Osteogenic oxysterols inhibit the adverse effects of oxidative stress on osteogenic differentiation of marrow stromal cells

The osteoporosis that occurs with aging is associated with reduced number and activity of osteoblastic cells. Aging, menopause, and osteoporosis are correlated with increased oxidative stress and reduced antioxidant defense mechanisms. We previously demonstrated that oxidative stress induced by a variety of compounds such as xanthine/xanthine oxidase (XXO) and minimally oxidized LDL (MM-LDL) inhibit the osteogenic differentiation of osteoprogenitor cells. Oxysterols are a family of products derived from cholesterol oxidation that have important biological activities. Recently, we reported that a specific oxysterol combination consisting of 22(S)- or 22(R)-hydroxycholesterol and 20(S)-hydroxycholesterol has potent osteogenic properties in vitro when applied to osteoprogenitor cells including M2-10B4 (M2) marrow stromal cells. We now demonstrate that this osteogenic combination of oxysterols prevents the adverse effects of oxidative stress on differentiation of M2 cells into mature osteoblastic cells. XXO and MM-LDL inhibited the osteogenic differentiation of M2 cells, demonstrated by the inhibition of markers of osteogenic differentiation: alkaline phosphatase activity, osteocalcin expression and mineralization. Treatment of M2 cells with osteogenic oxysterol combination 22(S)- and 20(S)-hydroxycholesterol both blocked and reversed the inhibition of osteogenic differentiation produced by XXO and MM-LDL in these cells. The protective effect of the oxysterols against oxidative stress was dependent on cyclooxygenase 1 and was associated with the osteogenic property of the oxysterols. These findings further demonstrate the ability of the osteogenic oxysterols to positively regulate osteogenic differentiation of cells, and suggests that the use of these compounds may be a novel strategy to prevent the adverse effects of oxidative stress on osteogenesis.     

Selenium suppresses oxidative-stress-enhanced vascular smooth muscle cell calcification by inhibiting the activation of the PI3K/AKT and ERK signaling pathways and endoplasmic reticulum stress

Vascular calcification is a prominent feature of many diseases, including atherosclerosis, and it has emerged as a powerful predictor of cardiovascular morbidity and mortality. A number of studies have examined the association between selenium and risk of cardiovascular diseases, but little is known about the role of selenium in vascular calcification. To determine the role of selenium in regulating vascular calcification, we assessed the effect of sodium selenite on oxidative-stress-enhanced vascular smooth muscle cell (VSMC) calcification and the underlying mechanism. Oxidative stress induced by xanthine/xanthine oxidase increased apoptosis, as determined by Hoechst 33342 staining and annexin V/propidium iodide staining, and it enhanced osteoblastic differentiation and calcification of VSMCs, on the basis of alkaline phosphatase activity, the expression of Runx2 and type I collagen, and calcium deposition. These effects of oxidative stress were significantly inhibited by selenite. The following processes may explain the inhibitory effects of selenite: (1) selenite significantly suppressed oxidative stress, as evidenced by the decrease of the oxidative status of the cell and lipid peroxidation levels, as well as by the increase of the total protein thiol content and the activity of the antioxidant selenoenzyme glutathione peroxidase; (2) selenite significantly attenuated oxidative-stress-induced activation of the phosphatidylinositol 3-kinase/AKT and extracellular-signal-regulated kinase signaling pathways, resulting in decreased osteoblastic differentiation of VSMCs; (3) selenite significantly inhibited oxidative-stress-activated endoplasmic reticulum stress, thereby leading to decreased apoptosis. Our results suggest a potential role of selenium in the prevention of vascular calcification, which may provide more mechanistic insights into the relationship between selenium and cardiovascular diseases.     

Metallothionein protects bone marrow stromal cells against hydrogen peroxide-induced inhibition of osteoblastic differentiation

Metallothionein (MT), a cysteine-rich, metal-binding protein, is involved in homeostatic regulation of essential metals and protection of cells against oxidative injury. It has been shown that oxidative stress is associated with pathogenesis of osteoporosis and is capable of inhibiting osteoblastic differentiation of bone cells by nuclear factor-kappaB (NF-kappaB). In this study, the effect of MT on oxidative stress-induced inhibition of osteoblast differentiation was examined. 50-200 microM hydrogen peroxide-induced oxidative stress suppressed the osteoblastic differentiation process of primary mouse bone marrow stromal cells (BMSCs), manifested by a reduction in the differentiation marker alkaline phosphatase (ALP). The presence of exogenous MT (20-500 microM) or induction of endogenous MT by ZnCl2 (50-200 microM) could protect BMSCs against H2O2-induced inhibition of osteoblastic differentiation, manifested by a resumption of H2O2-inhibited ALP activity and ALP positive cells. Furthermore, adding exogenous MT or inducing endogenous MT expression impaired H2O2-stimulated NF-kappaB signaling. These data indicate the ability of MT to protect BMSCs against oxidative stress-induced inhibition of osteoblastic differentiation.     

