Induction of pulmonary connective tissue growth factor in heart failure is associated with pulmonary parenchymal and vascular remodeling

OBJECTIVES: Pulmonary remodeling is a well recognized consequence of heart failure (HF). However, the cellular and molecular mechanisms orchestrating the structural alterations of the lungs in HF are poorly understood. We have previously reported induction of the profibrotic peptide connective tissue growth factor (CTGF) in myocardial tissue of rats with HF, suggesting a role of CTGF during myocardial remodeling. The aim of the present study was to explore the potential role of CTGF in pulmonary remodeling in HF. METHODS: Pulmonary tissue samples were obtained from rats with myocardial infarction (MI) subsequent to ligation of the left coronary artery. Real-time quantitative RT-PCR was employed to investigate mRNA levels. The cellular distribution of CTGF was analysed by immunohistochemistry. RESULTS: Seven days after induction of myocardial infarction (MI) and HF in rats we found 2.3-fold and 1.9-fold increase of pulmonary transforming growth factor-beta1 and procollagen alpha1(I) mRNA levels, respectively, and typical morphological characteristics of pulmonary remodeling including interstitial fibrosis and medial thickening of pulmonary arteries. Pulmonary CTGF mRNA levels were substantially elevated in HF rats compared to sham-operated rats (4-fold; P<0.05) and corresponded with similar increase (3-fold; P<0.05) of pulmonary CTGF protein contents. Immunohistochemical analysis revealed increased pulmonary anti-CTGF immunoreactivity in HF, with immunostaining predominantly localized to alveolar macrophages and interstitial fibroblasts. Isolated alveolar macrophages from HF rats demonstrated substantial induction of CTGF mRNA expression (16-fold; p<0.05). Interestingly, platelets caused robust induction of CTGF mRNA expression in alveolar macrophages upon co-culture in vitro. CONCLUSION: Pulmonary CTGF was substantially increased in parallel with pulmonary remodeling in rats with HF. Our data indicate that alveolar macrophages are a major source of increased pulmonary CTGF in HF and that CTGF may be a player in the profibrotic mechanisms associated with HF.     

结缔组织生长因子和周期蛋白D1在烟雾暴露大鼠肺血管重塑中的表达变化

目的 探讨烟雾暴露致大鼠肺血管重塑中结缔组织生长因子(CTGF)和周期蛋白D1的表达变化及其意义.方法 健康雄性Wistar大鼠24只,按随机数字表法分为对照组,烟雾暴露2周、4周和8周组检测PaO2,HE染色和平滑肌细胞α肌动蛋白染色观察肺血管重塑情况,行免疫组织化学染色观察CTGF和周期蛋白D1在肺动脉平滑肌中的表达,实时定量逆转录聚合酶链反应(qRT-PCR)检测肺动脉CTGF mRNA和周期蛋白D1 mRNA表达,Western blot法检测CTGF和周期蛋白D1的蛋白表达量.结果 各组大鼠PaO2分别为(91±4),(92±5),(91±4),(93±4)mm Hg(1 mm Hg=0.133 kPa),差异均无统计学意义(F=0.15,P＞0.05).随烟雾暴露时间的延长,肌化血管比例、肺血管壁厚度和血管直径比值(W/T)、CTGF和周期蛋白D1的mRNA和蛋白表达均呈时间依赖性增加,各组间比较差异均有统计学意义(F=51.06～590.20,P＜0.05).CTGF与周期蛋白D1表达与肺W/T呈显著正相关(r=0.097～0.918,P＜0.01),二者的表达之间也呈显著正相关.结论 烟雾暴露大鼠的CTGF和cyclinD1表达量均随暴露时间延长而显著升高,二者可能与烟雾暴露致肺动脉平滑肌细胞异常增殖有关.     

