Marcel Liberman

Oxidant Generation Predominates Around Calcifying Foci and Enhances Progression of Aortic Valve Calcification

Objective—We hypothesized that reactive oxygen species (ROS) contribute to progression of aortic valve (AV) calcification/stenosis. Methods and Results—We investigated ROS production and effects of antioxidants tempol and lipoic acid (LA) in calcification progression in rabbits given 0.5% cholesterol diet +104 IU/d Vit.D2 for 12 weeks. Superoxide and H2O2 microfluorotopography and 3-nitrotyrosine immunoreactivity showed increased signals not only in macrophages but preferentially around calcifying foci, in cells expressing osteoblast/osteoclast, but not macrophage markers. Such cells also showed increased expression of NAD(P)H oxidase subunits Nox2, p22phox, and protein disulfide isomerase. Nox4, but not Nox1 mRNA, was increased. Tempol augmented whereas LA decreased H2O2 signals. Importantly, AV calcification, assessed by echocardiography and histomorphometry, decreased 43% to 70% with LA, but increased with tempol (P≤0.05). Tempol further enhanced apoptosis and Nox4 expression. In human sclerotic or stenotic AV, we found analogous increases in ROS production and NAD(P)H oxidase expression around calcifying foci. An in vitro vascular smooth muscle cell (VSMC) calcification model also exhibited increased, catalase-inhibitable, calcium deposit with tempol, but not with LA. Conclusions—Our data provide evidence that ROS, particularly hydrogen peroxide, potentiate AV calcification progression. However, tempol exhibited a paradoxical effect, exacerbating AV/vascular calcification, likely because of its induced increase in peroxide generation.

Redox processes underlying the vascular repair reaction.

Accumulating evidence indicates that vascular dysfunction in atherosclerosis, hypertension, and diabetes is either caused by or accompanied by oxidative stress in the vessel wall. In particular, the role of redox processes as mediators of vascular repair and contributors to post-angioplasty restenosis is increasingly evident. Yet the pathophysiology of such complex phenomena is still unclear. After vascular injury, activation of enzymes such as NADPH oxidase leads to a marked increase in superoxide generation, proportional to the degree of injury, which rapidly subsides. Such early superoxide production is significantly greater after stent deployment, as compared to balloon injury. Recent data suggest the persistence of low levels of oxidant stress during the vascular repair reaction in neointimal and medial layers. Despite the compensatory increase in expression of iNOS and nNOS, nitric oxide bioavailability is reduced because of increased reaction rates with superoxide, yielding as by-products reactive nitrogen/oxygen species that induce protein nitration. Concurrently, the activity of vascular superoxide dismutases exhibits a sustained decrease following injury. This decreased activity appears to be a key contributor to vasoconstrictive remodeling and a major determinant of the occurrence of nitrative/oxidative stress. Replenishment of superoxide dismutase (SOD), as well as treatment with vitamins C and E or the lipid-lowering drug probucol and its analogs, led to decrease in constrictive remodeling and improved vessel caliber. Better understanding of the redox pathophysiology of vascular repair should help clarify the pathogenesis of many other vascular conditions and may provide novel therapeutic strategies to prevent vascular lumen loss.

Bidirectional Translation in Cardiovascular Calcification

Over the past decade, we have witnessed an explosion of fundamental research aimed at understanding mechanisms contributing to cardiovascular calcification. As highlighted in recent reviews, numerous animal models and patient group studies have lent key insights into mechanisms and processes underlying pathological remodeling of soft tissues,1 including activation of signaling cascades related to bone morphogenetic proteins,2 Wnt/β-catenin,3,4 matrix γ-carboxyglutamate (Gla) protein (MGP),5,6 transforming growth factor (TGF)-β, phosphate signaling,7,8 and various downstream targets. Although there are compelling data supporting the biological importance of these pathways, harnessing these mechanisms for the development of therapeutics has not yet been realized. Many pathways play an integral role in bone homeostasis, making systemic targeting a nonviable therapeutic approach. In this Recent Highlights focused on cardiovascular calcification, we have drawn from the pool of recent publications in ATVB and other leading journals that focus on genetic and nongenetic upstream modulators of ectopic calcification pathways, and we posit that interventions aimed at reducing their impact may be more readily translated to clinical therapies for patients. A greater understanding of the key local and systemic cofactors, initiators, and outcomes will create a complementary approach to advancing both science and medicine. We further argue that identification of biomarkers that are prognostic not only for the presence of vascular calcification (VC) but also for the rate of progression of VC will be instrumental in the early identification and appropriate management of patients in the future. Unlike metastatic calcification—caused by elevated levels of calcium in the blood—cardiovascular calcification is most often attributed to injury or maladaptive cellular responses to stress. Although in vitro studies and genetically modified model organisms can serve as useful platforms to understand the biology of disease phenotypes, the reality is that >85% of drugs stemming from …

