Experimental Vein Grafts Research Articles

Experimental vein grafts in the rat: re-endothelialization and permeability to albumin.

Abstract Intimal thickening in vein grafts is one of the major factors leading to graft failure. Studies have suggested that delayed re-endothelialization and an enhanced permeability to proliferative factors (macromolecules) in the blood are associated with the development of intimal thickening in these vessels. The aim of this study was to determine the pattern of re-endothelialization of vein grafts and the permeability of the new endothelium to a large molecule. Iliolumbar vein to iliac artery grafts 5 mm long were inserted microsurgically into 32 male albino Wistar rats. Graft re-endothelialization and permeability, at times ranging from 2 to 140 days after grafting, were studied by scanning electron microscopy and vital staining with Evans Blue dye, which binds to plasma albumin and was used as a model of a large molecule. Re-endothelialization of the graft had commenced by 2 days after grafting and in most animals was completed by 15 days. Some grafts exhibited local areas devoid of endothelium up to 4 weeks after grafting. The mechanism of re-endothelialization involved the migration of a sheet of endothelium across the anastomosis from the adjacent artery. This sheet subsequently linked up with islands of cells which developed in the centre of the graft by 5 days. Evans Blue dye was detected in all areas where endothelium had been removed by the grafting operation. Permeability of the graft to the dye was seen during the 2–4 week period after grafting, a time at which previous studies have shown that intimal thickening develops in this vein grafting model. Thus, delayed re-endothelialization and prolonged permeability to a large molecule (albumin) does occur in some experimental vein grafts. This may be the basis for intimal thickening which is known to occur in vein grafts.

Influence of prazosin on experimental vein graft intimal thickening.

The influence of prazosin on experimental vein graft intimal thickening was studied in a rat model. The iliolumbar vein was grafted into the common iliac artery in a prazosin-treated group (n = 17) and a control group (n = 23). Three weeks after grafting the animals were killed and the grafts harvested. Longitudinal sections were prepared and the intimal thickness measured in the proximal, mid and distal graft. Intimal thickness varied considerably in both groups. Median (range) intimal thicknesses for the control group were: proximal 50 (10-120) microns; mid 30 (10-70) microns; and distal graft 30 (10-100) microns. Results for the prazosin-treated group were: proximal 70 (10-160) microns; mid 25 (5-60) microns; and distal graft 25 (5-135) microns. There was no statistically significant difference in median intimal thicknesses between the control and treated groups.

Perivascular treatment with azathioprine reduces neointimal hyperplasia in experimental vein grafts

Background: Azathioprine is an immunosuppressive and anti-inflammatory drug and it has been shown to induce apoptosis in human T lymphocytes. We investigated whether local treatment with azathioprine can inhibit neointimal hyperplasia in experimental vein grafts.

Radiation Therapy Induced Modulation of Wound Healing at Experimental Vein Graft Anastomoses

Objectives The aim of this study was to investigate if radiation therapy (RT) favorably modulates wound healing at vein graft anastomoses. Materials and methods Jugular vein grafts were sewn into carotid arteries in 32 rats which were randomly divided into two groups: RT (gamma source, 14 Gray, n=16) and control (C, sham irradiation, n=16). Grafts and adjacent arteries were analyzed at 2 ( n=8) and 8 weeks ( n=8) by histology, immunohistochemistry, and morphometry. Results Although, RT did not reduce the overall occurrence of intimal hyperplasia, the distribution differed. RT led to a reduction of intimal hyperplasia in arterial segments (median: C: 41.873 μm 2; RT: 6.452 μm 2, p<0.0007). In contrast, RT augmented intimal hyperplasia in vein grafts (median: C: 30.287 μm 2; RT: 90.455 μm 2, p<0.014). Vein graft diameters after RT were enlarged (median: C: 2.098 μm; RT: 3.381, p<0.031). Over 80% of the cells were of mesenchymal origin in both groups. Conclusions RT reduced intimal hyperplasia in arterial segments. However, RT led to graft dilatation and increased intimal hyperplasia in vein grafts. RT did not favorably modulate the vascular wound healing response in this model.

