Oren-gedoku-to inhibits calcification of human aortic valve interstitial cells in vitro and aortic valve in spontaneously hypertensive rats in vivo.

Preventative medicine: The next revolution in the treatment of aortic stenosis

Matrix Gla protein negatively regulates calcification of human aortic valve interstitial cells isolated from calcified aortic valves

In the current issue of Circulation , Ix et al demonstrate an inverse correlation between mitral and aortic valve calcification and serum fetuin-A levels in a cross-sectional study of 970 patients with coronary artery disease and without renal disease.1 Increased serum fetuin was significantly associated with diabetes mellitus, hypertriglyceridemia, serum albumin, and body mass index, with a weaker association with serum C-reactive protein, low-density lipoprotein cholesterol, and serum calcium. The link between aortic stenosis and fetuin held only in those without diabetes.1 The data highlight the complex interactions of fetuin with inflammation, insulin resistance, and tissue calcification. Furthermore, the results, though intriguing, underscore our lack of understanding of the cellular mechanisms of calcification in diverse pathological substrates, such as the degenerating aortic leaflet and mitral annulus. Article p 2533 Fetuin is a member of the cystatin superfamily of cysteine protease inhibitors, originally discovered as the major component of fetal bovine serum. It is a carrier for growth factors, binds to and inactivates transforming growth factor (TGF)–β and bone morphogenic protein, and is a major component of mineralized bone.2 Fetuin-A is also an acute-phase glycoprotein, produced in adults primarily in the liver, and is a powerful circulating inhibitor of hydroxyapatite formation. Mice that lack fetuin-A exhibit extensive soft-tissue calcification, which is accelerated on a mineral-rich diet, which suggests that fetuin-A acts to inhibit calcification systemically.3 In addition to its effects on mineralization, fetuin-A inhibits insulin receptor autophosphorylation and tyrosine kinase activity in vitro and in vivo. Fetuin-null knockout mice demonstrate improved insulin sensitivity and resistance to diet-induced obesity, and it has been postulated that the absence of fetuin in mice may contribute to the improvement of insulin sensitivity associated with aging.4 Clinically, serum fetuin-A is decreased in patients with moderate to severe chronic kidney disease, especially …

Role of CD34 in calcification of human aortic valve interstitial cells from patients with aortic valve stenosis

Tumor necrosis factor-α accelerates the calcification of human aortic valve interstitial cells obtained from patients with calcific aortic valve stenosis via the BMP2-Dlx5 pathway.

Bone morphogenic protein 2 induces Runx2 and osteopontin expression in human aortic valve interstitial cells: Role of Smad1 and extracellular signal-regulated kinase 1/2

Calcific aortic valve stenosis is the most common indication for surgical valve replacement. Inflammation appears to be one of the mechanisms involved in aortic valve calcification, and valve interstitial cells seem to contribute to that process. Although Toll-like receptors (TLRs) play an important role in the cellular inflammatory response, it is unknown whether human aortic valve interstitial cells (HAVICs) express functional TLRs. We examined the expression of TLR2 and TLR4 in human aortic valve leaflets and in isolated HAVICs and analyzed the response of cultured HAVICs to the TLR2 and TLR4 agonists peptidoglycan (PGN) and LPS. Abundant TLR2 and TLR4 proteins were found in human aortic valve leaflets and in isolated HAVICs, and both receptors were detected in the membrane and cytoplasm of cultured HAVICs. Stimulation by either PGN or LPS resulted in the activation of the NF-kappaB signaling pathway and the production of multiple proinflammatory mediators, including IL-6, IL-8, and ICAM-1. In addition, stimulation by either PGN or LPS upregulated the expression of bone morphogenetic protein-2 (BMP-2) and Runx2, factors associated with osteogenesis. This study demonstrates for the first time that HAVICs express TLR2 and TLR4 and that stimulation of HAVICs by PGN or LPS induces the expression of proinflammatory mediators and the upregulation of osteogenesis-associated factors. These results suggest that TLR2 and TLR4 may play a role in aortic valve inflammation and stenosis.

Warfarin, a vitamin K antagonist, is the most common anticoagulant used to prevent thromboembolisms associated with atrial fibrillation or following valvular surgery. Although several studies have revealed that long-term warfarin use accelerates aortic valve calcification and the development of aortic stenosis (AS), the detailed mechanism for this phenomenon remains unclear. Therefore, our aim was twofold: to establish the conditions for warfarin-induced calcification of human aortic valve interstitial cells (HAVICs) using high-inorganic phosphate (Pi) conditions and to investigate the underlying mechanism. We prepared and cultured HAVICs from aortic valves affected by calcific aortic valve stenosis (AS group) and aortic valves affected by aortic regurgitation but without any signs of calcification (non-AS group). Under Pi concentrations of 3.2mM, warfarin significantly increased the calcification and alkaline phosphatase (ALP) activity of AS but not non-AS group HAVICs. Furthermore, gene expression of bone morphogenetic protein 2 (BMP2), a calcigenic marker, was significantly increased following 7 days of warfarin treatment. Warfarin-induced calcification of AS group HAVICs at 3.2mM Pi was significantly inhibited by dorsomorphin, a Smad inhibitor, and the pregnane X receptor (PXR) inhibitors, ketoconazole and coumestrol, but was unaffected by SN-50, an NF-κB inhibitor. Warfarin was also able to increase BMP2 gene expression at a physiological Pi concentration (1.0mM). Furthermore, excess BMP2 (30ng/mL) facilitated warfarin-induced ALP upregulation and HAVIC calcification, an effect which was significantly reduced in the presence of coumestrol. Together, our results suggest that warfarin accelerates calcification of HAVICs from AS patients via the PXR-BMP2-ALP pathway.

