Rabbit Vein Graft Research Articles

Prolonged hypercholesterolemia-induced tissue factor expression in rabbit vein grafts: a potential mechanism for graft failure

To evaluate tissue factor (TF) expression in vein grafts interposed in the arterial circulation of hypercholesterolemic rabbits. Veins implanted in the arterial circulation of normocholesterolemic rabbits respond by inflammation and infiltration by monocytes with transient TF expression. In a hypercholesterolemic milieu these monocytes may differentiate into macrophages capable of enhanced TF synthesis, which may facilitate hyperplasia and thrombosis. Autologous jugular veins interposed in the carotid artery of hypercholesterolemic rabbits were harvested at 1, 2, 4, 6, and 8 weeks after surgery and examined for presence and localization of rabbit TF antigen. Protein extracted from vein segments was evaluated for procoagulant activity by bioassay and for TF protein content by western blotting. Rabbit TF antigen was observed mostly in the subendothelium of vein grafts. Peak TF procoagulant activity observed at 1-2 weeks postsurgery (2.3+/-1.8 pg/mg, P<0.006) declined to 0.9+/-0.5, 0.2+/-0.1, and 0.15+/-0.06 pg/mg at 4, 6, and 8 weeks, respectively (P<0.03). Western blotting showed a time-dependent pattern for rabbit TF protein with prolonged expression peaking at 6 weeks. Prolonged expression of biologically active rabbit TF and TF protein were shown within jugular vein grafts of hypercholesterolemic rabbits. This response, reported for the first time and attributed to increased cholesterol levels, may possibly contribute to enhanced hyperplasia. These results suggest that TF expression could serve as another mechanism underlying vein graft failure and that hypercholesterolemia in bypass patients should be treated aggressively beginning within the weeks after surgery.

The effects of the autologous venous external stents on intimal hyperplasia of the vein grafts in rabbits

To assess the effect of the autologous venous external stents on intimal hyperplasia of the vein grafts in rabbits. Thirty-six male New Zealand white rabbits, aged 5 months and weighing 2.8 to 3.0 kg, were randomly divided into 3 groups: group A, group B and group C, with 12 rabbits in each group. First, a section about 6 cm long of vein was cut from the right external jugular vein of each rabbit and severed to have 3 equal-length segments. Next, each distal segment prepared for anastomosis. The proximal segment invaginating middle segment in group A and only middle segment in group B were used for the external stent. Later, the left common carotid artery was separated from surrounding tissue, from it a section about 0.5 cm long was cut away. Finally, the vein graft was inverted and end-to-end anastomosed to the two ends of the artery with a 9-0 suture. After bloodstream re-established, the diameter of each vein graft was measured. At 2 and 4 weeks postoperative, the graft veins were cut off and histologically examined by the means of HE staining and Masson staining. The smooth muscle cells (SMC) proliferation was studied by the immunohistochemical detection of proliferating cell nuclear antigen. After bloodstream re-established, the diameters of vein graft of group A and group B and group C were (1.6 +/- 0.3) mm, (2.2 +/- 0.4) mm and (2.6 +/- 0.6) mm respectively (P < 0.05). At 4 weeks postoperative, the data of the ratio of intima to media thickness and the index of the proliferating cells of the intima were as follow: group A (1.01 +/- 0.07 and 6.84 +/- 1.98), group B (1.32 +/- 0.08 and 11.01 +/- 2.61), group C (1.55 +/- 0.03 and 14.96 +/- 4.14). Both the data of group A were obviously less than that in group B, and that of group B was less than group C (P < 0.05). The autologous venous two-layer external stents inhibit intimal hyperplasia of the vein grafts.

