Long-term inhibition of intimal hyperplasia using vascular photodynamic therapy in balloon-injured carotid arteries

Flexible treatments for intimal hyperplasia after angioplasty are still needed. The aim of this study was to demonstrate the long-term effects of vascular photodynamic therapy with talaporfin sodium on intimal hyperplasia following interventional injury. Intimal hyperplasia was induced by balloon distension injury to the carotid artery in 31 rabbits. Talaporfin, 5.0 mg/kg, was delivered systemically immediately after balloon injury. The injury site was irradiated with a diode laser light of wavelength 664 nm using a fluence of 50 J/cm² after 30 min. At day 3 and weeks 3, 6, 9, 15, and 25 after photodynamic therapy, the treated artery of each rabbit was excised and examined immunohistochemically. Thirty minutes after talaporfin administration, drug fluorescence was found only in the balloon-injured carotid artery wall. At 3 days, no smooth muscle cells were seen in the media of the photodynamic therapy-treated arterial segments. Intimal hyperplasia developed progressively in the balloon-injured and untreated segments; however, in the segments treated with photodynamic therapy, intimal hyperplasia was markedly suppressed until 25 weeks and the media was repopulated by smooth muscle cells without macrophages. Vascular photodynamic therapy with talaporfin may be used to inhibit restenosis after vascular intervention. An erratum to this article is available at .     

Human interleukin-10 gene inhibits acute rejection by triggering apoptosis in allograft vascular transplantation

The aim this study is to explore effect of IL-10 on apoptosis of VSMCs in allograft arterial transplantation rats, and to investigate mechanism. SD rats were divied into three groups, including control group (CN, with physiological saline), blank vector group (BV, with blank adenovirus) and combined gene group (CG, with adenovirus carried IL-10 gene). The isolated donor vascular was transfected with the adenovirus carried hIL-10 gene for 30 minutes by immersing method. Forty-five days post transplantation, the grafts were harvested. The allografts pathologioc changes were observed and the size of vascular intima and middle layer of allografts were measured. The expression of hIL-10 was detected by RT-PCR, ELISA and immunohistochemistry, respectively. The repression of Fas/Fasl in artery allografts was also examined by immunohistochemistry method. The results indicated that 45 days after transplantation, the intimal and middle hyperplasia ratio in CG group was significantly lower than that in CN and BV group (P < 0.05). The transgene expression of human interleukin-10 was significantly enhanced in CG group compared to CN and BV group by ELISA, RT-PCR and immunohistochemistry (P < 0.05). The expression of Fas/FasL was higher in CG group compared with the other groups (P < 0.05). The level of apoptotic smooth muscle cells were significantly increased in CG group compared to CN and BV group (P < 0.05). In conclusion, adenovirus mediated IL-10 expression could up-regulate Fas/FasL expression, induce smooth muscle cell apoptosis and alleviate angiosclerosis process. The IL-10 gene transfer to allograft artery could inhibit acute rejection reaction of allograft vascular transplantation.     

内皮型一氧化氮合酶基因转染对血管损伤后新生内膜增生的影响

背景：一氧化氮能够抑制血管平滑肌细胞的迁移和增殖,而一氧化氮合酶是其合成的关键酶,有关一氧化氮合酶基因体内转染对平滑肌细胞及动脉粥样硬化血管损伤后内膜增生影响少有报道。 目的：观察内皮型一氧化氮合酶 (endothelial nitric oxide synthase,eNOS)基因体内局部转染对动脉粥样硬化大鼠血管损伤后新生内膜增生的抑制作用。 方法：建立动脉粥样硬化Wistar大鼠颈动脉球囊损伤模型,建模后随机分成空白对照组、AdCMV-lacz对照组和AdCMV-eNOS组,分别将PBS,AdCMV-lacz和AdCMV-eNOS体内转染至以上3组大鼠的损伤血管壁。转染2周后培养并鉴定损伤局部平滑肌细胞,并用RT-PCR法检测各组损伤及转染后血管平滑肌细胞eNOS mRNA的表达,同时观察转染后不同时期新生内膜增生的影响。 结果与结论：AdCMV-eNOS组的颈总动脉血管平滑肌细胞可表达eNOS mRNA。3组大鼠转染后1和3个月,AdCMV-eNOS组内膜/中膜面积比值低于空白对照组和AdCMV-lacz对照组(P < 0.01)。结果显示,eNOS基因体内转染损伤后血管可以抑制血管新生内膜增生,减少再狭窄发生率。     

