Global Intracoronary Infusion of Allogeneic Cardiosphere-Derived Cells Improves Ventricular Function and Stimulates Endogenous Myocyte Regeneration throughout the Heart in Swine with Hibernating Myocardium

Background

Cardiosphere-derived cells (CDCs) improve ventricular function and reduce fibrotic volume when administered via an infarct-related artery using the “stop-flow” technique. Unfortunately, myocyte loss and dysfunction occur globally in many patients with ischemic and non-ischemic cardiomyopathy, necessitating an approach to distribute CDCs throughout the entire heart. We therefore determined whether global intracoronary infusion of CDCs under continuous flow improves contractile function and stimulates new myocyte formation.

Methods and Results

Swine with hibernating myocardium from a chronic LAD occlusion were studied 3-months after instrumentation (n = 25). CDCs isolated from myocardial biopsies were infused into each major coronary artery (∼33×10⁶ icCDCs). Global icCDC infusion was safe and while ∼3% of injected CDCs were retained, they did not affect ventricular function or myocyte proliferation in normal animals. In contrast, four-weeks after icCDCs were administered to animals with hibernating myocardium, %LADWT increased from 23±6 to 51±5% (p<0.01). In diseased hearts, myocyte proliferation (phospho-histone-H3) increased in hibernating and remote regions with a concomitant increase in myocyte nuclear density. These effects were accompanied by reductions in myocyte diameter consistent with new myocyte formation. Only rare myocytes arose from sex-mismatched donor CDCs.

Conclusions

Global icCDC infusion under continuous flow is feasible and improves contractile function, regresses myocyte cellular hypertrophy and increases myocyte proliferation in diseased but not normal hearts. New myocytes arising via differentiation of injected cells are rare, implicating stimulation of endogenous myocyte regeneration as the primary mechanism of repair.     

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.     

Intracoronary delivery of autologous bone marrow mononuclear cells radiolabeled by 18F-fluoro-deoxy-glucose: tissue distribution and impact on post-infarct swine hearts

Intracoronary injection of the bone marrow-derived mononuclear cells (MNCs) is emerging as a potentially novel therapy for ischemic heart failure. This study was aimed at assessing the efficacy of intracoronary MNC delivery in the myocardium. The in vivo distribution and myocardial homing of intracoronarily delivered MNCs in experimental Chinese swine with acute myocardial infarction (AMI) created by occlusion of left anterior descending (LAD) coronary artery for 90 min. MNCs radiolabeled with 18F-fluoro-deoxy-glucose (18F-FDG) were delivered using a coronary catheter into the infarct-related coronary artery 1 week after AMI. Dual-nuclide single photon emission computed tomography (SPECT) revealed that 1 h after cell infusion, 6.8 +/- 1.8% of 18F-FDG-labeled MNCs occurred in the infarcted myocardium with the remaining activity found primarily in the liver and spleen. In the heart, MNCs were detected predominantly in the under-perfused myocardium. The infused cells retained in the hearts at a rate highly correlated with the under-perfused lesional sizes. Pathological examination further demonstrated that 6 weeks after infusion, compared to controls, the hearts receiving MNCs exhibited less fibrosis and inflammatory infiltrate, more viable tissue, and higher vascular density. Cardiac function was significantly improved in the MNC-infused hearts. Thus, 18F-FDG labeling and dual-nuclide SPECT imaging is capable of monitoring in vivo distribution and homing of MNCs after intracoronary infusion. MNC coronary delivery may improve cardiac function and positive ventricular remodeling in the heart with AMI.     

Cardiac regenerative potential of cardiosphere‐derived cells from adult dog hearts

The regenerative potential of cardiosphere‐derived cells (CDCs) for ischaemic heart disease has been demonstrated in mice, rats, pigs and a recently completed clinical trial. The regenerative potential of CDCs from dog hearts has yet to be tested. Here, we show that canine CDCs can be produced from adult dog hearts. These cells display similar phenotypes in comparison to previously studied CDCs derived from rodents and human beings. Canine CDCs can differentiate into cardiomyocytes, smooth muscle cells and endothelial cells in vitro. In addition, conditioned media from canine CDCs promote angiogenesis but inhibit cardiomyocyte death. In a doxorubicin‐induced mouse model of dilated cardiomyopathy (DCM), intravenous infusion of canine CDCs improves cardiac function and decreases cardiac fibrosis. Histology revealed that injected canine CDCs engraft in the mouse heart and increase capillary density. Out study demonstrates the regenerative potential of canine CDCs in a mouse model of DCM.     

