Beneficial effects of edaravone, a novel antioxidant, in rats with dilated cardiomyopathy

Edaravone, a novel antioxidant, acts by trapping hydroxyl radicals, quenching active oxygen and so on. Its cardioprotective activity against experimental autoimmune myocarditis (EAM) was reported. Nevertheless, it remains to be determined whether edaravone protects against cardiac remodelling in dilated cardiomyopathy (DCM). The present study was undertaken to assess whether edaravone attenuates myocardial fibrosis, and examine the effect of edaravone on cardiac function in rats with DCM after EAM. Rat model of EAM was prepared by injection with porcine cardiac myosin 28 days after immunization, we administered edaravone intraperitoneally at 3 and 10 mg/kg/day to rats for 28 days. The results were compared with vehicle-treated rats with DCM. Cardiac function, by haemodynamic and echocardiographic study and histopathology were performed. Left ventricular (LV) expression of NADPH oxidase subunits (p47(phox), p67(phox), gp91(phox) and Nox4), fibrosis markers (TGF-β(1) and OPN), endoplasmic reticulum (ER) stress markers (GRP78 and GADD 153) and apoptosis markers (cytochrome C and caspase-3) were measured by Western blotting. Edaravone-treated DCM rats showed better cardiac function compared with those of the vehicle-treated rats. In addition, LV expressions of NADPH oxidase subunits levels were significantly down-regulated in edaravone-treated rats. Furthermore, the number of collagen-III positive cells in the myocardium of edaravone-treated rats was lower compared with those of the vehicle-treated rats. Our results suggest that edaravone ameliorated the progression of DCM by modulating oxidative and ER stress-mediated myocardial apoptosis and fibrosis.     

Quinapril inhibits progression of heart failure and fibrosis in rats with dilated cardiomyopathy after myocarditis

The cardioprotective properties of quinapril, an angiotensin-converting enzyme inhibitor, were studied in a rat model of dilated cardiomyopathy. Twenty-eight days after immunization of pig cardiac myosin, four groups rats were given 0.2 mg/kg (Q0.2, n = 11), 2 mg/kg (Q2, n = 11) or 20 mg/kg (Q20, n = 11) of quinapril or vehicle (V, n = 15) orally once a day. After 1 month, left ventricular end-diastolic pressure (LVEDP), ±dP/dt, area of myocardial fibrosis, and myocardial mRNA expression of transforming growth factor (TGF)-1, collagen-III and fibronectin were measured. Four of 15 (27%) rats in V and two of 11 (18%) in Q0.2 died. None of the animals in Q2 or Q20 died. The LVEDP was higher and ±dP/dt was lower in V (14.1 ± 2.0 mmHg and +2409 ± 150/–2318 ± 235 mmHg/sec) than in age-matched normal rats (5.0 ± 0.6 mmHg and +6173 ± 191/–7120 ± 74 mmHg/sec; all p < 0.01). After quinapril treatment, LVEDP was decreased and ±dP/dt was increased in a dose-dependent manner (10.8 ± 1.8 mmHg and +3211 ± 307/–2928 ± 390 mmHg/sec in Q0.2, 9.4 ± 1.5 mmHg and +2871 ± 270/–2966 ± 366 mmHg/sec in Q2, and 6.6 ± 1.5 mmHg, and +3569 ± 169/–3960 ± 203 mmHg/sec in Q20). Increased expression levels of TGF-1, collagen-III and fibronectin mRNA in V were reduced in Q20. Quinapril improved survival rate and cardiac function in rats with dilated cardiomyopathy after myocarditis. Furthermore, myocardial fibrosis was regressed and myocardial structure returned to nearly normal in animals treated with quinapril.     

