Experimental research about the treatment for postoperative pathological changes : control of endothelial activation using antisense adenovirus vector

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Activated vascular endothelial cells (ECs) produce various factors to contribute to the development of postoperative pathological changes. In this study, we investigated whether antisense adenovirus vector sgsist ICAM-1 mRNA could suppress ICAM-1 expression on activated ECs.DNAs complementary to ICAM-1, VCAM-1, E-selectin, and PDGF-A chain mRNAs were produced by RT/PCR of total cellular RNA from TNF-_<alpha> activated human umbilical vein ECs using respective sense and antisense primers. cDNAs were subcloned in plasmids and checked their sequences. Checked cDNAs were reversely ligated into the specific cosmids for generation of adenovirus vector. Antisense adenovirus vectors against ICAM-1, VCAM-1, and E-selection mRNAs was generated in 293 cells, resulting from the homologous recombination between cDNAs-reversely-ligated cosmids and adenovirus DNA.The adenovirus vectors were transfected into ECs and their transcripts were checked by RT/PCR.ICAM-1 pression was analyzed by cell-ELISA on lipopolysaccharide-activated ECs which was tranfected with antisense adenovirus vector against ICAM-1 mRNA.However, ICAM-1 expression inbuced by lipopolysaccharide could not be reduced by transfection of the antisense adenovirus vector. We considered that the reasons why the vector failed to reduced the ICAM-1 expression might be due the fact that the expression cassette of the vector included a short cDNA sequence as 143bp and a long mismatchcd sequence as 200bp. Moreover, we have experienced another failure of antisense adenovirus vector. Antisense adenovirus vector against insulin failed to reduce insulin secretion from the ECs transfected with insulin adenovirus vector. Since the vector contained 334 bp-reversed cDNA and 200bp-mismatched sequence, it is suggested that mismatched sequence may really inhibit the binding of antisense mRNA to sense mRNA or that antisense treatment may not be so effective as it has been reported.