血管平滑肌细胞凋亡在血管再狭窄中作用的研究进展

血管平滑肌细胞构成新生内膜增生的重要部分,且在血管腔内治疗术后再狭窄的心血管疾病的发生和发展中具有重要作用。血管平滑肌细胞凋亡能有效抑制血管球囊损伤和血管旁路移植术后新生内膜增生,从而可为血管术后再狭窄提供治疗手段。     

Fas-induced apoptosis, and diseases caused by its abnormality

Homeostasis of multicellular organisms is controlled not only by proliferation and differentiation of cells but also by cell death. Physiological cell death mostly proceeds by apoptosis, a process which includes condensation and segmentation of the nuclei and cytoplasm, and often extensive fragmentation of chromosomal DNA into nucleosome units. Molecular and cellular characterization of Fas, a cell-surface protein recognized by cytotoxic monoclonal antibodies, has led to its definition as a receptor for a Fas ligand (FasL). FasL binds to Fas, which results in target cell apoptosis. A family of cysteine proteases seems to be sequentially activated during the Fas-mediated apoptosis. The phenotypes of the loss-of-function mutants of Fas and FasL indicate that this system is involved in T cell-mediated cytotoxicity, in down-regulation of immune responses, and probably in the turnover of senescent cells. Malfunction of the Fas system causes hyperplasia in peripheral lymphoid organs and the liver, accelerating autoimmune diseases and tumorigenesis, whereas exaggeration of the system leads to tissue disruption.     

Death of solid tumor cells induced by fas ligand expressing primary myoblasts

Anticancer therapy for solid tumors suffers from inadequate methods for the localized administration of cytotoxic agents. Fas ligand (FasL) has been reported to be cytotoxic to a variety of cells, including certain tumor cell lines. We therefore postulated that myoblasts could serve as non-transformed gene therapy vehicles for the continuous localized delivery of cytotoxic anticancer agents such as FasL. However, contrary to previous reports, fluorescence activated cell sorting (FACS) analyses revealed that both primary mouse and human myoblasts express Fas, the receptor for FasL. To avoid self-destruction and test the cytotoxic potential of myoblasts, the cells were isolated from mice deficient in Fas (lpr/lpr), the mouse counterpart of human autoimmune lymphoproliferative syndrome (ALPS). These primary mouse myoblasts were transduced with a retroviral vector encoding mouse FasL and expression of a biologically active and soluble form of the molecule was confirmed by the apoptotic demise of cocultured Fas-expressing Jurkat cells, the standard in the field. To test whether the lpr myoblasts expressing FasL could be used in anticancer therapy, human rhabdomyosarcoma derived cell lines were assayed for Fas and then tested in the apoptosis coculture assay. The majority of Fas-expressingmuscle tumor cells were rapidly killed. Moreover, FasL expressing myoblasts were remarkably potent; indeed well characterized cytotoxic antibodies to Fas were only 20% as efficient at killing rhabdomyosarcoma cells as FasL expressing myoblasts. These findings together with previous findings suggest that primary myoblasts, defective in Fas but genetically engineered to express FasL, could function as potent anticancer agents for use in the localized destruction of solid tumors in vivo by three synergistic mechanisms: (1) directly via Fas/FasL mediated apoptosis, (2) indirectly via neutrophil infiltration and immunodestruction, and (3) as allogeneic inducers of a bystander effect via B and T cells.     