Rapamycin treatment is associated with an increased apoptosis rate in experimental vein grafts☆

Rapamycin is an immunosuppressive agent with marked antiproliferative properties and is effective in reducing in stent restenosis and vein graft neointimal hyperplasia. Apoptosis is one mechanism counterbalancing cellular proliferation. We therefore investigated the role of apoptosis in rapamycin treated vein grafts in a mouse model. C57BL6J mice underwent interposition of the inferior vena cava from isogenic donor mice into the common carotid artery using a cuff technique. In the treatment group 200 microg of rapamycin were applied locally in pluronic gel. The control group did not receive local treatment. Vein grafts were harvested at 4 weeks postoperatively and underwent morphometric analysis as well as immunohistochemical analysis for apoptosis (TUNEL). In grafted veins without treatment (controls) neointimal thickness was 50 (12-58) microm at 4 weeks postoperatively. In 200 microg rapamycin treated grafts the neointimal thickness was 17 (5-55) microm. Rapamycin treated vein grafts showed a significantly increased rate of apoptosis in the adventitia as compared with controls (P=0.032). In the neointima the apoptosis rate was lower in both groups with no significant difference between rapamycin treated grafts and controls. We conclude that treatment of experimental vein grafts with rapamycin is associated with an increased apoptosis rate in the vascular wall and a trend towards reduction of neointimal hyperplasia. These results suggest that apoptosis may be a beneficial antiproliferative component for the treatment of vein graft disease.

Modulation of vascular remodeling induced by a brief intraluminal exposure to the recombinant R7020 strain of Herpes simplex-1

Vascular remodeling in response to injury or low shear stress (or both) is characterized by neointimal hyperplasia and luminal contraction. When profound, the response leads to restenosis after percutaneous endovascular intervention as well as to de novo stenosis in vein grafts. It has recently been reported that exposure of vein patches to neurovirulence-attenuated Herpes simplex virus-1 (HSV-1) decreases neointimal hyperplasia and increases luminal area. This experiment tested the hypothesis that R7020, a more highly attenuated mutant of HSV-1, would modulate the vascular remodeling response of experimental vein grafts chronically exposed to low shear stress. The external jugular veins of 31 New Zealand white rabbits were clamped and intraluminally exposed to vehicle (phospate-buffered saline solution, n = 11), R7020 2.5 x 10(8) plaque forming units [PFU]/mL (n = 8), or R7020 2.5 x 10(9) PFU/mL (n = 12) for 10 or 30 minutes at an average pressure of 80 mm Hg. After exposure, an end-to-side distal external jugular-to-common carotid artery anastomosis was created, resulting in a widely patent arteriovenous fistula. The external jugular was suture-ligated just proximal to the thoracic inlet, distal to a small 10- to 50-microm venous tributary, creating a reversed vein "graft" segment immediately and abruptly exposed to arterial pressure (48 +/- 3 mm Hg) and low shear stress (0.12 +/- .02 dyne/cm(2)). In the 29 animals (N = 31) that survived to harvest, 26 grafts were found to be patent and were analyzed further. Nine grafts were harvested within the first week after operation, snap frozen in liquid nitrogen, and assayed for the presence of the Herpes viral immediate-response protein ICP0 by Western blot analysis. The 17 remaining grafts were perfusion-fixed, excised, stained, and analyzed morphometrically by digital planimetry. In patent grafts, the hemodynamic environment of low shear stress was maintained (shear stress at harvest, 0.26 +/- .06 dyne/cm(2)). Western blot analysis revealed the presence of ICP0 in R7020-exposed vein grafts after 2, 3, 7, and 14 days; ICP0 was not detected in unexposed vein grafts or adjacent carotid arteries. After 4 weeks, vein grafts exposed to R7020 exhibited a statistically significantly increased ratio of luminal radius to wall thickness, indicating altered remodeling (vehicle, 6.7 +/- 1.3; R7020 2.5 x 10(8), 9.1 +/- 1.3; R7020 2.5 x 10(9) ratio, 11.3 +/- 1.4; P < .05 for high dose compared with vehicle). A brief exposure of the neurovirulence-attenuated HSV-1 strain R7020 results in an increased ratio of luminal radius to wall thickness in experimental vein grafts chronically exposed to low shear stress.