Distributing and delivering vessels of the human heart

The branching characteristics of the right coronary artery, acute marginal, posterior descending, left anterior descending, circumflex, and obtuse marginal arteries are compared with those of diagonal branches, left and right ventricular branches, septal, and higher-order branches, to test a newly proposed functional classification of the coronary arteries in which the first group rank as distributing vessels and the second as delivering vessels. According to this classification, the function of the first type is merely to convey blood to the borders of myocardial zones, while the function of the second is to implement the actual delivery of blood into these zones. This functional difference is important in the hemodynamic analysis of coronary heart disease, as it provides an assessment of the role of a vessel within the coronary network and hence an assessment of the functional importance of that vessel in a particular heart. Measurements from casts of human coronary arteries are used to examine the relevant characteristics of these vessels and hence to test the basis of this classification.     

Catheter-mediated subselective intracoronary gene delivery to the rabbit heart: introduction of a novel method

BACKGROUND: Recent studies suggest that gene therapy using replication-deficient adenoviruses will benefit treatment of cardiovascular diseases including heart failure. A persistent hurdle is the effective and reproducible delivery of a transgene to the myocardium with minimal iatrogenic morbidity. In this study, we sought to design a relatively non-invasive percutaneous gene delivery system that would maximize cardiac transgene expression and minimize mortality after intracoronary adenovirus injection. METHODS: Adult rabbits received a left circumflex coronary artery (LCx) infusion of 5x10(11) total viral particles of an adenovirus containing the marker transgene beta-galactosidase (Adeno-betaGal) via either a continuous infusion method utilizing an oxygenated, normothermic, physiologic pH Krebs solution driven by a Langendorff apparatus (n=12) or a timed bolus and set concentration at a constant infusion rate to the LCx (n=12). Six rabbits underwent global transgene delivery via an invasive method involving intraventricular delivery and aortic root cross-clamping. The efficacy of transgene expression via these three distinct delivery methods was determined in the left ventricle at 5 days by histological staining and colorimetric quantification assay. RESULTS: While the open-chest, aortic cross-clamping method provides the highest level of gene expression throughout the heart, the morbidity of this procedure is clinically prohibitive. Percutaneous LCx delivery of Adeno-betaGal using the Langendorff apparatus was associated with the lowest morbidity and mortality while still supporting significant myocardial gene expression. CONCLUSIONS: Percutaneous delivery of an adenovirus solution using a continuous infusion of oxygenated Krebs solution via a Langendorff apparatus appears to be a gene delivery modality offering the best compromise of gene expression and clinical utility to maximize any potential therapeutic outcome.     

Vasculogenesis and angiogenesis: Extracellular matrix remodeling in coronary collateral arteries and the ischemic heart

Heart failure secondary to ischemic cardiomyopathy is the primary cause of cardiovascular mortality. The promise of the collateral circulation lies in its potential to alter the course of the natural history of coronary heart disease. The collateral circulation of the heart is responsible for supplying blood and oxygen to the myocardium at ischemic risk following severe stenosis and reduced vasoelasticity function of a major coronary artery. In response to flow, stress, and pressure, collateral vessels are restructured and remodeled. Vascular remodeling by its very nature implies synthesis and degradation of extracellular matrix components in the vessel wall. Under normal physiological conditions proteinases that break down the specialized matrix are tightly regulated by antiproteinases. The balance between proteinase and antiproteinase influences is discoordinated during collateral development which leads to adaptive changes in the structure, function, and regulation of extracellular matrix components in the vessel wall. The role of extracellular matrix components in coronary collateral vessel formation in a canine model of chronic coronary artery occlusion has been demonstrated. The role of matrix proteinases and antiproteinases in the collateral vessel play a significant role in the underlying mechanisms of collateral development. This review presents new and significant information regarding the role of extracellular matrix proteinases and antiproteinases in vascular remodeling, function, and collateral development. Such information will have a significant impact on the understanding of the basic biology of the vascular extracellular matrix turnover, remodeling, and function as well as on elucidating potential avenues for pharmacological approaches designed to increase collateral formation and optimize myocardial blood flow in the treatment of ischemic heart disease. J. Cell. Biochem. 65:388–394. © 1997 Wiley-Liss, Inc.     