Pretreatment with intraluminal rapamycin nanoparticle perfusion inhibits neointimal hyperplasia in a rabbit vein graft model

PurposePoly lactic-co-glycolic acid nanoparticles (PLGA-NP) are widely used as a biodegradable biomaterial in medicine. Rapamycin-eluting stents have been used for prevention of restenosis during surgery. This study investigated the effect of pretreatment with intraluminal perfusion of carbopol-encapsulated rapamycin-loaded PLGA nanoparticles (RAP-PLGA-NP) on neointimal hyperplasia in a rabbit vein graft model.MethodsA segment of common carotid artery was replaced with a segment of external jugular vein in 60 rabbits which were then separated into four treatment groups, ie, Group 1, in which vein grafts were pretreated with intraluminal RAP-PLGA-NP perfusion, Group 2 in which vein grafts underwent equivalent empty vehicle (PLGA-NP) perfusion, Group 3, in which vein grafts received no treatment, and Group 4, which served as a sham operation group receiving normal vein contrast. On postoperative day 28, the grafts and normal veins were harvested for histologic examination, flow cytometry analysis, and high-performance liquid chromatography measurement.ResultsCompared with Group 1, the intima of the grafts were thickened, the ratio of intimal area to vessel area increased, and the collagen volume index of the vein grafts increased significantly in Groups 2 and 3. The cell proliferation index in Group 1 (21.11 ± 3.15%) was much lower than that in Group 2 (30.35 ± 2.69%) and in Group 3 (33.86 ± 8.72%). By high-performance liquid chromatography measurement, retention of rapamycin was detected in Group 1 (11.2 ± 0.37 μg/10 mg) 28 days after single drug perfusion.ConclusionPretreatment with intraluminal RAP-PLGA-NP perfusion may inhibit neointimal hyperplasia in vein grafts by penetrating into local tissue and limiting cell proliferation.

Chronic treatment of hydroxytryptamine type 2a receptor antagonist sarpogrelate hydrochloride modulates the vasoreactivity of serotonin in experimental rabbit vein grafts

It has been suggested that 5-hydroxytryptamine (5-HT) plays a role in the pathogenesis of vein graft spasms. It is suggested that smooth muscle 5-HT(2A) and 5-HT(1B) receptors contribute to 5-HT-induced contraction, while endothelial 5-HT(1B) receptors contribute to the 5-HT-induced endothelium-mediated relaxation. We recently found that chronic administration of the selective 5-HT(2A) receptor antagonist sarpogrelate hydrochloride (SH) enhances the function of endothelium-derived nitric oxide (NO) in rabbit vein grafts. However, it is unknown if such treatment modulates 5-HT-induced vasospasm in vein grafts, and if so, what the underlying mechanisms are. Male rabbits were divided into two groups: a control group and an SH-treated group. The jugular vein was interposed in the carotid artery in reversed fashion. Isometric tension was examined using vein grafts after 4 weeks. 5-HT (10(-8) -10(-6) M)-induced contraction was obtained in each group in the absence or presence of the NO synthase inhibitor l-N(G)-nitroarginine (L-NNA). The expression of 5-HT(2A) and 5-HT(1B) receptors was examined immunohistochemically. The 5-HT induced a concentration-dependent contractions in both groups. L-NNA did not significantly modify the 5-HT-induced contraction in the control group but enhanced it in the SH group. The 5-HT(1B) receptor antagonist GR55562 inhibited the 5-HT-induced contraction in the control group, while it increased the sensitivity of contraction to 5-HT in the SH-treated group in the absence (but not in the presence) of L-NNA. Positive immunoreactivities against 5-HT(1B) and 5-HT(2A) receptors were identified in endothelial and medial regions of vein grafts in both groups, and the expression of 5-HT(2A) receptors (but not 5-HT(1B) receptors) was significantly less in the SH-treated group than in the control group. Chronically administered SH to rabbits upregulates the autoinhibitory mechanism by 5-HT through a release of NO from endothelium via an activation of endothelial 5-HT(1B) receptors, thus attenuating its own contraction in vein grafts. Furthermore, such SH treatment downregulates the expression of smooth muscle 5-HT(2A) receptors, thus further attenuating the 5-HT-induced contraction. These novel findings further support the clinical usefulness of SH in vein graft spasm after bypass grafting.