Apoptosis signal-regulating kinase-1 is involved in vascular endothelial and cardiac remodeling caused by nitric oxide deficiency

Long-term treatment with N(omega)-nitro-l-arginine methylester (l-NAME), an NO synthase inhibitor, induces hypertension and cardiovascular injury. However, its precise mechanism is unknown. Using apoptosis signal-regulating kinase-1 (ASK1)-deficient mice, we investigated the role of ASK1 in cardiovascular injury caused by l-NAME treatment. l-NAME was orally administered to ASK1-deficient and C57BL/6J (wild) mice for 8 weeks. l-NAME treatment increased blood pressure of wild and ASK1-deficient mice to a similar extent, indicating no role of ASK1 in NO-deficient hypertension. l-NAME treatment significantly impaired acetylcholine-induced carotid arterial relaxation in wild mice (P<0.01), being associated with the decreased endothelial NO synthase (eNOS) activity (P<0.01) and the increased disruption of eNOS dimer (P<0.01), whereas these changes by l-NAME were substantially attenuated in ASK1-deficient mice. Thus, ASK1 is involved in the impairment of vascular endothelial function by reducing eNOS activity and disrupting eNOS dimer. l-NAME treatment increased vascular reduced nicotinamide-adenine dinucleotide phosphate oxidase activity and superoxide in wild mice to a greater extent than in ASK1 deficient mice. l-NAME treatment in wild mice caused cardiac hypertrophy, myocyte apoptosis, macrophage infiltration, coronary arterial remodeling, interstitial fibrosis, and the expression of monocyte chemoattractant protein-1 and transforming growth factor-beta1, whereas these cardiac changes by l-NAME were absent in ASK1-deficient mice. Cardiac reduced nicotinamide-adenine dinucleotide phosphate oxidase activation and superoxide elevation by l-NAME were much less in ASK1-deficient mice than in wild mice. Our work provided the first evidence that ASK1 is implicated in vascular endothelial dysfunction and cardiovascular remodeling induced by NO deficiency by regulating eNOS and reduced nicotinamide-adenine dinucleotide phosphate oxidase.     

基质金属蛋白酶9及金属蛋白酶组织抑制剂1在肺血栓栓塞相关肺血管重构中的作用探讨

目的 通过颈外静脉注入自体血凝块并应用氨甲环酸建立大鼠肺血栓栓塞症(PTE)相关肺血管重构动物模型,探讨基质金属蛋白酶9(MMP-9)及金属蛋白酶组织抑制剂1(TIMP-1)在PTE相关肺血管重构中的作用.方法 将56只SD大鼠随机分为PTE组及假手术组(Sham组).PTE组用注入血栓及抑制纤溶方法 制作PTE模型.Sham组用生理盐水代替血栓及抗纤溶药物.每组分别于制模后1、3、7、14 d各取7只鼠处死,留血检测 MMP-9、TIMP-1 含量.取左肺做苏木素伊红染色、磷钨酸苏木素(PTAH)染色及α- 平滑肌肌动蛋白(α-SMA)免疫组织化学(IH)染色,并做 MMP-9、TIMP-1 IH 及原位杂交(ISH)染色.结果 ①PTE组制模第7天见到栓塞部位肺血管重构,14 d 时更加明显.②PTE组血清 MMP-9 浓度及 MMP-9/TIMP-1 比值均高Sham(P0.01),TIMP-1在1 d 及3 d 时显著高于Sham组(P＜0.05,P＜0.01),7 d与14 d 时降至 Sham 组水平.③PTE 组 IH 染色显示栓塞部位的血管及新生内膜中的平滑肌细胞内皮细胞及浸润的单核一巨噬细胞,MMP-9 染色阳性.栓塞部位的血管内皮细胞及平滑肌细胞 TIMP-1染色阳性.Sham 组 MMP-9及 TIMP-1 染色阴性或弱阳性.MMP-9 及 TIMP-1 ISH染色结果与 IH 染色结果 基本一致.④α-SMA染色显示平滑肌细胞是新生内膜的主要成分.结论 颈外静脉注入自体血栓并重复应用抗纤溶药物可成功建立大鼠PTE相关肺血管重构模型,PTE相关肺血管重构的主要病理改变为新生内膜生成.MMP-9/TIMP-1失调参与了PTE相关肺血管重构过程.     

