The activity of c-myb antisense oligonucleotide to prevent intimal hyperplasia is nonspecific.

BACKGROUND: We sought to determine the efficacy and specificity of a new c-myb antisense by inhibiting neointimal hyperplasia in a rat abdominal aorta injury model. Using c-myb antisense oligonucleotides, inhibition of vascular smooth muscle cell proliferation has been reported. METHODS: Sixty-six male Wistar rats had a de-endothelialization of the abdominal aorta. Following a double blind randomization protocol, F127 pluronic gel containing one of the five oligonucleotides or plain gel was applied around the aorta: 1) 18-mer c-myb antisense (AS18) with four contiguous guanosines (G-quartet); 2) 15-mer c-myb antisense (AS15) without G-quartet; 3) 1-bp mismatch AS15 without G-quartet (MM1); 4) an oligonucleotide with G-quartet (4G), whereas the other bases were chosen at random; 5) 1-bp mismatch 4G without G-quartet (MM2). After 21 days all rats were sacrificed and aortas harvested for histomorphometric evaluation. Four rats were given fluorescent-labeled oligonucleotides to study in vivo localization after local advential delivery. RESULTS: Morphometric analysis showed significant suppression of neointimal hyperplasia in AS18 and 4G and MM2 groups compared with GEL, AS15 and MM1 groups (p<0.05). The oligonucleotide-labeled aortas showed penetration of the oligonucleotides into the media which increased with time. CONCLUSIONS: Our findings pointed to the potential non specificity of the c-myb antisense oligonucleotide in vivo. Such results will minimize the importance of antisense strategy as a potential therapeutic for preventing neointimal hyperplasia. The two oligonucleotides with a G-quartet inhibited neointimal hyperplasia in our model. Exploring a non-antisense mechanism, G-quartet oligonucleotides as potential drugs to reduce neointimal hyperplasia is attractive.

[Effect of antisense oligonucleotides on myo-intimal hyperplasia in a model of abdominal aortic injury in the rat]. / Action des oligonucléotides antisens sur l'hyperplasie myo-intimale dans un modèle de traumatisme d'aorte abdominale de rat.

Restenosis at a rate > 30% at 6 months is the major complication of both coronary and peripheral arterial angioplasty. Restenosis is mainly due to proliferation of smooth muscle cells, extracellular matrix and collagen which form a neointima. The proto-oncogene c-myb is a gene with an immediate response which has been implicated in the proliferation and alteration of the phenotype of smooth muscle cells. The antisenses are molecules of single-helix DNA the sequence of which is inverse to that of messenger RNA of the target proto-oncogene. They therefore have the possibility of forming a double helix with the messenger RNA and of preventing its translation. The antisenses of c-myb have already been successfully tested in in vitro and in vivo models of neointimal proliferation. The aim of this study was to demonstrate the efficacy of c-myb antisenses on the proliferation of smooth muscle cells in a model of abdominal aortic injury in the rat. Thirty-five male Wistar rats with an average weight of 350 grams were operated. Smooth muscle cell proliferation was obtained by desendothelialisation of the abdominal aorta from the level of the left renal vein to the aortic bifurcation. Using a randomised, double-blind protocol, 17 rats were given 500 microliters of pluronic gel (control group), 9 a sense oligonucleotide of c-myb in 500 microliters of pluronic gel (sense group) and 9 a c-myb antisense oligonucleide in 500 microliters of pluronic gel (antisense group). Two rats were given fluorescinlabelled antisenses; one was sacrificed 4 hours and the other 24 hours later.(ABSTRACT TRUNCATED AT 250 WORDS)

[Effects of an anti-c-myb antisense oligonucleotide on myo-intimal proliferation. Specificity of action and consequences on vasoreactivity]. / Effets sur la prolifération myo-intimale d'un oligonucléotide antisens dirigé contre le c-myb. Spécificité d' action et conséquences sur la vasoréactivité.

