Increased peak wall stress correlates with change in VSMC towards a synthetic phenotype mediated by ROS accumulation via CTGF. Understanding the molecular mechanisms that regulate VSMC towards a synthetic phenotype could unveil new regulatory pathways of aortic homoeostasis and impact the risk-stratification tool for patients at risk of aortic dissection and rupture.

There is a significant interest in the standardized classification of human genetic variants. We used whole-genome sequence data from 10,495 unrelated individuals to contrast population frequency of pathogenic variants to the expected population prevalence of the disease. Analyses included the ACMG-recommended 59 gene-condition sets for incidental findings and 463 genes associated with 265 OrphaNet conditions. A total of 25,505 variants were used to identify patterns of inflation (i.e., excess genetic risk and misclassification). Inflation increases as the level of evidence supporting the pathogenic nature of the variant decreases. We observed up to 11.5% of genetic disorders with inflation in pathogenic variant sets and up to 92.3% for the variant set with conflicting interpretations. This improved to 7.7% and 57.7%, respectively, after filtering for disease-specific allele frequency. The patterns of inflation were replicated using public data from more than 138,000 genomes. The burden of rare variants was a main contributing factor of the observed inflation, indicating collective misclassified rare variants. We also analyzed the dynamics of re-classification of variant pathogenicity in ClinVar over time, which indicates progressive improvement in variant classification. The study shows that databases include a significant proportion of wrongly ascertained variants; however, it underscores the critical role of ClinVar to contrast claims and foster validation across submitters.

Elevated expression of Bmi-1 is associated with many cancers, including breast cancer. Here, we examined the oncogenic potential of Bmi-1 in MCF10A cells, a spontaneously immortalized, nontransformed strain of human mammary epithelial cells (HMEC). Bmi-1 overexpression alone in MCF10A cells did not result in oncogenic transformation. However, Bmi-1 co-overexpression with activated H-Ras (RasG12V) resulted in efficient transformation of MCF10A cells in vitro. Although early-passage H-Ras-expressing MCF10A cells were not transformed, late-passage H-Ras-expressing cells exhibited features of transformation in vitro. Early-and late-passage H-Ras-expressing cells also differed in levels of expression of H-Ras and Ki-67, a marker of proliferation. Subsets of earlypassage H-Ras-expressing cells exhibited high Ras expression and were negative for Ki-67, whereas most late-passage H-Ras-expressing cells expressed low levels of Ras and were Ki-67 positive. Injection of late-passage H-Ras-expressing cells in severe combined immunodeficient mice formed carcinomas with leiomatous, hemangiomatous, and mast cell components; these tumors were quite distinct from those induced by latepassage cells co-overexpressing Bmi-1 and H-Ras, which formed poorly differentiated carcinomas with spindle cell features. Bmi-1 and H-Ras co-overexpression in MCF10A cells also induced features of epithelial-to-mesenchymal transition. Importantly, Bmi-1 inhibited senescence and permitted proliferation of cells expressing high levels of Ras. Examination of various growth-regulatory pathways suggested that Bmi-1 overexpression together with H-Ras promotes HMEC transformation and breast oncogenesis by deregulation of multiple growth-regulatory pathways by p16INK4a -independent mechanisms. [Cancer Res 2007;67(21):10286-95]

Objective Accumulation of reactive oxygen species (ROS) and remodeling of the microstructure of the cusp characterize aortic valve sclerosis, the early phase of calcific aortic valve disease. These events are associated with activation of valvular interstitial cells (VICs) toward an osteogenic-like phenotype. Because ROS cause DNA damage and transcriptional activation we investigated the relationship between ROS, DNA damage response, and transdifferentiation of VICs. Methods and Results Human aortic valve cusps and patient-matched VICs were collected from 39 patients both with and without calcific aortic valve disease. VICs were exposed to hydrogen peroxide (0.1–1 mmol/L) after cell transduction with extracellular superoxide dismutase/catalase adenoviruses and characterized for DNA-damage response, osteogenic transdifferentiation, and calcification. ROS induce relocalization of phosphorylated γH2AX, MRE11, and XRCC1 proteins with expression of osteogenic signaling molecule RUNX2 via AKT. We report a sustained activation of γH2AX in aortic valve sclerosis-derived VICs suggesting their impaired ability to repair DNA damage. Adenovirus superoxide dismutase/catalase transduction decreases ROS-induced DNA damage and VIC transdifferentiation in aortic valve sclerosis-derived cells. Finally, adenoviral transduction with catalase reverts ROS-mediated calcification and cellular transdifferentiation. Conclusion We conclude that the ROS-induced DNA damage response is dysfunctional in early asymptomatic stages of calcific aortic valve disease. We unveiled an association among ROS, DNA-damage response, and cellular transdifferentiation, reversible by antioxidant enzymes delivery.

