Attenuation of matrix protein secretion by antisense oligodeoxynucleotides to the cyclin kinase inhibitor p21(Waf1/Cip1)

Progressive fibrosis in major organs, including the heart, the kidney and the vascular tree, plays an important role in mediating chronic disease and atherosclerosis. Production of extracellular matrix proteins, in many cases regulated by the growth factor TGF-beta is an essential component of this process. In a parallel manner to TGF-beta, the cyclin kinase inhibitors (CKIs; which are induced by TGF-beta) regulate transit through the cell cycle, and their effect on growth has been shown to be bimodal in the case of vascular smooth muscle (VSM) cells. Using an antisense oligodeoxynucleotide to the CKI p21(Waf1/Cip1), developed in our laboratory and shown to specifically inhibit p21(Waf1/Cip1) protein levels, we asked whether attenuation of the CKI p21(Waf1/Cip1) by transfection of this oligodeoxynucleotide results in the abolition of TGF-beta-mediated growth inhibition and/or diminished matrix protein production and secretion in the presence or absence of TGF-beta. Specific inhibition of p21(Waf1/Cip1) protein with the antisense oligodeoxynucleotide markedly reduces the production and secretion of the matrix proteins fibronectin and laminin, both in the presence and absence of TGF-beta stimulation, in VSM cells as observed by Western blotting of cell lysate and conditioned medium. In addition, TGF-beta-mediated cell growth inhibition, though attenuated by this oligo, is preserved. Due to the relative ease and safety of transfecting antisense oligodeoxynucleotides into VSM, we believe that this work unmasks a potentially powerful technique for inhibition of matrix protein synthesis in VSM and related cell lines, and may lead to new treatment strategies for atherosclerotic as well as other systemic diseases characterized by aberrant matrix protein secretion.