Intracoronary allogeneic cardiosphere‐derived stem cells are safe for use in dogs with dilated cardiomyopathy

Cardiosphere‐derived cells (CDCs) have been shown to reduce scar size and increase viable myocardium in human patients with mild/moderate myocardial infarction. Studies in rodent models suggest that CDC therapy may confer therapeutic benefits in patients with non‐ischaemic dilated cardiomyopathy (DCM). We sought to determine the safety and efficacy of allogeneic CDC in a large animal (canine) model of spontaneous DCM. Canine CDCs (cCDCs) were grown from a donor dog heart. Similar to human CDCs, cCDCs express CD105 and are slightly positive for c‐kit and CD90. Thirty million of allogeneic cCDCs was infused into the coronary vessels of Doberman pinscher dogs with spontaneous DCM. Adverse events were closely monitored, and cardiac functions were measured by echocardiography. No adverse events occurred during and after cell infusion. Histology on dog hearts (after natural death) revealed no sign of immune rejection from the transplanted cells.     

VEGF165 expressing bone marrow mesenchymal stem cells differentiate into hepatocytes under HGF and EGF induction in vitro

A short half-life and low levels of growth factors in an injured microenvironment necessitates the sustainable delivery of growth factors and stem cells to augment the regeneration of injured tissues. Our aim was to investigate the ability of VEGF₁₆₅ expressing bone marrow mesenchymal stem cells (BMMSCs) to differentiate into hepatocytes when cultured with hepatocyte growth factor (HGF) and epidermal growth factor (EGF) in vitro. We isolated, cultured and identified rabbit BMMSCs, then electroporated the BMMSCs with VEGF₁₆₅-pCMV6-AC-GFP plasmid. G418 was used to select transfected cells and the efficiency was up to 70%. The groups were then divided as follows: Group A was electroporated with pCMV6-AC-GFP plasmid + HGF + EGF and Group B was electroporated with VEGF₁₆₅-pCMV6-AC-GFP plasmid +HGF + EGF. After 14 days, BMMSCs were induced into short spindle and polygonal cells. Alpha-fetoprotein (AFP) was positive and albumin (ALB) was negative in Group A, while both AFP and ALB were positive in group B on day 10. AFP and ALB in both groups were positive on day 20, but the quantity of AFP in group B decreased with prolonged time and was about 43.5% less than group A. The quantity of the ALB gene was increased with prolonged time in both groups. However, there was no significant difference between group A and B on day 10 and 20. Our results demonstrated that VEGF₁₆₅-pCMV6-AC-GFP plasmid modified BMMSCs still had the ability to differentiate into hepatocytes. The VEGF₁₆₅ gene promoted BMMSCs to differentiate into hepatocyte-like cells under the induction of HGF and EGF, and reduced the differentiation time. These results have implications for cell therapies.     

Enhancement of angiogenic effect of co-transfection human NGF and VEGF genes in rat bone marrow mesenchymal stem cells

The current study explored the feasibility and efficacy of co-transfection of the human nerve growth factor (NGF) and vascular endothelial growth factor 165 (VEGF165) genes in rat bone marrow mesenchymal stem cells (BMSCs). The obtained hNGF and vascular endothelial growth factor (VEGF) cDNAs were cloned into the pEGFP-C1 expression vector to construct the recombinant vectors. Co-transfection in rat BMSCs was performed and the expressions of both genes were detected by RT-PCR, Western blot, and enzyme-linked immunospecific assay. The biological activity of recombinant NGF and VEGF proteins was confirmed using the Chick Chorioallantoic Membrane (CAM) assay. NGF and VEGF genes could be expressed successfully in rat BMSCs. The recombinant NGF and VEGF from the rat BMSCs showed a more significant synergetic biological activity compared with single recombinant NGF or VEGF. These findings demonstrate that the co-transfection of hNGF + VEGF genes can enhance the angiogenic effect in vivo.     