Established neointimal hyperplasia in vein grafts expands via TGF-β-mediated progressive fibrosis

In weeks to months following implantation, neointimal hyperplasia (NIH) in vein grafts (VGs) transitions from a cellularized to a decellularized phenotype. The inhibition of early cellular proliferation failed to improve long-term VG patency. We have previously demonstrated that transforming growth factor-beta(1) (TGF-beta(1))/connective tissue growth factor (CTGF) pathways mediate a conversion of fibroblasts to myofibroblasts in the early VG (<2 wk). We hypothesize that these similar pathways drive fibrosis observed in the late VG lesion. Within rabbit VGs, real-time RT-PCR, Western blot analysis, ELISA, and immunohistochemistry were used to examine TGF-beta/CTGF pathways in late (1-6 mo) NIH. All VGs exhibited a steady NIH growth (P = 0.006) with significant reduction in cellularity (P = 0.01) over time. Substantial TGF-beta profibrotic activities, as evidenced by enhanced TGF-beta(1) activation, TGF-beta receptor types I (activin receptor-like kinase 5)-to-II receptor ratio, SMAD2/3 phosphorylation, and CTGF production, persisted throughout the observation period. An increased matrix synthesis was accompanied by a temporal reduction of matrix metalloproteinase-2 (P = 0.001) and -9 (P < 0.001) activity. VG NIH is characterized by a conversion from a proproliferative to a profibrotic morphology. An enhanced signaling via TGF-beta/CTGF coupled with reduced matrix metalloproteinase activities promotes progressive fibrotic NIH expansion. The modulation of late TGF-beta/CTGF signaling may offer a novel therapeutic strategy to improve the long-term VG durability.

Late Neointimal Hyperplasia in Vein Grafts Expands via TGF‐β/CTGF Mediated Fibrosis

IntroductionIn weeks to months following implantation, neointimal hyperplasia (NIH) in vein grafts (VG) transitions from a cellularized to decellularized phenotype. Inhibition of early cellular proliferation failed to improve long‐term VG patency. We have previously demonstrated that TGF‐β /CTGF pathways mediate a conversion of fibroblasts to myofibroblasts in the early VG (&lt;2wks). We hypothesize that these similar pathways drive fibrosis observed in the late VG lesion.MethodsWithin rabbit VGs, real time RT‐PCR, Western blotting, and immunohistochemistry were used to examine TGF‐β /CTGF pathways in late (1‐6 month) NIH.ResultsAll VGs exhibited a steady NIH growth (p=0.006) with significant reduction in cellularity (p=0.01) over time. Substantial TGF‐β profibrotic activities, as evidenced by enhanced TGF‐β1 activation, TGF‐β types I to II receptor ratio, SMAD2/3 phosphorylation, and CTGF production, persisted throughout the observation period. Increased matrix synthesis was accompanied by a temporal reduction of MMP2 (p=0.001) and 9 (p&lt;0.001) activity.ConclusionVG NIH is characterized by a conversion from a pro‐proliferative to profibrotic morphology. Enhanced signaling via TGF‐β /CTGF coupled with reduced MMP activities promotes progressive fibrotic NIH expansion. Modulation of late TGF‐β /CTGF signaling may offer a novel therapeutic strategy to improve the long‐term VG durability.

Sarpogrelate hydrochloride reduced intimal hyperplasia in experimental rabbit vein graft

The selective 5-HT(2A) receptor antagonist sarpogrelate has been clinically used for treatment in atherosclerotic diseases. However, it remains unknown whether administration of sarpogrelate inhibits intimal hyperplasia seen in autologous vein grafts. Therefore, we sought to clarify this question using an experimental rabbit vein graft model. Male rabbits were divided into two groups: a control group and a sarpogrelate-treated group. The jugular vein was interposed in the carotid artery in reversed fashion for 4 weeks and intimal hyperplasia of the grafted vein was measured (n = 8, in each group). Acetylcholine (ACh)-induced endothelium-dependent relaxation was tested by precontraction with prostaglandin F(2alpha) (PGF(2alpha), 5 muM) (n = 5, in each). endothelial nitric oxide synthase (eNOS) protein expression and superoxide production of these veins were also assessed. The suppression of intimal hyperplasia was significantly greater in the sarpogrelate-treated group than in the control group. ACh induced an endothelium-dependent relaxation in the sarpogrelate-treated group (but not in the control group). In endothelium-intact strips from the sarpogrelate-treated group, the nitric oxide (NO) synthase inhibitor nitroarginine enhanced the PGF(2alpha)-induced contraction and blocked the ACh-induced relaxation. Immunoreactive eNOS protein expression was similar between the two groups but superoxide production (estimated from ethidium fluorescence) in endothelial cells was significantly smaller in the sarpogrelate-treated group. The present results indicate that in vivo blockade of 5-HT(2A) receptors leads to an inhibition of intimal hyperplasia in rabbit vein graft. It is suggested that an increased function of endothelium-derived NO through a reduction in endothelial superoxide production may be a possible underlying mechanism for this. These novel findings support the clinical usefulness of sarpogrelate for preventing intimal hyperplasia in vein graft after bypass grafting.

