Gregory J. Fulton

Efficient adenoviral gene transfer to early venous bypass grafts: comparison with native vessels.

OBJECTIVES Gene therapy may provide new approaches to reduce vein graft failure following coronary or peripheral bypass surgery. The aim of this study was to investigate the relative efficacy of intraoperative adenoviral gene transfer to vein grafts, comparing transgene expression in vein grafts with that in matched native vessels in the same animal. In addition, we assessed the impact of bypass grafting on the cellular targets of gene transfer. METHODS New Zealand White rabbits underwent interposition bypass grafting of the carotid artery, using the ipsilateral external jugular vein, which was infected with an adenovirus expressing beta-galactosidase immediately prior to bypass grafting (n = 16). The contralateral native jugular vein (n = 16) and carotid artery (n = 8) were infected concurrently with the same adenoviral preparation. After 3, 7 or 14 days, beta-galactosidase protein expression was quantified by ELISA, and specific cell types expressing beta-galactosidase were identified by X-Gal staining and by immunohistochemistry. RESULTS After 3 days, endothelial cells were efficiently transduced in all vessels; medial smooth muscle cells were transduced infrequently. In contrast to jugular veins after gene transfer, endothelium in vein grafts showed expression of VCAM-1 and ICAM-1, and intense inflammation with CD18+ leukocytes. Transgene expression in vein grafts at day 3 was maintained at levels approximately 50% of that in ungrafted jugular veins, but continued to decrease through day 7. CONCLUSIONS Although vascular injury in early venous bypass grafts reduces gene transfer efficacy, significant transgene expression is maintained for at least 7 days. These findings have important implications for intraoperative gene transfer strategies in vein grafts.

G protein signaling and vein graft intimal hyperplasia: Reduction of intimal hyperplasia in vein grafts by a G(βγ) inhibitor suggests a major role of G protein signaling in lesion development

Vein grafting results in the development of intimal hyperplasia with accompanying changes in guanine nucleotide-binding (G) protein expression and function. Several serum mitogens that act through G protein-coupled receptors, such as lysophosphatidic acid, stimulate proliferative pathways that are dependent on the G protein betagamma subunit (Gbetagamma)-mediated activation of p21ras. This study examines the role of Gbetagamma signaling in intimal hyperplasia by targeting a gene encoding a specific Gbetagamma inhibitor in an experimental rabbit vein graft model. This inhibitor, the carboxyl terminus of the beta-adrenergic receptor kinase (betaARK(CT)), contains a Gbetagamma-binding domain. Vein graft intimal hyperplasia was significantly reduced by 37% (P<0.01), and physiological studies demonstrated that the normal alterations in G protein coupling phenotypically seen in this model were blocked by betaARK(CT) treatment. Thus, it appears that Gbetagamma-mediated pathways play a major role in intimal hyperplasia and that targeting inhibitors of Gbetagamma signaling offers novel intraoperative therapeutic modalities to inhibit the development of vein graft intimal hyperplasia and subsequent vein graft failure.

Modulation of Tissue Factor Protein Expression in Experimental Venous Bypass Grafts

Vein graft failure is a major limitation of coronary artery and peripheral vascular surgery. Tissue factor (TF), a transmembrane glycoprotein, generates thrombin by initiating the extrinsic coagulation cascade and plays a major role in the response to arterial injury. This study was designed to examine changes in TF protein expression in response to venous bypass grafting. New Zealand White rabbits underwent interposition bypass grafting of the common carotid artery via the ipsilateral external jugular vein. The contralateral control jugular veins (n = 6), early vein grafts (1 or 3 days after grafting, n = 18), and late vein grafts (14 or 28 days after grafting, n = 8) were examined by immunohistochemistry. The presence or absence of TF immunostaining in the intima was assessed in each vessel quadrant. In control veins, intimal TF staining was present in 5 of 24 vessel quadrants. In early vein grafts, TF staining was markedly increased in the intima (72 of 72 quadrants, P < .001 vs control veins), and TF immunostaining colocalized with CD18-positive leukocytes but not with endothelial cells, vascular smooth muscle cells, or RAM11-positive macrophages. In late vein grafts with intimal hyperplasia, TF expression was low or absent in the intima (6 of 32 quadrants, P < .001 vs early vein grafts; P = NS vs control veins), although medial smooth muscle cells expressed TF. Marked changes in TF expression occur in vein grafts. In early vein grafts, TF protein was greatly increased in the intima for at least 3 days and was associated with CD18-positive leukocytes. In late vein grafts with intimal hyperplasia, however, TF protein was not seen in the intima. These findings may have important implications for the development of therapeutic strategies to limit vein graft failure.

