Rationale: Calcification of heart valve structures is the most common form of valvular disease and is characterized by the appearance of bone-like phenotypes within affected structures. Despite the clinical significance, the underlying etiology of disease onset and progression is largely unknown and valve replacement remains the most effective treatment. The SRY-related transcription factor Sox9 is expressed in developing and mature heart valves, and its function is required for expression of cartilage-associated proteins, similar to its role in chondrogenesis. In addition to cartilage-associated defects, mice with reduced sox9 function develop skeletal bone prematurely; however, the ability of sox9 deficiency to promote ectopic osteogenic phenotypes in heart valves has not been examined. Objective: This study aims to determine the role of Sox9 in maintaining connective tissue homeostasis in mature heart valves using in vivo and in vitro approaches. Methods and Results: Using histological and molecular analyses, we report that, from 3 months of age, Sox9 fl/؉ ;Col2a1-cre mice develop calcific lesions in heart valve leaflets associated with increased expression of bone-related genes and activation of inflammation and matrix remodeling processes. Consistently, ectopic calcification is also observed following direct knockdown of Sox9 in heart valves in vitro. Furthermore, we show that retinoic acid treatment in mature heart valves is sufficient to promote calcific processes in vitro, which can be attenuated by Sox9 overexpression. Conclusions: This study provides insight into the molecular mechanisms of heart valve calcification and identifies reduced Sox9 function as a potential genetic basis for calcific valvular disease. (Circ Res. 2010;106:712-719.)Key Words: heart valves Ⅲ calcification Ⅲ Sox9 Ⅲ extracellular matrix Ⅲ mouse model C alcification of heart valve structures affects more than 27% of the US population over 65 years of age and is the major contributor of heart valve malfunction. 1 Despite the clinical significance, little is known about the mechanisms that underlie this multifactorial disease. Treatment options for valve calcification are limited, and no known therapies prevent disease progression. 2 Normal heart valve function requires organization of differentiated cell types and specialized extracellular matrix (ECM) within the valve leaflet is arranged according to blood flow. 3 This defined tissue architecture provides the mechanical resilience and compressibility required to open and close the valve orifices effectively during the cardiac cycle. 4 In diseased heart valves, loss of ECM organization is associated with changes in mechanical properties, ultimately leading to dysfunction. 5,6 One of the most striking alterations in valve ECM homeostasis is ectopic bone-like matrix mineralization observed in calcific valve disease. 7,8 At the functional level, this histopathologic alteration results in stiffened leaflets, narrowing of the valve opening, and impaired blood flow. 9 The mechanis...

Six broad-host-range plasmid vectors were developed to study gene expression in Bartonella henselae. The vectors were used to express a ␤-galactosidase reporter gene in B. henselae and to generate antisense RNA for gene knockdown. When applied to ompR, a putative transcription response regulator of B. henselae, this antisense RNA gene knockdown strategy reduced bacterial invasion of human endothelial cells by over 60%.

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