Superoxide production: A procalcifying cell signalling event in osteoblastic differentiation of vascular smooth muscle cells exposed to calcification media

Recent studies showed that hydrogen peroxide (H₂O₂) enhanced bone markers expression in vascular smooth muscle cells (VSMCs) implicated in osteoblastic differentiation. This study aimed at investigating the role of NAD(P)H oxidase in vascular calcification processes. A7r5 rat VSMCs were incubated with β-glycerophosphate (10 mm) or uremic serum to induce a diffuse mineralization. H₂O₂ production by VSMCs was determinated by chemiluminescence. NAD(P)H oxidase sub-unit (p22^phox), Cbfa-1, ERK phosphorylation and bone alkaline phosphatase (ALP) expressions were measured by Western blotting. VSMCs exhibited higher production of H₂O₂ and early expression of p22^phox with β-glycerophosphate or uremic serum within 24 h of treatment. β-glycerophosphate-induced oxidative stress was associated with Cbfa-1 expression followed by ALP expression and activity, meanwhile the VSMCs expressing ALP diffusely calcified their extracellular matrix. Interestingly, diphenyleneiodonium partly prevented the osteoblastic differentiation. Results from this model strongly suggest a major implication of vascular NAD(P)H oxidase in vascular calcification supported by VSMCs osteoblastic differentiation.     

Mechanical stimulation and mitogen-activated protein kinase signaling independently regulate osteogenic differentiation and mineralization by calcifying vascular cells

Ectopic calcification of vascular tissue is associated with several cardiovascular pathologies and likely involves active regulation by vascular smooth muscle cells and osteoblast-like vascular cells. This process often occurs in sites with altered mechanical environments, suggesting a role for mechanical stimuli in calcification. In this study, we investigated the effect of mechanical stimulation on the proliferation, osteogenic differentiation, calcification, and mitogen-activated protein kinase (MAPK) signaling in calcifying vascular cells (CVCs), a subpopulation of aortic smooth muscle cells putatively involved in vascular calcification. Application of equibiaxial cyclic strain (7%, 0.25 Hz) to CVCs had no effect on cell proliferation, but accelerated alkaline phosphatase expression and significantly increased mineralization by 3.1-fold over unstrained cells. Fluid motion in the absence of strain also enhanced mineralization, but to a lesser degree. Because MAPK pathways mediate mechanically regulated osteoblast differentiation, we tested whether similar signaling was involved in mineralization by CVCs. In static cultures, pharmacological inhibition of the extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and c-Jun N-terminal kinase pathways significantly attenuated mineral production by as much as -94%, compared with uninhibited CVCs. Strikingly, although mechanical stimulation activated each of the MAPK pathways, inhibition of these pathways had no effect on the mechanically induced enhancement of alkaline phosphatase activity or mineralization. These novel data indicate that mechanical signals regulate calcification by CVCs, and although MAPK signaling is critical to CVC osteogenic differentiation and mineralization, it is not involved directly in transduction of mechanical signals to regulate these processes under the conditions utilized in this study.     

Adiponectin attenuates the osteoblastic differentiation of vascular smooth muscle cells through the AMPK/mTOR pathway

Vascular calcification is common in patients with peripheral artery diseases and coronary artery diseases. The osteoblastic differentiation of vascular smooth muscle cells (VSMCs) contributes significantly to vascular calcification. Adiponectin has been demonstrated to exert a protective effect in osteoblastic differentiation of VSMCs through regulating mTOR activity. However, the upstream and downstream signaling molecules of adiponectin-regulated mTOR signaling have not been identified in VSMCs with osteoblastic differentiation. In this study, the VSMC differentiation model was established by beta-glycerophosphate (β-GP) induction. The mineralization was identified by Alizarin Red S staining. Protein expression and phosphorylation were detected by Western blot or immunofluorescence. Adiponectin attenuated osteoblastic differentiation and mineralization of β-GP-treated VSMCs. Adiponectin inhibited osteoblastic differentiation of VSMCs through increasing the level of p-AMPKα. Pretreatment of VSMCs with AMPK inhibitor blocked while AMPK activator enhanced the effect of adiponectin on osteoblastic differentiation of VSMCs. Adiponectin upregulated TSC2 expression and downregulated mTOR and S6K1 phosphorylation in β-GP-treated VSMCs. Adiponectin treatment significantly attenuates the osteoblastic differentiation and calcification of VSMCs through modulation of AMPK–TSC2–mTOR–S6K1 signal pathway.     

