Active macrophage-associated TGF-beta co-localizes with type I procollagen gene expression in atherosclerotic human pulmonary arteries.

Vascular remodeling in adult atherosclerotic pulmonary arteries is characterized by discrete areas of neointimal smooth muscle cell extracellular matrix gene expression in close proximity to non-foamy macrophages, suggesting regulation by local macrophage-associated factors. The purpose of these studies was to begin addressing the role of putative macrophage-associated factors such as transforming growth factor-beta (TGF-beta), by determining the spatial relationship between TGF-beta and neointimal matrix gene expression in human atherosclerotic pulmonary arteries. For example, the participation of TGF-beta in vascular remodeling could be inferred by its colocalization with non-foamy macrophages in areas of active matrix synthesis. In situ hybridization and immunohistochemistry demonstrated focal neointimal procollagen gene expression in close association with non-foamy but not foamy macrophages. Immunohistochemistry with isoform-specific anti-TGF-beta antibodies demonstrated all three isoforms of TGF-beta associated with non-foamy macrophages, but foamy macrophages were not immunoreactive. Neointimal and medial smooth muscle cells stained lightly. In contrast, intense TGF-beta immunoreactivity was also associated with medial smooth muscle cells in normal nonremodeling vessels. Immunohistochemistry with antibodies specific for latent TGF-beta was similar to immunohistochemistry for mature TGF-beta in both remodeling and nonremodeling vessels. Finally, using an antibody specific for active TGF-beta 1, immunoreactivity was only seen in non-foamy neointimal macrophages but not in foamy macrophages or medial smooth muscle cells from hypertensive or normal vessels. These observations suggest non-foamy macrophages may participate in modulating matrix gene expression in atherosclerotic remodeling via a TGF-beta-dependent mechanism.     

阿托伐他汀对大鼠自体移植静脉内膜增生的影响

目的： 探讨新型降脂药阿托伐他汀对自体移植静脉内膜增生的影响。 方法： 将Wistar大鼠颈外静脉移植于腹主动脉,建立大鼠自体静脉移植模型,实验分为3组：假手术组、移植对照组和移植实验组。自术后第1 d起,对移植实验组大鼠经胃管灌注给予阿托伐他汀(5 mg·kg-1·d-1)处理。干预4周后取移植静脉组织标本,制备4 μm厚组织切片,行病理组织学观察分析移植静脉内膜增生情况,行免疫组化染色分析新生内膜细胞SMα-actin和PCNA的表达情况。 结果： 移植对照组和实验组移植静脉内皮下层SMα-actin染色阳性平滑肌细胞大量增生,导致静脉内膜显著增厚,血管管腔明显狭窄。新生内膜定量分析显示移植实验组移植静脉内膜增生受到明显抑制,其新生内膜面积及新生内膜/中膜面积比均显著低于对照组(P<0.01);并且实验组移植静脉新生内膜细胞PCNA标记指数显著低于对照组(P<0.01)。 结论： 阿托伐他汀通过抑制新生内膜平滑肌细胞的增殖能有效抑制自体移植静脉内膜增生的发生发展,在防治血管重建术后再狭窄方面显示出良好的应用前景。     

Epithelial expression of profibrotic mediators in a model of allergen-induced airway remodeling

Airway remodeling, including subepithelial fibrosis, is a characteristic feature of asthma and likely contributes to the pathogenesis of airway hyperresponsiveness. We examined expression of genes related to airway wall fibrosis in a model of chronic allergen-induced airway dysfunction using laser capture microdissection and quantitative real-time PCR. BALB/c mice were sensitized and subjected to chronic ovalbumin exposure over a 12-wk period, after which they were rested and then harvested 2 and 8 wk after the last exposure. Chronic allergen-exposed mice had significantly increased indices of airway remodeling and airway hyperreactivity at all time points, although no difference in expression of fibrosis-related genes was found when mRNA extracted from whole lung was examined. In contrast, fibrosis-related gene expression was significantly upregulated in mRNA obtained from microdissected bronchial wall at 2 wk after chronic allergen exposure. In addition, when bronchial wall epithelium and smooth muscle were separately microdissected, gene expression of transforming growth factor-beta1 and plasminogen activating inhibitor-1 were significantly upregulated only in the airway epithelium. These data suggest that transforming growth factor-beta1 and other profibrotic mediators produced by airway wall, and specifically, airway epithelium, play an important role in the pathophysiology of airway remodeling.     

