Donald D. Heistad

Dysregulation of Antioxidant Mechanisms Contributes to Increased Oxidative Stress in Calcific Aortic Valvular Stenosis in Humans

OBJECTIVESnThe aim of this study was to determine whether oxidative stress is increased in calcified, stenotic aortic valves and to examine mechanisms that might contribute to increased oxidative stress.nnnBACKGROUNDnOxidative stress is increased in atherosclerotic lesions and might play an important role in plaque progression and calcification. The role of oxidative stress in valve disease is not clear.nnnMETHODSnSuperoxide (dihydroethidium fluorescence and lucigenin-enhanced chemiluminescence), hydrogen peroxide H2O2 (dichlorofluorescein fluorescence), and expression and activity of pro- and anti-oxidant enzymes were measured in normal valves from hearts not suitable for transplantation and stenotic aortic valves that were removed during surgical replacement of the valve.nnnRESULTSnIn normal valves, superoxide levels were relatively low and distributed homogeneously throughout the valve. In stenotic valves, superoxide levels were increased 2-fold near the calcified regions of the valve (p < 0.05); noncalcified regions did not differ significantly from normal valves. Hydrogen peroxide levels were also markedly elevated in calcified regions of stenotic valves. Nicotinamide adenine dinucleotide phosphate oxidase activity was not increased in calcified regions of stenotic valves. Superoxide levels in stenotic valves were significantly reduced by inhibition of nitric oxide synthases (NOS), which suggests uncoupling of the enzyme. Antioxidant mechanisms were reduced in calcified regions of the aortic valve, because total superoxide dismutase (SOD) activity and expression of all 3 SOD isoforms was significantly decreased. Catalase expression also was reduced in pericalcific regions.nnnCONCLUSIONSnThis study provides the first evidence that oxidative stress is increased in calcified regions of stenotic aortic valves from humans. Increased oxidative stress is due at least in part to reduction in expression and activity of antioxidant enzymes and perhaps to uncoupled NOS activity. Thus, mechanisms of oxidative stress differ greatly between stenotic aortic valves and atherosclerotic arteries.

Calcific Aortic Valve Stenosis in Old Hypercholesterolemic Mice

Background— Hypercholesterolemia and old age are clinical risk factors for development of aortic valve stenosis, and hypercholesterolemia is a putative therapeutic target. We tested the hypothesis that calcification and aortic valve stenosis would develop in genetically hypercholesterolemic old mice. Methods and Results— Twenty-four low-density lipoprotein receptor–deficient apolipoprotein B-100–only (LDLr−/−ApoB100/100)mice were fed normal chow from weaning until age 20.1±0.5 months (mean±SE; range 17 to 22 months). Twenty-one age-matched (20.8±0.9 months, range 17 to 25 months) C57Bl/6 mice served as controls. Echocardiographic imaging was used to assess morphology and function of the aortic valve and left ventricle. A subset of 12 mice underwent invasive hemodynamic assessment of aortic valve function. Functionally significant aortic stenosis, with >75% reduction in valve area, occurred in 8 of 24 LDLr−/−ApoB100/100 mice and in 0 of 21 controls (P=0.01). In the subset that underwent catheterization, mice with echocardiographic evidence of aortic stenosis had a systolic transvalvular gradient of 57±6 mm Hg. In the group of all LDLr−/−ApoB100/100 mice with aortic stenosis, left ventricular mass was increased by 67% (P=0.001) and ejection fraction was decreased by 30% (P=0.004) compared with LDLr−/−ApoB100/100 without aortic stenosis. Von Kossa staining of the aortic valve demonstrated abundant mineralization in LDLr−/−ApoB100/100 mice but not in control mice. Superoxide (oxyethidium fluorescence) was present in valve tissue from all 3 groups of mice and was more abundant in mice with aortic stenosis. Conclusions— Hypercholesterolemic LDLr−/−ApoB100/100 mice are prone to develop calcification and oxidative stress in the aortic valve, with functional valvular heart disease, mimicking the clinical syndrome. This discovery holds promise for elucidation of the pathophysiology of aortic valve disease mechanisms and for the design of effective nonsurgical treatment.