Lipoic acid, but not tempol, preserves vascular compliance and decreases medial calcification in a model of elastocalcinosis

Vascular calcification decreases compliance and increases morbidity. Mechanisms of this process are unclear. The role of oxidative stress and effects of antioxidants have been poorly explored. We investigated effects of the antioxidants lipoic acid (LA) and tempol in a model of atherosclerosis associated with elastocalcinosis. Male New Zealand white rabbits (2.5-3.0 kg) were fed regular chow (controls) or a 0.5% cholesterol (chol) diet+104 IU/day vitamin D2 (vitD) for 12 weeks, and assigned to treatment with water (vehicle, n=20), 0.12 mmol·kg-1·day-1 LA (n=11) or 0.1 mmol·kg-1·day-1 tempol (n=15). Chol+vitD-fed rabbits developed atherosclerotic plaques associated with expansive remodeling, elastic fiber disruption, medial calcification, and increased aortic stiffness. Histologically, LA prevented medial calcification by ∼60% and aortic stiffening by ∼60%. LA also preserved responsiveness to constrictor agents, while intima-media thickening was increased. In contrast to LA, tempol was associated with increased plaque collagen content, medial calcification and aortic stiffness, and produced differential changes in vasoactive responses in the chol+vitD group. Both LA and tempol prevented superoxide signals with chol+vitD. However, only LA prevented hydrogen peroxide-related signals with chol+vitD, while tempol enhanced them. These data suggest that LA, opposite to tempol, can minimize calcification and compliance loss in elastocalcionosis by inhibition of hydrogen peroxide generation.

Circulating osteogenic proteins are associated with coronary artery calcification and increase after myocardial infarction

Background Coronary artery calcification (CAC) and atherosclerotic inflammation associate with increased risk of myocardial infarction (MI). Vascular calcification is regulated by osteogenic proteins (OPs). It is unknown whether an association exists between CAC and plasma OPs and if they are affected by atherothrombotic inflammation. We tested the association of osteogenic and inflammatory proteins with CAC and assessed these biomarkers after MI. Methods Circulating OPs (osteoprotegerin, RANKL, fetuin-A, Matrix Gla protein [MGP]) and inflammatory proteins (C-reactive protein, oxidized-LDL, tumoral necrosis factor-α, transforming growth factor [TGF]-β1) were compared between stable patients with CAC (CAC ≥ 100 AU, n = 100) and controls (CAC = 0 AU, n = 30). The association between biomarkers and CAC was tested by multivariate analysis. In patients with MI (n = 40), biomarkers were compared between acute phase and 1–2 months post-MI, using controls as a baseline. Results MGP and fetuin-A levels were higher within individuals with CAC. Higher levels of MGP and RANKL were associated with CAC (OR 3.12 [95% CI 1.20–8.11], p = 0.02; and OR 1.75 [95% CI 1.04–2.94] respectively, p = 0.035). After MI, C-reactive protein, OPG and oxidized-LDL levels increased in the acute phase, whereas MGP and TGF-β1 increased 1–2 months post-MI. Conclusions Higher MGP and RANKL levels associate with CAC. These findings highlight the potential role of these proteins as modulators and markers of CAC. In addition, the post-MI increase in OPG and MGP, as well as of inflammatory proteins suggest that the regulation of these OPs is affected by atherothrombotic inflammation.

Endothelium-Dependent Vasodilation: Nitric Oxide and Other Mediators

Abstract Vasodilation is the archetypal function of the endothelial cell and the discovery of paracrine-dependent vasorelaxation by endothelium-derived production of the gaseous mediator nitric oxide (NO) was revolutionary. NO mediates its regulatory vasorelaxing effects through guanilyl cyclase activation. Also, thiol S-nitrosation by NO is increasingly evident as an effector mechanism. Another important NO-related chemistry is its reaction with superoxide radicals, yielding peroxynitrite and related oxidant and nitrating species associated with toxic effects. Nitrogen oxides are storage forms of NO which can exert vasodilation in the presence of hemeproteins. NO generation is mediated by NO synthase enzymes (endothelial, neuronal, and inducible isoforms), which depict complex regulation dependent on cofactors. The absence of such cofactors can uncouple NO generation from electron transfer, generating superoxide. The endothelium additional promotes vasodilation, mainly of small resistance arteries, through endothelium-derived hyperpolarizing factor(s) such as hydrogen peroxide, epoximetabolites of arachidonic acid, and gap junctions. Hydrogen sulfide is a novel gaseous endothelium-derived vasodilator. Together, these mechanisms compose an integrative platform providing an endothelium-associated dilator tone.