis [20]. The clinical signs of colorectal angiosarcomas are similar to common colorectal cancers. Mostly, these tumors have been treated like colorectal carcinomas. In most cases, primary surgical resection was performed. Classification and nomenclature of these rare malignancies have been changed often over recent years and, therefore, a comparison of the reported cases is problematic. The first two cases of rectal hemangiosarcoma were published in 1932 by Morgan [18] and Norbury [19]. Histologically, they described an “endothelioma” arising from the vascular endothelium. A 42-year-old male consulted his urologist because of sacral pain while sitting. On digital rectal examination, a small area (0.5 cm) of induration 4 cm from the anal verge was noted. Rectoscopy showed normal mucosa. Endosonography detected a 3×5-mm submucosal bulge of the anterior rectal wall, which was considered to be a postinfectious residue. His sacral pain was thought to originate from a lumbosacral deformity, and physical therapy was initiated. Twentymonths later, the patient was admitted to our department because of 2 weeks of episodic rectal bleeding. In addition, he had a history of episodes of swollen cervical lymph nodes for many years. Nevertheless, he felt well and had no weight loss or anorexia. Laboratory analysis demonstrated mild anemia, an increased fibrinogen level of 5.4 g/l, antithrombin III (ATIII) of 128%, gamma-glutamyltransferase (GGT) 1.22 μmol/l, alkaline phosphatase (AP) 6.01 μmol/l, C-reactive protein (CRP) 27.1 mg/l; other parameters, including tumor markers, were within the normal ranges. At this time, digital rectal palpation revealed a tumor of the anterior rectal wall, located by rectoscopy 3–5 cm from the linea dentata, and characterized as an ulcerated polypoid tumor. Using endosonography, an irregular contour of the tumor and an intact adventitia were detected. Microscopic examination of mucosal biopsies revealed an undifferentiated malignant, epithelioid neoplasm. The patient underwent a total mesorectal excision with colo-anal pouch anastomosis and a protective ileostomy. I. Hinterseher (*) . S. Pistorius . H. Bergert Department of Visceral, Thoracic and Vascular Surgery, Universitätsklinikum Carl Gustav Carus, Technical University of Dresden, Fetscherstrasse 74, 01307 Dresden, Germany e-mail: Irene. Hinterseher@uniklinikum-dresden.de Tel.: +49-351-4582863

Arterial Injuries Combined with Open Fractures - Management and Therapy

Vascular injuries are an uncommon finding. In times of peace vascular injuries occur in approximately 1-4 % during traffic accidents. Especially challenging is the treatment of open fractures combined with arterial lesions. These fractures are usually accompanied with severe soft tissue damage and injuries to neurological structures. The overall prognosis of these trauma patients is dependent on fast and sufficient diagnostics and therapy. In particular, for unstable patients time-consuming diagnostics can be dispensed and a primarily operative therapy should be targeted. Vascular reconstruction by direct suture is sometimes only possible with interposition and should be the primary goal. Interposition should be performed with autologous vein material because of the high risk of infection. Here we demonstrate on the basis of our patients the interdisciplinary -management of such trauma patients in our hospital.

Beans in the pericardium

A 73-year old woman was admitted from another hospital for suspected ST-segment elevation myocardial infarction (STEMI). She had chest pain and dyspnoea. She had a history of treated hypertension and of gastro-oesophageal refl ux disease (GORD) with Barrett’s metaplasia which was diagnosed 3 months previously. ECG showed ST-segment elevation in leads I, II, aVL, and V4–V6, ST-segment depression in III and aVR, and atypical high R spikes in V2–V4 (fi gure A). Cardiac auscultation was normal. Troponin I testing was positive. To fulfi l the German guidelines for STEMI (door-to-vessel time of less than 30 min in patients with STEMI admitted from another hospital), the patient was transferred to the catheter isation laboratory without any further tests. Coronary angiography was started 22 min after admission. During catheterisation, an extensive pneumopericardium was diagnosed (fi gure B); the coronary arteries were normal. Chest radiography confi rmed the pneumo pericardium and excluded a concomitant pneumonia. Echocardiography showed an on-off phenomenon ie, with good diastolic visibility and complete systolic disap pear ance of the heart because of the pneumo pericardium promoting ultrasound refl ection in systole. CT showed air in the anterior part of the pericardium with no evidence of a malignant process. A fi bre-optic bronchoscopy excluded additional perforations as well as aspiration. Given the patient’s history of GORD and despite the potential risk of a tamponade caused by air infl ation in the pericardium, an oesophagogastroscopy was done. A circular defect of 2·5 cm in the distal oesophagus was evident with a direct view to the rhythmically moving, pale pericardium. The patient became haemodynamically unstable, with signs of septic shock, increased infl ammatory markers (leucocytes 18 000×109/L, CRP 296 mg/L, procalcitonin 48·7 ng/mL), and low fi lling pressures. Clinical examination showed no evidence of congested jugular veins. Echocardiography ruled out tamponade. Stabilisation was achieved with catecholamines and fl uids. The multidisciplinary team decided to operate urgently. However, an oesophageal stent graft was not feasible because the distal landing zone for the stent was deemed too small, and a covered stent could be interfered with by the rhythmically moving heart. Therefore, a right-sided thoracotomy with intermittent single lung ventilation was done. The intraoperative situs showed a 2·5 cm defect of the oesophagus with penetration to the pericardium (fi gure C). The pericardium was debrided and purulent fl uid was drained; seven haricot beans from the last meal were removed. Plastic patching of the oesophagus was done, and drains for continuous pericardial irrigation were placed. Cultures of the pericardial fl uid confi rmed a purulent pericarditis (Escherichia coli and Proteus mirabilis). In the postoperative course, the patient experienced septic shock and multiple complications; she died 7 days after admission. Barrett’s metaplasia is an important complication of GORD. Symptoms of GORD can be similar to those for acute coronary syndromes. Treatment comprises lifestyle changes, pharmacological treatment, and endoscopic/ surgical procedures. The goal is to prevent compli cations. Pericardial perforation is a very rare complication of Barrett’s metaplasia. In our patient we opted for highrisk surgery because given the clinical and investigational fi ndings, we thought an endoscopic approach would not control the sepsis caused by purulent mediastinitis. An oesophageal stent combined with pericardial irrigation was an alternative treatment option; although less invasive, this treatment lacks the possibility of debriding the pericardium. Our patient presented a complex emergency situation, and, unfortunately, our prompt interventions proved too late.

Pulmonary Artery Pseudoaneurysm in a Patient With Aortic Valve Stenosis

A 77-year-old, high-risk woman with symptomatic aortic valve stenosis (aortic valve area 0.77 cm(2)) underwent coronary artery catheterization and right heart catheterization. After catheterization, she suddenly developed hemoptysis, and became hypoxic and hypotonic. She was intubated and the bleeding was stopped using positive end-expiratory pressure. Chest X-ray and computed tomography showed a pulmonary artery (PA) pseudoaneurysm with a maximum diameter of 40 mm at the right middle lobe. Endovascular treatment approaches by coil embolization failed, so surgical resection was indicated. In preparation for the procedure and to reduce perioperative risk, transapical aortic valve implantation was performed. The operation took about 40 minutes and the intraoperative activated clotting time was controlled at 180-200 sec. After successful transapical aortic valve implantation, aneurysmectomy was performed. Intraoperatively, the PA pseudoaneurysm was found to occupy nearly the entire middle lobe. A right middle lobectomy was performed. The operative course was uneventful. Transapical aortic valve implantation may have eliminated the risk of rupture or re-bleeding in such bleeding-prone patient.