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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 (SMCs PCSK9 ), cultured in a pro-calcific environment (2.0 mM or 2.4 mM inorganic phosphate, P i ) showed a significantly higher extracellular calcium (Ca 2+ ) 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 hSMCs PCSK9 . Under pro-calcific conditions, SMCs PCSK9 released a higher number of extracellular vesicles (EVs) positive for three tetraspanin molecules, such as CD63, CD9, and CD81. EVs derived from SMCs PCSK9 tended to be more enriched in calcium and alkaline phosphatase (ALP), compared to EVs from control SMCs. In addition, PCSK9 has been detected in SMCs PCSK9 -derived EVs. Finally, SMCs PCSK9 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 Ca 2+ and ALP., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Essentials Increase in serum uric acid (SUA) levels has been widely associated with higher risk of cardiovascular disease. We investigated the link between SUA levels and the risk of venous thromboembolism (VTE) recurrence. Patients with SUA levels ≥ 4.38 mg/dL showed a three-fold increase in the risk of VTE recurrence. Elevated SUA levels are associated with increased risk of recurrent VTE independently from traditional risk factors. ABSTRACT: Background The link between serum uric acid (SUA) and the risk of cardiovascular disease is well established. However, the impact of SUA levels on the risk of venous thromboembolism (VTE) recurrence is unknown. Objectives To investigate the association between SUA and the risk of VTE recurrence. Patients and Methods We performed a monocenter, prospective study on 280 patients with a previous episode of VTE that completed the oral anticoagulant period. SUA levels at enrollment were correlated with the risk of VTE recurrence (mean follow-up 71.1 ± 29.2 months). Results Patients were stratified according to SUA tertiles distribution at baseline (tertiles cut-off: I ≤ 4.37 mg/dL, II 4.38--5.54 mg/dL, III ≥ 5.55 mg/dL). Fifty episodes of VTE recurrence occurred during the follow-up and Kaplan-Meier survival analysis showed that subjects in the lower tertile of SUA distribution had significantly lower risk of future VTE recurrence (P = .003). No differences were seen among patients belonging to the second and the third tertile of SUA distribution. A multivariate Cox regression analysis showed that higher tertiles of SUA distribution had about three-fold increase in the risk of VTE recurrence as compared to subjects with SUA ≤ 4.37, independently from potential confounders (hazard ratio [HR] 3.04, 95% confidence interval [CI] 1.15--8.05 P = .025). Moreover, we observed that the adjusted hazard of VTE recurrence increased by 30% for each additional unit of SUA (mg/dL; HR 1.30, 95% CI 1.01--1.22, P = .040). Conclusion Elevated SUA levels are associated with increased risk of future VTE recurrence independently from traditional risk factors., (© 2020 International Society on Thrombosis and Haemostasis.)

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., Competing Interests: Declaration of competing interest The authors declared they do not have anything to disclose regarding conflict of interest with respect to this manuscript., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objectives: Pyrophosphate (PPi) is a potent inhibitor of ectopic mineralization but its role during aortic valve calcification is not known., Methods: Anti-calcific effect of PPi was investigated by using an in vitro model of serum-driven calcification of collagen sponges and decellularized porcine aortic valve leaflets. Bovine interstitial valve cells (VIC), seeded either within the collagen matrices or in transwell chambers, were used to test cellular ability to inhibit serum-induced calcification. PPi metabolism was investigated in clonal VIC harboring different calcifying potential., Results: In a cell-free system, high serum levels induced a dose-dependent calcification of type I collagen matrices which was prevented by PPi and ATP supplementation. Blockade of serum-driven calcification by PPi and ATP was also observed when using decellularized porcine aortic valve leaflets. A similar anti-calcific effect was also seen for bovine VIC, either statically seeded into the collagen matrices or co-cultured by using a transwell system. However, when we performed co-culture experiments by using clonal VIC harboring different calcifying potential, we observed that the subset of cells acquiring a pro-calcific profile lost the ability to protect the collagen from serum-driven calcification. Pro-calcific differentiation of the clonal VIC was accompanied by increase in ALP along with significant reduction in NPP activity and ATP/PPi extracellular accumulation. These changes were not observed in the clonal subtype with lower propensity towards calcification., Conclusions: We showed that PPi and ATP are potent inhibitors of serum-driven calcification of collagen matrix and that their extracellular accumulation is reduced in calcifying VIC., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Objective: We investigated the association between cholesterol across the LDL density range and in the VLDL and IDL particles with the prevalence of inflammatory cells in plaques of patients with severe carotid artery stenosis., Methods: Forty-five patients undergoing carotid endarterectomy were studied. Plaque specimens were analyzed for cellular composition by immunocytochemistry using monoclonal antibodies. Lipoprotein subclasses were separated by gradient ultracentrifugation., Results: We found no correlations between LDL-C, HDL-C and plasma triglyceride levels with plaque cellular composition. On the other hand, macrophage content was significantly related to cholesterol in the dense LDL subclasses (r = 0.30, p < 0.01) and in the triglyceride-rich lipoprotein remnants, namely dense VLDL and IDL particles (r = 0.46, p < 0.01). HDL subclasses were not correlated with plaque cellular composition. In a mirror manner, smooth muscle cells were inversely associated with cholesterol levels of the dense LDL subclasses (r = -0.32, p < 0.01 fraction 10; r = -0.26, p < 0.05 fraction 11) while only a non-significant trend was observed with the cholesterol in the VLDL-IDL fractions. These results provide the pathophysiological background to account for the relevance of non-HDL-C as the only lipid parameter, aside LDL density, significantly associated (β = 0.351, p = 0.021) with carotid plaque macrophage content., Conclusions: We provide evidence that lipoprotein subclasses, specifically cholesterol in the dense LDL fractions and in the triglyceride-rich lipoprotein remnants, significantly affect carotid plaque cellular composition, in particular macrophages content., (© 2013 Elsevier Ireland Ltd. All rights reserved.)