Calcific Aortic Valve Stenosis: Methods, Models, and Mechanisms

Calcific aortic valve stenosis (CAVS) is a major health problem facing aging societies. The identification of osteoblast-like and osteoclast-like cells in human tissue has led to a major paradigm shift in the field. CAVS was thought to be a passive, degenerative process, whereas now the progression of calcification in CAVS is considered to be actively regulated. Mechanistic studies examining the contributions of true ectopic osteogenesis, nonosseous calcification, and ectopic osteoblast-like cells (that appear to function differently from skeletal osteoblasts) to valvular dysfunction have been facilitated by the development of mouse models of CAVS. Recent studies also suggest that valvular fibrosis, as well as calcification, may play an important role in restricting cusp movement, and CAVS may be more appropriately viewed as a fibrocalcific disease. High-resolution echocardiography and magnetic resonance imaging have emerged as useful tools for testing the efficacy of pharmacological and genetic interventions in vivo. Key studies in humans and animals are reviewed that have shaped current paradigms in the field of CAVS, and suggest promising future areas for research.

Gene Transfer of Extracellular Superoxide Dismutase Reduces Arterial Pressure in Spontaneously Hypertensive Rats: Role of Heparin-Binding Domain

Abstract— Oxidative stress may contribute to hypertension. The goals of this study were to determine whether extracellular superoxide dismutase (ECSOD) reduces arterial pressure in spontaneously hypertensive rats (SHR) and whether its heparin-binding domain (HBD), which is responsible for cellular binding, is necessary for the function of ECSOD. Three days after intravenous injection of an adenoviral vector expressing human ECSOD (AdECSOD), mean arterial pressure (MAP) decreased from 165±4 mm Hg (mean±SE, n=7) to 124±3 mm Hg (n=7) in adult anesthetized SHR (P <0.01) but was not altered in normotensive Wistar-Kyoto rats. Cardiac output was not changed in SHR 3 days after AdECSOD. Gene transfer of ECSOD with deletion of the HBD (AdECSOD&Dgr;HBD) had no effect on SHR MAP, even though plasma SOD activity was greater after AdECSOD&Dgr;HBD than after AdECSOD. Immunohistochemistry revealed intense staining for ECSOD in blood vessels and kidneys after AdECSOD but not after AdECSOD&Dgr;HBD. Impaired relaxation of the carotid artery to acetylcholine in SHR was significantly improved after AdECSOD. Cumulative sodium balance in SHR was reduced by AdECSOD compared with AdECSOD&Dgr;HBD. Gene transfer of ECSOD also reduced MAP in conscious SHR, although the effect was not as profound as in anesthetized SHR. In summary, gene transfer of ECSOD, with a strict requirement for its HBD, reduces systemic vascular resistance and arterial pressure in a genetic model of hypertension. This reduction in arterial pressure may be mediated by vasomotor and/or renal mechanisms.

Lowering Plasma Cholesterol Levels Halts Progression of Aortic Valve Disease in Mice

Background— Treatment of hyperlipidemia produces functional and structural improvements in atherosclerotic vessels. However, the effects of treating hyperlipidemia on the structure and function of the aortic valve have been controversial, and any effects could be confounded by pleiotropic effects of hypolipidemic treatment. The goal of this study was to determine whether reducing elevated plasma lipid levels with a “genetic switch” in Reversa mice (Ldlr−/−/Apob100/100/Mttpfl/fl/Mx1-Cre+/+) reduces oxidative stress, reduces pro-osteogenic signaling, and retards the progression of aortic valve disease. Methods and Results— After 6 months of hypercholesterolemia, Reversa mice exhibited increases in superoxide, lipid deposition, myofibroblast activation, calcium deposition, and pro-osteogenic protein expression in the aortic valve. Maximum aortic valve cusp separation, as judged by echocardiography, was not altered. During an additional 6 months of hypercholesterolemia, superoxide levels, valvular lipid deposition, and myofibroblast activation remained elevated. Furthermore, calcium deposition and pro-osteogenic gene expression became more pronounced, and the aortic cusp separation decreased from 0.85±0.04 to 0.70±0.04 mm (mean±SE; P<0.05). Rapid normalization of cholesterol levels at 6 months of age (by inducing expression of Cre recombinase) normalized aortic valve superoxide levels, decreased myofibroblast activation, reduced valvular calcium burden, suppressed pro-osteogenic signaling cascades, and prevented reductions in aortic valve cusp separation. Conclusions— Collectively, these data indicate that reducing plasma lipid levels by genetic inactivation of the mttp gene in hypercholesterolemic mice with early aortic valve disease normalizes oxidative stress, reduces pro-osteogenic signaling, and halts the progression of aortic valve stenosis.