Cadaveric cardiosphere-derived cells can maintain regenerative capacity and improve the heart function of cardiomyopathy

Objective: Cardiosphere-derived cells (CDCs) improve cardiac function and attenuate remodeling in ischemic and non-ischemic cardiomyopathy, and are currently obtained through myocardial biopsy. However, there is not any study on whether functional CDCs may be obtained through cadaveric autopsy with similar benefits in non-ischemic cardiomyopathy. Methods: Cardiac tissues from human or mouse cadavers were harvested, plated at 4°C, and removed at varying time points to culture human CDCs (CLH-EDCs) and mouse CDCs (CM-CDCs). The differentiation and paracrine effects of CDCs were also assessed. Furthermore, intramyocardial injection of cadaveric CM-CDCs was performed in an induced dilated cardiomyopathy (DCM) model. Results: With the extension of post mortem hours, the number of CLH-EDCs and CM-CDCs harvested from autopsy specimens decreased. The expressions of von Willebrand factor (VWF) and smooth muscle actin (SMA) on CDCs were gradually reduced, however, cardiac troponin I (TNI) expression increased in the 24 h group compared to the 0 h group. CLH-EDCs were also found to have similar paracrine function in the 24 h group compared to 0 h group. 8 weeks after CM-CDCs transplantion to the injured heart, mean left ventricular ejection fraction increased in both 0 h (64.99 ± 3.4%) and 24 h (62.99 ± 2.8%) CM-CDCs-treated groups as compared to the PBS treated group (53.64 ± 5.6 cm), with a decrease in left ventricular internal diastolic diameter (0.29 ± 0.08 cm and 0.32 ± 0.04 cm in 0 h and 24 h groups, vs. 0.41 ± 0.05 cm in PBS group). Conclusion: CDCs from cadaveric autopsy are highly proliferative and differentiative, and may be used as a source for allograft transplantation, in order to decrease myocardial fibrosis, attenuate left ventricular remodeling, and improve heart function in doxorubicin-induced non-ischemic cardiomyopathy.     

Long-term follow-up of patients with myocarditis and idiopathic dilated cardiomyopathy after immunomodulatory therapy

Abstract The randomized clinical trial with interferon-α (IFN) or thymic hormones versus conventional therapy was conducted in patients with myocarditis and idiopathic dilated cardiomyopathy (IDC). We enrolled 180 patients to receive IFN (3–5 million units per day) for 3 months, thymomodulin (10 mg three times per week) for 2 months, or conventional therapy alone. Patients were followed-up for 7 years after the end of treatment. Left ventricular function, exercise tolerance and survival rate were significantly better at long-term follow-up in patients treated with IFN or thymomodulin, than in conventionally treated patients. These results implicate that immune modulating therapy might represent important contribution in the treatment of myocarditis and IDC.     

Differential expression profiles of plasma exosomal microRNAs in dilated cardiomyopathy with chronic heart failure

As one of the most prevalent heritable cardiovascular diseases, dilated cardiomyopathy (DCM) induces cardiac insufficiency and dysfunction. Although genetic mutation has been identified one of the causes of DCM, the usage of genetic biomarkers such as RNAs for DCM early diagnosis is still being overlooked. In addition, the alternation of RNAs could reflect the progression of the diseases, as an indicator for the prognosis of patients. Therefore, it is beneficial to develop genetic based diagnostic tool for DCM. RNAs are often unstable within circulatory system, leading to the infeasibility for clinical application. Recently discovered exosomal miRNAs have the stability that is then need for diagnostic purpose. Hence, fully understanding of the exosomal miRNA within DCM patients is vital for clinical translation. In this study, we employed the next generation sequencing based on the plasma exosomal miRNAs to comprehensively characterize the miRNAs expression in plasma exosomes from DCM patients exhibiting chronic heart failure (CHF) compared to healthy individuals. A complex landscape of differential miRNAs and target genes in DCM with CHF patients were identified. More importantly, we discovered that 92 differentially expressed miRNAs in DCM patients undergoing CHF were correlated with several enriched pathways, including oxytocin signalling pathway, circadian entrainment, hippo signalling pathway-multiple species, ras signalling pathway and morphine addiction. This study reveals the miRNA expression profiles in plasma exosomes in DCM patients with CHF, and further reveal their potential roles in the pathogenesis of it, presenting a new direction for clinical diagnosis and management of DCM patients with CHF.     

VEGF165 induces differentiation of hair follicle stem cells into endothelial cells and plays a role in in vivo angiogenesis

Within the vascular endothelial growth factor (VEGF) family of five subtypes, VEGF165 secreted by endothelial cells has been identified to be the most active and widely distributed factor that plays a vital role in courses of angiogenesis, vascularization and mesenchymal cell differentiation. Hair follicle stem cells (HFSCs) can be harvested from the bulge region of the outer root sheath of the hair follicle and are adult stem cells that have multi‐directional differentiation potential. Although the research on differentiation of stem cells (such as fat stem cells and bone marrow mesenchymal stem cells) to the endothelial cells has been extensive, but the various mechanisms and functional forms are unclear. In particular, study on HFSCs’ directional differentiation into vascular endothelial cells using VEGF165 has not been reported. In this study, VEGF165 was used as induction factor to induce the differentiation from HFSCs into vascular endothelial cells, and the results showed that Notch signalling pathway might affect the differentiation efficiency of vascular endothelial cells. In addition, the in vivo transplantation experiment provided that HFSCs could promote angiogenesis, and the main function is to accelerate host‐derived neovascularization. Therefore, HFSCs could be considered as an ideal cell source for vascular tissue engineering and cell transplantation in the treatment of ischaemic diseases.     

Combined treatment with systemic resveratrol and resveratrol preconditioned mesenchymal stem cells,maximizes antifibrotic action in diabetic cardiomyopathy

Wnt/β-catenin signaling pathway plays a crucial role in diabetic cardiomyopathy (DCM), thus we aimed at investigating the effect of one therapeutic approach with resveratrol (RSV) given systemically and combined treatment of RSV with mesenchymal stem cells (MSCs) that was either RSV-preconditioned or not on Wnt/β-catenin signaling pathway in streptozotocin-induced DCM, and to evaluate effects of RSV preconditioning on MSCs therapeutic potential. The rats were divided into control (C, n = 8), diabetic (DM, n = 8), diabetic treated with systemic RSV (DM-RSV, n = 8), diabetic treated with RSV and nonconditioned MSCs (DM-RSV-MSCs, n = 8), diabetic treated with RSV and RSV-incubated with MSCs (DM-RSV-MSCc, n = 8) and diabetic treated with RSV-conditioned MSCs (DM-MSCc, n = 8). Echocardiography (Echo) showed significant improvement of cardiac functions in all groups treated with RSV either systemic or added in culture media. Data of ejection fraction (EF%) of DM-RSV-MSCc (81.50; interquartile range [IQR], 80.00–83.00) was comparable to both DM-RSV-MSCs (77.50; IQR, 71.50–79.00), and DM-MSCc (71.50; IQR, 70.00–74.50). Histological examination of the left ventricles was performed for all groups. DM group revealed significant myocardial hypertrophy, apoptosis, interstitial fibrosis, and microvascular affection. All treated groups were associated, in variable degrees, with attenuation of cardiac hypertrophy and fibrosis, decreased area% for cardiac immunostaining of secreted frizzled-related protein (sFRP2) and Wnt/β-catenin and improvement of the microvasculature. In conclusion, MSCs pretreated with resveratrol for 7 days showed increased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and combined use of RSV (systemically and in culture media) significantly could improve cardiac remodeling capacity of MSCs via attenuation of sFRP2-mediated fibrosis and the downstream Wnt/β-catenin pathway.     

Autophagic vacuoles in cardiomyocytes of dilated cardiomyopathy with initially decompensated heart failure predict improved prognosis

Autophagy is a process of bulk protein degradation and organelle turnover, and is a current therapeutic target in several diseases. The present study aimed to clarify the significance of myocardial autophagy of patients with dilated cardiomyopathy (DCM). Left ventricular endomyocardial biopsy was performed in 250 consecutive patients with DCM (54.9±13.9 years; male, 79%), initially presenting with decompensated heart failure (HF). The association of these findings with HF mortality or recurrence was examined. Myofilament changes, which are apparent in the degenerated cardiomyocytes of DCM, were recognized in 164 patients (66%), and autophagic vacuoles in cardiomyocytes were identified in or near the area of myofilament changes in 86 patients (34%). Morphometrically, fibrosis (odds ratio [OR], 0.96; 95% confidence interval [CI], 0.93 to 0.99) and mitochondrial abnormality (OR, 2.24; 95% CI, 1.23 to 4.08) were independently related with autophagic vacuoles. During the follow-up period of 4.9±3.9 y, 24 patients (10%) died, including 10 (4%) who died of HF, and 67 (27%) were readmitted for HF recurrence. Multivariate analysis identified a family history of DCM (hazard ratio [HR], 2.117; 95% CI, 1.199 to 3.738), hemoglobin level (HR, 0.845; 95% CI, 0.749 to 0.953), myofilament changes (HR, 13.525; 95% CI, 5.340 to 34.255), and autophagic vacuoles (HR, 0.214; 95% CI, 0.114 to 0.400) as independent predictors of death or readmission due to HF recurrence. In conclusion, autophagic vacuoles in cardiomyocytes are associated with a better HF prognosis in patients with DCM, suggesting autophagy may play a role in the prevention of myocardial degeneration.     