Plasticity of CD133+ cells: role in pulmonary vascular remodeling

OBJECTIVE: Studies in pulmonary arteries (PA) of patients with chronic obstructive pulmonary disease (COPD) suggest that bone marrow-derived endothelial progenitor cells (CD133(+)) may infiltrate the intima and differentiate into smooth muscle cells (SMC). This study aimed to evaluate the plasticity of CD133(+) cells to differentiate into SMC and endothelial cells (EC) in both cell culture and human isolated PA. METHODS: Plasticity of granulocyte-colony stimulator factor (G-CSF)-mobilized peripheral blood CD133(+) cells was assessed in co-cultures with primary lines of human PA endothelial cells (PAEC) or SMC (PASMC) and in isolated human PA. We also evaluated if the phenotype of differentiated progenitor cells was acquired by fusion or differentiation. RESULTS: The in vitro studies demonstrated CD133(+) cells may acquire the morphology and phenotype of the cells they were co-cultured with. CD133(+) cells co-incubated with human isolated PA were able to migrate into the intima and differentiate into SMC. Progenitor cell differentiation was produced without fusion with mature cells. CONCLUSIONS: We provide evidence of plasticity of CD133(+) cells to differentiate into both endothelial cells and SMC, reinforcing the idea of their potential role in the remodeling process of PA in COPD. This process was conducted by transdifferentiation and not by cell fusion.     

血管内皮生长因子与慢性阻塞性肺疾病的肺血管重构

血管内皮生长因子（VEGF）具有促进血管内皮细胞增殖和血管生成的作用。研究表明，VEGF在慢性阻塞性肺疾病（COPD）的肺血管重构中起着重要作用，进而加重其气道阻力和气道炎症，影响COPD的预后。如何使VEGF保持一个合适的水平有待于我们进一步的研究。     

凝血酶及凝血因子Xa诱导血管平滑肌细胞中植物凝集素样氧化型低密度脂蛋白受体-1表达的实验研究

目的研究血管平滑肌细胞中凝血酶及凝血因子xa对新型的氧化型低密度脂蛋白的清道夫受体,即植物凝集素样氧化型低密度脂蛋白受体-1（LOX-1）表达的影响。方法培养的牛主动脉平滑肌细胞,予凝血酶及凝血因子xa刺激后,用鼠抗LOX-1单克隆抗体,对细胞裂解液和浓缩的培养基进行Western blot分析,观察LOX-1表达的变化。结果在凝血酶2．0U／ml及凝血因子Xa 50 nmol／L时,可观察到细胞膜结合型LOX-1表达明显增加。凝血酶3．0U／ml和凝血因子Xa100nmol／L刺激14h后,细胞培养基中可溶型LOX-1表达明显升高。对平滑肌细胞给予1．0U／ml凝血酶及100nmol／L凝血因子xa刺激,4h后LOX-1表达开始增加,12h后达高峰。AGl478是表皮生长因子受体相关酪氨酸激酶抑制剂。用指定浓度AGl478预刺激后,再予凝血酶和凝血因子xa,然后对细胞裂解液进行Western blot分析。AGl478可显著抑制凝血酶及凝血因子xa导致的LOX-1表达增加。结论凝血酶及凝血因子Xa可诱导LOX-1表达增加,此作用由表皮生长因子受体介导。     

The first armadillo repeat is involved in the recognition and regulation of beta-catenin phosphorylation by protein kinase CK1

