2'-Fluoropyrimidine RNA-based aptamers to the 165-amino acid form of vascular endothelial growth factor (VEGF165). Inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain.

Vascular endothelial growth factor (VEGF) has been implicated in the pathological induction of new blood vessel growth in a variety of proliferative disorders. Using the SELEX process (systematic evolution of ligands by exponential enrichment), we have isolated 2'-F-pyrimidine RNA oligonucleotide ligands (aptamers) to human VEGF165. Representative aptamers from three distinct sequence families were truncated to the minimal sequence capable of high affinity binding to VEGF (23-29 nucleotides) and were further modified by replacement of 2'-O-methyl for 2'-OH at all ribopurine positions where the substitution was tolerated. Equilibrium dissociation constants for the interaction of VEGF with the truncated, 2'-O-methyl-modified aptamers range between 49 and 130 pM. These aptamers bind equally well to murine VEGF164, do not bind to VEGF121 or the smaller isoform of placenta growth factor (PlGF129), and show reduced, but significant affinity for the VEGF165/PlGF129 heterodimer. Cysteine 137 in the exon 7-encoded domain of VEGF165 forms a photo-inducible cross-link to a single uridine residue in each of the three aptamers. The aptamers potently inhibit the binding of VEGF to the human VEGF receptors, KDR and Flt-1, expressed by transfected porcine aortic endothelial cells. Furthermore, one of the aptamers is able to significantly reduce intradermal VEGF-induced vascular permeability in vivo.

The bacteriophage T4 regB ribonuclease. Stimulation of the purified enzyme by ribosomal protein S1.

Infection of Escherichia coli by bacteriophage T4 induces a mRNA ribonuclease activity that shows specificity for cleavage within the sequence GGAG. Substrates of the activity in vivo include a number of phage mRNAs that are cleaved at GGAGs within the Shine/Dalgarno domains of their translation initiation regions. Induction of the ribonuclease depends on the product of the T4 gene regB. We describe here the overproduction and extensive purification of the RegB protein. RegB precisely co-purifies with an activity that cleaves within the sequence GGAG in oligonucleotide and polynucleotide RNAs and is therefore likely to constitute the sequence-specific catalytic component of the observed activity. We further report that the low cleavage rate observed with our preparations of purified RegB is substantially increased (1-2 orders of magnitude) by the addition of E. coli ribosomal protein S1. We discuss the implications of this observation for the mechanism of action of the RegB ribonuclease in vitro and in vivo.

Phenotypic stability and variation in cells of the porcine aorta: collagen and elastin production.

The extracellular matrix of the developing vasculature varies in composition as a function of time and position. Cellular models of vascular biology and pathology depend on the assumption that stable phenotypic characteristics of vascular cells can be propagated through several generations of in vitro cultivation. We show that the positional and developmental heterogeneity of matrix phenotypes in the porcine aorta are expressed by explanted vascular smooth muscle cell (SMC) and adventitial cell populations for a limited number of passages. Elastin was expressed most highly by thoracic SMC while interstitial collagen production was usually maximal in abdominal segments. Parallel gradients of collagen types I, III and V, detected by specific ELISA assays, were expressed in early-passage SMC. Adventitial cell populations from the abdominal aorta of the neonatal pig accumulated significant levels of collagen, while these fibroblasts produced less than 10% of the elastin made by SMC. All cell populations expressed alpha-smooth muscle actin in vitro. Gradients of collagen and elastin expression were evident for no more than three passages, and direct outgrowth of cells without limited digestion of the matrix further reduced phenotypic stability. Variation and decline of the elastin phenotype could be due to hypermethylation of regulatory sequences in the elastin gene or trans-acting factors, but elastin production was dose-dependently stimulated to a similar extent (100%; 10 microM 5-azacytidine) in all segmental SMC populations at early (p1) and late (p3) passage. These data indicated that faithful reflection of in vivo SMC behavior was limited to a few population doublings, at least under standard culture conditions. Modification of the cellular environment by reducing serum factors, changing matrix, or adding mechanical stimulation may increase phenotypic stability.

Identification of a T4 gene required for bacteriophage mRNA processing.

A ribonucleolytic activity that cleaves within the Shine/Dalgarno sequences of the bacteriophage T4 motA and ORF2 mRNAs was recently described. We have identified additional sites of processing within several other ribosome binding sites, including two sites in the polycistronic frd transcript. Deletion mutants (farP) that overproduce the product of frd are defective in this mRNA processing. The mutants were used to identify processing events dependent on the T4 activity including attack at nuclease-sensitive sites within the coding sequences of some genes and within the intercistronic region 5' of gene 43. All known processing sites lie within similar sequences. Another mutant in mRNA processing carries a point mutation in one of the open reading frames (orf61.9) removed by the farP deletions. Introduction of a cloned copy of this open reading frame into a unique site in the chromosome of farP phage is sufficient to restore mRNA processing capability. The open reading frame probably encodes the T4 regB protein.