Learning from vascular remodelling

The positioning of a hollow silicone collar around the carotid artery of a rabbit induces many changes of early atherosclerosis including intimal proliferation of smooth muscle cells. This occurs below an intact endothelium indicating that endothelial damage is not necessary for smooth muscle cell proliferation. The endothelium may in fact produce substances that control processes occurring in the intima. Vascular endothelial growth factor (VEGF) is an angiogenic agent that is produced by cultured vascular smooth muscle cells. The combination of hypoxia and factors such as platelet-derived growth factor, tumour necrosis factor alpha, basic fibroblast growth factor, and interleukin-1β lead to synergistic production of VEGF by cultured smooth muscle cells. VEGF receptors are present predominantly on the endothelium and may be an important target for modulating the response to damage, hypoxia and inflammation. Transfection of the gene for VEGF resulted in inhibition or regression of intimal hyperplasia induced by the silicone collar in the rabbit. Studies suggest that the two mediators responsible for this inhibition of smooth muscle cell proliferation are nitric oxide and prostacyclin, which are produced by cultured endothelial cells incubated with VEGF. Thus, VEGF produced by smooth muscle cells in response to hypoxia, damage or inflammation, acts on specific endothelial receptors to produce nitric oxide and prostacyclin, which inhibit smooth muscle cell proliferation. Failure of this process could give rise to intimal hyperplasia. Early clinical studies of VEGF transfection from the outside of human arteries using a biodegradable collar are in progress.     

Learning from rejection: What transplantation teaches us about (other) vascular pathologies

Allograft vasculopathy is an accelerated intimal hyperplastic lesion leading to progressive vascular stenosis; it represents the major long-term limitation to successful solid organ transplant. Although allograft vasculopathy is not formally an autoimmune disease, nor does it constitute a major cause of cardiovascular disease on a purely numerical basis, its pathogenesis provides an important window on the mechanisms by which immune injury can drive more common vascular pathologic entities. Thus, insights gleaned from vascularized solid organ transplants can shed new mechanistic (and therapeutic) light on: 1) the intimal vascular responses accompanying typical atherosclerosis and other inflammatory vessel diseases (e.g., scleroderma); 2) the pathogenesis of vascular stenosis versus aneurysm formation; 3) the sources of intimal smooth muscle cells in the healing of any vascular injury; and 4) the mechanisms by which smooth muscle cells are recruited into intimal lesions. Indeed, research on allograft vasculopathy has led to the understanding that interferon-γ plays a similar pathogenic role in a host of vascular stenosing lesions—and that Th2 cytokines can drive vascular remodeling and aneurysm formation. Moreover, circulating precursors (and not just medial smooth muscle cells) contribute to the intimal hyperplasia seen in atherosclerosis and in-stent restenosis. That non-vessel smooth muscle cells can be recruited to sites of vessel injury further suggests that chemokine and adhesion molecule interactions may be viable targets to limit vascular stenosis in a wide range of vascular lesions. This review will describe the pathogenesis of allograft vasculopathy, and will relate how understanding the underlying pathways informs our understanding of both human transplant-associated disease, as well as other human vascular pathologies.     

CD4+ mononuclear cells induce cytokine expression, vascular smooth muscle cell proliferation, and arterial occlusion after endothelial injury.

Studies of T cell-deficient or immunosuppressed animals undergoing arterial endothelial denudation have yielded conflicting results as to the contribution of the immune system to neointimal vascular smooth muscle cell accumulation and proliferation. We investigated the cell types and cytokine expression associated with intimal hyperplasia occurring 14 days after balloon angioplasty of the carotid artery in Sprague-Dawley rats. Immunohistological studies using monoclonal antibodies showed that the carotid luminal occlusion observed was associated with smooth muscle cell proliferation and neointimal accumulation of large numbers of CD4+, ED1+ mononuclear cells but no T cells. There was also wide-spread staining for the inflammatory cytokine interleukin-1B (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and IL-8, as well as dense expression of the potent smooth muscle mitogens platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta), and protein S. The relationship of smooth muscle cell proliferation to monocyte/macrophage accumulation and cytokine expression was tested by daily intraperitoneal administration for 14 days of a rat CD4-specific monoclonal antibody, BWH-4 (500 micrograms/day). Morphometric analysis at day 14 showed that the intimal area of animals treated with CD4 monoclonal antibody comprised 7% +/- 4% of the arterial wall compared with 50% +/- 6% in control animals (n = 6/group, P < 0.001). In addition, immunohistological studies showed that CD4 monoclonal antibody treatment markedly reduced the intimal accumulation of mononuclear and smooth muscle cells and essentially abrogated expression of the cytokines PDGF-BB, TGF-beta, IL-1 beta, TNF-alpha, and IL-8, plus the anticoagulant molecule, protein S. Our results document the extensive expression in vivo of cytokines that in vitro promote vascular smooth muscle cell proliferation, and suggest that CD4+ mononuclear cells or their secreted products play a key role in the pathogenesis of intimal hyperplasia after endothelial injury. Furthermore, these observations may have clinical relevance in the development of novel strategies to prevent arteriosclerosis.     

