Apocynin抑制慢性心力衰竭兔心肌纤维化的实验研究

目的:探讨NADPH氧化酶抑制剂Apocynin对心衰兔心肌纤维化的影响及其机制。方法:32只兔随机分为假手术安慰剂组、假手术Apocynin组、心衰安慰剂组、心衰Apocynin组。通过容量超负荷联合压力超负荷建立家兔心衰模型。安慰剂或Apocynin以15mg/天口服。8周后,行超声心动图检查,观察动物的心脏结构和功能变化。通过细胞色素C还原试验测定心肌NADPH氧化酶活性。通过组织形态学分析心肌纤维化程度。运用Real-timePCR检测心肌转化生长因子β(TGF-β)、结缔组织生长因子(CTGF)、基质金属蛋白酶-2(MMP-2)和MMP-9基因表达。结果:与假手术安慰剂组比较,心衰安慰剂组心脏明显扩大,心功能明显降低(P<0.05),心肌NADPH氧化酶活性增加(P<0.05),心肌纤维化显著(P<0.05),TGF-β、CTGF、MMP-2和MMP-9表达上调(P<0.05)。与心衰安慰剂组比较,心衰Apocynin组心功能显著改善(P<0.05),心肌NADPH氧化酶活性明显降低(P<0.05),心肌纤维化显著减轻(P<0.05),心肌TGF-β、CTGF、MMP-2和MMP-9表达明显降低(P<0.05)。结论:Apocynin可通过下调心肌TGF-β、CTGF、MMP-2和MMP-9基因表达,改善心衰兔心肌纤维化。     

Cardiomyopathy in dystrophin-deficient hearts is prevented by expression of a neuronal nitric oxide synthase transgene in the myocardium

Null mutation of dystrophin causes the lethal pathology of Duchenne muscular dystrophy (DMD) in which there is progressive pathology of skeletal and cardiac muscles. A large proportion of DMD patient deaths are attributable to cardiac dysfunction associated with ventricular fibrosis, arrhythmias and conduction abnormalities, although the relationships between the dystrophin mutation and the cardiac defects are unknown. Here, we tested whether cardiac pathology in dystrophin-deficient mdx mice can be corrected by the elevated production of nitric oxide (NO) by the myocardium. Dystrophin-deficient mdx mice were produced in which there was myocardial expression of a neuronal nitric oxide synthase (nNOS) transgene. Expression of the transgene prevented the progressive ventricular fibrosis of mdx mice and greatly reduced myocarditis. Electrocardiographs (ECG) attained by radiotelemetry of freely ambulatory mice showed that mdx mice displayed cardiac abnormalities that are characteristic of DMD patients, including deep Q-waves, diminished S:R ratios, polyphasic R-waves and frequent premature ventricular contractions. All of these ECG abnormalities in mdx mice were improved or corrected by nNOS transgene expression. In addition, defects in mdx cardiac autonomic function, which were reflected by decreased heart rate variability, were significantly reduced by nNOS transgene expression. These findings indicate that increasing NO production by dystrophic hearts may have therapeutic value.     

T and B lymphocyte depletion has a marked effect on the fibrosis of dystrophic skeletal muscles in the scid/mdx mouse

Abnormal connective tissue proliferation following muscle degeneration is a major pathological feature of Duchenne muscular dystrophy (DMD), a genetic myopathy due to lack of the sarcolemmal dystrophin protein. Since this fibrotic proliferation is likely to be a major obstacle to the efficacy of future therapies, research is needed to understand and prevent the fibrotic process in order to develop an effective treatment. Murine muscular dystrophy (mdx) is genetically homologous to DMD, and histopatological alterations are comparable to those of the muscles of patients with DMD. To investigate the development of fibrosis, we bred the mdx mouse with the scid immunodepressed mouse and analysed fibrosis histologically; we used ELISA analysis to determine TGF-beta1 expression. Significant reduction of fibrosis and TGF-beta1 expression was found in the muscles of the scid/mdx mice. However, we observed similar centrally located nuclei, necrosis, muscle degeneration and muscle force compared to the mdx animals. These data demonstrate a correlation between the absence of B and T lymphocytes and loss of fibrosis accompanied by reduction of TGF-beta1, suggesting the importance of modulation of the immune system in DMD.     