IL-8 promotes the calcification of human aortic valve interstitial cells, which is prevented through antagonizing CXCR1 and CXCR2 receptors

IL-8 promotes the calcification of human aortic valve interstitial cells, which is prevented through antagonizing CXCR1 and CXCR2 receptors

Aortic valve stenosis (AVS) is a common heart valve disease in elderly people, and is mostly accelerated by ectopic aortic valve calcification (AVC). We recently demonstrated that tumor necrosis factor-α (TNF-α) induces calcification of human aortic valve interstitial cells (HAVICs) obtained from AVS patients. In this study, we investigated the inhibitory effect of Orengetokuto (OGT) on TNF-α-induced calcification of HAVICs. OGT is composed of four crude drugs (Coptodis rhizoma, Scutellariae radix, Phellodendri cortex, and Gardeniae fructus) and is used for hypertension, palpitation and others. HAVICs isolated from aortic valves were cultured in α-MEM including 10% FBS, and calcification was induced by TNF-α (30 ng/μL). Gene expression of BMP2 and ALP activity as typical calcification markers, were significantly increased in response to TNF-α. To confirm the effect of OGT on TNF-α-induced calcification, HAVICs were cultured in medium containing OGT and TNF-α. We found that OGT inhibited TNF-α induced calcification confirmed by Alizarin Red S stain. Increasing BMP2 gene expression and ALP activity induced by TNF-α were also inhibited by OGT. These results suggest that OGT can be used for treatment of AVS by inhibiting acceleration of AVC.

The cellular mechanisms that induce calcific aortic stenosis are yet to be unraveled. Wnt signaling is increasingly being considered as a major player in the disease process. However, the presence of Wnt Frizzled (Fzd) receptors and co-receptors LRP5 and 6 in normal and diseased human aortic valves remains to be elucidated. Immunohistochemistry and quantitative polymerase chain reaction were used to determine Fzd receptor expression in normal and calcified human aortic valve tissue, as well as human aortic valve interstitial cells (HAVICs) isolated from calcified and normal human aortic valves. There was significantly higher mRNA expression of 4 out of the 10 Fzd receptors in calcified aortic valve tissues and 8 out of the 10 in HAVICs, and both LRP5/6 co-receptors in calcified aortic valves (P < 0.05). These results were confirmed by immunohistochemistry, which revealed abundant increase in immunoreactivity for Fzd3, 7, and 8, mainly in areas of lipid core and calcified nodules of diseased aortic valves. The findings of abundant expression of Fzd and LRP5/6 receptors in diseased aortic valves suggests a potential role for both canonical and noncanonical Wnt signaling in the pathogenesis of human aortic valve calcification. Future investigations aimed at targeting these molecules may provide potential therapies for aortic valve stenosis.

Values of osteoprotegerin in aortic valve tissue differ significantly between calcified aortic valve stenosis and normal aortic valve and are influenced by the presence of coronary atherosclerosis

Pathological significance of lipoprotein(a) in aortic valve stenosis

Development of calcific aortic valve stenosis involves multiple signaling pathways, which may be modulated by peroxisome proliferator-activated receptor-γ). This study tested the hypothesis that pioglitazone (Pio), a ligand for peroxisome proliferator-activated receptor-γ, inhibits calcification of the aortic valve in hypercholesteremic mice. Low density lipoprotein receptor(-/-)/apolipoprotein B(100/100) mice were fed a Western-type diet with or without Pio (20 mg/kg per day) for 6 months. Pio attenuated lipid deposition and calcification in the aortic valve, but not aorta. In the aortic valve, Pio reduced levels of active caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Valve function (echocardiography) was significantly improved by Pio. To determine whether changes in gene expression are associated with differential effects of Pio on aortic valves versus aorta, Reversa mice were fed Western diet with or without Pio for 2 months. Several procalcific genes were increased by Western diet, and the increase was attenuated by Pio, in aortic valve, but not aorta. Pio attenuates lipid deposition, calcification, and apoptosis in aortic valves of hypercholesterolemic mice, improves aortic valve function, and exhibits preferential effects on aortic valves versus aorta. We suggest that Pio protects against calcific aortic valve stenosis, and Pio or other peroxisome proliferator-activated receptor-γ ligands may be useful for early intervention to prevent or slow stenosis of aortic valves.