Risk factors for fibrocalcific aortic valve disease (FCAVD) are associated with systemic decreases in bioavailability of endothelium-derived nitric oxide (EDNO). In patients with bicuspid aortic valve (BAV), vascular expression of endothelial nitric oxide synthase (eNOS) is decreased, and eNOS(-/-) mice have increased prevalence of BAV. The goal of this study was to test the hypotheses that EDNO attenuates profibrotic actions of valve interstitial cells (VICs) in vitro and that EDNO deficiency accelerates development of FCAVD in vivo. As a result of the study, coculture of VICs with aortic valve endothelial cells (vlvECs) significantly decreased VIC activation, a critical early phase of FCAVD. Inhibition of VIC activation by vlvECs was attenuated by N(G)-nitro-l-arginine methyl ester or indomethacin. Coculture with vlvECs attenuated VIC expression of matrix metalloproteinase-9, which depended on stiffness of the culture matrix. Coculture with vlvECs preferentially inhibited collagen-3, compared with collagen-1, gene expression. BAV occurred in 30% of eNOS(-/-) mice. At age 6 mo, collagen was increased in both bicuspid and trileaflet eNOS(-/-) aortic valves, compared with wild-type valves. At 18 mo, total collagen was similar in eNOS(-/-) and wild-type mice, but collagen-3 was preferentially increased in eNOS(-/-) mice. Calcification and apoptosis were significantly increased in BAV of eNOS(-/-) mice at ages 6 and 18 mo. Remarkably, these histological changes were not accompanied by physiologically significant valve stenosis or regurgitation. In conclusion, coculture with vlvECs inhibits specific profibrotic VIC processes. In vivo, eNOS deficiency produces fibrosis in both trileaflet and BAVs but produces calcification only in BAVs.

BackgroundThere are no rigorously confirmed effective medical therapies for calcific aortic stenosis. Hypercholesterolemic Ldlr −/− Apob 100/100 mice develop calcific aortic stenosis and valvular cardiomyopathy in old age. Osteoprotegerin (OPG) modulates calcification in bone and blood vessels, but its effect on valve calcification and valve function is not known.ObjectivesTo determine the impact of pharmacologic treatment with OPG upon aortic valve calcification and valve function in aortic stenosis-prone hypercholesterolemic Ldlr −/− Apob 100/100 mice.MethodsYoung Ldlr −/− Apob 100/100 mice (age 2 months) were fed a Western diet and received exogenous OPG or vehicle (N = 12 each) 3 times per week, until age 8 months. After echocardiographic evaluation of valve function, the aortic valve was evaluated histologically. Older Ldlr −/− Apob 100/100 mice were fed a Western diet beginning at age 2 months. OPG or vehicle (N = 12 each) was administered from 6 to 12 months of age, followed by echocardiographic evaluation of valve function, followed by histologic evaluation.ResultsIn Young Ldlr −/− Apob 100/100 mice, OPG significantly attenuated osteogenic transformation in the aortic valve, but did not affect lipid accumulation. In Older Ldlr −/− Apob 100/100 mice, OPG attenuated accumulation of the osteoblast-specific matrix protein osteocalcin by ∼80%, and attenuated aortic valve calcification by ∼ 70%. OPG also attenuated impairment of aortic valve function.ConclusionsOPG attenuates pro-calcific processes in the aortic valve, and protects against impairment of aortic valve function in hypercholesterolemic aortic stenosis-prone Ldlr −/− Apob 100/100 mice.

ObjectiveSustained hemodynamic stress mediated by high blood flow promotes arteriogenesis, the outward remodeling of existing arteries. Here, we examined whether Ca2+/calmodulin-dependent kinase II (CaMKII) regulates arteriogenesis.Methods and ResultsLigation of the left common carotid led to an increase in vessel diameter and perimeter of internal and external elastic lamina in the contralateral, right common carotid. Deletion of CaMKIIδ (CaMKIIδ−/−) abolished this outward remodeling. Carotid ligation increased CaMKII expression and was associated with oxidative activation of CaMKII in the adventitia and endothelium. Remodeling was abrogated in a knock-in model in which oxidative activation of CaMKII is abolished. Early after ligation, matrix metalloproteinase 9 (MMP9) was robustly expressed in the adventitia of right carotid arteries of WT but not CaMKIIδ−/− mice. MMP9 mainly colocalized with adventitial macrophages. In contrast, we did not observe an effect of CaMKIIδ deficiency on other proposed mediators of arteriogenesis such as expression of adhesion molecules or smooth muscle proliferation. Transplantation of WT bone marrow into CaMKIIδ−/− mice normalized flow-mediated remodeling.ConclusionCaMKIIδ is activated by oxidation under high blood flow conditions and is required for flow-mediated remodeling through a mechanism that includes increased MMP9 expression in bone marrow-derived cells invading the arterial wall.

