Vimentin regulates Notch signaling strength and arterial remodeling in response to hemodynamic stress.

The intermediate filament (IF) cytoskeleton has been proposed to regulate morphogenic processes by integrating the cell fate signaling machinery with mechanical cues. Signaling between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) through the Notch pathway regulates arterial remodeling in response to changes in blood flow. Here we show that the IF-protein vimentin regulates Notch signaling strength and arterial remodeling in response to hemodynamic forces. Vimentin is important for Notch transactivation by ECs and vimentin knockout mice (VimKO) display disrupted VSMC differentiation and adverse remodeling in aortic explants and in vivo. Shear stress increases Jagged1 levels and Notch activation in a vimentin-dependent manner. Shear stress induces phosphorylation of vimentin at serine 38 and phosphorylated vimentin interacts with Jagged1 and increases Notch activation potential. Reduced Jagged1-Notch transactivation strength disrupts lateral signal induction through the arterial wall leading to adverse remodeling. Taken together we demonstrate that vimentin forms a central part of a mechanochemical transduction pathway that regulates multilayer communication and structural homeostasis of the arterial wall.

Melanocortin overexpression limits diet-induced inflammation and atherosclerosis in LDLR-/- mice.

Atherosclerosis is a chronic inflammatory disease of the arteries. The disease is initiated by endothelial dysfunction that allows the transport of leukocytes and low-density lipoprotein into the vessel wall forming atherosclerotic plaques. The melanocortin system is an endogenous peptide system that regulates, for example, energy homeostasis and cardiovascular function. Melanocortin treatment with endogenous or synthetic melanocortin peptides reduces body weight, protects the endothelium and alleviates vascular inflammation, but the long-term effects of melanocortin system activation on atheroprogression remain largely unknown. In this study, we evaluated the effects of transgenic melanocortin overexpression in a mouse model of atherosclerosis. Low-density lipoprotein receptor-deficient mice overexpressing alpha- and gamma3-MSH (MSH-OE) and their wild-type littermates were fed either a regular chow or Western-style diet for 16 weeks. During this time, their metabolic parameters were monitored. The aortae were collected for functional analysis, and the plaques in the aortic root and arch were characterised by histological and immunohistochemical stainings. The aortic expression of inflammatory mediators was determined by quantitative PCR. We found that transgenic MSH-OE improved glucose tolerance and limited atherosclerotic plaque formation particularly in Western diet-fed mice. In terms of aortic vasoreactivity, MSH-OE blunted alpha1-adrenoceptor-mediated vasoconstriction and enhanced relaxation response to acetylcholine, indicating improved endothelial function. In addition, MSH-OE markedly attenuated Western diet-induced upregulation of proinflammatory cytokines (Ccl2, Ccl5 and Il6) that contribute to the pathogenesis of atherosclerosis. These results show that the activation of the melanocortin system improves glucose homeostasis and limits diet-induced vascular inflammation and atherosclerotic plaque formation.

Melanocortin 1 Receptor Deficiency Promotes Atherosclerosis in Apolipoprotein E-/- Mice.

OBJECTIVE: The MC1-R (melanocortin 1 receptor) is expressed by monocytes and macrophages where it mediates anti-inflammatory actions. MC1-R also protects against macrophage foam cell formation primarily by promoting cholesterol efflux through the ABCA1 (ATP-binding cassette transporter subfamily A member 1) and ABCG1 (ATP-binding cassette transporter subfamily G member 1). In this study, we aimed to investigate whether global deficiency in MC1-R signaling affects the development of atherosclerosis. APPROACH AND RESULTS: Apoe-/- (apolipoprotein E deficient) mice were crossed with recessive yellow (Mc1re/e) mice carrying dysfunctional MC1-R and fed a high-fat diet to induce atherosclerosis. Apoe-/- Mc1re/e mice developed significantly larger atherosclerotic lesions in the aortic sinus and in the whole aorta compared with Apoe-/- controls. In terms of plaque composition, MC1-R deficiency was associated with less collagen and smooth muscle cells and increased necrotic core, indicative of more vulnerable lesions. These changes were accompanied by reduced Abca1 and Abcg1 expression in the aorta. Furthermore, Apoe-/- Mc1re/e mice showed a defect in bile acid metabolism that aggravated high-fat diet-induced hypercholesterolemia and hepatic lipid accumulation. Flow cytometric analysis of leukocyte profile revealed that dysfunctional MC1-R enhanced arterial accumulation of classical Ly6Chigh monocytes and macrophages, effects that were evident in mice fed a normal chow diet but not under high-fat diet conditions. In support of enhanced arterial recruitment of Ly6Chigh monocytes, these cells had increased expression of L-selectin and P-selectin glycoprotein ligand 1. CONCLUSIONS: The present study highlights the importance of MC1-R in the development of atherosclerosis. Deficiency in MC1-R signaling exacerbates atherosclerosis by disturbing cholesterol handling and by increasing arterial monocyte accumulation.