Local effects of nitric oxide supplementation and suppression in the development of intimal hyperplasia in experimental vein grafts

OBJECTIVES The universal response of vein grafts after insertion into the arterial circulation is the development of intimal hyperplasia; smooth muscle cell proliferation and connective tissue deposition, which may be modulated in part by dysfunctional endothelial nitric oxide (NO) metabolism. This study examines the effects of single dose, local application by pluronic gel of a NO donor, S-nitroso-N-acetylpenicillamine (SNAP) and an NO synthase inhibitor nitro-L-arginine methyl ester (L-NAME) on the formation of intimal hyperplasia. MATERIALS Forty New Zealand white rabbits underwent jugular vein interposition grafting of the common carotid artery. DESIGN Ten animals were controls, 10 animals had the outer surface of the vein graft coated with 30% pluronic gel (2.5 ml), and 10 each were immersed for 15 min prior to insertion in Ringer lactate containing 10(-3) M of SNAP or L-NAME and then had their vein grafts coated with 2.5 ml of gel containing either SNAP (10(-3) M) or L-NAME (10(-3) M), which allows for sustained delivery for up to 6 h. On the 28th post operative day, the animals were sacrificed and vein grafts were harvested for morphology by electron microscopy (SEM and TEM) and dimensional analysis by videomorphometry. RESULTS All vein grafts developed intimal hyperplasia. On SEM the vein grafts had a confluent layer of endothelial cells with multiple layers of smooth muscle cells representing intimal hyperplasia in TEM. There were no demonstrable morphological differences between the four groups. Local treatment with SNAP produced a significant 36% decrease in mean intimal thickness (72 +/- 4 microns vs. 45 +/- 4 microns; mean +/- S.E.M.; p < 0.01) without a change in medial thickness compared to gel-only treated groups (58 +/- 6 microns vs. 61 +/- 7 microns; p = ns). Inhibition of NO synthase by L-NAME had no effect on the development of intimal hyperplasia (72 +/- 4 microns vs. 79 +/- 10 microns; p = ns); medial thickness was also unchanged. CONCLUSION These data confirm the protective effect of NO in vascular injury and suggest that NO synthase activity is either absent or reduced to such a level that further inhibition in this short time course is not relevant to the pathophysiology of vein graft intimal hyperplasia.

G Protein Signaling and Vein Graft Intimal Hyperplasia: Reduction of Intimal Hyperplasia in Vein Grafts by a Gbg Inhibitor

Vein grafting results in the development of intimal hyperplasia with accompanying changes in guanine nucleotide-binding (G) protein expression and function. Several serum mitogens that act through G protein-coupled receptors, such as lysophosphatidic acid, stimulate proliferative pathways that are dependent on the G protein betagamma subunit (Gbetagamma)-mediated activation of p21ras. This study examines the role of Gbetagamma signaling in intimal hyperplasia by targeting a gene encoding a specific Gbetagamma inhibitor in an experimental rabbit vein graft model. This inhibitor, the carboxyl terminus of the beta-adrenergic receptor kinase (betaARK(CT)), contains a Gbetagamma-binding domain. Vein graft intimal hyperplasia was significantly reduced by 37% (P<0.01), and physiological studies demonstrated that the normal alterations in G protein coupling phenotypically seen in this model were blocked by betaARK(CT) treatment. Thus, it appears that Gbetagamma-mediated pathways play a major role in intimal hyperplasia and that targeting inhibitors of Gbetagamma signaling offers novel intraoperative therapeutic modalities to inhibit the development of vein graft intimal hyperplasia and subsequent vein graft failure.

Localized versus systemic angiotensin II receptor inhibition of intimal hyperplasia in experimental vein grafts by the specific angiotensin II receptor inhibitor L158,809

