Inflammatory cytokines promote mesenchymal transformation in embryonic and adult valve endothelial cells.

OBJECTIVE: Inflammatory activation of valve endothelium is an early phase of aortic valve disease pathogenesis, but subsequent mechanisms are poorly understood. Adult valve endothelial cells retain the developmental ability to undergo endothelial-to-mesenchymal transformation (EndMT), but a biological role has not been established. Here, we test whether and how inflammatory cytokines (tumor necrosis factor-α and interleukin-6) regulate EndMT in embryonic and adult valve endothelium. METHODS AND RESULTS: Using in vitro 3-dimensional collagen gel culture assays with primary cells, we determined that interleukin-6 and tumor necrosis factor-α induce EndMT and cell invasion in dose-dependent manners. Inflammatory-EndMT occurred through an Akt/nuclear factor-κB-dependent pathway in both adult and embryonic stages. In embryonic valves, inflammatory-EndMT required canonical transforming growth factor-ß signaling through activin receptor-like kinases 2 and 5 to drive EndMT. In adult valve endothelium, however, inflammatory-induced EndMT still occurred when activin receptor-like kinases 2 and 5 signaling was blocked. Inflammatory receptor gene expression was significantly upregulated in vivo during embryonic valve maturation. Endothelial-derived mesenchymal cells expressing activated nuclear factor-κB were found distal to calcific lesions in diseased human aortic valves. CONCLUSIONS: Inflammatory cytokine-induced EndMT in valve endothelium is present in both embryonic and adult stages, acting through Akt/nuclear factor-κB, but differently using transforming growth factor-ß signaling. Molecular signatures of valve EndMT may be important diagnostic and therapeutic targets in early valve disease.

Performance comparison of 6 in-hospital patient monitoring systems in the detection and alarm of ventricular cardiac arrhythmias.

Background: Patient monitoring devices are critical for alerting of potential cardiac arrhythmias during hospitalization; however, there are concerns of alarm fatigue due to high false alarm rates. Objective: The purpose of this study was to evaluate the sensitivity and false alarm rate of hospital-based continuous electrocardiographic (ECG) monitoring technologies. Methods: Six commonly used multiparameter bedside monitoring systems available in the United States were evaluated: B125M (GE HealthCare), ePM10 and iPM12 (Mindray), Efficia and IntelliVue (Philips), and Life Scope (Nihon Kohden). Sensitivity was tested using ECG recordings containing 57 true ventricular tachycardia (VT) events. False-positive rate testing used 205 patient-hours of ECG recordings containing no cardiac arrhythmias. Signals from ECG recordings were fed to devices simultaneously; high-severity arrhythmia alarms were tracked. Sensitivity to true VT events and false-positive rates were determined. Differences were assessed using Fisher exact tests (sensitivity) and Z-tests (false-positive rates). Results: B125M raised 56 total alarms for 57 annotated VT events and had the highest sensitivity (98%; P <.05), followed by iPM12 (84%), Life Scope (81%), Efficia (79%), ePM10 (77%), and IntelliVue (75%). B125M raised 20 false alarms, which was significantly lower (P <.0001) than iPM12 (284), Life Scope (292), IntelliVue (304), ePM10 (324), and Efficia (493). The most common false alarm was VT, followed by nonsustained VT. Conclusion: We found significant performance differences among multiparameter bedside ECG monitoring systems using previously collected recordings. B125M had the highest sensitivity in detecting true VT events and lowest false alarm rate. These results can assist in minimizing alarm fatigue and optimizing patient safety by careful selection of in-hospital continuous monitoring technology.

Valvular heart diseases in the developing world: developmental biology takes center stage.

Heart valve disease is a significant and increasing global problem of which, in the developing world, the primary sufferers are the children and young adults regarded as the critical 'engine' of future economic growth. Yet, up to 10 times the current number of known sufferers remain undiagnosed in these countries. Among the most prevalent and neglected diseases are rheumatic heart disease and endomyocardial fibrosis. The etiologies of these diseases can be described in part as a dysregulation or reactivation of developmental biology pathways. Consequently, connecting mechanisms of valvulogenesis and disease etiology may represent an excellent strategy to identify therapeutic targets. These local diseases require local solutions tailored to local resources; therefore, collaboration with experienced research groups should be encouraged as a way of accelerating the creation of relevant knowledge, and its clinical translation.