冠心病患者介入治疗前后血清IL-18、sCD40L 和hs-CRP 水平的 变化及其意义

目的:探讨冠心病患者血清白介素18(Interleukin-18,IL-18)、白细胞分化抗原40配体(CD40L)、及高敏C反应蛋白(hs-CRP)水平在经皮冠状动脉介入术治疗前后的变化和意义。方法:选择经冠状动脉造影确诊的冠心病患者85例,根据病变程度分为单支病变组(n=32)、双支病变组(n=28)和多支病变组(n=25),采用双抗体夹心ELISA法测定PCI术前术后血清IL-18、CD40L和hs-CRP水平。结果:血清IL-18水平测定结果:多支病变组高于双支病变组,双支病变组高于单支病变组;支架置入术后显著高于术前,差异均有统计学意义(P<0.01)。血清CD40L水平测定结果:多支病变组高于双支病变组和单支病变组,差异均有统计学意义(P<0.01),双支病变组与单支病变组间差异无统计学意义(P>0.05);支架置入术后较术前显著升高,差异有统计学意义(P<0.01)。血清hs-CRP水平测定结果:多支病变组高于双支病变组,双支病变组高于单支病变组;支架置入术后显著高于术前,差异均有统计学意义(P<0.01)。结论:冠心病患者血清IL-18、CD40L和hs-CRP与冠脉病变程度密切相关,介入治疗可使冠心病患者血清IL-18、CD40L和hs-CRP水平升高,监测血清中IL-18、CD40L和hs-CRP水平变化可了解治疗效果和炎症程度。     

Daily exercise enhances coronary resistance vessel sensitivity to pharmacological activation

The effect of daily exercise on the coronary resistance vessel sensitivity to intracoronary infusion of several pharmacological agents was assessed in 12 conscious adult mongrel dogs. alpha-Adrenergic receptor agonists (norepinephrine and phenylephrine) significantly decreased coronary blood flow velocity. beta 2-Adrenergic receptor agonists (isoproterenol and zinterol) and a metabolic vasodilator (adenosine) significantly increased coronary blood flow velocity. These responses occurred without altering factors that influence myocardial metabolism. Daily exercise significantly enhanced the coronary vascular sensitivity to each of the pharmacological agents. These results suggest that a nonspecific potentiation to pharmacological activation occurs after daily exercise. After left stellate ganglionectomy, intracoronary infusions of each pharmacological agent had similar effects on coronary blood flow velocity as presented for the intact dogs; however, daily exercise did not enhance the coronary vascular sensitivity to the pharmacological agents. These results demonstrate the need for an intact nervous system for the vascular adaptations associated with daily exercise.     

冠心病患者介入治疗前后血清IL-18、sCD40L和hs-CRP水平的变化及其意义

目的：探讨冠心病患者血清白介素18（Interleukin-18,IL-18）、白细胞分化抗原40配体（CD40L）、及高敏C反应蛋白（hs-CRP）水平在经皮冠状动脉介入术治疗前后的变化和意义。方法：选择经冠状动脉造影确诊的冠心病患者85例,根据病变程度分为单支病变组（n=32）、双支病变组（n=28）和多支病变组（n=25）,采用双抗体夹心ELISA法测定PCI术前术后血清IL-18、CD40L和hs-CRP水平。结果：血清IL-18水平测定结果：多支病变组高于双支病变组,双支病变组高于单支病变组;支架置入术后显著高于术前,差异均有统计学意义（P〈0.01）。血清CD40L水平测定结果：多支病变组高于双支病变组和单支病变组,差异均有统计学意义（P〈0.01）,双支病变组与单支病变组间差异无统计学意义（P〉0.05）;支架置入术后较术前显著升高,差异有统计学意义（P〈0.01）。血清hs-CRP水平测定结果：多支病变组高于双支病变组,双支病变组高于单支病变组;支架置入术后显著高于术前,差异均有统计学意义（P〈0.01）。结论：冠心病患者血清IL-18、CD40L和hs-CRP与冠脉病变程度密切相关,介入治疗可使冠心病患者血清IL-18、CD40L和hs-CRP水平升高,监测血清中IL-18、CD40L和hs-CRP水平变化可了解治疗效果和炎症程度。     

Effects of cordanum and propranolol on coronary resistance

The effects of cordanum and propranolol on the coronary resistance were studied in isolated cat hearts. The infusion of cordanum and propranolol may be followed by both reduction or enlargement of the coronary resistance. The alterations of the coronary resistance are in most cases parallel with the weakening of the isolated heart function. In some cases cordanum infusion is followed by a short-term reduction in the coronary resistance, which precedes the alteration of the heart function and thus may be due to direct cordanum effect on the coronary vessels.     

c-kit+ Cardiac Stem Cells Alleviate Post-Myocardial Infarction Left Ventricular Dysfunction Despite Poor Engraftment and Negligible Retention in the Recipient Heart