Local application of rapamycin inhibits neointimal hyperplasia in experimental vein grafts

Background Rapamycin is an immunosuppressive agent which also exhibits marked antiproliferative properties. Rapamycin coated stents have been demonstrated to suppress restenosis in experimental and clinical studies of percutaneous coronary catheter intervention. We investigated whether rapamycin can reduce neointima formation in a mouse model of vein graft disease. Methods C57BL6J mice underwent interposition of the inferior vena cava from isogenic donor mice into the common carotid artery using a previously described cuff technique. In the treatment group, 100 μg or 200 μg of rapamycin was applied locally in pluronic gel. The control group did not receive local treatment. Grafts were harvested at 1, 2, 4, and 6 weeks and underwent morphometric analysis as well as immunohistochemical analysis. Results In grafted veins without treatment (controls), median intimal thickness was 9.6 (6.4 to 29)μm, 11.9 (7.9 to 39.9)μm, 46.6 (12.4 to 57.7)μm, and 57.5 (32.5 to 71.1)μm after 1, 2, 4, and 6 weeks, respectively. Treatment with 100 μg or 200 μg rapamycin showed a dose dependant reduction of intimal thickness. In the 200 μg rapamycin treatment group the intimal thickness was 4.3 (3.4 to 5.6)μm, 3.8 (3.2 to 6.3)μm, 17.1 (4.8 to 63)μm, and 33.9 (11.3 to 80.3)μm after 1, 2, 4, and 6 weeks, respectively. This difference of intimal thickness of 200 μg treated animals compared with controls was statistically significant at 1 and 2 weeks. Immunohistochemically the reduction of intimal thickness was associated with a decreased amount of infiltration of CD-8 positive cells and a decreased amount of metallothionein positive cells in the rapamycin treated grafts. Conclusions We conclude that perivascular application of rapamycin inhibits neointimal hyperplasia of vein grafts in a mouse model. These results suggest that rapamycin may have a therapeutic potential for the treatment of vein graft disease.

Degradation of alpha-actin filaments in venous smooth muscle cells in response to mechanical stretch.

Mechanical stretch has been shown to induce the degradation of alpha-actin filaments in smooth muscle cells (SMC) of experimental vein grafts. Here, we investigate the possible role of ERK1/2 and p38 MAPK in regulating this process using an ex vivo venous culture model that simulates an experimental vein graft. An exposure of a vein to arterial pressure induced a significant increase in the medial circumferential strain, which induced rapid alpha-actin filament disruption, followed by degradation. The percentage of SMC alpha-actin filament coverage was reduced significantly under arterial pressure (91 +/- 1%, 43 +/- 13%, 51 +/- 5%, 28 +/- 3%, and 19 +/- 5% at 1, 6, 12, 24, and 48 h, respectively), whereas it did not change significantly in specimens under venous pressure at theses times. The degradation of SMC alpha-actin filaments paralleled an increase in the relative activity of caspase 3 (3.0 +/- 0.7- and 1.7 +/- 0.4-fold increase relative to the control level at 6 and 12 h, respectively) and a decrease in SMC density (from the control level of 1,368 +/- 66 cells/mm(2) at time 0 to 1,205 +/- 90, 783 +/- 129, 845 +/- 61, 637 +/- 55, and 432 +/- 125 cells/mm(2) at 1, 6, 12, 24, and 48 h of exposure to arterial pressure, respectively). Treatment with a p38 MAPK inhibitor (SB-203580) significantly reduced the stretch-induced activation of caspase 3 at 6 h (from 3.0 +/- 0.7- to 2.2 +/- 0.3-fold) in conjunction with a significant rescue of alpha-actin filament degradation (from 43 +/- 13% to 69 +/- 15%) at the same time. Treatment with an inhibitor for the ERK1/2 activator (PD-98059), however, did not induce a significant change in the activity of caspase 3 or the percentage of SMC alpha-actin filament coverage. These results suggest that p38 MAPK and caspase 3 may mediate stretch-dependent degradation of alpha-actin filaments in vascular SMCs.

Endothelial healing in vein grafts: proliferative burst unimpaired by genetic therapy of neointimal disease.

