Adenoviral-mediated expression of antisense RNA to basic fibroblast growth factor reduces tangential stress in arterialized vein grafts Abigail K. Hanna,

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Adenoviral-mediated expression of antisense RNA to basic fibroblast growth factor reduces tangential stress in arterialized vein grafts Abigail K. Hanna, MD, Walter N. Durán, PhD, Isabelle Leconte, PhD, Jonathan C. Fox, MD, PhD, David G. Neschis, MD, Robert W. Hobson, MD, Michael A. Golden, MD Journal of Vascular Surgery Volume 31, Issue 4, Pages 770-780 (April 2000) DOI: 10.1067/mva.2000.103239 Copyright © 2000 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 1 RT-PCR for bFGF RNA in normal carotid artery (lane 1), native jugular vein (lane 2), 2-day-old vein graft (lane 3), 1-week-old vein graft (lane 4), 2-week-old vein graft (lane 5), and 4-week-old vein graft (lane 6). Note that bFGF RNA is not detectable in native jugular vein and in the 2-day-old graft. It is present at 1 week and thereafter. The results are not quantitative because the goal of the study was simply to demonstrate the detection of or the lack of RNA (cDNA products). Lane overloading is responsible for the cDNA product smear observed in some lanes. Journal of Vascular Surgery 2000 31, 770-780DOI: (10.1067/mva.2000.103239) Copyright © 2000 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 2 A , Luminal view of the Ad.lacZ-treated vein graft at 4 days after grafting (original magnification, ×3). The carotid artery is above the anastomosis; the graft is below. Note the dense blue patches on the luminal surface in the vein graft after X-gal chromagen staining, which indicates the expression of the β-galactosidase transgene. B , Cross-sectional view of an Ad.lacZ-treated vein graft. Dark blue inclusions demonstrate β-galactosidase staining, which is transmural in some regions. Sixty-five percent of vein graft wall cells express the β-galactosidase transgene (original magnification, ×250). Nuclei were counterstained with hematoxylin. Journal of Vascular Surgery 2000 31, 770-780DOI: (10.1067/mva.2000.103239) Copyright © 2000 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 3 The expression of ASbFGF RNA in 3-day-old Ad.ASbFGF-treated vein grafts. RT was performed on total RNA extracted from Ad.ASbFGF-, Ad.LacZ-, and PBS-treated vein grafts. Only Ad.ASbFGF-treated grafts are depicted here with positive and negative controls. Lane 1 contains a base-pair ladder. Lane 3 contains the positive control and demonstrates the cDNA product obtained by the use of the ASbFGF plasmid as a PCR template. This plasmid was used to make the Ad.ASbFGF vector. Lane 4 contains the amplification cDNA product of 3-day-old Ad.ASbFGF-infected vein grafts. The primary product (arrow ) is the same (428 bp) as the plasmid positive control. Lane 5 demonstrates that amplification of the ASbFGF transgene in ungrafted veins yields no product. Lane 6 shows no ASbFGF transgene in RNAse-free water. PCR cDNA products are visualized on agarose gels stained with ethidium bromide. Lane 2 is empty. Journal of Vascular Surgery 2000 31, 770-780DOI: (10.1067/mva.2000.103239) Copyright © 2000 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 4 Immunohistochemistry for basic FGF protein in 3-week-old and 2-month-old vein grafts. At 3 weeks the thickened wall of vein grafts treated with Ad.ASbFGF shows minimal staining for bFGF protein (A ), although the Ad.lacZ and PBS grafts stain heavily for bFGF protein (B ; original magnification, ×125). At 2 months, an appreciable reduction in bFGF staining persists in the Ad.ASbFGF-treated grafts (C ) compared with control grafts (D ), although the difference is not as notable (original magnification, ×250). All nuclei were counterstained with hematoxylin. Journal of Vascular Surgery 2000 31, 770-780DOI: (10.1067/mva.2000.103239) Copyright © 2000 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 5 Histologic comparison of PBS- (A ), Ad.lacZ- (B ), and Ad.ASbFGF-treated (C ) AVGs at 2 months after the grafting. In these cross section samples, the black-staining SMC nuclei and elastin fibers in the control grafts (A and B ) have an arbitrary orientation to each other and the endothelial cells lining the lumen. The SMCs and elastin fibers of the Ad.ASbFGF-treated grafts (C ) are parallel to each other and circumferentially arrayed relative to the lumen in cross section. The criterion for the determination of the medial/adventitial border is the presence of capillaries and other vascular structures that are located only in the adventitia (elastin-collagen stain; original magnification, ×250). Journal of Vascular Surgery 2000 31, 770-780DOI: (10.1067/mva.2000.103239) Copyright © 2000 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 6 Cross section samples of Ad.ASbFGF-treated grafts at 2 months show the circumferential alignment of elastin fibers (A ) and SMC nuclei (B ). Heavy deposition of black elastin fibers is noted near the lumen (A ; elastin collagen stain). Nuclei stained with hematoxylin demonstrate the array of the SMCs (B ; original magnification, ×250). Journal of Vascular Surgery 2000 31, 770-780DOI: (10.1067/mva.2000.103239) Copyright © 2000 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions