Rajamannan, Nalini M.

We have previously demonstrated that β-catenin plays important roles in valve calcification with a specific osteogenic phenotype defined by increased bone mineral content and overall valve thickening. Recent studies indicate that KLF10 may be involved in mediating the Wnt signaling pathway in bone, which is known to play critical roles in osteoblast differentiation and mineralization. Therefore, we sought to test the role of KLF10 in mediating Wnt signaling, as well as differentiation and mineralization, in valve interstitial cells (VICs) isolated from porcine valves. Exposure of VICs to differentiation media led to increased expression of Runx2, Sox9 and osteocalcin. Differentiated cells also stained positive with Von Kossa while undifferentiated cells stained negative confirming the induction of an osteogenic phenotype. As expected, over-expression of both Lef1 and β-catenin led to activation of the top-flash reporter when transfected into VICs. Interestingly, over-expression of KLF10 also significantly up-regulated the top-flash reporter alone and further enhanced the activity of both Lef1 and β-catenin when co-transfected. We further confirmed the role of TIEG in an atherosclerotic mouse model ApoE/LRP5 double KO and found a two-fold increase in KLF10, Lrp6, and Runx2 expression in the cholesterol treated aortic valves as compared to controls. These data suggested that KLF10, Lef1 and β-catenin interact with each other to form a transcriptionally active protein complex leading to enhanced Wnt signaling in VICs. This possibility was further confirmed by the observation that KLF10 and β-catenin co-localize with one another in the nucleus of VICs following stimulation with LiCl and/or TGF-β. Taken together, these data implicate an important role for KLF10 in mediating Wnt signaling and Lef1 transcriptional activity in VICs, and implicate a potential role for canonical Wnt signaling in the observed osteogenic bone phenotype of cardiac aortic valves.

Background— Calcific aortic valve disease is the most common indication for surgical valve replacement in the United States. The cellular mechanisms of valve calcification are not well understood. We have previously shown that cellular proliferation and osteoblastogenesis are important in the development of valvular heart disease. Lrp5, a known low-density receptor-related protein, plays an essential role in cellular proliferation and osteoblastogenesis via the β-catenin signaling pathway. We hypothesize that Lrp5 also plays a role in aortic valve (AV) calcification in experimental hypercholesterolemia. Methods and Results— We examined the effects of cholesterol and atorvastatin in Watanabe rabbits (n=54). Group I (n=18) received a normal diet, group II (n=18) a 0.25% cholesterol diet, and group III (n=18) a 0.25% (w/w) cholesterol diet with atorvastatin for the development of calcification. The AVs were examined for cellular proliferation, Lrp5/β-catenin, and bone matrix markers. Bone formation was assessed by micro-computed tomography, calcein injection, and osteopontin expression. Low-density lipoprotein with and without atorvastatin was also tested in AV myofibroblasts for cellular proliferation and regulation of the Lrp5/β-catenin pathway. Our results demonstrate that the cholesterol diet induced complex bone formations in the calcified AVs with an increase in the Lrp5 receptors, osteopontin, and p42/44 expression. Atorvastatin reduced bone formation, cellular proliferation, and Lrp5/β-catenin protein levels in the AVs. In vitro analysis confirmed the Lrp5/β-catenin expression in myofibroblast cell proliferation. Conclusion— Hypercholesterolemic AV calcification is attenuated by atorvastatin and is mediated in part by the Lrp5/β-catenin pathway. This developmental pathway may be important in the signaling pathway of this disease.

