doi: 10.1161/01.RES.0000231258.23378.a6
© 2006 American Heart Association, Inc.
Editorials |
Vascular Cell Locomotion
Osteopontin, NADPH Oxidase, and Matrix Metalloproteinase-9
From the Hypertension and Vascular Research Division (P.J.P.) and the Department of Surgery (M.J.H.), Henry Ford Research Institute and Hospital, Detroit, Mich.
Correspondence to Patrick J. Pagano, Hypertension and Vascular Research Division, Henry Ford Research Institute and Hospital, ER 7044; Henry Ford Hospital, 2799 West Grand Blvd, Detroit, MI 48202. E-mail PPAGANO1{at}hfhs.org
See related article, pages 1479–1489
Key Words: osteopontin • MMPs • superoxide • adventitia • myofibroblasts • neointima • vascular cell motility
Since the discovery of osteopontin (OPN) in the vasculature, vascular biologists have been intrigued by its potential role in vascular arteriosclerosis and vessel calcification during the aging process.1 Osteopontin is a phosphorylated acidic glycoprotein adhesion molecule, originally cloned from bone; and as in bone, its cardiovascular functions include cellular migration, adhesion, and spreading.2 The most salient features of this intriguing calcium-regulating molecule are its ability to interact with 
vß3 and other integrins on the surface of cells. Because this ligand–receptor complex is known to be involved in cell migration, it is not surprising that an antibody to the ß3 subunit of this receptor effectively reduced restenosis in patients.3 Interestingly, angiotensin II and other growth factors, including platelet-derived growth factor and transforming growth factor-ß, regulate OPN gene expression in vascular smooth muscle cell (VSMC) cultures and fibroblasts.1,4 Cardiac fibroblasts have been extensively reported to produce OPN in response to these growth factors, resulting in fibroblast proliferation. On the one hand, OPN is thought to cause vascular dystrophy by altering extracellular matrix formation; on the other hand, cellular motogenesis is a hallmark of this emerging vascular cytokine. Its cellular expression includes a variety of inflammatory cells, such as T cells and macrophages, as well as mesenchymal cells of the vasculature including adventitial myofibroblasts. Moreover, OPN has both pro- and antiinflammatory properties. As a proinflammatory agent it can attract and modulate the function of the above mentioned cell types. Of seemingly major importance, OPN interacts with intracellular signaling agents to promote cell migration and matrix metalloproteinases, both of which are required for cell tunneling through dense and elastin-rich vascular tissue. In fibroblasts these agents appear to include cell surface CD44 and ERM proteins located just below the plasma membrane (at filopodia-like structures).5 These motility-promoting signaling agents are consequently controlled by a variety of tyrosine and serine/threonine kinases.
For some time, it has been established that vascular mesenchymal cells (VSMCs and AMFs) are committed into mitogenic and migratory action by NADPH oxidase activation.6–10 Earlier reports demonstrated that OPN produced by fibroblasts mediates growth factor–induced DNA synthesis.2 Although the cascade leading from oxidase activation to transcription factor upregulation, cell proliferation, and migration has been extensively characterized,11–13 a multitude of upstream and downstream players are also likely to emerge as initiators and terminal regulators of this process. Based on previous reports of submembranal c-Src and its substrate c-Abl being activated by NADPH oxidase–derived reactive oxygen species, leading to progrowth and promigratory signaling cascades, it is tempting to speculate that OPN, either directly or indirectly via vß3 and other integrins, may activate one or both of these molecules.5,14,15
In this issue of Circulation Research, Lai and coworkers16 elegantly and comprehensively demonstrate that OPN via activation of NADPH oxidase–derived superoxide anion and oxylipid formation17,18 promotes preferential upregulation of MMP-9 in primary aortic myofibroblasts and immortal VSMCs (A7r5) under recreated glucose-elevated conditions in vitro. As the authors illustrate, OPN upregulated pro–MMP-9 activity measured by zymography, and these data were confirmed via a lack of upregulation in OPN–/– mice. Interestingly, they demonstrated the ability of glucose, OPN, TNF-, and angiotensin II to induce pro–MMP-9 activity in A7r5 VSMCs. The authors point out that aortic fibroblasts and myofibroblasts (AMFs) have higher levels of NADPH oxidase activity as has been indicated by other reports8,9,19–21 and exhibit "robust" metabolic responses in diabetes, dyslipidemia, and hypertension. In fact, many reports over the past 35 years have indicated an early and marked proliferation of adventitial cells in various disease and injury models.22–26 Periaortic AMFs express high levels of OPN in the early stages of diabetes. Thus the authors decided to carry out most of their experiments in these cells. In one of these experiments, OPN knockout was used to confirm the essential role of OPN in glucose- and cytokine-induced pro–MMP-9 in AMFs. Although these experiments were in vitro and involved isolated cells, one significant limitation may be that OPN–/– mice have abnormal macrophage activity at sites of injury and infection under in vivo conditions.27 Because macrophages express MMP-9, an in vivo role of OPN would likely include these important pluripotent infiltrating leukocytes.