Cerebral Vascular Dysfunction During Hypercholesterolemia

Background and Purpose— Studies of peripheral arteries in hypercholesterolemic animals suggest that increased generation of superoxide contributes to endothelial dysfunction, especially in the presence of atherosclerotic lesions. We tested the hypothesis that vasomotor function is impaired in cerebral arterioles during hypercholesterolemia through a mechanism that involves oxidative stress. Methods— Apolipoprotein E–deficient (apoE−/−) mice were fed a normal or a high-fat diet for >6 months. ApoE+/− mice fed a normal diet were used as normocholesterolemic controls. Responses of cerebral arterioles were examined in open cranial windows in vivo in anesthetized mice. Results— In apoE−/− mice, intimal area was increased only in the proximal aorta on the normal diet and also markedly increased in the distal aorta on the high-fat diet. There were no increases in intimal area in the aortas of control mice or in the cerebral arterioles in any group. The dilator response of cerebral arterioles to ACh (10 &mgr;mol/L) in control mice (26±4% increase in diameter) was reduced in apoE−/− mice on either the normal (13±2%) or the high-fat (13±3%) diet (P<0.05 vs control). NADPH (10 &mgr;mol/L), a substrate for NADPH oxidase, produced dilator responses in control mice (8±4%) that were significantly increased in apoE−/− mice on the high-fat diet (16±2%, P<0.05 vs control). Tempol, a superoxide scavenger, and apocynin, an inhibitor of NADPH oxidase, significantly increased vasodilator responses to ACh and decreased vasodilation to NADPH in apoE−/− mice on the high-fat diet. Nitroprusside produced a similar dilatation in the cerebral arterioles of all groups. Conclusions— Hypercholesterolemia is associated with oxidative stress and endothelial dysfunction in cerebral arterioles, despite the absence of atherosclerotic lesions.

Mechanisms of Inducible Nitric Oxide Synthase–Mediated Vascular Dysfunction

Objective—Inducible nitric oxide synthase (iNOS) is expressed in arteries during inflammation and may contribute to vascular dysfunction. Effects of gene transfer of iNOS to carotid arteries were examined in vitro in the absence of systemic inflammation to allow examination of mechanisms by which iNOS impairs contraction and relaxation. Methods and Results—After gene transfer of iNOS with an adenovirus (AdiNOS), constrictor responses to phenylephrine (PE) and U46619 were impaired. After AdiNOS, inhibition of soluble guanylate cyclase (sGC) with 1H-[1,2,4]oxadiazolo-[4,3,2]quinoxalin-1-one (ODQ) reduced the EC50 for PE from 4.33±0.78 &mgr;mol/L to 1.15±0.43 &mgr;mol/L (mean±SEM). These results imply that iNOS impairs contraction by activation of the NO/cGMP pathway. Relaxation to acetylcholine (ACh) also was impaired after AdiNOS. Sepiapterin (300 &mgr;mol/L), the precursor for tetrahydrobiopterin (BH4), improved relaxation to Ach. Because BH4 is an essential cofactor for production of NO by both iNOS and endothelial nitric oxide synthase (eNOS), these results suggest that iNOS may reduce production of NO by eNOS by limiting availability of BH4. Next, we examined effects of expression of iNOS in endothelium and adventitia. Selective expression of iNOS in endothelium, but not adventitia, impaired contraction to phenylephrine and relaxation to acetylcholine. Conclusions—We conclude that: (1) iNOS may impair contraction in part by activation of sGC; (2) iNOS impairs relaxation, at least in part, by limiting availability of BH4; and (3) expression of iNOS in endothelium may be a more important mediator of vascular dysfunction than expression of iNOS in adventitia.

