The Research Of Effect Of Adenovirus On Vein Autografts In Rat

IntroductionIn vivo gene transfer into the arterial wall is a powerful experimental ap-proach by which one may both investigate the function of individual genes in the development of arterial pathology and attempt to manipulate the arterial wall for therapeutic purposes. Recently, replication-defective adenovirus has become the arterial gene transfer vector-of-choice for a majority of investigators. The advantages of adenovirus as an arterial gene transfer vector include the ease of preparation of high-titer virus ( > 10 plaque forming units [ pfu ] per ml) , and the ability of adenovirus to infect nondividing cells with high efficiency , in-cluding the endothelial cells and smooth muscle cells ( SMC) of the arterial wall. A potential disadvantage of adenovirus as an arterial gene transfer vector is that, despite the El gene deletion that renders the virus defective for replication, the remaining virus genome contains numerous open reading frames encoding vi-ral proteins. Expression of viral proteins by transduced cells elicits both humoral and cellular immune responses in animals and could conceivably provoke arterial cell activation, inflammation, and proliferation, as has been reported for wild-typeviruses.Despite the potential for adenovirus vectors to produce deleterious effects on the arterial wall, no study has yet examined the effects of these vectors on the arterial phenotype. Primary effects of adenovirus vectors (i.e. , effects unrelat-ed to any inserted transgene) would be a major concern in the design of biologi-cal experiments and could also confound the design and interpretation of thera-peutic experiments. For example, in arterial gene transfer experiments with a therapeutic endpoint, it is generally perceived that the adenovirus acts as an in- ert carrier of a therapeutic gene, and that the achievement of a beneficial end-point is neither moderated nor prevented by independent effects of the vector. Certainly, widely held current views of adenovirus vectors do not envisage the possibility that they could have primarily detrimental effects on the arterial wall.MethodsTo simulate the clinic operation of vein autografting, we inter (planted the cephalic vein, which is one branch of the jugular vein of Wister rat, to its com-mon carotid, so establishing a animal model. At 3(?)10 and 30 days postopera-tively, we got the samples. Routine HE stain and Verheoff( Van Gieson stain were made to measure the thickness with computer ( assisted image analyzer. We also used PCNA immunohistochemical stain by SABC way to find the expression quantity, which is prone to the proliferation state of vascular smooth muscle cells. We use microwave to repair the PCNA antigen before stain. GFP was de-tected by fluorescence microscope. The expression of ICAM-1 mRNA(?)VCAM-1 mRNA and protein were detected by RT-PCR and Western blot.Results1(?)At 3 day postoperatively, the expression of GFP reached the peak (26．4 (?)3．6% ) ; decrease at 10 day postoperative ( 14．5 (?) 2．1 % ) ; is very rare at 30 day postoperative (0．81(?)0．2 %).2(?)At 10 day postoperatively, the hyperplasia of vein intimal of control group was obvious. At 30 day postoperatively, the hyperplasia of vein intimal of control group was less than 10 day postoperative. Proliferation of VSMC and deposition of excelluler matrix were the main pathologic changes. There was sig-nificant difference (P<0．05) between the Ad5 group(?)the control group and the normal vein group, there was no significant difference between the Ad5 group and the control group.3(?)PCNA can present the proliferation rate of VSMC. PCNA locates in cell nuclear, and the positive cell nucleas are stained to be yellow or brown. We found that PCNA is very rare in normal vein walls, showing VSMCs are in static phase. But PCNA presentation increases obviously in control group, showing the high proliferating rate of VSMCs. The positive rate of PCNA is associated with the thickness of intimal.4(?)We found that the expression of ICAM-1 mRNA and VCAM-1 mRNA is very rare in normal vein walls. At 3 day postoperatively, The mRNA and pro-tein expression of ICAM-1 mRNA and VCAM-1 appear, and reached the peak at 10 day postoperative, then keep higher at 30 day postoperative. There was significant difference (P<0．05) between the Ad5 group(?)the control group and the normal vein group, there was no significant difference between the Ad5 group and the control group.Discussionour data suggest that ICAM-(?) and VCAM-1 expression are upregulated in SMC not by direct infection but rather by exposure to T cell-derived cyto-kines. Two other mechanisms for the observed upregulation of SMC ICAM-I and VCAM-1 are: (a) a primary effect of adenovirus on adhesion molecule ex-pression in infected SMC; ( b) upregulation of cytokine expression in adenovirus-infected SMC with consequent cytokine-induced adhesion molecule expres-sion.Our finding of increased neointima formation was unanticipated in light of current attempts to use adenovirus vectors to prevent arterial proliferative dis-ease. The stimulus for neointimal growth that results from adenovirus exposure is not clear, but could include direct mitogenic effects of the virus or indirect effects via infiltrating T cells.In conclusion, we demonstrate that the ability of adenovirus vectors to a-chieve high efficiency in vivo vascular gene transfer is tempered by the occur-rence of vascular cell activation, inflammation, and neointimal proliferation in normal rabbit arteries. Efforts at vector development should be directed at mini-mizing these effects so that the potential of adenovirus vectors to serve as investi-gational and therapeutic tools in vascular biology may be realized. Conclusion1．Adenovims-mediated gene transfer into surgically exposed normal rat vein results in: efficient, relatively endothelial-specific gene transfer with tran-sient recombinant gene expression; prolonged activation of vas-Vascular Acti-vation After Adenovirus Gene Transfer; crease intimal hyperplasia of vein auto-grafts.2．The ability of adenovirus vectors to achieve high efficiency in vivo vas-cular gene transfer is tempered by the occurrence of vascular cell activation, in-flammation, and neointimal proliferation in normal rabbit arteries. Efforts at vec-tor development should be directed at minimizing these effects so that the poten-tial of adenovirus vectors to serve as investigational and therapeutic tools in vas-cular biology may be realized.