Lead Acetate-Injected Mice is an Animal Model for Extrapolation of Calcifying Response to Humans Due to Low Involvement of Bone Resorption.

Vascular calcification affects the prognosis of patients with renal failure. Bisphosphonates are regarded as candidate anti-calcifying drugs because of their inhibitory effects on both calcium-phosphate aggregation and bone resorption. However, calcification in well-known rodent models is dependent upon bone resorption accompanied by excessive bone turnover, making it difficult to estimate accurately the anti-calcifying potential of drugs. Therefore, models with low bone resorption are required to extrapolate anti-calcifying effects to humans. Three bisphosphonates (etidronate, alendronate, and FYB-931) were characterised for their inhibitory effects on bone resorption in vivo and calcium-phosphate aggregation estimated by calciprotein particle formation in vitro. Then, their effects were examined using two models inducing ectopic calcification: the site where lead acetate was subcutaneously injected into mice and the transplanted, aorta obtained from a donor rat. The inhibitory effects of bisphosphonates on bone resorption and calcium-phosphate aggregation were alendronate > FYB-931 > etidronate and FYB-931 > alendronate = etidronate, respectively. In the lead acetate-induced model, calcification was most potently suppressed by FYB-931, followed by alendronate and etidronate. In the aorta-transplanted model, only FYB-931 suppressed calcification at a high dose. In both the models, no correlation was observed between calcification and bone resorption marker, tartrate-resistant acid phosphatase (TRACP). Results from the lead acetate-induced model showed that inhibitory potency against calcium-phosphate aggregation contributed to calcification inhibition. The two calcification models, especially the lead acetate-induced model, may be ideal for the extrapolation of calcifying response to humans because of calcium-phosphate aggregation rather than bone resorption as its mechanism.

In vitro Evaluation of the Calcification Inhibitory Properties of Policosanol, Genistein, and Vitamin D (Reduplaxin®) either Alone or in Combination.

INTRODUCTION: The process of vascular calcification has severe clinical consequences in a number of diseases, including diabetes, atherosclerosis, and end-stage renal disease. In the present study, we investigated the effect of policosanol (Poli), genistein (Gen), and vitamin D (VitD) separately and in association to evaluate the possible synergistic action on inorganic phosphate (Pi)-induced calcification of vascular smooth muscle cells (VSMCs). METHODS: Primary human VSMCs were cultured with either growth medium or growth medium supplemented with calcium and phosphorus (calcification medium) in combination with Poli, Gen, and VitD. Alizarin Red staining, mineralization, and the protein expression of RUNX2 and superoxide dismutase-2 (SOD2) were investigated. RESULTS: All three substances tested were effective at reducing osteogenic differentiation of VSMCs in a dose-dependent manner. Poli+Gen, Poli+VitD, Gen+VitD treatment induced a greater inhibition of calcification and RUNX2 expression compared to single compounds treatments. Moreover, the association of Poli+Gen+VitD (Reduplaxin®) was more effective at inhibiting VSMCs mineralization and preventing the increase in RUNX2 expression induced by calcification medium but not modified SOD2 expression. CONCLUSIONS: The association of Pol, Gen, and VitD (Reduplaxin®) has an additive inhibitory effect on the calcification process of VSMCs induced in vitro by a pro-calcifying medium.

New insight into air pollution-related cardiovascular disease: an adverse outcome pathway framework of PM2.5-associated vascular calcification.

Despite the air quality has been generally improved in recent years, ambient fine particulate matter (PM2.5), a major contributor to air pollution, remains one of the major threats to public health. Vascular calcification is a systematic pathology associated with an increased risk of cardiovascular disease. Although the epidemiological evidence has uncovered the association between PM2.5 exposure and vascular calcification, little is known about the underlying mechanisms. The adverse outcome pathway (AOP) concept offers a comprehensive interpretation of all of the findings obtained by toxicological and epidemiological studies. In this review, reactive oxygen species generation was identified as the molecular initiating event (MIE), which targeted subsequent key events (KEs) such as oxidative stress, inflammation, endoplasmic reticulum stress, and autophagy, from the cellular to the tissue/organ level. These KEs eventually led to the adverse outcome, namely increased incidence of vascular calcification and atherosclerosis morbidity. To the best of our knowledge, this is the first AOP framework devoted to PM2.5-associated vascular calcification, which benefits future investigations by identifying current limitations and latent biomarkers.

High Phosphate-Induced JAK-STAT Signalling Sustains Vascular Smooth Muscle Cell Inflammation and Limits Calcification.

Vascular calcification (VC) is an age-related complication characterised by calcium-phosphate deposition in the arterial wall driven by the osteogenic transformation of vascular smooth muscle cells (VSMCs). The JAK-STAT pathway is an emerging target in inflammation. Considering the relationship between VC and inflammation, we investigated the role of JAK-STAT signalling during VSMC calcification. Human aortic smooth muscle cells (HASMCs) were cultured in high-inorganic phosphate (Pi) medium for up to 7 days; calcium deposition was determined via Alizarin staining and colorimetric assay. Inflammatory factor secretion was evaluated via ELISA and JAK-STAT members' activation using Western blot or immunohistochemistry on HASMCs or calcified aortas of Vitamin D-treated C57BL6/J mice, respectively. The JAK-STAT pathway was blocked by JAK Inhibitor I and Von Kossa staining was used for calcium deposits in murine aortic rings. During Pi-induced calcification, HASMCs released IL-6, IL-8, and MCP-1 and activated JAK1-JAK3 proteins and STAT1. Phospho-STAT1 was detected in murine calcified aortas. Blocking of the JAK-STAT cascade reduced HASMC proliferation and pro-inflammatory factor expression and release while increasing calcium deposition and osteogenic transcription factor RUNX2 expression. Consistently, JAK-STAT pathway inhibition exacerbates mouse aortic ring calcification ex vivo. Intriguingly, our results suggest an alternative link between VSMC inflammation and VC.

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.