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Background: Calcific aortic valve disease (CAVD), a major cause for surgical aortic valve replacement, currently lacks available pharmacological treatments. Cadherin-11 (Cad11), a promising therapeutic target, promotes aortic valve calcification in vivo, but direct Cad11 inhibition in clinical trials has been unsuccessful. Targeting of downstream Cad11 effectors instead may be clinically useful; however, the downstream effectors that mediate Cad11-induced aortic valve cellular pathogenesis have not been investigated., Approach and Results: Immunofluorescence of calcified human aortic valves revealed that GTP-Rac1 is highly upregulated in calcified leaflets and is 2.15 times more co-localized with Cad11 in calcified valves than GTP-RhoA. Using dominant negative mutants in porcine aortic valve interstitial cells (PAVICs), we show that Cad11 predominantly regulates Runx2 nuclear localization via Rac1. Rac1-GEF inhibition via NSC23766 effectively reduces calcification in ex vivo porcine aortic valve leaflets treated with osteogenic media by 2.8-fold and also prevents Cad11-induced cell migration, compaction, and calcification in PAVICs. GTP-Rac1 and Trio, a known Cad11 binding partner and Rac1-GEF, are significantly upregulated in Nfatc1Cre; R26-Cad11Tg/Tg (Cad11 OX) mice that conditionally overexpress Cad11 in the heart valves by 3.1-fold and 6.3-fold, respectively. Finally, we found that the Trio-specific Rac1-GEF inhibitor, ITX3, effectively prevents Cad11-induced calcification and Runx2 induction in osteogenic conditions., Conclusion: Here we show that Cad11 induces many cellular pathogenic processes via Rac1 and that Rac1 inhibition effectively prevents many Cad11-induced aortic disease phenotypes. These findings highlight the therapeutic potential of blocking Rac1-GEFs in CAVD., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Objective: Calcific aortic valve disease (CAVD) is an actively regulated degenerative disease process. Clinical lesions exhibit marked 3D complexity not represented in current in vitro systems. We here present a unique mechanically stressed 3D culture system that recapitulates valve interstitial cell (VIC) induced matrix calcification through myofibroblastic activation and osteoblastic differentiation. We test the hypothesis that valve endothelial (VEC) - interstitial collaborative interactions modulate the risk and complexity of calcific pathogenesis within mechanically stressed and pro-inflammatory environments., Approach and Results: Porcine aortic valve endothelial and interstitial cells (VEC and VIC) were seeded in a mechanically constrained collagen hydrogels alone or in co-culture configurations. Raised 3D VIC-filled lesions formed within 7 days when cultured in osteogenic media (OGM), and surprisingly exacerbated by endothelial coculture. We identified a spatially coordinated pro-endochondral vs. pro-osteogenic signaling program within the lesion. VEC underwent Endothelial-to-Mesenchymal Transformation (EndMT) and populated the lesion center. The spatial complexity of molecular and cellular signatures of this 3D in vitro CAVD system were consistent with human diseased aortic valve histology. SNAI1 was highly expressed in the VEC and subendothelial direct VIC corroborates with human CAVD lesions. Spatial distribution of Sox9 vs. Runx2 expression within the developed lesions (Sox9 peri-lesion vs. Runx2 predominantly within lesions) mirrored their expression in heavily calcified human aortic valves. Finally, we demonstrate the applicability of this platform for screening potential pharmacologic therapies through blocking the canonical NFκB pathway via BAY 11-7082., Conclusions: Our results establish that VEC actively induce VIC pathological remodeling and calcification via EndMT and paracrine signaling. This mechanically constrained culture platform enables the interrogation of accelerated cell-mediated matrix remodeling behavior underpinned by this cellular feedback circuit. The high fidelity of this complex 3D model system to human CAVD mechanisms supports its use to test mechanisms of intercellular communication in valves and their pharmacological control., (Copyright © 2021. Published by Elsevier Ltd.)

Background: Celecoxib, a selective cyclooxygenase-2 inhibitor, was recently associated with increased incidence of aortic stenosis and found to produce a valvular calcification risk in vitro. Several cyclooxygenase-2 independent celecoxib derivatives have been developed and identified as possible therapies for inflammatory diseases due to their cadherin-11 inhibitory functions. Potential cardiovascular toxicities associated with these cyclooxygenase-2 independent celecoxib derivatives have not yet been investigated. Furthermore, the mechanism by which celecoxib produces valvular toxicity is not known., Methods and Results: Celecoxib treatment produces a 2.8-fold increase in calcification in ex vivo porcine aortic valve leaflets and a more than 2-fold increase in calcification in porcine aortic valve interstitial cells cultured in osteogenic media. Its cyclooxygenase-2 independent derivative, 2,5-dimethylcelecoxib, produces a similar 2.5-fold increase in calcification in ex vivo leaflets and a 13-fold increase in porcine aortic valve interstitial cells cultured in osteogenic media. We elucidate that this offtarget effect depends on the presence of either of the two media components: dexamethasone, a synthetic glucocorticoid used for osteogenic induction, or cortisol, a natural glucocorticoid present at basal levels in the fetal bovine serum. In the absence of glucocorticoids, these inhibitors effectively reduce calcification. By adding glucocorticoids or hydrocortisone to a serum substitute lacking endogenous glucocorticoids, we show that dimethylcelecoxib conditionally induces a 3.5-fold increase in aortic valve calcification and osteogenic expression. Treatment with the Mitogen-activated protein kinase kinase inhibitor, U0126, rescues the offtarget effect, suggesting that celecoxib and dimethylcelecoxib conditionally augment Mitogen-activated protein kinase kinase/extracellular-signal-regulated kinase activity in the presence of glucocorticoids., Conclusion: Here we identify glucocorticoids as a possible source of the increased valvular calcification risk associated with celecoxib and its cyclooxygenase-2 independent derivatives. In the absence of glucocorticoids, these inhibitors effectively reduce calcification. Furthermore, the offtarget effects are not due to the drug's intrinsic properties as dual cyclooxygenase-2 and cadherin-11 inhibitors. These findings inform future design and development of celecoxib derivatives for potential clinical therapy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Monocytes and macrophages are critical components of the myeloid niche of the innate immune system. In addition to traditional roles as phagocytes, this subsection of innate immunity has been implicated in its ability to regulate tissue homeostasis and inflammation across diverse physiological systems. Recent emergence of discriminatory features within the monocyte/macrophage niche within the last 5 years has helped to clarify specific function(s) of the subpopulations of these cells. It is becoming increasingly aware that these cells are likely implicated in valve development and disease. This review seeks to use current literature and opinions to show the diverse roles and potential contributions of this niche throughout valvulogenic processes, adult homeostatic function, valve disease mechanisms, and tissue engineering approaches.