Matrix metalloproteinase-2 ablation in dystrophin-deficient mdx muscles reduces angiogenesis resulting in impaired growth of regenerated muscle fibers

Matrix metalloproteases (MMPs) are a family of endopeptidases classified into subgroups based on substrate preference in normal physiological processes such as embryonic development and tissue remodeling, as well as in various disease processes via degradation of extracellular matrix components. Among the MMPs, MMP-9 and MMP-2 have been reported to be up-regulated in skeletal muscles in the lethal X-linked muscle disorder Duchenne muscular dystrophy (DMD), which is caused by loss of dystrophin. A recent study showed that deletion of the MMP9 gene in mdx, a mouse model for DMD, improved skeletal muscle pathology and function; however, the role of MMP-2 in the dystrophin-deficient muscle is not well known. In this study, we aimed at verifying the role of MMP-2 in the dystrophin-deficient muscle by using mdx mice with genetic ablation of MMP-2 (mdx/MMP-2(-/-)). We found impairment of regenerated muscle fiber growth with reduction of angiogenesis in mdx/MMP-2(-/-) mice at 3 months of age. Expression of vascular endothelial growth factor-A (VEGF-A), an important angiogenesis-related factor, decreased in mdx/MMP-2(-/-) mice at 3 months of age. MMP-2 had not a critical role in the degradation of dystrophin-glycoprotein complex (DGC) components such as β-dystroglycan and β-sarcoglycan in the regeneration process of the dystrophic muscle. Accordingly, MMP-2 may be essential for growth of regenerated muscle fibers through VEGF-associated angiogenesis in the dystrophin-deficient skeletal muscle.     

Diaphragm rescue alone prevents heart dysfunction in dystrophic mice

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused, in most cases, by the complete absence of the 427 kDa cytoskeletal protein, dystrophin. There is no effective treatment, and affected individuals die from respiratory failure and cardiomyopathy by age 30. Here, we investigated whether cardiomyopathy could be prevented in animal models of DMD by increasing diaphragm utrophin or dystrophin expression and thereby restoring diaphragm function. In a transgenic mdx mouse, where utrophin was over expressed in the skeletal muscle and the diaphragm, but not in the heart, we found cardiac function, specifically right and left ventricular ejection fraction as measured using in vivo magnetic resonance imaging, was restored to wild-type levels. In mdx mice treated with a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) that resulted in high levels of dystrophin restoration in the skeletal muscle and the diaphragm only, cardiac function was also restored to wild-type levels. In dystrophin/utrophin-deficient double-knockout (dKO) mice, a more severely affected animal model of DMD, treatment with a PPMO again produced high levels of dystrophin only in the skeletal muscle and the diaphragm, and once more restored cardiac function to wild-type levels. In the dKO mouse, there was no difference in heart function between treatment of the diaphragm plus the heart and treatment of the diaphragm alone. Restoration of diaphragm and other respiratory muscle function, irrespective of the method used, was sufficient to prevent cardiomyopathy in dystrophic mice. This novel mechanism of treating respiratory muscles to prevent cardiomyopathy in dystrophic mice warrants further investigation for its implications on the need to directly treat the heart in DMD.     

An immunohistochemical study of feline myocardial fibrosis

The aim of the present study was to investigate the pathology of feline myocardial fibrosis. The hearts from 40 cats with myocardial fibrosis were compared with the hearts from 25 normal cats. Clinical data were available in 11 cases. Hearts with myocardial fibrosis were hypomotile and there were hyperechoic areas in the ventricular wall on echocardiography. The presence of myocardial fibrosis was correlated significantly with hypertrophy of the ventricles, atrial dilation and angiosclerosis. Immunohistochemical studies demonstrated that normal feline cardiomyocytes expressed matrix metalloproteinase (MMP)-2, MMP-9, MMP-14, tissue inhibitor of matrix metalloproteinase (TIMP)-2 and transforming growth factor (TGF)-β2. Fibroblasts in normal hearts expressed only TIMP-2. In the hearts with myocardial fibrosis, expression of MMP-2, TIMP-3 and TGF-β2 by cardiomyocytes was significantly increased, but TIMP-2 expression was diminished. Fibroblasts in the affected hearts showed expression of MMP-14 in several cases. These findings suggest that a complex fibrotic remodelling of the feline myocardium occurs in this disease and that cardiomyocytes are involved in this process.     