Multiple phosphorylation of beta-catenin by glycogen synthase kinase 3 (GSK3) in the Wnt pathway is primed by CK1 through phosphorylation of Ser-45, which lacks a typical CK1 canonical sequence. Synthetic peptides encompassing amino acids 38-64 of beta-catenin are phosphorylated by CK1 on Ser-45 with low affinity (K(m) approximately 1 mM), whereas intact beta-catenin is phosphorylated at Ser-45 with very high affinity (K(m) approximately 200 nM). Peptides extended to include a putative CK1 docking motif (FXXXF) at 70-74 positions or a F74AA mutation in full-length beta-catenin had no significant effect on CK1 phosphorylation efficiency. beta-Catenin C-terminal deletion mutants up to residue 181 maintained their high affinity, whereas removal of the 131-181 fragment, corresponding to the first armadillo repeat, was deleterious, resulting in a 50-fold increase in K(m) value. Implication of the first armadillo repeat in beta-catenin targeting by CK1 is supported in that the Y142E mutation, which mimics phosphorylation of Tyr-142 by tyrosine kinases and promotes dissociation of beta-catenin from alpha-catenin, further improves CK1 phosphorylation efficiency, lowering the K(m) value to <50 nM, approximating the physiological concentration of beta-catenin. In contrast, alpha-catenin, which interacts with the N-terminal region of beta-catenin, prevents Ser-45 phosphorylation of CK1 in a dose-dependent manner. Our data show that the integrity of the N-terminal region and the first armadillo repeat are necessary and sufficient for high-affinity phosphorylation by CK1 of Ser-45. They also suggest that beta-catenin association with alpha-catenin and beta-catenin phosphorylation by CK1 at Ser-45 are mutually exclusive.     

Arterial endothelium-specific activin receptor-like kinase 1 expression suggests its role in arterialization and vascular remodeling

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular disorder characterized by epistaxis, mucocutaneous telangiectases, and arteriovenous malformations (AVM). Two genes are linked to HHT: endoglin (ENG) in HHT1 and activin receptor-like kinase 1 (ACVRL1; ALK1) in HHT2. Although both genes are involved in the transforming growth factor beta signaling pathways, the pathogenetic mechanisms for HHT remain elusive. It was shown that mutations in the Alk1 gene in mice and zebrafish resulted in an embryonic lethal phenotype due to severe dilation of blood vessels. We created a novel null mutant mouse line for Alk1 (Alk1lacZ) by replacing its exons, including the one that encodes the transmembrane domain, with the beta-galactosidase gene. Using Alk1lacZ mice, we show that Alk1 is predominantly expressed in developing arterial endothelium. Alk1 expression is greatly diminished in adult arteries, but is induced in preexisting feeding arteries and newly forming arterial vessels during wound healing and tumor angiogenesis. We also show that hemodynamic changes, which require vascular remodeling, may regulate Alk1 expression. Our studies suggest the role of Alk1 signaling in arterialization and remodeling of arteries. Contrary to the current view of HHT as venous disease, our findings suggest that the arterioles rather than the venules are the primary vessels affected by the loss of an Alk1 allele, and that blood vessels with reduction in Alk1 expression may harbor defects in responding to demands for vascular remodeling.     

Essential role of vascular endothelial growth factor in angiotensin II-induced vascular inflammation and remodeling

Angiotensin II (Ang II) upregulates vascular endothelial growth factor (VEGF) and activates vascular inflammation. However, the decisive role of VEGF in Ang II-induced vascular inflammation and remodeling has not been addressed. Ang II infusion to wild-type mice increased local expression of VEGF and its receptors in cells of aortic wall and plasma VEGF, and caused aortic inflammation (monocyte infiltration) and remodeling (wall thickening and fibrosis). Hypoxia-inducible factor-1alpha colocalized with VEGF-positive cell types. Blockade of VEGF by the soluble VEGF receptor 1 (sFlt-1) gene transfer attenuated the Ang II-induced inflammation and remodeling. The sFlt-1 gene transfer also inhibited the increased expression of VEGF and inflammatory factors such as monocyte chemoattractant protein-1. In contrast, sFlt-1 gene transfer did not affect Ang II-induced arterial hypertension and cardiac hypertrophy. VEGF is an essential mediator in Ang II-induced vascular inflammation and structural changes through its proinflammatory actions.     

