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

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.     

Transforming growth factor-beta1 induces apoptosis in vascular endothelial cells by activation of mitogen-activated protein kinase

BACKGROUND: Vascular endothelial cell apoptosis is central in atherosclerosis and intimal hyperplasia. Transforming growth factor (TGF)-beta1 induces endothelial cell apoptosis through unidentified mechanism(s). Although TGF-beta1 signals through the Smad proteins, in some nonendothelial cell types it also activates the mitogen-activated protein kinase (MAPK) (extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAPK [p38(MAPK)]). p38(MAPK) relays apoptotic signals in several cell types. We hypothesized that TGF-beta1 activates endothelial cell MAPKs and induces apoptosis through p38(MAPK) activation. METHODS: Human umbilical vein or bovine capillary endothelial cells were incubated with TGF-beta1 for 0.5 to 12 hours. MAPK activation was characterized by Western blotting with antibodies to phosphorylated extracellular signal-regulated kinase 1/2, p38(MAPK), or c-Jun N-terminal kinases 1/2. To study apoptosis, extracts of cells incubated with TGF-beta1 for 6 hours with or without MAPK inhibitors were characterized by Western blotting analysis of poly (ADP-Ribose) polymerase degradation. RESULTS: TGF-beta1 induced p38(MAPK), extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase 1/2 activation and increased apoptosis. Inhibition of p38(MAPK) significantly reduced TGF-beta1-induced apoptosis. In contrast, inhibition of other signaling pathways was ineffective. CONCLUSIONS: TGF-beta1 induces endothelial cell apoptosis through p38(MAPK) activation. Because TGF-beta1 is upregulated in vascular remodeling, p38(MAPK) is a potential target to prevent endothelial cell apoptosis during this process.     

Activation of mitogen-activated protein kinases during preparation of vein grafts and modulation by a synthetic inhibitor

OBJECTIVE: Long-term durability of saphenous vein grafts used for coronary artery bypass grafting is limited by neointimal formation. Arterial vascular injury is known to activate intracellular mitogen-activated protein kinases, including extracellular signal-regulated kinases and c-jun N-terminal kinases, that affect cell differentiation, proliferation, migration, and apoptosis. This study tests the hypothesis that these mitogen-activated protein kinases are activated in saphenous veins during preparation for coronary artery bypass grafting. METHODS: Saphenous veins were harvested from 10 patients undergoing coronary artery bypass grafting. A specimen from each vein was placed in ice-cold lysis buffer immediately after harvesting (t = 0). The remaining tissue was incubated at room temperature in normal saline, 0.1% dimethylsulfoxide (vehicle), or 50 mmol/L PD98059 (mitogen-activated protein kinase kinase-1/2 inhibitor) until the vein was grafted (mean 50 minutes). To study kinetics of intracellular signaling pathways, canine saphenous veins were harvested, and mitogen-activated protein kinases and PI-3 kinase pathways were studied after different incubation time intervals. Extracted proteins were analyzed by Western blotting or in vitro kinase assay. RESULTS: The human saphenous veins showed elevated levels of active extracellular signal-regulated kinase after harvesting (t = 0) and prior to implant (t = 1). Incubation with PD98059 resulted in decreased activation of extracellular signal-regulated kinase. Kinetics of canine saphenous veins showed extracellular signal-regulated kinase and c-jun N-terminal kinase activation, in a time-dependent manner, along with activation of the growth factor-regulated PI3 kinase pathway. CONCLUSIONS: This study characterizes activation of extracellular signal-regulated kinases and c-jun N-terminal kinases during vein graft preparation and demonstrates the ability to inhibit extracellular signal-regulated kinase activation by simple incubation with a specific inhibitor. Further studies are needed to evaluate the significance of these findings with respect to graft durability.     

Neurotoxicity of Lidocaine Involves Specific Activation of the p38 Mitogen-activated Protein Kinase, but Not Extracellular Signal-regulated or c-jun N-Terminal Kinases, and Is Mediated by Arachidonic Acid Metabolites

Background: Pharmacologic inhibition of the p38 mitogen-activated protein kinase (MAPK) leads to a reduction in lidocaine neurotoxicity in vitro and in vivo. The current study investigated in vitro the hypotheses that lidocaine neurotoxicity is specific for dorsal root ganglion cells of different size or phenotype, involves time-dependent and specific activation of the p38 MAPK, that p38 MAPK inhibitors are only effective if applied with local anesthetic, and that p38 MAPK activation triggers activation of lipoxygenase pathways.

