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Abstract-Vascular lesions resulting from injury are characterized by a thickening of the intima brought about in part through the production of increased amounts of extracellular matrix proteins by the vascular smooth muscle cells (VSMCs). In this study, we tested the hypothesis that cGMP-dependent protein kinase (PKG), an important mediator of NO and cGMP signaling in VSMCs, inhibits the production of two extracellular matrix proteins, osteopontin and thrombospondin, which are involved in the formation of the neointima. VSMCs deficient in PKG were stably transfected with cDNAs encoding either the holoenzyme PKG-I␣ or the constitutively active catalytic domain of PKG-I in order to directly examine the effects of PKG on osteopontin and thrombospondin production. Cells expressing either of the PKG constructs had dramatically reduced levels of osteopontin and thrombospondin-1 protein compared with control-transfected PKGdeficient cells. PKG transfection also altered the morphology of the VSMCs. These results indicate that PKG may be involved in suppressing extracellular matrix protein expression, which is one important characteristic of synthetic secretory VSMCs. Suppression of these matrix proteins may underlie the effects of NO-cGMP signaling to inhibit VSMC migration and phenotypic modulation. (Circ Res. 1998;82:139-146.)Key Words: nitric oxide Ⅲ phenotype Ⅲ matrix protein Ⅲ vascular disease Ⅲ atherosclerosis Ⅲ restenosis

The extracellular matrix of the normal adult brain lacks expression of most of the adhesive glycoproteins that are known to promote cell attachment, and it has been thought that the malignant invasion of astrocytoma tumor is mediated primarily by remodeling of the matrix by the tumor cells. It has been reported, however, that normal brain neuropil does contain a protein(s) that promotes cell attachment. Therefore, we explored the possibility that the cell attachment protein, osteopontin, is expressed in the normal human brain. Here, we report that osteopontin is expressed in the cortical gray and white matter of normal adult brain, with the levels of osteopontin expression being equivalent to those in malignant astrocytic tumor biopsies as assessed by Western blot analysis. Immunoblotting identified osteopontin polypeptides with relative molecular weights of 60-and 65-kDa in normal brain white matter and in astrocytic tumors, with an additional 70-kDa polypeptide being identified in normal cortical gray matter and in some astrocytic tumors. Recombinant osteopontin was found to promote attachment of U-251MG human malignant astrocytoma cells in a process that was inhibited by anti-integrin monoclonal antibodies anti-␣v␤3 (75%), anti-␣v␤5 (80%), and anti-␣5 (40%). On attachment, integrins ␣v␤5 and ␣v␤3 localized to focal adhesions, and there was an alteration in cell morphology with the formation of lamellae-like processes. The attachment was associated with activation of Rac in a slow and prolonged fashion and rapid activation of Rho. Similarly, integrins ␣v␤5 and ␣v␤3 localized to focal adhesions on attachment of the U-251MG cells to vitronectin, but on this substrate, the cells assumed a spread and flat morphology, and there was rapid activation of both Rac and Rho. Extracts of normal brain white matter were capable of promoting haptotactic migration, and this response was inhibitable by monoclonal antibodies anti-␣v␤3 and anti-␣5. Depletion of the osteopontin in these extracts abrogated the haptotactic response significantly (50%). These data indicate that the cell attachment protein, osteopontin, is expressed in the normal adult brain and that it has the potential to promote malignant astrocytoma cell invasion.

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