Melanocortin 1 Receptor Signaling Regulates Cholesterol Transport in Macrophages.

BACKGROUND: The melanocortin 1 receptor (MC1-R) is expressed by monocytes and macrophages, where it exerts anti-inflammatory actions on stimulation with its natural ligand α-melanocyte-stimulating hormone. The present study was designed to investigate the specific role of MC1-R in the context of atherosclerosis and possible regulatory pathways of MC1-R beyond anti-inflammation. METHODS: Human and mouse atherosclerotic samples and primary mouse macrophages were used to study the regulatory functions of MC1-R. The impact of pharmacological MC1-R activation on atherosclerosis was assessed in apolipoprotein E-deficient mice. RESULTS: Characterization of human and mouse atherosclerotic plaques revealed that MC1-R expression localizes in lesional macrophages and is significantly associated with the ATP-binding cassette transporters ABCA1 and ABCG1, which are responsible for initiating reverse cholesterol transport. Using bone marrow-derived macrophages, we observed that α-melanocyte-stimulating hormone and selective MC1-R agonists similarly promoted cholesterol efflux, which is a counterregulatory mechanism against foam cell formation. Mechanistically, MC1-R activation upregulated the levels of ABCA1 and ABCG1. These effects were accompanied by a reduction in cell surface CD36 expression and in cholesterol uptake, further protecting macrophages from excessive lipid accumulation. Conversely, macrophages deficient in functional MC1-R displayed a phenotype with impaired efflux and enhanced uptake of cholesterol. Pharmacological targeting of MC1-R in atherosclerotic apolipoprotein E-deficient mice reduced plasma cholesterol levels and aortic CD36 expression and increased plaque ABCG1 expression and signs of plaque stability. CONCLUSIONS: Our findings identify a novel role for MC1-R in macrophage cholesterol transport. Activation of MC1-R confers protection against macrophage foam cell formation through a dual mechanism: It prevents cholesterol uptake while concomitantly promoting ABCA1- and ABCG1-mediated reverse cholesterol transport.

Evaluation of the Effect of Fingolimod Treatment on Microglial Activation Using Serial PET Imaging in Multiple Sclerosis.

Traditionally, multiple sclerosis (MS) has been considered a white matter disease with focal inflammatory lesions. It is, however, becoming clear that significant pathology, such as microglial activation, also takes place outside the plaque areas, that is, in areas of normal-appearing white matter (NAWM) and gray matter (GM). Microglial activation can be detected in vivo using 18-kDa translocator protein (TSPO)-binding radioligands and PET. It is unknown whether fingolimod affects microglial activation in MS. The aim of this study was to investigate whether serial PET can be used to evaluate the effect of fingolimod treatment on microglial activation. Methods: Ten relapsing-remitting MS patients were studied using the TSPO radioligand 11C-(R)-PK11195. Imaging was performed at baseline and after 8 and 24 wk of fingolimod treatment. Eight healthy individuals were imaged for comparison. Microglial activation was evaluated as distribution volume ratio of 11C-(R)-PK11195. Results: The patients had MS for an average of 7.9 ± 4.3 y (mean ± SD), their total relapses averaged 4 ± 2.4, and their Expanded Disability Status Scale was 2.7 ± 0.5. The patients were switched to fingolimod because of safety reasons or therapy escalation. The mean washout period before the initiation of fingolimod was 2.3 ± 1.1 mo. The patients were clinically stable on fingolimod. At baseline, microglial activation was significantly higher in the combined NAWM and GM areas of MS patients than in healthy controls (P = 0.021). 11C-(R)-PK11195 binding was reduced (-12.31%) within the combined T2 lesion area after 6 mo of fingolimod treatment (P = 0.040) but not in the areas of NAWM or GM. Conclusion: Fingolimod treatment reduced microglial/macrophage activation at the site of focal inflammatory lesions, presumably by preventing leukocyte trafficking from the periphery. It did not affect the widespread, diffuse microglial activation in the NAWM and GM. The study opens new vistas for designing future therapeutic studies in MS that use the evaluation of microglial activation as an imaging outcome measure.

