**6. Conclusions and future directions**

duction and increased generation of reactive oxygen species (ROS) [223]. ROS activity is present in calcific lesions of human stenotic aortic valves, and it has been suggested that ROS activity may speed aortic valve calcification [144, 226]. In animal studies, rabbits fed a high cholesterol/high vitamin D diet develop aortic valve thickening, small deposits of valve calcification, and increased ROS activity in cells surrounding regions of valve calcification [226]. Furthermore, ROS activity was co-localized to clusters of cells expressing Runx2 and OPN, suggesting that ROS activity is associated with VICs displaying an osteogenic-like phenotype [226]. In VIC culture studies, TGFβ1 stimulation induces increased ROS activity, along with calcific nodule formation and ALP activity [227]. Increasing the availability of NO, via NO donors such as sodium nitroprusside, partially blocks both nodule formation and ALP activity, suggesting that NO levels are important for reducing ROS and inhibiting calcification in VICs [227]. There have been a number of small clinical studies investigating the levels of the NOS inhibitor, asymmetric dimethylarginine (ADMA), an indicator of en‐ dothelial cell dysfunction, in patients with moderate to severe AS [228-230]. In these studies, plasma levels of ADMA are significantly higher in patients with moderate to severe AS, compared to patients with mild AS or no disease, suggesting that NO production is disrupt‐ ed in CAVD/AS [228-230]. Combined, these studies suggest that increased ROS production is associated with aortic valve calcification and the induction of osteogenic gene expression. Thus, increasing the bioavailability of NO may be a potential therapeutic avenue to block

Previous reports have demonstrated that immune cell infiltration is common in CAVD [119, 132, 135]. Non-steroidal anti-inflammatory drugs (NSAIDS) are commonly used to treat pain and inflammation, and act by inhibiting the enzymes COX1 and/or COX2 [231-233]. These enzymes function by converting arachidonic acid to prostaglandins (PGs) [233, 234]. There has been one study conducted in human aortic VICs, demonstrating that stimulation of VICs with pro-inflammatory mimetics induces the expression of COX2 and the release of prostaglandins [235]. This study suggests that COX2 inhibition may be one way to treat the immune response associated with CAVD [235]. Interestingly, COX2 and PG signaling are al‐ so involved in bone formation as well as cellular responses to physical stress and strain, processes that also likely contribute to CAVD (reviewed in [131]). In bone, PG signaling has an anabolic effect, and PG treatment of osteoblast cultures results in increased expression of OCN, BMP2, Runx2, OPN, ALP, and BSP [236-244]. In osteoblast cultures, BMP2 stimulation induces COX2 expression through upregulation of Runx2, which binds to and activates the COX2 promoter [245, 246]. Downstream, PG signaling induces the expression of p38 MAPK through the activation of protein kinase A [243, 247]. Furthermore, fluid shear stress and other physical forces induce COX2 expression in osteoblast-like cells, suggesting that in‐ creased COX2 and PG signaling is a cellular response to altered mechanical forces [248, 249]. The combined results of these studies suggest that COX2/PG signaling may be an effective therapeutic target to treat CAVD progression, as COX2/PG signaling plays a role in inflam‐ mation, osteogenesis, and cellular response to physical strain, all of which are thought to be pathological mechanisms involved in CAVD [131, 243, 244, 248-251]. It would be interesting

ROS activity, and thereby disease progression, in CAVD.

**5.6. NSAIDs/COX2 inhibitors**

82 Calcific Aortic Valve Disease

There are numerous examples of shared molecular pathways between valvulogenesis, os‐ teogenesis, and disease pathogenesis of CAVD. In valvulogenesis, signaling factors involved in early cushion formation, such as BMP, Notch, and Wnt/β-catenin pathways are active in osteogenesis and in CAVD [7-9, 14]. Furthermore, transcription factors expressed in the ear‐ ly valve mesenchyme, such as Twist1, Msx2, and Sox9, can also be found in the primitive condensed bone mesenchyme and in the mesenchymal-like cells identified in diseased aortic valve tissues [8, 13, 80, 255]. In addition to signaling and transcription factors, molecular pathways governing ECM production and remodeling, such as the RANKL – NFATc1 – CtsK pathway are shared amongst valve progenitor, developing bone, and diseased valve tissues [11, 47, 103, 106, 193]. This commonality suggests that the mesenchymal cells found within these tissues are governed by common molecular pathways and that these develop‐ mental pathways are reactivated during disease. Additional parallels can be drawn between calcification of the embryonic bone tissues and calcification observed in diseased aortic valves. For example, the endochondral bone factors Runx2, OCN, and BSP are reactivated during aortic valve disease, suggesting that osteogenic molecular pathways are activated during CAVD and may contribute to pathogenic calcification [5, 13, 76, 80, 106, 152]. Effec‐ tive pharmacological therapies to treat CAVD remain elusive and identifying potential tar‐ gets for new pharmacotherapies is a priority, as the only effective treatment for CAVD with AS is valve replacement surgery [256]. Studies testing the effectiveness of statin therapy, in‐ hibitors of the renin-angiotensin-aldosterone, and bisphosphonates in slowing the progres‐ sion of CAVD have been disappointing (see therapeutic section). New therapeutic strategies are needed and, perhaps, targeted inhibition of BMP and Wnt signaling or maintenance of Notch signaling may provide new avenues for potential CAVD treatments.

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