Ultrastructural features of progressive intimal hyperplasia at the distal end-to-side anastomosis of vein grafts

The features of intimal hyperplasia at the distal end-to-side anastomosis of artertally implanted autovein bypass grafts in dogs were examined using light and transmission electron microscopy. The bypass grafting was done under conditions of reduced blood flow with an abnormal flow wave and high peripheral resistance. Anastomotic intimal hyperplasia was evident 14 to 31 days after implantation, then gradually increased, particularly at the toe portion of the anastomosis. From 6 to 12 months after implantation, the intimal hyperplasia was excessively increased and severe luminal stenosis had developed. The hyperplastic neointima consisted of two layers; a laminated superficial layer and a randomly arranged deeper layer. The superficial layer had three to four layers of smooth muscle cells, while the deeper layer was mostly fibrocollagenous tissues. Thus, the intimal hyperplasia at the distal end-to-side anastomosis of the arterially implanted autovein graft developed as a result of infiltration of fibroblast-like cells, presumably tissue-synthesizing mesenchymal cells. The neointima at the distal anastomosis, under conditions of reduced blood flow with high peripheral resistance, remained in an active phase of intimal hyperplasia even 12 months after bypass grafting.     

Therapeutics of vein graft intimal hyperplasia: 100 years on

Intimal hyperplasia is central to the pathology of vein graft re-stenosis, and despite considerable advances in our understanding of vascular biology since it was first described 100 years ago, it is still a significant clinical problem. Recent decades have seen the development of many new therapeutic agents aimed at treating this condition, but the successes of laboratory studies have not been replicated in the clinic yet. This review discusses these therapeutic agents, how their modes of action relate to the pathogenesis of vein graft intimal hyperplasia, and considerations of ways in which such therapy may be improved in the future.     

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.     

Photodynamic therapy reduces intimal hyperplasia in prosthetic vascular bypass grafts in a pig model.

BACKGROUND: Bypass surgery has a failing frequency of 30% during the first year, mainly due to intimal hyperplasia (IH). This negative effect is most pronounced in artificial grafts. Photodynamic therapy (PDT) is a technique in which light activates photosensitizer dyes to produce free-radicals resulting in an eradication of cells in the vascular wall. The aim of this study was to determine the effectiveness of PDT to reduce IH in a preclinical porcine PTFE bypass model. MATERIAL AND METHODS: Ten pigs were used. After a pilot PDT dosimetry study (n=3) PTFE grafts were bilaterally placed into the circulation as bypasses from the common to the external iliac arteries (n=7). The right sides served as controls (C). Before implantation of the left grafts, the arterial connecting sites of the left distal anastomoses were PDT-treated. The arteries were pressurized at 180 mmHg for 5 minutes with the photosensitizer Methylene Blue (330 microg/ml), and thereafter endoluminally irradiated with laser light (lambda = 660 nm, 100 mW/cm(2), 150 J/cm(2)). After 4 weeks the specimens were retrieved and formalin fixed. Cross sections through the midportions of the distal anastomoses and the grafts were used for histology, immunohistochemistry to identify inflammatory cells and morphometric evaluation (n=7). RESULTS: No systemic side effects and no graft occlusions were noted. PDT-treated anastomoses showed reduced IH in the mid-portions of the anastomoses (Area of IH: microm(2)/microm graft: C: 6970+/-1536, PDT: 2734+/-2560; P<0.005) as well as in the grafts (C: 5391+/-4031, PDT: 777+/-1331; P<0.02). The number of inflammatory cells per microscopic field was increased after PDT (C: 24+/-16, PDT: 37+/-15; P<0.009). CONCLUSIONS: Adjuvant PDT, performed in an endovascular fashion, was a safe method to reduce prosthetic graftstenosis in a preclinical setting. This study underscores the clinical potential of PDT to inhibit the development of clinical bypass graftstenosis.     

Heparin reduces the intimal hyperplasia seen in microvascular vein grafts.

The influence of heparin on microvascular vein graft intimal hyperplasia was studied in a rat model. The iliolumbar vein was grafted into the iliac artery in 80 rats. Heparin was delivered via a subcutaneous miniosmotic pump, starting either 2 days before grafting (early heparin group, n = 20) or immediately after grafting (heparin group, n = 30). Saline-containing pumps were placed in the control group (n = 30). Heparin activity was measured at 24 h, and again 3 weeks later when the animals were sacrificed. The grafts were harvested and prepared for histological examination. The intimal thickness was measured at the anastomoses and in the mid-graft region using an eye-piece graticule set at right angles to the graft internal elastic lamina. Heparin significantly reduced the intimal thickness at the anastomoses, from a median of 38 microns (range: 10-100 microns) in the control group to a median of 20 microns (range: 10-150 microns) in the heparin group. A similar reduction was seen in the mid-graft region. Although intimal thickening was reduced in the early heparin group, this reduction failed to reach statistical significance. The possible clinical application is discussed.     