Two-year retrospective cohort results on use of a dynamic unilateral brace for treatment of clubfoot: Can compliance and prevention of recurrence both be achieved?

Objectives: The Ponseti method has led to vast improvements in outcomes for infants born with clubfoot deformity, but challenges with compliance during the bracing phase of the protocol remain. Unilateral braces promise higher compliance but often have led to unacceptably high recurrence. Methods: We have developed a novel unilateral brace for clubfoot deformity that strategically applies patient-specific, anatomically-targeted forces to the lower limb to maintain correction. We retrospectively reviewed the cases of 26 patients with minimum follow-up of 24 months. The data were analyzed for recurrence rates, caregiver-reported compliance, and differences in Pirani score, dorsiflexion, abduction, hindfoot eversion, and resting rotation between initial and final follow-up. Results: Most patients (N = 23, 88%) were compliant with the bracing protocol. Two patients showed recurrence of deformity (8%). There were statistically significant improvements in Pirani score, dorsiflexion, abduction, hindfoot eversion, and resting external rotation. A subset of patients with sub-optimal correction at baseline showed improvement in all parameters across the course of bracing. Conclusions: This novel unilateral brace for maintenance of clubfoot correction after Ponseti treatment demonstrates rates of recurrence rates and caregiver-reported compliance at 2 years of follow up that are comparable to outcomes with traditional bilateral foot abduction orthoses.

OCT4-mediated inflammation induces cell reprogramming at the origin of cardiac valve development and calcification.

Cell plasticity plays a key role in embryos by maintaining the differentiation potential of progenitors. Whether postnatal somatic cells revert to an embryonic-like naïve state regaining plasticity and redifferentiate into a cell type leading to a disease remains intriguing. Using genetic lineage tracing and single-cell RNA sequencing, we reveal that Oct4 is induced by nuclear factor κB (NFκB) at embyronic day 9.5 in a subset of mouse endocardial cells originating from the anterior heart forming field at the onset of endocardial-to-mesenchymal transition. These cells acquired a chondro-osteogenic fate. OCT4 in adult valvular aortic cells leads to calcification of mouse and human valves. These calcifying cells originate from the Oct4 embryonic lineage. Genetic deletion of Pou5f1 (Pit-Oct-Unc, OCT4) in the endocardial cell lineage prevents aortic stenosis and calcification of ApoE−/− mouse valve. We established previously unidentified self-cell reprogramming NFκB- and OCT4-mediated inflammatory pathway triggering a dose-dependent mechanism of valve calcification.

Valve interstitial cell tensional homeostasis directs calcification and extracellular matrix remodeling processes via RhoA signaling.

AIMS: Valve interstitial cells are active and aggressive players in aortic valve calcification, but their dynamic mediation of mechanically-induced calcific remodeling is not well understood. The goal of this study was to elucidate the feedback loop between valve interstitial cell and calcification mechanics using a novel three-dimensional culture system that allows investigation of the active interplay between cells, disease, and the mechanical valve environment. METHODS & RESULTS: We designed and characterized a novel bioreactor system for quantifying aortic valve interstitial cell contractility in 3-D hydrogels in control and osteogenic conditions over 14 days. Interstitial cells demonstrated a marked ability to exert contractile force on their environment and to align collagen fibers with the direction of tension. Osteogenic environment disrupted interstitial cell contractility and led to disorganization of the collagen matrix, concurrent with increased αSMA, TGF-ß, Runx2 and calcific nodule formation. Interestingly, RhoA was also increased in osteogenic condition, pointing to an aberrant hyperactivation of valve interstitial cells mechanical activity in disease. This was confirmed by inhibition of RhoA experiments. Inhibition of RhoA concurrent with osteogenic treatment reduced pro-osteogenic signaling and calcific nodule formation. Time-course correlation analysis indicated a significant correlation between interstitial cell remodeling of collagen fibers and calcification events. CONCLUSIONS: Interstitial cell contractility mediates internal stress state and organization of the aortic valve extracellular matrix. Osteogenesis disrupts interstitial cell mechanical phenotype and drives disorganization, nodule formation, and pro-calcific signaling via a RhoA-dependent mechanism.

