K-Ras is the most prominent driver of oncogenesis and no effective K-Ras inhibitors have been established despite decades of intensive research. Identifying new K-Ras-binding proteins and their interaction domains offers the opportunity for defining new approaches in tackling oncogenic K-Ras. We have identified Galectin-8 as a novel, direct binding protein for K-Ras4B by mass spectrometry analyses and protein interaction studies. Galectin-8 is a tandem-repeat Galectin and it is widely expressed in lung and pancreatic carcinoma cells. siRNA-mediated depletion of Galectin-8 resulted in increased K-Ras4B content and ERK1/2 activity in lung and pancreatic carcinoma cells. Moreover, cell migration and cell proliferation were inhibited by the depletion of Galectin-8. The K-Ras4B–Galectin-8 interaction is indispensably associated with the farnesylation of K-Ras4B. The lysine-rich polybasic domain (PBD), a region that is unique for K-Ras4B as compared to H- and N-Ras, stabilizes the interaction and accounts for the specificity. Binding assays with the deletion mutants of Galectin-8, comprising either of the two carbohydrate recognition domains (CRD), revealed that K-Ras4B only interacts with the N-CRD, but not with the C-CRD. Structural modeling uncovers a potential binding pocket for the hydrophobic farnesyl chain of K-Ras4B and a cluster of negatively charged amino acids for interaction with the positively charged lysine residues in the N-CRD. Our results demonstrate that Galectin-8 is a new binding partner for K-Ras4B and it interacts via the N-CRD with the farnesylated PBD of K-Ras, thereby modulating the K-Ras effector pathways as well as cell proliferation and migration.

OBJECTIVES Patients with a bicuspid aortic valve (BAV) have an increased risk for developing thoracic aortic aneurysm, which is characterized by the destruction of the elastic media of the aortic wall. Several important enzymes have been characterized to play key roles in extracellular matrix homeostasis, namely matrix metalloproteinases (MMPs). In this study, we investigated MMP-2 levels and their epigenetic regulation via the miR-29 family. METHODS Aortic tissue samples from 58 patients were collected during cardiac surgery, of which 30 presented with a BAV and 28 with a tricuspid aortic valve. Polymerase chain reaction, western blot analysis and immunohistochemistry were performed to analyse MMP-2. In addition, enzyme-linked immunosorbent assay measurements were carried out to investigate both MMP-2 and tissue inhibitor of metalloproteinase-2 levels. To examine the epigenetic regulation of aortic extracellular matrix homeostasis, we furthermore studied the expression levels of miR-29 via qRT-PCR. RESULTS Patients with a BAV were significantly younger at the time of surgery, presented significantly less frequently with arterial hypertension and displayed more often with an additional valvular disease. On a molecular level, we found that MMP-2 is increased on gene and protein level in BAV patients. Tissue inhibitor of metalloproteinase-2 levels do not differ between the groups. Interestingly, we also found that only miR-29A is significantly downregulated in BAVs. CONCLUSIONS Our findings highlight the importance of MMP-2 in the context of extracellular matrix destruction in BAV patients. We present new evidence that miR-29A is a crucial epigenetic regulator of these pathomechanistic processes and might hold promise for future translational research.

OBJECTIVES The goal of therapy of proximal aortic aneurysms is to prevent an aortic catastrophe, e.g. acute dissection or rupture. The decision to intervene is currently based on maximum aortic diameter complemented by known risk factors like bicuspid aortic valve, positive family history or rapid growth rate. When applying Laplace’s law, wall tension is determined by pressure × radius divided by aortic wall thickness. Because current imaging modalities lack precision, wall thickness is currently neglected. The purpose of our study was therefore to correlate maximum aortic diameter with aortic wall thickness and known indices for adverse aortic events. METHODS Aortic samples from 292 patients were collected during cardiac surgery, of whom 158 presented with a bicuspid aortic valve and 134, with a tricuspid aortic valve. Aortic specimens were obtained during the operation and stored in 4% formaldehyde. Histological staining and analysis were performed to determine the thickness of the aortic wall. RESULTS Patients were 62 ± 13 years old at the time of the operation; 77% were men. The mean aortic dimensions were 44 mm, 41 mm and 51 mm at the aortic root, sinotubular junction and ascending aorta, respectively. Aortic valve stenosis was the most frequent (49%) valvular dysfunction, followed by aortic valve regurgitation (33%) and combined dysfunction (10%). The maximum aortic diameter at the ascending level did not correlate with the thickness of the media (R = 0.07) or the intima (R = 0.28) at the convex sample site. There was also no correlation of the ascending aortic diameter with age (R = −0.18) or body surface area (R = 0.07). The thickness of the intima (r = 0.31) and the media (R = 0.035) did not correlate with the Svensson index of aortic risk. Similarly, there was a low (R = 0.29) or absent (R = −0.04) correlation between the aortic size index and the intima or media thickness, respectively. There was a similar relationship of median thickness of the intima in the 4 aortic height index risk categories (P < 0.001). CONCLUSIONS Aortic diameter and conventional indices of aortic risk do not correlate with aortic wall thickness. Other indices may be required in order to identify patients at high risk for aortic complications.

The bicuspid aortic valve (BAV) is the most common cardiovascular congenital abnormality and is frequently associated with proximal aortopathy. We analyzed the tissues of patients with bicuspid and tricuspid aortic valve (TAV) regarding the protein expression of the receptor for advanced glycation products (RAGE) and its ligands, the advanced glycation end products (AGE), as well as the S100 calcium-binding protein A6 (S100A6). Since S100A6 overexpression attenuates cardiomyocyte apoptosis, we investigated the diverse pathways of apoptosis and autophagic cell death in the human ascending aortic specimen of 57 and 49 patients with BAV and TAV morphology, respectively, to identify differences and explanations for the higher risk of patients with BAV for severe cardiovascular diseases. We found significantly increased levels of RAGE, AGE and S100A6 in the aortic tissue of bicuspid patients which may promote apoptosis via the upregulation of caspase-3 activity. Although increased caspase-3 activity was not detected in BAV patients, increased protein expression of the 48 kDa fragment of vimentin was detected. mTOR as a downstream protein of Akt was significantly higher in patients with BAV, whereas Bcl-2 was increased in patients with TAV, assuming a better protection against apoptosis. The autophagy-related proteins p62 and ERK1/2 were increased in patients with BAV, assuming that cells in bicuspid tissue are more likely to undergo apoptotic cell death leading to changes in the wall and finally to aortopathies. We provide first-hand evidence of increased apoptotic cell death in the aortic tissue of BAV patients which may thus provide an explanation for the increased risk of structural aortic wall deficiency possibly underlying aortic aneurysm formation or acute dissection.