Aspirin and dipyridamole decrease intimal hyperplasia in experimental vein grafts

Release from platelets of a factor mitogenic for smooth muscle cells is a postulated mechanism for the pathogenesis of vascular intimal hyperplasia. In this study the effect of antiplatelet therapy was evaluated. Aspirin (165 mg twice daily) and dipyridamole (25 mg twice daily) were administered to six rhesus monkeys and six were given placebo only. Bilateral vein bypass grafts were placed in the iliac arteries. In addition, to evaluate the relative contribution of adventitial dissection and intimal injury, on one side the carotid artery and femoral vein were stripped of adventitia and on the other side the intima of these vessels were injured by the single passage of an inflated balloon tipped catheter. Animals were killed after 16 weeks. In grafts relative luminal area was determined by a photographic gravimetric method at three standard locations. Femoral veins and carotid arteries were classified as histologically normal or as exhibiting hyperplasia. All vessels with adventitial stripping were normal. All vessels with intimal injury in the placebo group except one exhibited intimal hyperplasia compared to the drug treated group in which over half were normal. Relative intimal area was significantly less in grafts from drug treated animals at all three locations and luminal area greater in two. These data suggest that vascular intimal hyperplasia can be reduced by treatment with antiplatelet agents.     

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.)     

Photodynamic therapy inhibition of experimental intimal hyperplasia: Acute and chronic effects

Purpose: Intimal hyperplasia (IH) is a focal arterial problem that still eludes successful therapy. We have previously demonstrated the feasibility of use of photodynamic therapy (PDT) for the acute treatment of experimental IH with light to activate an otherwise biologically inert photosensitizer. The purpose of this study was to determine the acute and long-term effects of PDT inhibition of IH on the artery wall.Methods: Segmental IH was induced by balloon injury localized to the cervical common carotid artery of 33 rats. The photosensitizer chloroaluminum sulfonated phthalocyanine (5 mg/kg) for the experimental group or saline solution for the control group was administered intravenously. Twenty-four hours later, all instrumented portions of arteries were irradiated at 675 nm to induce cytotoxic injury in the PDT-treated arteries as compared with laser only – treated arteries for controls. Animals were killed at 1, 2, 4, and 16 weeks.Results: There were no untoward side effects in either group. All PDT-treated arteries were devoid of smooth muscle or inflammatory cells in the treated media. There was no evidence of arterial degeneration of PDT-treated arteries. Only three arteries in the PDT group developed IH, whereas it was universal in all controls. In control arteries, immunocytochemistry with bromodeoxyuridine revealed maximal intimal and medial cell proliferation at 1 week, and morphometric analysis demonstrated a maximal IH at 2 weeks. Immunocytochemistry staining for smooth muscle cell actin was positive for the IH in control and when present in PDT-treated arteries, whereas the adventitia of PDT-treated arteries were positive after 2 weeks. Electron microscopy demonstrated early myofibroblast migration to the adventitia, and at 16 weeks occasional myofibroblasts were noted in the media of PDT-treated arteries. There was complete reendothelial cell covering of the intima by 4 weeks.Conclusions: These in vivo data demonstrate that PDT is an effective local method for the treatment of experimental IH. There is no evidence of significant recurrence of IH or arterial degeneration. Further studies with PDT may provide novel approaches to the understanding and treatment of arterial IH. (J VASC SURG 1994;19:321-31.)     

吡格列酮对静脉旁路血管内膜增生的影响

目的 探讨吡格列酮(PIO)对静脉旁路血管内膜增生的影响及可能机制.方法 将32只SD大鼠随机分为2组,分别予(3mg·kg-1·d-1)的PIO或盐水灌胃,1周后行自体右颈外静脉-颈总动脉移植术,术后持续给药2、4周后取静脉旁路血管.应用图像分析软件计算内膜增厚情况,westernbolt检测ERK1/2激活情况.体外培养人大隐静脉平滑肌细胞,采用CCK-8法检测细胞增殖,TUNEL法检测细胞凋亡.结果 与同期对照组相比,PIO明显减少术后2周[(8.56±1.64)μm对(25.44±0.89) μm,P＜0.01]和4周[(10.51 ±1.47) μm对(35.69±1.07) μm,P＜0.01]静脉旁路血管内膜厚度,抑制ERK1/2活性.PIO显著抑制PDGF-BB诱导的细胞增殖并促进细胞凋亡.结论 PIO能有效抑制静脉旁路血管术后内膜增生,可能与其下调ERK1/2活性,抑制血管平滑肌细胞增殖并促进凋亡密切相关.     

Accelerated intimal hyperplasia in aortocoronary internal mammary vein grafts in minipigs

Background  

More than 50% of aortocoronary saphenous vein grafts are occluded 10 years after surgery. Intimal hyperplasia is the initial critical step in the progression toward occlusion. Internal mammary veins, which are physiologically prone to less hydrostatic pressure, may undergo an accelerated progression to intimal hyperplasia and thus be suitable for investigation of the mechanisms of aortocoronary vein graft disease.     