The authors proceed to demonstrate that inhibitors of NF-
B blocked TNF-–induced pro–MMP-9 activation, and they postulate that a scavenger of superoxide anion, N-acetyl cysteine (NAC), would prevent TNF-–induced pro–MMP-9. These authors tested this hypothesis further using an exogenous scavenger of superoxide, superoxide dismutase (SOD). Reportedly, SOD attenuated pro–MMP-9 activity in these cells. This finding, however, is inconsistent with the authors’ suggestion that intracellular superoxide anion is instrumental in this process. Irrespective of the fact that PEG-SOD inhibited dihydroethidium staining (measurement of intracellular superoxide anion), the authors have perhaps inadvertently overlooked a role of extracellular superoxide anion in OPN-mediated MMP-9 activation. Without data showing the magnitude of the effect of SOD versus PEG-SOD on MMP-9 activation, we are unable to assess the relative importance of extra- versus intracellular superoxide in this process.
The involvement of NADPH oxidase in OPN-induced pro–MMP-9 was confirmed by the use of diphenylene iodonium, apocynin, and illustrations of induction of nox2, p47phox, and p67phox, which are essential components of the classic NADPH oxidase, and nox1, which is the anchoring component of the nox1 oxidase system. Although the actual isoform involved in this process does not seem to be of utmost importance at this time, it appears from the blots in Figure 3E that OPN knockout more markedly decreased nox2 and one of its essential cytosolic components, p67phox. The authors then go on to show that that addition of oxylipid 8-F isoprostane induced pro–MMP-9 activity in VSMCs and AMFs and restored MMP-9 activity in a dose-dependent manner in the presence of an antibody to OPN. The authors compellingly illustrate that restoring OPN to knockout cells rescued superoxide detection as well as activation of pro–MMP-9, pointedly noting that the N terminus of cleaved OPN was the active mediator in this upregulation. The OPN N-terminal domain encodes motifs necessary for superoxide anion, oxylipid formation, and pro–MMP-9 activation, all of whose signals are attenuated with the addition of PEG-SOD. It would have been interesting to see whether adding OPN to the AMF would have increased both expression and activity of NADPH oxidase(s).
Although this article provides an in depth and encompassing view of the molecular mechanisms involved in OPN-induced MMP-9 activity, one limitation of these studies is that the authors did not extend their investigation to an in vivo animal model. Thus it is difficult at this point to assess the true impact of OPN on MMP-9 activity and cell invasion in vivo during diabetes. At this juncture, it is also unclear why both A7r5 and AMFs were used, because few experiments were done comparing parameters in both cell types. Moreover, the article could have been substantially strengthened by comparison of primary aortic VSMCs and AMFs, which would have provided a clearer insight into the role of OPN in 2 distinct vascular mesenchymal cells under diabetes-like conditions.
In vascular proliferative diseases, including diabetes-associated atherosclerosis,3 the varied upregulation of specific matricellular proteins, including OPN, appears to mediate PDGF-BB– and bFGF-directed migration of vascular mesenchymal cells. The multifunctional CamKII kinase is located downstream from OPN and vß3 and is seemingly critical to promotogenic activity of these cells, involving recruitment of ERK 1/2 and potentially other mitogen-activated protein kinases.29 This in and of itself, along with the described effect of OPN to activate rapid cellular calcium entry via L-type calcium channels and perhaps even voltage-operated calcium channels,28,30 could lead to unmitigated mitogenesis and migration. It has been previously implied that vascular cell migration is dependent on NADPH oxidase activity and MMP activity.31,32 In fact, one report in particular indicated that NADPH oxidase–derived reactive oxygen species directly influence vascular MMP-2 activity in vitro.32 The generally accepted notion that NADPH oxidases are integral and/or proximal to the cell membrane would imply that, at the very least, OPN could induce immediate vascular cell invasion. Yet the subcellular circuitry related to mito- and motogenesis would be expected to be far more complex, including (but apparently not limited to) activation of the progrowth and promigratory Rho–Rac–NADPH oxidase–MAPK signaling axis. The potential is there for exaggerated MMP activity to lead to a weakened and flaccid vascular wall, resulting in aneurysm formation. Because it is becoming increasingly apparent that NADPH oxidase is an important common link among a variety of inflammatory cardiovascular disease processes that lead to vascular remodeling21,33,34 in addition to distinct forms of neoplasia,35,36 the findings of this article may be considered to have broad significance for pathophysiology. Finally, under conditions in which OPN levels may rise dramatically, it is not difficult to envisage exacerbated vascular dystrophy as a consequence of a potential by rampant calcium uptake and calcification of arteries1,30,37 characteristic of the aging process and arteriosclerosis.
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Acknowledgments |
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The authors wish to thank Drs Noelia Ardanaz and M. Eugenia Cifuentes for their critiques and suggestions.
Sources of Funding
This work was supported by NIH grant HL55425 (to P.J.P.). P.J.P. is an Established Investigator of the American Heart Association.
Disclosures
None.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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Related Article:
Circ. Res. 2006 98: 1479-1489.
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