Photodynamic therapy of vein grafts: Suppression of intimal hyperplasia of the vein graft but not the anastomosis

Purpose: There is no clinically useful therapy for the suppression of vein bypass graft intimal hyperplasia (IH). Photodynamic therapy (PDT), a technique that uses light to activate otherwise biologically inert photosensitizers to produce cytotoxic effects, has been demonstrated to successfully inhibit experimental IH in balloon-injured arteries. The purpose of this study was to investigate the efficacy of PDT as a method to reduce vein graft IH.Methods: Reversed external jugular vein bypass grafts of the common carotid artery were performed in 28 male Sprague-Dawley rats. The animals received either chloroaluminum sulfonated phthalocyanine (2.5 mg/kg intravenously) 24 hours before the ex vivo irradiation of the vein grafts (VG) with 100 joule/cm² at 675 nm (PDT VG) or saline solution as control (CON VG). Preharvest bromodeoxyuridine was administered to label proliferating cells. All vein grafts were perfusion fixed within 96 hours for a pilot study or at 2 and 4 weeks for the main study. Histology, immunohistochemistry, and morphometric analysis were performed.Results: There was no acute thrombus formation in the hypocellular PDT VG with occasional platelets but no leukocytes adherent to the luminal surface. Intimal areas of the PDT VG were 18% at 2 weeks and 53% at 4 weeks of the CON VGs (p < 0.05). Medial areas and percent of stenoses were also significantly less in PDT than in CON VG. However, intimal hyperplasia noted in the longitudinal sections within 2 mm of the anastomoses did not demonstrate a difference between PDT and CON VG. Intimal hyperplasia of both PDT and CON VG consisted of smooth muscle cells, verified by immunohistochemistry. Bromodeoxyuridine-labeled cells were more abundant in 2-week than in 4-week specimens, were found most frequently in the intimal areas of the CON VG body, and were equivalent in the anastomoses of PDT VG and CON VG.Conclusions: These data suggest that PDT of vein grafts suppresses the development of IH in the body of the vein graft but does not affect IH adjacent to the anastomoses. The artery may be the source of proliferating smooth muscle cells that contribute to the anastomotic vein graft IH. (J VASC SURG 1995;21:882-90.)     

Effect on vein graft intimal hyperplasia of nuclear factor-kB decoy transfection using the second generation of HVJ vector

AIM: Vein graft stenosis due to intimal hyperplasia (IH) is the main cause of graft failure. We examined possibilities of nuclear factor-kB (NF-kB) expression in vein grafts, and inhibitive effects of NF-kB decoy on the gene expression and subsequent vein graft IH. METHODS: Fifteen mongrel dogs underwent femoral artery replacement with autogenous vein grafts. Group I: grafts were retrieved at a predetermined time and subjected to NF-kB binding activity assay; Groups II and III: grafts were transfected with scrambled (II-a, III-a) or NF-kB (II-b, III-b) decoy using hemagglutinating virus of Japan envelope before implantation. Grafts were retrieved 7 days after implantation for evaluation of intercellular adhesion molecule-1 (ICAM-1) mRNA expression (Group II) and 4 weeks after implantation for comparison of IH by morphometric analysis (Group III). RESULTS: NF-kB binding activity was increased in a time-dependent manner, with a peak 2 days after implantation. The ratio between ICAM-1 and glyceraldehyde-3-phosphate dehydrogenase mRNA expression in II-b was significantly lower than that in II-a (0.347 +/- 0.07 versus 0.612+/-0.08; P = 0.047). The ratio of intimal cross-section area to luminal cross-section area of III-b was significantly lower than that of the III-a (0.096+/-0.03 versus 0.461+/-0.11; P = 0.048). CONCLUSION: NF-kB binding activity in vein grafts increases after implantation, and transfection of NF-kB decoy before implantation may reduce IH through the inhibition of ICAM-1 expression.     

An ultrastructural study of progressive intimal hyperplasia in rat vein grafts

Purpose: Intimal hyperplasia (IH) poses the greatest challenge for vein graft success. This fibroproliferative disorder causes obliterative stenosis and frequent graft occlusion. Although its causes remain poorly understood, it has been proposed that IH begins as a wound-healing response that cascades into a chronic state of unchecked proliferation. In this ultrastructural study, IH development and concomitant cell changes were evaluated in rat vein grafts.Methods: Epigastric vein–to–femoral artery grafts were placed in Lewis rats using standard microsurgical techniques. At various time points, grafts were harvested and processed for transmission electron microscopic, histologic, and immunohistochemical analyses. The proximal region, which displayed the most marked IH, was assessed for ultrastructural changes.Results: Our findings showed: (1) regeneration of the damaged endothelium by cells displaying an activated appearance; (2) early and complete smooth muscle cell death, with subsequent replacement by myofibroblastic cells; (3) extensive and sustained graft infiltration by monocytes/macrophages; and (4) intramural fibrin deposition.Conclusions: The rat vein graft wall was substantially altered after implantation into the arterial circulation. During and after IH development, the cells in the graft did not resemble cells that are present in the nongrafted epigastric vein. Marked cell death, mononuclear cell infiltration, and the presence of myofibroblastic cells suggest a state of aberrant wound healing. (J Vasc Surg 1997;26:94-103.)     

The protective effect of vein cuffed anastomoses is not mechanical in origin

Purpose: Intimal hyperplasia (IH) is a proliferative process of vascular smooth muscle cells that occurs after an arterial injury, particularly at outflow anastomoses of prosthetic bypass grafts. IH causes stenosis that leads ultimately to graft flow reduction and thrombosis. We have demonstrated previously that vein cuff interposition between an expanded polytetrafluoroethylene (e-PTFE) graft and artery at distal anastomoses diminished IH formation in the arterial outflow as compared with noncuffed anastomoses. Improved long-term patency rates associated with the placement of an interposition vein cuff at the distal anastomosis of e-PTFE grafts to infrageniculate arteries have also been demonstrated clinically. This study examined the mechanical factors that may contribute to the protective effect of cuffed anastomoses. These factors include the expansibility of the vein cuff as compared with e-PTFE, as well as the angle of the cuffed anastomosis.Methods: Compatible animals were selected by use of platelet aggregation studies. Nine dogs, group A, received a 4 mm e-PTFE graft plus a 1 cm long interposition vein cuff at the distal anastomosis in the left carotid artery. The same procedure was done on the right side, and in addition the vein cuff was encircled by an e-PTFE jacket incorporated into the anastomosis to prevent the expansion of the vein cuff with arterial pulsation. To study the effect of distal anastomotic angle and geometry on the formation of IH, five dogs, group B, received a 4 mm e-PTFE graft in both sides. On the left, the distal anastomosis was performed between the graft and the artery at an acute angle as it is commonly done when a bypass graft is placed. On the right side a 1 cm long, 6 mm diameter e-PTFE segment was interposed between the artery and the graft at a perpendicular angle. This geometry mimicked the right angle of a vein cuff - to-artery anastomosis. After 10 weeks the grafts were harvested, and the thickness of IH was measured with an ocular micrometer under light microscopy.Results: In group A, one dog had bilateral graft thrombosis (12%), and these grafts were discarded. In the remaining eight dogs there was no statistically significant difference in the thickness of IH between the right (jacketed group) and the left side (nonjacketed/control group), showing that vein cuff expansibility did not play a role in protecting against the formation of IH. In group B, bilateral graft thrombosis occurred in four of five dogs (80%), suggesting that the perpendicular anastomotic angle was not protective.Conclusion: These results suggested that the protective effect of the vein cuff is not mechanical in origin. (J VASC SURG 1995;21:558-66.)     

Vein interposition cuffs decrease the intimal hyperplastic response of polytetrafluoroethylene bypass grafts

Purpose: The modification of the distal anastomosis of polytetrafluoroethylene (PTFE) bypass grafts with vein interposition cuffs (VCs) has been reported to increase graft patency. However, the mechanisms that are responsible for this improved patency are unclear. Because intimal hyperplasia (IH) is a primary cause of prosthetic graft failure, we hypothesized that VCs affect the distal anastomosis by decreasing the IH response of the outflow artery. Methods: Twenty-three female domestic Yorkshire pigs (mean weight, 35 kg) underwent 42 femoral PTFE bypass grafting procedures. The PTFE bypass grafts were separated into the following three groups according to distal anastomotic configuration: end-to-side anastomoses (ES), VCs, and cuffs constructed with PTFE (PCs). Four femoral arteries from two pigs served as healthy controls. At sacrifice, the grafts were perfusion fixed, and the distal anastomoses harvested at 1 and 4 weeks. The specimens were hemisected and serially sectioned to identify the heel, toe, and mid-anastomotic regions. The sections were cut into 5-μm segments and analyzed for intima and media thickness and area, intima/media area ratio, and the distribution of IH in the vein cuff. The roles of transforming growth factor–β1 and platelet-derived growth factor–BB in IH development were assessed with immunohistochemistry. Results: IH development was significantly lower at all areas of the anastomosis, with VCs compared with ES and PCs at 4 weeks (P ≤ .001). IH decreased in VCs from 1 to 4 weeks in all areas of the anastomosis (P ≤ .001). PCs showed pronounced IH at the mid-anastomosis as compared with VCs and ES (P ≤ .001). IH was most pronounced at the toe with ES and PCs (P ≤ .001). Qualitatively, VCs altered the site of IH development, sparing the recipient artery with preferential thickening of the vein cuff and formation of a pseudointima at the vein-PTFE interface. Immunohistochemistry results showed positive staining for transforming growth factor–β1, platelet-derived growth factor–BB, and smooth muscle α-actin in the hyperplastic intima. Conclusion: PTFE bypass grafts with VCs had less IH develop than did grafts with ES and PC anastomoses. IH regression in VCs at 4 weeks suggests compensatory vessel wall remodeling mediated by the presence of the VC. Furthermore, VCs caused a redistribution of hyperplasia to the vein-PTFE interface, delaying IH-induced outflow obstruction in the recipient artery. The marked increase in IH with PCs, despite a similar geometric configuration to VCs, suggests that the biologic properties of autogenous tissue dissipate IH development. Similarly, the flow patterns in PCs and VCs should be identical, which suggests a less important role of hemodynamic forces in VC-mediated protection. (J Vasc Surg 2000;31:69-83.)     

Prophylactic gamma radiation of unaffected vein grafts failed to prevent vein graft disease in a chronic hypercholesterolemic porcine model.

OBJECTIVE: The value of prophylactic brachytherapy on vein graft disease is unknown. METHODS AND RESULTS: Vein bypass grafts in 23 hypercholesterolemic pigs after ex vivo gamma irradiation of the vein grafts (10, 20, and 40Gy) and 16 control veins were analyzed regarding: (1) expression of platelet-derived growth factor (PDGF-AA and -BB, ELISA); (2) smooth muscle cell (SMC) proliferation/cell death (double-immunohistochemistry Mib-1/TUNEL/SMC alpha-actin); and (3) vessel wall dimensions. Planimetric data on vessel wall dimensions revealed no positive effect of gamma radiation on neointima formation and inner lumen diameter. On the contrary, vein grafts subjected to 40Gy were significantly more likely to be occluded and to have reduced inner lumen and increased neointima formation. Radiation therapy had no effect on PDGF expression and SMC proliferation/cell death. The mean inner lumen diameter decreased as PDGF-AA expression increased. CONCLUSIONS: Prophylactic gamma radiation of unaffected vein grafts failed to prevent vein graft disease in a hypercholesterolemic porcine model. High-dose radiation (40Gy) resulted in more frequent graft occlusion and vein sclerosis.     

Macrophage depletion alters vein graft intimal hyperplasia.

BACKGROUND: The principal cause of vein graft failure is intimal hyperplasia (IH); however, its etiology remains unclear. In a rat model of vein graft IH we have observed prolonged transmural macrophage infiltration, leading us to hypothesize that these cells regulate IH. To test this, we used liposome-encapsulated dichloromethylene bisphosphonate (L-Cl2MBP) to deplete rat macrophages and observed the effects on IH. METHODS: Epigastric vein-to-femoral artery grafts were microsurgically placed in male Lewis rats that had been intravenously injected with L-Cl2MBP, phosphate-buffered saline solution liposomes, or phosphate-buffered saline solution alone 2 days before surgery. Several animals in each group received a second equivalent dose at 2 weeks. Grafts, contralateral epigastric veins, spleens, and livers were harvested at 1, 2, and 4 weeks for histologic examination, immunohistochemistry, and transmission electron microscopy. RESULTS: In the L-Cl2MBP-treated animals splenic and hepatic macrophages were greatly reduced, confirming the efficacy of the agent. At 1 to 2 weeks graft macrophages were significantly decreased, and there was a trend toward decreased IH. At 4 weeks macrophage numbers were normal and IH development had resumed. In contrast, the 4-week grafts treated with 2 doses of L-Cl2MBP had fewer macrophages and displayed severely attenuated IH. CONCLUSIONS: The results indicate a suppression of IH as macrophages are depleted, with a resumption of the process as macrophages repopulate the graft.     

金属蛋白酶1组织抑制因子在大鼠自体移植静脉中的表达研究

目的 研究金属蛋白酶1组织抑制因子(tissue inhibitor of metalloproteinase-1,TIMP-1)在大鼠自体移植静脉血管平滑肌细胞中的表达和动态变化的意义.方法 建立大鼠自体血管移植模型,移植后不同时间分别切取移植静脉,应用HE染色、免疫组织化学和原位杂交方法动态观察TIMP-1的表达和变化情况.结果 移植血管组织病理学改变:血管移植后1周可见内膜增生(intimal hyperplasia:IH),2～4周达到高峰.原位杂交结果:TIMP-1mRNA在移植后24 h出现细胞阳性表达,72 h阳性表达明显增多,1-2周时表达达到高峰,与移植后1 d相比,差异有统计学意义(P<0.01).免疫组织化学染色:静脉移植后72 h出现TIMP-1的表达,1周时增加明显,2周时表达最强,与移植后1 d相比,差异有统计学意义(P<0.01).结论 在自体移植静脉中存在TIMP-1的激活.自体静脉移植于动脉后,内源性TIMP-1的分泌增加不足以抑制内膜增生.     

Antisense to transforming growth factor-beta1 messenger RNA reduces vein graft intimal hyperplasia and monocyte chemotactic protein 1

BACKGROUND: Autogenous vein grafts are commonly used for arterial reconstructive procedures. Their success is limited by the development of intimal hyperplasia (IH), a fibroproliferative disease that predisposes the grafts to occlusive stenosis. Mesenchymal cell proliferation and the deposition of an extracellular matrix characterize neointimal development. Increasing evidence suggests that, regardless of blood vessel type, IH results from complex interactions among vessel wall cells, infiltrating leukocytes, and cytokines. Transforming growth factor-beta1 (TGF-beta1) is a pleiotropic cytokine with powerful effects on inflammatory cell chemotaxis; smooth muscle cell, fibroblast, and endothelial cell proliferation; and extracellular matrix synthesis. METHODS: Epigastric vein to common femoral artery interposition grafts were placed in male Lewis rats and harvested at 1, 2, 4, and 12 weeks after surgery. We used replication-defective adenoviruses to deliver a control reporter gene for the enzyme beta-galactosidase (Ad-GAL), empty virus (Ad-CMVpLpA), or the sequence encoding the antisense strand of TGF-beta1 (Ad-AST). The vein graft was transduced passively in medium containing 10 7 plaque-forming units per milliliter of Ad-GAL, Ad-CMVpLpA, or Ad-AST for 20 minutes at room temperature. The adenovirus-treated grafts were compared with grafts treated with medium without virus (sham). RESULTS: The Ad-GAL control grafts showed beta-galactosidase activity from 3 days to 4 weeks. Twenty percent of cells were positive out to 2 weeks, at which time the number of cells positive for beta-galactosidase activity began to decline. Treatment with Ad-AST resulted in a significant reduction vs sham, Ad-CMVpLpA, and Ad-GAL in TGF-beta1 messenger RNA, total TGF-beta1 protein, and bioactive TGF-beta1 protein. Neointimal area was significantly reduced in the Ad-AST group vs Ad-GAL at 4 weeks, vs Ad-CMVpLpA at 4 and 12 weeks, and vs sham at 2 and 4 weeks. The medial/adventitial layer was significantly thicker in the Ad-AST group than the Ad-GAL group at 12 weeks. In addition, we studied the effect of Ad-AST on monocyte chemotactic protein 1 (MCP-1). Although the reduction in TGF-beta1 resulted in a reduction of MCP-1 messenger RNA in whole-graft homogenates and MCP-1 protein-positive staining in histologic sections from the perianastomotic region, no reduction in the number of ED1-positive cells (monocytes and macrophages) was observed. CONCLUSIONS: Perioperative antisense TGF-beta1 treatment of the vein to be used in arterial reconstructions resulted in a prolonged diminution of IH; this emphasizes the importance of TGF-beta1 in neointimal thickening and indicates that ex vivo gene therapy can reduce the vessel's predisposition to IH. CLINICAL RELEVANCE: The main cause of occlusion and graft failure after peripheral and cardiac arterial reconstruction is IH. The study of the mechanisms and mediators of IH, including TGF-beta1, should lead to future gene therapies to prevent or limit IH. The clinical effect of such treatments would be enormous, because they would increase graft longevity, thereby enhancing quality of life and enabling patients to live without the threat of limb loss or recurrent heart attack.     