Endothelial-derived oxidative stress drives myofibroblastic activation and calcification of the aortic valve.

AIMS: Oxidative stress is present in and contributes to calcification of the aortic valve, but the driving factors behind the initiation of valve oxidative stress are not well understood. We tested whether the valve endothelium acts as an initiator and propagator of oxidative stress in aortic valve disease. METHODS AND RESULTS: Calcified human aortic valves showed side-specific elevation of superoxide in the endothelium, co-localized with high VCAM1 expression, linking oxidative stress, inflammation, and valve degeneration. Treatment with inflammatory cytokine TNFα increased superoxide and oxidative stress and decreased eNOS and VE-cadherin acutely over 48 hours in aortic valve endothelial cells (VEC) and chronically over 21 days in ex vivo AV leaflets. Co-treatment of VEC with tetrahydrobiopterin (BH4) but not apocynin mitigated TNFα-driven VEC oxidative stress. Co-treatment of ex vivo AV leaflets with TNFα+BH4 or TNFα+peg-SOD rescued endothelial function and mitigated inflammatory responses. Both BH4 and peg-SOD rescued valve leaflets from the pro-osteogenic effects of TNFα treatment, but only peg-SOD was able to mitigate the fibrogenic effects, including increased collagen and αSMA expression. CONCLUSIONS: Aortic valve endothelial cells are a novel source of oxidative stress in aortic valve disease. TNFα-driven VEC oxidative stress causes loss of endothelial protective function, chronic inflammation, and fibrogenic and osteogenic activation, mitigated differentially by BH4 and peg-SOD. These mechanisms identify new targets for tailored antioxidant therapy focused on mitigation of oxidative stress and restoration of endothelial protection.

Heterogeneous susceptibility of valve endothelial cells to mesenchymal transformation in response to TNFα.

Lack of understanding of the early mechanisms of aortic valve stenosis and calcification hinders the development of diagnostic and therapeutic intervention strategies. Inflammation is a known component of early aortic valve disease and can induce mesenchymal transformation in a subset of aortic valve endothelial cells. Here we present a three-dimensional culture system that allows transforming and non-transforming cells to be independently isolated and analyzed. We have used the system to identify and characterize the dynamic invasion and phenotypic transition of two distinct subsets of endothelial cells: those that invade and transform under TNFα treatment, and those that resist mesenchymal transformation and remain endothelial. We determine that non-transformed cells maintain control levels of endothelial genes VE-cadherin and eNOS, while transformed cells lose these endothelial characteristics and upregulate α-smooth muscle actin. Both subsets of cells have an inflammatory phenotype marked by increased ICAM-1, but transformed cells have increased MMP-9, Notch1, TGF-ß, and BMP-4, while non-transformed cells do not. Transformed cells also have distinct effects on alignment of collagen fibers as they invade the hydrogel system, which is not found in control endothelial or interstitial valve cells. Understanding the role of transforming and non-transforming endothelial cells in valve disease will provide an important pathological link between early inflammation and later stages of disease. Discovery of the molecular signature of transformation-resistant endothelial cells could inform development of treatment strategies that promote survival of the valve endothelium.

The mechanobiology of mitral valve function, degeneration, and repair.

In degenerative valve disease, the highly organized mitral valve leaflet matrix stratification is progressively destroyed and replaced with proteoglycan rich, mechanically inadequate tissue. This is driven by the actions of originally quiescent valve interstitial cells that become active contractile and migratory myofibroblasts. While treatment for myxomatous mitral valve disease in humans ranges from repair to total replacement, therapies in dogs focus on treating the consequences of the resulting mitral regurgitation. The fundamental gap in our understanding is how the resident valve cells respond to altered mechanical signals to drive tissue remodeling. Despite the pathological similarities and high clinical occurrence, surprisingly little mechanistic insight has been gleaned from the dog. This review presents what is known about mitral valve mechanobiology from clinical, in vivo, and in vitro data. There are a number of experimental strategies already available to pursue this significant opportunity, but success requires the collaboration between veterinary clinicians, scientists, and engineers.