Deficiency in Melanocortin 1 Receptor Signaling Predisposes to Vascular Endothelial Dysfunction and Increased Arterial Stiffness in Mice and Humans.

OBJECTIVE: The melanocortin 1 receptor (MC1-R) is expressed by vascular endothelial cells and shown to enhance nitric oxide (NO) availability and vasodilator function on pharmacological stimulation. However, the physiological role of MC1-R in the endothelium and its contribution to vascular homeostasis remain unresolved. We investigated whether a lack of functional MC1-R signaling carries a phenotype with predisposition to vascular abnormalities. APPROACH AND RESULTS: Recessive yellow mice (MC1R(e/e)), deficient in MC1-R signaling, and their wild-type littermates were studied for morphology and functional characteristics of the aorta. MC1R(e/e) mice showed increased collagen deposition and arterial stiffness accompanied by an elevation in pulse pressure. Contractile capacity and NO-dependent vasodilatation were impaired in the aorta of MC1R(e/e) mice supported by findings of decreased NO availability. These mice also displayed elevated levels of systemic and local cytokines. Exposing the mice to high-sodium diet or acute endotoxemia revealed increased susceptibility to inflammation-driven vascular dysfunction. Finally, we investigated whether a similar phenotype can be found in healthy human subjects carrying variant MC1-R alleles known to attenuate receptor function. In a longitudinal analysis of 2001 subjects with genotype and ultrasound data (The Cardiovascular Risk in Young Finns Study), weak MC1-R function was associated with lower flow-mediated dilatation response of the brachial artery and increased carotid artery stiffness. CONCLUSIONS: The present study demonstrates that deficiency in MC1-R signaling is associated with increased arterial stiffness and impairment in endothelium-dependent vasodilatation, suggesting a physiological role for MC1-R in the regulation of arterial tone.

Pharmacological activation of the melanocortin system limits plaque inflammation and ameliorates vascular dysfunction in atherosclerotic mice.

OBJECTIVE: Melanocortin peptides have been shown to elicit anti-inflammatory actions and to promote vascular endothelial function by activating type 1 and 3 melanocortin receptors. Here, we addressed whether these favorable properties of melanocortins could reduce atherosclerotic plaque inflammation and improve vasoreactivity in atherosclerotic mice. APPROACH AND RESULTS: Low-density lipoprotein receptor-deficient mice expressing only apolipoprotein B100 were fed a high-fat diet for 8 or 16 weeks and treated with either vehicle or a stable melanocortin analog, melanotan II (MT-II, 0.3 mg/kg per day, 4 weeks). We determined plaque uptake of fluorine-18-labeled fluorodeoxyglucose as a surrogate marker for atherosclerotic plaque inflammation and vascular function of the aorta by ex vivo analyses. MT-II had no effect on body weight or composition, or plasma cholesterol levels in atherosclerotic mice. Without attenuating atherosclerotic lesion size or lesional macrophage accumulation, MT-II treatment reduced fluorine-18-labeled fluorodeoxyglucose uptake in the atherosclerotic plaques. Resident macrophages in the lesions of MT-II-treated mice were polarized toward the anti-inflammatory M2 phenotype. Systemic inflammation was also attenuated by MT-II intervention as evidenced by decreased plasma levels of proinflammatory cytokines. In terms of aortic vasoreactivity, MT-II-treated mice showed enhanced endothelium-dependent relaxations, as well as promotion of vascular sensitivity to nitric oxide-mediated vasodilation, which were markedly impaired in control mice after prolonged duration of diet exposure. CONCLUSIONS: The present study demonstrates that pharmacological activation of the melanocortin system has therapeutic benefits in pre-established atherosclerosis by limiting plaque inflammation and promoting vascular endothelial function, which may provide a novel therapeutic approach for atherosclerosis.