A novel approach based on DNA-cleaving deoxyribozymes (DNAzymes) was developed to control expression of ␤ 1 and ␤ 3 integrins in endothelial cells. To engineer a specific cleavage site in mRNA, the flanking domains of DNAzymes were derived from oligodeoxynucleotides complementary to sequences corresponding to 1053-1070 and 1243-1267 in ␤ 1 and ␤ 3 mRNA, respectively. Phosphorothioate analogues of these antisense oligodeoxynucleotides, designated ␤1-1053 and ␤3-1243, significantly inhibited expression of ␤ 1 and ␤ 3 integrin subunits in endothelial and K562 cells at the level of mRNA and protein synthesis. They also specifically decreased the cell surface expression of corresponding subunits in endothelial cells and K562 cells, as measured by flow cytometry. In functional tests, ␤1-1053 and ␤3-1243 markedly reduced adhesion of cells to fibronectin and vitronectin, respectively. We designed DNAzymes to ␤ 1 and ␤ 3 mRNAs containing a 15-deoxynucleotide catalytic domain that was flanked by two substrate recognition segments of 8 and 10 deoxynucleotides for ␤ 1 and ␤ 3 DNAzymes, respectively. Both DNAzymes in the presence of Mg 2؉ specifically cleaved their substrates, synthetic ␤ 1 and ␤ 3 mRNA fragments. Although DNAzymes were partially modified with phosphorothioate and with 2-O-methyl groups at both the 5 and 3 ends indicated similar kinetic parameters, they were significantly more potent than the unmodified DNAzymes because of their much higher resistance to nuclease degradation. Similar to the antisense oligonucleotides, DNAzymes abolished microvascular endothelial cell capillary tube formation in fibrin and Matrigel. In conclusion, DNAzymes to ␤ 1 and ␤ 3 mRNAs with 2-O-methyl modifications are potentially useful as gene-inactivating agents and may ultimately provide a therapeutic means to inhibit angiogenesis in vivo.Angiogenesis is a multistep sequence of cellular reactions beginning with degradation of extracellular matrix and then proliferation and migration of endothelial cells followed by lumen formation and maturation (1). The formation of new blood vessels is critical to the development of normal tissues as well as the growth of solid tumors and to a large extent depends on specific molecular interactions between vascular cells and extracellular matrix (2, 3). Aberrant angiogenesis is also a key process for progress of many disorders including atherosclerosis (4), diabetic retinopathy (5) and restenosis (6). Currently, two groups of integrin receptors are known to regulate adhesive interactions during angiogenesis. Integrins in the ␤ 1 subfamily, particularly ␣ 5 ␤ 1 and ␣ 2 ␤ 1 , which are substantially expressed in human angiogenic blood vessels, promote endothelial cell morphogenesis, migration, and tube formation (7,8). The second group is composed of vitronectin receptors ␣ V ␤ 3 and ␣ V ␤ 5 , which are poorly expressed in quiescent vasculature (9, 10) but can be substantially expressed by neoplastic vasculature and cells (11,12). Expression of ␣ V ␤ 3 and ␣ V ␤ 5 can be differentially up-regulate...

Mammalian cells increase net expression of 1,25(OH) 2D3 receptors after exposure to physiological concentrations of 1,25(OH) 2D3 in vitro. We examined specific binding of 1,25(OH) 2D3 by human monocytes before and after daily administration of 1.5-2 micrograms 1,25(OH) 2D3 p.o. for 3 days in 5 healthy normal D-replete probands. Median specific binding (Nmax) at baseline was 793 molecules/cell and 2052 or 2828 at 24h and 72h of 1,25(OH) 2D3 treatment respectively. The results suggest (a) upregulation of 1,25(OH) 2D3 receptors occurs in man and (b) monocyte preparations can be used to assess receptor regulation in vivo.