Methods: The authors used primary sensory neuron cultures and pheochromocytoma cell line cultures to detect time-dependent activation of the p38 MAPK or related pathways such as extracellular signal-regulated kinases and c-jun N-terminal kinases. Cells were divided by size or by immunoreactivity for calcitonin gene-related peptide or isolectin B4, indicative of nociceptive phenotype. The authors also investigated whether arachidonic acid pathways represent a downstream effector of the p38 MAPK in local anesthetic-induced neurotoxicity.

Results: All types of dorsal root ganglion cells were subject to neurotoxic effects of lidocaine, which were mediated by specific activation of the p38 MAPK but not extracellular signal-regulated kinases or c-jun N-terminal kinases. Neuroprotective efficacy of p38 MAPK inhibitors declined significantly when administered more than 1 h after lidocaine exposure. Activation of p38 MAPK preceded activation of arachidonic acid pathways. Neurotoxicity of lidocaine, specific activation of p38 MAPK, and neuroprotective effects of a p38 MAPK inhibitor were further confirmed in pheochromocytoma cell line cultures.     

Neurotoxicity of lidocaine involves specific activation of the p38 mitogen-activated protein kinase, but not extracellular signal-regulated or c-jun N-terminal kinases, and is mediated by arachidonic acid metabolites

BACKGROUND: Pharmacologic inhibition of the p38 mitogen-activated protein kinase (MAPK) leads to a reduction in lidocaine neurotoxicity in vitro and in vivo. The current study investigated in vitro the hypotheses that lidocaine neurotoxicity is specific for dorsal root ganglion cells of different size or phenotype, involves time-dependent and specific activation of the p38 MAPK, that p38 MAPK inhibitors are only effective if applied with local anesthetic, and that p38 MAPK activation triggers activation of lipoxygenase pathways. METHODS: The authors used primary sensory neuron cultures and pheochromocytoma cell line cultures to detect time-dependent activation of the p38 MAPK or related pathways such as extracellular signal-regulated kinases and c-jun N-terminal kinases. Cells were divided by size or by immunoreactivity for calcitonin gene-related peptide or isolectin B4, indicative of nociceptive phenotype. The authors also investigated whether arachidonic acid pathways represent a downstream effector of the p38 MAPK in local anesthetic-induced neurotoxicity. RESULTS: All types of dorsal root ganglion cells were subject to neurotoxic effects of lidocaine, which were mediated by specific activation of the p38 MAPK but not extracellular signal-regulated kinases or c-jun N-terminal kinases. Neuroprotective efficacy of p38 MAPK inhibitors declined significantly when administered more than 1 h after lidocaine exposure. Activation of p38 MAPK preceded activation of arachidonic acid pathways. Neurotoxicity of lidocaine, specific activation of p38 MAPK, and neuroprotective effects of a p38 MAPK inhibitor were further confirmed in pheochromocytoma cell line cultures. CONCLUSIONS: Specific and time-dependent activation of the p38 MAPK is involved in lidocaine-induced neurotoxicity, most likely followed by activation of lipoxygenase pathways.     

Reduction of intimal hyperplasia and enhanced reactivity of experimental vein bypass grafts with verapamil treatment.

Recent studies have shown that calcium antagonists exert an antiatherogenic effect in animals fed cholesterol. Vein graft intimal hyperplasia is believed to be an early event in atherosclerotic lesion formation, which is a significant cause of graft failure. Altered vasoreactivity has also been postulated in the etiology of vein graft failure. Therefore this study examined the effect of verapamil treatment on the development of intimal hyperplasia and the vasoreactivity of experimental vein bypass grafts. The right external jugular vein was grafted into the right carotid artery of 30 male New Zealand white rabbits fed normal rabbit chow. The left external jugular vein was used as the control vein. Fifteen animals received verapamil (1.25 mg/day for 28 days) via the femoral vein by means of an osmotic pump. In 15 control animals the pump contained saline. Plasma verapamil concentration was 50.9 +/- 13.2 ng/mL (x +/- SEM), a dose that showed no effect on either blood pressure, total serum cholesterol, or in vitro platelet aggregation to ADP. Fourteen of fifteen grafts were patent in each group, for a patency rate of 93%. Histologic examination using computer morphometry showed significant reduction of intimal hyperplasia at the proximal, middle, and distal graft segments (p less than 0.05). In addition in vitro isometric tension studies of the vein grafts and control veins showed that verapamil causes enhanced reactivity of both vein grafts and control veins in response to norepinephrine and histamine (p less than 0.05). Reactivity of vein grafts to serotonin was unaltered. While none of the normal veins in the control group responded to serotonin, normal veins treated with verapamil contracted readily in response to serotonin. Endothelial-dependent relaxation to acetylcholine was absent in both control and verapamil-treated vein grafts, while normal veins from both groups responded to the same extent to acetylcholine. Because we could not demonstrate any difference in platelet or endothelium function between untreated and verapamil-treated animals, we examined the direct effect of verapamil on smooth muscle. Verapamil significantly inhibited [3H]-thymidine incorporation into DNA in vascular smooth muscle cells in culture in a dose-dependent manner. Verapamil treatment significantly reduces intimal hyperplasia in experimental vein grafts and inhibits smooth muscle cell proliferation in culture. Furthermore the enhanced reactivity to norepinephrine and histamine in the verapamil-treated vessels has no detrimental effect on the patency rate at 4 weeks. Thus by inhibiting intimal hyperplasia, calcium antagonists may improve the long-term patency of vein bypass grafts.     

Mechanical endothelial damage results in basic fibroblast growth factor-mediated activation of extracellular signal-regulated kinases.

BACKGROUND: Endothelial damage, such as that associated with balloon angioplasty or preparation of veins for bypass grafts, results in intimal hyperplasia. Growth factors and cytokines that modulate endothelial cell functions through various intracellular signaling pathways mediate rapid endothelial repair, which may prevent or reduce restenosis. Here we investigated the effect of mechanical injury of endothelial cells on the mitogen-activated kinase signaling pathways, extracellular-signal-regulated kinases (ERKs), C-Jun N-terminal kinase (JNK/SAPK), and p38. METHODS: Confluent human umbilical vein endothelial cells or bovine aortic endothelial cells were wounded with a razor blade; mitogen-activated kinase activation was monitored by immunoblotting with antibodies to active ERK, JNK/SAPK, or p38. RESULTS: Wounding of human umbilical vein endothelial cell or bovine aortic endothelial cell monolayers resulted in rapid (5-minute) activation of ERK-1 and -2, which was abolished by monoclonal antibody to basic fibroblast growth factor (FGF-2). This antibody or an inhibitor of ERK activation, PD98059, also blocked endothelial cell migration after the wounding. Thus FGF-2-induced ERK activation mediates the endothelial response to wounding. CONCLUSIONS: ERK-1 and -2 are activated by FGF-2 released from endothelial cells in response to injury. Therapeutic strategies aimed at reducing FGF-2-induced intimal hyperplasia should preserve ERK activation in endothelial cells while abolishing it in smooth muscle cells.     

Reduction of lipid peroxidation with intraoperative superoxide dismutase treatment decreases intimal hyperplasia in experimental vein grafts.