Objective Hypercholesterolemia (HC) and hypertension (HT) are associated with aortic valve stenosis (AVS) in humans. We have examined aortic valve function, structure, and gene expression in HC/HT mice. Approach and Results Control, hypertensive (HT), hypercholesterolemic (Apoe−/−) (HC), and HC/HT mice were studied. Severe aortic stenosis (echocardiography) occurred only in HC/HT mice. There was minimal calcification of the aortic valve. Several structural changes were identified at the base of the valve. The intercusp raphe (or “seam” between leaflets) was longer in HC/HT mice than in other mice, and collagen fibers at the base of the leaflets were reoriented to form a mesh. In HC/HT mice, the cusps were asymmetrical, which may contribute to changes that produce AVS. RNA-Sequencing (RNA-Seq) was used to identify molecular targets during the developmental phase of stenosis. Genes related to structure of the valve were identified that differentially expressed before FAVS developed. Both RNA and protein of a profibrotic molecule, plasminogen activator inhibitor 1 (PAI-1), were increased greatly in HC/HT mice. Conclusions HC/HT mice are the first model of fibrotic AVS. HC/HT mice develop severe AVS in the absence of significant calcification, a feature which resembles AVS in children and some adults. Structural changes at the base of the valve leaflets include lengthening of the raphe, remodeling of collagen, and asymmetry of the leaflets. Genes were identified that may contribute to development of FAVS.

Objective We studied the mechanistic links between fibrocalcific changes in the aortic valve and aortic valve function in mice homozygous for a hypomorphic epidermal growth factor receptor mutation (Wave mice). We also studied myocardial responses to aortic valve dysfunction in Wave mice. Approach and Results At 1.5 months of age, prior to development of valve fibrosis and calcification, aortic regurgitation, but not aortic stenosis, was common in Wave mice. Aortic valve fibrosis, pro-fibrotic signaling, calcification, osteogenic markers, lipid deposition, and apoptosis increased dramatically by 6 and 12 months of age in Wave mice. Aortic regurgitation remained prevalent, however, and aortic stenosis was rare, at all ages. Proteoglycan content was abnormally increased in aortic valves of Wave mice at all ages. Treatment with pioglitazone prevented abnormal valve calcification, but did not protect valve function. There was significant left ventricular volume overload, hypertrophy, and fetal gene expression, at all ages in Wave mice with aortic regurgitation. Left ventricular systolic function was normal until 6 months of age in Wave mice, but became impaired by 12 months of age. Myocardial transverse tubules were normal in the presence of left ventricular hypertrophy at 1.5 and 3 months of age, but became disrupted by 12 months of age. Conclusions We present the first comprehensive phenotypic and molecular characterization of spontaneous aortic regurgitation and volume-overload cardiomyopathy in an experimental model. In Wave mice, fibrocalcific changes are not linked to valve dysfunction, and are epiphenomena arising from structurally incompetent “myxomatous” valves.

Meat consumption and public concern for farm animal welfare are increasing, despite limited public understanding of agriculture and animal welfare. Turkey is important in U.S. holiday meal traditions and turkey meat is a frequently consumed processed product (i.e., lunchmeat). However, little is known about public perceptions and knowledge of commercial turkeys. An online survey was administered to 1,695 respondents in November 2018 to examine U.S. (1) demographic factors affecting meat consumption, selection of labeled meat products, and concern for animal welfare, (2) public knowledge of turkeys, and (3) concerns regarding the welfare of turkeys and other species. A total of 95% of respondents consumed meat and 10% hunted for some of the meat they consumed. Meat consumption frequency depended on region of residence, income level, gender, age, and whether respondents hunted. Of the meat consumers, 86% purchased turkey products. More meat consumers looked for the USDA organic label (39%) and the Non Genetically Modified Organism (GMO) project label (38%) than animal-welfare food labels (14%) when buying meat products. More pet owners (39%) than non-pet owners (25%) looked for animal welfare food labels. Being a pet owner increased the probability of being concerned about farm animal welfare. Concern for the commercial turkey was similar to concern for other farm animal species; self-reported knowledge of turkey production was low (mean score 2.64; scale of 1 to 7, 7 = highest). Turkey welfare concerns (mean score; rank from 1 to 5; 5 = least concerning) included poor nutrition (2.471) and illness (2.508), followed by housing (2.732), hot or cold weather (3.308) and transportation (3.981). Turkey welfare attributes that respondents cared the most about (mean score; scale of 1–5, 5 = cared the least) included space to move around (2.366), followed by veterinary health and wellness (2.680), ability to perform natural behavior (2.812), no feather loss or visible injuries (3.304), and decreased aggression (3.837). Demographic factors are important determinants of meat consumption and animal welfare concern. Public knowledge of turkey production is limited, despite a large percentage of the population purchasing turkey products.

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