Molecular mechanisms in intimal hyperplasia

Intimal hyperplasia is the process by which the cell population increases within the innermost layer of the arterial wall, such as occurs physiologically during closure of the ductus arteriosus and during involution of the uterus. It also occurs pathologically in pulmonary hypertension, atherosclerosis, after angioplasty, in transplanted organs, and in vein grafts. The underlying causes of intimal hyperplasia are migration and proliferation of vascular smooth muscle cells provoked by injury, inflammation, and stretch. This review discusses, at a molecular level, both the final common pathways leading to smooth muscle migration and proliferation and their (patho)-physiological triggers. It emphasizes the key roles played by growth factors and extracellular matrix-degrading metalloproteinases, which act in concert to remodel the extracellular matrix and permit cell migration and proliferation.     

Comparison of Artery Organ Culture and Co-culture Models for Studying Endothelial Cell Migration and Its Effect on Smooth Muscle Cell Proliferation and Migration

Arterial restenosis associated with intimal hyperplasia is the major cause of long-term failure of vascular interventions. Endothelium injury and the proliferation and migration of smooth muscle cells (SMC) are key events in the development of intimal hyperplasia. The objectives of this study were to develop an ex vivo artery injury model for studying endothelial cell (EC) migration and to compare it with an in vitro co-culture arterial wall injury model in terms of the effect of flow on EC migration and its effect on SMC migration and proliferation. Our results demonstrated that shear flow improves reendothelialization in the injured area by promoting EC migration. The migration distance of ECs is much smaller in the arteries than in an in vitro cell culture model (3.57 ± 1.29 mm vs. 5.2 ± 1.4 cm, p < 0.001). SMC proliferation was significantly less in the EC intact and reendothelialization areas than in the EC denuded areas indicating that reendothelialization suppresses SMC proliferation. Our models provide a new approach to study techniques to enhance endothelium healing.     

砷涂层支架置入后的血管组织相容性

背景：研究认为,三氧化二砷可以抑制血管平滑肌细胞的增殖,促进其凋亡,那么砷对血管平滑肌细胞的增生是否也有同样的抑制作用,砷涂层血管支架能否与血管组织相容,早期较好地被血管内膜覆盖或达到减少内膜过度增生的作用？ 目的：观察砷涂层血管支架的血管组织相容性。 方法：取大耳白家兔14只,随机分为2组,分别在腹主动脉处植入砷涂层316 L不锈钢支架和316 L不锈钢裸支架,植入28 d后结扎支架部位血管的远端和近端,取下支架部位的血管行苏木精-伊红染色,光镜检查。 结果与结论：①大体观察：支架处的血管外径稍大于相邻处血管的外径,呈扩张状态,无肉眼可见的血栓,切开支架,支架表面可见光滑的新生内膜形成,新生内膜表面光滑。②光镜观察：支架丝位于血管的中层,中层平滑肌被压,支架丝周边,血管内膜平滑肌增生,使血管内膜增厚。支架丝的血管腔面可见新生的血管内膜形成并覆盖支架丝,支架丝与血管组织之间可见一薄层黑色物质,为涂层药物砷及其化合物,证明砷涂层支架可以被血管组织覆盖,具有良好的血管组织相容性。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