BACKGROUND: Vein graft failure is commonly attributed to the development of intimal hyperplastic lesions. Oxidative stress has been implicated in the initiation and progression of atherosclerosis. In this study we examined the effects of local intraoperative treatment with polyethylene glycolated superoxide dismutase (PEG-SOD) on lipid peroxidation and on the development of intimal hyperplasia in experimental vein grafts. MATERIALS AND METHODS: Forty-one New Zealand White male rabbits had a right carotid interposition bypass graft using the ipsilateral reversed jugular vein. Sixteen animals received local PEG-SOD (4,100 units) treatment; 9 animals received the polyethylene glycol (PEG) vehicle without SOD; 16 animals were used as controls. Postoperatively, malondialdehyde (MDA, a product of lipid peroxidation) concentration and SOD activity were assessed in 3-day vein grafts by colorimetric spectrophotometry. To determine wall dimensions and vasomotor function, morphometric and isometric tension studies were performed on 28-day vein grafts. RESULTS: MDA concentration was increased 5. 7-fold (P < 0.05) in 3-day control vein grafts compared to ungrafted jugular veins. Intraoperative PEG-SOD treatment raised SOD activity 5.0-fold (P < 0.05) and reduced MDA concentration 8-fold (P < 0.05) in 3-day vein grafts compared to controls. At 28 days, intimal thickness was reduced by 35% with PEG-SOD treatment (54 +/- 4 vs 83 +/- 5; P < 0.001) compared to control vein grafts, without a change in medial thickness (77 +/- 4 vs 88 +/- 5; P = ns). The vasomotor functions of 28-day PEG-SOD-treated vein grafts to norepinephrine, serotonin, bradykinin, nitroprusside, and acetylcholine were not significantly changed when compared to controls. Treatment with PEG alone did not significantly alter lipid peroxidation, wall dimensions, or vasomotor function of vein grafts. CONCLUSION: This study demonstrates that intraoperative local treatment of vein grafts with PEG-SOD increases SOD activity and decreases lipid peroxidation for at least 3 days, resulting in reduced intimal hyperplasia at 28 days. These findings further implicate oxidative stress in the hyperplastic response of vein grafts and suggest a potential therapeutic role for PEG-SOD in the prevention of vein graft failure.     

Patterns of kinase activation induced by injury in the murine femoral artery

BACKGROUND: Intimal hyperplasia remains the principal lesion in the development of restenosis after vessel wall injury. Cell signaling in vascular smooth muscle cells remains a potential molecular target to modulate the development of intimal hyperplasia. The aim of this study was to define a baseline pattern of histological changes and kinase activation in a murine model. METHODS: The murine femoral wire injury model was used in which a microwire was passed through a branch of the femoral artery and used to denude the common femoral artery. Pluronic gel was used to apply mitogen-activated protein kinases (MAPK) inhibitors (PD98059, SB230580, and SP600125) on the exterior of the vessels. Specimens were perfusion-fixed and sections were stained for morphometry using an ImagePro system. Additional specimens of femoral artery were also harvested and snap frozen for Western blotting and zymography to allow for the study of kinase and protease activation. Contralateral vessels were used as controls. RESULTS: The injured femoral arteries developed intimal hyperplasia, which is maximal at 28 days and does not change substantially between day 28 and day 56. Sham-operated vessels did not produce such a response. Cell apoptosis peaked within 3 days and cell proliferation peaked at 7 days after injury. There is a time-dependent increase in kinase activity immediately after injury. MEK1/2 activation peaks at 20 min after injury and is followed by a peak in extracellular signal-regulated kinase-1/2 activation at 45 min. The stress kinases p38(MAPK) and JNK peak between 10 and 20 min. Activation of akt is later at 45 min and 120 min and activation of p70S6K was biphasic. There was a time-dependent increase in uPA/PAI-1 expression and activity after injury. Local application of MAPK inhibitors (PD98059, SB230580, and SP600125) within a pluronic gel reduced respective MAPK activity, decreased cell proliferation and enhanced cell apoptosis, increased PAI-1, and decreased uPA expression and activity; at 14 days there was a decrease in intimal hyperplasia. CONCLUSIONS: These data demonstrate that femoral wire injury in the mouse induces a consistent model of intimal hyperplasia and that it is associated with a time dependent increase in signaling kinase activity. Interruption of these pathways will interrupt the uPA/PAI-1 pathway and decrease intimal hyperplasia development. Accurate characterization of cell signaling is a necessary step in the development of molecular therapeutics.     