Although inhibition of neointimal hyperplasia by cell cycle gene blockade therapy results in improved endothelial cell function in experimental vein grafts, little is known either about endothelial healing immediately after vein grafting or about the effect of this therapy on the healing process. Scanning electron microscopy demonstrated an immediate decrease in vein graft endothelial cell density associated with vein graft wall stretch, followed by a return to baseline by postoperative day 3. En face detection of bromodeoxyuridine incorporation confirmed a rapid endothelial proliferation by 48 hours. Despite inhibition of underlying vascular smooth muscle cell proliferation, E2F decoy oligonucleotide did not inhibit either endothelial bromodeoxyuridine incorporation or the return to baseline cell density. This differential response to E2F decoy was also observed in human umbilical vein endothelial cell culture, which resisted the E2F decoy inhibition of cell growth that was observed in human umbilical artery smooth muscle cells, despite evidence for nuclear localized delivery of the oligonucleotide into both cell types. Furthermore, the reduction of E2F binding activity seen in a nuclear gel shift assay of cultured smooth muscle cells was not observed in endothelial cells. These results suggest a burst of graft endothelial cell proliferation that allows a rapid restoration of cell density in the monolayer. Additionally, there is a selective effect of E2F decoy gene therapy on target smooth muscle cells with sparing of this endothelial healing.

Inhibitory effect of prostaglandin E 1 on intimal thickening caused by poor runoff conditions in the canine autologous vein grafts.

The efficacy of ONO-1608, a newly developed liposomal formulation of prostaglandin E 1 prodrug, was evaluated on intimal hyperplasia of experimental canine autologous vein grafts under distal poor runoff conditions. The femoral vein was implanted into the femoral artery, preparing a distal poor runoff canine model. After 4 weeks of preparing the poor runoff model, the femoral vein was implanted into the femoral artery. They were then divided into two groups consisting of the control group and the ONO-1608 group. At 4 weeks, the grafts were harvested and intimal hyperplasia of the graft was measured with an ocular cytometer. Intimal cell proliferation was determined by bromodeoxyuridine incorporation 2 weeks after surgery. In addition, the effect of ONO-1608 on the proliferation of platelet-derived growth factor (PDGF)-stimulated human aortic smooth muscle cells (HASMCs) in culture was also investigated. At 4 weeks, the degree of intimal hyperplasia of the graft in the ONO-1608 group was significantly less than that of the control group. The bromodeoxyuridine labeling index 2 weeks after grafting was significantly lower in the ONO-1608 group compared with that in the control group. In addition, ONO-1608 significantly inhibited the proliferation of PDGF-stimulated HASMCs in culture. These results demonstrate the efficacy of ONO-1608 in reducing the degree of intimal hyperplasia of canine autogenous vein grafts under poor runoff conditions. The mechanism of reducing the intimal hyperplasia may be that ONO-1608 inhibited PDGF-stimulated proliferation of the smooth muscle cell. These results suggest that the administration of ONO-1608 may be beneficial in patients who have undergone gone arterial reconstruction.

The effects of extremely low shear stress on cellular proliferation and neointimal thickening in the failing bypass graft