Myxomatous Mitral valve prolapse (MVP) is the most common cardiac valvular abnormality in industrialized countries and a leading cause of mitral valve surgery for isolated mitral regurgitation. The key role of valvular interstitial cells (VICs) during mitral valve development and homeostasis has been recently suggested, however little is known about the molecular pathways leading to MVP. We aim to characterize Bone Morphogenetic Protein 4 (BMP4) as a cellular regulator of mitral valvular interstitial cell activation towards a pathologic synthetic phenotype and to analyze the cellular phenotypic changes and extracellular matrix (ECM) reorganization associated with the development of myxomatous mitral valve prolapse. Microarray analysis showed significant up regulation of BMP4-mediated signaling molecules in myxomatous MVP when compared to controls. Histological analysis and cellular characterization suggest that during myxomatous MVP development, healthy quiescent mitral VICs undergo a phenotypic activation via up regulation of BMP4-mediated pathway. In vitro hBMP4 treatment of isolated human mitral VICs mimics the cellular activation and ECM remodeling as seen in MVP tissues. The present study characterizes the cell biology of mitral VICs in physiological and pathological conditions and provides insights into the molecular and cellular mechanisms mediated by BMP4 during MVP. The ability to test and control the plasticity of VICs using different molecules may help in developing new diagnostic and therapeutic strategies for myxomatous MVP.

Our results demonstrate the significant clinical usefulness of telomerase activation and telomere length for head and neck cancer patients. These markers may be helpful in predicting the clinical course of the disease and thus in identifying the patients in need of a close follow-up and vigorous adjuvant treatment.

The bicuspid aortic valve (AV) is the most common cardiac congenital anomaly and has been found to be a significant risk factor for developing calcific AV disease. However, the mechanisms of disease development remain unclear. In this study we quantified the structure of human normal and bicuspid leaflets in the early disease stage. From these individual leaflet maps average fiber structure maps were generated using a novel spline based technique. Interestingly, we found statistically different and consistent regional structures between the normal and bicuspid valves. The regularity in the observed microstructure was a surprising finding, especially for the pathological BAV leaflets and is an essential cornerstone of any predictive mathematical models of valve disease. In contrast, we determined that isolated valve interstitial cells from BAV leaflets show the same in vitro calcification pathways as those from the normal AV leaflets. This result suggests the VICs are not intrinsically different when isolated, and that external features, such as abnormal microstructure and altered flow may be the primary contributors in the accelerated calcification experienced by BAV patients.

Background Calcific Aortic Valve Disease (CAVD) is the most common etiology of acquired valve disease. Initial phases of CAVD include thickening of the cusps, whereas advanced stages are associated with biomineralization and reduction of the aortic valve area. These conditions are known as Aortic Valve Sclerosis (AVSc) and Aortic Valve Stenosis (AVS), respectively. Due to its asymptomatic presentation, little is know about the molecular determinants of AVSc. The aim of this study is to correlate plasma and tissue Osteopontin (OPN) levels with echocardiographic evaluation for the identification of asymptomatic patients at risk of CAVD. In addition, we aim to analyze the differential expression and biological function of OPN splicing variants as biomarkers of early and late stages of CAVD. Methods From Jan 2010 to Feb 2011, 254 patients were enrolled in the study. Subjects were divided in three groups based on transesophageal echocardiographic (TEE) evaluation: Controls (56 subjects), AVSc (90), and AVS (164). Plasma and tissue OPN levels were measured by IHC, ELISA and qPCR. Results Patients with AVSc have and AVS higher OPN levels compared to controls. OPN levels are elevated in asymptomatic AVSc patients with no appearance of calcification during TEE evaluation. Osteopontin splicing variants -a, -b, and -c are differentially expressed during CAVD progression and are able to inhibit biomineralization in a cell-based biomineralization assay. Conclusions The analysis of the differential expression of OPN splicing variants during CAVD may help in developing diagnostic and risk stratification tools to follow the progression of asymptomatic AV degeneration.