Ghrelin attenuates the osteoblastic differentiation of vascular smooth muscle cells through the ERK pathway

Vascular calcification results from osteoblastic differentiation of vascular smooth muscle cells (VSMCs) and is a major risk factor for cardiovascular events. Ghrelin is a newly discovered bioactive peptide that acts as a natural endogenous ligand of the growth hormone secretagog receptor (GHSR). Several studies have identified the protective effects of ghrelin on the cardiovascular system, however research on the effects and mechanisms of ghrelin on vascular calcification is still quite rare. In this study, we determined the effect of ghrelin on osteoblastic differentiation of VSMCs and investigated the mechanism involved using the two universally accepted calcifying models of calcifying vascular smooth muscle cells (CVSMCs) and beta-glycerophosphate (beta-GP)-induced VSMCs. Our data demonstrated that ghrelin inhibits osteoblastic differentiation and mineralization of VSMCs due to decreased alkaline phosphatase (ALP) activity, Runx2 expression, bone morphogenetic protein-2 (BMP-2) expression and calcium content. Further study demonstrated that ghrelin exerted this suppression effect via an extracellular signal-related kinase (ERK)-dependent pathway and that the suppression effect of ghrelin was time dependent and dose dependent. Furthermore, inhibition of the growth hormone secretagog receptor (GHSR), the ghrelin receptor, by siRNA significantly reversed the activation of ERK by ghrelin. In conclusion, our study suggests that ghrelin may inhibit osteoblastic differentiation of VSMCs through the GHSR/ERK pathway.     

Necrotic and apoptotic cells serve as nuclei for calcification on osteoblastic differentiation of human mesenchymal stem cells in vitro

A close relationship between cell death and pathological calcification has recently been reported, such as vascular calcification in atherosclerosis. However, the roles of cell death in calcification by osteoblast lineage have not been elucidated in detail. In this study, we investigated whether cell death is involved in the calcification on osteoblastic differentiation of human bone marrow mesenchymal stem cells (hMSC) under osteogenic culture in vitro. Apoptosis and necrosis occurred in an osteogenic culture of hMSC, and cell death preceded calcification. The generation of intracellular reactive oxygen species, chromatin condensation and fragmentation, and caspase‐3 activation increased in this culture. A pan‐caspase inhibitor (Z‐VAD‐FMK) and anti‐oxidants (Tiron and n ‐acetylcysteine) inhibited osteogenic culture‐induced cell death and calcification. Furthermore, calcification was significantly promoted by the addition of necrotic dead cells or its membrane fraction. Spontaneously dead cells by osteogenic culture and exogenously added necrotic cells were surrounded by calcium deposits. Induction of localized cell death by photodynamic treatment in the osteogenic culture resulted in co‐localized calcification. These findings show that necrotic and apoptotic cell deaths were induced in an osteogenic culture of hMSC and indicated that both necrotic and apoptotic cells of osteoblast lineage served as nuclei for calcification on osteoblastic differentiation of hMSC in vitro. Copyright © 2013 John Wiley & Sons, Ltd.     

Oxidative stress inhibits osteoblastic differentiation of bone cells by ERK and NF-kappaB

