Rac1 mediates cadherin-11 induced cellular pathogenic processes in aortic valve calcification.

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.

Mechanistic insights into volatile anesthetic modulation of K2P channels.

K2P potassium channels are known to be modulated by volatile anesthetic (VA) drugs and play important roles in clinically relevant effects that accompany general anesthesia. Here, we utilize a photoaffinity analog of the VA isoflurane to identify a VA-binding site in the TREK1 K2P channel. The functional importance of the identified site was validated by mutagenesis and biochemical modification. Molecular dynamics simulations of TREK1 in the presence of VA found multiple neighboring residues on TREK1 TM2, TM3, and TM4 that contribute to anesthetic binding. The identified VA-binding region contains residues that play roles in the mechanisms by which heat, mechanical stretch, and pharmacological modulators alter TREK1 channel activity and overlaps with positions found to modulate TASK K2P channel VA sensitivity. Our findings define molecular contacts that mediate VA binding to TREK1 channels and suggest a mechanistic basis to explain how K2P channels are modulated by VAs.

Induction of aortic valve calcification by celecoxib and its COX-2 independent derivatives is glucocorticoid-dependent.

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.

Cadherin-11 Overexpression Induces Extracellular Matrix Remodeling and Calcification in Mature Aortic Valves.

OBJECTIVE: Calcific aortic valve (AoV) disease is a significant clinical problem for which the regulatory mechanisms are poorly understood. Enhanced cell-cell adhesion is a common mechanism of cellular aggregation, but its role in calcific lesion formation is not known. Cadherin-11 (Cad-11) has been associated with lesion formation in vitro, but its function during adult valve homeostasis and pathogenesis is not known. This study aims to elucidate the specific functions of Cad-11 and its downstream targets, RhoA and Sox9, in extracellular matrix remodeling and AoV calcification. APPROACH AND RESULTS: We conditionally overexpressed Cad-11 in murine heart valves using a novel double-transgenic Nfatc1(Cre);R26-Cad11(TglTg) mouse model. These mice developed hemodynamically significant aortic stenosis with prominent calcific lesions in the AoV leaflets. Cad-11 overexpression upregulated downstream targets, RhoA and Sox9, in the valve interstitial cells, causing calcification and extensive pathogenic extracellular matrix remodeling. AoV interstitial cells overexpressing Cad-11 in an osteogenic environment in vitro rapidly form calcific nodules analogous to in vivo lesions. Molecular analyses revealed upregulation of osteoblastic and myofibroblastic markers. Treatment with a Rho-associated protein kinase inhibitor attenuated nodule formation, further supporting that Cad-11-driven calcification acts through the small GTPase RhoA/Rho-associated protein kinase signaling pathway. CONCLUSIONS: This study identifies one of the underlying molecular mechanisms of heart valve calcification and demonstrates that overexpression of Cad-11 upregulates RhoA and Sox9 to induce calcification and extracellular matrix remodeling in adult AoV pathogenesis. The findings provide a potential molecular target for clinical treatment.