AbstractWe have previously demonstrated that Lrp5/6/β‐catenin plays an important role in valve calcification with a specific osteogenic phenotype defined by increased bone mineral content and overall valve thickening. Recent studies indicate that TIEG1 may be involved in mediating the Wnt signaling pathway in bone, which is known to play critical roles in osteoblast differentiation and bone mineralization. Therefore, we sought to test the role of TIEG1 in mediating Wnt signaling, in an established model of hypercholesterolemic valve disease. Our previous model treated null mice with cholesterol diets: Lrp5 −/−/ApoE −/− mice versus wild‐type control (n = 180). Group I (n = 60) normal diet, Group II (n = 60) 0.25% chol diet (w/w), and Group III (n = 60) 0.25% (w/w) chol diet + atorv was tested for gene expression for TIEG1, Lrp6, and Runx2. Real‐time polymerase chain reaction confirmed that there is upregulation of the gene expression for TIEG1 and Runx2 in the hypercholesterolemic double knockout and single knockout valves as compared with controls with a mild increase in Lrp6. To confirm the mechanism, coexpression of β‐catenin, TIEG1, and LEF1 in valve cells in vitro, led to the coactivation of the TOPFLASH reporter, which was further confirmed by the observation that TIEG1 and β‐catenin colocalize with one another in the nucleus of valvular interstitial cells (VICs) following stimulation with transforming growth factor‐β treatment, an established activator of TIEG1. Taken together, these data implicate an important role for TIEG1 in mediating valve osteogenesis.

Background— Rheumatic heart disease is the most common cause of valvular disease in developing countries. Despite the high prevalence of this disease, the cellular mechanisms are not well known. We hypothesized that rheumatic valve calcification is associated with an osteoblast bone formation and neoangiogenesis. Methods and Results— To test this hypothesis, we examined human rheumatic valves replaced at surgery (n=23), normal human valves (n=20) removed at cardiac transplantation, and degenerative mitral valve leaflets removed during surgical valve repair (n=15). Microcomputed tomography was used to assess mineralization fronts to reconstruct the extents of mineralization. Immunohistochemistry was used to localize osteopontin protein, α-actin, osteocalcin, vascular endothelial growth factor, von Willebrand factor, and CD68 (human macrophage). Microcomputed tomography demonstrated complex calcification developing within the heavily calcified rheumatic valves, not in the degenerative mitral valves and control valves. Immunohistochemistry localized osteopontin and osteocalcin to areas of smooth muscle cells within microvessels and proliferating myofibroblasts. Vascular endothelial growth factor was present in areas of inflammation and colocalized with the CD68 stain primarily in the calcified rheumatic valves. Alizarin red, osteopontin, and osteocalcin protein expression was upregulated in the calcified rheumatic valves and was present at low levels in the degenerative mitral valves. Conclusions— These findings support the concept that rheumatic valve calcification is not a random passive process but a regulated, inflammatory cellular process associated with the expression of osteoblast markers and neoangiogenesis.

Background— Calcific aortic stenosis is the third most common cardiovascular disease in the United States. We hypothesized that the mechanism for aortic valve calcification is similar to skeletal bone formation and that this process is mediated by an osteoblast-like phenotype. Methods and Results— To test this hypothesis, we examined calcified human aortic valves replaced at surgery (n=22) and normal human valves (n=20) removed at time of cardiac transplantation. Contact microradiography and micro-computerized tomography were used to assess the 2-dimensional and 3-dimensional extent of mineralization. Mineralization borders were identified with von Kossa and Goldner’s stains. Electron microscopy and energy-dispersive spectroscopy were performed for identification of bone ultrastructure and CaPO 4 composition. To analyze for the osteoblast and bone markers, reverse transcriptase–polymerase chain reaction was performed on calcified versus normal human valves for osteopontin, bone sialoprotein, osteocalcin, alkaline phosphatase, and the osteoblast-specific transcription factor Cbfa1. Microradiography and micro-computerized tomography confirmed the presence of calcification in the valve. Special stains for hydroxyapatite and CaPO 4 were positive in calcification margins. Electron microscopy identified mineralization, whereas energy-dispersive spectroscopy confirmed the presence of elemental CaPO 4 . Reverse transcriptase–polymerase chain reaction revealed increased mRNA levels of osteopontin, bone sialoprotein, osteocalcin, and Cbfa1 in the calcified valves. There was no change in alkaline phosphatase mRNA level but an increase in the protein expression in the diseased valves. Conclusions— These findings support the concept that aortic valve calcification is not a random degenerative process but an active regulated process associated with an osteoblast-like phenotype.