反义雷帕霉素靶蛋白基因局部转染对移植静脉内膜增生的影响

目的:观察以纳米粒子为载体的反义雷帕霉素靶蛋白(mTOR)基因局部转染对移植静脉内膜增生的影响。方法:应用聚乳酸聚乙醇酸共聚物(PLGA)和聚乙烯醇(PVA)包载mTOR基因,制备纳米级粒子混合物。检测其包埋率、体外释放情况及粒子大小。建立自体静脉移植模型,随机分成转基因组、空载体组、对照组。转基因组移植静脉转染以纳米粒子为载体的反义mTOR基因,空载体组单纯转染纳米粒子包载的空载体,对照组不予特殊处理。分别于术后3d、7d、14d、28d取材,常规HE、Verhoeff染色,RT-PCR、Westernblot检测mTOR基因的mRNA及蛋白的变化,TUNEL法观察血管平滑肌细胞(VSMC)凋亡的动态变化。结果:转基因组内膜中mTOR基因的mRNA及蛋白产物表达较其他两组明显减少(P<0.05);转基因组内膜增生厚度7d、14d、28d较其他组明显减少(P<0.01);转基因组凋亡细胞较其他组明显增高(P<0.05)。结论:纳米粒子可以作为转基因载体,反义mTOR基因的表达能够有效抑制自体移植静脉内膜的增生,促进VSMC的凋亡。     

The proteasome inhibitor bortezomib inhibits intimal hyperplasia of autologous vein grafting in rat model

OBJECTIVE: Increasing evidence indicates that inflammation plays an important role in intimal hyperplasia (IH) induced by autologous vein grafts. The proteasome inhibitor bortezomib shows anti-inflammatory effects, so we used an autologous vein transplantation model to test whether bortezomib inhibits neointimal formation in transplant-induced vasculopathy. MATERIALS AND METHODS: We subjected 88 rats to autologous external jugular vein grafting surgery randomly assigned to be treated with bortezomib or vehicle. After 24 or 72 hours, rats were humanely killed and vein grafts processed for real-time RT-PCR (24 and 72 hours), ELISA (24 hours), or neutrophil chemotaxis assay (24 hours). Subsequently, rats were humanely killed at 1 and 2 weeks after grafting with samples processed for morphometric analysis. RESULTS: Bortezomib significantly inhibited IH at 2 weeks compared with untreated controls (P < .05). Expression of mRNA for vascular cell adhesion molecule-1, intercellular adhesion molecule-1, cytokine-induced neutrophil chemoattractant 2beta, monocyte chemoattractant-1, interleukin (IL)-1, IL-6, and tumor necrosis factor-alpha markedly increased in injured vessels during the first day after surgery declining over the following 3 days. Bortezomib significantly attenuated gene expression and protein levels of most inflammatory mediators (P < .05), simultaneously inhibiting neutrophil chemotactic activity of vessel homogenates. CONCLUSIONS: Bortezomib inhibited neointimal formation at least partially by attenuating the inflammatory response in transplant-induced vasculopathy. It may become a novel vasoprotective agent in the clinical field.     