Background: The common -514 C-T promoter polymorphism of the hepatic lipase gene (LIPC) and the cholesteryl ester transfer protein (CETP) gene TaqIB polymorphism affect atherogenesis. We investigated the potential relationship between these polymorphisms and the maximum-intima-media thickness (M-IMT) after carotid endarterectomy., Methods: The LIPC and CETP genotypes were determined by PCR in 68 patients undergoing endarterectomy. Plaque specimens were analysed for cell composition by immunocytochemistry. Six month after surgery the M-IMT of the revascularized vessel was assessed by B-mode ultrasonography., Results: The CC carriers had denser LDL particles (p<0.0005), an abundance of macrophages (p<0.0005), fewer SMCs in the carotid plaque (p<0.0005), and higher prevalence of cerebrovascular events (72% versus 28%, p=0.002) compared to CT/TT carriers. After endarterectomy, CC carriers showed a lower M-IMT than the CT/TT group (1.36 mm versus 1.76 mm, p=0.04). No association between the CETP polymorphism and either carotid plaque cellular composition or M-IMT was observed. In a multivariate analysis, M-IMT was associated with plaque cell composition (macrophages, r=-0.39; SMC, r=0.44; p<0.005 for both) but not with pre-operative LDL-C, HDL-C, triglycerides, or LDL density., Conclusions: The LIPC promoter -514 C-T polymorphism is associated with a significantly reduced development of neointima after surgery. This effect seems to be mediated by scarcity of SMC in the plaque of CC carriers who display an excess prevalence of cerebrovascular events prior endarterectomy but are at low risk for restenosis. The pre-operative lipid phenotype plays a marginal role in the neointima formation.

Purpose: We investigated whether differences in cellular composition of the shoulder region of carotid plaque, a cell-rich, debris-free area, can be revealed with computer-driven analysis of ultrasound scans., Methods: In 26 patients referred for carotid endarterectomy, the shoulder region of plaque eligible for surgical removal was identified with ultrasound scanning. Digital images were obtained and evaluated with a specially developed computer-driven system (Medical Image Processing [MIP]). The gray level distribution of the region of interest (ROI), along with some statistical parameters exploring the spatial distribution of pixels, such as entropy and second angular moment, were analyzed. In the specimen retrieved at surgery, the area corresponding to the ROI was selected. Cryosections were tested at immunocytochemistry with monoclonal antibodies specific to smooth muscle cells (SMCs), macrophages), and lymphocytes. Computerized image analysis was performed to quantify each cellular component of the lesion., Results: Mean gray levels were related positively to the content of SMCs (r = 0.576, P =.002) and negatively to the content of macrophages (r = -0.555, P =.003). Lymphocytes did not show any correlation. Prevalence of SMCs, expressed as the ratio SMC/(SMC + macrophages), was related positively with entropy (r = 0.517, P =.007) and negatively with the second angular moment (r = -0.422, P =.032). The quartiles of gray level were useful for detecting significant differences in terms of cellular composition., Conclusions: Some cellular features of the shoulder region of plaque are associated with specific videodensitometric patterns evaluated with MIP. This approach enables in vivo noninvasive prediction and monitoring of cell composition of the shoulder region, and could be extended to study of the thickened intima.