黄芪注射液对缺血性心肌病大鼠心肌的保护作用*

目的: 探究黄芪注射液对缺血性心肌病大鼠心肌保护作用及其机制。方法: 将36只雄性鼠随机分成:对照组(12只)、缺血性心肌病组(12只)及黄芪注射液组(12只);缺血性心肌病组和黄芪注射液组的大鼠开胸结扎冠状动脉,建立缺血心肌病大鼠模型;建立心肌缺血模型后,黄芪注射液组术后注射黄芪注射液(每周一次,剂量:10 g/kg体重),共注射4次,其他两组腹腔均注射相同剂量的生理盐水;4周后给予3组大鼠麻醉后行心电图及心脏彩超后,处死大鼠取心肌标本行电镜检查,观察其心肌病理超微结构的变化,检测大鼠心肌细胞线粒体Ca²⁺浓度和心肌细胞线粒体融合蛋白mitofusin 1(Mfn1)及凋亡因子C/EBP 同源蛋白(chop)表达,以及黄芪注射液对大鼠心肌细胞ATP敏感钾通道电流的作用。结果: 与对照组比较,缺血性心肌病组中大鼠出现心律失常现象;心室扩大,EF值降低;心肌排列紊乱,线粒体空泡化严重;线粒体Ca²⁺浓度增加(P＜0.01);Mfn1表达减低(P＜0.05),chop表达增加(P＜0.01); 与缺血性心肌病组比较,黄芪注射液组中大鼠心律失常发生率明显减少,心肌细胞动作电位时程缩短,心脏彩超及心肌病理明显改善并存在大量线粒体融合,心肌线粒体Ca²⁺浓度和chop表达明显减少(P＜0.01),而Mfn1表达明显增加(P＜0.01),心肌细胞ATP敏感钾电流明显增加(P＜0.01),该作用可被ATP敏感钾通道特异性阻断剂格列本脲阻断。结论: 黄芪注射液明显减少缺血性心肌病大鼠心律失常的发生率,继而改善缺血性心肌病大鼠心脏功能、减轻心肌病理损伤,其作用机制可能通过心肌细胞ATP敏感钾通道所介导。     

Infusion of mesenchymal stem cells overexpressing GDNF ameliorates renal function in nephrotoxic serum nephritis

Nephrotoxic serum nephritis (NSN) is a well-established animal model of glomerulonephritis, a frequent clinical condition with a high mortality rate owing to the ineffectiveness of current therapies. Mesenchymal stem cells (MSCs) are adult stem cells with potential as novel therapies in regenerative medicine owing to the absence of allogenic rejection. Glial cell-derived neurotrophic factor (GDNF) acts as a morphogen in kidney development. The therapeutic effectiveness of bone marrow MSCs overexpressing GDNF (GDNF-MSCs) was evaluated in an NSN rat model. An adenoviral vector was used to transduce MSCs with GDNF and a green fluorescent protein reporter gene. Then, GDNF-MSCs were injected into NSN rats via the renal artery. The influence of GDNF on renal injury was assessed. The location of GDNF-MSCs in kidneys was detected using fluorescence microscopy, cells were counted, and kidney function was measured. Infusion of GNDF-MSCs enhanced the recovery of renal function in NSN rats. MSCs were detected in the kidney cortex after injection. Compared with control MSCs, GDNF-MSCs led to significantly better renal function and injury recovery in NSN rats. GDNF has a positive effect on MSC differentiation in renal tissue. Owing to their highly renoprotective capacity, GDNF-MSCs represent a possible novel cell-based paradigm for treatment of glomerulonephritis.     