Topical Mitogen-Activated Protein Kinases Inhibition Reduces Intimal Hyperplasia in Arterialized Vein Grafts

Vein graft arterialization results in activation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinases-1 and -2 (ERK1/2), which have been implicated in cell proliferation, migration, and apoptosis. The goal of our study was to characterize the effect of MAPK inhibition on intimal hyperplasia (IH) in arterialized vein grafts in hypercholesterolemic rabbits. Reversed bilateral jugular vein to common carotid artery interposition grafts were constructed in 16 New Zealand White rabbits. The veins were incubated for 30 min prior to grafting with either the synthetic ERK1/2 activation inhibitor UO126 or the control vehicle. Vein graft and control jugular vein were harvested 3 h, 1 d, and 28 d after arterialization for histological and biochemical analyses. Treatment with UO126 was associated with 31% reduction in mean intimal area (1.68 +/- 0.78 mm(2)versus 2.44 +/- 1.65 mm(2); mean +/- SD; P = 0.036) relative to controls. The intima-to-media ratio of UO126-treated vein grafts decreased by 29% (0.53 +/- 0.04 versus 0.74 +/- 0.06; mean +/- SD; P < 0.01) compared to controls, vehicle-treated vein grafts. There was also significant increase in apoptosis in UO126-treated vein graft medial cell layer at 1 d. Topical administration of UO126 before vein grafting significantly decreases IH in arterialized vein grafts in hypercholesterolemic rabbits. These results may have significant implications for the development of strategies aimed at blocking or reducing IH in bypass grafts. Therefore, further evaluation of this simple strategy to improve vein graft patency following coronary artery or peripheral vascular bypass surgery is warranted.

Midkine is expressed by infiltrating macrophages in in-stent restenosis in hypercholesterolemic rabbits

Neointimal hyperplasia is strikingly suppressed in an endothelium injury model in mice deficient in the growth factor midkine. Knockdown of midkine expression by means of antisense oligonucleotide or small interfering RNA has been shown to lead to suppression of neointimal hyperplasia in a balloon injury model and a rabbit vein graft model; therefore, midkine is an essential factor for neointimal hyperplasia. These findings, however, do not necessarily apply to the function of midkine in vascular stenoses such as in-stent restenosis, because human vascular stenosis is often accompanied by atherosclerosis. We investigated midkine expression in the neointima induced by implantation of a bare metal stent in the atheromatous lesions of hypercholesterolemic rabbits. We analyzed midkine expression during a THP-1 cell differentiation and in peritoneal macrophages exposed to low-density lipoprotein or oxidized low-density lipoprotein. Midkine expression reached the maximum level within 7 days after stenting and was detected in infiltrating macrophages. Differentiation of THP-1 cells to macrophage-like cells did not trigger midkine expression. Neither low-density lipoprotein nor oxidized low-density lipoprotein enhanced midkine expression in peritoneal macrophages that had been activated by thioglycollate, although these cells expressed a significant amount of midkine. The results indicate that macrophages are the major source of midkine in the atherosclerotic neointima. The amount of midkine expressed in macrophages may be sufficient (ie, further enhancement of the expression is not necessary) for the pathogenesis, because oxidized low-density lipoprotein stimulation did not induce the midkine expression. The growth factor midkine is induced during vascular stenosis in mouse and rat models with normal diet. Knockdown of midkine expression suppresses neointimal hyperplasia. The vascular response after stenting differs from that after balloon injury in that the inflammation is more prolonged and the accumulation of macrophages is more abundant in stent-injured vessel. We found here that macrophages are the major source of midkine in the atherosclerotic neointima of in-stent restenosis in hypercholesterolemic rabbits. Our data suggest that midkine has an important role in in-stent restenosis of atherosclerotic vessels and is a candidate molecular target to prevent in-stent restenosis.