牛磺酸对大鼠肢体缺血再灌注后肺损伤时细胞凋亡的影响

目的:从细胞凋亡的角度探讨肢体缺血再灌注(LIR)后急性肺损伤(ALI)的发病机制及牛磺酸的影响。方法:复制大鼠肢体缺血再灌注(LIR)损伤动物模型,采用TUNEL法、电泳法、半定量逆转录聚合酶链反应(SqRT-PCR)及免疫组织化学等技术观察LIR后肺损伤发生过程中,肺泡上皮及血管内皮细胞凋亡变化以及Fas/FasL系统蛋白质和mRNA表达的改变。结果:大鼠LIR后,肺泡上皮细胞和肺血管内皮细胞凋亡明显增加;肺组织Fas/FasLmRNA和蛋白质表达明显上调,DNA断链率、组织钙含量和活性氧(ROS)升高,且与肺泡上皮及血管内皮细胞凋亡的增加相一致。结论:肺泡上皮及血管内皮细胞凋亡以及Fas/FasL系统表达明显上调可能参与LIR后ALI的发生;牛磺酸可减少肺组织细胞凋亡,但并非通过影响Fas/FasL基因表达而实现其保护效应。     

Antigen-induced cell death of T effector cells in vitro proceeds via the Fas pathway,requires endogenous interferon-gamma and is independent of perforin and granzymes

Activation of resting T cells usually leads to their proliferation and differentiation into effector cells and a subsequent decline following elimination of the antigen. A situation of excessive antigen density may result in T cell receptor (TCR)-induced deletion of T effector cells, a process termed antigen-induced cell death (AgICD). Previous studies indicate that AgICD of cytotoxic T cells may be induced by either of the two key cytotoxic processes, granule exocytosis, including perforin and granzymes, or the Fas ligand (FasL)/Fas pathway. By using in vitro-polyclonally activated or ex vivo-derived virus-induced T cell populations from mice with mutations or targeted gene defects in one or more components of the two key cytolytic pathways we now show that TCR-induced apoptosis is only impaired in the absence of FasL and/or Fas, but not in the absence of perforin and/or granzymes. Furthermore, antibody-blockage of FasL alone is sufficient to inhibit early T cell death. Inhibition of both, FasL and tumor necrosis factor (TNF-alpha) is required to abrogate late apoptosis by AgICD. The fact that antibodies to IFN-gamma also inhibit AgICD suggests that the perforin plus granzyme-independent and FaSL and/or TNF-alpha facilitated process of AgICD of T effector cells is tightly regulated by endogenous IFN-gamma.     

Fas receptor‐mediated apoptosis: a clinical application?

Fas is a membrane protein belonging to the death receptor family. Cross-linking of Fas by its ligand, FasL, or agonistic anti-Fas antibodies, induces apoptosis of cells expressing Fas on the membrane by triggering a cascade of caspases. Since many different tumours express Fas on their membrane, targeting Fas-mediated apoptosis by anti-Fas antibodies may be a promising anticancer therapy. Unfortunately, not all Fas-expressing cells are sensitive to Fas-mediated apoptosis. This has resulted in the discovery of many different inhibition mechanisms of Fas-mediated apoptosis. In addition, mutations in the Fas or p53 gene can also influence the sensitivity for Fas-mediated apoptosis. However, the role of wild-type p53 in Fas expression is still controversial. Because several different cytotoxic drugs are able to induce Fas membrane expression, combination therapy of anticancer drugs with anti-Fas antibodies or FasL is conceivable as an anticancer strategy. The efficiency of the induction of Fas-mediated apoptosis by anti-Fas antibodies, FasL-expressing cells or recombinant FasL (rFasL) in tumours has been demonstrated in vivo in solid tumours implanted in mice. Unfortunately, systemic treatment with anti-Fas antibodies or rFasL causes severe damage to the liver, so most preclinical studies are now focusing on circumvention of this problem by local administration of FasL, or on the use of inducible FasL-expressing vectors as gene therapy.     