Objective: Previous studies demonstrating a correlation between low shear stress (τ = 5-15 dyne/cm2) and experimental vein graft neointimal thickening (NIT) support the role of low τ in vein graft failure. However, a simple linear relationship between low τ and NIT would underestimate the degree of NIT evident in high-grade occlusive lesions of failing human vein grafts. In this study we used a new experimental model that maintains patency at low τ (< 2 dyne/cm2), to delineate possible deviations from linearity in the low τ → NIT hypothesis. Methods: Thirty-two New Zealand White rabbits underwent creation of a common carotid vein patch with a segment of ipsilateral external jugular vein. Very low τ was created in 13 patches by ligation of the distal common carotid artery, leaving the only outflow through a small muscular branch. Normal τ was created in 11 patches by leaving the common carotid artery outflow intact. High τ was created in eight patches by ligation of the contralateral common carotid artery. Six patches were harvested after 2 weeks for measurement of cell cycle entry by proliferating cell nuclear antigen (PCNA) immunohistochemistry. The remaining 26 patches were harvested after 4 weeks, perfusion fixed, and excised for morphometric analysis. Results: Mean blood flow and τ at implantation ranged from 0.5 to 41 mL/min and 0.07 to 15 dyne/cm2, respectively. At the time of harvest, 30 of 32 patches remained patent, and the artificially created aberrations in blood flow were maintained (range, 0.7-41 mL/min). After 2 weeks PCNA immunohistochemistry showed a significantly higher level of cell cycling in patches exposed to low τ (40 ± 5 vs 1.6 ± 0.3 PCNA-positive cells per high-power field; P <.001), which is equivalent to approximately 20% of the total cells present. In patches harvested after 4 weeks, NIT ranged from 42 to 328 μm and significantly correlated with mean τ at implantation. Patches with very low τ exhibited histologic characteristics similar to those of failing human bypass grafts, including laminar thrombus and flow-limiting luminal stenosis. The relationship between τ and NIT was nonlinear in that extremely low τ (< 2 dyne/cm2) resulted in NIT beyond that predicted by a simple linear correlation (P =.003). Conclusion: Extremely low τ (< 2 dyne/cm2) stimulates high rates of smooth muscle cellular proliferation in arterialized vein patches. NIT is accelerated in these regions of low τ far beyond that predicted by a simple linear model. The nonlinear nature of the cellular proliferative response and NIT at τ less than 2 dyne/cm2 may explain the rapid progression of neointimal lesions in failing bypass grafts. (J Vasc Surg 2001;34:90-7.)

Adenoviral-mediated Gene Transfer in Human and Animal Vein Grafts Using Clinically Relevant Exposure Times, Pressures, and Viral Concentrations

This study examined the efficiency of adenoviral-mediated gene transfer in experimental vein grafts and cultured human saphenous vein under physiologic conditions using clinically relevant exposure times, pressures, and viral concentrations. The external jugular veins of 25 male New Zealand White rabbits were exposed to 0.5 mL of replication-deficient adenovirus vectors encoding beta-galactosidase (AdlacZ), control adenovirus (AdBg/II), or vehicle at pressures ranging from 0 to 120 mmHg for 10 min. Veins were excised and grafted into the carotid circulation. After 5 days, the vessels were reexposed, excised, and stained with X-gal chromagen for beta-galactosidase (beta-gal) activity. Gene transfer was also performed in 13 segments of human saphenous vein discarded at the time of bypass grafting. The veins were cultured for 0-21 days and assayed for beta-gal activity as above. Rabbit vein grafts exposed to high-pressure AdlacZ transfection showed significant transgene expression in 100% of grafts (39 +/- 2% positive cells/hpf) while only 60% of those transfected at low pressure expressed beta-gal (9 +/- 3% positive cells/hpf). All human veins exposed to AdlacZ expressed beta-gal to a variable degree (range 10-50% positive cells/hpf). No control grafts or veins expressed the transgene. Efficient adenoviral-mediated gene transfer in experimental vein grafts and human saphenous vein segments can be achieved using clinically feasible parameters of exposure time, pressure, and viral concentration.

Hemodynamically “insignificant” stenoses stimulate neointimal thickening in experimental vein grafts

Vein graft stenoses <50% are believed to be clinically “insignificant” because they cause little decrement in blood flow. However, the possible deleterious effects of minor flow disturbances on long-term cellular proliferation are unknown. The purpose of this study was to evaluate the effect of artificially created 50% stenoses on neointimal thickening in experimental vein grafts. Fifteen male New Zealand White rabbits underwent carotid interposition bypass grafting using the external jugular vein. Mid-graft 50% stenoses were created in eight grafts by the application of a two-millimeter internal diameter stainless steel clip. After four weeks, the grafts were perfusion-fixed, excised, and histologic sections were examined for neointimal thickening. Application of the clip caused a 50% reduction in external diameter (3.9 ± 0.4 mm to 2.0 mm), causing only slight perturbations in pressure gradient (Δp = 2.1 ± 1.1 to 2.3 ± 1.5 mmHg) and mean blood flow (flow 16 ± 3.0 to 14 ± 4.4 ml/min). After four weeks, four grafts had occluded (two from each group) and one clip had become dislodged, leaving ten grafts for analysis. Neointimal thickness was minimal in control grafts (88 ± 12 μm), but was significantly increased in stenotic grafts both immediately proximal (200 ± 39 μm; p = 0.03) and immediately distal to the stenosis (230 ± 50 μm; p = 0.02). Hemodynamically “insignificant” stenoses stimulate vein graft neointimal thickening. These results support the continued use of graft surveillance and an aggressive approach to the treatment of “minimal” vein graft lesions.