A variety of cytokines and immunologically relevant surface molecules are expressed by normal human skeletal muscle cells under proinflammatory stimuli

Muscle is an attractive target for gene therapy and for immunization with DNA vaccines and is also the target of immunological injury in myositis. It is important therefore to understand the immunologic capabilities of muscle cells themselves. In this study, we show that proinflammatory stimuli induce the expression of other cytokines such as IL-6, transforming growth factor-beta (TGF-β), and granulocyte-macrophage colony-stimulating factor (GM-CSF) by muscle cells themselves, as well as the up-regulation of human leucocyte antigen (HLA) class I, class II and intercellular adhesion molecule-1 (ICAM-1). Thus, muscle cells have an inherent ability to express and respond to a variety of cytokines and chemokines. The levels of HLA class I, class II and ICAM-1 in inflamed muscle may be affected by the secreted products of the stimulation.     

Time sequence of airway remodeling in a mouse model of chronic asthma: the relation with airway hyperresponsiveness

During the course of establishing an animal model of chronic asthma, we tried to elucidate the time sequence of airway hyperresponsiveness (AHR), airway inflammation, airway remodeling, and associated cytokines. Seven-week-old female BALB/c mice were studied as a chronic asthma model using ovalbumin (OVA). After sensitization, mice were exposed twice weekly to aerosolized OVA, and were divided into three groups depending on the duration of 4 weeks, 8 weeks, and 12 weeks. At each time point, airway responsiveness, inflammatory cells, cytokines in bronchoalveolar lavage fluids (BALF), serum OVA-specific IgE, IgG1, IgG2a, and histological examination were carried out. AHR to methacholine, increased levels of OVA-specific IgG1 and IgG2a, and goblet cell hyperplasia were continuously sustained at each time point of weeks. In contrast, we observed a time-dependent decrease in serum OVA-specific IgE, BALF eosinophils, BALF cytokines such as IL-13, transforming growth factor-beta1, and a time-dependent increase in BALF promatrix metalloproteinase-9 and peribronchial fibrosis. In this OVA-induced chronic asthma model, we observed airway remodelings as well as various cytokines and inflammatory cells being involved in different time-dependent manners. However, increased airway fibrosis did not directly correlate with a further increase in airway hyperresponsiveness.     

慢性肾功能衰竭促进小鼠动静脉瘘内膜增生

目的观察慢性肾功能衰竭对小鼠动静脉内瘘术后血管内膜增生的影响及单核细胞趋化蛋白-1(MCP-1)在内膜增生中的作用。方法将24只C57小鼠分为对照组(n=12)与实验组(n=12),实验组行左肾全切及右肾上极动脉结扎,对照组行假手术,术后6周行左侧颈总动脉-颈外静脉端吻合建立动静脉瘘(AVF)模型,AVF术后3周取瘘口静脉端血管组织。观察各组小鼠AVF内膜组织的病理改变及小鼠血液尿素氮(BUN)水平与内膜增生程度的关系;免疫组化法、RT-PCR及Western blot检测各组静脉组织α平滑肌肌动蛋白(α-SMA)、Ki-67、NF-κB及MCP-1的蛋白及mRNA的表达。结果 1)实验组较对照组小鼠血BUN水平明显上升(P0.05),静脉端内膜增生更显著,管腔更狭窄(P0.05)。2)实验组血管增生内膜α-SMA表达明显增加,血管平滑肌细胞(VSMC)增殖显著,NF-κB及MCP-1表达明显上升(均P0.05)。3)MCP-1促进体外培养VSMC增殖。结论慢性肾功能衰竭可明显增高NF-κB及MCP-1表达,从而促进动静脉瘘内膜增生及VSMC增殖。     

alpha8beta1 Integrin is up-regulated in the neointima concomitant with late luminal loss after balloon injury.