Although transplantation of c-kit+ cardiac stem cells (CSCs) has been shown to alleviate left ventricular (LV) dysfunction induced by myocardial infarction (MI), the number of exogenous CSCs remaining in the recipient heart following transplantation and their mechanism of action remain unclear. We have previously developed a highly sensitive and accurate method to quantify the absolute number of male murine CSCs in female recipient organs after transplantation. In the present study, we used this method to monitor the number of donor CSCs in the recipient heart after intracoronary infusion. Female mice underwent a 60-min coronary occlusion followed by reperfusion; 2 days later, 100,000 c-kit+/lin- syngeneic male mouse CSCs were infused intracoronarily. Only 12.7% of the male CSCs present in the heart immediately (5 min) after infusion were still present in the heart at 24 h, and their number declined rapidly thereafter. By 35 days after infusion, only ∼1,000 male CSCs were found in the heart. Significant numbers of male CSCs were found in the lungs and kidneys, but only in the first 24 h. The number of CSCs in the lungs increased between 5 min and 24 h after infusion, indicating recirculation of CSCs initially retained in other organs. Despite the low retention and rapid disappearance of CSCs from the recipient heart, intracoronary delivery of CSCs significantly improved LV function at 35 days (Millar catheter). These results suggest that direct differentiation of CSCs alone cannot account for the beneficial effects of CSCs on LV function; therefore, paracrine effects must be the major mechanism. The demonstration that functional improvement is dissociated from survival of transplanted cells has major implications for our understanding of cell therapy. In addition, this new quantitative method of stem cell measurement will be useful in testing approaches of enhancing CSC engraftment and survival after transplantation.     

Intracoronary Delivery of Human Mesenchymal/Stromal Stem Cells: Insights from Coronary Microcirculation Invasive Assessment in a Swine Model

Background

Mesenchymal stem/stromal cells have unique properties favorable to their use in clinical practice and have been studied for cardiac repair. However, these cells are larger than coronary microvessels and there is controversy about the risk of embolization and microinfarctions, which could jeopardize the safety and efficacy of intracoronary route for their delivery. The index of microcirculatory resistance (IMR) is an invasive method for quantitatively assessing the coronary microcirculation status.

Objectives

To examine heart microcirculation after intracoronary injection of mesenchymal stem/stromal cells with the index of microcirculatory resistance.

Methods

Healthy swine were randomized to receive by intracoronary route either 30x10⁶ MSC or the same solution with no cells (1% human albumin/PBS) (placebo). Blinded operators took coronary pressure and flow measurements, prior to intracoronary infusion and at 5 and 30 minutes post-delivery. Coronary flow reserve (CFR) and the IMR were compared between groups.

Results

CFR and IMR were done with a variance within the 3 transit time measurements of 6% at rest and 11% at maximal hyperemia. After intracoronary infusion there were no significant differences in CFR. The IMR was significantly higher in MSC-injected animals (at 30 minutes, 14.2U vs. 8.8U, p = 0.02) and intragroup analysis showed a significant increase of 112% from baseline to 30 minutes after cell infusion, although no electrocardiographic changes or clinical deterioration were noted.

Conclusion

Overall, this study provides definitive evidence of microcirculatory disruption upon intracoronary administration of mesenchymal stem/stromal cells, in a large animal model closely resembling human cardiac physiology, function and anatomy.     

不稳定心绞痛患者超敏C 反应蛋白水平与病变血管支数的关系

目的:探讨超敏c反应蛋白(hs-CRP)水平对于不稳定心绞痛(UA)患者多支血管病变的诊断价值。方法:回顾性选取不稳定心绞痛患者据其动脉造影结果分为单支病变组、多支病变组均行超敏c反应蛋白检测并比较两组的差异,在阳性结果基础上行ROC曲线分析。结果:1.单支病变组、多支病变组hs-CRP存在显著差异;2.ROC曲线下面积>0.5。结论:针对于不稳定心绞痛患者其超敏c反应蛋白水平与病变血管支数关系密切,对于临床诊断有一定价值。     

Myocardial gene transfer and long-term expression following intracoronary delivery of adeno-associated virus