Fibrinogen drives dystrophic muscle fibrosis via a TGFbeta/alternative macrophage activation pathway

In the fatal degenerative Duchenne muscular dystrophy (DMD), skeletal muscle is progressively replaced by fibrotic tissue. Here, we show that fibrinogen accumulates in dystrophic muscles of DMD patients and mdx mice. Genetic loss or pharmacological depletion of fibrinogen in these mice reduced fibrosis and dystrophy progression. Our results demonstrate that fibrinogen-Mac-1 receptor binding, through induction of IL-1beta, drives the synthesis of transforming growth factor-beta (TGFbeta) by mdx macrophages, which in turn induces collagen production in mdx fibroblasts. Fibrinogen-produced TGFbeta further amplifies collagen accumulation through activation of profibrotic alternatively activated macrophages. Fibrinogen, by engaging its alphavbeta3 receptor on fibroblasts, also directly promotes collagen synthesis. These data unveil a profibrotic role of fibrinogen deposition in muscle dystrophy.     

Antifibrotic response of cardiac fibroblasts in hypertensive hearts through enhanced TIMP-1 expression by basic fibroblast growth factor

BackgroundCardiac fibroblasts (CFs) play a pivotal role in the development of myocardial fibrosis. We previously demonstrated that direct injection of basic fibroblast growth factor (bFGF) into the hypertensive Dahl salt-sensitive (DS) rat heart prevented systolic dysfunction and left ventricular dilation effectively. However, the precise role played by bFGF in fibrotic response of CFs remains unclear. We suggested potential effects of bFGF on the fibrotic response of CFs in vitro.Methods and resultsHistopathologic assessment of cardiac fibrosis demonstrated a marked decline in the extent of perivascular and interstitial fibrosis in bFGF-injected hypertensive DS rat hearts. CFs harvested from the hearts of noninjected DS rats demonstrated a significantly increased messenger RNA (mRNA) expression of matrix metalloproteinase (MMP)-2, MMP-9, and both collagen I and III. In contrast, bFGF treatment in the CFs induced a marked increase in tissue inhibitor of MMP (TIMP)-1 expression and a marked decline in MMP-9 activation. bFGF also induced a decline in α-smooth muscle actin and collagen I and III mRNA expression in the CFs accompanied by inhibited differentiation of CFs into myofibroblasts. Small interfering RNA targeting FGF receptor 1 confirmed a specific interference of the mRNA expression changes elicited by bFGF. In vivo examination confirmed many TIMP-1-positive CFs in perivascular spaces of bFGF-injected hearts.ConclusionsUp-regulated TIMP-1 expression and down-regulated MMP-9 activation by bFGF in CFs could prevent excessive ECM degradation and collagen deposition in perivascular spaces effectively, leading to prevention of cardiac fibrosis during hypertensive heart failure.SummaryCardiac fibroblasts (CFs) play a pivotal role in myocardial fibrosis. The precise role of CFs in fibrotic response played by growth factors remains unclear. Our results indicates that basic fibroblast growth factor could up-regulate TIMP-1 expression and down-regulate MMP-9 activation in CFs in perivascular spaces, leading to inhibited progression of cardiac fibrosis during hypertensive heart failure.     