BACKGROUND This study examines the effect of the angiotensin II receptor (type 1) antagonist (L158,809) on the formation of vein graft intimal hyperplasia in vivo, by both localized and systemic administration. METHODS Forty New Zealand White rabbits underwent carotid interposition bypass grafting with the external jugular vein and were killed on postoperative day 28. To determine the effect of L158,809 on the development of intimal hyperplasia, 10 animals received long-term oral therapy with L158,809 (10 mg/kg/day, begun 5 days before operation and continued until harvest), 10 animals underwent coating of the grafts with a pluronic gel containing L158,809 (10(-5) mol/L), and 20 animals were controls (10 with and 10 without pluronic gel). These grafts were harvested for either histologic analysis (n = 6 per group) or in vitro isometric tension studies to angiotensin II (n = 4 per group). RESULTS Long-term oral treatment with L158,809 produced a 43% decrease in intimal thickness from 76 +/- 6 microns (mean +/- SEM) in the control animals to 43 +/- 7 microns in the treated vein grafts (p = 0.002). There was also a significant decrease (44%) in the medial thicknesses between the control (75 +/- 4 microns) and L158,809-treated (42 +/- 6 microns) vein grafts (p = 0.007). The contractile responses to angiotensin II were abolished in the vein grafts by long-term L158,809 therapy. Local application by gel of L158,809 produced a significant decrease (33%) in the intimal thickness (48 +/- 3 microns) but no change in medical thicknesses (76 +/- 6 microns) compared with control grafts. The contractile responses to angiotensin II were unchanged in the vein grafts by local L158,809 therapy. CONCLUSIONS This study shows that a local single application of L158,809 will reduce the intimal response but not the medial response in vein grafts, whereas long-term treatment will reduce intimal hyperplasia and the medial response in experimental vein grafts. Therefore angiotensin II acting through AT1 receptors mediates a significant part of the intimal hyperplastic response in vein grafts that appears to involve two phases: an acute intimal response requiring short-term therapy and a long-term medial response that requires prolonged therapy.

Time Course of the Regression of Intimal Hyperplasia in Experimental Vein Grafts

A previous study in which vein grafts were removed from the arterial circulation and reimplanted into the venous circulation of the same animal demonstrated regression of vein graft intimal hyperplasia and medial thickening within 14 days. The present study was designed to characterize the kinetics of the morphological and ultrastructural changes over this 14-day period. Twenty-one male New Zealand White rabbits received a reversed vein interposition bypass graft of the right common carotid artery. Fourteen days after the procedure, 21 vein grafts were isolated, removed, and reimplanted into the contralateral external jugular venous system as veno-venous interposition bypass grafts (reversal grafts). The grafts were harvested at 60 minutes, 1 day, 3 days, 5 days, 7 days, and 14 days after reversal. Before insertion into the venous circulation, the vein graft had a confluent endothelial cell surface with multiple layers of smooth muscle cells representing intimal hyperplasia. After 1 hour, the reversal graft retained an intact endothelial cell layer with no evidence of tissue edema or cellular disruption. By 24 hours, there were a few blood cells on the endothelial cell surface. There was no inflammatory infiltrate seen in the subendothelium, and the smooth muscle cells were unaltered. At 3 days, the endothelial cell lining remained intact with no polymorphonucleocytes in the subendothelium or within the graft wall. Underlying smooth muscle cells at this time were noted to contain cytoplasmic vacuoles. At 5 days, there were no inflammatory cells seen on the surface or within the vein graft wall, but many of the underlying smooth muscle cells within the intimal hyperplasia were noted to be fragmented and to have clumping of chromatin. After 7 days, the endothelial cells remained intact and there was widespread evidence of apoptosis beneath the subendothelium with highly fragmented smooth muscle cells, some of which were histologically in the process of breaking up. At 14 days, the grafts retained uniform endothelial cell surfaces. Most of the smooth muscle cells that composed the intimal hyperplasia seen before implantation as a reversal graft were gone. Areas of newly laid down collagen could be observed. There were no acute inflammatory cells but for some mast cells seen in the graft wall. This study demonstrates that in this model, regression of intimal hyperplasia was associated with apoptosis of the smooth muscle cells and the deposition of collagen. There was no evidence that this process is mediated by an acute inflammatory response. Regression therefore appears to be due to induction of smooth muscle cell apoptosis by either a reduction in pressure or flow or a combination of both factors. The findings will enable a systematic cellular and molecular analysis of the biology of regression, which may afford clues to better understand the biology of the developing intimal hyperplasia.