Adeno-Associated Virus-Mediated Gene Transfer in a Rabbit Vein Graft Model

Stenoses of venous grafts represent a major limitation in coronary artery bypass surgery. The use of viral vectors to facilitate over-expression of factors within the graft to promote long-term patency is a promising new therapeutic concept. One of the viral vector systems is the adeno-associated virus (AAV); a non-pathogenic single stranded DNA virus, which elicits only low immunological responses. Recombinant AAV vector coding for beta-galactosidase was produced and transferred ex vivo using intraluminal application to previously harvested rabbit internal jugular vein grafts (n = 8). The 30 min after application, an end-to-end anastomosis of each graft as a bypass to the carotid artery was performed in a previously established rabbit bypass model. X-Gal-staining of the grafts was performed after killing the animals to quantify gene expression. AAV transduction was successful in 100% of the grafts. After 30 days, beta-galactosidase gene expression could be assessed in the medial layer of the graft. Furthermore, no signs of inflammation could be detected. These findings suggest that recombinant AAV vectors are an alternative to the widely used adenoviral based vectors. These data support the further use of AAV vectors to overcome intimal hyperplasia after vein graft coronary artery bypass surgery.

Abstract 2057: Over-expression of p27 Inhibits Vascular Smooth Muscle Cells Proliferation and Neointima Formation in Rabbit Vein Graft Model

Neointima formation caused by vascular smooth muscle cells (VSMC) proliferation is an early event in vascular remodeling associated with arterialization of vein graft and the magnitude of this process may contribute for late failure. Cell cycle is tightly regulated and cyclin-dependent kinase inhibitor p27 kip1 plays a critical role in this process. We investigated whether overexpression of p27 can modulate this early cell proliferative response in rabbit vein grafts. First generation bicistronic adenovirus AdCMVp27hEGFP, (AV1) harbouring the human p27 sequence, and AdCMVLacz (AV2) were engineered. Overexpression of p27 was analyzed by immunofluorescence and the antiproliferative effect of AV1 was assessed in primary culture of VSMC followed by 1, 3, 5, and 9 days after AV1 transduction vs. AV2. A segment of jugular vein vas exposed to AV1 and AV2 (10 9 –10 11 pfu/mL) or placebo for 30 min and then interposed, end to end, to the carotid artery of adult male New Zealand rabbits. After 28 days the vein grafts were harvested and stained with Miller for neointima analyses. Imunofluorescence of VSMC showed positive cells 72 hs following AV1 transduction. Significant inhibition of cell proliferation was observed 7 and 9 days after the cells were exposed to AV1 compared to AV2 or control cells (7 days: 0.34 ± 0.15 × 10 5 vs. 1.5 ± 0.22 × 10 5 , 2.04 ± 0.29 × 10 5 cell/ml, p&lt;0.01; 9 days: 0.23 ± 0.1 × 10 5 vs. 1.49 ± 0.31 × 10 5 , 2.31 ± 0.41 × 10 5 cell/ml, p&lt;0.01). In vivo , transduction efficiency was evaluated by the expression of B-galactosidase 48 hs. after surgery in vein grafts exposed to AV2. Vein grafts transduced with AV1 showed significant reduction in neointima hyperplasia compared with AV2 or to control animals not exposed to any viral treatment (47.33 ± 5.36 vs. 112.83 ± 24.2 and 80.08 ± 10.51μm, p&lt;0.05). These results show that adenovirus mediated over expression of human protein p27 can inhibit VMSC proliferation in vitro and significantly impair the neointima formation associated with arterialization of vein graft after 28 days. It will important to establish the long term effects of this intervention specially when associated with other environment stimuli such as high fat that can clearly influence the outcome of this therapeutic intervention.

