ARMOR: Auto-Assembled Resilient Biomimetic Calcified Ornaments for Selective Cell Protection by Dual-Aptamer-Driven Hybridization Chain Reaction.

Unlike plant and microbial cells having cell walls, the outermost layer of mammalian cell is a delicate, two-layered structure of phospholipids with proteins embedded, which is susceptible to environmental changes. It is necessary to create an "armor" on cell surface to protect cell integrity. Here, we propose an Auto-assembled Resilient bioMimetic calcified ORnaments (ARMOR) strategy driven by dual-aptamer-based hybridization chain reaction (HCR) and Ca2+ assisted calcification for selective cell protection. This co-recognition design enhances the selectivity and leverages robust in situ signal amplification by HCR to improve the sensitivity. The calcified shell is cogenerated by crosslinking the alginate-HCR product with Ca2+ ion. ARMOR has high efficiency for shielding cells from environmental assaults, which can be applied to circulating tumor cell (CTC) protection, isolation, and identification, maintaining the native state and intact genetic information for downstream analysis.

[Effect and related mechanisms of RTA-408 on rat vascular smooth muscle cell calcification induced by advanced glycation end products].

Objective: To investigate the effect and related mechanisms of RTA-408 on rat vascular smooth muscle cells (VSMCs) calcification induced by advanced glycation end products(AGE). Methods: VSMCs were isolated from the aorta of Sprague Dawley rats and cultured in vitro. The fifth generation of VSMCs were randomly divided into 4 groups with random number table including control group(cells were incubated with normal medium for 2 days, then incubated with bovine serum albumin for 5 days),AGE group (cells were incubated with normal medium for 2 days, then incubated with 200 mg/L AGE for 5 days), experimental group(cells were incubated with 100 nmol/L RTA-408 for 2 days,then incubated with 200 mg/L AGE for 5 days),and RTA group(cells were incubated with 100 nmol/L RTA-408 for 2 days,then incubated with bovine serum albumin for 5 days). Cytosolic calciumin VSMC was measured using arsenazo Ⅲ assay. Von Kossa staining was utilized to detect the calcium deposition.The contents of malondialdehyde(MDA) and superoxide dismutase(SOD) in VSMCs were tested by appropriate kits.The protein expressions of osteopontin (OPN), alkaline phosphatase (ALP), nuclear factor E2 related factor 2(Nrf2), and NAD(P)H: quinone oxidoreductase 1(NQO1) were examined using Western blot. Results: (1) Cytosolic calciumconcentration was significantly higher in AGE group than in control group((2.43±0.15) mmol/L vs. (1.23±0.09) mmol/L, P<0.01), which was significantly reduced in experimental group((1.62±0.18) mmol/L,P<0.01 vs. AGE group). (2) Calcium deposition in VSMCs was significantly upregulated in AGE group than in control group(3.64±0.50 vs. 1.00±0.12, P<0.01), and was downregulated in experimental group (1.56±0.37, P<0.01 vs. AGE group). (3) The MDA contents were higher((3.79±0.27) nmol/mg prot vs.(1.99±0.15) nmol/mg prot, P<0.01), while the SOD activities were lower((308.45±14.28) U/mg prot vs. (428.58±11.00) U/mg prot, P<0.01) in AGE group than in control group. The MDA contents were lower((2.37±0.19) nmol/mg prot vs. (3.79±0.27) nmol/mg prot, P<0.01),while the SOD activities were higher((391.03±22.92) U/mg prot vs. (308.45±14.28) U/mg prot, P<0.05)in experimental group compared with AGE group. (4) The relative expressions of OPN and ALP were higher in AGE group than in control group(3.06±0.21 vs. 1.00±0.07, and 2.89±0.29 vs. 1.00±0.10,both P<0.01), both (OPN(1.15±0.12) and ALP(1.45±0.15)) were downregulated in experimental group (both P<0.01 vs. AGE group). (5) The relative protein expressions of Nrf2 and NQO1 in experimental group were higher than AGE group(2.37±0.17 vs. 1.17±0.09, and 3.91±0.18 vs. 1.05±0.08, both P<0.01). Conclusion: Activation of nrf2/NQO1 signaling pathway by RTA-408 can reduce the AGE-induced VSMC calcification through attenuating oxidative injury.

A facile chemical process to form an ultrathin hydroxyapatite layer with a customizable silver-releasing function on a titanium implant.