Role of tensile stress and strain in the induction of cell death in experimental vein grafts

Tensile stress and strain are known to induce vascular cell proliferation, a process that is physiologically counterbalanced by cell death. Here we investigate whether tensile stress and strain regulate vascular-cell death by using an end-to-end anastomosed rat vein graft model. In such a model, the circumferential tensile stress in the graft wall was increased by ∼140 times immediately after surgery compared with that in the venous wall. This change was associated with an increase in the percentage of TUNEL-positive cells at 1, 6, 24, 120, 240, and 720 h with two distinct peaks at 1 and 24 h (10.1±3.5 and 14.4±3.2%, respectively) compared with that in control jugular veins (0.4±0.5 and 0.5±0.5% at 1 and 24 h, respectively). When tensile stress and strain in the vein graft wall were reduced by using a biomechanical engineering approach, the rate of cell death was reduced significantly (3.6±1.1 and 1.6±0.5% at 1 and 24 h, respectively). Furthermore, DEVD-CHO, a tetrapeptide aldehyde that inhibits the activity of caspase 3, significantly suppressed this event. These results suggest that a step increase in tensile stress and strain in experimental vein grafts induces rapid cell death, which is possibly mediated by cell death signaling mechanisms.

Local treatment with recombinant tissue factor pathway inhibitor reduces the development of intimal hyperplasia in experimental vein grafts

Background: Tissue factor (TF)–initiated thrombin generation has been implicated in the development of intimal hyperplasia after arterial injury. An increase in intimal TF expression has been shown to precede the development of intimal hyperplasia in vein grafts. This study examines the effects of local treatment with recombinant human tissue factor pathway inhibitor (rTFPI) in experimental vein grafts. Methods: Thirty-six male New Zealand white rabbits underwent bypass grafting of the carotid artery by use of the reversed ipsilateral jugular vein and were divided into four groups. Twenty animals had ex vivo incubation with rTFPI treatment (50 μg · mL–1; n = 10) or placebo vehicle (control; n = 10). Sixteen animals received both ex vivo incubation and in vivo gel treatment with rTFPI (50 μg · mL–1; n = 8) or without rTFPI (gel-control; n = 8). After operation, vein grafts were harvested at 3 days for immunohistochemical and Western analyses and at 28 days for histomorphologic study. Results: Western analysis demonstrated a 6.2-fold reduction in the expression of TF protein with rTFPI treatment in comparison to without rTFPI treatment. CD-18 leukocyte staining was diminished, whereas Tie-2 endothelial staining was increased in all rTFPI-treated vein grafts, compared with control and gel-control vein grafts. Intimal thickness was reduced by 21% with ex vivo rTFPI treatment compared with placebo (69 ± 4 versus 87 ± 5 μm; P <.05) and by 30% with the addition of rTFPI in vivo compared with gel-control (60 ± 4 versus 86 ± 5 μm; P <.01). Conclusion: Local administration of rTFPI exerts early beneficial effects and limits the development of intimal hyperplasia in vein grafts. Therefore blocking TF-mediated pathway may offer new therapeutic options to reduce vein graft failure. (J Vasc Surg 2001;33:400-7.)

Enhanced Superoxide Production in Experimental Venous Bypass Graft Intimal Hyperplasia