Vascular Effects of the Human Extracellular Superoxide Dismutase R213G Variant

Background—Extracellular superoxide dismutase (ECSOD) is a major extracellular antioxidant enzyme. We have demonstrated that vascular effects of ECSOD require an intact heparin-binding domain. A common genetic variant with a substitution in the heparin-binding domain (ECSODR213G) was reported recently to be associated with ischemic heart disease. The goal of this study was to examine vascular effects of ECSODR213G. Methods and Results—A recombinant adenovirus (Ad) that expresses ECSODR213G was constructed. ECSODR213G and ECSOD proteins bound to collagen type I in vitro, but binding to aorta ex vivo was 10-fold greater with ECSOD than ECSODR213G. Three days after intravenous injection of AdECSODR213G or AdECSOD in spontaneously hypertensive rats (SHR), immunostaining demonstrated binding of ECSOD to carotid arteries and kidneys but minimal binding of ECSODR213G. Binding to aorta and carotid artery was 2.5- to 3-fold greater with ECSOD than ECSODR213G by immunoblotting. Arterial pressure was significantly reduced by AdECSOD but not by AdECSODR213G. Responses to acetylcholine and basal levels of nitric oxide in carotid arteries were impaired in SHR compared with normotensive Wistar-Kyoto rats and were improved after AdECSOD but not AdECSODR213G. Levels of superoxide and nitrotyrosine in aorta were higher in SHR than Wistar-Kyoto rats and were greatly reduced after AdECSOD but not AdECSODR213G. Conclusions—In contrast to ECSOD, ECSODR213G has no significant protective effect on arterial pressure, vascular function, or vascular levels of oxidative stress in SHR. These findings may provide a mechanistic basis for association studies that suggest that human beings carrying ECSODR213G are predisposed to vascular diseases.

Evidence That Acetylsalicylic Acid Attenuates Inflammation in the Walls of Human Cerebral Aneurysms: Preliminary Results

Background Inflammatory cells and molecules may play a critical role in formation and rupture of cerebral aneurysms. Recently, an epidemiologic study reported that acetylsalicylic acid (ASA) decreases the risk of aneurysm rupture. The goal of this study was to determine the effects of ASA on inflammatory cells and molecules in the walls of human cerebral aneurysms, using radiographic and histological techniques. Methods and Results Eleven prospectively enrolled patients harboring unruptured intracranial aneurysms were randomized into an ASA‐treated (81 mg daily) group (n=6) and an untreated (control) group (n=5). Aneurysms were imaged at baseline using ferumoxytol‐enhanced MRI to estimate uptake by macrophages. After 3 months, patients were reimaged before undergoing microsurgical clipping. Aneurysm tissues were collected for immunostaining with monoclonal antibodies for cyclooxygenase‐1 (COX‐1), cyclooxygenase‐2 (COX‐2), microsomal prostaglandin E2 synthase‐1 (mPGES‐1), and macrophages. A decrease in signal intensity on ferumoxytol‐enhanced MRI was observed after 3 months of ASA treatment. Expression of COX‐2 (but not COX‐1), mPGES‐1, and macrophages was lower in the ASA group than in the control group. Conclusions This study provides preliminary radiographical and histological evidence that ASA may attenuate the inflammatory process in the walls of human cerebral aneurysms. Clinical Trial Registration URL: http://www.clinicaltrials.gov. Unique identifier: NCT01710072.

Evidence for active regulation of pro-osteogenic signaling in advanced aortic valve disease.

Objective—To test the hypothesis that valvular calcium deposition, pro-osteogenic signaling, and function can be altered in mice with advanced aortic valve disease. Methods and Results—“Reversa” mice were given a Western-type diet for 12 months and screened for the presence of aortic valve stenosis. Mice with advanced valve disease were assigned to 1 of 2 groups: (1) those with continued progression for 2 months and (2) those with regression for 2 months, in which lipid lowering was accomplished by a genetic switch. Control mice were normocholesterolemic for 14 months. Mice with advanced valve disease had massive valvular calcification that was associated with increases in bone morphogenetic protein signaling, Wnt/&bgr;-catenin signaling, and markers of osteoblastlike cell differentiation. Remarkably, reducing plasma lipids with a genetic switch dramatically reduced markers of pro-osteogenic signaling and significantly reduced valvular calcium deposition. Nevertheless, despite a marked reduction in valvular calcium deposition, valve function remained markedly impaired. Phosphorylated Smad2 levels and myofibroblast activation (indexes of profibrotic signaling) remained elevated. Conclusion—Molecular processes that contribute to valvular calcification and osteogenesis remain remarkably labile during the end stages of aortic valve stenosis. Although reductions in valvular calcium deposition were not sufficient to improve valvular function in the animals studied, these findings demonstrate that aortic valve calcification is a remarkably dynamic process that can be modified therapeutically, even in the presence of advanced aortic valve disease.