Adeno-associated viral vectors (AAV) can direct long-term gene expression in post-mitotic cells. Previous studies have established that long-term cardiac gene transfer results from intramuscular injection into the heart. Cardiac gene transfer after direct intracoronary delivery of AAV in vivo, however, has been minimal in degree, and indirect intracoronary delivery, an approach used in an increasing number of studies, appears to be receiving more attention. To determine the utility of indirect intracoronary gene transfer of AAV, we used aortic and pulmonary artery cross clamping followed by proximal aortic injection of AAV encoding enhanced green fluorescent protein (AAV.EGFP) at 10(11) DNase resistant particles (drp; high-performance liquid chromatography (HPLC)-purified) per rat. Gene expression was quantified by fluorescent microscopy at four time points up to 1 year after vector delivery, revealing 20-32% transmural gene expression in the left ventricle at each time point. Histological analysis revealed little or no inflammatory response and levels of transgene expression were low in liver and undetectable in lung. In subsequent studies in pigs, direct intracoronary delivery into the left circumflex coronary artery of AAV.EGFP (2.64-5.28 x 10(13) drp; HPLC-purified) resulted in gene expression in 3 of 4 pigs 8 weeks following injection with no inflammatory response in the heart. PCR analysis confirmed AAV vector presence in the left circumflex perfusion bed. These data indicate that intracoronary delivery of AAV vector is associated with transgene expression in the heart, providing a means to obtain long-term expression of therapeutic genes.     

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.     

Bone marrow cell therapy after acute myocardial infarction: the HEBE trial in perspective, first results

During the last decennium, the role of bone marrow mononuclear cells (BMMC) has been underscored in the healing process after acute myocardial infarction (AMI). Although these cells improve left ventricular recovery after AMI in experimental studies, results from large-scale randomised trials investigating BMMC therapy in patients with AMI have shown contradictory results. To address this issue the HEBE study was designed, a multicentre, randomised trial, evaluating the effects of intracoronary infusion of BMMCs and the effects of intracoronary infusion of peripheral blood mononuclear cells after primary percutaneous coronary intervention. The primary endpoint of the HEBE trial is the change in regional myocardial function in dysfunctional segments at four months relative to baseline, based on segmental analysis as measured by magnetic resonance imaging. The results from the HEBE trial will provide detailed information about the effects of intracoronary BMMC therapy on post-infarct left ventricular recovery. In addition, further analysis of the data and material obtained may provide important mechanistic insights into the contribution of BMMCs to natural recovery from AMI as well as the response to cell therapy. This may significantly contribute to the development of improved cell-based therapies, aiming at optimising post-infarct recovery and preventing heart failure. (Neth Heart J 2008;16:436-9.)     

Effects of exercise training on coronary collateralization and control of collateral resistance

Coronary collateral vessels serve as a natural protective mechanism to provide coronary flow to ischemic myocardium secondary to critical coronary artery stenosis. The innate collateral circulation of the normal human heart is typically minimal and considerable variability occurs in extent of collateralization in coronary artery disease patients. A well-developed collateral circulation has been documented to exert protective effects upon myocardial perfusion, contractile function, infarct size, and electrocardiographic abnormalities. Thus therapeutic augmentation of collateral vessel development and/or functional adaptations in collateral and collateral-dependent arteries to reduce resistance into the ischemic myocardium represent a desirable goal in the management of coronary artery disease. Tremendous evidence has provided documentation for the therapeutic benefits of exercise training programs in patients with coronary artery disease (and collateralization); mechanisms that underlie these benefits are numerous and multifaceted, and currently under investigation in multiple laboratories worldwide. The role of enhanced collateralization as a major beneficial contributor has not been fully resolved. This topical review highlights literature that examines the effects of exercise training on collateralization in the diseased heart, as well as effects of exercise training on vascular endothelial and smooth muscle control of regional coronary tone in the collateralized heart. Future directions for research in this area involve further delineation of cellular/molecular mechanisms involved in effects of exercise training on collateralized myocardium, as well as development of novel therapies based on emerging concepts regarding exercise training and coronary artery disease.     

犬冠状动脉阻力的胆碱能调节

本实验在麻醉开胸犬,采用冠状动脉左旋支恒流灌注,于搏动的和心室纤颤(VF)的心脏,研究了电刺激迷走神经(VNS)及冠状动脉内注入乙酰胆碱(ACh)对冠状动脉阻力的影响。当 VNS 和冠脉内给 ACh 时,(1)心肌内小冠状动脉阻力显著减低,而心外膜大冠状动脉阻力并无明显变化;(2)冠状动脉左旋支总阻力的减低幅度在 VF 的心脏比在搏动的心脏显著减小。以上结果表明,迷走-ACh 扩张冠脉的作用主要是舒张心肌内小冠状动脉,并可通过减低心肌收缩力而间接降低冠状动脉阻力。