Several reports have shown that an 18-mere antisense oligonucleotide directed against c-myb (AS 18) inhibits the proliferation of smooth muscle. The aims of this study were to confirm the specificity of a new anti-c-myb antisense and to evaluate changes in vasoreactivity following treatment with a c-myb antisense. Five groups of rats. All underwent desendothelialisation of the abdominal aorta. A solution containing pluronic gel, or one of the following oligonucleotides: AS 18, 15 mere antisense directed against c-myb, an aleatory 4G sequence containing 4 consecutive guanosines, a 15 mere antisense mismatch (n = 11), was applied around the aorta. After 21 days, the thickness and mean surface areas of the media and intima were calculated. Four groups of rats were constituted for the vasoreactivity study: control (A), desendothelialisation (B), desendothelialisation + application of AS 18 (C) and application of AS 18 alone (D). One ring per aorta was sampled at the 21st day and analysed in an organ chamber. The following results were obtained: the thickness and average surface areas of the intima were smaller (p < 0.05) in the 4G and AS 18-groups; in group B, none of the 8 segments responded to acetylcholine; in group C, 6 out of 8 segments responded. The contraction study showed no differences between groups A and D or between groups B and C. The authors conclude that the mode of action of AS 18 antisense of c-myb is non-specific but due to the presence of 4 consecutive guanosines in the oligonucleotide. Oligonucleotide with this sequence inhibits myo-intimal hyperplasia and improves endothelium-dependent relaxation in this model without affecting the contraction.

[Effects on myointimal proliferation of an antisense oligonucleotide directed against c-myb: specificity of action and consequences on vasoreactivity]. / Effets sur la prolifération myo-intimale d'un oligonucléotide antisens dirigé contre le c-myb: spécificité d'action et conséquences sur la vasoréactivité.

Several reports have shown that an antisense oligonucleotide directed against c-myb (AS 18) inhibits the proliferation of smooth muscle cell. The aims of this study were to confirm the specificity of a new c-myb antisens and to evaluate changes in vasoreactivity following treatment with a c-myb antisense. Five groups of rats were constituted. All underwent desendothelialisation of the abdominal aorta. A solution containing pluronic gel, or one of the following oligonucleotides: AS 18, 15 mere antisense directed against c-myb, an aleatory 4G sequence containing 4 consecutive guanosines, a 15 mere antisense mismatch (n = 11), was applied around the aorta. After 21 days, the thickness and mean surface areas of the media and intima were calculated. Four groups of rats were constituted for the reactivity study: control (A), desendothelialisation (B), desendothelialisation + application of AS 18 (C), and application of AS 18 alone (D). One ring per aorta was sampled at the 21st day and analysed in an organ chamber. The following results were obtained: the thickness and average surface areas of the intima were smaller (p < 0.05) in the 4G and AS 18 groups; in group B, none of the 8 segments responded to acetylcholine; in group C, 6 out of 8 segments responded. The contraction study showed no difference between groups A and D or between B and C. The mode of action of AS 18 antisense of c-myb is non specific but due to the presence of 4 consecutive guanosines in the oligonucleotide. Oligonucleotide with this sequence inhibits myo-intimal hyperplasia and improves endothelium-dependent relaxation in this model without affecting the contraction.

An experimentally validated panel of subfamily-specific oligonucleotide primers (V alpha 1-w29/V beta 1-w24) for the study of human T cell receptor variable V gene segment usage by polymerase chain reaction.

We report here the characterization of a series of T cell receptor (TcR) V alpha or V beta subfamily-specific oligonucleotide primers. Criteria that have guided the design of each oligonucleotide include appropriate thermodynamic parameters as well as differential base-pairing scores with related and unrelated target sequences. The specificity of the oligonucleotides for each V alpha or V beta subfamily was tested by polymerase chain reaction (PCR) on both a series of TcR encoding plasmid DNA and clonal T cell populations. Unexpected cross-reactivities were observed with plasmid cDNA sequences corresponding to unrelated subfamily gene segments. This led to the synthesis of additional series of oligonucleotides to obtain a relevant panel. A series of V alpha 1-w29/V beta 1-w24 TcR subfamily-specific oligonucleotides was eventually selected which generates little, if any, cross-reactivity. The use of C alpha or C beta primers for the amplification of internal positive control templates (i.e. C beta for the V alpha series and C alpha for the V beta series) has been tested in PCR performed with cDNA derived from peripheral blood lymphocytes; it was shown not to alter the amplification of the V subfamily-specific DNA fragments. This panel of oligonucleotides will be helpful in the study of TcRV gene segment usage and, thus, may lead to a better characterization of T cell responses in physiological and pathological situations.