Major basic protein-1 promotes fibrosis of dystrophic muscle and attenuates the cellular immune response in muscular dystrophy

The immune response to dystrophin-deficient muscle promotes the pathology of Duchenne muscular dystrophy (DMD) and the mdx mouse model of DMD. In this investigation, we find that the release of major basic protein (MBP) by eosinophils is a prominent feature of DMD and mdx dystrophy and that eosinophils lyse muscle cells in vitro by the release of MBP-1. We also show that eosinophil depletions of mdx mice by injections of anti-chemokine receptor-3 reduce muscle cell lysis, although lysis of mdx muscle membranes is not reduced by null mutation of MBP-1 in vivo. However, ablation of MBP-1 expression in mdx mice produces other effects on muscular dystrophy. First, fibrosis of muscle and hearts, a major cause of mortality in DMD, is greatly reduced by null mutation of MBP-1 in mdx mice. Furthermore, either ablation of MBP-1 or eosinophil depletion causes large increases in cytotoxic T-lymphocytes (CTLs) in mdx muscles. The increase in CTLs in MBP-1-null mice does not reflect a general shift toward a Th1 inflammatory response, because the mutation had no significant effect on the expression of interferon-gamma, inducible nitric oxide synthase or tumor necrosis factor. Rather, MBP-1 reduces the activation and proliferation of splenocytes in vitro, indicating that MBP-1 acts in a more specific immunomodulatory role to affect the inflammatory response in muscular dystrophy. Together, these findings show that eosinophil-derived MBP-1 plays a significant role in regulating muscular dystrophy by attenuating the cellular immune response and promoting tissue fibrosis that can eventually contribute to increased mortality.     

Transgenic expression of Laminin α1 chain does not prevent muscle disease in the mdx mouse model for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder, and one of the most frequently encountered, but one for which there is as yet no treatment. Laminin-111 protein therapy was recently shown to be a promising approach to prevent muscle disease in the mdx mouse model of DMD. The present study demonstrated that transgenic expression of laminin α1 chain in mdx animals, resulting in laminin-111 heterotrimer formation in mdx muscle, does not improve the dystrophic phenotype. The mdx mice overexpressing laminin-111 (mdxLMα1) display features of mdx littermates: dystrophic pattern of muscle biopsy, elevated creatine kinase levels, reduced muscle strength, and decreased sarcolemmal integrity. Increased expression of integrin α7 is not beneficial for mdxLMα1 muscle, and components of the dystrophin-glycoprotein complex are not restored at the sarcolemma on laminin-111 overexpression. In summary, further studies are needed to verify the functionality of laminin-111 protein therapy in DMD and to describe the molecular events resulting from this approach.     

骨骼肌拉伤恢复进程中纤维化因子的变化

文题释义：纤维化：骨骼肌损伤修复进程包括依赖于成肌细胞激活和分化的肌纤维再生和胶原纤维进行持续合成和降解的结缔组织重塑两部分,这2个过程同时进行,相互支持又相互竞争制约。二者协调性是损伤肌肉修复质量的关键,当这2个过程速度吻合时,骨骼肌损伤完全修复;当结缔组织合成速度超过肌纤维再生时,形成瘢痕组织,骨骼肌纤维化。 纤维化因子：多种细胞因子在骨骼肌再生修复过程中调节纤维化的发生发展。转化生长因子(TGF-β)能够刺激成肌细胞转化为肌成纤维细胞,是促纤维化因子。结缔组织生长因子(CTGF)是介导转化生长因子β促纤维化作用的直接下游效应因子,主要参与纤维重塑。基质金属蛋白酶(MMPs)可以调控肌卫星细胞的增殖和迁移并调节细胞外基质中胶原的沉积和降解,是纤维化消除因子。基质金属蛋白酶组织抑制因子(TIMPs)是体内天然的基质金属蛋白酶家族抑制分子,被视作纤维化抑制因子。 背景：骨骼肌损伤是最常见的运动损伤,伤后可发展为纤维化等病理状态,损害肌肉功能。多种纤维化因子参与骨骼肌再生修复,调节组织纤维化进程。 目的：建立大鼠腓肠肌急性拉伤模型,观察骨骼肌伤后恢复进程中纤维化因子的表达变化,探讨其可能作用。 方法：112只雄性SD大鼠随机分为2组(n=56),对照组无干预,拉伤组用大鼠腓肠肌拉断仪拉伤左侧腓肠肌;每组再根据伤后取材时间随机分为7个亚组(n=8),即刻组、2 d组、4 d组、7 d组、14 d组、21 d组和28 d组。按既定时间取材,采用苏木精-伊红染色法观察骨骼肌肌纤维损伤及炎症变化,采用Western blot法检测转化生长因子β1、结缔组织生长因子、基质金属蛋白酶1、基质金属蛋白酶组织抑制因子1蛋白表达。实验通过北京体育大学运动科学实验伦理委员会批准。 结果与结论：①苏木精-伊红染色结果表明,拉伤组腓肠肌肌纤维排列紊乱,有炎性细胞聚集;②与对照组相比,拉伤后2 d起骨骼肌转化生长因子β1、结缔组织生长因子表达明显增加(P < 0.05),持续至后期仍有明显差异(P < 0.05);基质金属蛋白酶1表达在拉伤后2 d起明显增加(P < 0.05),7 d起与对照水平无差异(P > 0.05);基质金属蛋白酶组织抑制因子1表达在拉伤后7 d起明显增加(P < 0.05),并持续至28 d。提示：骨骼肌拉伤恢复进程中,转化生长因子β1/结缔组织生长因子蛋白通路被激活,趋于骨骼肌纤维化的发生发展;同时,基质金属蛋白酶1和基质金属蛋白酶组织抑制因子1先后被激活,参与骨骼肌的修复过程。 ORCID: 0000-0002-8450-0234(杨宁) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Change in the cells that express connective tissue growth factor in acute Coxsackievirus-induced myocardial fibrosis in mouse