Controlling Transplant Vasculopathy in Cryopreserved Vein Grafts with Polyethylene Glycol and Glutathione During Transport

BACKGROUND: the biological characteristics of cryopreserved allografts are poorly understood, although many factors are known to influence their outcome. This study examines the development of transplant vasculopathy in both fresh and cryopreserved vein allografts and specifically assesses the efficacy of a transport solution containing 10% polyethylene glycol and 10 microM glutathione (PEG/GSH). METHODS: jugular veins were harvested from control donor rabbits and transplanted as interposition carotid bypass grafts in 30 New Zealand White (NZW) rabbits. Ten received the fresh jugular veins (fresh). Ten animals received jugular veins which had been harvested, transported in a physiological solution, cryopreserved and stored in a standard fashion (cryopreserved). Ten animals received jugular veins which had been harvested, transported in the same solution with the addition of PEG/GSH, cryopreserved and stored in a standard fashion (PEG/GSH). Cryopreserved jugular veins were stored for 6 weeks before transplantation. All animals were sacrificed 28 days postoperatively. Vein grafts were perfusion-fixed and wall dimensions were determined by planimetry. RESULTS: all transplanted grafts were patent at harvest. The control cryopreserved vein grafts showed a 54% increase in mean intimal thickness (63+/-10 micron vs. 41+/-3 micron p<0.05) but no change in mean medial thickness (125+/-9 micron vs. 119+/-13 micron; p = N.S. ) compared to the fresh allograft. Transport of the grafts in PEG/GSH solution resulted in the abolition of the increase in intimal thickness (41+/-4 micron; p <0.01) associated with cryopreservation without a change in medial thickness (140+/-15 micron; p = N.S.) compared to the cryopreserved allograft. CONCLUSION: cryopreserved vein grafts develop significant intimal hyperplasia compared to freshly transplanted grafts. The use of PEG/GSH in the transport solution significantly reduces this transplant graft intimal hyperplasia to that which develops in fresh grafts and may lead to improvements in the clinical use of cryopreserved veins.     

Antisense oligonucleotides to c-fos and c-jun inhibit intimal thickening in a rat vein graft model.

BACKGROUND: C-fos and c-jun are 2 immediate early genes that have been implicated in the stimulation of vascular smooth muscle cell proliferation and migration. In previous experiments in our laboratory with a rat vein graft model a 2- to 3-fold increase of messenger RNA of c-fos and c-jun were noted 1 hour after vein graft perfusion. Because c-fos and c-jun are up-regulated after the perfusion of vein grafts, the purpose of this study was to delineate the temporal expression of c-fos and c-jun protein and to study the effect of antisense oligonucleotides (ASO) to c-fos and c-jun on intimal thickening observed in this model. METHODS: Sprague-Dawley rats underwent bilateral interposition femoral artery grafts with use of the superficial epigastric vein, which was harvested from 15 minutes up to 2 weeks and analyzed by Western blot for Fos and Jun protein. Additional rats underwent bypasses and at the time of the procedure 1 graft was treated with a pluronic gel containing an ASO to c-fos, c-jun, or sense and the contralateral side was treated with pluronic gel only. The vein grafts were harvested 2 weeks after the procedure and perfusion fixed. After longitudinal sectioning, the intimal and total wall thicknesses were measured in the perianastamotic and midgraft regions by a morphometric digitizing microscope and the statistics were analyzed by a paired Student's t test. RESULTS: Protein analysis by Western blot showed that c-fos levels rose quickly within 2 hours and leveled at 6 hours 40-fold above basal levels after vein graft perfusion. Similarly, c-jun levels rose 10-fold above basal levels after 15 minutes and peaked at 2 hours 120-fold above basal levels. The treatment of the vein grafts with these ASOs resulted in a reduction of about 30% in the thickness of the intimal layer and the total wall thickness in both the perianastomotic and the midgraft regions, which was statistically significant different from control veins. CONCLUSION: These results indicate a possible therapeutic role for ASO to immediate early genes in the treatment of vein graft intimal hyperplasia.     