A Canine Model of Intimal Hyperplasia (IH) in Autogenous Vein Grafting: A Preliminary Report

High failure rates (10–40% at 1 year and 2–6% per year thereafter) of autologous vein grafts in peripheral bypass surgery due to progressive intimal hyperplasia (IH) have prompted researchers to search for an animal model that develops IH in a relatively short period of time. This study summarizes our experiences in promoting IH in a canine model. Eight to ten centimeters of both jugular veins were exposed in 40 adult mongrel conditioned dogs. After division into 4–5-cm lengths, the segment of jugular vein most proximal was ballooned at 800–900 mm Hg with a modified 8F Fogarty catheter to induce intimal and medial layer injury. The distal segment was left nonballooned. Segments of these autologous vein grafts, 1.5 cm in length, both ballooned and nonballooned, were then anastomosed, end to end, into the carotid and femoral circulations. Six weeks postoperatively the grafts were perfusion-fixed, harvested, and histologically processed, and the amount of the lumen in midgraft sections occupied by IH was determined by image analysis. In all dogs, the degree of IH was significantly greater in the balloon catheterized vs noncatheterized graft segments. IH was more severe in the femoral than in the carotid arteries. The grafts that developed the most severe intimal hyperplasia were femoral grafts that had been balloon catheterized. We conclude that these protocols are effective in inducing IH in a canine model in short postoperative limes.     

核转录因子κB decoy寡核苷酸对移植静脉内膜增生的影响

目的 研究核转录因子κB(NFKB)decoy寡核苷酸(decoy ODNs)对移植静脉内膜增生的抑制作用,探讨其作用机制。方法 Wistar大鼠72只,建立自体静脉移植模型,术后随机分为：对照组,NFκB decoyODNs 50μg、200μg组,杂码decoyODNs 50μg、200μg组,lipofectin pluronic组等6个组,施加不同的处理方法,在移植后1、2周取材。组织形态学方法比较内膜增生程度,半定量RT-PCR方法检测细胞间黏附分子(ICAM)-1的mRNA表达,Western blot和免疫组化方法检测p65、ICAM-1的蛋白产物表达。结果 移植后1、2周内膜增生明显,局部应用50μg NFκB decoyODNs明显抑制内膜增生,抑制率为22％-31％;200μg组受抑制程度更为明显,抑制率达41％～53％,其他组内膜增生无明显变化。NFκB decoyODNs能够抑制：ICAM-1mRNA表达,p65和：ICAM-1的蛋白表达均显著低于各对照组,抑制率为30％-57％。结论 NFκB decoyODNs可显著抑制移植静脉的内膜增生,其作用可能是通过减少黏附分子的基因转录及蛋白产物表达而实现的。     

纳米粒子介导反义mTOR基因转染抑制移植静脉内膜增生

目的:观察以纳米粒子为载体的外源性反义雷帕霉素靶蛋白(mTOR)基因局部转染对移植静脉内膜增生的影响。方法:应用聚乳酸聚乙醇酸共聚物(PLGA)和聚乙烯醇(PVA)包载mTOR基因，制备纳米级粒子混合物。建立自体静脉移植模型72只，随机分成转基因组(转染以纳米粒子为载体的反义mTOR基因)，空载体组(单纯转染纳米粒子包载的空载体)和对照组(不予特殊处理)。分别于术后3，7，14，28d取材。检测mTOR基因的mRNA及蛋白产物表达，以及血管平滑肌细胞(VSMC)凋亡的动态变化。 结果:转基因组内膜中mTOR基因的mRNA及蛋白产物表达较其他两组明显减少(P<0.05)；转基因组内膜增生厚度于7，14，28d较其他组明显减少(P<0.01)；转基因组凋亡细胞较其他组明显增高(P<0.05)。结论:纳米粒子可以作为转基因载体。反义mTOR基因的表达能有效抑制自体移植静脉内膜的增生及促进VSMC凋亡。