NETosis in Acute Thrombotic Disorders.

The release of extracellular traps by neutrophils (NETs) represents a novel active mechanism of cell death that has been recently implicated in the pathogenesis of thrombotic disorders. The aim of this study was to investigate the generation of NETs in different groups of patients with acute thrombotic events (ATEs) and to establish whether NETs markers can predict the risk of new cardiovascular events. We performed a case-control study of patients with ATE, including acute coronary syndrome (n = 60), cerebrovascular accident (n = 50), and venous thromboembolism (n = 55). Control subjects (n = 70) were identified among patients admitted for acute chest pain and in which a diagnosis of ATE was excluded. Serum levels of NET markers and neutrophil activation, such as myeloperoxidase (MPO)-DNA complexes, neutrophil gelatinase-associated lipocalin, polymorphonuclear neutrophil elastase, lactoferrin, and MPO, were measured in each patient. We found that circulating levels of MPO-DNA complexes were significantly increased in patients with ATE (p < 0.001) compared with controls and that this association remained significant even after fully adjustment for traditional risk factors (p = 0.001). A receiver operating characteristics analysis of circulating MPO-DNA complexes in discriminating between controls and patients with ATE showed a significant area under the curve of 0.76 (95% confidence interval: 0.69-0.82). After a median follow-up of 40.7 (± 13.8) months, 24 out of the 165 patients with ATE presented a new cardiovascular event and 18 patients died. None of the markers under investigation influenced survival or the incidence of new cardiovascular events. In conclusion, we found that increase of markers of NETosis can be observed in acute thrombotic conditions, occurring both on the arterial and venous site. Nevertheless, the level of neutrophil markers measured during the ATE is not predictive of future risk of mortality and cardiovascular events.

PCSK9 promotes arterial medial calcification.

BACKGROUND AND AIMS: A complex interplay among chronic kidney disease (CKD), lipid metabolism and aortic calcification has been recognized. Here we investigated the influence of kidney function on PCSK9 levels and its potential direct action on smooth muscle cells (SMCs) calcification. METHODS AND RESULTS: In a cohort of 594 subjects, a negative association between glomerular filtration rate and plasma PCSK9 was found. Atherosclerotic cardiovascular disease, as co-morbidity, further increased PCSK9 plasma levels. Diet-induced uremic condition in rats led to aortic calcification and increased total cholesterol and Pcsk9 levels in plasma, livers, and kidneys. Both human and rat SMCs overexpressing human PCSK9 (SMCsPCSK9), cultured in a pro-calcific environment (2.0 mM or 2.4 mM inorganic phosphate, Pi) showed a significantly higher extracellular calcium (Ca2+) deposition compared to control SMCs. The addition of recombinant human PCSK9 did not increase the extracellular calcification of SMCs, suggesting the involvement of intracellular PCSK9. Accordingly, the further challenge with evolocumab did not affect calcium deposition in hSMCsPCSK9. Under pro-calcific conditions, SMCsPCSK9 released a higher number of extracellular vesicles (EVs) positive for three tetraspanin molecules, such as CD63, CD9, and CD81. EVs derived from SMCsPCSK9 tended to be more enriched in calcium and alkaline phosphatase (ALP), compared to EVs from control SMCs. In addition, PCSK9 has been detected in SMCsPCSK9-derived EVs. Finally, SMCsPCSK9 showed an increase in pro-calcific markers, namely bone morphogenetic protein 2 and ALP, and a decrease in anti-calcific mediator matrix GLA protein and osteopontin. CONCLUSIONS: Our study reveals a direct role of PCSK9 on vascular calcification induced by higher inorganic phosphate levels associated with renal impairment. This effect appears to be mediated by a switch towards a pro-calcific phenotype of SMCs associated with the release of EVs containing Ca2+ and ALP.

The Emerging Role of Nutraceuticals in Cardiovascular Calcification: Evidence from Preclinical and Clinical Studies.