血管内皮生长因子在肺动脉平滑肌细胞重构中的作用

目的 :探讨血管内皮生长因子 （VEGF）在野百合碱 （MCT）性肺动脉高压 （PH）中的作用。方法 :用 MCT复制大鼠慢性 PH病理模型 ,用免疫组化法和图像分析技术测定肺组织中 VEGF的表达。结果 :发现 VEGF可在正常大鼠肺血管平滑肌、支气管平滑肌和软骨组织中表达 ,并且 MCT组 VEGF表达的相对含量在肺血管平滑肌 （177±7）和支气管平滑肌 （172± 6）均较正常组 （血管平滑肌 13 6± 8,支气管平滑肌 13 8± 12 ）呈显著增强 （P<0 .0 5）。结论 :MCT性 PH中 VEGF在肺血管平滑肌的过度表达参与 MCT性 PH的发病过程     

The serum- and glucocorticoid-induced kinase is a physiological mediator of aldosterone action

Aldosterone plays a major role in regulating sodium and potassium flux in epithelial tissues such as kidney and colon. Recent evidence suggests that serum- and glucocorticoid-regulated kinase (SGK) is induced by aldosterone and acts as a key mediator of aldosterone action in epithelial tissues. Induction of SGK messenger RNA (mRNA) has previously been shown within 30 min of addition of supraphysiological doses of aldosterone to XENOPUS: A6 cells and within 4 h in rat kidney in vivo. In this study we determined the time course of SGK induction, at doses of aldosterone in the physiological range, in rat kidney and colon, using Northern and Western blot analyses and in situ hybridization and determined concurrent changes in urinary sodium and potassium excretion by Kagawa bioassay. On Northern blot analysis, SGK mRNA levels were significantly elevated in both kidney and colon 60 min after the injection of aldosterone. SGK protein in late distal colon was significantly elevated 2 and 4 h after aldosterone treatment. In situ hybridization showed SGK mRNA to be induced in renal collecting ducts and distal tubular elements in both cortex and medulla by doses of aldosterone of 0.1 microg/100 g BW or more within 30 min of steroid treatment. Significant changes in urinary composition were similarly seen with an aldosterone dose of 0.1 microg/100 g BW from 90 min after aldosterone injection. The early onset of SGK induction in kidney and colon and the correlation with urinary changes in terms of both time course and dose response suggest that SGK plays an important role in mediating the effects of aldosterone on sodium homeostasis in vivo.     

转化生长因子β1与一氧化氮合酶及成肌纤维细胞参与大鼠缺氧性肺血管重塑

目的观察转化生长因子β1(TGF-β1)与诱导型一氧化氮合酶(iNOS)基因动态表达变化及成肌纤维细胞形成在大鼠缺氧性肺血管重塑中的作用.方法40只成年雄性Wistar大鼠分成常氧组、缺氧3、7、14 d和21 d组,每组8只,测各组大鼠平均肺动脉压(mPAP)、血管形态学指标、右室肥大指数(RVHI);原位杂交和免疫组化检测TGF-β1、iNOS基因表达,透射电镜观察腺泡内血管壁细胞表型. 结果缺氧7 d后大鼠mPAP升高[(18.41±0.37)mm Hg,P<0.05].缺氧性肺血管重塑、右心室肥大于缺氧14 d后出现.透射电镜证实成肌纤维细胞含有特异性的微丝和丰富的粗面内质网.TGF-β1mRNA于缺氧3 d、7 d表达增高不明显,缺氧14d增高(0.385±0.028,P<0.01);TGF-β1蛋白在常氧组呈弱阳性,缺氧3 d表达增强(0.198±0.031,P<0.01),缺氧7 d组达高峰(0.267±0.035,P<0.01).对照组大鼠肺动脉壁iNOS mRNA弱阳性,缺氧3 d后表达增强(0.245±0.036,P<0.01),缺氧7 d达高峰水平(0.318±0.034,P<0.01),以后维持于高峰水平;iNOS蛋白在常氧组大鼠肺动脉中膜iNOS弱阳性,缺氧3 d始增高(0.225±0.030,P<0.01),缺氧7 d起稳定于高水平.结论缺氧诱导TGF-β1和iNOS动态表达变化及肺血管成肌纤维细胞的形成参与大鼠缺氧性肺血管重塑.