119. NOS2 Gene Transfer with a Synthetic Vector Inhibits Neointimal Hyperplasia in a Rabbit Vein Graft Model

119. NOS2 Gene Transfer with a Synthetic Vector Inhibits Neointimal Hyperplasia in a Rabbit Vein Graft Model

Angiotensin II and tumor necrosis factor-alpha upregulate survivin and Kruppel-like factor 5 in smooth muscle cells: Potential relevance to vein graft hyperplasia

Survivin (SVV) is a unique inhibitor of apoptosis protein (IAP) that regulates both apoptosis and mitosis. Recent work suggests that SVV plays a critical role in the vascular injury response, but the molecular pathways remain unclear. We examined the expression of SVV and the transcription factor, KLF5, in human venous smooth muscle cells (VSMC) by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis after treatment with angiotensin II (Ang II) or tumor necrosis factor-alpha (TNF-alpha). An adenoviral construct expressing SVV or GFP was also employed to assess effects on KLF5 expression. A rabbit carotid interposition vein graft model was used to assess the relevance of KLF5 to bypass graft healing. Stimulation of VSMC with Ang II and TNF-alpha led to a rapid upregulation of KLF5 expression, and a later increase in SVV, which was cell-cycle independent. Overexpression of SVV in VSMC led to an early and persistent induction of KLF5. KLF5 was upregulated in rabbit vein grafts early (1 day) after grafting. We speculate that SVV is a central point of convergence of multiple signaling pathways in vascular injury, and that it regulates the local amplification of these pathways in the vessel wall.

Inhibitory effects of NFκB decoy oligodeoxynucleotides on neointimal hyperplasia in a rabbit vein graft model

Autologous vein remains the commonly used conduit for bypass grafts; however, neointimal hyperplasia is known to be one of the major disease processes in vein graft failure. In this study, we focused on the important role of NFκB which controls the expression of numerous genes for various cytokines and adhesion molecules in the mechanism of graft failure. Thus, we investigated the inhibitory effect of NFκB decoy oligodeoxynucleotides (ODN) on vein graft failure in a rabbit hypercholesterolemic model. Jugular vein to carotid artery interposition grafts in rabbits were transfected intraoperatively with NFκB decoy ODN (40 μmol/l) by ex-vivo pressure-mediated transfection (300 mm Hg, 10 min). Treatment with NFκB decoy ODN significantly suppressed intimal hyperplasia 4 weeks after vein implantation as compared to scrambled decoy ODN, and increased medial thickness, leading to a significant reduction in intima-to-media ratio. Treatment with NFκB decoy ODN significantly inhibited the recruitment of macrophages and the proliferation of vascular smooth muscle cells (VSMC), while apoptosis in VSMC was significantly increased by NFκB decoy ODN. In addition, a study of vascular reactivity demonstrated that transfection of grafts with NFκB decoy ODN significantly improved endothelium-mediated vasorelaxation as compared to scrambled decoy ODN. Here, we demonstrated that inhibition of NFκB activation using decoy ODN inhibited the development of neointimal hyperplasia, followed by suppression of inflammatory changes and accumulation of VSMC in the neointima of rabbit vein grafts. The present study raises the possibility of a novel strategy for prevention of graft failure.

Long-term inhibition of Rho kinase suppresses intimal thickening in autologous vein grafts in rabbits

Rho kinase plays an important role in vascular smooth muscle cell (VSMC) contraction and other cellular functions, such as proliferation, migration, and apoptosis. Recent studies have demonstrated that long-term inhibition of Rho kinase suppresses coronary artery spasm and vascular lesion formation after arterial injury. In the cardiovascular surgery field, intimal thickening in vein grafts is the major cause of late graft failure, for which no effective treatment has yet been developed. In this study, we examined whether long-term inhibition of Rho kinase suppresses intimal thickening in autologous vein grafts in rabbits. Male rabbits were randomly divided into two groups and received normal chow (control group) or a special chow containing 0.09% fasudil (fasudil group). After oral administration, fasudil is metabolized to a specific Rho kinase inhibitor, hydroxyfasudil. Each group underwent reversed autologous vein graft surgery with the internal jugular vein into the left common carotid artery. At 1, 2, and 4 weeks after the operation, we examined the extent of intimal thickening of the graft and VSMC proliferation and apoptosis. The intimal thickening was significantly suppressed in the fasudil group compared with the control group at 2 and 4 weeks after the operation. In the fasudil group, VSMC proliferation was suppressed at 1 and 2 weeks after the operation, whereas VSMC apoptosis was enhanced at 2 weeks after the procedure. These results indicate that Rho kinase is substantially involved in the pathogenesis of intimal thickening of vein grafts and that it is an important therapeutic target for the prevention of graft failure.