Signaling pathways involved in oxidative stress-induced inhibition of osteoblast differentiation are not known. We showed in this report that H(2)O(2) (0.1-0.2mM)-induced oxidative stress suppressed the osteoblastic differentiation process of primary rabbit bone marrow stromal cells (BMSC) and calvarial osteoblasts, manifested by a reduction of differentiation markers including alkaline phosphatase (ALP), type I collagen, colony-forming unit-osteoprogenitor (CFU-O) formation, and nuclear phosphorylation of Runx2. H(2)O(2) treatment stimulated phospholipase C-gamma1 (PLC-gamma1), extracellular signal-regulated kinase 1/2 (ERK1/2), and NF-kappaB signaling but inhibited p38 mitogen-activated protein kinase (MAPK) activation. In the presence of 20microM PD98059 or 50microM caffeic acid phenethyl ester (CAPE), specific inhibitor for ERKs or NF-kappaB, respectively, could significantly reverse the decrease of above-mentioned osteoblastic differentiation markers elicited by H(2)O(2) (0.1mM). Furthermore, PD98059 also suppressed H(2)O(2)-stimulated NF-kappaB signaling in this process. These data suggest that ERK and ERK-dependent NF-kappaB activation is required for oxidative stress-induced inhibition of osteoblastic differentiation in rabbit BMSC and calvarial osteoblasts.     

Phosphate and pyrophosphate mediate PKA-induced vascular cell calcification

Vascular calcification is associated with increased cardiovascular risk and occurs by osteochondrogenic differentiation of vascular cells. Many of the same regulatory factors that control skeletal mineralization, including the complex metabolic pathway controlling levels of the activator, inorganic phosphate, and the potent inhibitor, pyrophosphate, also govern vascular calcification. We previously found that the cAMP/PKA signaling pathway mediates in vitro vascular cell calcification induced by inflammatory factors including tumor necrosis factor-alpha 1 and oxidized phospholipids. In this report, we tested whether this signaling pathway modulates phosphate and pyrophosphate metabolism. Treatment of primary murine aortic cells with the PKA activator, forskolin, significantly induced osteoblastic differentiation markers, including alkaline phosphatase (ALP), osteopontin, and osteocalcin as well as the pyrophosphate generator, ectonucleotide-pyrophosphatase/phosphodiesterase-1 (Enpp1) and the pyrophosphate transporter, ankylosis protein, but not the sodium/phosphate cotransporter, Pit-1. In the presence of a substrate for ALP, beta-glycerophosphate, which generates inorganic phosphate, forskolin also enhanced matrix mineralization. Inhibitors of ALP or Pit-1 abrogated forskolin-induced osteopontin expression and mineralization but not forskolin-induced osteocalcin or ALP. These results suggest that phosphate is necessary for PKA-induced calcification of vascular cells and that the extent of PKA-induced calcification is controlled by feedback induction of the inhibitor, pyrophosphate.     

Lipid peroxidation and cell cycle signaling: 4-hydroxynonenal,a key molecule in stress mediated signaling

Role of lipid peroxidation products, particularly 4-hydroxynonenal (4-HNE) in cell cycle signaling is becoming increasingly clear. In this article, recent studies suggesting an important role of 4-HNE in stress mediated signaling for apoptosis are critically evaluated. Evidence demonstrating the modulation of UV, oxidative stress, and chemical stress mediated apoptosis by blocking lipid peroxidation by the alpha-class glutathione S-transferases (GSTs) is presented which suggest an important role of these enzymes in protection against oxidative stress and a role of lipid peroxidation products in stress mediated signaling. Overexpression of 4-HNE metabolizing GSTs (mGSTA4-4, hGSTA4-4, or hGST5.8) protects cells against 4-HNE, oxidative stress (H(2)O(2) or xanthine/xanthine oxidase), and UV-A mediated apoptosis by blocking JNK and caspase activation suggesting a role of 4-HNE in the mechanisms of apoptosis caused by these stress factors. The intracellular concentration of 4-HNE appears to be crucial for the nature of cell cycle signaling and may be a determinant for the signaling for differentiation, proliferation, transformation, or apoptosis. The intracellular concentrations of 4-HNE are regulated through a coordinated action of GSTs (GSTA4-4 and hGST5.8) which conjugate 4-HNE to GSH to form the conjugate (GS-HNE) and the transporter 76 kDa Ral-binding GTPase activating protein (RLIP76), which catalyze ATP-dependent transport of GS-HNE. A mild stress caused by heat, UV-A, or H(2)O(2)with no apparent effect on the cells in culture causes a rapid, transient induction of hGST5.8 and RLIP76. These stress preconditioned cells acquire ability to metabolize and exclude 4-HNE at an accelerated pace and acquire relative resistance to apoptosis by UV and oxidative stress as compared to unconditioned control cells. This resistance of stress preconditioned cells can be abrogated by coating the cells with anti-RLIP76 antibodies which block the transport of GS-HNE. These studies and previous reports discussed in this article strongly suggest a key role of 4-HNE in stress mediated signaling.