Molecular mechanisms of cell death of mycophenolic acid-treated primary isolated rat islets: implication of mitogen-activated protein kinase activation

Optimal immunosuppression after pancreas islet transplantation has not yet been established to achieve long-term graft survival. Mycophenolic acid (MPA) is widely used as an immunosuppressive drug after transplantation including among recipients of pancreas islet cells. Previously, we reported MPA-induced islet apoptosis in the HIT-T15 cell line. In this study, we confirmed the effects of MPA on cell death and its potential implications on the mitogen-activated protein kinase (MAPK) family expression levels in primary isolated rat islets. Lewis islets isolated by collagenase digestion were purified by the density gradient method. Cell death was analyzed by methylthiazoletetrazolium assay. Activation of MAPK kinase 4 (MKK4), c-jun N-terminal protein kinase (JNK), p38 MAPK, and caspase-3 cleavage was examined by Western blot analyses. MPA treatments (>25 μmol/L) increased cell death significantly at 24 hours and in a dose-dependent manner activated MKK4, JNK, and p38 MAPK at 20 hours. Caspase-3 cleavage was also increased by MPA treatment. These results suggested that MPA induced significant cell death among primary isolated rat islets by activation of MKK4, JNK, and p38 MAPK, as well as caspase-3 cleavage.     

Prevention of smooth muscle cell phenotypic modulation in vein grafts: a histomorphometric study.

This is a prospective study of the relationship between graft preparation technique and the subsequent morphologic fate of vein grafts. Paired vein grafts (optimal vs injury prepared) were placed in a canine model and removed over time. Vein grafts intentionally injured by warm saline storage demonstrated endothelial and smooth muscle cell damage. In the acute postimplantation period, platelet adhesion and white cell infiltration of the graft were present. By 7 days, the endothelium had "healed", but the underlying smooth muscle cells had modulated and were of the transitional or synthetic phenotype. This persisted at 30 days, but by 60 days the graft wall had remodeled to a contractile smooth muscle cell phenotype. Changes in the extracellular matrix were greatest at 30 days corresponding to changes in the smooth muscle cell phenotype. None of these injurious responses were noted in optimally prepared, papaverine treated vein grafts. The combined intima and media (lumen to adventitial edge) was measured at baseline and at graft excision with use of digitized graphic techniques. The intimal/medial thickness of injured vein graft walls was always greater than that of pair-matched optimally treated vein grafts (p less than 0.01 analysis of variance). Optimal preparation of vein grafts is effective in minimizing endothelial and smooth muscle cell injury at the time of arterial reconstruction. This preservation of endothelial and smooth muscle cell integrity prevents subsequent morphologic changes associated with the "arterialization response".     

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 microm2; RT: 6.452 microm2, p < 0.0007). In contrast, RT augmented intimal hyperplasia in vein grafts (median: C: 30.287 microm2; RT: 90.455 microm2, p < 0.014). Vein graft diameters after RT were enlarged (median: C: 2.098 microm; 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.     

Cod-liver oil in the prevention of intimal hyperplasia in autogenous vein grafts used for arterial bypass

Cod-liver oil, rich in eicosapentaenoic acid, an unsaturated fatty acid, was administered to 14 mongrel dogs to determine if this acid would prevent platelet-mediated intimal hyperplasia. Twenty-eight 1 cm segments of undistended jugular vein were interposed between bilaterally divided femoral arteries. Seven control animals were fed a 2% cholesterol diet 1 week before and for 6 weeks after the operation. A further seven animals received cod-liver oil capsules containing 1.8 gm of eicosapentaenoic acid daily 1 week before and for 6 weeks after autogenous vein implantation, in addition to the lipid-supplemented diet. Baseline serum cholesterol was 4.6 +/- 0.4 mmol/L. The rise in serum cholesterol was similar in the two groups and increased to 7.4 +/- 0.6 mmol/L (control group) and to 6.8 +/- 0.2 mmol/L (eicosapentaenoic acid group) (p less than 0.001). Prothrombin time, partial thromboplastin time, bleeding time, and platelet counts were unchanged in the two groups. Vein grafts, harvested at 6 weeks, were fixed in formaldehyde. Mean intimal thickness was measured from multiple vein graft cross sections with a Zeiss computerized interactive image analyzing system. A mean of 140 +/- 11 measurements were computed from each graft. Marked intimal hyperplasia occurred in the control group and increased from 4.3 +/- 0.3 to 86.4 +/- 14 micron. In contrast, a high eicosapentaenoic acid diet inhibited intimal hyperplasia, with intimal thickness only increasing from 4.0 +/- 0.4 to 24.8 +/- 2.7 micron (p less than 0.001). These data indicate that eicosapentaenoic acid inhibits platelet-mediated intimal hyperplasia and suggest that cod-liver oil could be used to prevent intimal hyperplasia in vein grafts used for myocardial revascularization.     