INTRODUCTION: Constrictive remodeling of the neointima results in the late lumen loss and restenosis after balloon angioplasty. Intense expression of alpha8beta1 integrin in the contractile state of vascular smooth muscle cells (VSMCs) and in myofibroblasts led us to hypothesize that it might be involved in the process of late constrictive remodeling. METHODS AND RESULTS: Balloon injury was used to induce neointima formation in the rat carotid artery. Immunohistochemical analysis and immunoconfocal studies showed that late lumen narrowing was concomitant with the up-regulation of smooth muscle alpha-actin and alpha8 integrin in the neointima. The transforming growth factor-beta (TGF-beta)-induced contractile properties of fibroblasts and VSMCs populated in a three-dimensional collagen matrix was associated with up-regulation of alpha8 integrin. TGF-beta-induced myofibroblastic features in Rat1 fibroblasts were impaired in cells pretreated with a small interference RNA silencing the alpha8 integrin gene. CONCLUSION: The close correlation between alpha8 integrin up-regulation in the neointima and late luminal loss and alpha8 integrin being required for contractile properties induced by TGF-beta highlight a possible role for alpha8 integrin in postangioplasty restenosis.     

KCa3.1 Ca2+ activated K+ channels regulate human airway smooth muscle proliferation

Airway smooth muscle cell hyperplasia contributes to airway remodeling and hyperreactivity characteristic of asthma. Changes to potassium channel activity in proliferating human airway smooth muscle (HASM) cells have been described, but no regulatory role in proliferation has been attributed to them. We sought to investigate the expression of the intermediate conductance calcium-activated potassium channel K(Ca)3.1 in HASM cells and investigate its role in proliferation. Smooth muscle cells derived from human airways were grown in vitro and K(Ca)3.1 channel expression was measured using Western blot, RT-PCR, and patch clamp electrophysiology. Pharmacologic inhibitors of the channel were used in assays of cellular proliferation, and flow cytometry was used to identify cell cycle regulation. HASM cells expressed K(Ca)3.1 channel mRNA, protein, and activity with up-regulation evident after transforming growth factor-beta stimulation. Pharmacologic inhibition of K(Ca)3.1 led to growth arrest in cells stimulated to proliferate with mitogens. These inhibitors did not cause cellular toxicity or induce apoptosis. We have demonstrated, for the first time, the expression of K(Ca)3.1 channels in HASM cells. In addition, we have shown that K(Ca)3.1 channels are important in HASM cell proliferation, making these channels a potential therapeutic target in airway remodeling.     

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.     

A model of primary atherosclerosis and post-angioplasty restenosis in mice

Although mice deficient in various genes are providing greater insight into the mechanisms of restenosis after angioplasty, there have been limitations with murine models not simulating human vascular disease. To develop a more clinically applicable model of primary atherosclerosis and restenosis following angioplasty of the primary lesion, we fed apolipoprotein E-deficient mice a Western diet and occluded the left common carotid artery for 2 days. Three weeks after flow was restored, the temporarily occluded carotids demonstrated atherosclerotic lesions containing foam cells, cholesterol clefts, necrotic cores, and fibrous capsules. The atherosclerotic carotids in other animals underwent angioplasty with a beaded probe, resulting in plaque and medial layer disruption. Three weeks after angioplasty, although there was significant neointimal lesion formation, the luminal narrowing did not change significantly secondary to overall vessel enlargement (positive remodeling). Neointimal lesions were composed of smooth-muscle cells and extracellular matrix observed adjacent to the original atherosclerotic plaques. Similarly, even at 3 months after the angioplasty the lumen was maintained despite greater neointimal lesion formation caused by progressive positive remodeling. This new murine model of primary atherosclerosis and postangioplasty intimal hyperplasia and remodeling mimics the human disease pattern of postangioplasty intimal hyperplasia. Used in transgenic animals, this model will likely facilitate understanding of the mechanisms of restenosis in humans.     

Pirfenidone modulates airway responsiveness, inflammation, and remodeling after repeated challenge

We investigated the therapeutic potential of a newly developed antifibrotic agent, pirfenidone, to regulate airway remodeling and the development of allergic airway inflammation and airway hyperresponsiveness after chronic allergen challenge. Administration of pirfenidone after sensitization but during the period of ovalbumin challenge significantly prevented the development of airway hyperresponsiveness and prevented eosinophil and lymphocyte accumulation in the airways. IL-4, IL-5, and IL-13 levels in bronchoalveolar lavage fluid and ovalbumin-specific serum IgE antibody levels were also significantly reduced. Treatment with pirfenidone significantly reduced transforming growth factor-beta1 and platelet-derived growth factor levels in bronchoalveolar lavage fluid. Pirfenidone reduced the expression of transforming growth factor-beta1, the development of goblet cell hyperplasia and subepithelial collagenization, and the increases in contractile elements in the lung. These data indicate that pirfenidone may play an important role in the treatment of asthma and has the potential reduce or prevent airway remodeling.     

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。     

强力霉素影响基质金属蛋白酶活性和血管重构的实验研究

目的：观察强力霉素对损伤动脉组织中基质金属蛋白酶（MMP）活性的抑制作用，并探讨强力霉素对血管平滑肌细胞增殖、动脉内膜增生、管腔重构的影响。方法:球囊导管扩张动脉的方法建立大鼠颈总动脉损伤模型。治疗组用强力霉素30 mg·kg^-1·d^-1干预。明胶酶谱法测定损伤动脉组织中MMPs的活性。用HE染色、VVG染色、免疫组化标记α-actin和增殖细胞核抗原的方法观察损伤动脉内膜厚度、管腔重构及平滑肌细胞增殖的情况。结果:①强力霉素治疗组MMP-9活性在术后24 h、3 d分别比对照组低26.3％、34.5％（P<0.01）；MMP-2活性在术后7 d比对照组低40.0％（P<0.01）。②强力霉素治疗使术后7 d内膜平滑肌细胞增殖率（43.23％±1.06％）显著低于对照组（62.76％±1.02％）（P<0.01）；使术后14 d、28 d新生内膜厚度比对照组分别少32.0％、38.8％（P<0.01），而管腔面积比对照组多58.0％、90.4％（P<0.01） 。结论：强力霉素可以显著降低血管损伤后MMPs活性，抑制内膜平滑肌细胞的增殖、新生内膜增生以及管腔重构，提示它可能具有防治PTCA术后再狭窄的作用。     

CCR3 Blockade Attenuates Eosinophilic Ileitis and Associated Remodeling

Intestinal remodeling and stricture formation is a complication of inflammatory bowel disease (IBD) that often requires surgical intervention. Although eosinophils are associated with mucosal remodeling in other organs and are increased in IBD tissues, their role in IBD-associated remodeling is unclear. Histological and molecular features of ileitis and remodeling were assessed using immunohistochemical, histomorphometric, flow cytometric, and molecular analysis (real-time RT-PCR) techniques in a murine model of chronic eosinophilic ileitis. Collagen protein was assessed by Sircol assay. Using a spontaneous eosinophilic Crohn's-like mouse model SAMP1/SkuSlc, we demonstrate an association between ileitis progression and remodeling over the course of 40 weeks. Mucosal and submucosal eosinophilia increased over the time course and correlated with increased histological inflammatory indices. Ileitis and remodeling increased over the 40 weeks, as did expression of fibronectin. CCR3-specific antibody-mediated reduction of eosinophils resulted in significant decrease in goblet cell hyperplasia, muscularis propria hypertrophy, villus blunting, and expression of inflammatory and remodeling genes, including fibronectin. Cellularity of local mesenteric lymph nodes, including T- and B-lymphocytes, was also significantly reduced. Thus, eosinophils participate in intestinal remodeling, supporting eosinophils as a novel therapeutic target.     

Factor Xa stimulates proinflammatory and profibrotic responses in fibroblasts via protease-activated receptor-2 activation

Coagulation proteases have been suggested to play a role in the pathogenesis of tissue remodeling and fibrosis. We therefore assessed the proinflammatory and fibroproliferative effects of coagulation protease factor (F)Xa. We show that FXa elicits a signaling response in C2C12 and NIH3T3 fibroblasts. FXa-induced ERK1/2 phosphorylation was dependent on protease-activated receptor (PAR)-2 cleavage because desensitization with a PAR-2 agonist (trypsin) but not a PAR-1 agonist (thrombin) abolished FXa-induced signal transduction and PAR-2 siRNA abolished FXa-induced ERK1/2 phosphorylation. The PAR-2-dependent cellular effects of FXa led to fibroblast proliferation, migration, and differentiation into myofibroblasts, as demonstrated by the expression of alpha-smooth muscle actin and desmin, followed by the secretion of the cytokines monocyte chemotactic protein-1 and interleukin-6 as well as the expression of the fibrogenic proteins transforming growth factor-beta and fibronectin. To assess the relevance of FXa-induced proliferation and cell migration, we examined the effect of FXa in a wound scratch assay. Indeed, FXa facilitated wound healing in a PAR-2- and ERK1/2-dependent manner. Taken together, these results support the notion that, beyond its role in coagulation, FXa-dependent PAR-2 cleavage might play a role in the progression of tissue fibrosis and remodeling.     

低浓度脂多糖加速大鼠颈动脉球囊损伤后新生内膜的形成

目的：探讨低浓度脂多糖（LPS）刺激机体免疫系统对血管损伤后加速血管新生内膜增生的影响。方法：选用健康Wistar大鼠，静脉注入LPS后，行颈动脉球囊损伤术，建立血管内膜损伤模型。采用免疫荧光或组织化学染色观察内膜增生变化。Western blot 分析损伤组织特异性平滑肌细胞标志物和细胞凋亡表达。用ELISA测定血清白介素-1β(IL-1β)含量和流式细胞术分析CD14阳性细胞表达水平。结果：每只鼠注入LPS(50 ng)后循环血中单核细胞和IL-1β水平显著升高。血管损伤7 d后中膜平滑肌细胞增殖, 转化为合成表型，新生内膜逐渐形成，随时间延长，内膜增生加速，内膜厚度由(151.2±14.5)μm2增至(173.9±15.3)μm2。免疫荧光染色观察到增殖细胞核抗原及核因子-κB分别定位于新生内膜和外膜。Western blot分析显示新生内膜形成早期LPS组平滑肌特异性标志蛋白α-肌动蛋白多于对照组，caspase-3表达持续上调，细胞凋亡多于对照组。结论：炎症介质LPS刺激全身免疫系统导致血管损伤后新生内膜暂时性增生，表明炎症介质可以加剧血管损伤后再狭窄的形成。     

4种肺动脉高压动物模型肺血管重构模式的差异研究

目的：探讨4种肺动脉高压（PH）动物模型肺血管重构模式的差异。方法:雄性SD大鼠（350-400g），分别通过腹主动脉-腔静脉分流（A-VF, n=10）、左肺切除（PE, n=10）、野百合碱注射（MCT, n=10）、左肺切除+MCT（PE+MCT,n=12）4种方法建立PH模型。检测平均肺动脉压力(mPAP）、RV/(LV+S)重量比值、肺小动脉中膜厚度百分比（WT%）、无肌性动脉肌化程度和新生内膜(neointima)形成、新生内膜增殖度和血管阻塞计分（VOS）。结果:在PE+MCT组（肺切除术后5周，MCT注射后4周）右肺腺泡内血管出现了新生内膜病变，其它组均没有新生内膜病变形成。PE+MCT组的动物出现了严重的右心室肥大，动脉中膜明显增厚，平均肺动脉压（mPAP）和无肌性血管肌化程度显著增加；A-VF、PE和MCT组仅形成轻-中度的右心室肥大、mPAP升高和小动脉肌化。结论:左肺切除联合应用MCT能成功诱导大鼠PH新生内膜模型，该模型能更好地模拟人类严重PH的病理改变，是研究梗阻性PH更为适用的动物模型。     

Macrophage depletion impairs wound healing and increases left ventricular remodeling after myocardial injury in mice

Macrophages have been suggested to be beneficial for myocardial wound healing. We investigated the role of macrophages in myocardial wound healing by inhibition of macrophage infiltration after myocardial injury. We used a murine cryoinjury model to induce left ventricular damage. Infiltrating macrophages were depleted during the 1st week after cryoinjury by serial intravenous injections of clodronate-containing liposomes. After injury, the presence of macrophages, which secreted high levels of transforming growth factor-beta and vascular endothelial growth factor-A, led to rapid removal of cell debris and replacement by granulation tissue containing inflammatory cells and blood vessels, followed by myofibroblast infiltration and collagen deposition. In macrophage-depleted hearts, nonresorbed cell debris was still observed 4 weeks after injury. Secretion of transforming growth factor-beta and vascular endothelial growth factor-A as well as neovascularization, myofibroblast infiltration, and collagen deposition decreased. Moreover, macrophage depletion resulted in a high mortality rate accompanied by increased left ventricular dilatation and wall thinning. In conclusion, infiltrating macrophage depletion markedly impairs wound healing and increases remodeling and mortality after myocardial injury, identifying the macrophage as a key player in myocardial wound healing. Based on these findings, we propose that increasing macrophage numbers early after myocardial infarction could be a clinically relevant option to promote myocardial wound healing and subsequently to reduce remodeling and heart failure.