Silver-containing hydroxyapatite (Ag/HAp) layer on a bioinert material provides both bioactive and antibacterial properties; however, the Ag release duration needs to be customized to a patient's age and metabolism for minimizing the toxic effects. Herein, we present a facile chemical process to produce an ultrathin Ag/HAp layer on a Ti implant with a customized Ag-releasing profile. The process involves the following steps: preparation of a slurry-type reagent by mixing calcium phosphate powder with an aqueous AgNO3 solution, burying a Ti substrate in the slurry, and heating the slurry in air. An HAp layer, approximately 50 nm thick, with Ag particle deposits was obtained using this process. The Ag-particle content can be varied by adjusting the concentration of AgNO3 solution used for slurry preparation, resulting in different Ag-release profiles in a physiological solution. For instance, Ag release was retained for up to 30 days when 100 mM AgNO3 was used, whereas the release lasted 15 days when 10 mM AgNO3 was used. The duration of the antibacterial activity varied accordingly, but Ag-release-derived cytotoxicity was not observed irrespective of the AgNO3 concentration. In addition, differentiation of osteoblast-like cells was facilitated owing to the formation of the HAp layer. Thus, the chemical process presented in this study allows the production-at a clinical site-of an Ag/HAp layer customized to the patient's needs.

Zinc ameliorates human aortic valve calcification through GPR39 mediated ERK1/2 signalling pathway.

AIMS: Calcific aortic valve disease (CAVD) is the most common heart valve disease in the Western world. It has been reported that zinc is accumulated in calcified human aortic valves. However, whether zinc directly regulates CAVD is yet to be elucidated. The present study sought to determine the potential role of zinc in the pathogenesis of CAVD. METHODS AND RESULTS: Using a combination of a human valve interstitial cell (hVIC) calcification model, human aortic valve tissues, and blood samples, we report that 20 µM zinc supplementation attenuates hVIC in vitro calcification, and that this is mediated through inhibition of apoptosis and osteogenic differentiation via the zinc-sensing receptor GPR39-dependent ERK1/2 signalling pathway. Furthermore, we report that GPR39 protein expression is dramatically reduced in calcified human aortic valves, and there is a significant reduction in zinc serum levels in patients with CAVD. Moreover, we reveal that 20 µM zinc treatment prevents the reduction of GPR39 observed in calcified hVICs. We also show that the zinc transporter ZIP13 and ZIP14 are significantly increased in hVICs in response to zinc treatment. Knockdown of ZIP13 or ZIP14 significantly inhibited hVIC in vitro calcification and osteogenic differentiation. CONCLUSIONS: Together, these findings suggest that zinc is a novel inhibitor of CAVD, and report that zinc transporter ZIP13 and ZIP14 are important regulators of hVIC in vitro calcification and osteogenic differentiation. Zinc supplementation may offer a potential therapeutic strategy for CAVD.

Matrix stiffness determines the phenotype of vascular smooth muscle cell in vitro and in vivo: Role of DNA methyltransferase 1.

Cells perceive the physical cues such as perturbations of extracellular matrix (ECM) stiffness, and translate these stimuli into biochemical signals controlling various aspects of cell behavior, which contribute to the physiological and pathological processes of multiple organs. In this study, we tested the hypothesis that during arterial stiffening, vascular smooth muscle cells (SMCs) sense the increase of ECM stiffness, which modulates the cellular phenotype through the regulation in DNA methyltransferases 1 (DNMT1) expression. Moreover, we hypothesized that the mechanisms involve intrinsic stiffening and deficiency in contractility of vascular SMCs. Substrate stiffening was mimicked in vitro with polyacrylamide gels. A contractile-to-synthetic phenotypic transition was induced by substrate stiffening in vascular SMCs through the down-regulation of DNMT1 expression. DNMT1 repression was also observed in the tunica media of mice aortas in an acute aortic injury model and a chronic kidney failure model, as well as in the tunica intima of human carotid arteries with calcified atherosclerotic lesions. DNMT1 inhibition facilitates arterial stiffening in vivo and promotes osteogenic transdifferentiation, calcification and cellular stiffening of vascular SMCs in vitro. These effects may be attributable, at least in part, to the role of DNMT1 in regulating the promoter activities of Transgelin (SM22α) and α-smooth muscle actin (SMA) and the functional contractility of SMCs. We conclude that DNMT1 is a critical regulator that negatively regulates arterial stiffening via maintaining the contractile phenotype of vascular SMCs. This research may facilitate elucidation of the complex crosstalk between vascular SMCs and their surrounding matrix in healthy and in pathological conditions and provide new insights into the implications for potential targeting of the phenotypic regulatory mechanisms in material-related therapeutic applications.

Leptin can promote mineralization and up-regulate RANKL mRNA expression in osteoblasts from adult female SD rats.

Osteoblast-related bone formation is an indispensable part of bone remodeling. Osteoblasts can regulate osteoclast activity through the OPG/RANK/RANKL system. Therefore, studies focus on osteoblast proliferation, differentiation, mineralization, and regulation of osteoclasto genes are important. Leptin is a polypeptide encoded by the obesity gene. In the past, regulatory roles of leptin on sugar and lipid metabolism have been extensively studied. In recent years, leptin has been found to have multiple effects on bone metabolism. However, its role in osteoblasts has not been clarified. In order to investigate the effects of leptin on osteoblasts of adult female SD rats, primary cultured osteoblasts were passaged and divided into control and leptin groups. The effects of leptin on osteoblast proliferation, differentiation, mineralization, and OPG and RANKL mRNA expression were observed. Leptin significantly promoted mineralization of osteoblasts in adult female SD rats, and the mineralized area gradually increased with the increase of leptin concentration. When the concentration of leptin was 100 ng/ml, the mineralized area was the highest (P = 0.001). The expression of RANKL mRNA was also elevated by 10 ng/ml leptin. However, leptin had no significant effect on osteoblast proliferation, secretion of ALP, and expression of OPG mRNA in adult female SD rat osteoblast.

