Estrogen-stimulated endothelial repair requires osteopontin.

OBJECTIVE: Estradiol (E(2)) is known to accelerate reendothelialization and thus prevent intimal thickening and in-stent restenosis after angioplasty. Transplantation experiments with ERalpha(-/-) mice have previously shown that E(2) acts through local and bone marrow cell compartments to enhance endothelial healing. However, the downstream mechanisms induced by E(2) to mediate endothelial repair are still poorly understood. METHODS AND RESULTS: We show here that after endovascular carotid artery injury, E(2)-enhanced endothelial repair is lost in osteopontin-deficient mice (OPN(-/-)). Transplantation of OPN(-/-) bone marrow into wild-type lethally irradiated mice, and vice versa, suggested that osteopontin plays a crucial role in both the local and the bone marrow actions of E(2). In the vascular compartment, using transgenic mice expressing doxycyclin regulatable-osteopontin, we show that endothelial cell specific osteopontin overexpression mimics E(2)-enhanced endothelial cell migration and proliferation in the regenerating endothelium. In the bone marrow cell compartment, we demonstrate that E(2) enhances bone marrow-derived mononuclear cell adhesion to regenerating endothelium in vivo, and that this effect is dependent on osteopontin. CONCLUSIONS: We demonstrate here that E(2) acceleration of the endothelial repair requires osteopontin, both for bone marrow-derived cell recruitment and for endothelial cell migration and proliferation.

Autocrine expression of osteopontin contributes to PDGF-mediated arterial smooth muscle cell migration.

OBJECTIVE: Migration of smooth muscle cells (SMCs) from the media to the intima of arteries is involved in intimal thickening. The platelet-derived growth factor (PDGF) BB is recognized as a major migratory factor for arterial SMCs both in vitro and during neointima formation. Since PDGF acts in synergy with the matrix protein osteopontin (OPN) and also induces its expression, the present study was conceived to explore the role of the OPN produced in an autocrine fashion by PDGF-stimulated SMCs in the migration process and to define regulatory mechanisms of OPN expression. METHODS AND RESULTS: PDGF stimulation of quiescent rat aortic SMCs induced their migration (transfilter assays) and the increase of OPN expression (mRNA and protein assays). Blockade of either OPN expression by a specific short interference RNA (siRNA) or of its function by a blocking antibody decreased the PDGF-stimulated migration by about 70%, demonstrating that autocrine production and excretion of OPN are integral to the PDGF-induced SMC migration. In parallel, SMC stimulation by PDGF also activated the transcription factor CREB essentially through mitogen-activated protein kinase (MAPK) 1/2 and protein kinase A (PKA) pathways. Inhibition of either CREB expression (via siRNA) or function (via dominant-negative CREB) decreased both PDGF-induced SMC migration and OPN expression. SMC transfection with OPN promoter reporter constructs demonstrated that PDGF-induced OPN transcription is mediated by CREB binding to two functional sites of the OPN promoter: a CRE site located at -1403 and an AP-1 site located at -76. CONCLUSION: The present study demonstrates that the autocrine expression of OPN plays a major role in PDGF-induced SMC migration. It further shows that the transcription factor CREB, activated in PDGF-stimulated SMCs, plays a key role in PDGF-induced SMC migration, probably by regulating OPN expression.

Osteopontin expression in normal and fibrotic liver. altered liver healing in osteopontin-deficient mice.

BACKGROUND/AIMS: Osteopontin has been implicated in numerous physiopathological events. Osteopontin expression in normal and fibrotic liver and liver fibrogenesis in osteopontin-deficient mice were studied. METHODS: Fibrosis was induced in mice and rats by carbon tetrachloride (CCl4) treatment or bile duct ligation. The liver was used for conventional histology, osteopontin immunohistochemistry and in situ hybridization, or protein and RNA extraction. In mice, necrotic areas and fibrosis were evaluated by quantitative image analysis. RESULTS: In normal liver, osteopontin mRNA expression was very low. After CCl4 treatment or bile duct ligation, osteopontin mRNA expression was increased. Osteopontin was expressed by biliary epithelial cells in normal and fibrotic liver. Soon after the beginning of the CCl4 treatment, osteopontin was also present in inflammatory cells of the necrotic areas. In osteopontin-deficient mice, necrotic areas after a single dose of CCl4, and fibrosis after chronic CCl4 treatment were significantly increased as compared with wild-type treated mice. CONCLUSIONS: Our results show that osteopontin expression increases during liver fibrogenesis. Furthermore, osteopontin-deficient mice were more susceptible to CCl4 treatment, displaying more necrosis during the initial steps (probably due to a deficiency in nitric oxide production) and more fibrosis thereafter. The increase in osteopontin expression observed during liver fibrogenesis may play a protective role.