Antisense basic fibroblast growth factor alters the time course of mitogen-activated protein kinase in arterialized vein graft remodeling.

Neointimal hyperplasia (NIH) is complete by 3 weeks in rabbit vein grafts implanted into the arterial circulation. Activation of the mitogen-activated protein kinase (MAPK) family of protein kinases is thought to be critical in remodeling events such as cellular proliferation, differentiation, and migration, as found in NIH. We previously demonstrated that antisense basic fibroblast growth factor (ASbFGF) inhibited the synthesis of basic fibroblast growth factor (bFGF) in the balloon injury model of NIH. We examined the effect of ASbFGF on NIH and the time course of MAPK, extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal protein kinase (JNK), and p38 kinase activation in arterialized vein grafts. Carotid interposition of a vein bypass graft was performed in 75 New Zealand White rabbits. Segments of the external jugular vein were transfected with a replication-deficient adenovirus containing the messenger RNA sequence for rat ASbFGF at 1 x 10(10) plaque-forming units per milliliter; control animals were given phosphate-buffered saline solution (PBS) alone. Rabbits were killed at 30 minutes, 4 days, 7 days, and 21 days (n = 8). Four grafts in each group were fixed with formalin and embedded in paraffin, then processed with elastin-collagen and hematoxylin-eosin stains. The other four grafts were individually frozen, and total protein was extracted. Phosphorylation of MAPK, ERK1/2, JNK, and p38, was determined with Western blot analysis and immunohistochemistry. Groups were compared with analysis of variance. The thickness of neointima in the PBS group and the ASbFGF group at 21 days was 60.2 +/- 2.1 and 39.4 +/- 2.1 microm, respectively (P <.01). In both the control and ASbFGF groups, all 3 MAPKs demonstrated activation compared with preimplantation levels. However, when compared with the PBS group the ASbFGF group showed greater than 33% inhibition of all three MAPKs by day 4 and day 7 (P <.05), but no significant difference in any MAPK activation by day 21 (P >.05, all groups). Cells staining positive for activated MAPK were found in the neointima and adventitia of vein grafts in both the PBS and ASbFGF groups. MAPKs are activated during the first week after vein graft implantation. Grafts treated with ASbFGF demonstrated reduced MAPK activation and less neointimal thickening. These results suggest that the process of vein graft adaptation to the arterial circulation, and subsequent NIH, may depend on basic fibroblast growth factor activity, which is mediated, at least in part, by a MAPK-dependent mechanism.

Overexpression of G Protein-Coupled Receptor Kinase-2 in Smooth Muscle Cells Reduces Neointimal Hyperplasia

K. Peppel, L. Zhang, T. T. T. Huynh, X. Huang, A. Jacobson, L. Brian, S. T. Exum, P.-O. Hagen and N. J. Freedman. Overexpression of G Protein-Coupled Receptor Kinase-2 in Smooth Muscle Cells Reduces Neointimal Hyperplasia. Journal of Molecular Cellular Cardiology (2002) 34, 1399–1409. The activation of vascular smooth muscle cells (SMCs) in neointimal hyperplasia involves signaling through receptor tyrosine kinases as well as G protein-coupled receptors. Overexpression of G protein-coupled receptor kinase-2 (GRK2) in SMCs can attenuate mitogenic signaling and proliferation in response to not only several G protein-coupled receptor agonists, but also platelet-derived growth factor (PDGF). To test whether overexpression of GRK2 could inhibit other SMC responses implicated in neointimal hyperplasia, we assessed SMC chemotaxis and mitogenic signaling evoked by PDGF and Gq-coupled receptor agonists. To test the effects of GRK2 overexpression on neointimal hyperplasia in vivo, we employed a rabbit autologous vein graft model system. GRK2 overexpression reduced PDGF-promoted SMC chemotaxis by 85% (P<0.01), but had no effect on chemotaxis promoted by epidermal growth factor (EGF). Congruently, GRK2 overexpression reduced by ∼50% (P<0.05) the [3H]thymidine incorporation induced by combinations of PDGF and Gq-coupled receptor agonists, but had no effect on that induced by PDGF plus EGF. PDGF-, but not EGF-promoted phosphoinositide 3-kinase activity in SMCs was also inhibited by GRK2 overexpression. In rabbit vein grafts, we achieved GRK2 overexpression in medial SMCs, reduced cell proliferation during the first week after graft implantation, and reduced steady state neointimal thickness by 29% (P<0.01), without affecting medial thickness or potentiating SMC apoptosis. Because of its ability to dampen chemotactic and mitogenic signaling through PDGF and Gq-coupled receptors, GRK2 overexpression in SMCs may be a useful therapeutic approach for neointimal hyperplasia.