Cardiac fibrosis and inflammation are major pathologic conditions that result from viral myocarditis. Connective tissue growth factor (CTGF) stimulates fibroblast proliferation and induces production of extracellular matrix molecules. We studied the correlation between CTGF and cardiac fibrosis in an acute Coxsackievirus B3 (CVB3) myocarditis animal model. Eight-week-old BALB/c mice were infected intraperitoneally with 10(4) plaque forming units (PFU) of CVB3. Myocardial inflammation peaked on day 7 and decreased markedly by day 14 post-infection (pi); cardiac fibrosis was noted from day 7 and peaked on day 14. By contrast, CTGF was weakly expressed by the interstitial cells in uninfected control hearts and also in the hearts of day 3 pi. CTGF expression measured by real-time PCR was elevated on day 3 and peaked on day 7 pi. TGF-beta expression peaked at day 7 pi. The cell type of CTGF expression changed from interstitial cells to myocytes after virus infection. On day 7, CTGF was strongly expressed by myocytes and inflammatory cells surrounding calcified necrotic areas. In addition, cardiac myocytes expressed CTGF on day 14. Our results, based on an acute CVB3 model of myocarditis, provide evidence that CTGF may mediate the development of fibrosis after viral myocarditis, and that the cells expressed CTGF changes during the course of viral myocarditis.     

Connective tissue growth factor and cardiac fibrosis

Cardiac fibrosis is a major pathogenic factor in a variety of cardiovascular diseases and refers to an excessive deposition of extracellular matrix components in the heart, which leads to cardiac dysfunction and eventually overt heart failure. Evidence is accumulating for a crucial role of connective tissue growth factor (CTGF) in fibrotic processes in several tissues including the heart. CTGF orchestrates the actions of important local factors evoking cardiac fibrosis. The central role of CTGF as a matricellular protein modulating the fibrotic process in cardiac remodelling makes it a possible biomarker for cardiac fibrosis and a potential candidate for therapeutic intervention to mitigate fibrosis in the heart.     

