Original contribution
Oxidative stress modulates osteoblastic differentiation of vascular and bone cells

https://doi.org/10.1016/S0891-5849(01)00610-4Get rights and content

Abstract

Oxidative stress may regulate cellular function in multiple pathological conditions, including atherosclerosis. One feature of the atherosclerotic plaque is calcium mineral deposition, which appears to result from the differentiation of vascular osteoblastic cells, calcifying vascular cells (CVC). To determine the role of oxidative stress in regulating the activity of CVC, we treated these cells with hydrogen peroxide (H2O2) or xanthine/xanthine oxidase (XXO) and assessed their effects on intracellular oxidative stress, differentiation, and mineralization. These agents increased intracellular oxidative stress as determined by 2,7 dichlorofluorescein fluorescence, and enhanced osteoblastic differentiation of vascular cells, based on alkaline phosphatase activity and mineralization. In contrast, H2O2 and XXO resulted in inhibition of differentiation markers in bone osteoblastic cells, MC3T3-E1, and marrow stromal cells, M2-10B4, while increasing oxidative stress. In addition, minimally oxidized low-density lipoprotein (MM-LDL), previously shown to enhance vascular cell and inhibit bone cell differentiation, also increased intracellular oxidative stress in the three cell types. These effects of XXO and MM-LDL were counteracted by the antioxidants Trolox and pyrrolidinedithiocarbamate. These results suggest that oxidative stress modulates differentiation of vascular and bone cells oppositely, which may explain the parallel buildup and loss of calcification, seen in vascular calcification and osteoporosis, respectively.

Introduction

The development of atherosclerosis involves an increase in oxidative stress [1], [2]. Macrophages within the atherosclerotic lesion produce reactive oxygen species (ROS) such as hydrogen peroxide and superoxide anion [3]. Endothelial cells also release ROS into the artery wall in response to various agents and conditions, such as bradykinin, hypoxia, and hypercholesterolemia [4], [5], [6]. Furthermore, they respond to oxidized LDL and leptin by increasing intracellular ROS [7], [8]. Similarly, smooth muscle cells increase intracellular ROS generation, mostly by NADPH oxidase, in response to PDGF, TNF-α, and angiotensin II [9], [10], [11].

One feature of atherosclerotic lesions that has important clinical consequences is vascular calcification. As an in vitro model of vascular calcification, we have previously isolated and cloned a subpopulation of aortic medial smooth muscle cells, which undergo osteoblastic differentiation [12]. These cells, called calcifying vascular cells (CVC), express several markers of osteoblastic differentiation, such as collagen I, alkaline phosphatase, and osteocalcin, and produce hydroxyapatite mineral [12]. Furthermore, several oxidized lipids with atherogenic properties, including minimally oxidized low-density lipoprotein (MM-LDL), oxidized palmitoyl arachidonoyl phosphatidylcholine, and isoprostaglandin E2, were previously shown to induce differentiation of CVC [13].

Previous studies have shown that increased arterial calcification correlates with an increase in osteoporosis [14], [15]. In osteoporosis, bone loss involves both increased osteoclastic bone resorption and decreased osteoblastic bone formation [16], [17]. ROS enhance osteoclastic activity [18], [19], but the effect of ROS on osteoblastic function is unknown.

In this report we tested the hypothesis that oxidative stress modulates osteoblastic differentiation of vascular and bone cells. To determine the effect of oxidative stress on CVC, we treated the cells with hydrogen peroxide (H2O2) or xanthine/xanthine oxidase (XXO), a reaction in which xanthine oxidase converts xanthine to uric acid and generates superoxide anion. Both agents induced osteoblastic differentiation of CVC. We also treated MC3T3-E1, a preosteoblastic cell line, and M2-10B4, a marrow stromal cell line that undergoes osteoblastic differentiation [20], with XXO and H2O2. We found that, in contrast to CVC, both inducers of oxidative stress inhibited differentiation of both osteoblastic cell lines. Previously, we found that atherogenic oxidized lipids, such as MM-LDL, may contribute to vascular calcification and osteoporotic bone loss by differentially regulating the osteoblastic differentiation of vascular and bone cells. In this report, we demonstrate that MM-LDL also increased oxidative stress in all three cell types, while inducing a reciprocal response in vascular and bone cells. In addition, antioxidants inhibited the effects of XXO and MM-LDL, which suggests that production of ROS mediates the enhanced differentiation of vascular cells and inhibits the differentiation of osteoblastic cells in response to these agents.

Section snippets

Materials

[3H]Thymidine and 45CaCl2 were from Amersham Corp. (Piscataway, NJ, USA). Benzoic acid, DMSO, glucose oxidase, hydrogen peroxide (30% v/v), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, thiazoyl blue), xanthine, and xanthine oxidase, pyrrolidinedithiocarbamate (PDTC), and 2,7 dichlorofluorescein diacetate were from Sigma (St. Louis, MO, USA). Deferoxamine and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were from Calbiochem (San Diego, CA, USA).

Cell culture

CVC, the

Reactive oxygen species production in response to oxidative stress in vascular cells and bone cells

Intracellular ROS production by 25 μM xanthine and 25 mU/ml xanthine oxidase (XXO) and by 1 mM hydrogen peroxide (H2O2) was measured by DCF fluorescence in CVC and MC3T3-E1 over the course of 1 h. In response to XXO, DCF fluorescence increased in both cell types (2.2 ± 0.4-fold in CVC; 2.6 ± 0.4-fold in MC3T3-E1) compared to control at 1 h (Fig. 1A, C). In response to H2O2, the increase was 5.1 ± 1.0-fold and 7.1 ± 1.7-fold for CVC and MC3T3-E1 cells, respectively, compared to control at 1 h

Discussion

The present study suggests that oxidative stress has opposite effects on the differentiation of calcifying vascular cells and bone cells. Xanthine/xanthine oxidase and H2O2 increased differentiation in CVC while inhibiting differentiation in bone cells, as assessed by their effect on an early differentiation marker, alkaline phosphatase activity. Furthermore, XXO increased mineral formation, a late marker of differentiation, in CVC while inhibiting mineralization in M2-10B4 and MC3T3-E1 cells.

Acknowledgements

This research was supported by NIH grant HL30568 and the Laubisch Fund. N. Mody was also supported by the Jennifer Buchwald Endowment, and F. Parhami is a recipient of NIH grant AG10415 through the Claude D. Pepper Older American Independence Center at UCLA. We thank J. Berliner and B. Premack for their guidance, V. Le for technical assistance, and J. Bishop and A. Gasparyan for manuscript preparation.

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