-Vein graft intimal hyperplasia, due to smooth muscle cell (SMC) proliferation, remains a limiting factor in long-term vein graft patency. Increased superoxide production regulates SMC mitogenesis and contributes to reduced NO bioactivity in systemic models of vascular disease. We compared superoxide production in experimental venous bypass grafts with ungrafted veins and determined its enzymatic sources and cellular localization. Vascular superoxide production was measured in vein grafts and control jugular veins obtained from normocholesterolemic rabbits undergoing jugular vein-carotid artery interposition bypass grafting. Surgical isolation of the contralateral jugular vein, without bypass grafting, provided an additional control for the effects of surgical manipulation. Superoxide production was increased 3-fold in vein grafts compared with control veins. Systematic stimulation and inhibition of specific oxidases revealed that the major source of increased vein graft superoxide production was a membrane-associated NAD(P)H-dependent oxidase. Western blotting of vascular homogenates demonstrated corresponding increases in NAD(P)H oxidase p22phox (membrane-associated) and p67phox (cytosolic) subunits in vein grafts compared with jugular veins. There was marked intimal hyperplasia in vein grafts, and immunohistochemical staining of vessel cryosections revealed increased p22phox-expressing cells in vein grafts that were predominantly intimal SMCs. Superoxide generation is increased in experimental vein grafts compared with ungrafted veins. The principal source of increased superoxide generation in vein grafts is an NAD(P)H oxidase, expressed by intimal SMCs. These findings suggest a role for NAD(P)H oxidase-mediated superoxide production in the proliferative response to vascular injury in vein grafts.

Effect of external stenting and systemic hypertension on intimal hyperplasia in rat vein grafts.

Overdistention of vein grafts in arterial circulation and systemic hypertension are thought to be influential risk factors contributing to vein graft failures. This study tested the effects of external stenting in preventing systemic hypertension and overdistention of the rat vein graft in the long term. Jugular vein grafts were interposed into the carotid artery of normotensive (n = 39) and two-kidney, one-clip hypertensive (n = 30) rats. Jugular vein grafts wrapped with 1.5-mm-diameter polyester stents were used in normotensive (n = 26) and hypertensive (n = 25) rats. The vein grafts were harvested at 1, 2, 4, 8, 12, and 24 weeks after the grafting procedure. The neointimal area and wall thickness were measured by computerized planimetry, and Ki-67 immunohistochemistry was used to detect replicative smooth muscle cells in the graft wall. In each group, intimal hyperplasia was apparent at 1 week and increased gradually to 24 weeks. The number of Ki-67-positive cells was most increased at 2 weeks after the grafting procedure and gradually decreased thereafter. The numbers of Ki-67-positive cells and the extent of intimal hyperplasia were not significantly different between normotensive and hypertensive rats. Both neointima formation and cell proliferation in the graft wall were significantly reduced by external stenting as compared with the results with unstented grafts. Systemic hypertension by itself is not a risk factor for intimal hyperplasia and experimental vein graft failure in the long term. External stenting is effective against intimal hyperplasia, and it is possible to reduce the subsequent atherosclerotic change of the vein graft wall and improve the long-term patency of the vein graft with external stenting.

Focal expression of angiotensin II type 1 receptor and smooth muscle cell proliferation in the neointima of experimental vein grafts: relation to eddy blood flow.

Eddy flow has been shown to promote focal smooth muscle cell (SMC) proliferation and neointimal formation in experimental vein grafts. This study focuses on whether the angiotensin II type 1 (AT(1)) receptor mediates these events. Experimental vein grafts with and without eddy flow were created in the rat. Losartan was used to assess the influence of the AT(1) receptor on SMC proliferation. In vein grafts with eddy flow, apparent focal expression of AT(1) mRNA and protein was found in the leading region of the proximal focal neointima, where eddy flow occurred, but not in the trailing region, where eddy flow diminished, at days 5, 10, 20, and 30. The rate of SMC proliferation in the leading region (10.9+/-1.4%, 19.5+/-2.2%, 12.2+/-2.0%, and 6.9+/-1.3% at these times, respectively) was significantly higher than that in the trailing region (9.5+/-1.8%, 15.3+/-2.0%, 8.2+/-1.9%, and 3.2+/-0.7%) in these vein grafts. When eddy flow was prevented in engineered vein grafts, no apparent location difference was found in the distribution of AT(1) receptor mRNA and protein in the neointima, and the rate of SMC proliferation (5.3+/-1.0%, 5.8+/-0.9%, 3.4+/-1.0%, and 3.7+/-0.9% at days 5, 10, 20, and 30, respectively) was reduced significantly. In vein grafts with losartan, the rate of SMC proliferation in the leading region of the neointima (9.4+/-1.8%, 10.1+/-1.3%, 8.3+/-0.9%, and 4.2+/-0. 5% at days 5, 10, 20, and 30, respectively) was significantly lower than that in vein grafts without losartan. These results suggested that eddy flow upregulated the AT(1) receptor, which in turn mediated focal SMC proliferation in the neointima of experimental vein grafts.