Hydrophilic statin suppresses vein graft intimal hyperplasia via endothelial cell-tropic Rho-kinase inhibition

BACKGROUND: Recent studies suggest that statins can protect the vasculature in a manner that is independent of their lipid-lowering activity through inhibition of the small guanosine triphosphate-binding protein, Rho, and Rho-associated kinase. Little information is available on the inhibitory effect of statins on vein graft intimal hyperplasia, the main cause of late graft failure after bypass grafting. We therefore examined the effects of a hydrophilic statin on vein graft intimal hyperplasia in vivo and Rho-kinase activity in vitro. METHODS: In the first experiment, rabbits were randomized to a control group (n = 7) that was fed regular rabbit chow or to a pravastatin group (n = 7) that was fed regular rabbit chow supplemented with 10 mg/kg pravastatin sodium. The branches of the jugular vein were ligated and an approximately 3-cm segment of the jugular vein was taken for an autologous reversed-vein graft. The carotid artery was cut and replaced with the harvested autologous jugular vein. At 2 and 4 weeks after the operation, vein grafts in both groups were harvested, and intimal hyperplasia of the vein grafts was assessed. In the second experiment, human umbilical vein endothelial cells and vascular smooth muscle cells were cultured and then treated with 1 micromol/L and 30 micromol/L pravastatin for 24 hours and harvested. Immunoblotting was performed on the resulting precipitates. Quantitative evaluation of phosphorylated myosin binding subunit and endothelial nitric oxide synthase was performed by densitometric analysis. RESULTS: We demonstrated that oral administration of the hydrophilic statin pravastatin to normocholesterolemic rabbits inhibited intimal hyperplasia of carotid interposition-reversed jugular vein grafts 4 weeks after implantation (pravastatin group, 39.5 +/- 3.5 microm vs control group, 64.0 +/- 7.1 microm; n = 7; P < .05) and suppressed cell proliferation and apoptosis in the neointima 2 weeks after implantation. In addition, we found that pravastatin inhibited Rho-kinase activity and accelerated endothelial nitric oxide synthase expression in human umbilical vein endothelial cells but did not inhibit Rho-kinase activity in vascular smooth muscle cells. CONCLUSIONS: These novel findings clearly demonstrate that a hydrophilic statin can suppress intimal hyperplasia of the vein graft in vivo and also show endothelial cell-tropic inhibition of Rho-kinase in vitro. Furthermore, these results strongly support the clinical use of hydrophilic statins to prevent intimal hyperplasia of the vein graft after bypass grafting. CLINICAL RELEVANCE: Late graft failure caused by neointimal hyperplasia limits the efficacy of vein grafting. Various treatments were examined to reduce neointimal hyperplasia, but a standard clinical treatment has not yet been established. We report here the inhibitory effect of pravastatin on the development of vein graft intimal hyperplasia. In addition, we demonstrate that pravastatin showed endothelial cell-tropic benefits through both the inhibition of Rho-kinase activity and acceleration of eNOS expression in vitro. Because the clinical benefits and safety of pravastatin have been established to a certain extent through long-term clinical usage, pravastatin may soon become standard treatment after vein bypass grafting.     

大鼠自体移植静脉差异表达microRNA及其生物信息学分析

目的：通过分析大鼠自体移植静脉microRNA（miRNA）表达谱,探讨miRNA对移植静脉内膜增生的调控机制。 方法：将SD大鼠自体颈外静脉移植到肾下腹主动脉制作静脉移植模型,分别术后3、14 d取移植静脉,以正常SD大鼠颈外静脉为对照,提取总RNA后用高通量测序技术检测miRNA表达谱,筛选差异表达miRNA并预测其靶基因,进行靶基因的GO富集分析和KEGG富集分析。 结果：与正常静脉比较,术后14 d的移植静脉差异表达miRNA总数为265,其中表达上调的miRNA有183个,表达下调的miRNA有82个;术后14 d与术后3 d的移植静脉间比较,差异表达的miRNA总数158,其中表达上调的miRNA有94个,表达下调的miRNA有64个。miRNA靶基因GO功能分析显示,富集的基因主要参与了DNA转录过程的调节、细胞间信号转导的调控和蛋白质磷酸化过程的调节;KEGG通路分析显示,富集的信号通路主要有MAPK信号通路、cAMP信号通路、Wnt信号通路、紧密连接、cGMP-PKG信号通路。 结论：miRNA通过复杂的调控网络参与移植静脉内膜增生的生物学过程。所发现的miRNA及其调控网络可能为移植静脉内膜增生的机制研究与干预提供参考。     