Sex-specific pathways in early cardiac response to pressure overload in mice

Pressure overload (PO) first causes cardiac hypertrophy and then heart failure (HF), which are associated with sex differences in cardiac morphology and function. We aimed to identify genes that may cause HF-related sex differences. We used a transverse aortic constriction (TAC) mouse model leading to hypertrophy without sex differences in cardiac function after 2 weeks, but with sex differences in hypertrophy 6 and 9 weeks after TAC. Cardiac gene expression was analyzed 2 weeks after surgery. Deregulated genes were classified into functional gene ontology (GO) categories and used for pathway analysis. Classical marker genes of hypertrophy were similarly upregulated in both sexes (alpha-actin, ANP, BNP, CTGF). Thirty-five genes controlling mitochondrial function (PGC-1, cytochrome oxidase, carnitine palmitoyl transferase, acyl-CoA dehydrogenase, pyruvate dehydrogenase kinase) had lower expression in males compared to females after TAC. Genes encoding ribosomal proteins and genes associated with extracellular matrix remodeling exhibited relative higher expression in males (collagen 3, matrix metalloproteinase 2, TIMP2, and TGFbeta2, all about twofold) after TAC. We confirmed 87% of the gene expression by real-time polymerase chain reaction. By GO classification, female-specific genes were related to mitochondria and metabolism and males to matrix and biosynthesis. Promoter studies confirmed the upregulation of PGC-1 by E2. Less downregulation of metabolic genes in female hearts and increased protein synthesis capacity and deregulation of matrix remodeling in male hearts characterize the sex-specific early response to PO. These differences could contribute to subsequent sex differences in cardiac function and HF.     

Elevated levels of active matrix metalloproteinase-9 cause hypertrophy in skeletal muscle of normal and dystrophin-deficient mdx mice

Matrix metalloproteinases (MMPs) are a group of extracellular proteases involved in tissue remodeling in several physiological and pathophysiological conditions. While increased expression of MMPs (especially MMP-9) has been observed in skeletal muscle in numerous conditions, their physiological significance remains less-well understood. By generating novel skeletal muscle-specific transgenic (Tg) mice expressing constitutively active mutant of MMP-9 (i.e. MMP-9G100L), in this study, we have investigated the effects of elevated levels of MMP-9 on skeletal muscle structure and function in vivo. Tg expression of enzymatically active MMP-9 protein significantly increased skeletal muscle fiber cross-section area, levels of contractile proteins and force production in isometric contractions. MMP-9 stimulated the activation of the Akt signaling pathway in Tg mice. Moreover, expression of active MMP-9 increased the proportion of fast-type fiber in soleus muscle of mice. Overexpression of MMP-9 also considerably reduced the deposition of collagens I and IV in skeletal muscle in vivo. In one-year-old mdx mice (a model for Duchenne muscular dystrophy, DMD), deletion of the Mmp9 gene reduced fiber hypertrophy and phosphorylation of Akt and p38 mitogen-activated protein kinase. Collectively, our study suggests that elevated levels of active MMP-9 protein cause hypertrophy in skeletal muscle and that the modulation of MMP-9 levels may have therapeutic value in various muscular disorders including DMD.     

Increased expression of plasminogen activator inhibitor-1 in cardiomyocytes contributes to cardiac fibrosis after myocardial infarction

Plasminogen activator inhibitor-1 (PAI-1) plays a critical role in tissue fibrosis by inactivating matrix metalloproteinases, which might effect on the progression of left ventricular dysfunction. However, little has been known about the expression of PAI-1 during cardiac remodeling. We used a mouse model of myocardial infarction (MI) by coronary ligation, in which the progression of left ventricular remodeling was confirmed by echocardiography. Histological examination showed that interstitial and perivascular fibrosis progressed in the post-MI (PMI) heart at 4 weeks after the procedure. We observed the dramatic induction of cardiac PAI-1 mRNA and PAI-1 antigen in plasma in the PMI mice, as compared with the sham-operated (sham) mice. In situ hybridization analysis demonstrated that strong signals for PAI-1 mRNA were localized to cardiomyocytes in the border of infarct area and around fibrous lesions, and to perivascular mononuclear cells, which seemed to be mast cells, only in hearts of the PMI mice. Importantly, less development of cardiac fibrosis after MI was observed in mice deficient in PAI-1 as compared to wild-type mice. The mRNA expression of cytokines, transforming growth factor-beta, and tumor necrosis factor-alpha, was also increased in hearts of the PMI mice, but not in the sham mice. These observations suggest that cardiomyocytes and mast cells contribute to the increased PAI-1 expression, resulting in the development of interstitial and perivascular fibrosis in the PMI heart, and that the regional induction of cytokines may be involved in this process.