Characterisation of angiotensin II receptor mediated responses and inhibition of intimal hyperplasia in experimental vein grafts by the specific angiotensin II receptor inhibitor, L158,809

OBJECTIVES This study characterises pharmacologically the angiotensin II receptor in experimental vein grafts and examines the effect of the angiotensin II receptor (type 1) antagonist (L158,809) on the formation of vein graft intimal hyperplasia in vivo, as well as the in vitro physiological response to angiotensin II of vein grafts after chronic oral L158,809 treatment. MATERIALS Thirty New Zealand White rabbits had a right carotid interposition bypass graft using the external jugular vein and were killed on the 28th postoperative day. DESIGN To characterise the angiotensin II receptors, concentration response curves to angiotensin II were obtained in vitro in the presence or absence of L158,809. To determine the effect of L158,809 on the development of intimal hyperplasia, 10 animals received chronic oral therapy with L158,809 (10 mg/kg/day; begun 5 days before surgery and continued until harvest) and 10 animals received vehicle only as controls. These grafts were harvested either for histology (n = 6 per group) or for in vitro isometric tension studies to angiotensin II. RESULTS The monophasic contractile response to angiotensin II in the untreated vein grafts could be inhibited in a concentration dependent manner by L158,809 with first order kinetics. Chronic oral treatment with L158,809 produced a 48% decrease in intimal thickness from 82 +/- 1 micron (mean +/- S.E.M.) in the controls to 43 +/- 7 microns in the treated vein grafts (p = 0.002). There was also a significant decrease (45%) in the medial thickness between the control (76 +/- 6 microns) and L158,809 treated (42 +/- 6 microns) vein grafts (p = 0.007). The responses to angiotensin II were abolished in the vein grafts by chronic L158,809 therapy. CONCLUSIONS This study suggests that vein graft angiotensin II responses are mediated through a type 1 receptor and that chronic inhibition with L158,809, significantly reduces intimal hyperplasia and medial hypertrophy in experimental vein grafts and concomitantly abolishes the in vitro responses to angiotensin II. Therefore, angiotensin II acting through AT1 receptors mediates a significant part of the intimal hyperplastic response in vein grafts.

Preservation of the Endothelium in Venous Bypass Grafts: Relevance for Graft Patency

Abstract Veins continue to be a main choice for arterial bypass grafts, but postoperative graft occlusion rates continue to be a problem, with up to 40% of aortocoronary bypass operations in the U.S.A. now being “redo” or “revision” procedures. An understanding of the factors contributing to the problem of endothelial pathology, and possible strategies for preventing it, include surgical, pharmacological and molecular factors. The endothelial cell is central to vascular wall biology, coordinating vascular wall modelling through the synthesis of key elements which influence smooth muscle cell behaviour. Removal of the endothelium leads to an imbalance in the components of the vasculature, with proliferation of vascular smooth muscle cells resulting in intimal hyperplasia. Endeavours to preserve endothelial cell presence and function may minimise implantation injury (and subsequent early vein graft failure), and may also minimise intimal hyperplasia (the cause of short to intermediate term vein graft failure). The pathophysiology of vein graft failure is still poorly understood. With the advent of new technologies, it is expected that a rational approach to the prevention of intimal hyperplasia (through methods such as gene transfer) may soon be feasible. Even before the full potential of such new technologies is realised, meticulous and continuous efforts to preserve endothelium at the stages of vein harvest, storage and graft implantation should be implemented to maximise the patency of venous grafts.

Alterations in collagen subtype III and IV protein in experimental venous bypass grafting.

ObjectiveTo examine changes in collagen III and IV protein during the development of intimal hyperplasia in experimental vein grafts. MethodsSixteen New Zealand White rabbits underwent reversed, jugular vein, interposition grafting of the carotid artery. Vessels were harvested 3, 7 or 28 days after operation and subjected to immunohistochemical examination and gelatinase assays. ResultsIn control vein, collagen IV was expressed around adventitial blood vessels and throughout the endothelium. Compared with its presence in control veins, collagen IV protein was decreased in the endothelium in all 3-day vein grafts and undetectable in the endothelium and intima in 7-day vein grafts, but was present in the endothelium and intimai hyperplasia in 28-day vein grafts. In contrast, collagen III was absent from the endothelium of control vein and 3-day vein grafts, was present at low levels in the intima of 7-day vein grafts, but was absent from the endothelium and intimai hyperplasia in 28-day vein grafts. In 3-day vein grafts, areas of collagen IV loss colocalized to areas of leukocyte infiltration. Protein extracts from 3-day vein grafts contained a 72 kDa gelatinase. ConclusionsThe presence and alterations of collagen protein in veins and vein grafts are subtype specific. Collagen III does not appear to be a normal component of intimai hyperplasia in vein grafts. The decrease in collagen IV protein in the endothelium of veins may be a component of the endothelial changes that follow bypass grafting, mediated by leukocytes, the induction of gelatinase activity, or both.