抗血管平滑肌增生和迁移的药物涂层与支架置入后再狭窄

背景：抑制血管平滑肌细胞增殖和血管内膜的增生而不影响血管内皮愈合的药物涂层是研制新型药物洗脱支架和防治支架内再狭窄的新策略。 目的：综述抗血管平滑肌细胞增生和迁移药物涂层支架的研究进展。 方法：由第一作者应用计算机检索维普数据库中与抗血管平滑肌细胞增生和迁移药物涂层支架置入治疗心血管疾病有关的文献,检索时限1998-01/2011-10。关键词：心血管疾病;药物涂层支架;血管平滑肌;细胞增生;细胞迁移。对资料进行初审,并查看每篇文献后的引文。纳入相关文献25篇,中文15篇,英文10篇。 结果与结论：目前临床应用最广的抗血管平滑肌细胞增生和迁移的涂层药物是雷帕霉素和紫杉醇。研究表明,雷帕霉素支架表面携带的雷帕霉素和紫杉醇支架表面携带的紫杉醇均能有效地降低支架置入后再狭窄及临床靶血管血运重建率。目前国内大多数研究仅为小样本、单中心、非随机对照试验,其更长期的疗效和安全性有待更长期的随访和多中心随机对照研究来证明。     

Enhanced Intimal Proliferation upon Injury to Pre-Existing Neointima and Resistance of Neointimal Cells to Cell Death

Recent evidence supports a role for cell death and inflammation as etiologic factors in neointimal formation and restenosis after angioplasty. This study was undertaken to examine the pattern and intensity of the proliferative response, cell death, and activation of inflammatory, endothelial and smooth muscle cells (SMC) in a model of intimal reinjury. Two ballooning injuries were performed to rat aorta, the second one 14 days after the first injury. Our results demonstrate that ballooning injury to pre-existing neointima differs clearly from an injury to a normal aorta. First, ballooning injury to pre-existing neointima doubled the proliferative response of SMC and intimal thickening, but proliferation of SMC occurred only in the intima, and did not extend into the media. Second, within four hours after the first injury, the number of TUNEL-positive SMC in the media increased from 3% to 23%, but no such increase was found in the pre-existing neointima after the second injury. Third, the prompt proliferative response of intimal SMC after the second injury was linked with a significant increase in endothelial P-selectin and neointimal VCAM-1 immunoreactivity, compared to the first injury at corresponding time points, followed by high numbers of activated ED3+ macrophages and CD4+ T cells in the developing neointima. A balance in injury-induced cell death and proliferation obviously maintains stable cell numbers observed in the media, whereas in the neointima, the resistance of SMC to injury-induced cell death may contribute to a rapid lesion formation in restenosis.     

Photodynamic therapy induces apoptosis in intimal hyperplastic arteries

Photodynamic therapy (PDT) generates free radicals through the absorption of light by photosensitizers. PDT shows promise in the treatment of intimal hyperplasia, which contributes to restenosis, by completely eradicating cells in the vessel wall. This study investigates the mechanisms of PDT-induced cell death. PDT, using the photosensitizer chloroaluminum-sulfonated phthalocyanine (1 mg/kg) and laser light (lambda = 675 nm) 100 J/cm(2) was administered to rat carotid arteries after balloon injury-induced intimal hyperplasia. Apoptosis was determined by cell morphology with light microscopy and transmission electron microscopy, DNA cleavage by terminal dUTP nick-end labeling staining, and nucleosomal fragmentation (ladder pattern) by DNA agarose gel electrophoresis. Four hours after PDT, apoptosis was observed in vascular cells, as evidenced by terminal dUTP nick-end labeling staining and transmission electron microscopy. Within 24 hours no cells were present in the neointima and media. Immunofluorescence using an alpha-smooth muscle cell actin antibody confirmed the disappearance of all neointimal and medial cells within 24 hours. No inflammatory cell infiltrate was observed during this time frame. Apoptosis was sharply confined to the PDT treatment field. These data demonstrate that vascular PDT induces apoptosis as a mechanism of rapid, complete, and precise cell eradication in the artery wall. These findings and the lack of inflammatory reaction provide the basis for understanding and developing PDT for a successful clinical application in the treatment of hyperplastic conditions such as restenosis.