Partial prevention of monocyte and granulocyte activation in experimental vein grafts by using a biomechanical engineering approach

Leukocytes interact with endothelial cells and contribute to the development of vascular diseases such as thrombosis and atherosclerosis. These processes are possibly influenced by mechanical factors. This study focused on the role of mechanical stretch in the activation of monocytes and granulocytes in experimental vein grafts. Two models were created by using rats: a nonengineered vein graft with increased tensile stress, which was created by grafting a jugular vein into the abdominal aorta, and an engineered vein graft with reduced tensile stress, which was created by restricting the vein graft into a cylindrical sheath constructed by using fixative-treated intestinal tissue. The density of activated monocytes and granulocytes, which attached to the endothelium, and the distribution of the intercellular adhesion molecule (ICAM)-1 in endothelial cells were examined using immunohistological assays. It was found that, in nonengineered vein grafts, the density of activated monocytes and granulocytes increased significantly compared to that in normal jugular veins at day 1, 5, 10 and 20. At each observation time, the cell density in the proximal region of the nonengineered vein grafts was significantly higher than that in the middle and distal regions, and the cell density in the distal region was significantly higher than that in the middle region. These changes were associated with ICAM-1 clustering at day 1 and 5 and focal ICAM-1 un-regulation at day 10 and 20. In engineered vein grafts, the density of activated monocytes and granulocytes decreased significantly compared to that in nonengineered vein grafts at all observation times, although it was significantly higher than that in normal jugular veins. At each observation time, the cell density in the proximal and distal regions was significantly higher than that in the middle region, but no significant difference was found between the proximal and distal regions. ICAM-1 clustering along endothelial cell borders was found at day 1 and 5, but no apparent focal ICAM-1 up-regulation was found at day 10 and 20. These results suggested that mechanical stretch due to exposure to increased tensile stress contributed to the activation of monocytes and granulocytes in experimental vein grafts, and this event could be partially prevented by reducing tensile stress using a biomechanical engineering approach.

Ex-vivo gene therapy of human vascular bypass grafts with E2F decoy: the PREVENT single-centre, randomised, controlled trial

Cell-cycle blockade by ex-vivo gene therapy of experimental vein grafts inhibits the neointimal hyperplasia and subsequent accelerated atherosclerosis that lead to human bypass-graft failure. In a prospective, randomised, controlled trial, we investigated the safety and biological efficacy of intraoperative gene therapy in patients receiving bypass vein grafts. We studied gene therapy that uses decoy oligodeoxynucleotide, which binds and inactivates the pivotal cell-cycle transcription factor E2F. 41 patients were randomly assigned untreated (16), E2F-decoy-treated (17), or scrambled-oligodeoxynucleotide-treated (eight) human infrainguinal vein grafts. Oligonucleotide was delivered to grafts intraoperatively by ex-vivo pressure-mediated transfection. The primary endpoints were safety and inhibition of target cell-cycle regulatory genes and of DNA synthesis in the grafts. Analysis was by intention to treat. Mean transfection efficiency was 89.0% (SD 1.9). Proliferating-cell nuclear antigen and c-myc mRNA concentrations and bromodeoxyuridine incorporation were decreased in the EF2-decoy group by medians of 73% [IQR 53-84], 70% [50-79], and 74% [56-83], respectively) but not in the scrambled-oligodeoxynucleotide group (p<0.0001). Groups did not differ for postoperative complication rates. At 12 months, fewer graft occlusions, revisions, or critical stenoses were seen in the E2F-decoy group than in the untreated group (hazard ratio 0.34 [95% CI 0.12-0.99]). Intraoperative transfection of human bypass vein grafts with E2F-decoy oligodeoxynucleotide is safe, feasible, and can achieve sequence-specific inhibition of cell-cycle gene expression and DNA replication. Application of this genetic-engineering strategy may lower failure rates of human primary bypass vein grafting.