Effects of angiotensin-II receptor blocker candesartan cilexetil in rats with dilated cardiomyopathy

We examined effects of an angiotensin-II receptor blockers, candesartan cilexetil, in rats with dilated cardiomyopathy after autoimmune myocarditis. Candesartan cilexetil showed angiotensin-II blocking action in a dose-dependent manner in rats with dilated cardiomyopathy. Twenty-eight days after immunization, surviving Lewis rats were divided into four groups and given candesartan cilexetil at 0.05 mg/kg, 0.5 mg/kg or 5 mg/kg per day (Group-C0.05, n = 15, Group-C0.5, n = 15 and Group-C5, n = 15, respectively) or vehicle alone (Group-V, n = 15). After oral administration for 1 month, the left ventricular end-diastolic pressure and heart weight/body weight ratio were lower in Group-C0.05 (13.3± 1.1 mmHg and 3.7± 0.2 g/kg, respectively), in Group-C0.5 (8.0± 0.9 mmHg and 3.3± 0.1 g/kg, respectively) and in Group-C5 (5.5± 1 mmHg and 3.1± 0.1 g/kg, respectively) than in Group-V (13.5± 1.0 mmHg and 3.8± 0.2 g/kg, respectively). The area of myocardial fibrosis was also lower in Group-C0.05 (25± 3%), in Group-C0.5 (20± 3%), and in Group-C5 (12± 1%) than in Group-V (32± 4%). Furthermore, expressions of transforming growth factor-1 and collagen-III mRNA were suppressed in Group-C0.05 (349± 23% and 395± 22%, respectively), Group-C0.5 (292± 81% and 364± 42%, respectively) and in Group-C5 (204± 63% and 259± 33%, respectively) compared with those in Group-V (367± 26% and 437± 18%, respectively). These results suggest that candesartan cilexetil can improve the function of inefficient heart. (Mol Cell Biochem 269: 137–142, 2005)     

Influence of dilated cardiomyopathy and a left ventricular assist device on vortex dynamics in the left ventricle

Together with new developments in mechanical cardiac support, the analysis of vortex dynamics in the left ventricle has become an increasingly important topic in literature. The aim of this study was to develop a method to investigate the influence of a left ventricular assist device (LVAD) on vortex dynamics in a failing ventricle. An axisymmetric fluid dynamics model of the left ventricle was developed and coupled to a lumped parameter model of the complete circulation. Simulations were performed for healthy conditions and dilated cardiomyopathy (DCM). Vortex structures in these simulations were analysed by means of automated detection. Results show that the strength of the leading vortex ring is lower in a DCM ventricle than in a healthy ventricle. The LVAD further influences the maximum strength of the vortex and also causes the vortex to disappear earlier in time with increasing LVAD flows. Understanding these phenomena by means of the method proposed in this study will contribute to enhanced diagnostics and monitoring during cardiac support.     

Concentrations of myosin-activating protein kinases in the myocardium of patients with dilated cardiomyopathy and in animal heart

Skeletal myosin light chain kinase in the myocardia of various animal species was identified by immunoblotting. The myocardial concentrations of this protein and myosin-activating protein kinases (RhoA-activated kinase, integrin-linked kinase, and zipper-interacting kinase) were compared in healthy humans and patients with dilated cardiomyopathy. Skeletal myosin light chain kinase was detected in the human and chicken embryo hearts, rather than in the embryonic and adult rat hearts. In the myocardium of patients with dilated cardiomyopathy, the concentrations of myosin light chain kinase, RhoA-activated kinase, and integrin-linked kinase increase and the concentration of zipper-interacting kinase decreases. The results obtained are likely to characterize compensatory processes in cardiomyocytes in dilated cardiomyopathy that are aimed at increasing their viability and contractility.