Chronic ACE inhibition reduces intimal hyperplasia in experimental vein grafts.

Intimal hyperplasia is an important factor in the pathophysiology of vein graft failure. Local renin-angiotensin systems recently have been shown to modulate the development of intimal hyperplasia in arteries after intimal injury. The effect of chronic angiotensin-converting enzyme (ACE) inhibition on the development of intimal hyperplasia in experimental vein grafts was examined in this study. Ten New Zealand White rabbits received 10 mg/kg of captopril daily in their drinking water. One week later the right carotid artery was divided and bypassed with the reversed right external jugular vein in these rabbits and in 10 matched controls. Captopril was continued for 28 days after operation, when all the grafts were harvested. Five grafts from each group were perfusion fixed, and the intimal thickness in the proximal, middle, and distal segments was determined. Rings from the remaining grafts (n = 20 in each group) were studied in vitro under isometric tension, and their responses to norepinephrine (NE), histamine (HIST), serotonin (5-HT), angiotensin I (AI), and angiotensin II (AII) was measured. The intimal thickness of the proximal, middle, and distal segments of the captopril-treated grafts were significantly less than controls, being reduced in all segments by approximately 40% (p less than 0.0001). With regard to vasoreactivity, the captopril-treated grafts were hypersensitive to 5-HT (control ED50 5.5 +/- 0.5 X 10(-7) mol/L vs. captopril-treated 1.1 +/- 0.2 X 10(-6) mol/L; p less than 0.005) although the maximal response was significantly reduced (control 1.6 +/- 0.3 g vs. captopril-treated 0.8 +/- 0.1 g; p less than 0.05). There were no differences in sensitivity between control and captopril-treated rings with respect to NE, HIST, AI, or AII. Four of the ten captopril-treated segments, however, failed to respond to AI, and the maximal active tension of the responders was significantly reduced (control 0.47 +/- 0.06 g vs. 0.20 +/- 0.05 g; p less than 0.02). These results suggest that ACE is involved in the modulation of vein graft intimal hyperplasia, and that ACE inhibitors may have therapeutic applications in patients undergoing vein bypass procedures.     

反义寡核苷酸抑制Survivin基因表达对移植静脉内膜增生的影响

目的研究反义寡脱氧核苷酸（ASODN）抑制Survivin基因表达对移植静脉内膜增生的抑制作用。方法Wistar大鼠60只，建立自体静脉移植模型，术后随机分为：对照组、Survivin ASODN 50、200μg组、正义对照组、Lipofectin＋pluronic等五个组，施加不同的处理因素，在移植后1、2周取材。组织形态学方法比较内膜增生程度，逆转录．聚合酶链反应（RT-PCR）检测Survivin基因的mRNA表达，Westem blot检测Survivin基因的蛋白产物表达，免疫组织化学方法检测Survivin及增殖细胞核抗原（PCNA）的表达，脱氧核苷酸转移酶末端标记法（TUNEL）检测血管平滑肌细胞（VSMC）凋亡的变化。结果移植后1、2周内膜增生明显，局部转染50μg Survivin ASODN组内膜增生明显受抑制（P〈0．05），200μg组受抑制程度更为明显（P〈0．05）；与对照组相比，Survivln ASODN组Survivin的mRNA及蛋白产物表达显著减少（P〈0．05），PCNA阳性表达同时减少，而TUNEL阳性细胞却明显增加。结论Survivin ASODN可显著抑制移植静脉的内膜增生，其作用可能是通过抑制Survivin基因及其蛋白产物表达，从而抑制VSMC增殖、促进其凋亡而实现的。     

伊马替尼抑制大鼠静脉移植物内膜增生

目的 观察伊马替尼对自体移植静脉内膜增生的影响.方法 建立大鼠自体颈外静脉移植模型,实验分为4组:移植组、低剂量给药组、高剂量给药组及对照组.移植4周后取移植静脉,行病理学检查观察内膜增生情况,免疫组织化学及Western blot法检测PDGFRβ、ERK及P-PDGFRβ、P-ERK蛋白表达情况.结果与对照组比较,移植组和低剂量给药组内皮下平滑肌细胞大量增生,静脉内膜显著增厚,管腔明显狭窄,高剂量给药组内膜无明显增厚.Western blot结果显示,移植组血管P-PDGFRβ蛋白含量(P-PDGFRβ/GAPDH)较对照组明显增加(0.81±0.06比0.18±0.02,P<0.05),而高剂量给药组较移植组明显减少(0.32±0.03比0.81±0.06,P<0.05),低剂量给药组与移植组比较差异无统计学意义(P>0.05),P-ERK蛋白表达亦呈同样趋势变化.结论 高剂量伊马替尼能有效抑制自体移植静脉内膜增生,其机制可能与抑制PDGF信号通路蛋白磷酸化,从而抑制平滑肌细胞增殖有关.