Early loss of thrombomodulin expression impairs vein graft thromboresistance: implications for vein graft failure.

Thrombosis is the major cause of early vein graft failure. Our aim was to determine whether alterations in the expression of the anticoagulant proteins, thrombomodulin (TM) and the endothelial cell protein C receptor (EPCR), impair endothelial thromboresistance that may contribute to vein graft failure. Immunohistochemical staining of autologous rabbit vein graft sections revealed that the expression of TM, but not EPCR, was reduced significantly early after graft implantation. Western blot analysis revealed that TM expression was reduced by >95% during the first 2 weeks after implantation, with gradual but incomplete recovery by 42 days. This resulted in up to a 95% reduction in the capacity of the grafts to activate protein C and was associated with an increase in bound thrombin activity, which peaked on day 7 at 28.7 +/- 3.8 mU/cm(2) and remained elevated for more than 14 days. Restoration of TM expression using adenovirus vector-mediated gene transfer significantly enhanced the capacity of grafts to activate protein C and reduced bound thrombin activity on day 7 to levels comparable to that of normal veins (5.7 +/- 0.4 versus 5.2 +/- 1.1 mU/cm(2), respectively, P=0.74). Surprisingly, neointima formation was not affected by this inhibition of local thrombin activity. These data suggest that the early loss of TM expression significantly impairs vein graft thromboresistance and results in enhanced local thrombin generation. Although enhanced local thrombin generation may predispose to early vein graft failure due to thrombosis, it does not seem to contribute significantly to late vein graft failure due to neointimal hyperplasia.

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.

Long-term stabilization of vein graft wall architecture and prolonged resistance to experimental atherosclerosis after E2F decoy oligonucleotide gene therapy

Objective: We tested the hypothesis that a single intraoperative transfection of rabbit vein grafts with a decoy oligonucleotide that blocks cell-cycle gene transactivation by the transcription factor E2F induces long-term stable adaptation that involves medial hypertrophy and a resistance to neointimal hyperplasia and atherosclerosis. Methods: Jugular vein to carotid artery interposition vein grafts in hypercholesterolemic rabbits were treated, using pressure-mediated delivery, with either E2F decoy oligonucleotide, scrambled oligonucleotide, or vehicle alone. E2F decoy inhibition of cell-cycle gene expression was determined by measuring proliferating cell nuclear antigen upregulation and bromodeoxyuridine incorporation in vascular smooth muscle cells. Neointimal hyperplasia and atherosclerosis were compared between groups at 6 months after operation. Wall stress was derived from the ratio of luminal radius to wall thickness. Normal rabbits exposed to 6 weeks of diet-induced hypercholesterolemia starting 6 months after operation were analyzed in the same manner. Results: The E2F decoy oligonucleotide, but not scrambled oligonucleotide or vehicle alone, inhibited proliferating cell nuclear antigen expression and smooth muscle cell proliferation. Furthermore, this manipulation of cell-cycle gene expression yielded an inhibition of neointimal hyperplasia and atherosclerotic plaque formation throughout the 6 months of cholesterol feeding. In normocholesterolemic rabbits, vehicle-treated and scrambled oligonucleotide-treated vein grafts remain susceptible to diet-induced atherosclerosis as well, whereas resistance to this disease induction remained stable in genetically engineered grafts. Conclusion: A single intraoperative pressure-mediated delivery of E2F decoy effectively provides vein grafts with long-term resistance to neointimal hyperplasia and atherosclerosis. These findings suggest that long-term reduction in human vein graft failure rates may be feasible with this ex vivo gene therapy approach. (J Thorac Cardiovasc Surg 2001;121:714-22)