Reduction of vein graft intimal hyperplasia and preservation of endothelium-dependent relaxation by topical vascular endothelial growth factor

Purpose: Recent evidence suggests that vascular endothelial growth factor (VEGF), in addition to stimulating angiogenesis, also serves a repair/maintenance or survival function, modulating various aspects of endothelial cell function. This study was designed to examine the effect of VEGF pretreatment in a model of vein graft intimal hyperplasia. Methods: Reversed jugular vein–to–common carotid artery interposition grafts were constructed in New Zealand White rabbits. Vein conduits were immersed in solution containing 500 mg rhVEGF165 or saline solution for 20 minutes before implantation. Twenty-eight days later the vein grafts and contralateral control jugular veins were harvested for either histologic or isometric tension studies. Results: VEGF-treated vein grafts showed a 23% reduction in intimal area (0.76 ± 0.07 mm² vs 0.98 ± 0.06 mm²; p = 0.028) and a 30% reduction in intimal thickness (62 ± 6 μm vs 89 ± 5 μm; p = 0.001) when compared with control grafts. After precontraction with norepinephrine, the maximal relaxation to acetylcholine (endothelium-dependent, receptor-mediated agonist) for control vein grafts was 0%, whereas for VEGF-treated vein grafts it was 25% ± 9% (p < 0.05 vs control grafts). The maximal relaxation to the calcium ionophore A23187 (endothelium-dependent, receptor-independent agonist) was also greater in VEGF-treated grafts than in control grafts (172.3% ± 19.4% vs 122.5% ± 13.7%; p < 0.05). There was no difference in the response to sodium nitroprusside (endothelium-independent agonist) between the two groups. Conclusions: A single topical application of VEGF before implantation reduces intimal hyperplasia and improves endothelial function in a rabbit vein graft model. Further evaluation of this simple strategy to improve vein graft patency appears warranted. (J Vasc Surg 1998;27:167-73.)     

Effects of a p38 mitogen-activated protein kinase inhibitor as an additive to university of wisconsin solution on reperfusion injury in liver transplantation

BACKGROUND: Activation of p38 mitogen-activated protein kinase (MAPK) plays an important role in the development of ischemia/reperfusion injury in nonhepatic organs, such as the heart. However, the role of p38 MAPK activation in the liver is unclear. We examined the effects of FR167653, a novel p38 MAPK inhibitor, as an additive to University of Wisconsin (UW) solution in rat liver transplantation. METHODS: Rat orthotopic liver transplantation was performed after 30 hr of cold storage using UW solution with or without FR167653. Ten-day survival rates, serum alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) levels, liver tissue blood flow, histological findings, and activities of p38 MAPK and p46/p54 c-Jun N-terminal kinase (JNK) in liver grafts were evaluated. RESULTS: The addition of FR167653 significantly increased animal survival rates. FR167653 significantly suppressed serum ALT and LDH levels and improved liver tissue blood flow after transplantation. FR167653 also ameliorated histological damage to the liver graft. Neither p38 MAPK nor p46/p54 JNKs was activated during cold storage, whereas both were markedly activated within 30 min of reperfusion and remained activated until 60 min after reperfusion. FR167653 inhibited the activation of p38 MAPK both 30 and 60 min after reperfusion, but it did not affect the activation of p46/p54 JNKs. CONCLUSIONS: The addition of FR167653 to UW solution improved liver graft viability and animal survival rates associated with the inhibition of p38 MAPK activation. These results suggest that inhibiting the activation of p38 MAPK may attenuate ischemia/reperfusion injury in liver transplantation.