Osteoblastic differentiation and cell calcification of adamantinomatous craniopharyngioma induced by bone morphogenetic protein-2.

BACKGROUND: The calcification of adamantinomatous craniopharyngioma (ACP) often creates difficulties for surgical therapy. Nevertheless, the mechanism of ACP calcification is unclear. Our previous studies demonstrated that osteoblastic factors might play important roles in ACP calcification. OBJECTIVE: We examined the effects of recombinant human Bmp2 on ACP cell differentiation by testing osteoblastic proteins and calcium deposition. METHODS: The expression of osteoblastic factors including osteopontin (OPN), Runx2, and osterix in Bmp2-treated ACP cells was examined by western blot and/or real time PCR. ALP activity and calcium deposition after Bmp2 induction were also tested. RESULTS: Bmp2 significantly amplified the expression of Runx2, Osterix and OPN, as well as ALP activity. Both of these effects could be repressed by noggin treatment. Bmp2 also significantly induced the calcification of ACP, and noggin inhibited this calcium deposition. CONCLUSION: Our study demonstrated for the first time that ACP cells could differentiate into an osteoblastic lineage via induction by Bmp2. The mechanism of ACP calcification likely involves osteoblastic differentiation modulated by Bmp2. Further studies targeting Bmp2 cascades could result in novel therapeutic interventions for recurrent ACP.

Activation of peroxisome proliferator-activated receptor γ inhibits vascular calcification by upregulating Klotho.

Cardiovascular diseases are common in patients with chronic kidney disease. One of the key symptoms is the calcification of the vascular smooth muscle cells (VSMCs), which is induced by dysregulated mineral metabolism with high circulating levels of inorganic phosphate (Pi) and calcium. Klotho, which was originally identified as an aging suppressor gene, has been shown to be associated with vascular calcification. Since Klotho was recently identified as a target for nuclear receptor peroxisome proliferator-activated receptor (PPAR) γ, the present study aimed to determine whether PPARγ regulates VSMC calcification through modulating the expression levels of Klotho. It was demonstrated that the expression of PPARγ was downregulated during Pi-induced VSMC calcification. In addition, treatment with PPARγ agonists inhibited the calcification and enhanced the expression of Klotho in VSMCs in a PPARγ-dependent manner. Of note, loss of Klotho expression by RNA interference abolished the ability of PPARγ activation to inhibit VSMC calcification. Furthermore, activation of Klotho as well as PPARγ inhibited the expression of Pi transporter 1/2 and reduced Pi influx into VSMCs. To the best of our knowledge, the present study was the first to demonstrate that PPARγ regulates VSMC calcification through activating Klotho.

Activation of the FGF signaling pathway and subsequent induction of mesenchymal stem cell differentiation by inorganic polyphosphate.

Inorganic polyphosphate [poly(P)] is a biopolymer existing in almost all cells and tissues, although its biological functions in higher eukaryotes have not been completely elucidated. We previously demonstrated that poly(P) enhances the function of fibroblast growth factors (FGFs) by stabilizing them and strengthening the affinity between FGFs and their cell surface receptors. Since FGFs play crucial roles in bone regeneration, we further investigated the effect of poly(P) on the cell differentiation of human stem cells via FGF signaling systems. Human dental pulp cells (HDPCs) isolated from human dental pulp show the characteristics of multipotent mesenchymal stem cells (MSCs). HDPCs secreted FGFs and the proliferation of HDPCs was shown to be enhanced by treatment with poly(P). Cell surface receptor-bound FGF-2 was stably maintained for more than 40 hours in the presence of poly(P). The phosphorylation of ERK1/2 was also enhanced by poly(P). The effect of poly(P) on the osteogenic differentiation of HDPCs and human MSCs (hMSCs) were also investigated. After 5 days of treatment with poly(P), type-I collagen expression of both cell types was enhanced. The C-terminal peptide of type-I collagen was also released at higher levels in poly(P)-treated HDPCs. Microarray analysis showed that expression of matrix metalloproteinase-1 (MMP1), osteopontin (OPN), osteocalcin (OC) and osteoprotegerin was induced in both cell types by poly(P). Furthermore, induced expression of MMP1, OPN and OC genes in both cells was confirmed by real-time PCR. Calcification of both cell types was clearly observed by alizarin red staining following treatment with poly(P). The results suggest that the activation of the FGF signaling pathway by poly(P) induces both proliferation and mineralization of stem cells.