Cardiovascular calcification is the ectopic deposition of calcium-phosphate crystals within the arterial wall and the aortic valve leaflets. This pathological process leads to increased vascular stiffness, reduced arterial elasticity, and aortic valve stenosis, increasing the risk of cardiovascular diseases. Although cardiovascular calcification is an increasing health care burden, to date no medical therapies have been approved for treating or preventing it. Considering the current lack of therapeutic strategies and the increasing prevalence of cardiovascular calcification, the investigation of some nutraceuticals to prevent this pathological condition has become prevalent in recent years. Recent preclinical and clinical studies evaluated the potential anti-calcific role of nutraceuticals (including magnesium, zinc, iron, vitamin K, and phytate) in the progression of vascular calcification, providing evidence for their dietary supplementation, especially in high-risk populations. The present review summarizes the current knowledge and latest advances for nutraceuticals with the most relevant preclinical and clinical data, including magnesium, zinc, iron, vitamin K, and phytate. Their supplementation might be recommended as a cost-effective strategy to avoid nutritional deficiency and to prevent or treat cardiovascular calcification. However, the optimal dose of nutraceuticals has not been identified and large interventional trials are warranted to support their protective effects on cardiovascular disease.

Current Evidence and Future Perspectives on Pharmacological Treatment of Calcific Aortic Valve Stenosis.

Calcific aortic valve stenosis (CAVS), the most common heart valve disease, is characterized by the slow progressive fibro-calcific remodeling of the valve leaflets, leading to progressive obstruction to the blood flow. CAVS is an increasing health care burden and the development of an effective medical treatment is a major medical need. To date, no effective pharmacological therapies have proven to halt or delay its progression to the severe symptomatic stage and aortic valve replacement represents the only available option to improve clinical outcomes and to increase survival. In the present report, the current knowledge and latest advances in the medical management of patients with CAVS are summarized, placing emphasis on lipid-lowering agents, vasoactive drugs, and anti-calcific treatments. In addition, novel potential therapeutic targets recently identified and currently under investigation are reported.

l-Arginine prevents inflammatory and pro-calcific differentiation of interstitial aortic valve cells.

BACKGROUND AND AIMS: Reduced bioavailability of nitric oxide (NO) has been implicated in the pathogenesis of calcific aortic stenosis. Herein, we investigated the effects of l-Arginine, the main precursor of NO, on the osteogenic differentiation of aortic interstitial valve cells (VICs). METHODS: We isolated a clonal population of bovine VICs that expresses osteogenic markers and induces calcification of collagen matrix after stimulation with endotoxin (LPS 500 ng/mL). VICs were treated in vitro with different combinations of LPS ± l-Arginine (50 or 100 mM) and cell extracts were collected to perform proteomic (iTRAQ) and gene expression (RT-PCR) analysis. RESULTS: l-Arginine prevents the over-expression of alkaline phosphatase (ALP, p < 0.001) and reduces matrix calcification (p < 0.05) in VICs treated with LPS. l-Arginine also reduces the over-expression of inflammatory molecules induced by LPS (TNF-alpha, IL-6 and IL-1beta, p < 0.001). The proteomic analysis allowed to identify 49 proteins with an altered expression profile after stimulation with LPS and significantly modified by l-Arginine. These include proteins involved in the redox homeostasis of the cells (i.e. Xanthine Oxidase, Catalase, Aldehyde Oxidase), remodeling of the extracellular matrix (i.e. ADAMTSL4, Basigin, COL3A1) and cellular signaling (i.e. Fibrillin-1, Legumain, S100A13). The RT-PCR analysis confirmed the modifications of Fibrillin-1, ADAMTSL4, Basigin and Xanthine Oxidase, whose expression levels increase after stimulation with LPS and are reduced by l-Arginine (p < 0.05). CONCLUSIONS: l-Arginine prevents osteogenic differentiation of VICs and reduces matrix calcification. This effect is achieved through the modulation of proteins involved in the cellular redox system, remodeling of extracellular matrix and inflammatory activation of VICs.